4-oxoretinoate for use in post-myocardial infarction therapy

Abstract

The present invention relates to 4-oxoretinoate for use in treatment or prevention of cardiac dysfunction, heart failure, or adverse cardiac remodelling after myocardial infarction or impaired cardiac blood flow. The present invention also relates to 4-oxoretinoate for use in treatment of coronary artery disease, or of ischemia-reperfusion injury.

Description

[0001] 4-Oxoretinoate for Post-Myocardial Infarction Therapy

[0002] This application claims the right of priority of European Patent Application EP24222539.9 filed 20 December 2024, which is incorporated by reference herein.

[0003] Field

[0004] The present invention relates to 4-oxoretinoate for use in treatment or prevention of cardiac dysfunction, heart failure, or adverse cardiac remodelling after myocardial infarction or impaired cardiac blood flow. The present invention also relates to 4-oxoretinoate for use in treatment of coronary artery disease, or of ischemia-reperfusion injury.

[0005] Background

[0006] Myocardial infarction (Ml) presents a substantial global health issue. Post-MI survival and outcomes depend on acute compensatory responses, scar formation, and tissue remodelling in both the cardiac lesion and remote myocardium. Inflammation, which is crucial for post-MI healing and tissue remodelling, is driven by infiltrating leukocytes that coordinate processes like debris breakdown, collagen deposition, and neovascularization. Demand for these inflammatory leukocytes post injury is met by emergency haematopoiesis (EH). However, excessive EH has been linked to worse remodelling, cardiac dysfunction, and heart failure following Ml. Targeting systemic inflammation post-MI has led to inconclusive results. Thus, there is an urgent need for new and specific therapeutic approaches.

[0007] Positioned at the apex of the hematopoietic system, bone marrow (BM) quiescent hematopoietic stem cells (HSCs) have the ability to generate multipotent progenitors (MPPs), which can differentiate into lineage-committed progenitors and subsequently give rise to leukocytes. Dysregulation of HSC quiescence can lead to aberrant haematopoiesis such as clonal haematopoiesis and HSC exhaustion. We have previously shown in mice that active metabolites of vitamin A are potent modulators of HSC activity, and they effectively safeguard HSCs against activation upon non-physiological stimuli (Cabezas-Wallscheid N. et al., Cell, 169(5), 2017; Schbnberger K. et al., Cell Stem Cell, 29(1), 2022). It has been described that mouse HSCs proliferate and functionally decline upon Ml. Still, whether HSCs give rise to progeny during EH that infiltrate the heart upon Ml has not been proven. In fact, the contribution of HSCs upon stress conditions has been recently challenged.

[0008] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to prevent or treat cardiac dysfunction, or cardiac diseases. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification. Summary of the Invention

[0009] The invention relates to 4-oxoretinoate for use in treatment or prevention of certain cardiac indications, in particular cardiac dysfunction after myocardial infarction or impaired cardiac blood flow, myocardial infarction or impaired cardiac blood flow, adverse cardiac remodelling after myocardial infarction or impaired cardiac blood flow, coronary artery disease, or ischemiareperfusion injury.

[0010] Terms and definitions

[0011] General

[0012] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0013] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0014] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0015] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0016] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0017] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0019] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0020] As used herein, the term treating or treatment of any disease or disorder (e.g. cardiac dysfunction) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow.

[0021] As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier.

[0022] As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). The invention also encompasses nanoparticles, liposomes, or cellular carriers within the meaning of pharmaceutically acceptable carrier.

[0023] Detailed Description of the Invention

[0024] A first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of cardiac dysfunction after myocardial infarction. An alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of impaired cardiac blood flow. Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in prevention of heart failure after myocardial infarction. Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in prevention of heart failure after impaired cardiac blood flow.

[0025] Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of adverse cardiac remodelling after myocardial infarction. Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of adverse cardiac remodelling after impaired cardiac blood flow. In certain embodiments, adverse cardiac remodelling is manifested as ventricular dilation.

[0026] Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment of coronary artery disease. Coronary artery disease is a chronic inflammatory condition.

[0027] Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of ischemia-reperfusion injury. Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of ischemia-reperfusion injury after restoring blood flow. Another alternative of the first aspect of the invention relates to 4- oxoretinoate for use in treatment or prevention of ischemia-reperfusion injury after myocardial infarction. Another alternative of the first aspect of the invention relates to 4-oxoretinoate for use in treatment or prevention of ischemia-reperfusion injury after impaired cardiac blood flow.

[0028] In certain embodiments, the (above-mentioned) disease is associated with increased hematopoietic stem cell activity.

[0029] In certain embodiments, the (above-mentioned) disease is associated with increased hematopoietic stem cell proliferation.

[0030] In certain embodiments, increased hematopoietic stem cell proliferation or activity is manifested as emergency hematopoiesis. This emergency haematopoiesis may be a direct consequence of a myocardial infarction.

[0031] 4-Oxoretinoate

[0032] 4-oxoretinoate and 4-oxo-retinoic acid are used synonymously inside the present specification. 4- oxoretinoate has a pKa value of 4.76 (based on calculator plugins for pKa prediction and calculation, 2024, ChemAxon (http: / / www.chemaxon.com), https: / / qo.druqbank.com / metabolites / DBMET02550). Therefore, 4-oxoretinoic acid will likely dissociate to 4-oxo-retinoate inside most body fluids, as for example in the bloodstream (having a pH value of 7.4).

[0033] AII-trans-4-oxoretinoate and all-trans-4-oxo-retinoic acid:

[0034]

[0035] 9-cis-4-oxoretinoate and 9-cis-4-oxoretinoic acid:

[0036] 13-cis-4-oxoretinoate and 13-cis-4-oxoretinoic acid:

[0037] Endogenous 4-oxoretinoate is mainly present intracellularly as 4-oxoretinol. Vitamin A derivatives are stored in the liver as retinyl esters and, upon demand, mobilized into the bloodstream mainly in the form of retinol (main detectable vitamin A form in circulation). Retinol is taken up by cells and intracellularly metabolized to 4-oxo-retinoic acid (mainly detectable within cells).

[0038] In certain embodiments, 4-oxoretinoate is selected from the group of all-trans-4-oxoretinoate, 9- cis-4-oxoretinoate, and 13-cis-4-oxo-retinoic acid. In certain embodiments, 4-oxoretinoate is all- trans-4-oxoretinoate. In certain embodiments, 4-oxoretinoate is 9-cis-4-oxoretinoate. In certain embodiments, 4-oxoretinoate is 13-cis-4-oxoretinoate.

[0039] In certain embodiments, 4-oxoretinoic acid is selected from the group of all-trans-4-oxoretinoic acid (CAS No: 38030-57-8), 9-cis-4-oxoretinoic acid (CAS No: 150737-18-1), and 13-cis-4-oxo- retinoic acid (CAS No: 71748-58-8). In certain embodiments, 4-oxoretinoic acid is all-trans-4- oxoretinoic acid. In certain embodiments, 4-oxoretinoic acid is 9-cis-4-oxoretinoic acid. In certain embodiments, 4-oxoretinoic acid is 13-cis-4-oxoretinoic acid.

[0040] Administration regime

[0041] In certain embodiments, 4-oxoretinoate is administered for at most one month. In certain embodiments, 4-oxoretinoate is administered for at most one week. In certain embodiments, 4- oxoretinoate is administered for 2 to 3 days.

[0042] Pharmaceutical Compositions, Administration / Dosage Forms and Salts

[0043] According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0044] The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

[0045] In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.

[0046] The invention further encompasses a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0047] The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0048] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[0049] Method of Manufacture and Method of Treatment according to the invention

[0050] The invention further encompasses, as an additional aspect, the use of 4-oxoreti noate as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of cardiac dysfunction.

[0051] Similarly, the invention encompasses methods of treatment of cardiac dysfunction, comprising administering to a patient in need thereof a therapeutically effective amount of 4-oxoretinoate, or its pharmaceutically acceptable salt, as specified in detail herein.

[0052] Also, the invention encompasses a method of treatment of certain cardiac indications, in particular cardiac dysfunction after myocardial infarction or impaired cardiac blood flow, myocardial infarction or impaired cardiac blood flow, adverse cardiac remodelling after myocardial infarction or impaired cardiac blood flow, coronary artery disease, or ischemia-reperfusion injury.

[0053] Also, the invention encompasses a method of treatment of an indication associated with increased hematopoietic stem cell proliferation and / or activity.

[0054] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for an isomer may be combined with any of the alternative embodiments of an indication.

[0055] The specification further comprises the following items:

[0056] Items:

[0057] 1 . 4-Oxoretinoate for use in treatment or prevention of a cardiac condition associated with impaired coronary blood flow.

[0058] 2. 4-Oxoretinoate for use according to item 1 , wherein the cardiac condition is selected from: (a) coronary artery disease;

[0059] (b) myocardial ischemia;

[0060] (c) ischemia-reperfusion injury;

[0061] (d) myocardial infarction.

[0062] 3. 4-Oxoretinoate for use according to any of the preceding items, wherein the cardiac condition comprises a functional impairment following myocardial infarction or impaired coronary blood flow.

[0063] 4. 4-Oxoretinoate for use according to item 3, wherein the functional impairment is cardiac dysfunction.

[0064] 5. 4-Oxoretinoate for use according to item 3 or 4, wherein the functional impairment is heart failure.

[0065] 6. 4-Oxoretinoate for use according to any of items 1-5, wherein the condition comprises pathological structural remodeling of the heart.

[0066] 7. 4-Oxoretinoate for use according to item 6, wherein the pathological structural remodeling is adverse cardiac remodeling following myocardial infarction or impaired coronary blood flow.

[0067] 8. 4-Oxoretinoate for use according to item 7, wherein the adverse cardiac remodeling comprises ventricular dilation.

[0068] 9. 4-Oxoretinoate for use according to item 8, wherein the ventricular dilation is left-ventricular dilation.

[0069] 10. 4-Oxoretinoate for use according to any one of the preceding items, wherein the condition is associated with increased hematopoietic stem cell activity.

[0070] 11 . 4-Oxoretinoate for use according to any one of the preceding items, wherein the condition is associated with increased hematopoietic stem cell proliferation.

[0071] 12. 4-Oxoretinoate for use according to any one of the preceding items, wherein the 4- oxoretinoate is selected from the group of all-trans-4-oxo-retinoic acid, 9-cis-4-oxo-retinoic acid, and 13-cis-4-oxo-retinoic acid.

[0072] 13. 4-Oxoretinoate for use according to any one of the preceding items, wherein the 4- oxoretinoate is all-trans-4-oxo-retinoic acid.

[0073] 14. 4-Oxoretinoate for use according to any one of the preceding items, wherein 4-oxoretinoate is administered for at most one month, particularly for at most one week, more particularly for 2 to 3 days.

