Therapeutic and diagnostic methods for cerebral hypoperfusion and related conditions

Abstract

Various embodiments relate generally to the use of EPAS1 inhibitors for treating or preventing cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject. Moreover, various embodiments also relate to methods for diagnosing, or predicting a subject's risk of developing, cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, preferably a cerebrovascular disease, based upon identified biomarker.

Description

THERAPEUTIC AND DIAGNOSTIC METHODS FOR CEREBRAL HYPOPERFUSION AND RELATED CONDITIONSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore Patent Application No. 10202402859S filed 13 September 2024, the content of which being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] Various embodiments relate generally to the use of EPAS1 inhibitors for treating or preventing cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject. Moreover, various embodiments also relate to methods for diagnosing, or predicting a subject’s risk of developing, cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, based upon identified biomarkers.BACKGROUND

[0003] Vascular cognitive impairment is a debilitating condition caused by a reduction in cerebral blood flow, that may or may not be associated with a stroke. This affects cognitive abilities such as executive functioning, which results in functional impairment, thereby contributing to vascular dementia[1]. Chronic cerebral hypoperfusion is frequently observed with ageing and leads to progressive changes to brain vasculatures, including greater tortuosity of the arteries, increased collagen buildup in the veins, and a decline in capillary density[2]. Neuroinflammation has been recognized as a crucial pathological response to cerebral ischemia. However, our understanding of the molecular features of non-neuronal cells, as well as perturbations to the arteriovenous axis in the ischemic brain, remains limited. Endothelial cells maintain the integrity of and regulate the transport across the blood-brain barrier and control neurovascular coupling[3]. Moreover, endothelial cells interact with and influence surrounding parenchymal tissue on their abluminal side. As such, dysfunctional endothelial cells could have widespread consequences for the brain and serve as an initial driver of vascular cognitive impairment.

[0004] Cerebral endothelial cells exist along an arterial-venous transcriptional continuum of gradual phenotypic change along the arteriovenous axis[4]. Neurological diseases arising from vascular pathologies often manifest themselves in a zonation-dependent manner. In cerebral cavernous malformations, loss-of-function mutations in one of the CCM genes lead to vascular malformations primarily of venous origin[5] . Similarly, venous endothelial cells are the primary source of endothelial cells in arteriovenous malformations, where misdirected and highly proliferative venous endothelial cells form a direct shunt between arteries and veins, bypassing capillaries[6]. Venous endothelial cells appear as early responders to oxygen changes, with chronic hypoxia inducing endothelial proliferation and vascular leakiness primarily in post-capillary venules, while arterioles show nochanges in permeability!?]. Therefore, it is hypothesized that intracranial endothelial subtypes differentially affect the zonated patterns of cerebrovascular lesions in chronic cerebral hypoperfusion.

[0005] Currently, there are no approved therapies that directly target the vascular pathology or halt the progression of neurovascular injury driving cerebral hypoperfusion and cognitive decline in associated diseases such as cerebrovascular disorders and vascular cognitive impairment (VC I) . The current standard of care for VCI and vascular dementia focuses on managing underlying vascular risk factors to prevent further cerebrovascular damage. This includes optimal blood pressure control, lipid- lowering therapy (e.g., statins), glycemic control in diabetics, and lifestyle modifications such as smoking cessation, physical activity, and dietary interventions. Cognitive symptoms are managed symptomatically, often with cholinesterase inhibitors or memantine, although these are not approved specifically for VCI.

[0006] Therefore, there remains a need in the art to identify novel therapeutic targets for promoting vascular normalization, particularly for the treatment of cerebral hypoperfusion and associated conditions or diseases, including cerebrovascular disorders. There is also a need to identify biomarkers predictive or indicative of such conditions.SUMMARY

[0007] In one aspect, there is provided an endothelial PAS domain protein 1 (EPAS1) inhibitor for use in treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.

[0008] In various embodiments, the disease or condition associated with cerebral hypoperfusion is selected from the group consisting of cerebrovascular diseases, neuroinflammatory conditions, neurodegenerative disorders, cognitive and neuropsychiatric conditions, and systemic or developmental conditions involving secondary cerebral hypoperfusion.

[0009] In various embodiments, the disease or condition associated with cerebral hypoperfusion is neuroinflammation, or a cerebrovascular disease.

[0010] In various embodiments, the EPAS1 inhibitor is for use in treating cerebral hypoperfusion.

[0011] In various embodiments, the disease or condition associated with cerebral hypoperfusion is vascular cognitive impairment or vascular dementia arising from cerebral hypoperfusion.

[0012] In various embodiments, the EPAS1 inhibitor reduces vascular remodelling, angiogenic sprouting, vein-associated microglial activation, and / or restores cerebral blood flow to achieve vascular normalization.

[0013] In various embodiments, the EPAS1 inhibitor is a small molecule, peptide, nucleic acid-based inhibitor, or an antibody that specifically binds to EPAS1 or its regulatory components.

[0014] In various embodiments, the EPAS1 inhibitor is a small molecule selected from PT2385, PT2399, PT2977, or a pharmaceutically acceptable salt thereof.

[0015] In various embodiments, the EPAS1 inhibitor is PT2385, or a pharmaceutically acceptable salt thereof.

[0016] In another aspect, there is provided apharmaceutical composition comprising an EPAS1 inhibitor and a pharmaceutically acceptable carrier and / or excipient for use in treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.

[0017] In various embodiments of the pharmaceutical composition, the EPAS1 inhibitor is PT2385, or a pharmaceutically acceptable salt thereof.

[0018] In another aspect there is provided a method of treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject, the method comprising administering an effective amount of an EPAS1 inhibitor or pharmaceutical composition disclosed herein, to the subject.

[0019] In another aspect, there is provided the se of an EPAS1 inhibitor in the manufacture of a medicament for treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.

[0020] In various embodiments, the subject is a human subject.

[0021] In another aspect, there is provided method of monitoring the efficacy of an EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in a subject, comprising: (a) administering the EPAS1 inhibitor to the subject; (b) obtaining a biological sample from the subject; and (c) detecting and measuring the amount of CECs and / or ACKR1 + CECs, in the biological sample; and (d) comparing the measured amount of CECs and / or ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and / or ACKR1 + CECs measured in the sample relative to the reference amount is indicative of the efficacy of the EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in the subject, optionally steps (a)-(d) are repeated two or more times within a timeframe and the detection and measurement at each time point is compared against each other toassess the progression of the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion and efficacy of the treatment.

[0022] In various embodiments, the method further comprises comparing cerebral blood flow measurements of the subject obtained by magnetic resonance imaging using arterial spin labelling (ASL) readouts before initiation of treatment and over the course of treatment.

[0023] In another aspect, there is provided a method for diagnosing, or predicting a subject's risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, the method comprising: measuring the amount of circulating endothelial cells (CECs), and CECs that are positive for Atypical Chemokine Receptor f (ACKRf ) in the sample that has been obtained from the subject; and comparing the measured amount of CECs and ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and ACKR1 + CECs measured in the sample relative to the reference amount is indicative of the subject having, or the subject being at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0024] In various embodiments, the amount of CECs is measured by flow cytometry using the markers CD45- / CD31 + / CD133- / DNA+, and a percentage of ACKR1 + CECs is measured relative to the amount of CECs.

[0025] In various embodiments, the biological sample is a blood sample, preferably a PMBC sample.

[0026] In various embodiments, the differential amount comprises an increase in the percentage or absolute abundance of CECs and ACKR1 + CECs in the subject, which is indicative of the subject having, or the subject being at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0027] In various embodiments, the method further comprises administering an EPAS1 inhibitor to the subject for treating, preventing, or ameliorating the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion.

[0028] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a cerebrovascular disease.

[0029] In various embodiments, the condition or disease associated with cerebral hypoperfusion is endothelial dysfunction and / or venous dysfunction.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Various embodiments will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0031] FIG. 1A-1 F shows the phenotyping of vessel subtype-specific response to cerebral hypoperfusion: FIG. 1A Schematic of BOAS performed on a mouse. Created in BioRender. Wazny, V. (2025) https: / / BioRender.com / ujauv4d; FIG. 1 B A time course of percentage of cerebral blood flow changes relative to their respective baseline values of individual mice (n = 11 sham, n = 10 BCAS). Statistical significances reflect differences of sham versus BCAS: FIG. 1 C Representative immunofluorescence images of Pecaml -positive cerebral vessels acquired 10 days or 60 days post- BCAS surgery. Statistical significances reflect differences of day 10 versus day 60 post-BCAS. Scale bars, 100 pm; FIG. 1 D Three-dimensional images of cerebral vasculatures injected with 500 kDa FITC-conjugated dextran. Alpha-smooth muscle actin (aSMA, green) was used to distinguish arterial (striated) from venous (discontinuous aSMA) vessels. Quantitative analysis of the number of sprouts per 100 pm of vessel length was determined. White arrows indicate angiogenic sprouts. Statistical significances reflect differences of sham versus BCAS. Scale bars, 30 pm; FIG. 1 E Representative immunofluorescence images of arterial and venous vessels stained with Pecaml and aSMA (white) at 10 days and 60 days post-BCAS. Quantitative analysis of the lumen diameter measured by the shortest length across every vessel cross-section. Statistical significances reflect differences of sham versus BCAS. Scale bars, 20 pm; and FIG. 1 F Representative immunofluorescence images of eNOS (green) protein expression on Pecaml -positive cerebral vessels (red). Protein expression of eNOS was quantified by eNOS signal intensity per area of the vessel. Statistical significances reflect differences of sham versus BCAS. Scale bars, 20 pm. FIG. 1 B-1 F Data points represent individual animals in mean ± s.d.; unpaired t-test or Mann-Whitney test comparing two groups (two-sided). FIG. 1 C-1 F Sample size: 10 days [n = 12 sham (6 females, 6 males), n = 10 BCAS (5 females, 5 males)] or 60 days [n = 13 sham (6 females, 7 males), n = 10 BCAS (5 females, 5 males)]. Source data are provided as a Source Data file.

[0032] FIG. 2A-2B shows single-cell transcriptomic analysis of mouse prefrontal cortex; FIG. 2A Volcano plots for pseudobulk comparisons of BCAS endothelial subgroups relative to sham; and FIG. 2B Human phenotype ontology of significantly differentially expressed genes of BCAS endothelial subgroups relative to sham, sorted by p-value ranking.

[0033] FIG. 3A-3G shows profiling endothelial cell transcriptomes in response to cerebral hypoperfusion: FIG. 3A Schematic of the single-cell RNA-sequencing workflow on mouse prefrontal cortex; FIG. 3B UMAR visualization of single-cell transcriptomes from BCAS (n = 4, 2 males, 2 females) and sham (n = 4, 2 males, 2 females) mouse brains. Expression patterns of endothelial marker genes (Flt1 , Cldn5, Pecaml ) in dataset have been highlighted on UMAP plots; FIG. 3C Endothelial subtypes identified based on gene expression patterns of published curated dataset. Dotplot showing expression and proportion of cells per endothelial subtypes expressing their respective marker genes; FIG. 3D Proportions of endothelial subtypes obtained from sham versus BOAS groups. Chi-squared goodness-of-fit test (two-sided) was performed to assess the difference in frequencies between the groups; FIG. 3E Numbers of significantly upregulated and downregulated differentially expressed genes (DEGs) for each endothelial subtype in response to BCAS (P-adjusted value < 0.05, Benjamini-Hochberg method); FIG. 3F Bar graphs of top 5 enriched gene ontology biological process clusters, based on differentially upregulated genes for each endothelial subtype, sorted by P-value. T erms with a p-value < 0.01 , a minimum count of 3, and an enrichment factor > 1 .5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) are collected and grouped into clusters based on their membership similarities. More specifically, p-values are calculated based on the cumulative hypergeometric distribution, and q-values are calculated using the Benjamini-Hochberg procedure to account for multiple testings. Kappa scores are used as the similarity metric when performing hierarchical clustering on the enriched terms, and sub-trees with a similarity of > 0.3 are considered a cluster. The most statistically significant term within a cluster is chosen to represent the cluster; FIG. 3G Venn diagram showing the overlap of significantly upregulated DEGs (P-adjusted value < 0.05, Benjamini-Hochberg method) related to various biological processes in BCAS venous endothelial cells. Bolded genes refer to transcription factors.

[0034] FIG. 4A-4I shows single-cell transcriptomic analysis of mouse prefrontal cortex. Quality control (QC) metrics were used to assess the quality of single-cell RNA-seq data for all samples. QC thresholds were applied to filter out low-quality cells: FIG. 4A Total counts per cell, illustrating the relationship between the total number of counts (UMI) and the number of detected genes for each cell. Cells with low total counts or few detected genes were flagged as low quality: FIG. 4B Detected genes per cell versus mitochondrial gene percentage. Cells with high mitochondrial gene content (>20%) were flagged as low quality; FIG. 4C Violin plots of total counts per cell across samples; FIG. 4D Violin plots of total detected genes for cells across samples; FIG. 4E Violin plots of the percentage of mitochondrial gene content across samples; FIG. 4F Bar graphs of enriched gene ontology biological process clusters, based on differentially upregulated genes for each endothelial subtype, sorted by P-value; FIG. 4G Dot plot showing gene expression in venous endothelial cells in aged versus young mouse brains; FIG. 4H Dot plot showing expression of vasodilatory genes and proportion of positively expressing cells in venous population; FIG. 4I UMAP visualisation (without batch correction) of endothelial cells coloured according to experimental groups - sham and BCAS (left), as well as individual animals.

[0035] FIG. 5A-5F shows the differential responses of human arterial and venous endothelial cells to 1% oxygen: FIG. 5A Schematic of the endothelial differentiation system. Human pluripotent stem cells were differentiated into primitive streak, then dorsal lateral mesoderm, and subsequently into arterial and venous endothelial cells; FIG. 5B Heatmap showing the expression profile of upregulated genes associated with angiogenesis, blood vessel morphogenesis and hypoxia in BOAS venous cells,and mapped onto the single-cell transcriptomics of human pluripotent stem cells (H1), primitive streak, lateral mesoderm, pre-vein, and venous endothelial cell populations. The colour intensity indicates the expression level; FIG. 5C Time course of relative ID1 gene expressions comparing arterial and venous endothelial cells under 1 % oxygen conditions (n = 3 biological replicates); FIG. 5D Representative immunofluorescence images of arterial and venous endothelial cells stained for EPAS1 and nuclei (DAPI) in normoxia and after 4 hours of 1% oxygen exposure. Scale bar, 50 pm; FIG. 5E Quantification of the fold change of % EPAS1 colocalization with the nuclei over time comparing arterial and venous cells (n = 3 biological replicates, n = 3 field of view per replicate); and FIG. 5F Venous endothelial tube formation assay under 21 % and 1 % oxygen conditions, both untreated and treated with PT2385. Quantification was based on the number of endothelial tube-like structures (n = 3 biological replicates). Scale bar, 400 pm. FIG. 5C, 5E and 5F Data points represent mean ± s.d.; unpaired t-test comparing two independent groups (two-sided), one-way ANOVA for multiple comparisons (two-sided). Source data are provided as a Source Data file.

[0036] FIG. 6A-6H shows venous remodelling in response to cerebral hypoperfusion is pharmacologically reversible: FIG. 6A Timeline for BOAS / sham surgery, drug administration, and cerebrovascular phenotyping; FIG. 6B Percentage of cerebral blood flow changes relative to their respective baseline values of individual mice comparing sham + vehicle (n = 11 ), BOAS + vehicle (n = 10), and PT2385-treated BOAS (n = 12) mice at 40 days post-BCAS; FIG. 6C Representative immunofluorescence images of Pecaml -positive cerebral vessels acquired 60 days post-BCAS surgery. Scale bar, 100 pm; FIG. 6D Three-dimensional images of cerebral vasculatures injected with 500 kDa FITC-conjugated dextran (green). Alpha-smooth muscle actin (aSMA, green) was used to distinguish arterial (striated) from venous (discontinuous aSMA) vessels. Quantitative analysis of the number of sprouts per 100 pm of vessel length was determined. White arrows indicate angiogenic sprouts. Scale bars, 30 pm; FIG. 6E Representative immunofluorescence images of microglia stained with Iba1 (green) against Pecaml -positive vessels. Scale bars, 20 pm; FIG. 6F Circle plot showing the number of ligand-receptor (L-R) interactions between pairwise cell populations among the endothelial cell subtypes and microglial populations in sham and BCAS groups. aEC, arterial; capEC, capillary; vcapEC, venous capillary; vEC, venous endothelial cells; FIG. 6G Representative immunofluorescence images of Iba1 -positive microglia juxtaposed on arterial or venous structures. The average number of microglial cell bodies per vessel (three independent vessels) was measured in every animal, with at least 80% of each rendered spot of microglial cell body colocalizing with the vessels. Arrows indicate examples of vessel-associated microglia. Scale bars, 20 pm; and FIG. 6H Ramification index of vein- and artery-associated microglia to characterize microglia activation state. Scale bars, 5 pm. FIG. 6C-6E, 6G, 6HData points represent individual animals in mean ± s.d.; oneway ANOVA or Kruskal-Wallis test for multiple comparisons (two-sided). Sample size at 60 days post- BCAS: n = 13 sham + vehicle (6 females, 7 males), n = 10 BCAS + vehicle (5 females, 5 males) and n = 12 BCAS (7 females, 5 males). Source data are provided as a Source Data file.

[0037] FIG.7A-7D shows ACKR1 is a candidate marker for human cerebral venous endothelial cells: FIG. 7A Expression of ACKR1 in various cell types of human brain single-nucleus transcriptomes (accession code for top: GSE173731 , accession code for bottom: GSE163577). Data were generated with an associated web-based visualization tool; FIG. 7B UMAR visualization of prefrontal cortex cells (left), and Epasl expression from sham (n = 4) and BOAS (n = 4) mice (right); FIG. 7C Fold change of Epasl expression comparing the four endothelial subtypes; and FIG. 7D Epasl expression in microglia population of sham and BOAS mice.

[0038] FIG. 8A-8D shows behavioural assessments of cognitive and motor functions in sham and BOAS mice: FIG. 8A Percentage of spontaneous alternation in the Y-maze test to evaluate spatial working memory by measuring the tendency of mice to explore new arms of the maze; FIG. 8B Total time spent per alternation pattern in the Y-maze; FIG. 8C Total distance travelled in the open field test, assessing locomotor activity and general exploratory behavior; and FIG. 8D Number of corner zone immobile episodes in the open field test, representing anxiety-like behavior. Data points represent mean ± s.d. comparing sham + vehicle (n = 11 ), BCAS + vehicle (n = 10), and PT2385- treated BCAS (n = 12) mice; one-way ANOVA or Kruskal-Wallis test for multiple comparisons; *p<0.05 and ns, non-significant.

