Methods of treating cardiometabolic diseases

Abstract

This disclosure concerns methods of treating cardiometabolic diseases and conditions, in particular cardiovascular diseases conditions associated with metabolic disorders, using extracellular vesicles derived from milk.

Description

[0001] METHODS OF TREATING CARDIOMETABOLIC DISEASES

[0002] Technical field

[0003] The present invention relates, in general terms, to methods of treating cardiometabolic diseases, and more specifically to methods of treating metabolic cardiovascular diseases and conditions using extracellular vesicles derived from milk.

[0004] Background

[0005] Cardiometabolic diseases and disorders, such as obesity and other lifestyle-related metabolic disorders, continue to be among the leading causes of morbidity and mortality worldwide and a significant global health burden. Patients often develop metabolic syndrome, a cluster of conditions that includes hypertension, dyslipidemia and insulin resistance, which puts them at significant risk of developing various associated diseases and health complications. These diseases encompass diabetes and a range of conditions that affect the heart, blood vessels and nervous system, such as coronary artery disease, stroke, hypertension, heart failure, and neurodegenerative diseases.

[0006] Heart failure is a complex clinical syndrome characterised by the inability of the heart to pump blood effectively to meet the body’s demands. It can be classified into two main types based on the effect of the disease on the heart’s ejection fraction (i.e., the volume of blood pumped out by the heart with each beat). In heart failure with reduced ejection fraction (HFrEF), the heart’s ability to pump is reduced, leading to an ejection fraction below the normal range (typically less than 50%). Heart failure with preserved ejection fraction (HFpEF) is characterised by a near-normal ejection fraction, but the heart struggles to relax normally and to fill properly.

[0007] Heart failure is frequently associated with metabolic syndrome and is a common cause of hospitalisation for those above the age of sixty. The global prevalence of heart failure is increasing due to population aging. Metabolic disorders caused by unhealthy lifestyles aggravate the condition. Morbidity and mortality for heart failure patients remain high, but therapeutic options arc limited. Moreover, heart failure is a progressive condition closely related to underlying cardiovascular issues such as hypertension, atherosclerosis, and myocardial infarction. Chronic hypertension can strain the heart over time and lead to both systolic and diastolic dysfunction. Atherosclerosis can reduce blood flow to the heart, causing strain on cardiac muscle. Myocardial infarction can result in tissue damage and impair the systolic and diastolic function. The correlation between these disorders makes universal strategies for effective suppression of various heart disorders particularly important.

[0008] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0009] Summary

[0010] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising orally administering an effective amount of a milk- derived extracellular vesicle (mEV) composition to the subject to treat or prevent the cardiovascular disease or condition.

[0011] Disclosed herein is a method of treating or preventing metabolic heart failure in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat or prevent the heart failure.

[0012] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising: (a) detecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition; and (b) orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject found to be at risk of or likely to be suffering from a cardiovascular disease or condition to treat or prevent the cardiovascular disease or condition in the subject.

[0013] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising: (a) detecting increased intestinal permeability in the subject, wherein the subject is at risk of or suffering from a cardiovascular disease or condition, wherein an increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and (b) orally administering an effective amount of an mEV composition to a subject found suitable for treatment with mEV s to prevent or treat the cardiovascular disease or condition in the subject.

[0014] Disclosed herein is a method of selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition for treatment with rnEVs, the method comprising: (a) detecting an increased intestinal permeability in the subject, wherein an increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and (b) selecting the subject found likely to have increased intestinal permeability for treatment with mEVs.

[0015] Disclosed herein is a method of promoting gut health in a subject, the method comprising: (a) selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition; and (b) orally providing a milk-EV composition to the subject so as to promote gut health in the subject.

[0016] Disclosed herein is a method of treating gastrointestinal dysfunction in a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition, the method comprising administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat the gastrointestinal dysfunction in the subject.

[0017] Disclosed herein is a method of treating a cardiometabolic disease or disorder in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to prevent or treat the cardiometabolic disease or disorder.

[0018] Brief description of the drawings

[0019] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0020] Figure 1 shows that disorder of gut banier and gut microbiota appear s in a HFpEF mouse model. (A) Representative left ventricular M-modc echocardiographic tracings (top) and cardiac functional parameters determined by echocardiography (bottom). LVEF, left ventricular ejection fraction; E / A ratio, the ratio between mitral E wave and A wave; and E / E’, the ratio between mitral E wave and E’ wave. (B) Body weight and (C) glucose tolerance. (D) Representative images of colon sections stained with H&E. Scale bars, 100 pm. (E) Colon length. (F-H) mRNA expression levels of tight junction proteins ZO-1 and Occludin, and inflammatory cytokine TNF-a, in the colon. GAPDH was used as the reference gene. (I) Intestinal permeability assessed by fluorescein isothiocyanate (FITC)- dextran test. (J) Levels of endotoxin detected in the faeces (left) and plasma (right). (K) P- diversity evaluated using the weighted UniFrac -based PCA of gut microbiota. (L) Cladogram based on LEfSe analysis showing community composition of the mice gut microbiota. (M) Linear discriminant analysis (LDA) effect size (LEfSe) investigating bacterial community at the phylum level. LDA score higher than 3 indicates a higher relative abundance in the corresponding group than that in the other group. Data arc means ± SD; *P < 0.05, **P < 0.01 , ***P < 0.001 , and ***P < 0.0001 , one-way ANOVA. (N) Concentrations of endotoxin (left), LBP (middle, marker of microbial translocation) and FABP2 (right, marker of gut barrier dysfunction) in human plasma, n = 8-10 per group. (O) Feces were collected from HFpEF patients (n=15) and non-HFpEF volunteers (n=10). Bacterial DNA from feces was analyzed using 16S rRNA sequencing. Alpha diversity indexes were calculated with QIIME2 according to the number of ASV / OTU in each group.

[0021] Figure 2 shows that orally-administered mEVs systemically alleviate mice HFpEF progression. (A) Scheme of experimental design showing groups and durations (C57BL / 6J fed with normal chow diet or HFD plus L-NAME water at 8 weeks of age, diet treatment accompanied by oral gavage of PBS, supernatant 0.6 mg / kg / day, and mEVs 0.6 mgZkg / day) for 15 weeks. (B) Representative IV IS images of dissected organs from mice 24 hours after oral 81 administration of mEVs. (C) Body weight of mice. (D) Quantification of body weight of mice at week 15. (E, F) Systolic blood pressure (SBP) and diastolic blood pressure (DBP) of different experimental groups after 5 or 15 weeks of treatment. (G, H) Intraperitoneal insulin-tolerance test (ipITT) and glucose-tolerance test (ipGTT). (I) AUC quantification of ipGTT after 5 or 15 weeks of diet. (J) Representative images of PicroSirius red (PSR) staining in skeletal muscle of mice. (K) mRNA expression levels of MyHC-1 in the muscle (L) grip force measurements of mice from different experimental groups (M) Running distance during exercise exhaustion test, n = 7 mice per group. Supernatant, EV-depleted supernatants. Data arc means ± SD; *P < 0.05, **P < 0.01, ***? < 0.001, and ***P < 0.0001, one-way ANOVA. Figure 3 shows that orally-administered mEVs hinder heart abnormality in HFpEF. (A) Representative macroscopic images of the heart from mice of different experimental groups. Images are representative in each group. Scale bars, 5 mm. (B) Representative images of haematoxylin and eosin (H&E) and WGA staining in transversal sections of left ventricle of mice of different experimental groups. Images are representative in each group. Scale bars, 1000 pm (H&E) and 100 pm (WGA). (C) Ratio of heart weight to tibia length (HW / TL).

[0022] (D) WGA quantification of cardiomyocyte cross-sectional area. (E) Representative left ventricular M-mode echocardiographic tracings. Images are representative of 7 independent mice in each group. (F) Percentage of LVEF. (G) Ratio between mitral E wave and A wave (E / A). (H) Ratio between mitral E wave and E’ wave (E / E’). (I) Ratio between wet and dry lung weight (J, K) mRNA expression levels of the UPR marker XBPls and of iNOS in the heart. GAPDH was used as the reference gene, n = 7 mice per group. Supernatant, EV- depleted supernatants. *P < 0.05, **P < 0.01, < 0.001, and ****p < 0.0001, one-way

[0023] ANOVA. (L) mRNA expression levels of TNF-a. GADPH was used as the reference gene. (M) Levels of endotoxin detected in the heart, n = 7 mice per group. Data was analysed by one-way ANOVA and presented as mean ± SD; < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0024] Figure 4 shows that mEVs reverse the disruption of gut barrier and gut microbiota in HFpEF mice. Mice were treated according to the schedule illustrated in Figure 2A. n = 7 mice per group. (A) Representative images of colon sections stained with H&E to assess histological score. Scale bars, 100 pm. (B) Quantification of colon length from mice of different experimental groups. (C, D) mRNA expression levels of the inflammatory cytokine, TNF- a, and tight junction protein, ZO-1, in the colon. GAPDH was used as the reference gene.

[0025] (E) Intestinal permeability of mice of different groups assessed by fluorescein isothiocyanate (FITC)-dextran test. (F, G) Levels of endotoxin detected in faeces and plasma. Bacterial DNA from mice faeces was analysed using 16S rRNA gene sequencing (n = 5). (H, I) Alpha diversity indexes at week 15 calculated with QIIME2 according to AS V / OTU numbers of each group. (J) Abundance of 3 primary genera in the phylum Bacteroidota and Verrucomicrobiota (abundance values range from 0.00 95 to 0.20). (K) Heatmap illustrating altered relative abundance of mouse gut bacteria at the genus 253 level. Statistical significance was determined for all pairwise comparisons using Spearman’s method, p < 0.05. 254 Spearman r values range from -1.5 (blue) to 1.5 (red). (L) The top 12 abundant microRNAs (miRNAs) in mEVs identified through RNA sequencing. (M) Transepithelial electrical resistance (TEER) values were determined at 20h by the Millicell ERS-2 Voltohmmeter. (N) Western blot analysis of the tight junction protein ZO-1 upon DSS treatment at 4 hours. Supernatant, EV-depleted supernatants. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, one-way ANOVA. (L) The top 12 abundant microRNAs (miRNAs) in mEVs were identified through RNA sequencing. (M) Transepithelial electrical resistance (TEER) values were determined at 20h by the Millicell ERS-2 Voltohmmeter. (N) Western blot analysis of the tight junction protein ZO-1 upon DSS treatment at 4 hours.