[0074] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. of the Figures

[0075] Fig. 1 shows 4-oxo-RA safeguards HSC functionality and dampens HSC activity upon Ml a. 4-oxo-RA reduces differentiation of HSCs towards myeloid cells and emergency haematopoiesis in the bone marrow in HSC lineage tracing experiments. Flow cytometry-based plots, illustrating the percentage of HSC-traced (dTomatopos) cell frequencies for Ml+vehicle and MI+4-oxo-RA conditions in the BM upon Ml. Cell frequencies are normalized to the vehicle condition. Data is presented as mean with standard deviation. Ordinary two-way ANOVA. n = 6-8. b. 4-oxo-RA dampens activation of HSCs post-MI and protects HSCs from cell cycle entry. Flow cytometrybased analysis of HSC cell cycle of sham, Ml+vehicle and MI+4-oxo-RA conditions. The percentage of cell cycle phases (GO, G1 , and G2 / S / M) is shown. Depicted p values correspond to the percentage of cells in the GO phase. Statistics denote comparisons between Ml+vehicle or MI+4-oxo-RA condition and sham condition. Ordinary two-way ANOVA. n = 7-11 . c. 4-oxo-RA dampens proliferation of HSCs post- MI. Single cell HSC division assay in sham, Ml+vehicle and MI+4-oxo-RA HSCs. The percentage of cells is shown. Depicted p values correspond to the percentage of nondivided cells. Statistics denote comparisons between vehicle or 4-oxo-RA condition and the sham condition. Ordinary two-way ANOVA. n = 7-11 . d. 4-oxo-RA counteracts loss of HSC self-renewal post-MI. HSC Colony Forming Unit assay comparing sham, vehicle and 4-oxo-RA conditions upon Ml. Ordinary one-way ANOVA. n = 7-11 .

[0076] Fig. 2 shows 4-oxo-RA preserves long-term cardiac function and reduces scar formation in myocardium post-MI. a. 4-oxo-RA dampens recruitment of infiltrating inflammatory myeloid cells from bone marrow to heart. Flow cytometry-based plots of HSC lineage tracing experiments, illustrating the percentage of HSC-traced (dTomatopos) cell frequencies for the Ml+vehicle and MI+4-oxo-RA condition in the myocardium upon Ml. Cell frequencies are normalized to the vehicle condition. Data is presented as mean with standard deviation. Ordinary two-way ANOVA. n = 6-8. b. 4-oxo-RA reduces infiltration of pro-inflammatory myeloid cells to the heart post-MI. Left panel: UMAP plot of 4-oxo-RA mouse cardiac CD11 bposcells single cell RNA-sequencing (scRNA-seq) upon Ml, based on projection of vehicle scRNA-seq. Colors indicate the predicted cell type annotation, n = 2-3. Right panel: UMAP density plots of vehicle and projected 4- oxo-RA mouse cardiac CD11 bposcells scRNA-seq upon Ml depicting relative cell abundance. Lower panel: Bar plot of quantified relative cluster abundance in Ml+vehicle and projected MI+4-oxo-RA mouse cardiac CD11 bposcells scRNA-seq. Fisher tests, c. 4-oxo-RA reduces cardiac inflammation. 4-oxo-RA mitigates expression profiles of inflammatory cytokines in the myocardium in Ml+vehicle and MI+4-oxo-RA conditions (reparative phase at day 10 post Ml). Normalized mean relative to Oaz1 expression and relative to sham surgery is shown. Values are relative to the average per gene, n = 3-4. d. 4-oxo-RA reduces detrimental scar formation in the myocardium. Masson's Trichrome staining for overall collagen deposition and in myocardial zones in Ml+vehicle and MI+4-oxo-RA. Lines highlight the separated areas in representative images. Two-tailed unpaired t-test. n = 6. RZ, Remote zone; BZ, Border zone; IZ, Infarct zone. e. 4-oxo-RA preserves cardiac function post-MI. Cardiac functional assessment by echocardiography (heart function post-MI). Quantification of echocardiographic parameters: ejection fraction and stroke volume, comparing the quality control at day one to the chronic phase function at day 28 across treatment conditions post Ml. Ordinary one-way ANOVA. n = 7-13. LV, left ventricle.

[0077] Fig. 3 Rarp is indispensable for 4-oxo-RA’s beneficial effects on HSCs and heart function post-MI. a. Rarp expression is specific for bone marrow HSCs. qPCR expression profile of Rarb in bone marrow and cardiac populations in control and Ml mice. Normalized mean relative to Oaz1 housekeeper expression is shown, n = 3-4. b. 4- oxo-RA does not reduce post-MI proliferation of Rarp knockout HSCs. Single cell HSC division assay in sham, Ml+vehicle and MI+4-oxo-RA Rarp knockout HSCs. The percentage of cells is shown. Statistics denote comparisons between Ml+vehicle or MI+4-oxo-RA condition and sham condition. Ordinary two-way ANOVA. n = 5-6. c. 4- oxo-RA fails to maintain HSC self-renewal in absence of Rarp. Colony Forming Unit assay comparing sham, Ml+vehicle and MI+4-oxo-RA condition. Ordinary two-way ANOVA. n = 6. d. 4-oxo-RA relies on Rarp in HSCs to preserve cardiac function post- MI. Cardiac functional assessment by echocardiography in Rarp knockout mice (heart function post-MI). Quantification of ejection fraction, comparing the quality control at day one to the chronic phase function at day 28 across treatment conditions post Ml. Ordinary one-way ANOVA. n = 7-10. LV, left ventricle.

[0078] Fig. 4 shows : a. 4-oxo-RA restores human HSC function and counteracts human HSC activity post Ml. Single cell HSC division assay in Ml HSCs after in vitro treatment with 4-oxo-RA or control (DMSO). The percentage of cells is shown. Depicted p values correspond to the percentage of non-divided cells. Ordinary two-way ANOVA. n = 3. b. Human Ml HSC Colony Forming Unit assay after in vitro treatment with 4-oxo-RA or control (DMSO). Unpaired t-test. n = 6.

[0079] Fig. 5 The figure shows a single-cell division assay of mouse bone marrow hematopoietic stem cells cultured in vitro under activating conditions. Treatment with 4-oxo-retinoic acid significantly increased the proportion of non-dividing HSCs compared to control, demonstrating preserved quiescence. In contrast, treatment with the structurally distinct RARp agonist AC-261066 resulted in a significantly lower proportion of nondividing cells under identical conditions. Data are presented as mean ± SD; n = 3; unpaired two-tailed t-test. In this preclinical study, we demonstrate for the first time that Ml induces significant detrimental transcriptional and functional alterations in human BM HSCs. Further, we show that HSCs contribute to the generation of inflammatory cardiac-infiltrating leukocytes upon Ml through lineage tracing experiments. We propose a novel therapeutic approach by exclusively modulating the root of EH with vitamin A metabolites, specifically forcing HSC quiescence in the aftermath of Ml, to dampen the excessive EH and ultimately improve long-term cardiac function. We found all-trans retinoic acid (at-RA), a well-established clinical agent, to negatively impact local cardiac healing post-MI, whereas its downstream metabolite, 4-oxo-RA, bypassed these drawbacks and demonstrated improved cardiac recovery upon Ml.

[0080] Example 1: Ml leads to persistent activation and impaired functionality of human HSCs

[0081] We collected sternal BM biopsies from >150 patients undergoing cardiac surgery. Patients with a history of cancer, hematological diseases, prior chemotherapy, clonal haematopoiesis or radiation therapy, infectious diseases and acute infections were then excluded. We then selected patients with coronary artery bypass grafting (CABG) surgery and excluded cases with heart failure by means of reduced left ventricular ejection fraction (EF < 45%) or other signs of cardiac congestion (e.g. proBNP > 1000 pg / ml). A total of 49 biopsies were used in the study with patients falling into the category of either (1) control samples from patients with chronic coronary artery disease (CAD) but no history of Ml, and (2) samples from patients with a history of Ml. To ensure comparability between the control and Ml group, clinical patient characteristics such as age, gender, and relevant health metrics were matched.

[0082] To determine the consequences of Ml on human BM HSCs, we conducted single-cell RNA sequencing (scRNA-seq) on human stem and progenitor cells (HSPCs) (Lineageneg, CD38neg, CD34pos) isolated from 7 Ml (MI-HSPCs) and 6 control (control-HSPCs) patients. Annotation of scRNA-seq clusters revealed 3 major cell populations including HSCs, MPPs, and multi-lymphoid primed progenitors (MLPs). Comparing the relative cell numbers between control and Ml samples within each cluster, we found a significant decrease in HSCs, accompanied by increases in MPPs and MLPs, following Ml. Gene set enrichment scoring showed that sternness-associated gene signatures were downregulated, whereas cell cycle activation related terms were upregulated in HSCs upon Ml. Gene ontology (GO) analysis highlighted upregulation of processes such as Regulation of inflammatory response and Positive regulation of cytokine production, collectively suggesting increased cellular activity and differentiation in HSCs after Ml.

[0083] To assess the in vitro function of MI-HSPCs, we performed serial colony forming unit (CFU) assays. MI-HSPCs showed increased colony output in the first plating, which was consistent with their higher level of activation and cell-cycle priming. Conversely, MI-HSPCs showed reduced colony formation in the second plating, indicating an impaired self-renewal capacity. To assess the in vivo functional potential, we transplanted human MI-HSPCs and their respective control into humanized mice NBSGW. Human chimerism was monitored in peripheral blood (PB) for a period of 6 months, allowing the evaluation of long-term self-renewal capacity. While both groups initially exhibited similar engraftment, MI-HSPCs demonstrated a reduced capacity to sustain long-term engraftment. These findings confirm an impaired functionality of human stem cells following Ml.

[0084] GO term analysis showed that human MI-HSPCs were molecularly primed towards leukocyte activation, particularly towards myeloid cells. To further investigate the downstream consequences of HSC activation post Ml, we focused on monocytes due to their crucial role in cardiac injury. We performed scRNA-seq analysis on BM monocytes (Lineageneg, CD45pos, HLA-DRpos) collected from acute (Ml < 7 days at BM isolation) and chronic (Ml > 12 months at BM isolation) Ml patients. RNA velocity and pseudotime analysis suggested that annotated cells followed a trajectory from common myeloid progenitors (CMPs) to cycling cells, classical, intermediate, and non-classical monocytes, successively. During the acute phase, CMP levels significantly rose, suggesting an increased demand for monocyte production in response to cardiac injury, while the chronic phase revealed a rise in the subsequent cycling subpopulation. Notably, Gene Set Enrichment Analysis (GSEA) showed a pro-inflammatory priming during the acute phase that persisted into the chronic phase of post-MI classical monocytes. These findings suggest that Ml leads to an inflammatory priming at the HSPC level, accompanied by chronic alterations in downstream monocyte progeny.