[0039] FIG. 9A-9E shows circulating venous endothelial cell levels are elevated in human subjects with cerebrovascular disease burden: FIG. 9A Schematic of study workflow. Human subjects underwent blood sample collection, magnetic resonance imaging and arterial spin labelling. Flow cytometry analysis on peripheral blood mononuclear cells (PBMCs) was used to identify circulating endothelial cells (CECs) based on the immunophenotypic markers of CD45- / CD31 + / CD133- / DNA+, followed by characterization with brain venous marker, ACKR1 ; FIG. 9B Spearman’s correlation analysis between cerebral tissue perfusion, measured by arterial spin labelling, with the number of CECs per million PBMCs. Subjects were grouped by Fazekas scores. Spearman's correlation coefficient r and p values (two-tailed test) are indicated; FIG. 9C Quantification of the number of CECs per million PBMCs (left), and percentage of ACKR1 + CECs (right) in subjects grouped by presence (Fazekas > 0, n = 39 individual participants) or absence (Fazekas = 0, n = 5 individual participants) of cerebrovascular disease burden. Data points represent mean ± s.e.m.; Mann-Whitney test (two- sided); *p < 0.05 and ns, non-significant; FIG. 9D Correlation analysis between the percentage of ACKR1 + CECs and Z-scores of various cognitive functions. Spearman’s correlation coefficient r and p values (two-tailed test) are indicated for executive function Z-scores. Pearson’s correlation coefficient r and p values (two-tailed) are indicated for language and global cognitive Z-scores; FIG. 9E Proportional analysis of zero (0), low (<95th percentile), and high (>95th percentile) percentages of ACKR1 + CECs in subjects grouped by negative and positive global cognitive Z-scores. Source data are provided as a Source Data file.

[0040] FIG. 10 shows gating for ACKR1 -positive circulating endothelial cells (CECs). Gating of ACKR1 + CECs based on fluorescence minus one (FMO) control (left). Example of stained sample with ACKR1 expressing CECs (right).DETAILED DESCRIPTION

[0041] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in context of the EPAS1 inhibitor and compositions are analogously valid for the respective methods, uses and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will prevail. "About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[0043] The present inventors discovered that endothelial PAS domain protein 1 (EPAS1 ), also known as hypoxia-inducible factor 2 alpha (Hif2a), may be targeted as a therapeutic strategy for vascular normalization in slowing the progression of cerebrovascular diseases. In particular, it was shown that the inhibition of EPAS1 mitigated vascular remodeling and dampened vein-associated microglial activation in a mouse model of chronic cerebral hypoperfusion induced by bilateral carotid artery stenosis (BCAS model) which is known to replicate key features of vascular dementia, including white matter changes, blood-brain barrier disruption, and cognitive impairment, suggesting that modulating chronic angiogenesis might have a role in reducing neuroinflammation. In particular, it was shown that BCAS induces chronic angiogenic sprouting, and EPAS1 was shown as the molecular driver of venous remodelling in vivo, as well as in in vitro human isogenic arterial and venous cellular system. Rescue experiments in BCAS mice using an inhibitor of EPAS1 was shown to reduce the chronic vascular remodelling, suppress vein-associated microglial activation and restore cerebral blood flow to the level of sham controls.

[0044] Notably, pharmacological inhibition of EPAS1 in vivo has been shown to mitigate chronic vascular sprouting, dampens vein-associated microglial activation and restores cerebral blood flow.Here, it was shown that EPAS1 represents a therapeutic target for vascular normalization to potentially alleviate neuroinflammation, cerebral hypoperfusion and related conditions or diseases.

[0045] In particular, the present inventors demonstrated that inhibiting EPAS1 , through use of a small molecule drug, PT2385 as a representative example, effectively reversed the venous response to low oxygen, both in vitro and in vivo, and specifically reduced the density and activation of vein- associated microglia. Vessel-associated microglia was shown to play a role in cerebral blood flow regulation during hypoperfusion. Microglial P2RY12 contributes to cerebrovascular adaptation during common carotid artery occlusion, and its blockade reduces cerebral blood flow without affecting neuronal responses. The microglial processes around capillaries correlate with cerebral blood flow levels during transient ischemia, in turn indicating blood flow's role in microglial activation. In systemic inflammation, microglia migrate towards cerebral vessels to initially protect their integrity, then transform into a reactive state that results in widespread neuroinflammation. In vitro evidence further suggests that damaged endothelium can promote microglia to transition toward an inflammatory phenotype. Thus, decreased vein-associated microglial density and activation following EPAS1 inhibition (e.g. using PT2385) here may be attributable to the resolution of venous dysfunction and impaired blood flow.

[0046] Accordingly, in one aspect, the present invention provides an EPAS1 inhibitor for use in treating, preventing, or ameliorating cerebral hypoperfusion in a subject. In another aspect, the present invention provides an EPAS1 inhibitor for use in treating, preventing, or ameliorating a disease or condition associated with cerebral hypoperfusion in a subject.

[0047] As used herein, the terms "treating" and "treatment" refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage. As used herein, the term "preventing” refers to the prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented and those in whom reoccurrence of the disorder needs to be prevented. In various embodiments, the terms “treating”, “ameliorating”, “delaying” or “preventing”, as used herein refer to achieving one or more of the following in the subject: (a) reducing the severity of a given condition; (b) limiting or preventing the development of a condition; (c) removing a given condition; (d) limiting or preventing the recurrence of a given condition; (e) alleviation of the condition and / or its symptoms; and (f) delay the onset of a condition In particular, the therapeutic terms “treating, ameliorating or preventing”, may refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention or treatment.

[0048] As used herein, the term "cerebral hypoperfusion" refers to a pathological condition characterized by a sustained or transient reduction in cerebral blood flow below the threshold required to maintain normal neuronal function and cerebral homeostasis. This reduction may be focal or global and may arise due to systemic, cardiac, or vascular factors. Cerebral hypoperfusion is a pathophysiological state that can contribute to, exacerbate, or result from various neurological and vascular disorders. In various embodiments, cerebral hypoperfusion leads to metabolic stress, neuroinflammation, white matter injury, demyelination, or neuronal loss, and is implicated in the development or progression of cerebrovascular diseases, neurodegenerative disorders with vascular involvement, and other hypoxia-related brain conditions.

[0049] As used herein, the term “a disease or condition associated with cerebral hypoperfusion” refers to any pathological state or condition / disease in which a sustained or transient reduction in cerebral blood flow contributes to the onset, progression, exacerbation, or clinical manifestation of disease. Such diseases or conditions may be broadly classified into one or more of the following categories: (i) cerebrovascular diseases, including ischemic stroke, watershed infarcts, transient ischemic attacks (TIAs), cerebral small vessel disease, chronic cerebral ischemia, carotid artery stenosis, vascular dementia, post-stroke cognitive impairment, and vascular cognitive impairment (VCI); (ii) endothelial and vascular dysfunctions, including endothelial dysfunction, impaired cerebrovascular autoregulation, venous dysfunction (e.g., venous outflow impairment, venous congestion, or venous insufficiency), microvascular rarefaction, and blood-brain barrier disruption, which contribute to or result from cerebral hypoperfusion; (Hi) neuroinflammatory conditions associated with or exacerbated by impaired perfusion, such as hypoperfusion-related neuroinflammation, white matter lesions, white matter hyperintensities, age-related microglial activation, and inflammation-driven cognitive decline; (iv) neurodegenerative disorders with vascular or inflammatory components, including Alzheimer's disease with a vascular component, mixed dementia, and Lewy body dementia with cerebral hypoperfusion; (v) cognitive and neuropsychiatric conditions, such as mild cognitive impairment with vascular features, vascular depression, and postoperative cognitive dysfunction; and (vi) systemic or developmental conditions involving secondary cerebral hypoperfusion, including congestive heart failure-related cerebral hypoperfusion, hypotension-induced brain injury, obstructive sleep apnea, periventricular leukomalacia (PVL), and neonatal hypoxic-ischemic encephalopathy (HIE). In various embodiments, the disease or condition may be characterized by cognitive impairment, neuronal loss, demyelination, neuroinflammation, or other pathological changes resulting from reduced cerebral blood flow.

[0050] In various embodiments, the disease or condition associated with cerebral hypoperfusion is selected from the group consisting of cerebrovascular diseases, neuroinflammatory conditions, neurodegenerative disorders, cognitive and neuropsychiatric conditions, and systemic or developmental conditions involving secondary cerebral hypoperfusion.

[0051] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a cerebrovascular disease. As used herein, the term “cerebrovascular disease” refers to any disorder, condition, or pathology primarily involving the blood vessels of the brain or leading to disrupted cerebral blood flow, including both ischemic and haemorrhagic mechanisms. Cerebrovascular diseases may arise from vessel occlusion, rupture, malformation, inflammation, or dysfunction, and encompass clinical syndromes associated with acute or chronic vascular compromise. In various embodiments, cerebrovascular disease includes, but is not limited to: ischemic stroke, haemorrhagic stroke, transient ischemic attacks (TIAs), cerebral small vessel disease, vascular dementia, cerebral amyloid angiopathy, carotid artery stenosis, chronic cerebral hypoperfusion syndromes, arteriovenous malformations (AVMs), moyamoya disease, and cerebral venous sinus thrombosis. These conditions may present with imaging-detected vascular lesions, cognitive impairment, focal neurological deficits, or white matter changes, and may be associated with risk factors such as hypertension, diabetes mellitus, atherosclerosis, and chronic heart failure.

[0052] In various embodiments, the cerebrovascular disease is vascular dementia. In various embodiments, the cerebrovascular disease is cerebral small vessel disease, which is strongly linked to chronic cerebral hypoperfusion and is characterized by abnormal angiogenesis, venous remodelling, and white matter changes.

[0053] In various embodiments, the disease or condition associated with cerebral hypoperfusion may be vascular cognitive impairment or vascular dementia associated with cerebral hypoperfusion in a subject. In various embodiments, the EPAS1 inhibitor may be used for treating, preventing, or ameliorating vascular cognitive impairment or vascular dementia arising from cerebral hypoperfusion in a subject. In various embodiments, the disease or condition associated with cerebral hypoperfusion may be vascular cognitive impairment that may lead to vascular dementia.

[0054] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a neuroinflammatory condition. The treatment of neuroinflammation associated with cerebral hypoperfusion, may be carried out by dampening activation of vein-associated microglia. Microglial activation in response to hypoperfusion contributes to blood-brain barrier disruption and white matter injury.

[0055] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a neurodegenerative disorder.

[0056] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a cognitive and neuropsychiatric condition.

[0057] In various embodiments, the disease or condition associated with cerebral hypoperfusion isa systemic or developmental conditions involving secondary cerebral hypoperfusion.

[0058] In various embodiments, inhibition of EPAS1 used for the therapeutic benefit in the treatment, prevention, or amelioration of cerebral hypoperfusion, or diseases or conditions associated with cerebral hypoperfusion, may have one or more of the following effects:(i) Restoring cerebrovascular homeostasis by normalizing cerebral blood flow, reducing pathological angiogenesis, and resolving venous endothelial dysfunction; and / or(ii) Suppressing microglial activation, particularly of vein-associated microglia, thereby reducing neuroinflammatory damage that contributes to cognitive impairment: and / or(iii) Mitigating vascular remodelling and abnormal angiogenic sprouting, stabilizing the cerebral vasculatures, and preventing white matter damage and disease progression, thereby preserving cognitive function; and / or(iv) Decreases circulating endothelial cell (CEC) levels and ameliorates venous dysfunction, offering a vascular-protective strategy for hypoperfusion-associated diseases.

[0059] The above effects may collectively target key pathological consequences of impaired cerebral hypoperfusion. In various embodiments, the EPAS1 inhibitor reduces vascular remodelling, angiogenic sprouting, vein-associated microglial activation, and / or restores cerebral blood flow to achieve vascular normalization in the subject.

[0060] The term EPAS1 , as used herein, may also refer to Hif2a, and thus to the same gene and protein product. The EPAS1 may be of any species of origin, and is preferably a human EPAS1. Nucleic acid and amino acid sequences for EPAS1 are known in the art. See, for example, Homo sapiens endothelial PAS domain protein 1 (EPAS1 ), mRNA NCBI Reference Sequence: NM_001430.4; NCBI Reference Sequence: NP_001421.2; and UniProtKB Q99814 (EPAS l_HUMAN). In various embodiments, the EPAS1 polypeptide or nucleic acid comprises or consists of a sequence of the human HIF-2a protein sequence set forth in NCBI Accession No. NP 001421.2 or UniProt Accession No. Q99814, or the coding nucleotide sequence of NCBI Accession No. NM 001430.5, or functional variants thereof.

[0061] Generally, the term “functional variant” covers such EPAS1 polypeptides and encoding nucleic acid that have at least 80%, or at least 90% sequence identity with the reference amino acid or nucleotide sequence over their entire length, preferably at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity. The identity of amino acid or nucleotide sequences is generally determined by means of a sequence comparison. This sequence comparison is based on the BLAST algorithm that is established in the existing art and commonly used (cf. e.g. Altschul et al. (1990) “Basic local alignment search tool”, J. Mol. Biol. 215:403-410, and Altschul et al. (1997): “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”; Nucleic Acids Res., 25, p. 3389-3402) and is effected in principle by mutually associatingsimilar successions of amino acids in the amino acid sequences or nucleotides in the nucleotide sequence, respectively. A tabular association of the relevant positions is referred to as an "alignment." Sequence comparisons (alignments), in particular multiple sequence comparisons, are commonly prepared using computer programs which are available and known to those skilled in the art.

[0062] In various embodiments, the EPAS1 inhibitor may be any compound, agent, or composition capable of inhibiting the expression, translation, stabilization, dimerization, DNA binding, or transcriptional activity of EPAS1. Suitable EPAS1 inhibitors include, but are not limited to, small molecules, peptides, nucleic acid-based inhibitors (e.g., antisense oligonucleotides, siRNAs, shRNAs, or CRISPR-based gene editing constructs), or antibodies that specifically bind to EPAS1 or its regulatory components. In various embodiments, the EPAS1 inhibitor may be termed as an EPAS1 antagonist, referring to a molecule or compound that prevents, inhibits or reduces EPAS1 activation and function.

[0063] In various embodiments, the EPAS1 inhibitor may be a small molecule inhibitor. The small molecule inhibitor may be organic or inorganic compounds, and may be selected from PT2385, PT2399, and PT2977 (also known as belzutifan or MK-6482), which disrupt the PAS-B domain of EPAS1 and prevent dimerization with ARNT, thereby inhibiting hypoxia-inducible gene transcription: FM19G1 1 (a dual HIF-1a / HIF-2a inhibitor), HIF2VI (a structure-guided small molecule that binds the PAS-B domain of HIF-2a to prevent dimerization with ARNT), PT3400074, C76, atractylenolide I (ATL-I), IPHBA, YC-1 , acriflavine, NSC-134754, digoxin; and 4-(2-(4- isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA); and compounds as disclosed in WO2016 / 196239 A1 and WO2019 / 100053 A1 .

[0064] In various embodiments, the EPAS1 inhibitor is PT2385. PT2385 disrupts EPAS1 function by binding to a ligand-accessible cavity within its PAS-B domain, thereby preventing heterodimerization with ARNT. The compound PT2385 has the chemical formula:

[0065] The small molecule PT2385 may be defined as having the CAS No: 1672665-49-4; formula: C17H12F3NO4S, and the IUPAC Name: (S)-3-((2,2-difluoro-1-hydroxy-7-(methylsulfonyl)-2,3- dihydro-1H-inden-4-yl)oxy)-5-fluorobenzonitrile.

[0066] In various embodiments, the EPAS1 inhibitor is PT2399. The compound PT2399 has the chemical formula:

[0067] The small molecule PT2399 may be defined as having the CAS No: 1672662-14-4: formula: C17H10F5NO4S, and the IUPAC Name: 3-[[(1 S)-7-(d!fluoromethylsulfonyi)-2,2-difluoro-1 - hydroxy-1 ,3-dihydroinden-4-yi]oxy]-5- fluorobenzonitrile.

[0068] In various embodiments, the EPAS1 inhibitor is PT2977. PT2977 is an orally administrable selective small molecule HIF-2a inhibitor with improved potency compared to PT2385. The compound PT2977 has the chemical formula:

[0069] The small molecule PT2977 may be defined as having the CAS No: 1672668-24-4; formula: C17H12F3NO4S, and the IUPAC Name: 3-[[(1 S,2S,3fl)-2,3-difluoro-1-hydroxy-7- methylsulfonyl-2,3-dihydro-1 H-inden-4-yl]oxy]-5-fluorobenzonitrile.

[0070] In various embodiments, analogues, derivatives, stereoisomers (including enantiomers and diastereomers), tautomers, salts, solvates, hydrates, polymorphs, and prodrugs of PT2385, PT2399, and PT2977 are also encompassed within the scope of suitable EPAS1 inhibitors. These include compounds with chemical or structural modifications that retain the ability to bind the PAS-B domain of EPAS1 and inhibit its function. In various embodiments, the EPAS1 inhibitor may be a racemic mixture, a single enantiomer, or an optically pure isomer.

[0071] In various embodiments, the EPAS1 inhibitor may be a nucleic acid-based inhibitor. Thenucleic acid-based inhibitor may be an EPAS1 -targeting antisense oligonucleotide (ASOs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and CRISPR-based genome or transcriptome editing constructs that inhibit EPAS1 expression at the mRNA or genomic level. An example may be EZN-2968 (an ASO originally developed for HIF-1a) but designed to specifically hybridize to EPAS1 transcripts. In various embodiments, such nucleic acid-based inhibitors are delivered using gene therapy vectors, including viral vectors (such as adeno-associated virus (AAV), lentivirus, or adenovirus) or non-viral delivery systems (such as lipid nanoparticles (LNPs)), wherein the vector comprises a nucleotide sequence encoding or enabling expression of the EPAS1 -targeting agent.

[0072] In various embodiments, the EPAS1 inhibitor may be a peptide or peptidomimetic inhibitor. The peptide or peptidomimetic inhibitor may interfere with EPAS1 protein interactions, stability, DNA binding activity, its nuclear localization, or prevent binding to hypoxia response elements (HREs). Peptides mimicking interaction motifs (e.g., EPAS1-ARNT dimerization domain or EPAS1 -cofactor interfaces) can be designed to competitively inhibit binding. Such peptides may require cellpenetrating modifications (like TAT-fusion, stapled peptides, or nanoparticle delivery).

[0073] In various embodiments, the EPAS1 inhibitor may be an antibody-based inhibitor (anti- EPAS1 antibody). The antibody-based inhibitor may comprise monoclonal antibodies or antibody fragments that bind EPAS1 or its interaction partners (e.g. ARNT) to inhibit EPAS1 function. In various embodiments, the antibody-based inhibitor may be intrabodies that are engineered antibodies expressed inside cells capable of binding EPAS1 intracellularly. In various embodiments, the antibody-based inhibitor may be antibody-drug conjugates (ADCs) or nanobody fusions.