[0026] Figure 5 shows that mEVs restore the abundance of Lachnospiraceae and its metabolite SCFAs on week 15. Bacterial DNA from faeces was analysed using 16S rRNA gene sequencing (n = 5). (A) Linear discriminant analysis (LDA) effect size (LEfSc) method to investigate bacterial community at the species level at week 15. LDA score higher than 3 indicates a higher relative abundance in the corresponding group than that in other groups. (B) The relative abundance of primary genus in the phylum Firmicutes, Desulfobacterota, and Actinobacteriota (abundance values range from 0.00 to 0.13) at week 15. (C) The abundance of primary genus in the phylum Firmicutes and Bacteroidota (abundance values range from 0.00 to 0.26). (D) Changes of the OTUs of Lachnospiraceae_FSC020 at the genus level at week 15. (E) Relative abundance of Lachnospiraceae_bacterium at the species level at week 15. (F) Cladogram based on LEfSe analysis showing community composition of the mice gut microbiota. Metabolites in plasma of HFpEF mice with and without mEVs treatment were analysed using metabolomics analysis (n = 5). (G) -diversity evaluated using the weighted UniFrac-based PCA of gut microbiota. (H) Abundance of metabolites with marked diversity (abundance values range from 1 to 4). (1) Circos plot of Spearman correlations between microbes and metabolites, n = 5 mice per group. Data are presented as mean ± SD; < 0.05, < 0.01, ***p < 0.001, and ****p < 0.0001. 259

[0027] Supernatant, refers to mEVs-depleted supernatant.

[0028] Fig- 6 shows that orally-administrated mEVs systemically alleviate HFpEF progression in mice. (A) Photographs showing the body size of mice in different treatment groups. (B) Quantification of body weight of mice at week 15. (C) The trend of body weight change over 15 weeks. (D) Intraperitoneal glucose-tolerance test (ipGTT) after 15 weeks of diet. (E) Systolic blood pressure (SBP) and (F) diastolic blood pressure (DBP) of mice in different groups after 15 weeks of treatment. (G) Running distance during exercise exhaustion test. (H) Grip force measurements of mice in different groups. (1) Scheme showing the design of metabolic cage, n = 4 mice per 370 group. (J) Daily food intake. (K) Activity levels measured by beam breaks. (L) Energy expenditure. (M) VO2 value. (N) Ratio of lean mass to total body weight. Data was analysed by one-way ANOVA (B, E, F H, and J-N), and by two- way ANOVA (C, D). Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p 373 < 0.0001.

[0029] Fig. 7 shows that Lachnospiraceae relevant metabolites ameliorate HFpEF progression. (A) Scheme of experimental design, n = 5 mice per group. (B) Photographs showing the body size of mice in different treatment groups. (C) Quantification of body weight of mice on week 15. (D) Intraperitoneal glucose-tolerance test (ipGTT) after 15 weeks of diet. (E) Representative macroscopic images of the heart from mice of different experimental groups (Scale bars, 5 mm) and representative images of Masson’s trichrome (MT) staining in transversal sections of the mice hearts in different experimental groups (scale bars, 1000 pm). (F) The ratio of heart weight to tibia length (HW / TL). (G) Ratio between mitral E wave and A wave (E / A) on week 15. (H) Ratio between mitral E wave and E’ wave (E / E’) on week 15. (I— J) ELISA results showing cGMP and PKG protein levels. (K) Heart levels of nitric oxide. (L) One-week food intake at week 15. (M) Intraperitoneal insulin-tolerance test (ipITT) after 15 weeks of model generation. (N, O) Systolic blood pressure (SBP) and diastolic blood pressure (DBP) of different experimental groups after 15 weeks of treatment. (P) Ratio between wet and dry lung weight (W / D) at week 15. (Q) Running distance during exercise exhaustion test. (R) Grip force measurements of mice of different experimental groups. n=5. Data was analysed by one-way ANOVA (C, E, F, H-L, N-R), or by two-way ANOVA (D, M). Data are presented as mean ± SD; *p < 0.05, **p < 0.01, and ****p < 0.0001.

[0030] Detailed description

[0031] The present disclosure provides a method of treating or preventing a cardiovascular disease or condition in a subject by oral administration of extracellular' vesicles derived from milk (mEVs) to the subject. The inventors have found that oral administration of milk EVs (such as EVs derived from bovine milk) can slow or reverse progression of heart failure. Milk EVs have the capacity to influence gut immunity and gut microbiomc composition in inflammatory bowel disease, and can also act on other organs (e.g., liver) and influence overall metabolism by modulating gut barrier integrity.

[0032] Without being bound by theory, mEVs may be able to deliver lipids, proteins and importantly microRNAs which have protective effects on certain tissues or organs, e.g., by modulating intestinal inflammation and improving tight junction formation. In particular, oral administration of purified mEVs derived from bovine milk may influence cardiovascular disease progression by modulating three aspects of gastrointestinal health: intestinal inflammation, intestinal barrier integrity, and gut microbiome. From the intestinal inflammation aspect, oral administration of mEVs tend to be taken up by intestinal immune cells and may hinder inflammation and immune cell infiltration. From the epithelial barrier aspect, miRNAs in mEV s interact with intestinal epithelial cells and protect the epithelium from toxin-induced tight junction disruption and promote mucus secretion. This protection, by oral administration of mEVs, prevents gut toxins and harmful bacteria from entering the blood stream, thus ameliorating chemical- or diet-induced metabolic syndromes or diseases. From the gut microbiota aspect, oral administration of mEVs promotes flora diversity and increases the abundance of microorganisms producing short-chain fatty acids (SCFAs). This may increase levels of butyric acid in blood circulation and improve overall metabolic health. The inventors have shown that, in an animal model of heart failure with preserved ejection fraction (HFpEF), oral administration of mEVs halts or reverses obesity, insulin resistance, heart dysfunction, pulmonary hypertension, and skeletal muscle dysfunction. Oral administration of mEVs can reduce gut inflammation, gut barrier disruption, and dysbiosis, and these factors together can alleviate the progression of metabolic heart failure.

[0033] Accordingly, disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject.

[0034] General definitions

[0035] The term “cardiometabolic disease” refers to a group of interrelated metabolic and cardiovascular disorders that collectively increase the risk of atherosclerosis, coronary artery disease, stroke, heart failure, myocardial infarction, insulin resistance and diabetes. Cardiometabolic disease is characterised by dysrcgulatcd metabolic processes that contribute to vascular dysfunction, atherosclerosis, insulin resistance, obesity, dyslipidemia, hypertension, and inflammation, ultimately leading to cardiovascular complications.

[0036] The terms “cardiovascular disease”, “cardiovascular condition”, “disorder of the cardiovascular system” and “condition of the cardiovascular system” refer to diseases, disorders and conditions affecting the heart and blood vessels and include, for example: heart failure, myocardial infarction (heart attack), hypertension (high blood pressure), vascular diseases (e.g., atherosclerosis or vascular calcification), coronary heart disease, peripheral artery disease, cerebrovascular disease, stroke, stable and unstable angina pectoris, myocardial insufficiency, abnormal heart rhythms (or arrhythmias), persistent ischemic dysfunction (“hibernating myocardium”), temporary post-ischemic dysfunction (“stunned myocardium”), disturbances of peripheral blood flow, acute coronary syndrome, and heart muscle disease (cardiomyopathy).

[0037] The term “metabolic cardiovascular disease or condition” refers to a disease or condition in which blood circulation through the vasculature or the heart’s ability to pump blood efficiently is compromised due to underlying metabolic abnormalities. Metabolic cardiovascular diseases may be associated with or may arise from various metabolic disorders such as dyslipidemia, insulin resistance, glucose intolerance, diabetes, obesity, or metabolic syndrome. These conditions can directly affect the heart muscle or contribute to other risk factors such as hypertension and atherosclerosis, which further strain the heart. Non-limiting examples of metabolic cardiovascular diseases or conditions include heart failure, myocardial infarction, atherosclerosis, microvascular dysfunction, coronary heart disease, cerebrovascular disease, stroke, diabetic cardiomyopathy, and non-alcoholic fatty liver disease (NAFLD)-associated cardiovascular conditions.

[0038] The term “heart failure” includes both acute and chronic manifestations of heart failure, as well as more specific or related types of disease, such as advanced heart failure, post-acute heart failure, cardio-renal syndrome, heart failure with impaired kidney function, chronic heart failure, chronic heart failure with mid-range ejection fraction (HFmEF), compensated heart failure, decompensated heart failure, right heart failure, left heart failure, global heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart valve defects, heart failure associated with heart valve defects, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valve insufficiency, heart failure associated with combined heart valve defects, myocardial inflammation (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, heart failure associated with cardiac storage disorders, diastolic heart failure, systolic heart failure, acute phases of worsening heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmEF), chronic heart failure with reduced ejection fraction (HFrEF), chronic heart failure with preserved ejection fraction (HFpEF), chronic heart failure with mid-range ejection fraction (HFmEF), post myocardial remodeling, angina, hypertension, pulmonary hypertension and pulmonary artery hypertension.

[0039] “Dysbiosis” is defined herein as a physiological state in which a subject carries a microbiota profile that differs significantly from a corresponding microbiota profile that is typical of a healthy subject (e.g., a subject who is not at risk of or not suffering from a cardiovascular and / or metabolic disease). Dysbiosis may also be defined in terms of the extent of the perturbation in the relative levels, or the metabolic phenotype, of the microorganisms or groups of microorganisms contributing to the profile, i.e., by a measure of how different a microbiota profile is from a normal microbiota profile or by how much a microbiota profile deviates from a normal microbiota profile.

[0040] As used herein, an “extracellular vesicle (EV)” refers to a lipid bilayer-delimited nano- or micro-sized non-replicative particle secreted or shed from a cell. Extracellular vesicles may range in size from about 10 nm to about 10 pm. They carry a cargo of proteins, nucleic acids, lipids, metabolites and / or organelles from the parent cell, either within the vesicle or attached to the lipid membrane. EVs may be classified based on size, biochemical composition, cell of origin, biogenesis or condition under which they are released. Examples of EVs include but are not limited to ectosomes (released from the plasma membrane), exosomes (derived from endosomes), apoptotic bodies (release by cells undergoing apoptosis), oncosomes (released by cancer cells) and migrasomes (released by migrating cells).

[0041] As used herein, an “exosome” refers to an EV with a diameter of about 30 nm to about 300 nm. It is believed that materials cndocytoscd by a cell or other cellular components may be sorted into endosomal compartments, forming intraluminal vesicles within multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane may then release these vesicles into the extracellular environment as exosomes. Exo somes may be suitably identified by detecting known protein markers such as CD9, CD63, CD81, HSP70, HSP90, Alix, TsglOl, Annexin (such as Annexin I, Annexin II or Annexin V), Flotillin (such as Flotillin-1 or Flotillin-2), and others.

[0042] The term “sample” as used herein includes any biological specimen that may be extracted, untreated, treated, diluted or concentrated from a subject. A sample includes within its scope a collection of similar fluids, cells, or tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), isolated from a subject, as well as fluids, cells, or tissues present within a subject. In some embodiments the sample is a biological fluid. Biological fluids arc typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Certain biological fluids derive from particular tissues, organs or localized regions and certain other biological fluids may be more globally or systemically situated in a subject or biological source. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. Biological fluids may also include liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like. The term “sample” as used herein encompasses materials removed from a subject or materials present in a subject.

[0043] A “reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell”, or “control tissue”, as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased pail of the body (e.g., tissue or cells) of the same subject or individual. For example, healthy and / or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to an inflamed tissue). In another embodiment, a reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cell of the body of an individual who is not the subject or individual.