[0085] Collectively, our data provide compelling evidence of the activation and functional decline of human HSCs post Ml.

[0086] Example 2: HSCs contribute to inflammatory myeloid cell infiltration in the heart

[0087] Several studies have shown a correlation of mouse HSC proliferation and increased immune cells in the heart upon Ml. Previously, we observed that systemic anti-inflammatory treatment with IL- 1 P inhibitors was also associated with reduced HSC proliferation post Ml. However, it is still unknown if these activated HSCs directly contribute to the immune progeny that infiltrates the heart tissue, and therefore it is unclear whether targeting HSC activation would be a reasonable therapeutic strategy for Ml. To investigate whether HSCs are responsible for the production of infiltrating immune cells in the heart upon Ml, we made use of the well-established Fgd5CreERT2 / + HSC fate mapping mouse model which has been employed in numerous studies (Gazit R. et al., Journal of Experimental Medicine, 211 (7), 2014). This mouse model harbors a ZsGreen-2A-CreERT2 cassette within the native Fgd5 locus and is crossed with Rosa26-Lox- Stop-Lox-Tomato mice (Rosa26lsl-Tomato / +). Tamoxifen administration results in a permanent dTomato label on HSCs that is inherited by their progeny allowing lineage tracing. In the BM, Fgd5 is highly expressed in HSCs when compared to downstream hematopoietic populations, while in the cardiac tissue we only found marginal expression in endothelial cells, validating its suitability for tracking HSC progeny in the context of Ml. Upon label induction and following a standard equilibrium time of four weeks to minimize labeling variability in the HSC compartment, we subjected mice to Ml by performing left anterior descending artery (LAD) ligation. Sham surgery, which mimics the stress of the surgical procedure without inducing ischemic injury, served as control. Additional control groups included mice without any surgery or treatment, which served as a baseline for label equilibrium comparison.

[0088] Following Ml, we observed a significant increase in the proportion of dTomato-labeled (dTomatopos; HSC-derived cells) MPPs (Lineageneg, cKitpos, Sca-1 pos (LKS) CD48pos, CD150neg) and myeloid progenitors (MyP; LKSneg) in the BM, as shown by flow cytometry. We employed a mixed-effects linear model as previously reported, to evaluate the impact of Ml on HSC differentiation across baseline, sham, and Ml conditions, with the baseline serving as the reference (see Methods). To this end, data was normalized to the labeling in the HSC compartment. The sham condition showed no significant deviation from the baseline, suggesting that the observed variations in sham mice align with the expected range of the model, including random effects (indicated by a coefficient of 0.004; p-value=0.865). In contrast, the Ml condition showed a significant difference from the baseline, suggesting that Ml drives a distinct differentiation response in cell compartments that cannot be solely attributed to the model and random effects (indicated by a coefficient of 0.083; p-value=0.001). Subsequent analysis to determine which specific cell compartment contributes to this variation yielded non-significant results (p-value>0.05) confirming that the observed changes in differentiation are attributable to a response from the source HSCs, rather than from other compartments (MPPs and MyP). These results suggest that Ml increases the differentiation of HSCs through an MPP-MyP-myeloid trajectory. Further, we observed increased absolute frequencies of dTomatopos leukocytes (CD45pos), particularly myeloid cells (CD45pos, CD11 bpos) upon Ml, while lymphoid cells (CD45pos, B220pos or CD3pos) did not significantly change.

[0089] In the myocardium, we detected dTomatopos cells by immunofluorescence staining, which particularly infiltrated the infarct zone post-MI. Furthermore, flow cytometry-based quantification showed increased dTomatopos leukocytes, especially myeloid cells including neutrophils (Lineageneg, CD45pos, CDU bpos, Ly6Gpos), macrophages (Lineageneg, CD45pos, CDU bpos, Ly6Gneg, F4 / 80pos) and inflammatory monocytes (Lineageneg, CD45pos, CDU bpos, Ly6Gneg, F4 / 80neg, Ly6Chigh).

[0090] These findings indicate that BM HSC activation after Ml triggers the production and subsequent infiltration of myeloid cells into the cardiac tissue.

[0091] To minimize downstream labeling of HSC-derived cells at the time of Ml, we shortened the equilibrium time (10 days) after labeling induction. We then investigated HSC-derived cardiac myeloid infiltration by performing scRNA-seq on myeloid (CDU bpos) dTomatopos and dTomatoneg cells, both isolated from the myocardium. We first assessed the overall gene expression changes between dTomatopos and dTomatoneg myeloid cells. Interestingly, we observed an upregulation of inflammation and cardiac infiltration-related genes, such as S100a8 and Ly6c1 , in dTomatopos myeloid cells, suggesting that they contribute to myocardial inflammation post Ml. We then annotated the distinct myeloid subsets based on published population markers and gene signatures. Quantification of cell abundance of each cluster showed that dTomatopos cells are predominantly inflammatory monocytes and neutrophils (e.g. high levels of Tnf, 111 b, S100a8), known for their central contribution to inflammation within the myocardium post Ml. Notably, this cluster as well as reparatory macrophages showed the most transcriptomic differences, with a significant upregulation of pro-inflammatory genes such as Ly6c1 and Cxcl12 and downregulation of healing-related genes such as Ccl24 and Mrc2 in HSC- derived dTomatopos infiltrating cells.

[0092] Overall, we identified BM HSCs as pivotal contributors to the inflammatory myeloid cell infiltration in the myocardium after Ml. These findings underscore the potential for targeted intervention at the HSC level to beneficially shape the subsequent immune response.

[0093] Example 3: Regulation of MI-HSCs by vitamin A metabolites

[0094] To uncover potential regulators of MI-HSC activation, we performed GO term analysis of differentially expressed genes (DEGs) and identified a significant downregulation of retinoic acid (RA)Zvitamin A receptor binding in human MI-HSCs. We have recently shown that RA metabolites such as all-trans-retinoic acid (at-RA) and its downstream metabolite 4-oxo-retinoic acid (4-oxo- RA) positively modulate mouse HSC function by maintaining quiescence under stress conditions (Cabezas-Wallscheid N. et al., 2017 ibid Schbnberger K. et al., 2022 ibid). To explore the role of RA signaling as a modulator of human MI-HSCs, we first treated healthy human BM HSCs (Lineageneg, CD38neg, CD34pos, CD45RAneg) in vitro with at-RA or 4-oxo-RA and performed RNA-seq, CFU assays, and single-cell division assays. In line with our mouse study, at-RA and 4- oxo-RA treatment increased transcriptional signatures associated with HSC features, enhanced in vitro self-renewal capacity and reduced the proportion of HSCs undergoing cell division. These findings show that RA metabolites positively regulate human HSC function. Given the observed dysregulation of RA signaling in human MI-HSCs, we hypothesized that these metabolites would be modulators of HSCs post Ml.

[0095] Due to at-RA’s clinical availability, we first investigated whether at-RA can modulate HSC activation in vivo upon Ml and consequently reduce downstream immune cell production to ultimately enhance cardiac healing. We performed LAD ligation in mice followed by either at-RA or DMSO (vehicle control) treatment for two consecutive days post-MI. Sham surgery served as a non-ischemic control. We then analyzed HSC activation in the acute phase post Ml. GSEA revealed that HSCs were transcriptionally activated in vehicle-treated mice following Ml compared to sham controls, whereas at-RA counteracted this activation and preserved sternness signatures in comparison to vehicle-treated mice. Cell cycle analysis, ex-vivo single-cell HSC (scHSC) division and CFU assays showed enhanced quiescence and in vitro self-renewal capacity of HSCs after Ml upon at-RA treatment. Overall, these findings demonstrate that at-RA effectively counteracts the functional decline of HSCs following Ml.

[0096] We next assessed whether the beneficial effects of at-RA on HSCs extend to modulating the downstream immune response. We observed decreased leukocyte numbers in the BM, and myocardium in the acute phase of Ml, including Ly6Chi monocytes. In contrast, immunohistochemistry staining of the myocardium revealed an accumulation of myeloid cells during the chronic phase post Ml especially in remote areas distant to the initial lesion. While echocardiographic analysis one day after surgery confirmed equal cardiac dysfunction between Ml and at-RA treated groups, cardiac function did not show any improvement upon at-RA treatment in the chronic phase after Ml.

[0097] At-RA controls gene expression via the transcriptional activation of the three Retinoic Acid Receptor (Rar) types Rara, Rarb, and Rarg exerting distinct transcriptional responses. To understand the mechanisms underlying at-RA’s adverse effects upon Ml, we investigated the expression of RA receptors in the hematopoietic system (HSCs, MPPs, myeloid, T- and B-cells), the BM niche (endothelial cells, osteoblasts, and mesenchymal stem cells) and the cardiac tissue (fibroblasts, endothelial cells, monocytes, and macrophages). qPCR analysis showed that Rara and Rarg were highly expressed in several hematopoietic and BM niche populations, while Rarb was exclusively expressed in HSCs. RNA-seq of cardiac cell populations showed high Rara expression levels in monocytes and macrophages. Considering Rara's established role in driving pro-inflammation and the accumulation of cardiac myeloid cells upon at-RA treatment, activating the at-RA-Rara axis in myeloid cells may counteract the beneficial effects of reducing HSC- induced EH on cardiac remodelling. In line, after treating wildtype mice with at-RA and performing RNA-seq on isolated cardiac monocytes and macrophages, we observed a global inflammatory priming in both populations.

[0098] Altogether, at-RA dampens HSC activation and reduces myelopoiesis upon Ml while inducing a pro-inflammatory phenotype in cardiac myeloid cells, limiting the improvement in myocardial remodelling. Thus, the lack of specificity of at-RA precludes its usability to improve Ml outcomes. Given that at-RA is a well-established clinical agent for hematological malignancies like acute promyelocytic leukemia and dermatological conditions, our findings underscore the importance of excluding its use in Ml patients.

[0099] Example 4: 4-oxo-RA safeguards HSC functionality upon Ml

[0100] We previously showed that 4-oxo-RA, a downstream metabolite of at-RA, promotes HSC quiescence through Rarp binding (Schbnberger K. et al., 2022 ibid). Rarb is not expressed in cardiac cells, while in the BM Rarb is exclusively expressed in HSCs when compared to downstream progenitors, all differentiated blood cells including monocytes, and BM niche cells, as assessed by both transcriptomics and qPCR. Additionally, its expression is not affected upon Ml. Based on this, we hypothesized that 4-oxo-RA may circumvent at-RA’s adverse cardiac effects by selectively targeting HSCs in the BM. Indeed, and in sharp contrast to at-RA, in vivo administration of 4-oxo-RA had no significant impact on the myocardium (macrophages, monocytes, fibroblasts, and endothelial cells), as shown by GSEA and differentially expressed genes.