[0074] In various embodiments, the EPAS1 inhibitor or a pharmaceutically acceptable salt thereof, may be used for said treatment.

[0075] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the parent compound (i.e. EPAS1 inhibitor) without being toxic to the subject. Such salts include, but are not restricted to: (1 ) an acid addition salt which is obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like, preferably hydrochloric acid or (L)-malic acid; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e. g., an alkali metal ion, such as sodium or potassium, an alkaline earth ion, such as magnesium or calcium, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. For more specific,non-limiting examples see, for instance, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1 -19).

[0076] In this regard, the invention also relates to the use of the EPAS1 inhibitor as a pharmaceutical. The EPAS1 inhibitor is thus contemplated for use as a pharmaceutical in treating or preventing cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject. Accordingly, there is also provided a pharmaceutical composition comprising the EPAS1 inhibitor, and a pharmaceutically acceptable carrier and / or excipient. A "pharmaceutical composition" refers to a mixture of one or more of the compounds described herein, or physiologically / pharmaceutically acceptable salts thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the EPAS1 inhibitor to the subject.

[0077] The pharmaceutical composition may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, caplets, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington: The Science and Practice of Pharmacy, cited above. In various embodiments, the pharmaceutical composition may be formulated as a tablet to be administered orally.

[0078] Pharmaceutical compositions comprising a EPAS1 inhibitor in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in any conventional manner by mixing, granulating, or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and / or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host.

[0079] In various embodiments, the composition may be formulated for oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), intranasal, transdermal, intracerebroventricular, or intrathecal administration, depending on the formulation and therapeutic objective.

[0080] In various embodiments, the EPAS1 inhibitor, or composition, is to be administered in a therapeutically effective amount sufficient to reduce or inhibit EPAS1 expression or activity in a subject in need thereof, for the treatment or prevention of cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion.

[0081] An “effective amount”, as used herein, relates to an amount that is sufficient to provide a desired effect, including preventing, reducing the risk of being afflicted by, alleviating and abating a disease and / or its attendant symptoms. This applies to terms used herein, such as “therapeutically effective amount” (alleviating and abating a disease and / or its attendant symptoms) and “prophylactically effective amount” (preventing, reducing the risk of being afflicted by a disease and / or its attendant symptoms).

[0082] Accordingly, in one aspect, there is provided a method of treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject, the method comprising administering an effective amount of the EPAS1 inhibitor or pharmaceutical composition disclosed herein, to the subject. There is also provided the use of the EPAS1 inhibitor in the manufacture of a medicament or pharmaceutical composition, for treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in the subject.

[0083] The EPAS1 inhibitor or composition may be administered to the subject orally, parenterally, rectally, vaginally, buccally, sublingually, nasally, by inhalation, topically, transdermally, or via an implanted reservoir in dosage forms containing conventional non-toxic pharmaceutically acceptable carriers and excipients. The term "parenteral" as used herein is intended to include subcutaneous, intravenous, and intramuscular injection. The amount of the EPAS1 inhibitor administered will, of course, be dependent on the particular active agent, the condition or disorder being treated, the severity of the condition or disorder, the subject's weight, the mode of administration and other pertinent factors known to the prescribing physician. In various embodiments, the EPAS1 inhibitor or composition may be administered in a solid dosage form (e.g., tablet, capsule), liquid solution, injectable formulation, or sustained-release system. The dosing regimen may be adjusted based on age, body weight, renal or hepatic function, and plasma drug concentrations.

[0084] In various embodiments, the EPAS1 inhibitor may be administered continuously or intermittently over a period of 1 day to 6 months, such as 7 to 90 days, or 2 to 12 weeks, dependingon disease progression and patient response. The treatment may be repeated in cycles, or maintained as a long-term therapy. The timing of the administration of the EPAS1 inhibitor or composition will also depend on the formulation and / or route of administration used. The EPAS1 inhibitor or composition may be administered once daily, but may also be administered two, three or four times daily, or every other day, or once or twice per week. For example, the subject can be administered one or more treatments 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours, days, weeks, or months apart. In various embodiments, the EPAS1 inhibitor may be administered once a day, however, it will be appreciated that administration of the EPAS1 inhibitor may be continued daily until an improvement of the treatment is apparent or an unacceptable toxicity is detected in the subject.

[0085] The precise dosage of the EPAS1 inhibitor will vary according to a variety of factors including but not limited to the inhibitor that is selected and subject- dependent variables (e.g., age, immune system health, clinical symptoms etc.). In cases of a solid dosage form, examples of daily dosages of the EPAS1 inhibitor which can be used are an effective amount within the dosage range of about 0.001 mg to about 2 mg per kilogram of body weight, about 0.001 mg to about 5 mg per kilogram of body weight, about 0.001 mg to about 10 mg per kilogram of body weight, about 0.001 mg to about 20 mg per kilogram of body weight, about 0.001 mg to about 50 mg per kilogram of body weight, about 0.001 mg to about 100 mg per kilogram of body weight, about 0.001 mg to about 200 mg per kilogram of body weight, or about 0.001 mg to about 300 mg per kilogram of body weight.

[0086] In various embodiments, when administered orally or by inhalation, examples of daily dosages are an effective amount within the dosage range of about 0.1 mg to about 10 mg, or about 0.1 mg to about 20 mg, or about 0.1 mg to about 30 mg, or about 0.1 mg to about 40 mg, or about0.1 mg to about 50 mg, or about 0.1 mg to about 60 mg, or about 0.1 mg to about 70 mg, or about0.1 mg to about 80 mg, or about 0.1 mg to about 90 mg, or about 0.1 mg to about 100 mg, or about 0.1 mg to about 200 mg, or about 0.1 mg to about 300 mg, or about 0.1 mg to about 400 mg, or about0.1 mg to about 500 mg, or about 0.1 mg to about 600 mg, or about 0.1 mg to about 700 mg, or about0.1 mg to about 800 mg, or about 0.1 mg to about 900 mg, or about 0.1 mg to about 1 g, or about 20 mg to 300 mg, or about 20 mg to 500 mg, or about 20 mg to 700 mg, or about 20 mg to 1000 mg, or about 50 mg to 1500 mg, or about 50 mg to 2000 mg.

[0087] In various embodiments, the EPAS1 inhibitor may be administered in daily doses of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, or about 2000 mg, independently of body weight. However, it is understood that pediatric patients may requiresmaller dosages, and depending on the severity of the disease and condition of the patient, dosages may vary.

[0088] In various embodiments, when formulated as a liquid, the concentration of the EPAS1 inhibitor may be about 0.01 mg / ml to about 0.1 mg / ml or about 0.1 mg / ml to about 1 mg / ml, but can also be about 1 mg / ml to about 10 mg / ml or about 10 mg / ml to about 100 mg / ml. The liquid formulation could be a solution or a suspension. When formulated as a solid, for example as a tablet or as a powder for inhalation, the concentration, expressed as the weight of a compound divided by total weight, will typically be about 0.01% to about 0.1%, about 0.1% to about 1%, about 1 % to about 10%, about 10% to about 20%, about 20% to about 40%, about 40% to about 60%, about 60% to about 80%, or about 80% to about 100%.

[0089] In various embodiments, the EPAS1 inhibitor and compositions may be formulated for extended release. For example, the EPAS1 inhibitor or composition can be suitable for administration once daily or less. In various embodiments, the EPAS1 inhibitor or composition may be only administered to the subject once every 24-48 hours. In various embodiments, administration of the EPAS1 inhibitor or composition will be given as a long-term treatment regimen whereby pharmacokinetic steady state conditions will be reached.

[0090] In various embodiments, the EPAS1 inhibitor is a small molecule, such as PT2385, and may be administered orally, once or twice daily, at a dose ranging from about 5 mg to about 1000 mg per day, such as from about 10 mg to about 500 mg per day, or more typically from about 50 mg to about 200 mg per day. In various embodiments, the EPAS1 inhibitor is PT2385, administered at a dose of approximately 100 mg once daily, or 50 mg twice daily, based on clinical studies showing tolerability and blood-brain barrier penetration.

[0091] In various embodiments, the method for treating cerebral hypoperfusion or a disease or condition associated with cerebral hypoperfusion in a subject, may comprise: (i) obtaining a biological sample from the subject; (ii) detecting and / or quantifying the expression level of EPAS1 in the sample: and (iii) administering to the subject an effective amount of an EPAS1 inhibitor, wherein the administration is based at least in part on the detected and / or quantified level of EPAS1 expression. In various embodiments, the EPAS1 expression level is indicative of suitability for EPAS1 inhibitor treatment, such that elevated EPAS1 expression is predictive of a therapeutic response.

[0092] In various embodiments, the biological sample may comprise brain tissue, cerebrospinal fluid, or a peripheral blood sample, including isolated circulating endothelial cells (CECs), peripheral blood mononuclear cells (PBMCs), or endothelial progenitor cells. EPAS1 expression may be measured at the mRNA level using quantitative reverse transcription PCR (qRT-PCR), digital droplet PCR, or RNA sequencing. Alternatively, EPAS1 expression may be assessed at the protein level usingimmunohistochemistry ( I HC) , immunofluorescence, Western blotting, or flow cytometry with EPAS1 - specific antibodies. In various embodiments, in situ hybridization (ISH) or multiplex immunoassay platforms may be used for tissue-based or cell-based detection.

[0093] In various embodiments, the EPAS1 expression level may also be monitored before and after treatment to evaluate the efficacy of the EPAS1 inhibitor. A reduction in EPAS1 expression following administration may be indicative of a favorable therapeutic response. In various embodiments, EPAS1 expression may be evaluated alongside other biomarkers (e.g., CEC, and / or ACKR1+CEC amounts) to stratify patients, guide dosing regimens, or monitor disease progression.

[0094] In various embodiments, the EPAS1 inhibitor may be administered alone or in combination with one or more additional therapeutic agents for the treatment, prevention, or modulation of cerebral hypoperfusion, a disease or condition associated with cerebral hypoperfusion, such as vascular dysfunction, or neuroinflammation. Accordingly, in various embodiments, the EPAS1 inhibitor or composition may be co-administered with an additional therapeutic agent, such as an antiinflammatory agent, neuroprotective agent, antioxidant, or a vascular remodelling modulator.

[0095] The terms “co-administration" or “combined administration” or the like as utilized herein are meant to encompass administration of the selected therapeutic agents (EPAS1 inhibitor) to a single subject and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

[0096] As used herein, the term “subject" is used interchangeably with "individual" and "patient" herein and refers to a warm-blooded animal, preferably a mammal, more preferably a human. Said subject may be awaiting or receiving medical care or is or will become the subject of a medical procedure or is being monitored for the development of cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion. In various embodiments, the subject is a human.

[0097] In various embodiments, the subject is a human who has been diagnosed with cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, and has either received treatment or is treatment-naive. In various embodiments, the subject has been diagnosed with cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion prior to the method of treatment disclosed herein being carried out. The subject may have either received treatment or is treatment-naive or treated with therapies excluding EPAS1 inhibitors.

[0098] In various embodiments, analysis of circulating endothelial cells (CECs) from routine blood samples of a subject may offer a minimally invasive strategy for earlier detection of endothelial and / or vascular dysfunction, prior to the onset of irreversible cognitive decline. The correlation between venous-derived CEC levels and cognitive performance supports the utility of CECs as clinicallyrelevant biomarkers. These biomarkers may serve as early indicators of vascular pathology, enabling timely intervention, and may also be used as stratification tools in clinical trials to identify patients most likely to benefit from vascular-protective therapies, such as EPAS1 inhibition. Furthermore, CEC levels may function as predictive or pharmacodynamic biomarkers to mechanistically interpret treatment responses and outcomes of EPASf inhibition and / or other therapeutic agents.

[0099] In particular, the present inventors showed that damaged venous endothelium has been linked to elevated levels of circulating endothelial cells (CECs) and poor cognitive outcomes. Thus, CECs of venous origin may serve as minimally invasive biomarkers to identify a subject’s risk of cognitive decline, cerebral hypoperfusion, or any condition or disease associated with cerebral hypoperfusion. Moreover, damaged CECs reveal that venous endothelial dysfunction is more pronounced in human subjects with cerebrovascular disease and correlates with poorer cognitive performance.

[0100] Thus, CECs as a biomarker may be useful in selecting suitable subjects for treatment with the EPAS1 inhibitor disclosed herein as well as monitoring a subject’s therapeutic response to EPAS1 inhibition. In various embodiments, the invention provides methods for diagnosis, patient stratification / selection, and treatment monitoring based on the detection and quantification of venous- derived CECs in a biological sample.

[0101] In various embodiments, the subject may be selected for treatment with an EPAS1 inhibitor based on the detection and measurement of CECs in a sample that has been obtained from the subject. The amount of CECs measured in the sample may be compared to a reference amount to provide an indication on if the subject may be selected for said treatment and likely be responsive thereto. In various embodiments, subject may be selected for treatment with an EPAS1 inhibitor based on the magnetic resonance imaging (MRI), in addition to the detection and measurement of CECs in a sample that has been obtained from the subject.

[0102] The term “sample,” as used herein, refers to a composition obtained or derived from a subject that contains a cellular and / or molecular entity to be detected, characterized, and / or quantified based on physical, biochemical, chemical, and / or physiological characteristics. In the context of the present invention, the sample may be used to detect circulating endothelial cells (CECs), particularly of venous origin. The phrase “disease sample” and variations thereof refer to any sample obtained from a subject that is expected or known to contain CECs or other relevant analytes for diagnostic, prognostic, or treatment-monitoring purposes. Suitable biological samples include, but are not limited to, whole blood, plasma, serum, and fractions thereof enriched for peripheral blood mononuclear cells (PBMCs), which can be processed to isolate and quantify CECs. Other suitable samples may include cerebrospinal fluid, urine, vitreous fluid, or tissue biopsies, depending on the anatomical location of interest. For example, brain surgeons may retrieve intracranial blood aspirate or cerebralarterial / venous blood during acute stroke treatment. Hepatologists may retrieve portal and hepatic venous blood during liver surgery. Samples may also encompass cultured cells, cell lysates, tissue homogenates, and combinations thereof, where appropriate.

[0103] In various embodiments, the sample is a blood-derived sample, such as whole blood, plasma, or a fraction enriched for peripheral blood mononuclear cells (PBMCs). Preferably, the sample is a PBMC preparation isolated from whole blood using density gradient centrifugation or similar methods.

[0104] Accordingly, in various embodiments, the subject to be treated with the EPAS1 inhibitor may have been diagnosed with, or predicted to be at risk of developing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, through a method comprising:(i) detecting and measuring the amount of circulating endothelial cells (CECs) in a sample that has been obtained from the subject; and(ii) comparing the measured amount of CECs in the sample to a reference amount, wherein a differential amount of CECs measured in the sample relative to the reference amount is indicative of the subject having cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, or the subject being at risk of developing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0105] In various embodiments, a subject may be diagnosed, or predicted to be at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion according to the above method independently of any therapeutic intervention involving EPAS1 inhibition disclosed herein. Thus, in one aspect, there is also provided a method for diagnosing, or predicting a subject's risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, the method comprising:(i) detecting and measuring the amount of circulating endothelial cells (CECs) in a sample that has been obtained from the subject; and(ii) comparing the measured amount of CECs in the sample to a reference amount, wherein a differential amount of CECs measured in the sample relative to the reference amount is indicative of the subject having, or the subject being at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0106] In various embodiments, the subject may be diagnosed, or predicted to be at risk of developing vascular cognitive impairment and vascular dementia due to cerebral hypoperfusion.

[0107] In various embodiments, the condition or disease associated with cerebral hypoperfusion may be endothelial dysfunction, such that endothelial dysfunction may be detected in a subject based on measured CECs in the subject as a biomarker for said endothelial dysfunction. Accordingly, in various embodiments, endothelial dysfunction may be detected in the subject to be treated with theEPAS1 inhibitor, comprising the steps of:(i) detecting and measuring the amount of circulating endothelial cells (CECs) in a sample that has been obtained from the subject; and(ii) comparing the measured amount of CECs in the sample to a reference amount, wherein a differential amount of CECs measured in the sample relative to the reference amount is indicative of the subject having endothelial dysfunction.

[0108] In various embodiments, endothelial dysfunction may be detected in a subject independently of any therapeutic intervention involving EPAS1 inhibition disclosed herein. Thus, in various embodiments, detection and measurement of CECs may serve as the diagnostic step itself, providing an indication of endothelial dysfunction. In other embodiments, such biomarker detection may be used as a stratification tool following an established diagnosis of cerebral hypoperfusion or related diseases or conditions, to guide patient selection and monitoring for EPAS1 inhibitor treatment. Thus, detection and measurement of CECs may serve directly as a diagnostic step indicating endothelial dysfunction, whereas the treatment method may be directed to administering an EPAS1 inhibitor, optionally after CEC biomarker-based stratification.

[0109] As used herein, the terms “amount” or “level” of circulating endothelial cells (CECs) refer to a detectable quantity of CECs present in a biological sample obtained from a subject, such as a whole blood sample or a PBMC-enriched fraction. The amount may be expressed as the number of CECs per million peripheral blood mononuclear cells (PBMCs), as an absolute number of CECs per unit volume (e.g., cells per mL) and termed as an absolute abundance, or as a percentage of total nucleated cells or CEC subpopulations, depending on the detection method used.

[0110] In various embodiments, the step of detecting and measuring the amount of CECs in a sample obtained from a subject may include one or more preparatory and analytical procedures known to those skilled in the art. In various embodiments, the sample is a blood-derived specimen and may be processed to isolate CECs using techniques such as density gradient centrifugation, immunomagnetic bead enrichment, or microfluidic separation. Detection and quantification may be carried out using flow cytometry, based on immunophenotyping of cells that exhibit a defined surface marker profile (e.g. combined immunophenotypic profile), to distinguish mature endothelial cells from hematopoietic and progenitor cells.

[0111] In various embodiments, the CECs in the sample may be detected, and measured, by flow cytometry using the markers CD45- / CD31 + / CD133- / DNA+, that represent a combined immunophenotypic profile. In this regard, CECs may be distinguished from other peripheral blood cells by their expression of specific surface and nuclear markers. In particular, CECs are characterized by being CD45-negative (CD45-), indicating the absence of hematopoietic lineage markers; CD31 -positive (CD31+), reflecting the expression of platelet endothelial cell adhesionmolecule-1 (PECAM-1 ), a pan-endothelial marker; CD133-negative (CD133-), distinguishing mature endothelial cells from circulating endothelial progenitor cells; and DNA-positive (DNA+), confirming the presence of an intact nucleus and viability of the detected cells. In addition, VE-Cadherin can also be used as a pan-endothelial marker of CECs.