[0044] The term “treating” as used herein may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and / or (4) causing a decrease in the severity of one or more symptoms of the disorder.

[0045] The term “effective amount” as defined herein is meant the administration of an amount of agent to an individual in need thereof, either in a single dose or as part of a series, that is effective for that elicitation, treatment or prevention. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0046] The term “subject” as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. The “subject” may include a person, a patient or individual, and may be of any age or gender.

[0047] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0048] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0049] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of’, and variations such as “consists essentially of’ will be understood to indicate that the recited clcmcnt(s) is / arc essential i.c. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0050] Cardiometabolic diseases

[0051] Compositions and methods herein may be used to treat various cardiometabolic and cardiovascular diseases or conditions, including but not limited to heart failure, atherosclerosis, coronary heart disease, myocardial insufficiency, myocardial infarction, abnormal heart rhythms (arrhythmias), persistent ischemic dysfunction, cardiomyopathy (including diabetic cardiomyopathy), non-alcoholic fatty liver disease (NAFLD)-associated cardiovascular conditions, coronary artery disease, peripheral artery disease, hypertension, atherosclerosis, vascular calcification, cerebrovascular disease, obesity and diabetes.

[0052] In some embodiments, the cardiometabolic disease or condition is obesity, diabetes, metabolic cardiovascular disease, or a neurological disease or condition. The diabetes may be type II diabetes.

[0053] In some embodiments, the neurological disease or condition is dementia or stroke. The dementia may be Alzheimer’s disease, Huntington’s disease, frontotemporal dementia, Parkinson’s disease, Lewy body dementia or vascular dementia. In some embodiments, the cardiovascular disease or condition is a metabolic cardiovascular disease or condition, i.e., one that arises from or is associated with one or more underlying metabolic dysfunctions such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance, glucose intolerance, and dyslipidemias. Metabolic cardiovascular diseases and conditions include, for example, metabolic heart failure, coronary heart disease, diabetic cardiomyopathy, myocardial infarction, atherosclerosis, diabetes and obesity.

[0054] In one embodiment, the metabolic cardiovascular disease or condition is metabolic heart failure.

[0055] The term “metabolic heart failure” refers to heart failure that arises from underlying metabolic abnormalities and includes, but is not limited to, heart failure with reduced ejection fraction (HFrEF), heart failure with preserved ejection fraction (HFpEF), heart failure with mid-range ejection fraction (HFmEF), heart failure secondary to myocardial infarction, heart failure secondary to hypertension (e.g., systemic or pulmonary arterial hypertension), ischemic heart failure and diabetic heart failure. Metabolic heart failure includes both acute and chronic manifestations of heart failure. The heart failure may be leftsided, right-sided, biventricular or biatrial.

[0056] HFpEF is characterised by a left ventricular ejection fraction (LVEF) of > 50% with signs of diastolic dysfunction. HFrEF is characterised by a left ventricular ejection fraction (LVEF) of < 40% with signs of systolic dysfunction. HFmEF is an intermediate condition characterised by a left ventricular ejection fraction (LVEF) of 41-49%, often with diastolic dysfunction and borderline systolic dysfunction.

[0057] In one embodiment, the metabolic heart failure is heart failure with preserved ejection fraction (HFpEF). In one embodiment, the metabolic heart failure is heart failure with reduced ejection fraction (HFrEF).

[0058] In one embodiment, the metabolic cardiovascular disease or condition is obesity or diabetes. In one embodiment, the metabolic cardiovascular' disease or condition is atherosclerosis. In other embodiments, the metabolic cardiovascular disease or condition is myocardial infarction (MI). In some embodiments, the cardiovascular disease or condition is associated with a gastrointestinal dysfunction, such as gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability. The cardiovascular disease or condition may be, for example, caused by or exacerbated by the gastrointestinal dysfunction.

[0059] Extracellular vesicles herein may be isolated from bovine, human, goat or camel milk. In one embodiment, the mEVs are isolated from bovine milk. In another embodiment, the mEVs are isolated from human milk. The bovine or human milk from which mEVs axe isolated may be colostrum, transitional milk or mature milk.

[0060] In one embodiment, the milk-derived extracellular vesicles are exosomes.

[0061] Extracellular vesicles may be isolated from milk using any suitable method. In some embodiments, mEV isolation comprises incubating the milk with chymosin (0.03-0.15% w / v) and calcium chloride (0.2-1% w / v), subjecting the mixture to a combination of serial ultracentrifugation at progressively higher speeds (c.g., between 15,000 to 150,000 g), ultrafiltration, and size exclusion chromatography (SEC) to extract the mEVs.

[0062] In one embodiment, mEV isolation comprises:

[0063] (a) adding 0.03-0.15% w / v chymosin and 0.2-1% w / v calcium chloride (CaCh) to milk to obtain a mixture and incubating the mixture at 3-10 °C for 15-60 min;

[0064] (b) performing ultracentrifugation of the mixture at 15,000-18,000 g for 20-60 min;

[0065] (c) collecting supernatant from step (b) and centrifuging the supernatant at 60,000-20,000 g for 30-120 min at 3-10°C;

[0066] (d) collecting supernatant from step (c) and centrifuging the supernatant at 90,000-150,000 g for 60-120 min at 3-10 °C;

[0067] (e) resuspending a pellet obtained from step (d) in phosphate-buffered saline and washing the pellet by ultracentrifugation at 90,000-150,000 g for 30-120 min at 3-10 °C;

[0068] (f) performing ultra-filtration of the pellet obtained from step (e) using 100 kDa filters; and

[0069] (g) performing size exclusion chromatography. In one particular example, 0.05% w / v chymosin and 0.3% w / v CaCh are incubated with the milk at 4°C for 30 min. Afterward, the samples are centrifuged at 16,500 g to remove fat, casein and residual chymosin. The supernatant is collected and then centrifuged at 90,000 g for 60 min at 4°C to remove large particles and debris. Next, aliquots of the supernatant are centrifuged at 135,000 g at 4°C for 90 min. The pellet is resuspended in phosphate-buffered saline (PBS) and washed by ultracentrifugation at 135,000 g for 60 min. The pellet containing mEVs is then transferred onto 100 kDa filters and centrifuged twice at 3,000 g for 30 min to improve mEV purity, and subjected to size exclusion chromatography.

[0070] Isolated extracellular vesicles may be detected using known markers that are specific to a class of EVs. For instance, known exosomal markers include but are not limited to protein markers of the cndosomal pathway (c.g., CD9, CD63, CD81), heat shock proteins (c.g., Hsp70 and Hsp90) and multivesicular body (MVB) synthesis proteins (e.g., Alix and TsglOl). These protein markers may be detected via western blotting or other known means of detecting proteins. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and NanoSight LM10 analysis can also be used to analyse the presence and purity of isolated EVs.

[0071] The size of bovine mEVs may be about 30 nm to about 200 nm. The size of human mEVs may be about 30 nm to about 600 nm. For example, milk-derived exosomes may be about 100 nm in diameter.

[0072] Milk-derived EVs are highly resistant to degradation while in transit in the digestive system, and are well preserved in the digestive system in terms of morphology, size, quantity, and function. Milk-derived EVs also contain abundant bioactive microRNAs (miRNAs) and proteins. In one example, the miRNA in the mEVs includes, but is not limited to, miR-148a, miR-21, miR-30a, miR-30d, miR-26a, miR-200a, miR-200b, miR-200c, let-7a, let-7g, miR- 99a, miR-101, and combinations thereof.

[0073] This disclosure also provides compositions comprising milk-derived extracellular vesicles (mEV s) and acceptable carriers, diluents and adjuvants. In a preferred embodiment, the mEV composition is an oral composition. Oral compositions may be in the form of solid preparations (e.g., powders, tablets, capsules) or liquid preparations (e.g., suspensions) for direct consumption or for supplementation into food or beverages.

[0074] Formulations for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and / or time delay agents. Suitable binders include gum acacia, gelatin, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose and polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include com starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents which delay disintegration include glyceryl monostearate or glyceryl distcaratc.

[0075] In some embodiments, the mEV composition is formulated as a food product or a food additive using standard techniques well known to one of ordinary skill in the art. For example, the composition may be directly added to an edible material or may be used to prepare an intermediate composition (e.g., a additive or premix) suitable to be subsequently added to the edible material.

[0076] Examples of food products include, but are not limited to, milk powder, fermented dairy products such as cheeses and yogurt, butter, meat, poultry, fish, eggs, cereals and cereal- derived products such as noodles and porridge, legumes, pulses, beverages (e.g., tea, coffee, juice, broth, etc.), flour products, baked foods, confectionery, candy, fermented foods (e.g., kimchi, sauerkraut, miso, tempeh, kombucha), animal feeds, health foods, infant foods, and dietary supplements. In some embodiments, the mEV composition is a nutraceutical composition for health promotion in healthy subjects, in particular for preventing gastrointestinal dysfunction associated with metabolic cardiovascular diseases and conditions. The nutraceutical composition may include naturally derived or synthetic ingredients and can be delivered in various forms such as capsules, tablets, powders, liquids, or functional foods. In some embodiments, the nutraceutical composition comprises, in addition to mEVs, ingredients that promote gut health, including but not limited to probiotics, prebiotics (e.g., resistant starches, fructooligosaccharides and galactooligosaccharides), postbiotics (e.g., probiotic exopolysaccharides), polyphenols (e.g., curcumin, epigallocatechin gallate, quercetin and cocoa), and dietary fibres (e.g., psyllium husks, flaxseed or chia seed fibres, beta-glucans).

[0077] In some embodiments, the mEV composition is a pharmaceutical composition. The pharmaceutical composition may comprise pharmaceutically acceptable carriers, diluents or adjuvants, non-limiting examples of which include demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or isopropanol; lower aryl-alcohols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar: gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0078] Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

[0079] Methods of treatment and prevention Methods are provided herein for treating and preventing cardiometabolic and cardiovascular diseases and conditions by oral administration of milk-derived extracellular vesicles (mEVs).

[0080] Disclosed herein is a method of treating or preventing a cardiometabolic disease or disorder in a subject, the method comprising orally administering an effective amount of a milk- derived extracellular vesicle (mEV) composition to the subject to prevent or treat the cardiometabolic disease or disorder.

[0081] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising orally administering an effective amount of a milk- derived extracellular vesicle (mEV) composition to the subject to treat or prevent the cardiovascular disease or condition.

[0082] Disclosed herein is a milk-derived extracellular vesicle (mEV) composition, for use in treating or preventing a cardiometabolic or cardiovascular disease or condition in a subject, wherein an effective amount of the mEV composition is to be orally administered to the subject.

[0083] Disclosed herein is the use of a milk-derived extracellular vesicle (mEV) composition in the manufacture of a medicament for treating or preventing a cardiometabolic or cardiovascular disease or condition in a subject, wherein an effective amount of the mEV composition is to be orally administered to the subject.

