Epidrugs in the clinical management of atherosclerosis: Mechanisms, challenges and promises.

Atherosclerosis is a complex and multigenic pathology associated with significant epigenetic reprogramming. Traditional factors (age, sex, obesity, hyperglycaemia, dyslipidaemia, hypertension) and nontraditional factors (foetal indices, microbiome alteration, clonal hematopoiesis, air pollution, sleep disorders) induce endothelial dysfunction, resulting in reduced vascular tone and increased vascular permeability, inflammation and shear stress. These factors induce paracrine and autocrine interactions between several cell types, including vascular smooth muscle cells, endothelial cells, monocytes/macrophages, dendritic cells and T cells. Such cellular interactions lead to tissue-specific epigenetic reprogramming regulated by DNA methylation, histone modifications and microRNAs, which manifests in atherosclerosis. Our review outlines epigenetic signatures during atherosclerosis, which are viewed as potential clinical biomarkers that may be adopted as new therapeutic targets. Additionally, we emphasize epigenetic modifiers referred to as 'epidrugs' as potential therapeutic molecules to correct gene expression patterns and restore vascular homeostasis during atherosclerosis. Further, we suggest nanomedicine-based strategies involving the use of epidrugs, which may selectively target cells in the atherosclerotic microenvironment and reduce off-target effects.

DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition.

Extrinsic and intrinsic pathological stimuli in vascular disorders induce DNA methylation based epigenetic reprogramming in endothelial cells, which leads to perturbed gene expression and subsequently results in endothelial dysfunction (ED). ED is also characterized by release of exosomes with altered proteome leading to paracrine interactions in vasculature and subsequently contributing to manifestation, progression and severity of vascular complications. However, epigenetic regulation of exosome proteome is not known. Hence, our present study aimed to understand influence of DNA methylation on exosome proteome composition and their influence on endothelial cell (EC) function. DNMT isoforms (DNMT1, DNMT3A, and DNMT3B) were overexpressed using lentivirus in ECs. Exosomes were isolated and characterized from ECs overexpressing DNMT isoforms and C57BL/6 mice plasma treated with 5-aza-2'-deoxycytidine. 3D spheroid assay was performed to understand the influence of exosomes derived from cells overexpressing DNMTs on EC functions. Further, the exosomes were subjected to TMT labelled proteomics analysis followed by validation. 3D spheroid assay showed increase in the pro-angiogenic activity in response to exosomes derived from DNMT overexpressing cells which was impeded by inclusion of 5-aza-2'-deoxycytidine. Our results showed that exosome proteome and PTMs were significantly modulated and were associated with dysregulation of vascular homeostasis, metabolism, inflammation and endothelial cell functions. In vitro and in vivo validation showed elevated DNMT1 and TGF-ß1 exosome proteins due to DNMT1 and DNMT3A overexpression, but not DNMT3B which was mitigated by 5-aza-2'-deoxycytidine indicating epigenetic regulation. Further, exosomes induced ED as evidenced by reduced expression of phospho-eNOSser1177. Our study unveils epigenetically regulated exosome proteins, aiding management of vascular complications.

Neutrophil (dys)function due to altered immuno-metabolic axis in type 2 diabetes: implications in combating infections.

Metabolic and inflammatory pathways are highly interdependent, and both systems are dysregulated in Type 2 diabetes (T2D). T2D is associated with pre-activated inflammatory signaling networks, aberrant cytokine production and increased acute phase reactants which leads to a pro-inflammatory 'feed forward loop'. Nutrient 'excess' conditions in T2D with hyperglycemia, elevated lipids and branched-chain amino acids significantly alter the functions of immune cells including neutrophils. Neutrophils are metabolically active cells and utilizes energy from glycolysis, stored glycogen and ß-oxidation while depending on the pentose phosphate pathway for NADPH for performing effector functions such as chemotaxis, phagocytosis and forming extracellular traps. Metabolic changes in T2D result in constitutive activation and impeded acquisition of effector or regulatory activities of neutrophils and render T2D subjects for recurrent infections. Increased flux through the polyol and hexosamine pathways, elevated production of advanced glycation end products (AGEs), and activation of protein kinase C isoforms lead to (a) an enhancement in superoxide generation; (b) the stimulation of inflammatory pathways and subsequently to (c) abnormal host responses. Neutrophil dysfunction diminishes the effectiveness of wound healing, successful tissue regeneration and immune surveillance against offending pathogens. Hence, Metabolic reprogramming in neutrophils determines frequency, severity and duration of infections in T2D. The present review discusses the influence of the altered immuno-metabolic axis on neutrophil dysfunction along with challenges and therapeutic opportunities for clinical management of T2D-associated infections.

Distinct metabolic signatures in blood plasma of bisphenol A-exposed women with polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is a complicated endocrinopathy with an unclear etiology that afflicts fertility status in women. Although the underlying causes and pathophysiology of PCOS are not completely understood, it is suspected to be driven by environmental factors as well as genetic and epigenetic factors. Bisphenol A (BPA) is a weak estrogenic endocrine disruptor known to cause adverse reproductive outcomes in women. A growing relevance supports the notion that BPA may contribute to PCOS pathogenesis. Due to the indeterminate molecular mechanisms of BPA in PCOS endocrinopathy, we sought liquid chromatography with tandem mass spectrometry (LC-MS/MS), a metabolomics strategy that could generate a metabolic signature based on urinary BPA levels of PCOS and healthy individuals. Towards this, we examined urinary BPA levels in PCOS and healthy women by ELISA and performed univariate and chemometric analysis to distinguish metabolic patterns among high and low BPA in PCOS and healthy females, followed by pathway and biomarker analysis employing MetaboAnalyst 5.0. Our findings indicated aberrant levels of certain steroids, sphingolipids, and others, implying considerable disturbances in steroid hormone biosynthesis, linoleic, linolenic, sphingolipid metabolism, and various other pathways across target groups in comparison to healthy women with low BPA levels. Collectively, our findings provide insight into metabolic signatures of BPA-exposed PCOS women, which can potentially improve management strategies and precision medicine.