[0112] Flow cytometric detection may typically involve staining peripheral blood mononuclear cells (PBMCs) isolated from whole blood using density gradient centrifugation. Fluorescently labelled monoclonal antibodies targeting CD45, CD31 , and CD133 can be used in combination with a nuclear dye (e.g., DAPI or Hoechst) to facilitate multi-parametric analysis. Data are acquired using a flow cytometer capable of detecting multiple fluorophores and analysed using gating strategies that exclude debris, doublets, and non-viable cells. The defined profile of CD45- / CD317CD133- / DNA+allows for the specific identification of mature, non-hematopoietic, nucleated endothelial cells in circulation. This approach provides a method for assessing CEC levels in subject samples, and may be used for diagnostic purposes, monitoring of disease progression, and evaluation of therapeutic responses, particularly in the context of cerebrovascular disease and cerebral hypoperfusion.

[0113] In various embodiments, CECs expressing atypical chemokine receptor 1 (ACKR1 ) may be selectively detected and measured, in addition to the amount of CECs as outlined above. ACKR1 expression on CECs may be used to distinguish a subpopulation of damaged endothelial cells of venous origin, which are observed to increase in subjects with cerebrovascular disease. In various embodiments, detection and quantification of the amount of ACKR1+CECs may be performed using antibodies or other binding reagents specific for the extracellular domain of the ACKR1 protein, allowing for the quantification of this CEC subpopulation by flow cytometry or immunoassays. The detection and measurement of ACKR1+CECs may be integrated into the flow cytometry panel by including fluorophore-conjugated anti-ACKR1 antibodies, allowing quantification of this subset within the previously gated CEC population. Alternatively, detection of ACKR1+CECs may be performed using immunomagnetic separation or immunoassay-based approaches (e.g., ELISA, CyTOF, or mass cytometry), provided that the assay distinguishes ACKR1+ CECs from other ACKR1 - expressing cell types such as erythroid-lineage cells.

[0114] In various embodiments, detection methods for ACKR1+CECs may be configured to distinguish this subpopulation from other ACKR1 -expressing cell types, such as erythroid lineage cells. Such distinction may be achieved by immunophenotyping within a gated CEC population (e.g., CD45 / CD31 7CD133 / DNA1), thereby ensuring that ACKR1 positivity is specifically attributed to circulating endothelial cells rather than erythroid cells.

[0115] The proportion of ACKR1+circulating endothelial cells (CECs) may be quantified relative to the total CEC population by flow cytometric analysis using defined immunophenotypic markers (e.g., CD457CD317CD1337DNA+). The results can be reported as the percentage of ACKR1+CECs withinthe overall CEC population. Alternatively, the absolute abundance of ACKR1+CECs can be expressed as the number of ACKR1+CECs per million peripheral blood mononuclear cells (PBMCs).

[0116] As used herein, atypical chemokine receptor 1 (ACKR1) refers to a non-classical chemokine receptor also historically known as Duffy antigen receptor for chemokines (DARC). The ACKR1 may be a human ACKR1 , where the human ACKR1 gene is located on chromosome 1 (1 q23) and is referenced in the NCBI Gene database under Gene ID: 2532. The corresponding RefSeq mRNA accession number is NM 001122951.3, and the RefSeq protein accession number is NP_001116423.1 . The UniProtKB entry for the human ACKR1 protein is Q16570. ACKR1 expression on CECs, particularly when elevated in peripheral blood, may be used herein as a surrogate biomarker of venous endothelial injury.

[0117] In various embodiments, the disease or condition associated with cerebral hypoperfusion is a cerebrovascular disease, and the human subject may be diagnosed with the cerebrovascular disease based, at least in part, on the detection and quantification of ACKR1+CECs in a biological sample obtained from the subject. The amount of ACKR1 + CECs in the sample may be correlated with decreases in cognitive performance, particularly in executive function and language skills.

[0118] Accordingly, in various embodiments, the subject to be treated with the EPAS1 inhibitor may be diagnosed, or predicted to be at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, through a method comprising: measuring the amount of circulating endothelial cells (CECs), and / or CECs that are positive for Atypical Chemokine Receptor 1 (ACKR1 ) in the sample that has been obtained from the subject; and comparing the measured amount of CECs and / or ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and / or ACKR1 + CECs measured in the sample relative to the reference amount is indicative of the subject having, or the subject being at risk of developing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0119] In various embodiments, a subject may be diagnosed, or predicted to be at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion according to the above method independently of any therapeutic intervention involving EPAS1 inhibition disclosed herein. Thus, there is also provided a method for diagnosing, or predicting a subject's risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, the method comprising:(i) detecting and measuring the amount of circulating endothelial cells (CECs), and / or CECs that are positive for Atypical Chemokine Receptor 1 (ACKR1 ) in a sample that has been obtainedfrom the subject; and(ii) comparing the measured amount of CECs and / or ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and / or ACKR1 + CECs measured in the sample relative to the reference amount is indicative of the subject having, or the subject being at risk of developing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

[0120] In various embodiments, the subject may be diagnosed, or predicted to be at risk of developing cerebrovascular disease.

[0121] In various embodiments, the subject may be diagnosed, or predicted to be at risk of developing vascular cognitive impairment and vascular dementia due to the cerebral hypoperfusion.

[0122] In various embodiments, the condition or disease associated with cerebral hypoperfusion may be venous dysfunction, such that vascular dysfunction may be detected in a subject based on measured CECs, and / or ACKR1 + CECs, in the subject as a biomarker for said vascular dysfunction. Accordingly, in various embodiments, endothelial dysfunction and / or vascular dysfunction may be detected in the subject to be treated with the EPAS1 inhibitor, comprising the steps of:(i) detecting and measuring the amount of circulating endothelial cells (CECs), and / or ACKR1 + CECs, in a sample that has been obtained from the subject; and(ii) comparing the measured amount of CECs and / or ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and / or ACKR1+ CECs measured in the sample relative to the reference amount is indicative of the subject having endothelial dysfunction and / or vascular dysfunction.

[0123] In various embodiments, endothelial dysfunction and / or venous dysfunction may be detected in a subject independently of any therapeutic intervention involving EPAS1 inhibition disclosed herein. Thus, in various embodiments, detection and measurement of CECs and / or ACKR1+CECs may serve as the diagnostic step itself, providing an indication of endothelial dysfunction and / or vascular dysfunction. In other embodiments, such biomarker detection may be employed as a stratification tool following an established diagnosis of cerebral hypoperfusion or related diseases or conditions, to guide patient selection and monitoring for EPAS1 inhibitor treatment. Thus, detection and measurement of CECs and / or ACKR1+CECs may serve directly as the diagnostic step (indicating endothelial and / or vascular dysfunction), whereas the treatment method is directed to administering an EPAS1 inhibitor, optionally after CECs and / or ACKR1+CECs biomarker-based stratification.

[0124] As used herein, “endothelial injury” refers to structural damage or loss of endothelial cells, often leading to release of circulating endothelial cells (CECs). “endothelial dysfunction” refers toUimpaired endothelial function (e.g., abnormal vasodilation, barrier integrity, or signalling), which may arise from or occur independently of endothelial injury, and “vascular dysfunction” may encompass endothelial dysfunction as well as other abnormalities of vascular structure or regulation, including arterial, capillary, or venous impairment.

[0125] In various embodiments, the method may comprise detecting and measuring both the amount of CECs, and / or ACKR1 + CECs in the sample, wherein the percentage of ACKR1 + CECs relative to the total amount of CECs is determined.

[0126] In various embodiments, the measured / quantified amount of the CECs, and / or ACKR1 + CECs, is used to derive an evaluation index when compared to a reference amount. As used herein, the term “evaluation index” refers to a quantitative or qualitative value, score, classification, or parameter derived from one or more measured biological variables, such as the level of CECs, or ACKR1 + CECs, that is used to assess, indicate, predict, stratify, or interpret a biological state or clinical condition. The evaluation index may represent a direct numerical measurement (e.g., CECs per million PBMCs, or % population of ACKR1 + CECs), a categorical classification (e.g., low, moderate, high), or a value derived from computational models, algorithms, or threshold-based rules. In the context of the present invention, the evaluation index may be calculated from the absolute or relative amount of CECs and optionally integrated with clinical variables (e.g., cognitive Z-scores or imaging-derived scores such as the Fazekas score) to provide an objective, standardized indication of vascular health, disease burden, or therapeutic efficacy. This approach enables consistent interpretation across subjects, improves diagnostic accuracy, and facilitates clinical decision-making by transforming raw biomarker measurements into clinically actionable outputs.

[0127] The term “reference amount” refers to a comparative benchmark or control value representing the expected or baseline level of CECs in a subject population. This value may be derived from a healthy (non-diseased) reference population, a population with known clinical characteristics (e.g., mild vs. severe cerebrovascular disease), or from historical clinical datasets. The reference amount may reflect a normal range of CEC levels observed in individuals without cerebral hypoperfusion, or it may correspond to stratified reference values associated with different stages of vascular disease or cognitive function. In various embodiments, the reference amount may be obtained from a matched control group, a longitudinal baseline sample from the same subject, or data derived from publicly available clinical databases or studies. The reference amount may be presented as a median, mean ± standard deviation, percentile-based range, or predefined diagnostic threshold for CEC, or ACKR1 + CEC, counts. It serves as a standard against which the amount of CECs, or ACKR1 + CECs, detected in a test sample is compared to determine whether the subject exhibits a differential amount / level indicative of cerebral hypoperfusion or a related condition, or the efficiency of a treatment for monitoring purposes.

[0128] The term “differential amount”, as used herein, refers to a significant deviation in the measured amount / level of CECs, or ACKR1 + CECs, in a subject's sample relative to the reference amount. This deviation may be either an increase (e.g., elevated CEC counts associated with endothelial damage) or a decrease, and may indicate an aberrant vascular condition or risk thereof, such as cerebral hypoperfusion or associated cerebrovascular disease. A differential amount may be defined statistically (e.g., exceeding a normal range, surpassing the 95th percentile, or showing a p- value < 0.05 using a Mann-Whitney or t-test), or by applying predefined fold-change thresholds (e.g., >1.3x increase) or percent change cutoffs (e.g., >30% difference relative to baseline). The actual threshold used to define a differential amount may vary based on the detection method (e.g., flow cytometry, imaging cytometry), the specific CEC subpopulation analysed (e.g., ACKR1+CECs), and the context of use (e.g., diagnosis, risk stratification, or treatment monitoring). In various embodiments, a “differential amount” of CECs correlates with a clinical phenotype, such as reduced cerebral perfusion, increased white matter hyperintensity burden (e.g., Fazekas score >1 ), or decreased cognitive function (e.g., global or domain-specific Z-scores). Accordingly, a differential amount may be used to infer the presence, severity, or risk of developing cerebral hypoperfusion or a condition associated therewith, or if a treatment being administered to the subject is effective in treating a related condition or disease.

[0129] In various embodiments, the amount of ACKR1 + CECs detected and measured in the sample, is correlated with cognitive performance of the subject, particularly in executive function and language skills. In particular, a significant correlation is observed between a higher percentage of ACKR1 + CECs and a decrease in global cognition. The quantification of ACKR1 + CECs, thus provides a minimally invasive, blood-based biomarker for assessing cerebrovascular integrity and potentially stratifying patients for clinical intervention and selection of suitable treatment regimens thereof.

[0130] In various embodiments, the different amount comprises elevated levels of CECs and / or ACKR1+CECs, which are indicative of endothelial injury or vascular dysfunction and have been associated with cerebral hypoperfusion and cerebrovascular disease, including cerebral small vessel disease, vascular dementia, and white matter pathology.

[0131] In various embodiments, the differential amount comprises an increase in the percentage of ACKR1 + CECs in the sample relative to the reference amount. Such an increase is indicative of the subject having, or being at risk of developing, cerebral hypoperfusion, or any condition or disease associated with cerebral hypoperfusion. In various embodiments, the percentage of ACKR1+CECs is calculated as the number of CECs expressing ACKR1 divided by the total number of CECs identified, multiplied by 100. Alternatively, the absolute abundance of ACKRT CECs can be expressed as the number of ACKR1+CECs per million peripheral blood mononuclear cells (PBMCs).In various embodiments, this percentage or absolute abundance is compared to a reference percentage or absolute abundance of ACKR1+CECs derived from a healthy control population or a predefined clinical baseline.

[0132] In various embodiments, the increased percentage is indicative of the subject having a cerebrovascular disease, or the subject being at risk of developing a cerebrovascular disease, such as small vessel disease or vascular cognitive impairment. In various embodiments, the percentage of ACKR1+CECs is inversely correlated with cognitive performance, particularly in domains such as executive function, language, and global cognition. Higher levels of ACKR1+CECs are associated with lower cognitive Z-scores, supporting their utility as prognostic biomarkers of cognitive decline in cerebrovascular conditions.

[0133] In various embodiments, the subject may be stratified based on CEC, and / or ACKR1 + CEC, levels into normal, elevated, or high-risk categories, using population-derived percentiles or statistically defined thresholds. For example, CEC, and / or ACKR1+CECs, counts above the 95th percentile of a control population may be classified as elevated and indicative of underlying vascular pathology.

[0134] In various embodiments, subjects may be grouped based on cerebrovascular disease severity as determined by the Fazekas scale, a standardized scoring system for white matter hyperintensity burden on neuroimaging. Subjects with absent-to-mild cerebrovascular disease are defined as those with Fazekas scores ranging from 0 to 4, whereas those with moderate-to-severe disease have scores from 5 to 12. In this regard, an increased percentage of ACKRT CECs is observed in subjects with moderate-to-severe cerebrovascular disease compared to those with absent or mild disease. This association supports the use of ACKR1+CECs as a biomarker of venous vascular injury in the brain.

[0135] In various embodiments, an inverse correlation is observed between total CEC, amounts and cerebral perfusion in subjects with Fazekas scores of 0-4 (absent-to-mild disease), indicating that increasing CEC counts reflect worsening cerebrovascular disease burden. However, this relationship is not evident in subjects with severe disease (Fazekas > 5), suggesting a plateau or saturation of endothelial damage in later stages of disease. In various embodiments, the subject has a Fazekas score greater than 0.

[0136] In various embodiments, subjects may be stratified based on their percentage of ACKR1+CECs into three categories: zero (0%), low (<95th percentile), and high (>95th percentile). Individuals with negative global cognitive Z-scores, reflecting below-average performance, are significantly more likely to have high proportions of ACKR1+CECs, whereas those with positive Z-scores morefrequently exhibit low or undetectable levels. This relationship underscores the clinical value of ACKR1+CECs for both risk stratification and monitoring of neurovascular health, with particular utility in cerebrovascular disease.

[0137] In various embodiments, the diagnostic / predictive method may further comprise selecting a treatment for the subject based on an indication, grouping and / or stratification provided by the methods described herein.

[0138] In various embodiments, the selected treatment may be a vascular-protective therapy or treatment. As used herein, the term “vascular-protective therapy” refers to any pharmacological, biological, or non-pharmacological intervention that maintains, restores, or improves vascular structure and / or function. Such therapies act, for example, by preserving endothelial integrity, enhancing nitric oxide bioavailability, reducing oxidative stress or inflammation, improving vascular tone, limiting thrombosis, stabilizing the blood-brain barrier, or preventing maladaptive vascular or venous remodelling.

[0139] In various embodiments, vascular-protective therapies may include, but are not limited to: antihypertensive agents (e.g., ACE inhibitors, angiotensin receptor blockers, calcium channel blockers, beta blockers); lipid-lowering drugs (e.g., statins, PCSK9 inhibitors); antiplatelet or anticoagulant agents (e.g., aspirin, clopidogrel, warfarin, direct oral anticoagulants); nitric oxide donors and phosphodiesterase inhibitors; endothelin receptor antagonists; VEGF inhibitors or modulators; anti-inflammatory or antioxidant agents; and EPAS1 (HIF-2a) inhibitors as disclosed herein. In various embodiments, vascular-protective therapies may comprise non-pharmacological interventions such as dietary modification, physical exercise, cognitive training, or management of vascular risk factors. In various embodiments, vascular-protective therapies encompass treatments for cerebral hypoperfusion and for any disease or condition associated with cerebral hypoperfusion as defined herein.

[0140] In various embodiments, the treatment may include nitric oxide donors, endothelin receptor antagonists, phosphodiesterase inhibitors, VEGF inhibitors, anti-inflammatory agents, or other pharmacological modulators of vascular homeostasis. In various embodiments, subjects may be selected for non-pharmacological interventions such as dietary modification, physical exercise, cognitive training, or management of vascular risk factors, either alone or in combination with pharmacological therapy. In various embodiments, the subject treatment selected may comprise therapeutic agents for the treatment, prevention, or modulation of cerebral hypoperfusion, a disease or condition associated with cerebral hypoperfusion, endothelial and / or vascular dysfunction, or neuroinflammation.

[0141] In various embodiments, the treatment may comprise administration of an EPAS1 inhibitor, alone or in combination with one or more vascular-protective therapies. Thus, the method may further comprise administering the EPAS1 inhibitor of claim 1 to the subject for treating, preventing, or ameliorating the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion.

[0142] Accordingly, in various embodiments, the method of treating or preventing cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject, may comprise: diagnosing, or predicting a subject's risk of developing, a cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion using the method disclosed herein; and administering the EPAS1 inhibitor or composition to the subject, based on the diagnosis or prediction.

[0143] In various embodiments, the method may further comprise classifying the likelihood of the subject being responsive or non-responsive to the administration of the EPAS1 inhibitor based on the measured amount of the CECs, and / or ACKRT CECs, in the subject's sample relative to a reference amount. In the event that the subject may be determined, or predicted, to be responsive or non- responsive, a suitable treatment regimen may be selected for the subject.

[0144] In various embodiments, changes in CEC, and / or ACKR1+ CEC, levels over time may be used to evaluate disease progression or the efficacy of treatments administered to the subject.

[0145] Accordingly, in one aspect, there may be provided a method of monitoring the efficacy of the EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in a subject, comprising:(a) administering the EPAS1 inhibitor to the subject;(b) obtaining a biological sample from the subject; and(c) detecting and measuring the amount of circulating endothelial cells (CECs), and / or ACKR1 + CECs, in the biological sample; and(d) comparing the measured amount of CECs, and / or ACKR1 + CECs, in the sample to a reference amount, wherein a differential amount of CECs, and / or ACKR1+ CECs, measured in the sample relative to the reference amount is indicative of the efficacy of the EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in the subject.

[0146] In various embodiments, steps (a)-(d) are repeated two or more times within a time-frame and the detection and measurement at each time point is compared against each other to assess the progression of the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion and efficacy of the treatment.

[0147] In various embodiments, the monitoring method may further comprise obtaining cerebral blood flow (CBF) measurements by magnetic resonance imaging (MRI), for example using arterial spin labelling (ASL) sequences, prior to initiation of treatment and at one or more time points during the course of treatment. The ASL readouts provide a quantitative and non-invasive measure of regional and global CBF, and may be compared within the same subject. A relative increase in CBF following administration of the EPAS1 inhibitor, compared to the pre-treatment baseline measurement, may be indicative of improvement of cerebral hypoperfusion and therapeutic efficacy. Conversely, a lack of increase, or a decline in CBF relative to baseline, may indicate insufficient therapeutic response. Thus, in various embodiments, the monitoring method may further comprise comparing cerebral blood flow measurement of the subject by MRI brain imaging arterial spin labelling readouts before and / or over the course of treatment.