[0084] In some embodiments, the mEVs are administered orally at a concentration of about 0.01 mg / kg to about 6 mg / kg. In some embodiments, the mEVs arc administered orally at a concentration of about 0.01 mg / kg to about 0.1 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 0.1 mg / kg to about 0.5 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 0.5 mg / kg to about 1 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 1 mg / kg to about 2 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 2 mg / kg to about 3 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 3 mg / kg to about 4 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 4 mg / kg to about 5 mg / kg. In some embodiments, the mEVs are administered orally at a concentration of about 5 mg / kg to about 6 mg / kg. For example, the mEVs may be administered orally at a concentration of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 0.7 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, or about 6 mg / kg.

[0085] In some embodiments, the mEVs are administered orally at a frequency of once a day to once a week. In one embodiment, the mEVs are administered at a frequency of once a day. In another embodiment, the mEVs are administered at a frequency of once every two days. In another embodiment, the mEVs are administered at a frequency of once every three days. In another embodiment, the mEVs arc administered at a frequency of once every four days. In another embodiment, the mEVs are administered at a frequency of once every five days. In another embodiment, the mEVs are administered at a frequency of once every six days. In another embodiment, the mEVs are administered at a frequency of once a week.

[0086] The mEVs may be administered for any duration appropriate to prevent or treat the cardiovascular disease or condition. In some embodiments, the mEVs are administered to the subject orally for a duration of at least 1 week, such as for a duration of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or longer than 15 weeks. In one embodiment, the mEVs are administered for a duration of at least about 5 weeks. In one embodiment, the mEVs are administered for a duration of about 5 weeks to about 15 weeks.

[0087] Subjects may be selected for treatment having been determined to be at risk of or likely to be suffering from the cardiovascular disease or condition. In some embodiments, subjects are selected for treatment having been determined to have gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability. In yet other embodiments, subjects are selected for treatment having been determined to be at risk of or likely to be suffering from the cardiovascular disease or condition, and having been determined to have gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability. Accordingly, in some embodiments, methods herein may further comprise a step of selecting a subject for treatment with mEVs. For example, in one embodiment, methods may comprise detecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition, and administering mEVs to subjects found to be at risk of or likely to be suffering from a cardiovascular disease or condition to treat or prevent the cardiovascular disease or condition in the subject. In another embodiment, methods may comprise detecting gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability in a subject, and administering mEVs to subjects with gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability to prevent or treat the cardiovascular disease or condition in the subject. In a further embodiment, methods may comprise selecting subjects for treatment with mEVs based on detecting a risk or presence of cardiovascular disease, and detecting the presence of gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability.

[0088] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising: (a) detecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition; and (b) orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject found to be at risk of or likely to be suffering from a cardiovascular disease or condition to treat or prevent the cardiovascular disease or condition in the subject.

[0089] Methods known in the art may be used to detect cardiovascular diseases or conditions, such as methods based on echocardiography (ECHO), electrocardiogram (ECG), and measurements of biomarkers such as troponins, lipids and C-reactive protein (CRP) in a blood sample from the subject. Methods of detecting a subject who is at risk of or likely to be suffering from a cardiomctabolic disease or metabolic cardiovascular disease or condition may further include detecting one or more of the following in the subject: insulin resistance, glucose intolerance, high blood pressure, high cholesterol, high triglyceride and / or high body mass index (BMT).

[0090] Heart failure may be detected using a combination of echocardiography (e.g., to evaluate left ventricular' ejection fraction and left ventricular remodelling), Doppler echocardiography (e.g., to measure E / A and E / c’ ratios), and Speckle Tracking (e.g., to assess global longitudinal strain). Such techniques are well known to one of ordinary skill in the art.

[0091] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising: (a) detecting gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability in a sample from the subject, wherein the presence of gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and (b) orally administering an effective amount of an mEV composition to a subject found suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject.

[0092] Disclosed herein is a method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising: (a) detecting increased intestinal permeability in a sample from the subject, wherein an increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and (b) orally administering an effective amount of an mEV composition to a subject found suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject.

[0093] The sample may be, but is not limited to, any fluid or solid taken from the lumen or surface of the gastrointestinal tract or any sample of any of the tissues that form the organs of the gastrointestinal tract. Thus, the sample may be any luminal content of the gastrointestinal tract, particularly the intestinal tract (e.g., stomach contents, intestinal contents, caecal contents, mucus and facccs / stool, or combinations thereof), as well as samples obtained mechanically from the gastrointestinal tract e.g., by swab, rinse, aspirate or scrape of a gastrointestinal tract cavity or surface or by biopsy of a gastrointestinal tract tissue or organ.

[0094] The sample may be used in the form in which it was initially retrieved. Alternatively, the sample may have undergone some degree of manipulation, refinement or purification before being used in the methods of this disclosure. Thus, the term “sample” also includes preparations thereof, e.g., relatively pure or partially purified starting materials, such as semi-pure preparations of the above-mentioned samples. Further included is the product of the microbial culture of said sample.

[0095] The purification may be slight, for instance amounting to no more than the concentration of the solids, or cells, of the sample into a smaller volume or the separation of cells from some or all of the remainder of the sample. In certain embodiments, a preparation of the nucleic acids from the samples may be used, e.g., to determine the relative levels of gastrointestinal tract microbiota in the sample. It may be advantageous if the predominant nucleic acid of the nucleic acid preparation is DNA, however, RNA may be the predominant nucleic acid in other embodiments. These preparations include relatively pure or partially purified nucleic acid preparations.

[0096] Gastrointestinal inflammation may be detected by endoscopy or histology according to methods well known in the art. For example, endoscopy can provide direct visualisation of inflammation, ulcers, and erosions, and histopathology can confirm the presence of, e.g., infiltrating immune cells (neutrophils, lymphocytes, eosinophils) in endoscopic biopsy samples. Alternatively, blood or stool biomarkers may be used to detect gastrointestinal inflammation, such biomarkers including but not limited to calprotectin, lactoferrin, zonulin, C-rcactivc protein and the scrum cytokines IL-6, TNF-a, and IL- Ip, which arc all elevated under inflammatory conditions.

[0097] Dysbiosis may be detected by profiling of gastrointestinal tract microbiota (i.e., the preparation of a gastrointestinal tract microbiota profile). Microbiota profiling may involve any known and convenient means by which the levels of microorganisms or groups of microorganisms in the gastrointestinal tract of a subject may be quantified, typically from a sample taken from the gastrointestinal tract. For example, profiling may involve nucleic acid analysis (e.g., nucleic acid sequencing, oligonucleotide hybridisation probe-based approaches, primer-based nucleic acid amplification approaches, etc.), antibody or other specific affinity ligand-based approaches, or proteomic or metabolomic approaches.

[0098] In some embodiments, dysbiosis is indicated by a decrease in the overall diversity of the intestinal microbiota compared to the microbiota of a healthy subject (i.e., a subject without the cardiomctabolic or cardiovascular disease). In some embodiments, dysbiosis is indicated by a decrease in the levels of Bacteroides, Lactobacillus, Parabacteroides, Proteobacteria, Blautia, Lachnospiraceae (e.g., Lachnobacterium, Lachnospirales or Lachnoclostridium), and / or Muribaculaceae bacteria in the intestinal microbiota. In some embodiments, the dysbiosis is indicated by an increase in the levels of Akkermansia, Desulf bbacterota, Deferribacterota, Ileibacterium and Campilobacterota bacteria in the intestinal microbiota.

[0099] Increased intestinal permeability may be detected using methods well known in the art. In one embodiment, detecting increased intestinal permeability comprises orally administering to the subject a label and detecting uptake of the label by the subject, wherein an increased level of uptake of the label as compared to a reference indicates that the subject is likely to be suffering from a gastrointestinal dysfunction. As a non-limiting example, the relative uptake of the sugars mannitol and lactulose can be detected. Mannitol is readily absorbed by the gastrointestinal tract while lactulose is only slightly absorbed and serves as a marker for mucosal integrity. An elevated lactulose to mannitol absorption ratio, as determined by measuring levels of the two sugars excreted in urine following oral administration, is an indicator of intestinal barrier dysfunction.

[0100] Increased intestinal permeability may also be detected by detecting changes in levels of biomarkers in blood and / or faecal samples. For example, D-lactate, the endotoxin lipopolysaccharide (LPS), lipopolysaccharide-binding protein (LBP) and fatty-acid-binding protein (FABP) are blood markers that reflect translocation of bacteria or their metabolic products across the intestinal barrier. Elevated levels of D-lactate, endotoxin, LBP or FABP indicate intestinal barrier dysfunction and bacterial overgrowth in the gut. Thus, in another embodiment, detecting increased intestinal permeability comprises detecting a level of endotoxin in a sample obtained from the subject, wherein an increased level of endotoxin as compared to a reference indicates that the subject is likely to be suffering from a gastrointestinal dysfunction. Alternative biomarkers include but arc not limited to the tight junction proteins zonulin and claudin-3. Increased levels of zonulin in the blood and increased expression of claudin-3 in intestinal epithelial cells are indicative of increased intestinal permeability.

[0101] The level of uptake of a label and the level of a biomarker such as endotoxin may be compared to a reference to determine the presence of increased intestinal permeability in the subject. The reference may be, for example, the corresponding level (i.c., level of uptake or biomarker level) from a sample from a subject of the same species without the cardiovascular disease, or an average level in samples from a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without cardiovascular disease. Alternatively, the reference may be the corresponding level in a sample from the same subject before the onset or suspected onset of a metabolic disease, before the start of a treatment regimen (e.g., treatment with mEVs), or at a different time-point during the course of a cardiovascular disease or during the course of treatment for the cardiovascular disease.

[0102] Advantageously, the mE V s can prevent or improve gastrointestinal dysfunction through one or more of the following: normalising or maintaining gastrointestinal epithelial barrier function; normalising or maintaining gastrointestinal mucosal barrier function; reducing gastrointestinal inflammation; regulating gastrointestinal microflora; protecting against infection by pathogenic gastrointestinal bacteria; and promoting probiotic colonisation of the intestinal tract. Tn particular, the mEVs may promote the growth of intestinal bacteria (e.g., Lachnospiraceae) that produce bioactive short chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid. Prevention and / or amelioration of the gastrointestinal dysfunction in turn prevents and / or treats the cardiovascular disease.

[0103] Disclosed herein is a method of selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition for treatment with mEVs, the method comprising: (a) detecting an increased intestinal permeability in the subject, wherein an increased intestinal permeability indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition; and (b) selecting a subject found to have increased intestinal permeability for treatment with mEVs.

[0104] The method may further comprise orally administering an effective amount of an mEV composition to the subject.

[0105] Disclosed herein is a method of promoting gut health in a subject, the method comprising a) selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition and b) orally providing a milk-EV composition to the subject so as to promote gut health in the subject. The cardiovascular disease or condition may be one that is associated with gastrointestinal dysfunction. Disclosed herein is a method of treating or preventing a gastrointestinal dysfunction (such as gastrointestinal inflammation, dysbiosis or increased intestinal permeability) in a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition, the method comprising administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat the gastrointestinal dysfunction in the subject. The cardiovascular disease or condition may be one that is associated with the gastrointestinal dysfunction.