[0101] To explore the potential of 4-oxo-RA, in not only circumventing at-RA’s adverse effects but also favorably modulating the immune response post Ml, we induced Ml in mice followed by 4-oxo-RA (or vehicle) treatment for two consecutive days.

[0102] In the acute phase of Ml, we isolated BM HSPCs (Lineageneg, Scal pos, cKitpos) to conduct scRNA-seq analysis. We grouped cells into 3 distinct major annotations based on molecular signatures: HSCs, MPPs, and highly cycling MPPs (MPP-cyc). Similar to our human data, we confirmed a significant reduction in the relative HSC numbers along with an expansion of MPP- cyc in vehicle-treated Ml mice. Importantly, the 4-oxo-RA-treated group showed similar HSC and MPP-cyc percentages upon Ml when compared to sham control, suggesting mitigated HSC activation. GSEA showed that 4-oxo-RA treatment conserves HSC quiescence-related gene signatures and suppresses activated MPP signatures, underlining its potential to preserve HSC identity and counteract Ml-induced HSC activation. HSC lineage tracing using the Fgd5CreERT2 mouse model further demonstrated that 4-oxo-RA treatment significantly reduced the release of circulating dTomatopos myeloid (CD11 bpos) cells in PB during the 3-day time course. We also observed reduced differentiation of HSCs towards the myeloid compartment (Fig. 1 a). Of note, no significant effect was observed in lymphoid cells. Mechanistically, 4-oxo-RA treatment maintained HSC quiescence post Ml, as evidenced by cell cycle analysis and ex vivo scHSC division assays (Fig. 1 b and c). In addition to enforcing quiescence, 4-oxo-RA treatment also maintained the selfrenewal capacity of HSCs, as shown by in vitro CFU (acute phase) and in vivo HSC transplantation (chronic phase) assays (Fig. 1d). Collectively, these results demonstrate that 4- oxo-RA can counter the functional decline of HSCs after Ml.

[0103] HSCs migrate from the BM to the spleen in response to Ml, where they contribute to extramedullary hematopoiesis. We therefore conducted scRNA-seq analysis on splenic HSPCs and identified two major clusters in the spleen progenitor compartment: HSCs and MPPs. 4-oxo- RA-treated mice showed an increased relative abundance of HSCs and a reduction in MPPs, suggesting dampened HSC activation in the spleen. This was further supported by GSEA results, which showed higher cell-cycle priming in the vehicle-treated compared to the 4-oxo-RA-treated group in spleen HSCs. These findings demonstrate that post-MI HSC activation occurs in the BM and spleen and can be mitigated by 4-oxo-RA treatment. Example 5: 4-oxo-RA preserves long-term cardiac function upon Ml

[0104] HSC lineage tracing with the Fgd5CreERT2 model showed that 4-oxo-RA treatment reduced the proportion of dTomatopos myeloid (CD11 bpos) cells in the myocardium as measured by flow cytometry. Specifically, we observed a reduced contribution of HSCs into Ml-induced neutrophils, monocytes, and macrophages infiltrating the myocardium, including the inflammatory dTomatopos Ly6Chi monocyte population (Fig. 2a). We next performed scRNA-seq analysis of HSC-traced myeloid (CD11 bpos) cells in the myocardium and compared vehicle to 4-oxo-RA- treated mice using our refined short equilibrium tracing approach. Overall comparison of cardiac dTomatopos myeloid cells between 4-oxo-RA and vehicle-treated mice showed a downregulation of inflammation-related GO terms upon 4-oxo-RA treatment. We then projected 4-oxo-RA cells onto our Ml scRNA-seq clusters and found a relative enrichment of reparatory macrophages, which are defined by high levels of Arg1 and enrichment of reparative processes such as ECM and Wound healing (Fig. 2b). In line, we observed an upregulation of these healing-related terms in 4-oxo-RA treated in dTomatopos cells. Pro-inflammatory monocytes and neutrophils were the most affected populations, showing a downregulation of proinflammatory genes such as Cxcll upon 4-oxo-RA treatment. Accordingly, we observed reduced expression of inflammatory cytokines in the overall infarct tissue during the reparative phase upon Ml as shown by qPCR (Fig. 2c). Additionally, we quantified reduced collagen deposition, particularly in the remote zone of the heart, along with decreased expression of collagen-related genes in cardiac fibroblasts, collectively indicating a dampened scar formation upon 4-oxo-RA (Fig. 2d).

[0105] Immunohistochemistry stainings showed reduced myeloid infiltration in the myocardium during the chronic phase. Stroke volume and EF of the left ventricle were significantly preserved in the chronic phase post Ml demonstrating a significant improvement in heart function upon 4-oxo-RA treatment (Fig. 2e). Further, 4-oxo-RA treatment also showed a slight improved (non-significant) survival upon Ml compared to vehicle-treated mice.

[0106] These results provide strong evidence that 4-oxo-RA intervenes at the HSC level, altering their differentiation trajectory in the BM. This intervention preserves HSC functionality and effectively modulates the BM-heart myeloid axis. By reshaping both the quantitative and qualitative dynamics of myeloid infiltration into the cardiac tissue, 4-oxo-RA contributes to a suppressed inflammatory state and thus demonstrates promising therapeutic potential in enhancing post-MI cardiac function.

[0107] Example 6: RarB is indispensable for 4-oxo-RA’ s beneficial effects

[0108] To mechanistically investigate the role of Rarp in facilitating 4-oxo-RA's beneficial effects on myocardial outcomes, we subjected Rarp knockout mice to Ml followed by two day 4-oxo-RA treatment. Of note, Rarp expression is exclusive for BM HSCs and not expressed in other hematopoietic cells, the bone marrow niche or cardiac cells (Fig. 3a). In the absence of Rarp, 4- oxo-RA treatment did not maintain HSC function and neither mitigated the downstream myeloid response in the BM and myocardium as shown by scRNA-seq analysis, scHSC division, CFU assays and flow cytometry analysis (Fig. 3b, c). Ultimately, 4-oxo-RA proved ineffective in preserving cardiac function in the chronic phase of Ml in Rarp knockout mice (Fig. 3d). Similarly, we transplanted Rarp knockout BM cells into wildtype recipient mice, which then underwent Ml via LAD ligation. In these Rarp KO chimeras, treatment with 4-oxo-RA showed no improvement in the cardiac function, highlighting the lack of a beneficial effect when Rarp is specifically deleted in the BM. In conclusion, 4-oxo-RA mediates its beneficial effect on HSC protection and cardiac remodelling through Rarp at the BM level.

[0109] Example 7: RA metabolites restore the function of human HSPCs impaired by Ml

[0110] Considering that modulating HSC-dependent inflammatory myelopoiesis improves cardiac function in the mouse, we next addressed whether RA metabolites can restore human HSCs following Ml-induced impairment. To do this, we isolated human HSPCs from Ml patients and treated them with the RA metabolites 4-oxo-RA and at-RA.

[0111] Using population RNA-seq and GSEA, we observed a significant enrichment of gene signatures linked with human quiescent HSCs, indicating a preserved HSC identity upon culture. This was accompanied by the upregulation of processes related to cell adhesion and negative regulation of cell activation and inflammatory response. Notably, treatment with RA metabolites also downregulated differentiation signatures, indicating their role in suppressing human HSC differentiation in culture. Functionally, both metabolites attenuated HSPC proliferation in singlecell division assays (Fig. 4a). Post-treatment CFU assays demonstrated that both RA metabolites maintained in vitro self-renewal capacity (Fig. 4b). To further investigate the translatability of RA signaling to human, we assessed the differential expression of the human dataset and our previously published mouse data upon RA treatment (Schbnberger K. et al., 2022 ibid). We found a notable overlap of upregulated genes related to HSC quiescence and of downregulated genes associated with cell cycle and differentiation upon RA treatment in both species. Additionally, direct target genes of RA in mouse (Schbnberger K. et al., 2022 ibid) were significantly enriched in human RA treated HSPCs, indicating a conserved RA signaling among both species.

[0112] Finally, we aimed to investigate the effect of retinoids in human monocytes. Indeed, human BM monocytes express RARA and not RARB, in line with the mouse data. Thus, we isolated human PB monocytes from healthy donors and treated them with both RA metabolites. RNA-seq analysis revealed an upregulation of IFN-y response following at-RA treatment, which was attenuated by 4-oxo-RA. This regulatory pattern was consistent across various inflammatory cytokines, as determined by a cytokine secretion assay. Additionally, 4-oxo-RA treatment resembled control-treated monocytes, showing low expression of ICAM-1 , a key molecule in leukocyte recruitment, and reduced ROS levels that are associated with monocyte activation, when compared to at-RA treatment.

[0113] In conclusion, 4-oxo-RA effectively counteracted the impairment of human HSC function following Ml and bypassed at-RA’s inflammatory effects on human monocytes. These findings underscore its potential in therapeutic interventions at human HSC level upon Ml to prevent excess myelopoiesis in patients.

[0114] Example 8: Comparison to another RAR / 3 agonist

[0115] Under in vitro culture conditions that promote HSC activation and proliferation, treatment with 4- oxo-retinoic acid significantly increased the fraction of non-dividing HSCs compared to control (Figure 5). In contrast, the RARp agonist AC-261066 showed a significantly reduced ability to maintain HSC quiescence, as reflected by an increased proportion of dividing cells compared to 4-oxo-RA. These results demonstrate that preservation of HSC quiescence is not a general property of RARp agonists but is specifically associated with 4-oxo-retinoic acid.

[0116] Example 9: Discussion

[0117] In summary, we demonstrate that, upon Ml, 4-oxo-RA treatment enforces HSC quiescence, thereby mitigating EH and ultimately preserving cardiac function. Upon 4-oxo-RA treatment post Ml, we observed (ii) dampened HSC activation; (iii) a reduction in HSC-derived inflammatory myeloid cells infiltrating the heart; and (iv) no beneficial effects on HSCs and cardiac function in the Rarp KO mice and wildtype mice transplanted with Rarp KO BM. Further, we found (i) specific expression of the high affinity 4-oxo-RA receptor Rarb in BM HSCs compared to downstream hematopoietic cell populations, BM niche cells and cardiac tissue in homeostatic conditions and post Ml. Finally, (v) we show absence of transcriptional changes in cardiac cell populations upon 4-oxo-RA treatment, confirming a high therapeutic specificity to HSCs.