[0148] By obtaining CBF measurements by MRI ASL before and during treatment, and comparing the CBF values over time, an increase in CBF relative to baseline may further support the efficacy of the EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated therewith.

[0149] In this regard, the amount of CECs and / or ACKR1 + CECs detected and measured in a biological sample from a subject may be used as a biomarker in the preparation of a diagnostic product for (I) diagnosing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion in a subject, (ii) predicting a subject's risk of developing cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion (ill) predicting the efficacy of a treatment for cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, and / or (iv) monitoring the efficacy of a treatment for cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion. In various embodiments, the diagnostic product is selected from the group consisting of a kit, a diagnostic device and a computer system.

[0150] As used herein, the term “kit” relates to a kit-of-parts wherein the separate components of the kit are physically separated as individual components. Such kits typically comprise a number of agents and reagents useful and necessary to perform the methods of the invention, and the necessary steps of the methods disclosed herein.

[0151] In various embodiments, the kit may be configured for detecting and quantifying CECs and / or ACKR1+CECs in a biological sample, such as a blood sample. The kit may comprise one or more reagents for enriching or isolating CECs, including immunomagnetic beads or microfluidic chips coated with antibodies specific for endothelial markers and further comprising detection reagents specific for ACKR1. The kit may further include fluorescently labelled antibodies or secondary detection reagents suitable for use in flow cytometry, immunofluorescence, or immunohistochemistry, and may optionally comprise nucleic acid probes, primers, or amplification reagents for detectingACKR1 gene transcripts in isolated CECs by PCR or qPCR. In various embodiments, the kit may comprise buffers, fixatives, and wash solutions optimized for CEC stability and preservation, along with positive and negative control samples to calibrate assay performance. In various embodiments, the kit may include a microfluidic cartridge or chip pre-loaded with separation and detection reagents, designed to be operably linked to a diagnostic device or reader. Instructions for use, reference charts or digital calibration software, and a computer-readable medium storing algorithms for automated quantification and interpretation of CEC and ACKR1+CEC levels relative to reference values may also be included.

[0152] The invention is further illustrated by the following non-limiting examples and the appended claims.EXAMPLESMATERIALS AND METHODS

[0153] Human studies approval, subject enrolment, and sample collection: This study was approved by the Local Ethics Committee, namely Nanyang Technological University Institutional Review Board (IRB-2021 -1036). Each participant provided written informed consent after being informed about the study's nature and potential consequences. The study protocol is in accordance with the Helsinki Declaration. Human subjects were recruited as part of the Biomarkers and Cognition Study, Singapore (BIOCIS) of Dementia Research Centre Singapore. Baseline patient characteristics, comprising age, sex, ethnicity, and comorbidities, were collected through blood sampling at the time of enrolment (Table 1). For blood sample collection, 6-8 mL was collected from each participant via venepuncture and processed in the lab within 3 hours. Following Ficoll centrifugation of fresh blood, buffy coat layers containing PBMCs were isolated for analysis of circulating endothelial cells.

[0154] Table 1. Demographics of human subjects deeply phenotyped with functional brain MRI, cognitive assessment, and CEC profiling.All values are reported as N (%), where N indicated number of observations.

[0155] Animal studies approval and BCAS procedure: BCAS surgery was performed using an established protocol[8]. C57BL / 6J mice (aged 3-4 months) were anesthetized with 2.0% isoflurane before a midline cervical incision was made for tissue harvesting at 10 or 60 days post-BCAS. Common carotid arteries were exposed by careful separation of tissue layers and the vagal nerves. 0.18 mm diameter microcoils (Sawane Spring Co., Ltd) were applied by rotating them around each common carotid artery. Body temperature was monitored and kept within the range of 36.5-37.5 °C by using a heating blanket. After microcoil application, the incision was closed with a silk suture, and micewere left on the heating blanket after surgery. Sham mice underwent the same procedure without microcoil placement. Four sham and 4 BCAS mice (2 males and 2 females per experimental group) were used for transcriptomic studies. For histological and functional studies, 10-13 mice were used per experimental group with both sex representations.

[0156] Care provided to the animals followed institutional guidelines and was approved by the local Institutional Animal Care and Use Committee (IACUC # A18095). Animals are housed in individually ventilated cages on forced ventilation- based system, air is HEPAfiltered for supply to IVC cages, and HEPA filtered on exhaust. Exhaust air is not recycled. Room air change per hour set to >10. Animal holding room temperature is set at 22*C (+ / - 2*C), with humidity kept in 50% to 70% range. Holding rooms have surface mounted and water-resistant lighting fixtures, set to provide 12:12 hrs photoperiod (light:dark). Housing density follows the NACLAR guidelines. Animals are fed standard irradiated maintenance or breeding rodent diets. Water is supplied by standard water bottles. All animals are housed on autoclaved corn cob bedding and are provided with nesting material. Environmental enrichment program and strict social housing practice is in place.

[0157] Measurement of cerebral blood flow by laser-Doppler flowmetry: Under deep anesthesia (2% v / v isoflurane), a 1 cm midline scalp incision was made and retracted to expose the skull. A laser Doppler flowmetry probe (FLO-N1 , OMEGAWAVE, Inc.) was carefully positioned at the midpoint between bregma and lambda over the parietal cortex, a location that does not directly overlay large arterial or venous structures. A 3 mW, 780 nm laser was delivered through the probe to record CBF. Baseline CBF was recorded at the onset of surgery, following anesthesia induction. Probe positioning was standardized across animals, and the signal was allowed to stabilize within ±0.5 perfusion units before data collection. In each animal at every time point, three replicate readings were acquired and averaged to ensure consistency. Subsequent CBF readings were taken after microcoil implantation and at regular intervals, every 5 days up to day 30, or on day 40 for drug treatment studies. CBF values were expressed as a percentage of each animal's individual baseline. To minimize variability, physiological and environmental conditions were tightly controlled. Mice were maintained at 37 ± 0.5 °C using a feedback-regulated heating pad. All measurements were performed in a thermostat-controlled, windowless room with constant artificial lighting.

[0158] Histological analysis by immunofluorescence staining: Mice were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), and transcardially perfused with DPBS (SH3002803, Hyclone). Cerebral tissue was fixed in 4% paraformaldehyde (PFA; 09154-85, Nacalai Tesque) overnight at 4°C. The anterior 4 mm portion of the brain was cryoprotected in 30% (w / v) sucrose solution (Thermo Fisher Scientific, 57-50-1 ), embedded in Tissue-Tek optimal cutting temperature compound (Sakura, 4583), and flash frozen in liquid nitrogen. Coronal sections of 20 pm thickness were cut on a cryostat (Leica, CM3050 S) and mounted on glass slides. The slides were washed with DPBS, permeabilized with 0.1% Triton X-100 (omitted for CD45 staining), and blocked with 1% bovine serum albumin (BSA; 9998, Cell Signaling Technology). Primary antibody incubations were done overnight at 4°C. The slides were then washed and incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature in the dark. Sections were counter-stained with DAPI (1 :5000) and mounted using Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, F4680). Antibody details are provided in Table 2.

[0159] Table 2. List of antibodies for immunofluorescence and flow cytometry.

[0160] To discern the origins of endothelial cells in the expansion of the vascular network, aSMA staining patterns were used to distinguish smooth muscle cell-invested arterial (striated aSMA) vessels from venous (discontinuous aSMA) vessels. For lumen diameter analysis, a systematic approach was implemented to identify and consistently measure vessels of the same type across samples. Specifically, the pericallosal artery and vein was focused on, which supply the cingulate cortex, a region within the prefrontal cortex, allowing us to compare analogous vascular structures in both BOAS and sham groups. This approach minimized variance by avoiding misidentification between small veins and arterioles, which could otherwise introduce confounding factors. To characterize active vascular remodeling, wall-to-lumen ratio was derived to determine structural changes due to wall thickening.

[0161] Histological analysis by three-dimensional vascular visualization: Mice were transcardially perfused with DPBS, followed by 50 mg / mL 500 kDa FITC-Dextran (Sigma-Aldrich, FD500S). Brains were harvested and fixed in 4% PFA overnight at 4 °C. Subsequently, the prefrontal cortex tissue was used for tissue clearing. Pre-delipidation was done using 50% CUBIC-L (TCI, T3740) for 3 hours at room temperature, followed by further delipidation with CUBIC-L at 37°C for 5 days until optical transparency was achieved. CUBIC-L was refreshed on days 1 and 2, followed by every other subsequent day. The tissue was washed with DPBS, permeabilized with 0.2% Triton X- 100, and then blocked with 5% BSA. Incubation with conjugated-primary antibodies was done at 4°C for 5 days. Tissue was then washed and stored in DPBS at 4 °C until imaging. Antibody details are provided in Table 2.

[0162] Image acquisition and analysis: High-resolution images of alpha smooth muscle, microglia and arterial / venous sprouting from 1 mm thick brain slices were acquired using the inverted laser scanning confocal with Airy scan super-resolution capability (LSM800, ZEISS) at NTU-Optical BioImaging Centre (NOBIC). High-throughput imaging of multiple prefrontal 20 pm brain sections for vascular density and eNOS was carried out the Carl Zeiss Axio Scan.ZI slide scanner. Other images were acquired using the inverted widefield fluorescence microscope (CellDiscoverer7, ZEISS). Multichannel images were captured using the ZEN software (Blue Edition, Carl Zeiss). Imaris (v 9.9.1 ), lmageJ2 (v 2.14.0) and MatLab (R2023b) were used in the processing, analysis and quantification of images.

[0163] 1 . Microglial ramification analysis: a. Image Pre-processing and Analysis: Quantitative analysis of microglial association with vessels was performed using a custom MATLAB-based pipeline, designed for automated processing and extraction of microglial connectivity features. The pipeline included the following steps: b. Input Image Acquisition: “.tif” images containing immunolabeled microglia and vessels were loaded into the pipeline. Each image was pre-assigned to its respective experimental group (BCAS SHAM, BCAS Vehicle, or BCAS Drug) and vessel type (artery, vein, micro vessel). c. Microglial Connectivity Analysis: The green channel (corresponding to microglial labelling) was extracted from each image. Noise was reduced using a median filter, and microglial structures were enhanced using a Frangi vesselness filter for better detection of curvilinear features.Morphological operations, including binary thresholding and closing, were applied to create a connectivity map of microglial networks. Skeletonization: A skeletonized representation of microglial structures was generated using iterative thinning. This representation provided insights into microglial ramification patterns associated with vessels. Quantification of Ramification Index: The ramification index, a measure of microglial branching complexity, was calculated for each image using the formula:Here, perimeter and area represent the total length and coverage of microglial structures, respectively. Automated Output Generation: For each image, the pipeline automatically saved the following: Processed Images: Enhanced microglial connectivity maps and skeletonized networks. Quantitative Results: Ramification indices were compiled into a CSV file for statistical analysis. d. Data Processing and Statistical Analysis: Quantitative data were analysed to compare microglial ramification indices across experimental groups (BCAS SHAM, BCAS Vehicle, and BCAS Drug) and vessel types (arteries, veins, micro vessels). Statistical analyses were performed using R (v4.2.0) or GraphPad Prism (v9). Differences between groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. P-values < 0.05 were considered statistically significant.e. Pipeline Availability: The custom MATLAB pipeline used for this analysis, including the main script and supporting functions, is available on GitHub at https: / / github.com / aravind245 / microglial_ramification_pipeline. This repository includes sample images, processed results, and detailed instructions for reproducing the analysis.

[0164] 2. eNOS Signal Intensity Analysis a. Image Acquisition and Pre-processing: Fluorescence microscopy images of endothelial nitric oxide synthase (eNOS) were acquired as .tif files and stored in a designated directory for batch processing. The raw images were imported into MATLAB using the imread function. To minimize noise and enhance contrast, the green fluorescence channel (assumed to contain eNOS signal) was extracted, followed by contrast-limited adaptive histogram equalization (CLAHE) using imadjust. A median filter (medfilt2) with a kernel size of 2x2 pixels was applied to reduce speckle noise while preserving structural details. To improve segmentation accuracy, a morphological closing operation (imclose) with a 30-pixel disk-shaped structuring element was performed, enhancing continuity in the fluorescence signal. b. Segmentation and Feature Extraction: To isolate eNOS-positive regions, the pre-processed images were binarized using adaptive thresholding (imbinarize). Small artifacts and background noise were removed using area-based filtering (bwareaopen), retaining only objects with an area exceeding 1000 pixels. Border-connected regions were eliminated (imclearborder) to prevent artifacts from interfering with intensity calculations. The segmented mask was then applied to the original image, preserving only fluorescence-positive regions for intensity measurement. c. Quantification of eNOS Signal Intensity: The largest contiguous eNOS-positive region in each image was identified based on region properties analysis (regionprops). The mean fluorescence intensity of this region was computed as a surrogate marker for eNOS expression. If multiple regions were present, only the region with the largest area was selected to ensure signal specificity. The extracted intensity values were stored in a structured results table (struct2table) and exported as a .csv file (writetable) for downstream statistical analysis. d. Data Processing and Output: The script processed all “.tif” images in the input directory, performing batch analysis iteratively. Output results were stored in a dedicated results folder, with each image’s extracted intensity value recorded alongside its filename. The complete dataset of intensities was saved as intensities.csv, which could be integrated into statistical tools for further analysis.

[0165] This automated pipeline enabled quantitative, high-throughput analysis of eNOS fluorescence intensity, reducing manual bias and ensuring reproducibility across multiple image sets. This pipeline is fully available on GitHub at httos: / / aithub.com / aravind245 / enos signal intensity measurement.

[0166] Single-cell library preparation from mouse prefrontal cortex tissue: Mice were anesthetized with isoflurane and transcardially perfused with ice-cold, bubbled artificial cerebrospinal fluid (ACSF). The brain was dissected and mounted for sectioning. Coronal brain sections were generated with a vibratome (Leica VT 1200). The vibratome chamber was filled with a sucrose-based cutting solution, and sections were cut to a thickness of 250 pm. Sections were subsequently transferred into ice-cold ACSF-Trehalose (ACSFT). Prefrontal cortex regions were excised from individual brain sections. The Paxinos and Franklin’s mouse brain atlas was used as reference.

[0167] Tissue were digested with Pronase (Sigma-Aldrich, 537088; 2 mg / ml) diluted in bubbled ACSFT at room temperature for 45 minutes. Subsequently, tissue was washed with 1% FBS in ACSFT three times. Mechanical dissociation of tissue into a single-cell suspension was achieved by pipetting the samples using pipette tips of decreasing bore sizes (p1000, p200, p100, p20). The resultant cell suspensions were filtered through a 0.2 pm cell strainer.

[0168] Single-cell suspensions were loaded onto a 10X Genomics Chromium Controller chip by personnel at The Spatial and Single Cell Genomics Platform (S2GP), Genome Institute Singapore (GIS, A*STAR), targeting the recovery of 6,000 cells. Each animal was prepared as a unique singlecell library using the Chromium Single Cell 3’ Reagent Kit (10X Genomics, v. 3.1 ). Resulting libraries were quantified and evaluated for quality using the High Sensitivity DNA chip on Bioanalyzer Agilent 2100 (Agilent Genomics). Individual cDNA libraries were pooled equimolarly and sequenced by NovogeneAIT Genomics (Singapore) using the Illumina NovaSeq6000 platform.

[0169] Analysis of single-cell RNA sequencing data: NovogeneAIT Genomics (Singapore) processed our raw single-cell sequencing data using CellRanger (10X Genomics, v. 6.0.2) and mapped reads to the mouse genome assembly (mm10). The CellRanger software suite converted scRNA-seq data into a count matrix using STAR to align reads to the reference genome, thereby determining the count of unique molecular identifiers (UMIs) mapped to each gene. Count matrices processed via a pipeline built on R / Bioconductor (R 4.0.2 / Bioconductor 3.12) packages using the SingleCellExperiment class (v. 1.12.0). Analysis of the dataset was done as previously described

[0022] .

[0170] Samples from both sham and BCAS groups were processed under identical experimental conditions, including tissue collection, cell isolation, library preparation, and sequencing. This consistency minimizes technical variability, reducing the need for batch correction. It was aimed to capture biologically meaningful differences between sham and BCAS groups within Pecaml - expressing endothelial cells, a highly specific and clearly identifiable cell population. In the uncorrected UMAR plots of the Pecam1 + cell population, cells from both sham and BCAS conditions are intermixed without forming distinct clusters specific to either condition (FIG. 41). This indicates minimal technical variability attributable to batch effects. Consequently, it was decided to proceedwithout batch correction to avoid the risk of overcorrecting and potentially obscuring subtle, biologically meaningful differences related to the disease condition.

[0171] To assign cluster cell types, cluster-enriched genes were interpreted using findMarkers (scran). Assignments were cross-referenced with known prefrontal cortex cell type markers. To elucidate endothelial identities along the arteriovenous zonation, the AUCell package (v. 1.12.0) was employed in conjunction with the cluster marker genes . Raw counts were ranked within each cell, and the Areas Under the ROC Curves (AUC) were calculated for each marker set. The identity attributed to each cell was determined based on the highest AUC value, using the 10% top-ranking genes. The “AEC2” population was excluded due to low cell count, as well as the mixed artery-venous “AVEC” population.

[0172] edgeR tool was then used to perform pseudobulk differential expression analysis on endothelial subtypes. Differentially expressed genes with P adjust-value < 0.05 were considered significant. Metascape was used for biological process enrichment analysis. For membership gene list analysis, the search terms “angiogenesis”, “blood vessel morphogenesis”, “embryonic development”, and “hypoxia”, were applied to GO Biological Processes ontology, using Metascape.

[0173] For endothelial-microglial interactions, the CellChat (v1.6.1 ) package was employed to infer and analyze intercellular communication networks. For each dataset, the workflow began with extracting the expression matrix and metadata from the Seurat object, followed by generating a CellChat object using the “createCellChat” function. Ligand-receptor interactions were identified and quantified based on differentially overexpressed ligands and receptors (p < 0.05) using the CellChat mouse database and the “identifyOverExpressedlnteractions” function. Communication probabilities between cells were inferred by analyzing highly variable genes and pathways through the “computeCommunProb" function. To refine the analysis, interactions were filtered by applying the “filtercommunication” function with a threshold of a minimum of 10 cells per group to ensure robust detection of cell-cell communication signals. Next, pathway-level communication probabilities were computed using the “computeCommunProbPathway” function, which aggregated interaction probabilities across multiple ligands and receptors within each signaling pathway. Visualization of results was carried out using various functions provided by CellChat. Specifically, “netVisual_circle” was used to display the number of interactions between cell types, while “netVisual_chord_cell” illustrated cell-cell signaling pathways.