Ethnic disparities attributed to the manifestation in and response to type 2 diabetes: insights from metabolomics.

Type 2 diabetes (T2D) associated health disparities among different ethnicities have long been known. Ethnic variations also exist in T2D related comorbidities including insulin resistance, vascular complications and drug response. Genetic heterogeneity, dietary patterns, nutrient metabolism and gut microbiome composition attribute to ethnic disparities in both manifestation and progression of T2D. These factors differentially regulate the rate of metabolism and metabolic health. Metabolomics studies have indicated significant differences in carbohydrate, lipid and amino acid metabolism among ethnicities. Interestingly, genetic variations regulating lipid and amino acid metabolism might also contribute to inter-ethnic differences in T2D. Comprehensive and comparative metabolomics analysis between ethnicities might help to design personalized dietary regimen and newer therapeutic strategies. In the present review, we explore population based metabolomics data to identify inter-ethnic differences in metabolites and discuss how (a) genetic variations, (b) dietary patterns and (c) microbiome composition may attribute for such differences in T2D.

Mechanistic insights into glucose induced vascular epigenetic reprogramming in type 2 diabetes.

Endothelial cells lining the vessel wall regulate thrombosis, inflammation, angiogenesis and balance between vasoconstriction and vasodilatory functions. Subjects with Type 2 diabetes (T2D) accrue a multitude of vasculopathies causing high morbidity and mortality across the globe. High glucose and its modified products such as advanced glycation end products lead to a bidirectional activation of inflammatory and epigenetic machinery in endothelial cells resulting in a state of chronic inflammatory milieu and eventually into vascular complications. Clinical and experimental studies have shown that despite the therapeutic normalization of glucose levels, subjects with T2D overt to vascular complications through a process of metabolic memory which is associated with significant epigenetic reprogramming in endothelial cells. In normal physiological conditions, vascular endothelial cells display a quiescent state and only in response to either physiological or pathological response, endothelial cells undergo proliferation. During the pathogenesis of T2D, DNA methylation, histone marks and non-coding RNAs forming the epigenetic landscape are dysregulated and activate quiescent endothelial cells to switch on a diverse set of molecular activities and lead to endothelial dysfunction. In the present review, we provide a comprehensive overview of how hyperglycemia in T2D reprograms endothelial epigenome and lead to functional consequences in the pathogenesis of vascular complications. Further, we catalogue and discuss epi-drugs that may ameliorate endothelial functions during T2D.

Extrinsic and intrinsic factors influencing metabolic memory in type 2 diabetes.

Direct and indirect influence of pathological conditions in Type 2 Diabetes (T2D) on vasculature manifests in micro and/or macro vascular complications that act as a major source of morbidity and mortality. Although preventive therapies exist to control hyperglycemia, diabetic subjects are always at risk to accrue vascular complications. One of the hypotheses explained is 'glycemic' or 'metabolic' memory, a process of permanent epigenetic change in different cell types whereby diabetes associated vascular complications continue despite glycemic control by antidiabetic drugs. Epigenetic mechanisms including DNA methylation possess a strong influence on the association between environment and gene expression, thus indicating its importance in the pathogenesis of a complex disease such as T2D. The vascular system is more prone to environmental influences and present high flexibility in response to physiological and pathological challenges. DNA methylation based epigenetic changes during metabolic memory are influenced by sustained hyperglycemia, inflammatory mediators, gut microbiome composition, lifestyle modifications and gene-nutrient interactions. Hence, understanding underlying mechanisms in manifesting vascular complications regulated by DNA methylation is of high clinical importance. The review provides an insight into various extrinsic and intrinsic factors influencing the regulation of DNA methyltransferases contributing to the pathogenesis of vascular complications during T2D.

3D tumor angiogenesis models: recent advances and challenges.

The development of blood vessels, referred to as angiogenesis, is an intricate process regulated spatially and temporally through a delicate balance between the qualitative and quantitative expression of pro and anti-angiogenic molecules. As angiogenesis is a prerequisite for solid tumors to grow and metastasize, a variety of tumor angiogenesis models have been formulated to better understand the underlying mechanisms and associated clinical applications. Studies have demonstrated independent mechanisms inducing angiogenesis in tumors such as (a) HIF-1/VEGF mediated paracrine interactions between a cancer cell and endothelial cells, (b) recruitment of progenitor endothelial cells, and (c) vasculogenic mimicry. Moreover, single-cell sequencing technologies have indicated endothelial cell heterogeneity among organ systems including tumor tissues. However, existing angiogenesis models often rely upon normal endothelial cells which significantly differ from tumor endothelial cells exhibiting distinct (epi)genetic and metabolic signatures. Besides, the existence of intra-individual variations necessitates the development of improved tumor vascular model systems for personalized medicine. In the present review, we summarize recent advancements of 3D tumor vascular model systems which include (a) tissue engineering-based tumor models; (b) vascular organoid models, and (c) organ-on-chips and their importance in replicating the tumor angiogenesis along with the associated challenges to design improved models.

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis.

BACKGROUND: Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. METHODS: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. RESULTS: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially ß chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. CONCLUSION: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)