[0118] Our study bridges a critical translational gap in the BM-heart axis and demonstrates HSC activation and loss of functionality in human patients post Ml, consistent with previous studies performed in mice (24: Dutta, P. et al. Myocardial Infarction Activates CCR2+ Hematopoietic Stem and Progenitor Cells. Cell Stem Cell 16, 477-487 (2015)). These findings highlight a detrimental and persistent impact on the BM HSC pool that is independent of chronic inflammation observed in heart failure patients (45-46; Marvasti, T. B. et al. Heart Failure Impairs Bone Marrow Hematopoietic Stem Cell Function and Responses to Injury. J Am Heart Assoc 12, 27727 (2023); Hoffmann, J. et al. Post-myocardial infarction heart failure dysregulates the bone vascular niche. Nat Commun 12, 3964 (2021)). We show that Ml patients display a myeloid- priming signature at the HSPC level that is accompanied by a persistent inflammatory state and distinct temporal distribution of monocytes in the BM. Indeed, it has been reported that elevated levels of circulating monocytes correlate with worsened ventricular remodelling in Ml patients (47- 49; Maekawa, Y. et al. Prognostic significance of peripheral monocytosis after reperfused acute myocardial infarctions possible role for left ventricular remodelling. J Am Coll Cardiol 39, 241 — 246 (2002); Mariani, M. et al. Significance of total and differential leucocyte count in patients with acute myocardial infarction treated with primary coronary angioplasty. Eur Heart J 27 , 2511-2515 (2006); Tsujioka, H. et al. Impact of Heterogeneity of Human Peripheral Blood Monocyte Subsets on Myocardial Salvage in Patients With Primary Acute Myocardial Infarction. J Am Coll Cardiol 54, 130-138 (2009).). This implies that reduced monocytes could be beneficial for cardiac recovery and could thus dampen the risk of subsequent ischemic events. Due to the lack of tracing-tools, we and others could not provide any evidence of a direct contribution from BM Ml- activated HSCs to cardiac infiltrating monocytes (23, 24, 50; Heidt, T. et al. Chronic variable stress activates hematopoietic stem cells. Nat Med 20, 754-758 (2014); Dutta, P. et al. Myocardial Infarction Activates CCR2+ Hematopoietic Stem and Progenitor Cells. Cell Stem Cell 16, 477-487 (2015); Heidt, T. et al. Differential Contribution of Monocytes to Heart Macrophages in Steady-State and After Myocardial Infarction. Circ Res 115, 284-295 (2014)). Indeed, the role of HSCs in producing differentiated cells upon EH has been recently challenged (25, 27; Munz, C. M. et al. Regeneration after blood loss and acute inflammation proceeds without contribution of primitive HSCs. Blood 141 , 2483-2492 (2023); Fanti, A. K. et al. Flt3- and Tie2-Cre tracing identifies regeneration in sepsis from multipotent progenitors but not hematopoietic stem cells. Cell Stem Cell 30, 207-218. l (2023)). Using an HSC mouse lineage-tracing approach (Gazit R. et al., 2014 ibid', Bowling et al.; Cell, 2020), we have shown the rapid contribution of BM HSCs to myeloid cell production in the acute phase following Ml. Our study delineates a possible differentiation pathway of HSCs, progressing from MPPs to MyP in the BM, ultimately leading to the presence of inflammatory myeloid cells within the infarcted heart. It is important to note that our tool cannot rule out partial contributions from other compartments. Despite this limitation, the Fgd5 allele is the most specific model that still allowed us to trace enough cells into the heart for profiling, which confirmed the inflammatory properties of HSC-derived myelopoiesis in the myocardium.

[0119] Targeting systemic inflammation has been suggested as a potential Ml treatment. We previously showed that systemic inhibition of IL-1 p, known to be produced by cardiac myeloid cells, reduced the myeloid cells and general cardiac inflammatory response (14: Sager, H. B. et al. Targeting interleukin-1 p reduces leukocyte production after acute myocardial infarction. Circulation 132, 1880-1890 (2015).). However, this reduction occurs in a non-selective manner with multiple unknown effects in other organs. This might explain why human clinical studies targeting overall inflammation have led to inconsistent outcomes (10-13; Ridker, P. M. et al. Low-Dose Methotrexate for the Prevention of Atherosclerotic Events. New England Journal of Medicine 380, 752-762 (2019); Ridker, P. M. et al. Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease. New England Journal of Medicine 377, 1119-1131 (2017); Nidorf, S. M. et al. Colchicine in Patients with Chronic Coronary Disease. New England Journal of Medicine 383, 1838-1847 (2020); Brown, E. J. et al. Scar thinning due to ibuprofen administration after experimental myocardial infarction. Am J Cardiol 51 , 877-883 (1983)). We therefore propose to target the root of the inflammatory events by modulating specifically HSC activity and thus downstream myeloid production, limiting disease progression.

[0120] We previously showed that active metabolites of vitamin A play a crucial role in modulating in vivo mouse HSC quiescence (Schbnberger K. et al., 2022 ibid; Cabezas-Wallscheid, N. et al. Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy. Cell 169, 807-823. e19 (2017).). Here we reveal the potential of at-RA, extensively used in different clinical applications, in inhibiting human HSC activation and mitigating emergency myelopoiesis. However, we also observe a pro-inflammatory response in cardiac myeloid cells that may counteract cardiac remodelling benefits. This may be due to at-RA’s non-selective binding within RA receptors, leading to diverse cellular responses (Michaille, et al.; Developmental Dynamics 1994; Rowe et al.; Development 1991 ; Bushue & Wan. Adv. Drug Deliv. Rev. 2010). In line with our observations, previous studies have reported contradictory roles for at-RA in cardiac repair following Ml. Some studies suggest cardio-protective effects of at-RA, such as the reduction of cardiac hypertrophy (56; Paiva, S. A. R. et al. Retinoic acid supplementation attenuates ventricular remodelling after myocardial infarction in rats. J Nutr 35, 2326-2328 (2005) and anti- apoptotic effects after Ml. However, other studies indicate adverse effects on Ml outcomes (59- 60; Danzl, K. et al. Early inhibition of endothelial retinoid uptake upon myocardial infarction restores cardiac function and prevents cell, tissue, and animal death. J Mol Cell Cardiol 126, 105-117 (2019); Silva, R. A. C. et al. Cardiac Remodelling Induced by All-Trans Retinoic Acid is Detrimental in Normal Rats. Cellular Physiology and Biochemistry 43, 1449-1459 (2017). Our study underlines the broad impact of at-RA upon systemic administration and might contribute to clarify these conflicting results. Importantly, our findings also highlight the importance of avoiding at-RA treatment, which is used in various clinical contexts, in post-MI patients.

[0121] In contrast, 4-oxo-RA exhibits higher binding specificity for Rarp (Silva et al.; Cell. Physiol. Biochem. 2017; Idres, N., Marill, J., Flexor, M. A. & Chabot, G. G. Activation of retinoic acid receptor-dependent transcription by all-trans-retinoic acid metabolites and isomers. Journal of Biological Chemistry 277 , 31491-31498 (2002).) which is exclusively expressed in HSCs among all cell populations present in BM and not in cardiac tissue (19,62-65; Cabezas-Wallscheid, N. et al. Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy. Cell 169, 807-823. e19 (2017); Cabezas-Wallscheid, N. et al. Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 15, 507-522 (2014).; Dahlin, J. S. et al. A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice. Blood 131 , e1-e11 (2018); Qian, P. et al. Retinoid-Sensitive Epigenetic Regulation of the Hoxb Cluster Maintains Normal Haematopoiesis and Inhibits Leukemogenesis. Cell Stem Cell 22, 740-754. e7 (2018); Fei, L. et al. Systematic identification of cell-fate regulatory programs using a single-cell atlas of mouse development. Nature Genetics 2022 54:754, 1051-1061 (2022). Here we demonstrate that 4-oxo-RA treatment specifically attenuates MI-HSC activation, thereby reducing myelopoiesis and off-target effects on myeloid cells. In the myocardium, we observe a reduced monocyte recruitment and an enrichment of reparatory-type macrophages. A decrease in cardiac inflammation may also establish a microenvironment that leads to a differential programming of infiltrating monocytes towards a more reparative state. Taken together, this may contribute to the reduction of fibrosis and observed improvement of cardiac function upon 4-oxo-RA treatment. In contrast to systemic anti-inflammatory approaches, treatment with 4-oxo-RA represents a highly specific modulation of EH, which subsequently impacts the function of distal organs like the heart.

[0122] Example 10: Materials and Methods

[0123] Mouse models

[0124] Mice used in this study were bred in-house at the Max Planck Institute of Immunology and Epigenetics (MPI-IE) and housed in individually ventilated cages (IVCs), with the exception of C57BI6J wildtypes with induced myocardial infarction (Ml), which were purchased from Janvier and Charles River. All mice with induced Ml were conventionally housed at the Freiburg Center for Experimental Models and Transgenic Service. Only female mice aged 6 to 12 weeks were used. Euthanasia followed German guidelines using cervical dislocation or C02 gas inhalation. Animal procedures followed approved protocols by German authorities and Regierungsprasidium Freiburg, under §4 (3) of the German Animal Protection Act, with animal protocol numbers 35- 9185.81 / G-19 / 32, 35-9185.81 ZG-19 / 112, 35-9185.81 / G21 / 063 and 35-9185.81 / G-22 / 102. Lineage tracing experiments were performed under protocol 23-048-PIL approved by the CEEA committee of Parc Cientific de Barcelona.

[0125] Transplantation models

[0126] Female mice, either B6Ly5.1 (CD45.1) or C57BL / 6J x B6Ly5.1 (CD45.1 / 2), were used as recipients for transplantation experiments and / or as supportive or competitive donors. Recipient mice were aged between 6 and 12 weeks. Supportive or competitive donors were age-matched to their respective control group.

[0127] Rar[3 knockout mouse model

[0128] Mice lacking the Rarp gene were purchased from The Jackson Laboratory (Jax Stock Rarptm1Vgi / HsvJ, stock No 022999). Breeding was conducted at MPI-IE. Experimental procedures involved exclusively female mice aged 6 to 12 weeks.

[0129] NBSGW

[0130] Female NBSGW mice (Jax Stock NOD.Cg-KitW-41 J Tyr+ Prkdcscid Il2rgtm1 Wjl / ThomJ, stock No 026622), aged 6 to 12 weeks, were used as transplantation recipients for human HSPCs (Lineageneg, CD34pos).

[0131] Fgd5 lineage tracing (Fgd5-CreERT2 x Rosa26-LSL-tdTomato)

[0132] Fgd5-CreERT2 mice express a tamoxifen-inducible Cre recombinase and green fluorescent protein (ZsGreen) in the Fgd5 locus being active in HSCs (C57BL / 6N-Fgd5tm3(cre / ERT2)Djr / J; stock No 027789). This model was crossed with Rosa26-LSL-tdTomato mice (B6.Cg- Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J, stock No 007914). Expression of red fluorescent protein (tdTomato) is controlled by a loxP-flanked STOP cassette. Upon Cre-mediated recombination, robust tdTomato fluorescence is observed, allowing lineage tracing from HSCs into downstream compartments (Sawen P. et al., Elife, 7(e41258), 2018).