[0174] In vivo drug administration of PT2385: Following BCAS surgery, mice were given a 10-day recovery period prior to the initiation of drug or vehicle administration. PT2385 (MCE, HY-12867) was prepared at a concentration of 6 mg / mL using a vehicle of 0.5% methyl cellulose (Sigma-Aldrich, M7027) and 0.5% Tween-80 (Sigma-Aldrich, P5188). Mice were dosed at 30 mg / kg body weight. We have carefully referenced established literature to determine the optimal dosage, administrationmethod, and duration for effective and selective EPAS1 inhibition. PT2385 has been consistently reported in the literature to demonstrate specificity for EPAS1 inhibition at doses ranging from 10 to 60 mg / kg, with durations from a few days up to 12 weeks. Following the recovery period, BCAS- operated mice were randomly assigned to two groups: one receiving the vehicle n = 10 (5 females, 5 males), and another receiving PT2385 n = 12 (7 females, 5 males). The sham-operated group received the vehicle only n = 13 (6 females, 7 males). All administrations were performed via oral gavage once daily for a total of 30 days. The health and behavior of all animals were closely monitored throughout the study period.

[0175] Behavioral assessments: The open field test was performed in a square white box 40 x 40 x 30 (height) cm for 8 min. The chamber was divided into a central field (center, 20 cm 20 cm) and an outer field (10 cm border from walls). The mouse’s movement was recorded using a video camera, and analyzed using the AnyMaze video tracking system. The center of the animal's body was used as the reference point. The Y-maze comprised three equally spaced arms (120° apart, 35 cm long, and 10 cm wide). The mice were placed in one of the arms (the start arm) and were allowed to explore in the Y-maze for 8 min with access to all three arms. Animals were tracked using AnyMaze software, and the total time and the percent time spent in each arm were analyzed.

[0176] Differentiation of arterial and venous endothelial cells from human pluripotent stem cells: A previously established differentiation protocol of arterial and venous endothelial cells was established

[0012] , H9 embryonic stem cells were propagated in mTeSR medium (StemCell Technologies, 85850) on plates coated with Matrigel (Corning, 356234). To seed for endothelial differentiation, H9 were dissociated with accutase (Gibco, A1110501 ) into single cells, then plated onto Matrigel-coated plates at a density of 50,000 cells / cm2in mTeSRI medium supplemented with 5 pM ROCK inhibitor, Y-27632 (STEMCELL Technologies, 72304). 24 hours later, H9 cells were induced towards the mid-primitive streak differentiation pathway with 40 ng / ml recombinant human BMP-4 (R&D Systems, 314-BP / CF), 6 pM CHIR99021 (Sigma-Aldrich, SML1046), 4 pg / ml recombinant human FGF2 (R&D Systems, 233-FB / CF), PIK-90 (Sigma-Aldrich, 528117), recombinant human activin A (R&D Systems, 338-AC / CF) in Chemically Defined Medium (CDM) made up of 50% Ham’s F12 (Gibco, 31765092), 50% Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, 31980097), 1 mg / ml polyvinyl alcohol (Sigma-Aldrich, P8136), 1% chemically defined lipid concentrate (Gibco, 11905031 ), 450 pM 1 -thioglycerol (Sigma-Aldrich, M6145), 15 pg / ml transferrin (Roche, 10652202001 ) and 0.7 pg / ml recombinant human insulin (Roche, 11376497001 ). After 24 hours, mid-primitive streak cells were differentiated into dorsal lateral mesoderm in CDM, supplemented with 40 ng / mL BMP4, 2.5 pM GDC-0941 (STEMCELL Technologies, 73152), 10 pM Forskolin (Tocris, 1099), 2 pM SB505124 (Sigma-Aldrich, S4696), 100 ng / ml VEGF (R&D Systems, 293-VE / CF), 1 pM XAV939 (Tocris, 3748) and 200 pg / mL L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, A8960) for 24 hours. Following that, the dorsal lateral mesoderm was further differentiated towards either arterial endothelial induction or pre-vein induction,with a duration of 24 hours. For arterial induction, CDM supplemented with 15 ng / ml_ recombinant human activin A, 250 nM DMH-1 (Tocris, 4126), 2.5 pM GDC-0941 , 100 ng / mL VEGF, 1 pM XAV939 and 200 pg / ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate was used. For pre-vein induction, CDM comprises 2 pM SB505124, 250 nM DMH1 , 2 pM RO4929097 (MedChemExpress, HY-11102), 100 ng / ml VEGF, 1 pM XAV939 and 200 pg / ml L-Ascorbic acid 2-phosphate sesquimagnesium. For pre-vein induction, cells were dissociated using accutase and reseeded on Matrigel pre-coated plates at a density of 79,000 cell / cm2. Pre-vein cells were differentiated into venous endothelial cells using CDM comprises 2 pM SB505124, 2 pM RO4929097, 500 nM PD0325901 (Tocris, 4192), 1 pM CHIR99021 and 200 pg / mL L-Ascorbic acid 2-phosphate sesquimagnesium, with a duration of 24 hours. Notably, cells were briefly washed with Dulbecco's Phosphate Buffered Saline (DPBS) (Cytiva, SH30028.03) to eliminate any residual traces of their respective induction medium before proceeding to the next stage of differentiation. Following differentiation, arterial endothelial cells were maintained in EGM-2 media (Lonza, cat no. CC-3162), and venous endothelial cells were maintained in EGM-2 media supplemented with 2 pM SB505124 and 2 pM RO4929097.

[0177] Differentiated arterial and venous endothelial cells were characterized by flow cytometry analysis. Cells were washed with DPBS and dissociated with accutase. Dissociated cells were centrifuged for 5 min at 300 xg in DPBS, then re-suspended in DPBS and the primary antibodies for arterial and venous endothelial characterization. Cells were stained at 4°C for 30 min in the dark. Stained cells were washed and resuspended in DPBS, then analyzed with BD LSRFortessa X-20 Cell Analyzer (BD Biosciences), and data acquisition was performed on FACSDiva software (BD Bioscience). Data analysis was performed with FlowJo software v 10.7.1 software (Becton Dickinson). Differentiated arterial and venous endothelial cells with at least 80% and 70% efficiency respectively were used directly for subsequent experiments.

[0178] Experimental settings for in vitro cell culture: Endothelial cells were cultured under 1% oxygen condition using a Hypoxic Incubator Chamber (StemCell Technologies, 27310) placed within a standard incubator. The chamber was purged at a flow rate of 20 L / min for 5 minutes with a gas mixture of 1% O2 and 5% CO2, balanced with N2. For in vitro cell staining for EPAS1 , endothelial cell monolayers were fixed with 4% PFA (09154-85, Nacalai Tesque) at 4°C, then permeabilized and blocked in 0.1% Triton-X100 and 1% BSA at 4 °C. Primary antibody incubation was done overnight at 4 °C. Secondary antibody staining was done for 1 hour at room temperature in the dark. Nuclear counterstaining was performed using DAPI (1 :5000). For in vitro PT2385 treatment, endothelial cells were treated with EGM-2 media supplemented with 10 pmol / L PT2385 (abeam, ab235501 ) dissolved in DMSO. Cells were treated with PT2385 during the seeding of cells for tube formation.

[0179] Venous endothelial tube formation: H9-derived venous endothelial cells were thawed and allowed to recover in EGM-2 media (Lonza, cat no. CC-3162) supplemented with 2% heat-inactivatedFBS, 1 pM ROCK inhibitor, 2 pM SB505124, 2 pM RO4929097. Venous endothelial cells were then cultured in EGM-2 supplemented with 10% FBS (heat-inactivated) for 24 hours prior to dissociating them for tube formation assay. Cells were reseeded onto Geltrex (Al 413302, Thermo Fisher Scientific) coated plates at a density of 6x104cells / cm2in EGM-2 supplemented with 10% FBS and monitored hourly for formation of tube-like structures. Number of tube-like structures was quantified based on the number of well-formed loops per field of view based on reported method

[0023] ,

[0180] Quantitative real-time polymerase chain reaction: Cells were lysed with RLT Buffer, and RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106) according to manufacturer instructions. cDNA synthesis was performed using LunaScript RT SuperMix kit (E3010, New England BioLabs) incubated in a thermal cycler at 25 °C for 2 minutes, followed by 45 minutes at 55°C, and a final step of 1 minute at 95 °C. Quantitative PCR (qPCR) was performed with cDNA and the Luna Universal qPCR Master Mix (M3003, New England BioLabs) according to manufacturer instructions. The qPCR reactions were performed using QuantStudio 6 (4485697, Thermo Fisher Scientific). All gene expression data were normalized to the 18S rRNAgene levels. Please refer to Table 3 for primer sequences.

[0181] Table 3. qPCR Primer sequences.

[0182] Hi-C visualization: In situ Hi-C sequencing data for human umbilical vein endothelial cells, human aortic endothelial cells (Telo HAEC), and prefrontal cortex were obtained from the GEO repository (accession numbers: GSE63525, GSE126200, GSE87112, respectively). Visualization of the Hi-C data was performed using the 3D Genome Browser at a resolution of 10 kb covering the region chr2:44760000-47770000.

[0183] Profiling of circulating damaged endothelial cells in human subjects: Human subject PBMCs were stained with antibodies in the dark for 10 min at room temperature, followed by 20 min at 4 °C. Following incubation, cells were washed and resuspended in DPBS with 1% BSA. Flow cytometry was performed using BD LSRFortessa X-20 Analyzer (BD Biosciences), and data acquisition was performed on FACSDiva software (BD Bioscience). Spectral overlap between INDO- 1 , APC, PE, PE-Cy7, FITC, channels was calculated using a single stain for each channel. Analysis of each subject included at least included 1 million events. Acquired data were analyzed using FlowJo software v 10.7.1 software (Becton Dickinson). CECs were detected by a combined immunophenotypic profile of CD45- / CD31 + / CD133- / DNA+ and were further characterized for theexpression of ACKR1. Fluorescence minus one (FMO) controls were used to identify the ACKR1 positive cell population (FIG. 10). Antibody details are provided in Table 2.

[0184] Functional MRI imaging of cerebrovascular disease burden in human subjects: Patients underwent neuroimaging using MRI. T1 - and T2-FLAIR images were used for visual rating of scans based on the modified Fazekas scale for WMH severity. The modified Fazekas scale was employed to quantify white matter lesions in four brain regions: right periventricular, left periventricular, right deep subcortical, and left deep subcortical, resulting in a score range of 0-12.

[0185] Human cerebral tissue perfusion by analysis of arterial spin labeling: Two-dimensional pulsed arterial spin labeling (ASL) data were acquired and processed, generating measurements in ml_ / 100 g / min. Briefly, ASL data acquisition utilized FSL’s Bayesian Inference for ASL MRI (BASIL) toolbox, and cerebral blood flow was quantified using the Buxton ASL kinetic model. Generated cerebral blood flow images were corrected for partial volume effects using BASIL’S adaptive spatial prior approach.

[0186] Cognitive assessments in human subjects: The following tests were used for cognitive assessments:Episodic Memory a.Rey Auditory Verbal Learning Test (RAVLT) Delayed b.Anna Tan Delayed (Wechsler Memory Scale (WMS)-IV Logical Memory) c.Rey-Osterrieth Complex Figure (ROCF) DelayedExecutive Function a. Trial Making Test B b. Colour Trials 2 c. Digit Span BackwardsProcessing Speed a. Colour Trials 1 b.WAIS CodingVisuospatial a.Rey-Osterrieth Complex Figure (ROCF) copy b.WAIS Block design (time bonus)Language a.Naming Test (MoCa Naming + Visual Cognitive Assessment Test (VCAT) Naming) b. Semantic fluency animals

[0187] Z-scores were derived through the following formula: [(Participant score - mean of cognitively normal) / cognitively normal SD)]. The global cognitive score was derived by combining all neuropsychological assessments to get an average score.

[0188] Statistical analysis: The data analysis, excluding the single-cell RNA sequencing data, was performed using GraphPad Prism (v. 10.2.0). Data were tested for normality using the Shapiro- Wilk test. P-values for data with a single factor were obtained using an unpaired t-test (parametric) or Mann-Whitney test (non-parametric). P-values for data with multiple comparisons were assessed using a one-way ANOVA (parametric) or Kruskal-Wallis (non-parametric) test. Error bars represent s.d..; *p < 0.05, **p < 0.01 , ***p < 0.001 ,m*p < 0.0001 ; ns, non-significant.

[0189] Data availability: The authors declare that all data supporting the findings of this study are available within the paper and supplementary information. The raw and processed single-cell RNA sequencing data from this study have been deposited in the NCBI Gene Expression Omnibus database (accession number: GSE263191 ). Source data are provided with this paper. GSE263191 [https : / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE263191]

[0190] Code availability: The codes for image analysis can be accessed from Github repository. Microglia ramification index [httos: / / doi.org / 10.5281 / zenodo.15493406] eNOS signal intensity measurement [https: / / doi.org / 10.5281 / zenodo.15493432]RESULTSExample 1 : Differential arteriovenous responses to cerebral hypoperfusion

[0191] Using laser Doppler flowmetry, the baseline cerebral blood flow readings were measured prior to BOAS and sham surgeries in each animal. In the BCAS model, a 0.18 mm (external diameter) microcoil is permanently fixed around both carotid arteries (FIG. 1A), as described previously

[0010] . The longitudinal data was quantified relative changes in cerebral blood flow over time, normalized to each animal's baseline measurement, ensuring that the analysis accounted for inter-individual variability in initial flow rates. Immediately following this procedure, cerebral blood flow was reduced to 60-70%, followed by a partial recovery by day 5 that was sustained for up to 30 days post-BCAS (FIG. 1 B).

[0192] Given that BCAS treatment selectively affects working memory, cerebrovascular phenotyping on the brain's frontal lobes was focused upon. Overall vessel density was determined by imaging endothelial cells expressing Pecaml . In sham-operated mice, vessel density remained unchanged between day 10 and day 60 post-BCAS (FIG. 1 C), indicating vascular stability under normal conditions. In contrast, BCAS mice exhibited a significant rise in vessel density by day 60, suggesting a gradual response of angiogenesis to chronic hypoperfusion. Frontal lobes largely consist of grey matter, which may be impacted later than white matter, explaining the prolonged angiogenicresponse. A previous study reported early venous branch growth taking place at 6 days post-BCAS, involving changes in vessel calibre[9]. To study arteriovenous responses to cerebral hypoperfusion, the origins of FITC-Dextran labelled blood vessels was discerned using alpha smooth muscle actin (aSMA) staining patterns to distinguish smooth muscle cell-invested arterial (striated aSMA) vessels from venous (discontinuous aSMA) vessels. Indeed, it was found that BCAS mice exhibited earlier sprouting of venous vessels compared to sham on day 10 that was sustained till day 60 (FIG. 1 D). On the other hand, arterial vessels demonstrated a delayed angiogenic response on day 60. Thus, vascular remodelling under chronic hypoperfusion was characterized by an initial angiogenic response predominantly from venous structures, followed by continued venous expansion and delayed arterial angiogenic adaptation.

[0193] Furthermore, in the analysis of vascular lumen size, anatomically equivalent vascular structures was focused on in both BCAS and sham groups - the pericallosal arteries that supply the cingulate cortex, a region within the prefrontal cortex, as well as the sagittal sinus veins that drain from the prefrontal cortex. The results demonstrated that in the BCAS model, veins exhibited significantly increased lumen diameter compared to sham controls, while arteries showed reduced lumen diameter, indicative of constriction on 60 days post-BCAS (FIG. 1 E). These findings might suggest compensatory mechanisms in response to chronic hypoperfusion. Arterial constriction reflected the brain's autoregulatory effort to optimize perfusion pressure. On the other hand, venous dilation likely compensated for reduced outflow efficiency, facilitating the drainage of deoxygenated blood.

[0194] To discern whether vein lumen changes were due to active vasodilation or passive adjustments in vascular tone secondary to altered intraluminal pressure, brain tissues were stained for endothelial nitric oxide synthase (eNOS). eNOS protein levels were assessed at 60 days post- BCAS, a timepoint representing prolonged cerebral hypoperfusion. Increased eNOS protein expression in venous segments was observed, whereas arterial eNOS expression was decreased (FIG. 1 F). While eNOS-derived nitric oxide is well known to mediate transient vasodilation in response to acute shear stress, its expression under chronic hypoperfusion may reflect sustained endothelial signalling. To further evaluate structural remodelling, changes in vessel architecture was examined. A significant increase in the wall-to-lumen ratio in veins of BCAS mice (FIG. 2A) was observed, indicative of venous wall thickening. This was accompanied by elevated collagen IV staining (FIG. 2B), suggesting extracellular matrix remodelling.

[0195] In summary, it was found that the cerebrovascular response to hypoperfusion primarily involved venous angiogenic sprouting and vasodilation, highlighting structural and functional adaptations.Example 2: Single-cell transcriptomics reveal pro-angiogenic venous cells

[0196] Next, the transcriptomic basis of dysfunctional endothelial subtypes along the arteriovenous axis by examining genome-wide expression changes in endothelial cells was determined. Single-cell transcriptomics was performed on prefrontal cortex tissue obtained from BCAS and sham mice 10 days post-surgery to capture early transcript changes (FIG. 3A). Following quality control filtering (FIG. 4A-4E) and unsupervised clustering, individual clusters were annotated based on the expression of cell type-specific markers[4], allowing for the identification of major cell types present in the brain (FIG. 3B). Particular interest was taken in the endothelial cell population with highly enriched expression of well-known endothelial-specific marker genes, including Flt1, Cldn5 and Pecaml. AUCell (v. 1 .12.0) was employed to assign individual endothelial cells into distinct subtypes based on expression profiles of arteriovenous zonation-dependent genes[4]. This approach identified sub-populations of arterial, capillary, venous-capillary, and venous endothelial cells expressing their respective subtype-specific markers (FIG. 3C). In the proportional analysis of endothelial subtypes, both sham and BCAS groups exhibited a predominance of cells from small vessels, primarily capillary and venous-capillary endothelial cells, followed by arterial and venous endothelial cells (FIG. 3D). This distribution aligned with the expected proportions of brain vasculature[4]. Notably, the BCAS group showed a significant increase in the proportion of venous and venous-capillary endothelial cells compared to the sham group. This observation was consistent with phenotypic evidence of more apparent venous angiogenic expansion in BCAS mice FIG. 1A, suggesting endothelial subtypespecific dynamics in brain ischemia.