[0106] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0107] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0110] EXAMPLES

[0111] Methods

[0112] TEER measurement The apical and basolateral compartments of the Transwells were kept in complete DMEM, which was 535 refreshed every 2 days. The measurement and recording of TEER (trans- epithelial electrical resistance) values 536 were performed using a Millicell ERS-2 resistance system (EMD Millipore Corporation, Burlington, MA). TEER was calculated using the formula: TEER = (Rm - Ri) * A, where Rm represents the transmembrane resistance, Ri signifies the intrinsic resistance of the cell medium, and A denotes the surface area of the Transwell membrane.

[0113] Experimental animals

[0114] For the HFD+L-NAME model, 8-week-old wild-type male mice of C57BL / 6J background were grouped randomly and maintained on a 12-hour light and dark cycle for 15 weeks. The mice had unrestricted access to food (#2916, Tcklad for CHOW groups and D12492, Research Diet Inc. for the HFpEF groups) and water. N[w]-nitro-l-arginine methyl ester (L- NAME; 0.5 g / L, Sigma Aldrich) w as supplied in the drinking water for the indicated periods of time, after adjusting the pH to 7.4 for the HFpEF groups. For mEVs study, mice were orally administered with 1.2 mg / kg mEVs every other day starting at week 0 feeding on the special diet and water. Saline and EV-depleted supernatant ( 1.2 mg / kg every' other day) were used as negative controls. For metabolites study, HFD / L-NAME-fed C57BL / 6J mice were separated into 4 groups and treated with saline (i.p. injection), butyric acid (i.p. injection, 3.1 mg / kg / day), azelaic acid (i.p. injection, 80 mg / kg / day), and g-caprolactone (i.p. injection, 70 mg / kg / day), respectively, for 15 weeks. Body weight was assessed and blood was collected through cheek pouch for all groups. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National University of Singapore and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIIH Publication, 8th Edition, 2011).

[0115] Metabolic cage study

[0116] Male C57BL / 6 mice (8 weeks old, n=4 per group) w ere individually housed and maintained on a 12-hour light / dark cycle. Mice were randomly assigned to groups and subjected to HFD combined with L-NAME treatment for 3 weeks. Starting at week 0, mice were orally administered 1.2 mg / kg of milk-derived extracellular vesicles (mE V s) every other day, while saline and EV-depleted supernatant (1.2 mg / kg every other day) were used as negative controls. The mice were acclimatized to metabolic cages at a controlled temperature of 22 °C for one week prior to the initiation of the study. Following two days of baseline data collection, the ambient temperature was increased by 2°C increments daily at 0600 h (the start of the light phase). Throughout the study, key metabolic parameters, including food intake, energy expenditure, oxygen consumption (VO2), and physical activity, were continuously monitored and recorded.

[0117] Conventional echocardiography and Doppler imaging

[0118] As previously described, Transthoracic echocardiography was conducted using a VisualSonics Vevo 2100 system with an MS400 transducer (VisualSonics Inc, Toronto, Canada). Briefly, in conscious, gently restrained mice, systolic function indices were obtained from mid-ventricular M-mode scans, while diastolic function measurements were taken in anesthetized mice using apical 4-chamber views, pulsed-wave, and tissue Doppler at the mitral valve level. During the echocardiogram, isofluranc was used to induce anesthesia, and the concentration was adjusted to maintain a heart rate of 415-460 beats per minute under controlled body temperature. Multiple parameters are collected: heart rate (HR), left ventricular ejection fraction (LVEF), peak Doppler blood inflow velocity across the mitral valve during early diastole (E), peak Doppler blood inflow velocity across the mitral valve during late diastole (A), peak tissue Doppler of myocardial relaxation velocity at the mitral valve annulus during early diastole (E’), peak tissue Doppler of myocardial relaxation velocity at the mitral valve annulus during late diastole (A’), and left ventricular global longitudinal strain (GLS). Following the procedures, all mice recovered from anesthesia without complications. Each parameter was measured at least three times, and the averages were reported.

[0119] Tail cuff blood pressure recordings

[0120] Noninvasive measurement of systolic and diastolic blood pressure was conducted in conscious mice using the tail-cuff method and a CODA instrument (Kent Scientific Corporation). Mice were separated into individual holders on a temperature-controlled platform set at 37°C and tails were fixed to maintain steady-state conditions during the recordings. Prior to the actual testing, all mice underwent a training period to familiarise them with short-term restraint. Blood pressure was recorded for 5 consecutive days at each time point and readings were averaged from at least 8 measurements per session.

[0121] Exercise exhaustion test Mice were acclimatized to treadmill exercise for three days before the exercise exhaustion test. The animals were made to run uphill (20°) on the treadmill (Columbus Instruments, Columbus, USA). The test began with a warm-up speed of 5 m / min for 4 minutes, followed by an increase to 14 m / min for 2 minutes. Every subsequent 2 minutes, the speed was raised by 2 m / min until the animal reached a state of exhaustion. Exhaustion was defined as the inability of the animal to resume running within 10 seconds of direct contact with an electric stimulus grid. The running time was recorded, and the running distance was calculated.

[0122] Grip strength test

[0123] Muscle strength was evaluated using a grip strength test. The mice were positioned horizontally along a straight line to eliminate the influence of body weight differences. They were allowed to grip a pull-bar assembly connected to a grip-strength meter (Columbus Instruments, Columbus, USA). Then the mice were gently pulled by the tail until their grip was released, and the peak force in grams shown by the sensor was recorded. The test was considered complete when 6 trials were recorded for each pair of limbs (5 trials for the forelimbs and 5 trials for the hindlimbs). All measurements were performed in a blinded manner.

[0124] Intraperitoneal glucose and insulin tolerance test

[0125] After fasting for 6 hours, mice were intraperitoneally injected with 2 g / kg glucose saline or 1.5 U / kg insulin saline, and tail blood glucose levels (unit: mg / dL) were measured with a blood glucose meter before administration (0 min) and 30, 60, and 90 min after administration.

[0126] Biodistribution in mice

[0127] To monitor the biodistribution of mEVs in healthy and HFpEF mice, mEVs were labeled with Cy5 fluorescence dyes (Cyanine 5 NHS ester, Lumiprobe, 43020) through a click chemistry method. Briefly, 300 pg / l mEVs were incubated with 1 pl of Cy5 (5 mg / ml) at 4 °C for 4 hours, followed by ultrafiltration to get rid of free dye. The labeled mEVs were administered through oral gavage while the same amount of free Cy5 dyes in PBS was administered to mice as a control. After that, mice were anesthetized and imaged over a 24- hour period using IVIS Spectrum In Vivo Imaging System (PerkinElmer, Boston, USA). Organs were collected at the 24-hour time point and imaged using IVIS. Histology

[0128] Colon, heart, lung, and muscle tissues were fixed in a 10% formalin solution (Sigma- Aldrich, HT501128) and embedded in paraffin. Paraffin-embedded tissue was cut into 5- pm-thick sections and stained with hematoxylin (Sigma-Aldrich, H9627) and eosin (Merck, E4382), Masson’s Trichrome (Solarbio, G1346), and PicroSirius red (Abeam, abl50681). Wheat germ agglutinin (WGA) staining was used to measure cardiomyocyte cross-sectional area (CSA), and antigens were retrieved from heart sections by boiling them in sodium citrate buffer (pH 6.0, Antigen Retrieval Citra, BioGenex). To block endogenous peroxidase activity, tissue sections were incubated in a 3% hydrogen peroxide solution (Sigma- Aldrich, 516813). Subsequently, to prevent nonspecific binding, the sections were treated with 3% bovine serum albumin (BSA) for 30 minutes at room temperature. Heart tissue was stained with diluted WGA Fluorescein (Vector Laboratories, FL- 1021) overnight. After the washing steps, the tissue was stained with 10 pg / mL Lectin from triticum vulgaris FITC conjugate (L4895, Sigma-Aldrich, L4895) in the dark for 1 hour. After washing steps, the sections were coverslipped with a water-soluble DAPT mounting medium (Invitrogen). Images were taken under an eclipse TI2-E inverted research microscope (Nikon). Cardiomyocyte size, fibrosis area, and capillary numbers were quantified with ImageJ software version 2.0 (NIH, Bethesda, MD) (three microscopic fields per heart ).

[0129] Immunofluorescence staining

[0130] Cells and colon tissue 'ere fixed with 4% paraformaldehyde. For colon tissue, it was dehydrated with 30% sucrose solution at 4°C overnight, embedded in Frozen Section Compound (Leica), and cut into 5-pm cryoscctions. After that, cells and sections were incubated in the blocking buffer for 1 hour, followed by incubation with primary antibodies, including Occludin (Abeam, ab216327) and ZO-1 (Abeam, ab221547) at 4°C overnight. After washing with PBS, samples were stained using fluorescent dye-conjugated secondary antibodies for 1 hour in the dark at room temperature. Finally, samples were washed with PBS and mounted with DAPI. Images were taken under an eclipse TI2-E inverted research microscope (Nikon) and analyzed with ImageJ software (NIH, Bethesda, MD).

[0131] Gut permeability assays

[0132] Gut permeability was assessed by orally administering FITC-dextran (0.6 mg / g; Sigma- Aldrich, 46944) to the mice. 4 hours after administration, blood was collected through check pouch and plasma was obtained by centrifugation at 2000 rpm for 15 min. Fluorescence measurements were performed using a microplate reader (Thermo Fisher Scientific, USA) with excitation at 485 nm and emission at 530 nm. The intensity of fluorescence was expressed in fluorescence intensity and used to quantify gut permeability.

[0133] Quantitative real-time polymerase chain reaction

[0134] Cells and tissue were subjected to total RNA extraction through the RNeasy Mini Kit (QIAGEN, 74106), followed by quantification using Nanodrop (Thermo Fisher Scientific). After extraction, a total of 500 ng of RNA was reverse transcribed into cDNA using the Reverse Transcription Kit (QIAGEN, 205313). After that, cDNA was mixed with the PowerUp SYBR Green Master Mix (Applied Biosystems, 01151389) and measured using Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, USA). The relative expression of genes was determined using the -2AACt method, with GADPH serving as the housekeeping gene.

[0135] Western blot

[0136] Western blot was conducted as previously reported. Briefly, proteins of the colon were extracted and separated by SDS-polyacrylamide gel electrophoresis. Then proteins were transferred to a polyvinylidene difluoride membrane and were blocked using 5% BSA in tris-buffered saline with 0.1% Twccn-20 detergent at room temperature for 1 hour. The membrane was probed with different primary antibodies, including ZO-1 (Abeam, ab221547) and Occludin (Abeam, ab216327). Secondary antibodies were used for protein visualisation. Proteins were quantified with Image.! software version (NTH, Bethesda, MD).

[0137] RNA isolation and qPCR

[0138] Total RNA was extracted from mice heart, colon, and muscle using TRIzol reagent or RNeasy Mini Kit (QIAGEN, 74104). A total of 500 ng RNA was used for reverse transcription using QuantiTect Rev. Transcription Kit (QIAGEN, 205311). qPCR was performed in triplicate with SYBR master mix (Bio-Rad, 1725270) with specific primers for target sequences. The 2-AACT method of relative quantification was employed to assess the of target mRNA in samples. GADPH was used for normalization. Fold ratio was then computed in comparison to mRNA expression levels observed in control samples.