[0133] For labeling induction, mice received intraperitoneal injections of tamoxifen dissolved in oil at 75 mg / kg body weight once daily for 5 days, between 5 and 10 weeks of age. After a 4-week period to verify successful labeling (using platelets as reference), Ml surgery was performed on mice, followed by treatment with either 4-oxo-RA or a control vehicle, as detailed elsewhere. The mice used in the study were bred at IRB Barcelona under protocol 22-001 -ARF.

[0134] Labeling propagation analysis was performed using the HSC population as the top reference compartment (CD48neg, CD150pos, LSK), the MPP (CD48pos, CD150neg, LSK), and MyP (LS- K) populations as downstream compartments. Data were analyzed using a mixed-effects model using the percentage of tdTomato as dependent variable, and the population and the treatment as independent variables.

[0135] 1. Model Equation:

[0136] Y_ij = p0+ PiXi_ij + p2X2_ij + ... + P_nX_n_ij + u_i + E_ij

[0137] - Y_ij is the dependent variable (e.g., % of tdTomato) for the i-th mouse in the j-th condition.

[0138] - p0is the intercept.

[0139] - Pi, p2, ..., p_n are the fixed effect coefficients for each independent variable Xi, X2, ..., X_n (e.g., treatment conditions like baseline, sham, and Ml, and populations).

[0140] - u_i is the random effect for the i-th mouse, capturing individual variability.

[0141] - E_ij is the random error term for the i-th mouse in the j-th condition.

[0142] 2. Random Effects: u_i ~ N(0, o2_u)

[0143] N(0, o2_u) denotes a normal distribution with mean 0 and variance o2_u.

[0144] 3. Error Term:

[0145] E_ij ~ N(0, O2_E)

[0146] N(0, O2_E) denotes a normal distribution with mean 0 and variance O2_E.

[0147] Experimental in vivo mouse experiments

[0148] Ml surgery - operative left anterior descending artery (LAD)

[0149] Ml was induced in female C57BL / 6J or Rarp knockout mice aged from 6 to approximately 12 weeks through permanent occlusion of the left anterior descending artery (LAD) ligation. Anesthesia was induced by intraperitoneal (i.p.) injection of 100 mg / kg ketamine (Zoetis) and 10 mg / kg xylazine (Bayer Vital). Analgesia was initiated around 30 minutes prior to surgery through subcutaneous (s.c.) injection of 0.1 mg / kg buprenorphine. To account for perioperative dehydration resulting from blood loss and perspiration, 20 ml / kg of isotonic 5% glucose injection solution (Glucosteril; Fresenius Kabi Deutschland GmbH) in 0.9% NaCI (9 mg / ml) was administered by i.p. injection. A small animal respirator (MiniVent ventilator for mice model 845; Hugo Sachs Elektronik) was used with ventilation set at a positive end-inspiratory pressure (PEEP) of 5 mbar, a respiratory rate of 110 / min, and an inspiration / expiration ratio of 1 / 1 .5. Throughout the procedure, oxygen saturation, heart rate, and respiratory rate were monitored using the MouseOX system (Starr Life Sciences). Anesthesia was sustained by the addition of 0.5-2% isoflurane (AbbVie) during surgery. Following right lateral positioning of the animal, shaving, and skin disinfection, a left lateral thoracotomy was conducted between the third and fourth rib. The pericardium was opened to identify the LAD coronary artery. Permanent LAD ligation was executed using a single suture in the proximal middle third of the LAD, utilizing an 8- 0 prolene suture (Ethicon). Upon evacuating the pneumothorax, closure of chest and skin wounds was accomplished with a 5-0 or 6-0 prolene suture (Ethicon).

[0150] Ml experiments in RarB KO chimeras

[0151] Female wild-type C57BL / 6 (CD45.1) recipient mice were subjected to cumulative, lethal irradiation with a total dose of 9.5 Gy. Hematopoietic reconstitution was achieved by transplanting 5 millions of total BM cells from Rarp KO donor mice via tail vein injection. Four weeks after transplantation, PB was collected via puncture of the facial vein to assess chimerism and confirm complete hematopoietic reconstitution of the wild-type C57BL / 6 (CD45.1) BM with that from Rarb KO mice. LAD ligation or sham surgery was induced as described and at least six weeks posttransplantation. During the chronic phase of myocardial healing (at day 28), cardiac function was evaluated by echocardiography.

[0152] Retinoic acid / vitamin A in vivo treatments post Ml

[0153] C57BL / 6J or Rarp KO mice were intraperitoneally injected on the first and second days after Ml surgery with either 30 mg / kg body weight at-RA (Sigma-Aldrich), 30 mg / kg body weight 4-oxo-RA (Sigma-Aldrich), or with the corresponding amount of DMSO in PBS. Mice were euthanized during the acute phase of Ml (days 2 and 3), the reparative phase (day 7), as well as the chronic phase of Ml (days 21 and 28). Following euthanasia, HSCs were isolated and underwent further analysis, including assessments of cell cycle, single-cell division assays, and colony-forming unit assays (as detailed below).

[0154] Echocardiography

[0155] Echocardiography was conducted following the methodology previously published in (Heidt et al., Circulation Research, 115(12), 2014). Parameters including left ventricular ejection fraction, end- systolic and end-diastolic volume, and stroke volume were evaluated.

[0156] Downstream characterization of mouse models

[0157] Histology

[0158] For immunohistochemistry analysis, hearts were harvested and embedded in O.C.T. compound (Sakura Finetek), then snap-frozen on dry ice. Sections of 5 pm thickness were stained with antibodies against CD11 b. Staining was followed by biotinylated secondary antibody. We employed the VECTASTAIN Elite ABC HRP kit and ImmPACT AMEC Red Peroxidase (HRP) substrate (Vector Laboratories, Inc.) for color development. Heart sections from Fgd5CreERT2 mice were stained with DAPI, and analysis was conducted using the particle analyzer feature in Imaged. dTomatopos counts were normalized to DAPIpos counts for quantification.

[0159] Image analyses post Ml

[0160] Tissue sections were imaged and subsequently analyzed using Fiji / lmageJ software. To assess collagen deposition in different cardiac regions, a color deconvolution algorithm was applied to separate specific staining components. The deconvoluted images were then thresholded to segment areas of interest based on pixel intensity values corresponding to collagen.

[0161] Regions of interest (ROIs) corresponding to the infarct zone, border zone and remote zone of the heart were manually defined and adjusted for each sample. These ROIs were used to measure collagen content within the designated zones. Thresholding was performed to ensure accurate quantification of collagen-positive areas. All measurements were taken across predefined regions and were standardized for comparison across samples.

[0162] In the case of dTomato-positive cell analysis in cardiac tissue, sections were also stained with DAPI to visualize nuclei. This allowed for normalization of dTomato-positive cell counts relative to the total number of DAPI-positive cells, providing accurate assessment of cell abundance within the tissue.

[0163] Flow cytometric heart analysis post Ml

[0164] Following euthanasia of the mice, hearts were extracted to analyze the infarcted myocardium. Infarcted myocardial tissue was excised, minced, and subjected to enzymatic digestion using a mixture of collagenase I (450 U / ml), collagenase XI (125 U / ml), DNase I (26 U / ml), and hyaluronidase (60 U / ml) (all from Sigma-Aldrich). Enzymatic reaction was incubated at a temperature of 37°C and a rotation speed of 600 rpm for a duration of 1 hour and subsequently stopped by the addition of 30 ml of FACS buffer consisting of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 1% fetal bovine serum.

[0165] Cardiac cell suspension was stained using the following antibodies: Lineage-BV605 (CD19, CD90, CD4, CD8a, NK1 .1 , Teri 19, CD49b), Ly6C-FITC, CD115-BV711 , Ly6G-APC, CD11 b- APC-Cy7, CD45.2-PB, and F4 / 80-Pe-Cy7.

[0166] Flow cytometric blood, spleen, and whole BM analysis post Ml

[0167] Following the extraction of venous blood via tail vein puncture, mice were euthanized to collect spleen, femurs, tibiae, and pelvis for BM analysis.

[0168] Venous blood was collected using 5 mM EDTA (Sigma-Aldrich). The spleen was filtered through a 40 pm cell strainer to create a single-cell suspension. The BM was flushed and then filtered through a 40 m cell strainer to create a single cell suspension. Cell suspensions were treated with 1x red blood cell lysis buffer (BioLegend) for subsequent staining.

[0169] Spleen, blood, and whole BM were stained using the following antibodies: Ly6C-FITC, CD115- BV711 , B220-BV650, CD3-PerCP-Cy5.5, Ly6G-APC, CD11 b-APC-Cy7 and CD45.2-PB. Additionally, in a separate staining procedure, spleen, blood, and BM were stained using the following antibodies: Lineage-BV650 (Gr1 , CD11 b, B220, Ter119, CD4, CD8a), cKit-BV711 , Sca1-APC-Cy7, CD150-PeCy5, CD48-BV421 , CD16 / 32-APC, and CD127-Pe-Cy7.

[0170] Enrichment of mouse HSPCs and isolation of HSCs

[0171] Murine BM cells were isolated from the femur, tibia, hip bone, and vertebrae by gentle crushing with mortar and pestle in PBS. Red blood cell lysis was performed with ACK Lysing Buffer (Thermo Fisher Scientific) for 5 minutes at room temperature (RT). Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen) was used for lineage negative enrichment according to the manufacturer’s protocol. Briefly, the BM was stained with 1 :4 dilution of the Lineage Cocktail for 30-60 minutes at 4 °C on a rotating wheel. Labelled cells were then incubated for 20 minutes with 400 pl of washed Dynabeads coated with polyclonal sheep anti-rat IgG per sample. Depletion of lineage cells was performed using a magnet. Lineage-depleted BM cells were stained with lineage markers (Gr1 , CD11 b, B220, Ter119, CD4, CD8a), ckit, Seal , CD150, CD48 and CD34. Sorting was then performed using a FACS Aria II, III or FACSymphony (Becton Dickinson). Subsequently, cells were collected into ice-cold PBS for reconstitution assays, Complete Stem Cell Medium (StemPro-34 SFM, Life Technologies, supplemented with 50 ng / ml SCF, 25 ng / ml TPO, 30 ng / ml Flt3-Ligand [all from Preprotech], 100 pg / ml Penicillin / Streptomycin, and 2 mM L- Glutamine [both from Gibco]) for experiments for in vitro culture, PBS with 2% BSA for scRNA- seq, RNA lysis buffer (Arcturus PicoPure RNA Isolation Kit (Applied Biosystems)) for population RNA-seq and stored at -80°C.