[0197] To investigate the functional relevance of gene expression changes, we performed pseudobulk differential expression analysis to obtain differentially expressed genes (P-adjusted value < 0.05, Benjamini-Hochberg method) in BCAS mice versus sham mice for each of the endothelial subtypes. Among the endothelial subtypes, venous-capillary and capillary cells had the largest number of significant differentially regulated genes (FIG. 3E). Gene ontology (GO) analysis identified top enriched processes across all endothelial subtypes, highlighting vascular structural remodeling in response to cerebral hypoperfusion. These included focal adhesion assembly in arterial endothelial cells, supramolecular fiber organization in capillary endothelial cells, cell morphogenesis and angiogenesis in venous-capillary endothelial cells, and cell junction assembly and angiogenesis in venous endothelial cells (FIG. 3F). Interestingly, GO analysis also revealed upregulated biological processes related to synapse organization and synaptic signaling across multiple endothelial subtypes (FIG. 4F). This finding reflects the organotypic traits of specialized endothelial cells in different tissues, as reported in previous studies

[0010] . For instance, brain endothelial cells express genes associated with synaptic vesicle function, while heart endothelial cells express cardiac contractile genes. Notably, in the BCAS enriched genes, we observed evidence of endothelial cell de-differentiation, with enriched processes related to blood vessel development in arterial cells, developmental maturation in capillary cells, and embryonic development in venous cells (FIG. 4F). This suggests a potential regression to a more plastic or progenitor-like state, which may contribute to vascular remodeling in response to cerebral hypoperfusion.

[0198] To investigate the molecular drivers of venous remodeling in response to cerebral hypoperfusion, venous-enriched genes associated with blood vessel morphogenesis were analyzed , angiogenesis, hypoxia, and embryonic development. The common molecular drivers identified included AcvrH, Edn1, Epasl, and Kdr, with Epasl encoding a transcription factor (FIG. 3G). Notably, Epasl (also known as Hif2a) is a well-established regulator of angiogenesis and could potentially confer venous endothelial cells with heightened sensitivity to hypoxia, promoting their pro-angiogenic phenotype. Further supporting this notion, patients with EPAS1 gain-of-function mutations exhibit abnormal vascular patterns, including venous dilation, tortuosity, and postcapillary venule leakage. These venous cells also exhibited developmental hallmarks, such as the expression of Kdr (vascular endothelial growth factor receptor 2, Vegfr2), a known downstream target of Epasl .

[0199] Since age-related declines in cerebral blood flow are well-documented in humans

[0011] , it was examined whether venous endothelial signatures identified in the BOAS model were also evident in the aged brain. A list of endothelial molecular signatures associated with BCAS was mapped onto a published aged mouse brain endothelial single-cell transcriptomic dataset. Notably, upregulation of several key genes implicated in angiogenic and developmental pathways was observed, such as Epasl, ld1, Kdr, Klf2, and AcvrH, in aged brain endothelial cells (FIG. 4G). These findings suggest a shared endothelial adaptation to cerebral hypoperfusion and vascular aging, involving a reactivation of developmental programs in venous endothelial cells, promoting angiogenic responses.

[0200] As evidenced in FIG. 1A-1 F, BCAS venous endothelial cells displayed vasoactive properties. Key signaling pathways associated with active vasodilation were further examined, specifically focusing on molecules involved in regulating vascular tone, such as nitric oxide, prostacyclin, and G- protein-coupled receptors. This included analyzing genes linked to the eNOS pathway, calcium signaling, and vasodilatory molecules like cyclic GMP. Analysis showed that, compared to sham control, BCAS condition significantly elevated the expression levels of these vasoactive molecules in venous cells (FIG. 4H). This provides insight that venous endothelial cells could be engaged in active vasodilatory responses, in addition to passive adjustments to hemodynamic changes in the BCAS model.

[0201] An interactive browser has been made available that allows the exploration of single-cell RNA-seq profiles of BCAS and sham endothelial cells at httDs: / / christinecheunalab.shinvaoDs.io / mouse bcas ec / .Example 3: EPAS1 preferentially regulates venous angiogenic response to hypoxia

[0202] To determine how endothelial cell state could mediate the preferential angiogenic phenotype of venous cells, an in vitro system of human arterial and venous cells was leveraged (FIG. 5A) using an established protocol for endothelial differentiation from human pluripotent stem cells

[0012] , As mostprimary endothelial cells lose their subtype specificity after being sub-cultured in vitro, human arterial and venous endothelial systems offered the advantage of isogenic cell subtypes differentiated using chemically defined conditions to drive their subtype commitment. The arterial and venous cell differentiation could be reproduced with high efficiency (-90% arterial and -80% venous specification). Several venous genes differentially upregulated during BCAS that were associated with angiogenesis, blood vessel morphogenesis and hypoxia were mapped onto single-cell transcriptomes

[0012] of different stages of human venous differentiation. Most of these genes had enriched expressions in the prevein cell population that is a precursor to venous endothelial cells (FIG. 5B). Therefore, these BCAS-induced genes could potentially represent a dedifferentiated endothelial state, conferring angiogenic capability in venous cells.

[0203] To model the differential effects of cerebral hypoperfusion on endothelial subtypes, both arterial and venous endothelial cells were subjected to low oxygen condition (1%) for up to 48 hours (FIG. 5A). The absence of glucose deprivation was acknowledged. As the differentiation protocol relied on chemically defined conditions, including glucose in the basal media, removing glucose would disrupt the established protocol and potentially impact differentiation efficiency and cell fate commitment. Therefore, reduced oxygen as the stress paradigm was applied for this model. Because ID1 (also known as Inhibitor Of Differentiation 1 ) maintains multipotency in stem / progenitor cells, the gene expression of ID1 was tracked in arterial and venous endothelial cells exposed to 1 % oxygen. In arterial cells, ID1 expression levels were sharply induced during the first 2 hours of 1% oxygen exposure, and then rapidly declined (FIG. 5C). In contrast, expressions of ID1 in venous cells remained significantly elevated for up to 48 hours of exposure to 1 % oxygen. A similar reactivation of the developmental program has been documented in the oncofetal reprogramming of endothelial cells during tumour angiogenesis

[0013] . These finding highlights the plasticity of venous cells in response to low oxygen and the involvement of developmental genes in chronic angiogenic responses.

[0204] Hypoxia-inducible factors play a crucial role in embryonic vascular development. Activation of HIF-1 supports vasculogenesis under intense hypoxic conditions, while the dependence on HIF-2 is essential for the remodelling of newly formed vasculatures. Correspondingly, Epasl (Hif2a) was significantly upregulated in our BCAS venous population that was actively remodelling. Hence, we compared the dynamics of EPAS1 induction in human arterial and venous endothelial cells exposed to 1 % oxygen. Nuclear translocation of EPAS1 , a key step in the activation of its downstream hypoxia response genes, was analysed by measuring colocalization of EPAS1 with nuclei stained with DAPI (FIG. 5D). EPAS1 was translocated into the nuclei of venous cells up to 4-fold within 6 hours of 1% oxygen exposure and this was sustained up to 48 hours (FIG. 5E). On the other hand, induction of EPAS1 nuclear translocation in arterial cells was milder, reaching 2-fold after 10 hours and remaining elevated afterward, albeit at significantly lower levels than observed for venous cells. These expression changes concurred with the temporal regulation of HIFs where EPAS1 drives chronic hypoxia response (> 24 hours).

[0205] Iterative structure-based design utilizing the heterodimer of PAS-B domains of EPAS1 and ARNT lead to the discovery of PT2385 - a potent, selective, and orally active small-molecule inhibitor against EPAS1

[0014] . An endothelial tube formation assay were performed in venous cells and found that 1% oxygen significantly increased the density of tube-like structures in comparison to 21% oxygen (FIG. 5F). Further, the inhibition of EPAS1 by PT2385 abolished the low oxygen-induced tube formation by venous cells. While 21 % O2served as a baseline normoxic condition in this study, it was recognized that it might not reflect the physiological PO2experienced by endothelial cells in vivo, particularly for venous endothelium where PO2levels are typically closer to 40 mmHg. Future studies could refine this model by incorporating PO2values more reflective of in vivo conditions.Example 4: EPAS1 inhibition restores cerebral blood flow and dampens vein-associated microglial activation

[0206] The marked effect of PT2385 in vitro led to examining the actions of this drug in the BOAS model. PT2385 has been used in human clinical trials because of its high penetration of the bloodbrain barrier. It was hypothesized that EPAS1 inhibition by PT2385 could reverse venous remodeling induced by cerebral hypoperfusion in vivo. This drug was administered after a 10-day recovery period post-BCAS to allow physiological compensatory angiogenesis to take place (FIG. 6A). Mice then received daily doses of PT2385 for 30 days, followed by harvesting of brain frontal lobe tissue to characterize arteriovenous and microglial properties. In BOAS mice, cerebral blood flow was restored to levels comparable to sham controls following PT2385 treatment, whereas cerebral blood flow was significantly reduced in untreated BOAS mice (FIG. 6B). To assay overall cerebrovascular density, we visualized Pecaml -expressing endothelial structures. Remarkably, the increase in vessel density produced by hypoperfusion in BCAS was reversed back to control levels following PT2385 treatment (FIG. 6C). When arterial and venous vessels were distinguished by co-staining FITC-Dextran labeled vessels with aSMA, it was found that PT2385 treatment reversed the increased vascular sprouting produced by BCAS (FIG. 6D). This emphasized the importance of EPAS1 for hypoperfusion-induced angiogenic sprouting.

[0207] Disruption of the integrity of cerebrovascular structure by pathophysiological conditions - such as ischemic trauma, brain tumors, and neurodegenerative diseases - are associated with the accumulation of activated microglia. Similarly, the brains of BCAS mice had a significantly higher density of ionized calcium binding adaptor molecule 1 (Iba1 )-expressing microglia, an effect that was eliminated by treatment with PT2385 (FIG. 6E). Analysis of our single-cell data revealed distinct endothelial-microglial communication patterns across endothelial subtypes, shedding light on gliovascular interactions. In the sham control, ligand-receptor interactions were observed between microglia and arterial, capillary, and venous-capillary endothelial cells but were absent with venous endothelial cells (FIG. 6F). Under BCAS conditions, new interactions emerged between venous endothelial cells and microglia, involving Sema3c-Nrp1 / Nrp2 / Plxna4, Ptn-Ncl, and Nampt-lnsr pairs(FIG. 7A-7D). Endothelial-derived semaphorin 3C (Sema3c) likely served as a guidance cue to regulate microglial migration, positioning, and activation, with Nrp1 / Nrp2 and Plxna4 acting as coreceptors to fine-tune these responses. Similarly, endothelial-derived pleiotrophin (Ptn) might act as a neurotrophic and immunomodulatory factor to modulate microglial activation and phagocytosis through nucleolin (Nel). The Nampt-lnsr pair, which is unique to venous endothelial cells, suggest a specialized mechanism where Nampt, an enzyme in NAD+ biosynthesis, signals through the insulin receptor (Insr) on microglia to regulate energy homeostasis and stress responses.

[0208] A growing number of studies have highlighted the involvement of vessel-associated microglia in angiogenesis, vascular maintenance, and potentially the regulation of cerebral blood flow

[0015] . These microglia are primarily distinguished from parenchymal microglia by their close proximity to blood vessels. To explore the distinct roles of vein- and artery-associated microglia, their distribution following BOAS was assessed. Analysis revealed that BOAS significantly increased the physical association of microglia with veins, an effect that was attenuated by PT2385 treatment (FIG. 6G). In contrast, the levels of microglial association with arteries remained unchanged (FIG. 6G). Next, to assess microglial activation, morphological analysis was performed using a ramification index. With this index, long, thin processes yield a high index that reflects resting microglia, while a lower index indicates the more ameboid shape of activated microglial. Following BCAS, vein-associated microglia showed a significant increase in activation (FIG. 6H), whereas artery-associated microglia remained unaffected (FIG. 6H). Treatment with PT2385 selectively reduced the activation of vein-associated microglia, suggesting a specific, EPAS1 -mediated response of these microglia to BCAS-induced venous stress.

[0209] Gene expression analysis revealed that Epasl expression in the prefrontal cortex was localized to the endothelial cell cluster, with venous endothelial cells showing a higher relative change in expression compared to other endothelial subtypes (FIG. 7B, 7C). However, Epasl expression was negligible in the microglial cluster and was not induced in microglia by the BCAS procedure (FIG. 7C). This indicated that EPAS1 inhibition by PT2385 likely reduced microglial activation via venous endothelial-microglial interactions. These findings point to dynamic communication between endothelial cells and microglia, which may play a role in inflammatory responses during brain ischemia. Further studies will be required to understand the mechanisms underlying such interactions.

[0210] BCAS impairs spatial working memory, reference memory, and cognitive function. To assess the behavioral impact of PT2385 treatment, spatial working memory was evaluated by using a Y- maze and general locomotor activity in an open field. In the Y-maze, while spontaneous alternations (a measure of working memory) did not differ significantly across groups, the total time taken per alternation pattern was significantly shorter in treated BCAS mice compared to untreated mice (FIG. 8A, 8B). This finding suggests that PT2385 treatment may improve cognitive processing speed orefficiency. In the open field test, the total distance traveled, a measure of general locomotion, remained relatively consistent across all groups (FIG. 8C). However, in the analysis of corner zone immobile episodes, a marker of anxiety-like behavior, treated BOAS mice showed a lowered trend and appeared to normalize to sham control levels (FIG. 8D). This suggests that PT2385 may alleviate anxiety-like behaviors.Example 5: Venous dysfunction is associated with human cerebrovascular lesions and poorer cognitive function

[0211] Finally, BCAS-related brain venous pathology was sought to be connected to cerebral blood flow, cerebrovascular burdens, and cognitive functions in humans. Participants (n = 47, demographics detailed in Table 1 ) were recruited through the Dementia Research Centre, Singapore, to undergo brain magnetic resonance imaging (MRI), cerebral blood flow analysis, and cognitive assessments (FIG. 9A). The limited availability of primary endothelial cells obtained from patient brain biopsies constituted a bottleneck that we circumvented by using a surrogate biomarker of vascular injury, circulating endothelial cells (CECs). CECs are cells dislodged from damaged endothelia into the bloodstream and serve as a direct indicator of in situ vascular injury or aberrant vascular remodeling. CECs have been validated as a cell-based biomarker for vascular dysfunction across various diseases, including cardiovascular disease, COVID-19, metabolic disorders, preeclampsia, and sickle cell anemia

[0016] . Thus, blood was collected from every subject and then processed to retrieve the peripheral blood mononuclear cell (PBMC) fraction. CECs were then detected in PBMC samples by flow cytometry using a combined immunophenotypic profile of CD457CD31+ / CD133 DNA+(FIG. 9A).

[0212] MRI white matter hyperintensity, a measure of cerebrovascular disease, was quantified by a modified Fazekas scale. White matter hyperintensity was measured in four brain regions: right periventricular, left periventricular, right deep subcortical, and left deep subcortical. Absent-to-mild cerebrovascular disease was defined as a score ranging from 0-4 on the total Fazekas scale, whereas individuals with scores of 5-12 were classified as having moderate-to-severe cerebrovascular disease. Visual ratings were carried out independently by two trained raters, and any substantial discrepancies in scores were resolved through consensus. To measure cerebral perfusion, cerebral blood flow was determined by 2D pulsed arterial spin labeling data from brain MRI images.

[0213] Participants with mild cerebrovascular disease (Fazekas score 1 -4) exhibited a significant inverse correlation between the number of CECs and cerebral perfusion (FIG. 9B), indicating a decline of cerebral blood flow is associated with endothelial damage. However, this trend was only apparent in the absent-to-mild cerebrovascular disease groups (Fazekas score 0-4), rather than in severe cerebrovascular disease (Fazekas score > 5). This suggests that cerebral hypoperfusion gave rise to early vascular perturbations. In cases of severe white matter lesions, factors such as neuronal deficits, glial dysfunction, and neuroinflammation may have confounded the correlation betweencerebral perfusion and endothelial changes.

[0214] To identify the subset of CECs originating from venous structures, an additional marker was required that fulfilled both organotypic signature and venous subtype of interest. Venous specificity of ACKR1 expression in human is evident from brain single-nucleus transcriptomes

[0017] (FIG. 7A). It was recognized that ACKR1 protein might not be exclusively expressed in brain endothelial cells or venules; hence more precise markers for selective identification of damaged venous cells in the brain would be desirable. Nonetheless, ACKR1 expression was used to identify damaged CECs that originated from venous vessels (FIG. 9A). When the human subjects were divided into two groups based on the presence (Fazekas score > 0) or absence (Fazekas score = 0) of cerebrovascular disease, there was an upward trend in CEC levels (FIG. 9C, left), and a significant increase in the percentage of ACKR1+ CECs in subjects with cerebrovascular disease (FIG. 9C, right). Hence, white matter lesions seen in patients with vascular cognitive impairment might be associated with venous dysfunction.

[0215] Increases in white matter hyperintensity and cerebrovascular burden are associated with poorer cognition. In the subject cohort, the levels of ACKR1 + CECs were correlated with decreases in cognitive performance, particularly in executive function and language skills (FIG. 9D). In neuropsychological examinations, both single test scores and entire performance profiles can be used to identify cognitive deficits. An average global cognitive score was further derived - based on executive function, language, visuospatial, episodic memory abilities, and processing speed - to compare to CEC biomarkers. A significant correlation was observed between a higher percentage of ACKR1 + CECs and a decrease in global cognition (FIG. 9D). When the subjects were subset according to zero, low or high (> 95thpercentile) percentage of ACKR1 + CECs, individuals with less than average global cognitive performance (negative Z-score) sustained greater proportion of detectable ACKR1 + CECs, especially ‘high’ levels of ACKR1 + CECs (FIG. 9E). Taken together, we conclude that venous endothelial damage was associated with poorer cognitive functions in humans.

[0216] In human genetic studies of cognitive performance, the intergenic single nucleotide polymorphism rs34743896 has been associated with a change in attention score. Interestingly, the closest gene to rs34743896 is EPAS1 (~30 kb away). Data mining of in situ Hi-C sequencing datasets were performed that map genome-wide chromatin contacts in human umbilical vein endothelial cells, human aortic endothelial cells, and human brain prefrontal cortex (FIG. 8A-8D). Given the proximity of rs34743896 and EPAS1, both were indeed found in regions of intensified chromatin interactions, and potentially within the same topographically associating domains. While it is known that genetic variants can act distally, to affect regulatory elements of genes within the same topographically associating domains where regions of chromatin physically interact more frequently, further investigation is required to define the relationship between EPAS1 and cognition.DISCUSSION

[0217] This study delineated the responses of specific subtypes of endothelial cells to cerebral hypoperfusion at the molecular, structural, and functional levels, cross-species convergence of pathways in venous susceptibility to cerebral hypoperfusion in a clinically relevant BOAS mouse model of vascular dementia, as well as in deeply phenotyped human subjects was pinpointed. Human analyses linked hallmarks of venous endothelial dysfunction to the risk of cognitive impairment. In line with observations in the BCAS mouse model, the well-described loss of blood-brain barrier integrity seen in cerebral hypoperfusion and inflammatory demyelinating diseases have been related to venular leakiness and venous association with leukocyte infiltration. These findings also suggest that venous normalization as a therapeutic strategy to moderate vein-associated microglial activation and potentially reduce neuroinflammation in brain ischemia.

