Cellular and Molecular Mechanisms of Endothelial Dysfunction in Diabetic Cardiomyopathy

Type 2 diabetes (T2D) is associated with cardiac microvascular dysfunction, which is thought to contribute to the development of diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms responsible for HFpEF remain unclear, and no effective diagnostics or main-stay treatments are available. To gain insight into biomarkers and disease mechanisms we measured the microRNA content of circulating extracellular vesicles (EVs) during pathogenesis in two animal models of T2D-associated HFpEF (i.e., obese mouse [Lepr-/-] and lean rat [Goto Kakizaki]). We found that miR-30d and miR-30e were increased prior to echocardiographic evidence of diastolic dysfunction in T2D mice, and they were also elevated in T2D rats with established diastolic dysfunction. These microRNAs may serve as biomarkers of cardiac microvascular dysfunction as they are upregulated in the endothelial cells (ECs) of the left ventricle of the heart, but not other organs. Furthermore, the miR-30 family is secreted in response to activation of senescence pathways, a characteristic feature of diabetic ECs. Assessment of pathways regulated by miR-30d/e revealed a large number of target genes involved in fatty acid biosynthesis and metabolism. Importantly, over-expression of miR-30e in ECs increased fatty acid oxidation and the production of reactive oxygen species, while inhibiting the miR-30 family decreased fatty acid oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to dramatically down-regulate the expression of eNOS, an important regulator of microvascular and cardiomyocyte function. Thus, miR-30d/e may represent early biomarkers of diastolic dysfunction that reflect altered fatty acid metabolism and microvascular dysfunction in the heart. Furthermore, the pathways regulated by miR-30 may represent therapeutic targets for diabetes-associated HFpEF.Ph.D

Extracellular Vesicles as Protagonists of Diabetic Cardiovascular Pathology

Extracellular vesicles (EVs) represent an emerging mechanism of cell–cell communication in the cardiovascular system. Recent data suggest that EVs are produced and taken up by multiple cardiovascular cell types, influencing target cells through signaling or transfer of cargo (including proteins, lipids, messenger RNA, and non-coding RNA). The concentration and contents of circulating EVs are altered in several diseases and represent explicit signatures of cellular activation, making them of particular interest as circulating biomarkers. EVs also actively contribute to the progression of various cardiovascular diseases, including diabetes-related vascular disease. Understanding the relationships between circulating EVs, diabetes, and cardiovascular disease is of importance as diabetic patients are at elevated risk for developing several debilitating cardiovascular pathologies, including diabetic cardiomyopathy (DCM), a disease that remains an enigma at the molecular level. Enhancing and exploiting our understanding of EV biology could facilitate the development of effective non-invasive diagnostics, prognostics, and therapeutics. This review will focus on EV biology in diabetic cardiovascular diseases, including atherosclerosis and DCM. We will review EV biogenesis and functional properties, as well as provide insight into their emerging role in cell–cell communication. Finally, we will address the utility of EVs as clinical biomarkers and outline their impact as a biomedical tool in the development of therapeutics

Diabetes affects extracellular vesicle content and function

Introduction: The role of circulating extracellular vesicles in cardiovascular diseases remains incompletely understood.We previously demonstrated that extracellular vesicles circulating in plasma of healthy mice can suppress monocyte activation through transfer of anti-inflammatory microRNAs. Here we set out to determine the effect of diabetes on the function of plasma extracellular vesicles since diabetes is known to negatively affect vascular function, playing a contributory role in cardiovascular diseases. Methods: Circulating plasma extracellular vesicles were isolated from mouse and rat models of type 2 diabetes. Extracellular vesicles were characterised with nanoparticle tracking analysis. Furthermore, qPCR and RNA-sequencing approaches were used to characterise vesicle content and function. Results: We found that vesicle abundance and size were increased in mouse and rat models of type 2 diabetes. MicroRNAs in plasma extracellular vesicles were dysregulated during the progression of diabetes in thesemodels. Finally, we demonstrate that vesicles isolated from diabetic plasma can activate inflammatory pathways in endothelial cells. Current studies are seeking to determine the contribution of microRNA transfer to endothelial dysfunction. Conclusions: These studies suggest that the microRNA content and function of extracellular vesicles are dysregulated during diabetes. Advancements in this area could facilitate the development of more effective non-invasive diagnostics, prognostics, and therapeutics. Funding: Supported by funding from the Canadian Vascular Network and the Canadian Institutes of Health Research