[0139] Biochemical assays Mouse colon tissue, heart tissue, and plasma were collected for biochemical assays. Heart homogenate was centrifuged at 3000 g for 10 min. Supernatants were collected to determine cGMP and PKG levels with an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bio, Nanjing, CN). Human serum levels of gut barrier dysfunction markers, I- FABP and LBP, were measured using ELISA kits (Nanjing Jiancheng Bio). Plasma levels of NO were determined using a Nitric Oxide assay kit according to the manufacturer’s protocol (Abeam, ab272517).

[0140] Endotoxin assay

[0141] The endotoxin level of mice feces, plasma, and heart was measured using Pierce™ Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific, A39552) following the instructions of the protocol. Briefly, samples and standards were added to a 96-wcll plate prewarmed to 37°C, and lysate was added to each well. After incubation at 37°C for 14 minutes, chromogenic substrate was added to each well and the mixture was incubated at 37°C for 6 minutes. Finally, stop solution was added to each well and the absorption at 405nm was determined using microplate reader (Thermo Fisher Scientific, USA).

[0142] Microbiota 16S rRNA sequencing 686

[0143] Fecal genomic DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN Inc., Netherlands). The V4-V5 region of bacterial 16S rRNA genes were amplified using the forward primer 515F (5’-688 GTGCCAGCMGCCGCGGTAA-3’) and the reverse primer 907R (5’-CCGTCAATTCMTTTRAGTTT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The resulting PCR amplicons were purified, quantified, and pooled in equal amounts. Paired-end sequencing with 2x300 bp reads was performed on the Illumina MiSeq platform using the MiSeq Reagent Kit v3 at Beijing Novogcnc Technology Co., Ltd (Beijing, China). High-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity using UCLUST70. Sequence data analysis was conducted using QUME and R packages (v3.2.0). The taxonomy compositions and relative abundances were visualized using MEGAN and GraPhlAn. To identify differentially abundant taxa across groups, the Linear discriminant analysis effect size (LEfSe) was employed with default parameters.

[0144] Metabolomics Liquid chromatography-mass spectrometry (LC-MS) was utilized for the quantification of metabolites in mice plasma using the SCIEX QTRAP 6500 LC-MS / MS system. Sample preparation included thawing the samples on ice, followed by extraction with methanol and the addition of an internal standard. Liquid chromatography was performed using the Waters ACQUITY UPLC HSS T3 Column (1.8 pm, 2.1 mmx 100 mm). The mobile phase consisted of water with 0.1% (v / v) formic acid (A) and acetonitrile with 0.1% (v / v) formic acid (B). Multiple reaction monitoring was employed to analyse metabolites in the samples. Data processing and quantification were conducted using the MultiQuant software. OPLS-DA analysis was implemented using the ropls R package.

[0145] RNA sequencing

[0146] Total RNA was extracted from mice’s hearts using RNAiso Plus. mRNA enrichment, fragmentation, reverse transcription, library construction, and sequencing were performed using the Novaseq 6000 (Illumina) platform. The resulting clean reads were aligned to the mouse genome (mm 10) using TopHat v2.0.1 17 with default options and a TopHat transcript index built from Ensembl_GRCm38 as the reference. As previously described, pathway enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID).

[0147] Example 1: Extraction of mEVs from bovine milk

[0148] According to Nutrition in the Prevention and Treatment of Disease (Fourth Edition, 2017), bovine milk contains 4-5% lactose. According to McCance and Widdowson's The composition of Foods (Sixth summary ed. 2002), bovine full fat milk contains 3.9% fat (2.5% saturated fatty acids, 1.2% unsaturated fatty acids). The present mEV isolation protocol results in less than 0.1% lactose and fat in the enriched fraction of mEVs.

[0149] EVs were isolated from bovine milk by pre-treating the milk with chymosin and CaCh, followed by serial ultracentrifugation, ultra-filtration, and size exclusion chromatography (SEC). In brief, 0.05% chymosin and 0.3% CaCh were added to bovine milk and incubated at 4°C for 30 min. After the curd formed, the samples were centrifuged at 16,500 g to eliminate fat, casein, and remaining chymosin. The whey was collected and further centrifuged at 90,000 g for 60 minutes at 4°C using a BECKMAN Optima XE-100 Ultracentrifuge with a Type 70 Ti alloy 508 fixed angle rotor (Beckman Coulter Inc., Brea, USA) to remove large particles and debris. Subsequently, 15 ml portions of the supernatant were centrifuged at 135,000 g for 90 minutes at 4°C, and the resulting pellet was resuspended using phosphate-buffered saline (PBS) and washed by ultracentrifugation at 135,000 g for 60 minutes. The microvesicles (mEVs) were then collected and transferred to 100-kDa Amicon® ultra centrifugal filters (Sigma-Aldrich, USA) and centrifuged twice at 3000 g for 30 minutes to enhance purity. The supernatant without mEVs was collected following the ultracentrifugation step at 135,000 g. The protein concentrations of both mEVs and mEV- depleted supernatant were determined using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The comprehensive isolation protocol of mEVs has been submitted to the EV-TRACK database under the ID EV20001827.

[0150] For SEC isolation of mEVs, 0.5 ml of the resuspended pellet was loaded onto a home-made SEC column packed with Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) in a Telos SPE column (Kinesis, Cambridgeshire, UK). 0.5 ml fractions were collected once the sample was added. In total, 30 0.5 ml-fractions were collected using 0.22 pm-filtered PBS as the elution buffer. The Bradford assay was used to determine protein concentration.

[0151] The objectives of this study arc 1) to establish a therapeutic approach, i.c., oral administration of mEVs, for protection of gut microenvironment; and 2) to develop functional food for health promotion in healthy subjects; and 3) to develop oral therapeutics (and medical nutrition formula) for the treatment of heart failure patients. Further, the oral administration of mEVs approach may be applied in other metabolic diseases such as obesity and diabetes.

[0152] Example 2: Disorder of gut barrier and gut microbiota appears in HFpEF

[0153] Disruption of gut barrier and gut microbiota tends to appear in a variety of CVDs and metabolic diseases and is considered to be related to its pathogenesis. Given that HFpEF is a typical CVD that associated to metabolic syndrome, it is hypothesised that gut barrier and gut microbiota disruption occurs in HFpEF progression. To verify this, a HFpEF mouse model was established by feeding 8-week-old C57BL / 6J mice with high fat diet and water with L-NAME (HFD+L-NAME) for 15 weeks. Eongitudinal echocardiographic evaluation showed preservation of left ventricular ejection fraction (LVEF) but increased E / A and E / E’ ratios at 15 weeks in the HFD+L-NAME-induced HFpEF model (Fig. 1A). These mice also exhibited significant weight gain and impaired glucose tolerance compared to the untreated healthy control group (Fig. IB, C). The results confirmed the successful generation of HFpEF model with systemic metabolic disorder.

[0154] Compared to healthy control, the HFpEF mice exhibited distorted colonic morphology with mucosal inflammatory infiltration and shortened length (Fig. ID, E). Also, qPCR result showed that the expression of the pro-inflammatory marker TNF-a was markedly upregulated in the HFpEF mice, alongside reduced expression 154 of tight junction proteins Occludin and ZO-1 (Fig. 1F-H).

[0155] To quantify the gut barrier dysfunction, FITC-dcxtran was administered via oral gavage at week 15. At 4 hours post-gavage, the plasma fluorescence in HFpEF mice was two-fold higher than that in healthy control, indicating increased gut permeability (Fig. II). Additionally, elevated faeces and plasma endotoxin levels further confirmed gut barrier disruption in HFpEF (Fig. 1J).

[0156] Analysis of the gut microbiota composition in the HFpEF mice using 16S rRNA sequencing showed that there was reduced gut microbial diversity, as reflected by significant decreases in a-diversity indices, including Observed_OTUs and Shannon index. PCA and PCoA analyses further revealed significant disruption in gut microbial diversity in the HFpEF mice (Fig. IK). Furthermore, the composition and relative abundance of gut microbiota differed significantly between the healthy control and the HFpEF mice (Fig. IL, M). At the genus level, the relative abundance of Lachnospiraceae , a key fatty acid producer, was reduced in the HFpEF mice. This shift suggests a loss of beneficial microbes that support gut health and an increase in inflammatory or pathogenic taxa, potentially contributing to metabolic dysfunction and HFpEF progression.

[0157] To investigate if the same alterations in gut barrier and microbiota are present in HF patients, a total of fifteen HFpEF patients and ten non-HFpEF donors were examined clinically. Patients with HFpEF had a higher E / E’ ratio (13.5±6.8 versus 6.3±2.1 , p= 0.01) and elevated plasma levels of CK-MB, a biomarker for the diagnosis of cardiac muscle damage (2.3+1.2 ng / ml versus 1.2±0.5, p-0.02). Other clinical parameters, such as blood pressure, body mass index, and some cardiac indices (left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular posterior wall (LVPW), were comparable between HFpEF patients and non-HFpEF subjects.

[0158] To evaluate the gut barrier status in HFpEF patients, plasma levels of three gut barrier integrity markers, endotoxin, gut lipopolysaccharide-binding protein (LBP) and fatty-acid- binding protein (I-FABP), were measured (Fig. IN). Compared to non-HFpEF subjects, all three markers are significantly elevated in the HFpEF patients, indicating the presence of gut barrier dysfunction.

[0159] Potential alterations in gut microbiota in HFpEF patients was examined by 16S rRNA sequencing from fecal samples. Compared to the non-HFpEF subjects, the HFpEF patients displayed a reduced diversity of gut microbiota, as reflected by lower a-diversity indices, including ACE, Chao, Shannon, and Simpson indices (Fig. 10). The Microbial Dysbiosis Index (MDI) was elevated, while the Gut Microbiome Health Index (GMHI) was reduced, indicating the presence of gut dysbiosis. The dysbiosis was further confirmed by the fldiversity indices of gut microbiota assessed by the weighted UniFrac-based principal component analysis (PCA) of the operational taxonomic unit (OTU) composition. In addition, the relative abundance of specific bacterial populations was altered in HFpEF patients. It was observed that there was a depletion of Proteobacteria and increased relative abundance of Desulfobacterota, Deferribacterota, and Campilobacterota at the phylum level. At the genus level, Ileibacterium was enriched, whereas Blautia was depleted. These findings highlight the onset of gut dysbiosis associated with HFpEF pathogenesis.

[0160] Example 3: Orally-administered mEVs systemically alleviate HFpEF progression

[0161] It has previously been shown that orally-administrated mEVs can treat metabolic disorders, such as non-alcoholic steatohepatitis (NASH), through modulating the gut environment. Metabolic disruption in HFpEF leads to changes in the immune system, creating a vicious cycle that exacerbates the underlying factors driving HFpEF pathogenesis.