[0172] Cell cycle analysis

[0173] Following lineage depletion, erythrocyte-lysed BM was stained for HSC markers (Lineageneg, cKitpos, Sca-1 pos, CD150pos, CD48neg, CD34neg). Subsequently, cells were fixed for 10 minutes at 4°C using BD Cytofix / Cytoperm Buffer (Becton Dickinson and Company) and intracellular Ki-67 (BD Biosciences) staining was performed using PermWash solution for at least 45 minutes at 4°C (Becton Dickinson and Company). Before proceeding with flow cytometry analysis, the cells were stained with DAPI (Sigma-Aldrich) at RT for a minimum of 20 minutes.

[0174] Mouse single cell division assay

[0175] Individual HSCs (Lineageneg, cKitpos, Sca-1 pos, CD150pos, CD48neg, CD34neg) were sorted into 72-well Terasaki plates (Greiner Bio-One) containing Complete Stem Cell Medium (as specified above). Following a 48-hour incubation period, each well was manually examined to determine the number of cell divisions: 1 cell indicated no division, 2 cells indicated 1 division, and more than 2 cells indicated more than 1 division. Mouse serial colony-forming-unit assays (CFU)s

[0176] A total of 300 mouse HSCs (Lineageneg, cKitpos, Sca-1 pos, CD150pos, CD48neg, CD34neg) were sorted into 1 ml of MethoCult M3434 (StemCell Technologies) and plated for subsequent culture. After 7 days post plating, the number of colonies was counted (colonies were defined as consisting of > 300 cells). For second and third platings, 104 cells were re-plated in MethoCult M3434 (StemCell Technologies). Colony formation resulting from these subsequent platings was assessed approximately 3 and 5 days after the second and third rounds, respectively.

[0177] HSC transplantation experiments

[0178] 500 HSCs (Lineageneg, cKitpos, Sca-1 pos, CD150pos, CD48neg, CD34neg) were obtained from CD45.2pos mice that had experienced Ml surgery, along with their respective control group (Sham surgery). These cells were transplanted into fully irradiated CD45.1 (Ly5.1) mice (cumulative dose of 4.5 Gy + 5 Gy), together with 4x105 supportive spleen cells from age- matched CD45.1 / 2 mice. Transplantation was performed by tail vein injection within 24 hours after irradiation. CD45.2-donor cell contribution was monitored in PB samples collected from the submandibular vein at 4, 8, 12 and 16 weeks post transplantation. CD45.2 chimerism was assessed using flow cytometry with the following monoclonal antibodies: anti-CD45.1 (A20)- FITC, anti-CD45.2 (104)-PB, anti-CD11 b (M1 / 70)-APCCy7, anti-GR1 (RB6.8C5)-APC, anti-CD8a (53.6.7)- PECy5, anti-CD4 (GK1 ,5)-PECy5, and anti-B220 (RA3.6B2)-AF700. After 16 weeks, the mice were euthanized and their BM (hip bone, tibia, and femur) was subjected to analysis.

[0179] Characterization of human HSPCs

[0180] Myocardial infarction patient samples

[0181] Sternal BM was collected during surgical procedures. Exclusion criteria comprised patients with active cancer or hematological disorders, individuals who had received chemotherapy or radiation therapy, as well as those with acute infections. The inclusion criteria included patients scheduled for CABG surgery who had a preserved left ventricular EF greater than 45% and showed no signs of heart failure, e.g. proBNP > 1000 pg / ml. The filtered patients were categorized into two groups: (i) Ctrl patients with no history of Ml, and (ii) Ml patients with a documented history of Ml. Clinical characteristics such as age, gender, left ventricular EF, smoking status and proBNP were closely matched between the two groups.

[0182] Ethical considerations

[0183] This study was conducted in accordance with the ethical standards and guidelines established by the Ethical Review Board of Freiburg. Ethical approval for the study protocol (Ethics Approval Number: 388 / 19) was obtained from the Ethical Review Board of Freiburg on January 16, 2020. All experimental procedures involving human patients were performed in compliance with the relevant laws and institutional guidelines. Informed consent was obtained from all human participants involved in the study. Human single cell division assays on in vitro treated cells

[0184] Individual HSPCs (Lineageneg, CD38neg, CD34pos) or HSCs (Lineageneg, CD38neg, CD34pos, CD45RAneg) were sorted into 72-well Terasaki plates (Greiner Bio-One) containing StemSpan SFEM II media (StemCell Technologies) supplemented with StemSpan CD34pos Expansion Supplement (StemCell Technologies). For in vitro cell cultivation, at-RA (2.5 pM final concentration), 4-oxo-RA (2.5 pM final concentration), or the respective volume of DMSO was added to the culture medium. Following a 48-hour incubation period, each well was manually examined to determine the number of cell divisions: 1 cell indicated no division, 2 cells indicated 1 division, and more than 2 cells indicated more than 1 division.

[0185] Human serial colony-forming-unit assays (CPUs)

[0186] A total of 1000 HSPCs (Lineageneg, CD38neg, CD34pos) were sorted into 1 ml of MethoCult H4435 (StemCell Technologies) and plated for subsequent culture. After ? days post plating, the number of colonies was counted (colonies were defined as consisting of > 300 cells). 104 cells were replated in MethoCult H4435 (StemCell Technologies) and quantified after ? days.

[0187] Human CPU assays on in vitro treated human HSPCs

[0188] A total of 1000 HSPCs (Lineageneg, CD38neg, CD34pos) or HSCs (Lineageneg, CD38neg, CD34pos, CD45RAneg) were sorted into 96-well low attachment plates containing StemSpan SFEM II media (StemCell Technologies) supplemented with StemSpan CD34pos Expansion Supplement (StemCell Technologies). For in vitro treatments, cells were cultured for a period of 72 hours using a concentration of at-RA (2.5 pM final), 4-oxo-RA (2.5 pM final), or the corresponding volume of DMSO. Subsequently, cells were transferred into 1 ml of MethoCult H4435 (StemCell Technologies) and serial CFU assay was performed according to the procedures outlined as described above.

[0189] In vitro treatments of human monocytes

[0190] Human peripheral blood mononuclear cells (PBMCs) were processed using the EasySep Human Monocyte Enrichment Kit (StemCell Technologies) to isolate monocytes for culture. Cells were cultured in RPMI 1640 medium supplemented with 1x L-Glutamine, 10% FBS and 1x penicillinstreptomycin (PenStrep). For in vitro treatments, human monocytes were cultured in a medium containing a final concentration of 0.2 pM at-RA or 4-oxo-RA. After 16 hours of culture, culture supernatant was collected for the cytokine secretion assay (human inflammatory cytokine panel - BioLegend). For flow cytometry analysis and bulk RNA-seq analysis, cells were cultured for 24 hours.

[0191] CellROX Staining

[0192] Cells were incubated at 37°C with CellROX Deep Red (1 :500, Invitrogen) in their respective media for 30 minutes, according to the manufacturer's instructions. Cells were washed three times with PBS and subsequently stained for FACS analysis on the BD LSRFortessa Cell Analyzer (BD Biosciences). NBSGW transplantations

[0193] In total, 10,000 BM HSPCs (Lineageneg, CD34pos) were sorted and injected via tail vein into 6- to 12-week-old female NBSGW mice. Recipient mice were irradiated with 1 Gy and injected within 24 hours post irradiation. CD45.2-donor cell contribution was monitored in PB collected from the submandibular vein at 6, 12, 18, 24 and 36 weeks post transplantation. Human CD45.2 chimerism was assessed using flow cytometry with the following monoclonal antibodies: anti- CD45-FITC (human), anti-CD45-PE (mouse). After 24 weeks, the mice were euthanized, and their BM (hip bone, tibia, and femur) was subjected to analysis. A mouse was classified as engrafted if the human CD45 positive percentage was greater than or equal to 0.3%.

[0194] Bulk RNA-seq:

[0195] Nucleic acid extraction protocol

[0196] Cells were sorted into RNA lysis buffer (Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) and then stored at -80°C until further use. RNA isolation was conducted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) following the manufacturer's guidelines. DNase treatment was carried out employing the RNase-Free DNase Set (Qiagen). The resulting total RNA was utilized for generating cDNA libraries.

[0197] Nucleic acid library construction protocol cDNA libraries were generated using SMARTseq v4 (Takara Bio). Amplification cycles were adjusted accordingly to RNA input amount. For HSCs, 13 cycles of amplification were performed. For cardiac cell populations, 12 to 14 cycles of amplification were performed. To produce uniquely and dually barcoded sequencing libraries from the cDNA libraries, the NEBNext Ultra II FS DNA library kit was utilized. This involved fragmenting 5 ng of the cDNA library for 22.5 minutes, followed by adaptor ligation and library amplification using cycle numbers determined by amount of input material.

[0198] Nucleic acid sequencing

[0199] Libraries underwent sequencing on the Illumina NovaSeq platform, generating 45-55 million reads depth with 100bp paired-end sequencing

[0200] Population RNA-seq analysis method: low-level processing

[0201] Raw FASTQ files underwent alignment against the mm10 or hg38 reference genome using the mRNA-seq tool of the bioinformatics pipeline snakePipes v.2.5.2 (Bhardwaj V. et al., Bioinformatics, 35(22), 2019). Within this tool, the Alignment mode was utilized for mapping the sequenced reads via STAR (STAR_2.7.4a) (Dobin A. et al., Bioinformatics, 29(1), 2013), and expression counts were quantified using featurecounts (Liao Y. et al., Bioinformatics, 30(7), 2014). Data quality was evaluated by Deeptools QC v3.3.2 (Ramirez F. et al., Nucleic Acids Res., 44(W1), 2016). Genes with an average expression exceeding 100 counts in at least one condition were specifically selected for further analysis. To assess differential expression, DESeq2 was employed (Love M. et al., Genome Biology, 15(12), 2014) and results were considered statistically significant at a false discovery rate (FDR) of 0.1 .