[0218] Emerging data suggest that venous endothelial cells are unique in their ability to serve as the primary source of endothelial cellular mass during both developmental and pathological angiogenesis. During assembly of the vascular network within the zebrafish hindbrain, the arterial system is predominantly derived from the sprouting and migration of endothelial cells from preexisting veins. Similarly, in mouse embryonic development, widespread capillary arterialization occurs from venous-features plexus during the expansion of intra-embryonic vasculatures. During postnatal vascular development of the mouse retina of neural crest origin, endothelial tip cells that are derived from veins differentiate into other endothelial subtypes[6]. Such developmental plasticity of venous cells appears to translate to an angiogenic phenotype in diseases of the brain. Here, the molecular signatures of BCAS venous cells matched the dedifferentiated characteristics of venous precursor cells in a human endothelial differentiation system

[0012] . Angiogenic endothelial subpopulations have also been commonly identified in the human brain single-cell transcriptomes of arteriovenous malformations

[0018] and Alzheimer's disease patients

[0019] , harboring pathological hallmarks for angiogenesis and immune cell reactivity respectively. We believe that venous endothelial cells exhibit a propensity for reactivation of their developmental programs, leading to chronic vascular remodeling and subsequent cerebrovascular instability.

[0219] EPAS1 plays both essential and pathophysiological roles in angiogenesis and vascular remodeling

[0020] . Epas / -deficient mouse embryos develop severe vascular defects and die in utero. While blood vessels were formed by vasculogenesis, they either fused improperly or failed to assemble into larger vessels, suggesting that EPAS1 plays an important role at post-vasculogenesis stages and is required for the remodeling of the primary vascular network into a mature hierarchy pattern. Here, it was uncovered that EPAS1 (HIF-2a) could be a key driver of venous remodeling preferentially. Human venous endothelial cells, when subjected to low oxygen (1 %) condition in vitro, responded with more pronounced and sustained EPAS1 signaling, unlike the arterial endothelial cells. Both ID1 and EPAS1 revealed similar expression dynamics in venous endothelial cells under low oxygen conditions. Id1 is highly expressed in the endothelial cells of developing vessels and tumorvasculatures, and the knock-out of Id1 results in embryonic lethality due to vascular malformations and premature neuronal differentiation. ID1 encodes a helix-loop-helix (HLH) protein devoid of DNA binding activity but can inhibit the DNA binding and transcriptional activation ability of the proteins it interacts with. While the direct interaction between ID1 and EPAS1 is unknown, a close member of the HLH family, ID2, can interact with the von Hippel-Lindau (VHL) ubiquitin ligase complex, consequently inhibiting the ubiquitylation and degradation of EPAS1 . Regarding the more well-studied HIF-1 a, ID1 protein enhances its protein stability through reduced association of VHL with HIF-1a, thus interfering with proteasomal degradation of HIF-1a. In fact, EPASf and HIF- a share a 48% amino acid sequence identity and possess similar protein structures. Despite their unique regulatory pathways, both proteins undergo oxygen-dependent degradation facilitated by VHL, highlighting a common regulatory mechanism. Therefore, the interplay between I Df and EPAS1 might be facilitated via ID1 's interaction with VHL.

[0220] To understand whether venous response is adaptive or maladaptive under blood flow impairment, it is believed that Epasl could initially drive compensatory angiogenesis from venous endothelial cells. Arterial constriction has been observed to regulate flow and venous dilation to manage outflow, highlighting the hemodynamic implications of cerebrovascular responses to hypoperfusion. Unlike HIF-1 that controls acute response to hypoxia, EPAS1 mediates chronic response. Hence, sustained imbalances of arteriovenous responses in brain ischemia could transition to maladaptation. While venous pathology has not been known to directly cause chronic hypoperfusion, the absence of valves in cerebral veins permits bidirectional blood flow, potentially exacerbating blood outflow. Hence, venous cells would be exposed to more metabolic by-products due to impaired drainage, exacerbating vascular remodeling. Elevated CEC levels in human subjects, correlated with reduced cerebral blood flow and cognitive impairment, could indicate endothelial cells that have been shed from dysfunctional blood vessels, often because of endothelial damage or stress. This underscores the need for therapeutic interventions to alleviate venous dysfunction and restore cerebral blood flow.

[0221] The finding of venous dependency on EPAS1 -driven angiogenesis draws parallels with a gain-of-function EPAS1 mutation in human patients, which results in a spectrum of venous anomalies and malformations that are attributed to failure of developmental vascular regression. A mutation in the oxygen degradation domain of EPAS1 hinders its hydroxylation by prolyl hydroxylase domaincontaining protein 2 and its subsequent binding to the VHL protein. This leads to stabilization of EPAS1 and lack of response to increasing oxygen tension. EPAS1 stability, in turn, contributes to chronic venous remodeling. Interestingly, genetic adaptations involving EPAS1 in high-altitude populations, notably in Tibetan and Andean highlanders, reduce hypoxia-induced responses such as excessive erythrocytosis, which can strain the cardiovascular system. In Tibetans, EPAS1 adaptations, mainly missense mutations and regulatory changes, moderate the hypoxia response, preventing extreme red blood cell increases and likely supporting cognitive stability through consistentcerebral oxygenation. Conversely, Andeans, who have adaptations that result in higher hematocrit levels, may experience a greater risk of hypoxia-related complications, including potential subtle cognitive effects with prolonged altitude exposure. These differences highlight how EPAS1 adaptations contribute to resilience against low oxygen but suggest that cognitive outcomes are likely shaped by other genetic and environmental factors.

[0222] Developing therapeutic interventions targeting venous maladaptation to low oxygen, as well as neuroinflammation, will be beneficial. This study demonstrated that inhibiting EPAS1 through a small molecule drug, PT2385, effectively reversed the venous response to low oxygen, both in vitro and in vivo, and specifically reduced the density and activation of vein-associated microglia. Vessel- associated microglia appear to play a role in cerebral blood flow regulation during hypoperfusion. Microglial P2RY12 contributes to cerebrovascular adaptation during common carotid artery occlusion, and its blockade reduces cerebral blood flow without affecting neuronal responses. The microglial processes around capillaries correlate with cerebral blood flow levels during transient ischemia, in turn indicating blood flow's role in microglial activation. In systemic inflammation, microglia migrate towards cerebral vessels to initially protect their integrity, then transform into a reactive state that results in widespread neuroinflammation. In vitro evidence further suggests that damaged endothelium can promote microglia to transition toward an inflammatory phenotype. Thus, decreased vein-associated microglial density and activation following PT2385 treatment here may be attributable to the resolution of venous dysfunction and impaired blood flow.

[0223] This study bridges findings from animal models to human conditions, aligning cerebrovascular and cognitive parameters where feasible. In mice, cerebral blood flow was quantified using Doppler flowmetry, providing real-time measurement of blood flow changes. For human subjects, MRI arterial spin labeling was employed, offering a non-invasive assessment of cerebral perfusion. Although distinct methods, both techniques allow us to infer cerebral blood flow, enabling meaningful cross-species comparison of perfusion dynamics. In terms of cerebrovascular phenotyping, in vivo analysis of cerebrovascular structure in our mouse model included quantitative measurements of sprouting and vasodilation in arteriovenous vessels, markers directly relevant to vascular remodeling and endothelial function. In humans, MRI imaging of white matter hyperintensities serves as an indirect marker of small vessel disease. To assess cognitive function and its relation to vascular changes behavioral tests were performed in mice, focusing on working memory and motor function, which approximate the executive function, language, and memory assessments conducted in our human cohort. This approach aims to achieve an integrative, crossspecies analysis. Specific markers of vascular integrity that are readily obtainable in the mouse model cannot be directly measured in human subjects. In the human cohort, a composite cognitive score, derived from assessments with distinct testing formats, was utilized to approximate functional domains close to those evaluated in mouse behavioral tests.

[0224] In conclusion, targeting EPAS1 as a therapeutic strategy for vascular normalization slows the progression of cerebrovascular disease. EPAS1 inhibition mitigated vascular remodeling and dampened vein-associated microglial activation in the BCAS model, suggesting that modulating chronic angiogenesis has a role in reducing neuroinflammation. Currently, diagnosis of vascular dementia relies on neuroimaging and assessment of cognitive function impairment

[0021] . With this criterion, however, patients are only diagnosed after the onset of cognitive decline when the disease is irrevocably developed, as there is yet no effective treatment. Analysis herein of patient CECs, as part of a minimally invasive routine blood test, will translate to accurate and transferable endpoints in early clinical assessment of vascular health. The proof-of-concept in correlating the levels of CECs with cognitive functions in patients may have applications in stratification-based clinical trials for selecting patients for vascular-protective therapy, and as treatment-prediction biomarkers to interpret clinical trial findings mechanistically.

[0225] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

[0226] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The EPAS1 inhibitors, compositions, methods, kits and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0227] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0228] The content of all documents and patent documents cited herein is incorporated by referencein their entirety.References:1 . Wiesmann, M., Kiliaan, A. J. & Claassen, J. A. Vascular aspects of cognitive impairment and dementia. J Cereb Blood Flow Metab 33, 1696-1706 (2013). https: / / doi.org:10.1038 / icbfm.2Q13.1592. Brown, W. R., Moody, D. M., Challa, V. R., There, C. R. & Anstrom, J. A. Venous collagenosis and arteriolar tortuosity in leukoaraiosis. J Neurol Sol 203-204, 159-163 (2002). httPs: / / doi.org:10.1016 / s0022-510x(02)00283-63. Quick, S., Moss, J., Rajani, R. M. & Williams, A. A Vessel for Change: Endothelial Dysfunction in Cerebral Small Vessel Disease. Trends Neurosci 44, 289-305 (2021 ). https: / / doi.org:10.1016 / i.tins.2020.11 .0034. Vanlandewijck, M. etal. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475-480 (2018). https : / / doi.org:10.1038 / nature257395. Fischer, A., Zalvide, J., Faurobert, E., Albiges-Rizo, C. & Tournier-Lasserve, E. Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis. Trends Mol Med 19, 302- 308 (2013). https: / / doi.org:10.1016 / i.molmed .2013.02.0046. Lee, H. W. et al. Role of Venous Endothelial Cells in Developmental and PathologicAngiogenesis. Circulation 144, 1308-1322 (2021 ). https : / / doi.oro:10.1161 / CIRCULATIONAHA.121.0540717. Halder, S. K. & Milner, R. The impact of chronic mild hypoxia on cerebrovascular remodelling; uncoupling of angiogenesis and vascular breakdown. Fluids Barriers CNS 18, 50 (2021 ). https: / / doi.org:10.1186 / S12987-021 -00284-x8 Nishio, K. ef al. A mouse model characterizing features of vascular dementia with hippocampal atrophy. Stroke 41 , 1278-1284 (2010). https: / / doi.org:10.1161 / STROKEAHA.110.5816869 Srinivasan, V. J. et al. Micro-heterogeneity of flow in a mouse model of chronic cerebral hypoperfusion revealed by longitudinal Doppler optical coherence tomography and angiography. J Cereb Blood Flow Metab 35, 1552-1560 (2015). https: / / doi.oro:10.1038 / icbfm.2015.17510 Barnett, S. N. et al. An organotypic atlas of human vascular cells. Nat Med 30, 3468-3481 (2024). https: / / doi.org :10.1038 / S41591 -024-03376-x1 1 Mokhber, N. et al. Cerebral blood flow changes during aging process and in cognitive disorders: A review. Neuroradiol J 34, 300-307 (2021). https: / / doi.org:10.1177 / 1971400921100277812 Ang, L. T. etal. Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell 185, 2523-2541 e2530 (2022). https: / / doi.org:10.1016 / i.cell.2022.05.02413 Schwab, M. et al. Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature. JCI Insight 8 (2023). https : / / doi.org:10.1172 / ici. insight.14307114 Wallace, E. M. et al. A Small-Molecule Antagonist of HIF2alpha Is Efficacious in Preclinical Models of Renal Cell Carcinoma. Cancer Res 76, 5491 -5500 (2016). httos: / / doi.org:10.1158 / 0008- 5472.CAN-16-047315 Chen, S. et al. Microglia and macrophages in the neuro-glia-vascular unit: From identity to functions. Neurobiol Dis 179, 106066 (2023). https : / / doi.org:10.1016 / i.nbd.2023.10606616 Ng, C. Y. & Cheung, C. Origins and functional differences of blood endothelial cells. Semin Cell Dev Biol (2023). https: / / doi.org:10.1016 / i.semcdb.2023.05.00117 Yang, A. C. etal. A human brain vascular atlas reveals diverse mediators of Alzheimer's risk. Nature 603, 885-892 (2022). https : / / doi.org:10.1038 / s41586-021 -04369-318 Winkler, E. A. etal. A single-cell atlas of the normal and malformed human brain vasculature. Science 375, eabi7377 (2022). https : / / doi.oro:10.1126 / science.abi737719 Lau, S. F., Cao, H., Fu, A. K. Y. & Ip, N. Y. Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer's disease. Proc Natl Acad Sci U S A 117, 25800-25809 (2020). https: / / doi.org:10.1073 / pnas.200876211720 Hashimoto, T. & Shibasaki, F. Hypoxia-inducible factor as an angiogenic master switch. Front Pediatr3, 33 (2015). https: / / doi.org:10.3389 / fped.2015.0003321 Gorelick, P. B. et al. Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association / american stroke association. Stroke 42, 2672-2713 (2011 ). https : / / doi.org:10.1161 / STR.0b013e318229949622 Basnakova, A. eta / . The habenula clock influences response to a stressor. Neurobiol Stress 15, 100403 (2021 ). https : / / doi.org:10.1016 / j.Ynstr.2O21 .10040323 Bae, E. et al. Integrin alpha3beta1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis. Nat Common 13, 4268 (2022). https : / / doi.org:10.1038 / s41467-022-31981 -2

Claims

CLAIMS1. An endothelial PAS domain protein 1 (EPAS1 ) inhibitor for use in treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.

2. The EPAS1 inhibitor of claim 1 , wherein the disease or condition associated with cerebral hypoperfusion is selected from the group consisting of cerebrovascular diseases, neuroinflammatory conditions, neurodegenerative disorders, cognitive and neuropsychiatric conditions, and systemic or developmental conditions involving secondary cerebral hypoperfusion.

3. The EPAS1 inhibitor of claim 1 , wherein the disease or condition associated with cerebral hypoperfusion is neuroinflammation, or a cerebrovascular disease.

4. The EPAS1 inhibitor of claim 1 , for use in treating cerebral hypoperfusion.

5. The EPAS1 inhibitor of claim 1 , wherein the disease or condition associated with cerebral hypoperfusion is vascular cognitive impairment or vascular dementia arising from cerebral hypoperfusion.

6. The EPAS1 inhibitor of claim 1 , wherein the EPAS1 inhibitor reduces vascular remodelling, angiogenic sprouting, vein-associated microglial activation, and / or restores cerebral blood flow to achieve vascular normalization.

7. The EPAS1 inhibitor of claim 1 , wherein the EPAS1 inhibitor is a small molecule, peptide, nucleic acid-based inhibitor, or an antibody that specifically binds to EPAS1 or its regulatory components.

8. The EPAS1 inhibitor of claim 1 , wherein the EPAS1 inhibitor is a small molecule selected from PT2385, PT2399, PT2977, or a pharmaceutically acceptable salt thereof.

9. The EPAS1 inhibitor of claim 1 , wherein the EPAS1 inhibitor is PT2385, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition comprising an EPAS1 inhibitor and a pharmaceutically acceptable carrier and / or excipient for use in treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.1 1. The pharmaceutical composition of claim 10, wherein the EPAS1 inhibitor is PT2385, or a pharmaceutically acceptable salt thereof.

12. A method of treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject, the method comprising administering an effective amount of an EPAS1 inhibitor of claim 1 or pharmaceutical composition of claim 10, to the subject.

13. Use of an EPAS1 inhibitor in the manufacture of a medicament for treating, preventing, or ameliorating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion, in a subject.

14. The EPAS1 inhibitor of claim 1 , the pharmaceutical composition of claim 10, the method of claim 12, or the use of claim 13, wherein the subject is a human subject.

15. A method of monitoring the efficacy of an EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in a subject, comprising:(a) administering the EPAS1 inhibitor to the subject;(b) obtaining a biological sample from the subject; and(c) detecting and measuring the amount of CECs and / or ACKR1 + CECs, in the biological sample; and(d) comparing the measured amount of CECs and / or ACKR1 + CECs in the sample to a reference amount, wherein a differential amount of CECs and / or ACKR1 + CECs measured in the sample relative to the reference amount is indicative of the efficacy of the EPAS1 inhibitor in treating cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion in the subject, optionally steps (a)-(d) are repeated two or more times within a time-frame and the detection and measurement at each time point is compared against each other to assess the progression of the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion and efficacy of the treatment.

16. The method of claim 15, further comprising comparing cerebral blood flow measurements of the subject obtained by magnetic resonance imaging using arterial spin labelling (ASL) readouts before initiation of treatment and over the course of treatment.

17. A method for diagnosing, or predicting a subject's risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion, the method comprising: measuring the amount of circulating endothelial cells (CECs), and CECs that are positive for Atypical Chemokine Receptor 1 (ACKR1 ) in the sample that has been obtained from the subject; and comparing the measured amount of CECs and ACKR1 + CECs in the sample to a reference amount,wherein a differential amount of CECs and ACKR1+ CECs measured in the sample relative to the reference amount is indicative of the subject having, or the subject being at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

18. The method of claim 17, wherein the amount of CECs is measured by flow cytometry using the markers CD45- / CD31 + / CD133- / DNA+, and a percentage of ACKR1+ CECs is measured relative to the amount of CECs.

19. The method of claim 17, wherein the biological sample is a blood sample, preferably a PMBC sample.

20. The method of claim 17, wherein the differential amount comprises an increase in the percentage or absolute abundance of CECs and ACKR1 + CECs in the subject, which is indicative of the subject having, or the subject being at risk of developing, cerebral hypoperfusion or any condition or disease associated with cerebral hypoperfusion.

21. The method of claim 17, wherein the condition or disease associated with cerebral hypoperfusion is endothelial dysfunction and / or venous dysfunction.

22. The method of claim 17, further comprising administering the EPAS1 inhibitor of claim 1 to the subject for treating, preventing, or ameliorating the cerebral hypoperfusion, or a disease or condition associated with cerebral hypoperfusion.

23. The EPAS1 inhibitor of claim 1 , the pharmaceutical composition of claim 10, the method of claim 12, 15 or 17, or use of claim 13, wherein the disease or condition associated with cerebral hypoperfusion is a cerebrovascular disease.

Images