[0162] To evaluate if mEVs have similar systemic modulation effect on metabolic disorders and heart abnormalities in HFpEF mice, HFD+L-NAME C57BL / 6J mice were grouped and orally treated with mEVs, EV-dcplctcd supernatant, or saline, respectively, for 15 weeks. Normal chow diet-fed C57BL / 6J mice were used as control (Fig. 2A). mEVs were administered at an oral dose of 1.2 mg / kg every other day for a duration of 15 weeks.

[0163] The biodistribution of the orally administered mEVs was first examined. After administration, mEVs were detected in the colon but not in distal organs such as liver, heart, spleen, kidney (Fig. 2B). Notably, the HFpEF mice exhibited enhanced colonic accumulation of mEVs compared to the healthy controls, suggesting a disease- specific gut microenvironment that potentiated mEV s retention.

[0164] Compared with untreated mice and supernatant-treated mice (negative control), mEV treatment significantly reduced weight increase in the HFpEF mice (Fig. 2C, D). mEV treatment also reduced systolic and diastolic blood pressure (SBP and DBP) to normal levels (Fig. 2E, F). Moreover, glucose intolerance and insulin intolerance in HFpEF were significantly reversed upon mEV treatment (Fig. 2G-I). Apart from systemic indicators, leg muscle and exercise tolerance characterisation further proved the effect of mEVs on hindering progression of systemic disorder. Specifically, while there were no major differences in histopathology (Fig. 2J) and expression of molecular markers of skeletal muscle (Fig. 2K) in the four groups, mice from HFpEF group and supernatant group displayed an obvious reduction in grip strength and running distance compared to the other cohorts (Fig. 2L, M). These results indicated that the reduction in exercise tolerance in the HFpEF model is due to excess weight instead of any deficit in skeletal muscle function, and mEVs can reverse this alteration, possibly by modulating body weight. These results showed that orally -administrated mEVs can modulate systemic indicators of metabolic disorder in HFpEF progression.

[0165] Apart from the systemic effect of mEVs, the effect of mEVs on heart abnormality in HFpEF progression was also monitored. Cardiac hypertrophy was prominent in the HFpEF mice as demonstrated from morphometric analysis and histological analysis of tissue sections, which was alleviated by oral mEVs treatment, whereas mEVs-depleted supernatant had little effect (Fig. 3A, B). The heart weight to tibia length ratio also confirmed the effect of mEVs in alleviating hypertrophy (Fig. 3C). Enlarged cardiomyocyte cross-sectional areas were detected in the HFpEF mice based on WGA staining, which was also alleviated by mEVs treatment (Fig. 3B, D). The results further showed that mEVs hindered cardiomyocytc hypertrophy. Longitudinal echocardiographic evaluation revealed that there was preservation of the left ventricular ejection fraction (LVEF) in all four groups (Fig. 3E, F). However, HFpEF mice exhibited signs of diastolic dysfunction and elevated left ventricular filling pressure as measured using non-invasive Doppler imaging (Fig. 3G, H). Notably, these abnormal indices showed improvements after mEV treatment (Fig. 3G, H), as early as 5 weeks after first administration. In addition, lung weight was significantly increased in the HFpEF mice which, combined with lung histology, may serve as an indicator of pulmonary edema in heart failure. After mEV treatment, lung weight returned to normal and lung congestion was reduced (Fig. 31).

[0166] Accumulation of misfolded proteins occurs in clinical HFpEF and inactivation of the IREla-XBPl signaling pathway has been reported in the HFpEF mice model. The decrease in XBP1 results from increased activity of inducible nitric oxide synthase (iNOS), whose transcript level also increased in HFpEF mouse hearts and dropped upon mEV treatment (Fig. 3J, K). Moreover, cardiac inflammation was effectively alleviated (Fig. 3L), and there was reduced endotoxin level in the mEV treated HFpEF hearts (Fig. 3M), suggesting restoration of the gut barrier integrity.

[0167] Example 4: mEVs restore gut barrier integrity and gut microbiota in HFpEF mice

[0168] The 12 most abundant miRNAs in mEVs were screened for their effects on barrier integrity in Caco-2 191 cells (Fig. 4L). Transepithelial electrical resistance analysis showed that 3% DSS-exposure resulted in disruption of barrier integrity in Caco-2 epithelial monolayers (Fig. 4M). Compared with the scramble-miRNA (negative control), pretreatment with mEV- enriched miR-148a, miR-21-5p, miR-200b, let-7a-5p, 194 miR-200c, miR-101, or miR-30d preserved barrier integrity due to upregulation of intestinal tight junction proteins such as ZO-1 (Fig. 4N). These findings highlight the potential of mEVs in maintaining epithelial barrier integrity.

[0169] Disruption of gut barrier integrity has been shown to occur in HFpEF mice (see Example 1 ). It was previously shown that mEVs had gut barrier protection effect in both colitis and NASH mouse models, thus the effect of mEV s on gut barrier protection in HFpEF mice was also evaluated. Mice were characterised every five weeks and mice colons were collected at the end point. HE staining of the colon sections demonstrated that mEV treatment but not supernatant treatment reduced colonic epithelium disruption in HFpEF model (Fig. 4A). Surprisingly, when measuring the length of the harvested colons, it was noticed that colonshortening occurred in HFpEF mice and oral-administration of mEVs can attenuate the trend (Fig. 4B). At molecular level, mEV treatment reduced colonic TNF-a levels and increased the expression of tight junction proteins such as ZO-1 (Fig. 4C, D). Function-wise, mEV treatment decreased HFpEF-induced gut hyperpermeability, as evidenced by reduced FFTC- dextran fluorescence and endotoxin in plasma (Fig. 4E). The faeces and plasma endotoxin levels of the four groups were measured, and there was a marked increase of endotoxin in both faeces and plasma of HFpEF and supernatant groups, but not in the mEV-treated group (Fig. 4F, G). These findings demonstrated that oral administration of mEVs can effectively alleviate gut barrier dysfunction in HFpEF by reducing inflammation and restoring epithelial tight junctions.

[0170] Gut microbiota dysbiosis was observed in HFpEF mice. To evaluate whether mEVs can restore gut microbiota balance in HFpEF, fecal samples were collected from mice at weeks 5 and 15 of HFpEF development, and a-diversity indices such as Peilou evenness, Simpson index, and Chaol index were analysed using 16S rRNA sequencing. By week 15, there was a marked decline in a-diversity in untreated HFpEF mice, reflecting progressive dysbiosis (Fig. 4H, I). Notably, mEV treatment partially preserved microbial diversity, suggesting its potential to mitigate gut dysbiosis in HFpEF (Fig. 4H, I).

[0171] Clear differences in the composition of the gut microbiota were observed among the four groups (Fig. 4.T). The heatmap suggested that, at the genus level, the abundance of the short chain fatty acid (SCFA) producers, Muribaculaceae and Blaulia, recovered upon oral administration of mEVs. The abundance of the genus Alistipes from the Bacteroidota phylum slightly increased in the HFpEF group and the supernatant-treated group, while the level dropped markedly in the mEVs-trcatcd group (Fig. 4K). Similarly, the level of the genus Akkermansia from the Verrucomicrobiota phylum showed a notable increase in the HFpEF and the supernatant groups, and a notable decrease upon mEV treatment. In contrast, the level of the genus Muribaculaceae decreased in the untreated group and the supernatant- treated group and recovered in the mEV-treated group, which agreed with the heatmap result. Overall, the results showed that oral administration of mEV s can reverse gut leakage and dysbiosis in HFpEF mice. Example 5: mEVs alleviate HFpEF by restoring the abundance of Lachnospiraceae and promoting microbial production of butyric acid, azelaic acid and g-caprolactone

[0172] To further explore the specific gut microbial changes associated with HFpEF model generation and mEV treatment, linear discriminant analysis (LDA) was conducted to identify differentially enriched bacteria. In mEV-treated mice, Lachnospiraceae was increased at both family and genus levels. Lachnospiraceae_bacterium at the species level and Lachnospirales at the order level were also increased (Fig. 5A). Consistent with LDA results, mEV treatment elevated the abundance of the genera Lachnoclostridium and Lachnospiraceae_NK4A136_group (Fig. 5B, C). The relative abundance and LEfSe analysis also suggested that mEV treatment can increase the OTU number of the group Lachnospiraccac_FCS020 and the relative abundance of the species Lachnospiraceae_bacterium (Fig. 5D-F). These results suggest that Lachnospiraceae may be a key gut microbe accounting for the therapeutic effects of mEVs in HFpEF.

[0173] To investigate the therapeutic effects of mEVs on the heart via gut microbiota in HFpEF, metabolomics was performed on the mouse plasma to analyse the alteration of metabolites. In the HFpEF mice, principal component analysis (PCA) revealed obvious changes in the overall plasma metabolite profile compared to the healthy mice, indicating a significant metabolic disturbance (Fig. 5G). However, this metabolic disturbance was less pronounced in the mEV-treated mice (Fig. 5G). Hierarchical clustering heatmaps and univariate analysis further confirmed the reversal of key metabolite changes such as azelaic acid, butyric acid, and g-caprolactone after mEVs treatment (Fig. 5H), suggesting the role of mEVs in restoring metabolic homeostasis.

[0174] The abundance of Lachnospiraceae bacterium is associated with the production of shortchain fatty acids (SCFAs). SCFAs have been reported to have positive effect on heart diseases and metabolic disorders. The metabolomics analysis showed a significant upregulation of circulating butyric acid, azelaic acid, and g-caprolactone in the mEV-treated compared to the untreated HFpEF mice (Fig. 5H). Correlation analysis indicated a close association between Lachnospiraceae abundance and these metabolites, especially butyric acid and azelaic acid, further linking gut microbiota shifts to metabolic reprogramming in HFpEF (Fig. 51). Taken together, these findings confirm the potential link between the beneficial effects of rnEVs on HFpEF progression and the modulation of Lachnospiraceae metabolites.

[0175] To directly validate the regulatory effects of mEVs-mediated gut microbiota metabolites on HFpEF progression, HFD+L-NAME-induced HFpEF mice were randomly divided into four treatment arms which received an i.p. injection of saline (200 pL, 25.7 NaCl mg / kg / day), butyric acid (200 pL. 3.1 mg / kg / day), azelaic acid (200 pL, 80 mg / kg / day), or g-caprolactone (200 pL, 70 mg / kg / day) (Fig. 7A). Although food intake remained stable, clear differences in body size were observed among the four groups at week 15 (Fig. 7B, L). Compared to the HFpEF group, the butyric acid- or azelaic acid-treated group showed significantly reduced body weight, highlighting the anti-obesity effects of these metabolites (Fig. 7C). These fatty acids also enhanced glucose tolerance and insulin sensitivity in the HFpEF mice, while g- caprolactone failed to exert similar effects (Fig. 7D, M). Fatty acid administration showed an effect in reducing blood pressure and improving lung edema in the HFpEF mice (Fig. 7N-P). Tn addition, butyric acid and azelaic acid attenuated cardiac hypertrophy and fibrosis in HFpEF mice (Fig. 7E, F). Importantly, both fatty acids improved the cardiac diastolic function, as reflected by lower the E / A and E / E’values (Fig. 7G, H), similar to the effect of mEV treatment. This improvement was also associated with enhanced exercise capacity (Fig. 7Q, R). Importantly, butyrate or azelaic acid treatment increased cGMP and PKG protein levels, indicating that cGMP-PKG pathway was activated, but g-caprolactone had no such effect (Fig. 71, J). The NO levels in HFpEF hearts also demonstrated a similar trend (Fig. 7K). These results reaffirm the therapeutic effect of mEVs on HFpEF, potentially by modulating Lachnospiraceae metabolite-mediated NO-cGMP-PKG pathway activation.