[0202] Population RNA-seq analysis method: downstream analysis

[0203] The expression of previously published gene signatures was assessed by Gene Set Enrichment Analysis (GSEA) by using the fgseaMultilevel function from the fgsea package with default parameters (Sergushichev A. et al., bioRxiv, 060012, 2016), filtering significant pathways at a FDR equal to 0.1 . Specific signature enrichment profiles were generated with the gseaplot2 function (Yu G. et al., R Package Version, 1.8.1 , 2020). The resulting Normalized Enrichment Score (NES) and P-adjusted value of selected signatures were plotted using ggplot2 (Wickham H. et al., Springer, 2009). In mouse-derived datasets, the enrichment of Reactome pathways (Jassal B. et al., Nucleic Acids Research, 48(D1), 2020), HSC / MPP (Cabezas-Wallscheid N. et al., 2014 ibid), Activated / Dormant HSC (Cabezas-Wallscheid et al., 2017 ibid), MolO / NoMo HSC (Wilson N. et al., Cell Stem Cell, 16(6), 2015), Retinoic acid metabolism (Schbnberger K. et al., 2022 ibid), Inflammatory / Reparatory-macrophage (Nahrendorf et al.; Circulation 2010), Angiogenesis Hallmark, Wound healing (G0:0042060), Extracellular matrix (G0:0031012) and Inflammatory response (G0:0006954) signatures in pairwise comparisons was assessed. In the analysis of human data, LT (long-term)-HSC / ST(short-term)-HSC (Martinez F. et al., The Journal of Immunology, 177(10), 2006), quiescent / activated LT-HSC (Kaufmann K. et al., Nature Immunology, 22(6), 2021), HSC differentiation (G0:1902036), cell cycle (hsa04110), IFN-g response (M5913) and Inflammatory / Reparatory-macrophage (Buscher K. et al., Nature Communications, 14(19), 2017) signatures were evaluated. In the dataset of cardiac differentiated cell populations, the average VST-transformed expression values of selected genes were represented using the pheatmap package (Kolde R. et al., CRAN, 2015.). For the RA signaling translatability analysis, DEGs from our previously published mouse 4-oxo-RA and at-RA HSC treatments (P-adjusted < 0.1) (Schbnberger K. et al., 2022 ibid) were translated to human gene symbols and depicted in volcano plots of our human data, when commonly up- or downregulated. GSEA of mouse direct target genes of both RA metabolites (Schbnberger K. et al., 2022 ibid) was also performed in our human dataset.

[0204] The raw data were deposited in ArrayExpress and are available under the accession numbers [E- MTAB-13505, E-MTAB-13506, E-MTAB-13508, E-MTAB-14660],

[0205] ScRNA-sequencing

[0206] Nucleic acid library and sequencing

[0207] ScRNA-seq experiments were performed on 25,000 mouse BM HSPCs (Lineageneg, Scal pos, cKitpos) or cardiac myeloid cells, 7,000 to 10,000 human BM HSPCs (Lineageneg, CD38neg, CD34pos) or 3,000 to 20,000 human BM monocytes. Sorted cells in PBS supplemented with 2% BSA were pelleted at 300g for 5 minutes at 4 °C and washed once in ice-cold PBS containing 0.5% BSA. Cells were resuspended in PBS with 0.5% BSA to a maximum cell concentration of 390 cells / pl and placed on ice. Cell viability was assessed by trypan blue staining. A cell suspension aliquot (2 pl) was diluted 1 :2 in 0.4% trypan blue solution, incubated for 2 minutes on a microscope slide and analyzed under a microscope. All samples for scRNA-seq analysis showed more than 90% viable cells. ScRNA-seq was performed on the 10X Genomics platform using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 dual index (10x Genomics, PN- 1000268) following the manufacturer's instructions. A total of 43 pl of cell suspension was loaded onto a chip G according to the manufacturer’s instructions, aiming for a targeted cell recovery of up to 10,000 cells. PCR cycles for cDNA and final library generation were adjusted according to the target cell recoveries. The quality of the obtained cDNA and final libraries was assessed by capillary electrophoresis (Fragment analyzer, HS NGS Fragment Kit, Agilent). Sequencing was performed on a NovaSeq9000 device (Illumina) with a read length of 28-10-10-90 bp (R1-i5-i7- R2), aiming for a minimum sequencing depth of 20,000 read pairs per cell.

[0208] ScRNA-seq analysis method: low-level processing

[0209] Raw UMI-based data files underwent mapping against the mm10 or hg38 reference genome using the scRNA-seq tool of the bioinformatics pipeline snakePipes v.2.5.2 with the 10xV3 mode (Bhardwaj V. et al., 2019 ibid). With this tool, the reads were i) mapped, ii) UMI-deduplicated, and iii) counted using STARsolo, creating BAM files and a Seurat object containing the gene counts. Deeptools QC was employed to assess the quality of this data (Ramirez F. et al., 2016 ibid).

[0210] Following data preprocessing, scRNA-seq analysis was performed with the R package Seurat (Satija R. et al., Nature Biotechnology, 33(5), 2015). The Seurat object was imported and different cell filtering criteria, including a minimum number of counts and expressed genes per cell, were applied depending on the dataset to avoid empty droplets. Conversely, low-quality and dying cells with a percentage of mitochondrial mRNA exceeding 20% in HSPC datasets and 5% in Cd11 b pos datasets were excluded. Doublets were removed using the doubletFinder_v3 function from the DoubletFinder package (McGinnis C. et al., Cell Systems, 8(4), 2019), assuming a maximum doublet formation rate of 7.6% (pK < 0.076).

[0211] ScRNA-seq analysis method: downstream analysis

[0212] A log transformation and normalization of the data were implemented before integrating the different samples to eliminate potential batch effects. Integration was performed using the functions SelectlntegrationFeatures, FindlntegrationAnchors, and IntegrateData (Stuart T. et al., Cell, 177(7), 2019). Linear dimensional reduction of the integrated dataset was performed through Principal Component Analysis (PCA). After applying the Jackstraw and Elbow plot methodologies from Seurat, a clustering analysis was carried out, selecting the initial 20 PCs. The identification of cell clusters was accomplished using the FindClusters method, and the results were visualized using the Uniform Manifold Approximation and Projection (UMAP) technique (Becht E. et al., Nature Biotechnology, 37(1), 2018). Clusters with a high doublet scoring were identified and filtered out using the doubletcells function from the scran package (Lun A. et al., Genome Biology, 17, 2016). Additionally, potential contaminations in the HSPC datasets were evaluated with the enrichment of LS-K signatures (Klimmeck D. et al., Stem Cell Reports, 3(5), 2014) and differentiated cell markers, using AddModuleScore and FindAIIMarkers, respectively. To annotate the filtered good quality clusters, enrichment of previously published human BM HSPC markers (Klimmeck D. et al., Stem Cell Reports, 3, 2014; Pellin D. et al., Nature Communications, 10(1), 2019, Karamitros D. et al., Nature Communications, 19(1), 2017), mouse BM HSPC signatures (Sommerkamp P. et al., Blood, 137(23), 2021), human BM monocyte markers (Oetjen K. et al., JCI Insight, 3, 2018; Baccin C. et al., Nature Cell Biology, 22(1), 2019, Triana S. et al., Nature Immunology, 22(12), 2021), and mouse heart monocyte markers (Martini E. et al., Circulation, 140, 2019, Van Berio J. et al., Circ Res, 129(12), 2021) was assessed. In the cardiac 4-oxo-RA monocyte dataset of the Fgd5CreERT2 mouse model, filtered good quality cells were projected against the previously analyzed Vehicle data using the FindTransferAnchors and MapQuery functions from Seurat. Predicted cell annotations were then represented in the Vehicle UMAP reduction as reference.

[0213] The relative percentage of cells in each condition per cluster was quantified and visualized in a barplot, calculating significance on these frequencies using Fisher’s exact tests (Lopez D. et al., Cell Rep, 41 (8), 2022). Briefly, we calculate the percentage of cells per annotated cluster relative to each condition and perform statistics in a separate manner to assess if there is a statistically significant association between cluster and condition. For this aim, a contingency table per cluster is created, reporting the relative percentage of cells from each condition in the cluster of interest and the respective percentage of cells that belong to all other clusters. A two-sided Fisher's exact test is then applied to these contingency matrices and resulting P-values are adjusted by the Bonferroni method. Cell density per separate condition was depicted using the MASS package (Venables W. et al., 2002 ibid). To conduct RNA velocity analysis, spliced and unspliced reads were counted, and the dynamical model of the python package scVelo was applied, as depicted in Bergen V. et al., Nature Biotechnology, 38(12), 2020.

[0214] The AddModuleScore function was employed to illustrate the previously mentioned signature enrichment score. Gene expression and signature enrichment were represented using FeaturePlot, DimPlot, and dotplot functions. Lists of cluster markers generated by the FindMarkers function (min. pct = 0.25, logfc.threshold = 0) were utilized for GSEA, as previously explained. Volcano plots of the differentially expressed genes (DEGs) were represented with EnhancedVolcano (Blighe K. et al., R Package Version, 1 .6.0, 2020). Gene ontology (GO) enrichment analysis of DEGs was performed with the enrichGO or compareCluster functions from the clusterProfiler package (Wu T. et al., Innovation, 2(3):100141 , 2021).

[0215] Single hematopoietic stem cell division assay

[0216] Individual mouse hematopoietic stem cells (HSCs; defined by Lineage-, cKit+, Sca-1+, CD150+, CD48-, CD34- marker expression) were single-cell sorted into 72-well plates containing complete stem cell medium. Cells were cultured for 48 hours in the presence of control (DMSO), 4-oxo- retinoic acid, or the RARp agonist AC-261066 (SML2667; Merck) and the number of cell divisions was determined. Wells containing a single cell were scored as non-dividing, whereas wells containing two or more cells were scored as divided. The percentage of non-dividing cells was used as a measure of HSC quiescence.

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Claims

Claims1 . 4-Oxoretinoate for use in treatment or prevention of the condition of cardiac dysfunction after myocardial infarction, or after impaired cardiac blood flow.

2. 4-Oxoretinoate for use in prevention of the condition of heart failure after myocardial infarction, or after impaired cardiac blood flow.

3. 4-Oxoretinoate for use in treatment or prevention of the condition of adverse cardiac remodelling after myocardial infarction, or after impaired cardiac blood flow, particularly for use in treatment or prevention of ventricular dilation after myocardial infarction, or after impaired cardiac blood flow.

4. 4-Oxoretinoate for use in treatment of the condition of coronary artery disease.

5. 4-Oxoretinoate for use in treatment or prevention of the condition of ischemia-reperfusion injury.

6. 4-Oxoretinoate for use according to any one of the preceding claims, wherein the condition is associated with increased hematopoietic stem cell activity.

7. 4-Oxoretinoate for use according to any one of the preceding claims, wherein the condition is associated with increased hematopoietic stem cell proliferation.

8. 4-Oxoretinoate for use according to any one of the preceding claims, wherein the 4- oxoretinoate is selected from the group of all-trans-4-oxo-retinoic acid, 9-cis-4-oxo- retinoic acid, and 13-cis-4-oxo-retinoic acid.

9. 4-Oxoretinoate for use according to any one of the preceding claims, wherein the 4- oxoretinoate is all-trans-4-oxo-retinoic acid.

10. 4-Oxoretinoate for use according to any one of the preceding claims, wherein 4- oxoretinoate is administered for at most one month, particularly for at most one week, more particularly for 2 to 3 days.

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