[0176] Example 6: mEVs modulate metabolic activity in HFpEF

[0177] Metabolic alteration is a key risk factor to HFpEF, therefore, the effects of mEV treatment on the overall metabolic activity in HFpEF mice was evaluated. HFpEF mice exhibited apparent obesity compared to the healthy controls (Fig. 6A, B), and mEV treatment effectively prevented weight gain during HFpEF progression (Fig. 6C). Correspondingly, mEV treatment also attenuated glucose intolerance and insulin resistance in the treated HFpEF mice (Fig. 6D). Although 5-week mEV treatment had minimal therapeutic effects on hypertension, another hallmark of HFpEF, extended administration led to significant reduction in both systolic and diastolic blood pressure at 15 weeks (Fig. 6E, F). Additionally, exercise tolerance of HFpEF mice were assessed with different treatments. While skeletal muscle histology and molecular features showed no differences across groups, mEV treatment improved running distance and grip strength in HFpEF mice (Fig. 6G, H). These results suggest that mEV treatment can improve exercise capacity in HFpEF primarily due to reduced obesity and enhanced cardiac function, rather than direct impact on muscle function.

[0178] To exclude the possibility that changes in body weight and metabolic profile were confounded by food intake, metabolic cage experiments were conducted to monitor energy balance and metabolic activity (Fig. 61). Key parameters such as food consumption, physical activity, energy expenditure, and oxygen consumption (VOz), were accurately recorded starting from week 3, which coincided with the onset of distinct weight gain in HFpEF mice (Fig. 6C). No significant differences in food intake were observed among mEV-treated, supernatant-treated, and untreated HFpEF mice (Fig. 6J), indicating that weight changes were not due to differences in caloric intake. However, mEV-treated mice exhibited greater physical activity, higher energy expenditure (-25% increase), and elevated VO2 levels compared to untreated HFpEF mice (Fig. 6K-M).

[0179] Despite increased energy expenditure, mEV treatment resulted in a higher lean mass-to-body mass ratio compared to the untreated or the supernatant-treated mice (Fig. 6N), suggesting a shift in body composition and improved metabolic flux. These results indicate that mEV enhance metabolic activity, contributing to reduced body weight and improved metabolic profiles in HFpEF mice independently of food intake. By promoting energy expenditure and systemic metabolic health, oral administration of mEV s demonstrates therapeutic potential for targeting metabolic dysfunctions in HFpEF.

[0180] Example 7: Discussion

[0181] Although HFpEF is often associated with systemic inflammation and metabolic dysregulation, it has rarely been linked to gut dysfunction. In this study, it was found that there is profound gut barrier disruption and gut dysbiosis in both HFpEF patients and preclinical models, revealing a possible gut-heart axis mechanism contributing to HFpEF metabolic stress. In the intestines of HFpEF patients and HFpEF mouse models, a reduction in beneficial microbes such as Lachnospiraceae was seen. Lachnospiraceae produces butyrate acid and azelaic acid, and these fatty acids have been reported to alleviate metabolic stress and exert cardiac protective effects, but their effects on HFpEF have not been reported. The current findings demonstrated the pathogenic roles of gut barrier dysfunction and gut dysbiosis in HFpEF, as well as the feasibility of treating HFpEF through modulating gut microenvironment.

[0182] Oral administration of mEVs provided a promising therapeutic approach for HFpEF patients through restoring gut barrier integrity and improving microbiota homeostasis. Specifically, mEV-enriched miRNAs, such as miR-200b, miR-200c, miR-101, and miR-30d, enhance the expression of tight junction proteins and repair gut leakage, thereby reducing translocation of endotoxins into the bloodstream and the heart. This gut barrier-stabilizing effect could effectively reduce inflammation in HFpEF hearts, providing an effective avenue for cardiovascular protection.

[0183] Tt is also demonstrated that oral administration of mEV in HFpEF restores gut microbiota homeostasis, which is essential for human health. Gut dysbiosis manifests as decreased bacterial diversity and dominance of pathogenic species. mEV treatment effectively increased the relative abundance of beneficial Lachnospiraceae and their metabolites, including butyrate acid and azelaic acid. These metabolites can serve as energy sources for gut epithelial cells and stabilize tight junctions, which may help improve gut barrier function to alleviate HFpEF.

[0184] Due to the modulatory effects on the gut barrier and gut microbiota, mEV treatment consequently improved the diastolic dysfunction and cardiac hypertrophy in HFpEF. mEV treatment was also effective in modulating the metabolic activities in HFpEF mice, including reducing obesity, improving glucose tolerance and insulin sensitivity, as well as enhancing exercise capacity.

[0185] Overall, the results showed that orally-administrated mEVs 1) were able to overcome the harsh environment of the GI tract to reach the colon; 2) were able to reverse gut inflammation, barrier dysfunction, and microbial dysbiosis; and 3) prevented HFpEF development in mouse models. mEVs thus have the potential to be used as naturally-sourced oral drugs for gut protection and as adjunctive therapy for HFpEF and other metabolic diseases. It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat or prevent the cardiovascular disease or condition.

2. The method of claim 1, wherein the cardiovascular disease or condition is a metabolic cardiovascular disease or condition.

3. The method of claim 2, wherein the metabolic cardiovascular disease or condition is metabolic heart failure.

4. The method of claim 3, wherein the metabolic heart failure is heart failure with preserved ejection fraction (HFpEF).

5. The method of claim 2, wherein the metabolic cardiovascular disease or condition is obesity or diabetes.

6. The method of claim 2, wherein the metabolic cardiovascular disease or condition is atherosclerosis.

7. The method of claim 1, wherein the cardiovascular disease or condition is myocardial infarction (Ml).

8. The method of any one of claims 1 to 7, wherein the subject is found to be at risk of, or likely to be suffering from the cardiovascular disease or condition.

9. The method of any one of claims 1 to 8, wherein the subject is found to have gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability as compared to a reference.

10. The method of claim 9, wherein the subject is found to have increased intestinal permeability as compared to a reference.

11. The method of any one of claims 1 to 10, wherein the mEVs are derived from bovine, human, goat or camel milk.

12. The method of any one of claims 1 to 11, wherein the mEV composition is a food, nutraceutical or pharmaceutical composition.

13. The method of any one of claims 1 to 12, wherein the mEVs are administered at a concentration of about 0.01 mg / kg to about 6 mg / kg.

14. The method of any one of claims 1 to 13, wherein the mEVs are administered at a frequency of once every two days.

15. The method of any one of claims 1 to 14, wherein the mEVs are administered for a duration of at least about 5 weeks.

16. The method of claim 15, wherein the mEVs are administered for a duration of about 5 weeks to about 15 weeks.

17. A method of treating or preventing metabolic heart failure in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat or prevent the heart failure.

18. The method of claim 17, wherein the metabolic heart failure is heart failure with preserved ejection fraction (HFpEF).

19. The method of claim 17 or 18, wherein the subject is found to have gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability as compared to a reference.

20. The method of claim 19, wherein the subject is found to have increased intestinal permeability as compared to a reference.

21. A method of treating or preventing a cardiovascular disease or condition in a subject, the method comprising:(a) detecting a subject who is at risk of or likely to be suffering from the cardiovascular disease or condition; and(b) orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject found to be at risk of or likely to be suffering from a cardiovascular disease or condition to treat or prevent the cardiovascular disease or condition in the subject.

22. The method of claim 21, wherein the cardiovascular disease or condition is a metabolic cardiovascular disease or condition.

23. The method of claim 22, wherein the metabolic cardiovascular disease or condition is metabolic heart failure.

24. The method of claim 22, wherein the metabolic cardiovascular disease or condition is obesity or diabetes.

25. The method of claim 22, wherein the metabolic cardiovascular disease or condition is atherosclerosis.

26. The method of claim 21 , wherein the cardiovascular disease or condition is myocardial infarction (MI).

27. The method of any one of claims 21 to 26, wherein the subject is found to have gastrointestinal inflammation, dysbiosis and / or increased intestinal permeability as compared to a reference.

28. The method of claim 27, wherein the subject is found to have increased intestinal permeability as compared to a reference.

29. A method of treating or preventing a cardiovascular disease or condition in a subject who is at risk of or likely to be suffering from the cardiovascular disease or condition, the method comprising:(a) detecting increased intestinal permeability in the subject, wherein an increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and(b) orally administering an effective amount of an mEV composition to a subject found suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject.

30. The method of claim 29, wherein detecting increased intestinal permeability in step (a) comprises orally administering to the subject a label and detecting uptake of the label by the subject, wherein an increased level of uptake of the label as compared to a reference indicates that the subject is likely to be suffering from a gastrointestinal dysfunction.

31. The method of claim 29, wherein detecting increased intestinal permeability in step (a) comprises detecting a level of endotoxin in a sample obtained from the subject, wherein an increased level of endotoxin as compared to a reference indicates that the subject is likely to be suffering from a gastrointestinal dysfunction.

32. The method of any one of claims 29 to 31, wherein the cardiovascular disease or condition is a metabolic cardiovascular disease or condition.

33. The method of claim 32, wherein the metabolic cardiovascular disease or condition is metabolic heart failure.

34. The method of claim 32, wherein the metabolic cardiovascular disease or condition is obesity or diabetes.

35. The method of claim 32, wherein the metabolic cardiovascular disease or condition is atherosclerosis.

36. The method of any one of claims 29 to 31, wherein the cardiovascular disease or condition is myocardial infarction (MI).

37. A method of selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition for treatment with mEVs, the method comprising:(a) detecting increased intestinal permeability in the subject, wherein an increased intestinal permeability as compared to a reference indicates that the subject is suitable for treatment with mEVs to prevent or treat the cardiovascular disease or condition in the subject; and(b) selecting the subject found likely to have increased intestinal permeability for treatment with mEVs.

38. A method of promoting gut health in a subject, the method comprising: (a) selecting a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition; and (b) orally providing a milk-EV composition to the subject so as to promote gut health in the subject.

39. A method of treating gastrointestinal dysfunction in a subject who is at risk of or likely to be suffering from a cardiovascular disease or condition, the method comprising administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to treat the gastrointestinal dysfunction in the subject.

40. A method of treating a cardiometabolic disease or disorder in a subject, the method comprising orally administering an effective amount of a milk-derived extracellular vesicle (mEV) composition to the subject to prevent or treat the cardiometabolic disease or disorder.

41. The method of claim 40, wherein the cardiometabolic disease or condition is obesity, diabetes or a neurological disease or condition.

42. The method of claim 41, wherein the neurological disease or condition is dementia or stroke.

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