Circulating Extracellular Vesicles From Mouse and Rat Models of Diabetes Reveal Specific Microrna Signatures as Biomarkers of Diabetic Cardiomyopathy

Background: Type-2 diabetes (T2D) is associated with both reduced and preserved ejection fraction heart failure. Obese db/db mice and Goto Kakizaki (GK) rats represents animals models of T2D that develop cardiac dysfunction similar to human diabetic cardiomyopathy, in which dominant early findings are of diastolic (and not systolic) dysfunction. Circulating extracellular vesicles (EV) contain microRNAs (miR) that can be transferred to recipient cells to modulate their function. We explored whether analysis of EV content from animals models of T2D would inform on the pathophysiology, diagnosis and therapeutic targets of cardiac dysfunction. Hypothesis: EV from animal models of T2D will have altered miR content that contributes to the pathophysiology of diabetic cardiomyopathy. Methods & Results: miR qPCR arrays on circulating EV isolated from plasma of db/db mice reveal several miR (-7, -15, -25, -30e, -148a, -150, -195) modulated during disease progression. These changes in miR content occur prior to echocardiographic evidence of diastolic dysfunction, including global longitudinal strain and strain rate. Among circulating EV miR from the GK rat model, miR-30 was also upregulated (1.42 fold, p=0.03) compared to Wistar rat. In GK rat left ventricle, and in H9C2 rat cardiac myoblast cultured in 25 mM high glucose media, mass spectrometry revealed proteins that were overexpressed in the diabetic heart including oxidative phosphorylation, glycolysis, fatty acid degredation and the citrate cycle. Using a bioinformatics approach, we next identified metabolic pathways affected by miR-30. Based on these findings, in vivo therapy with antagomiR and mimics of miR-30 are underway to test causality and reversibility of the observed cardiomyopathy. Conclusion: EV from animal models of T2D have altered miR content, including miR-30. We also identify alterations in the expression of a network of metabolism genes in the heart, which are implicated in diabetic cardiomyopathy. If causality is supported by experiments that enhance or block miR-30 expression in these models of disease, we will have identified a novel biomarker and therapeutic target for diabetic cardiomyopathy

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

peer reviewedThe transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis

MiR-30 promotes fatty acid beta-oxidation and endothelial cell dysfunction and is a circulating biomarker of coronary microvascular dysfunction in pre-clinical models of diabetes.

[en] BACKGROUND: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. METHODS: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). RESULTS: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid β-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid β-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. CONCLUSIONS: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart

Search for intermediate mass black hole binaries in the first observing run of Advanced LIGO

International audienceDuring their first observational run, the two Advanced LIGO detectors attained an unprecedented sensitivity, resulting in the first direct detections of gravitational-wave signals produced by stellar-mass binary black hole systems. This paper reports on an all-sky search for gravitational waves (GWs) from merging intermediate mass black hole binaries (IMBHBs). The combined results from two independent search techniques were used in this study: the first employs a matched-filter algorithm that uses a bank of filters covering the GW signal parameter space, while the second is a generic search for GW transients (bursts). No GWs from IMBHBs were detected; therefore, we constrain the rate of several classes of IMBHB mergers. The most stringent limit is obtained for black holes of individual mass 100  M⊙, with spins aligned with the binary orbital angular momentum. For such systems, the merger rate is constrained to be less than 0.93  Gpc−3 yr−1 in comoving units at the 90% confidence level, an improvement of nearly 2 orders of magnitude over previous upper limits

First low-frequency Einstein@Home all-sky search for continuous gravitational waves in Advanced LIGO data

International audienceWe report results of a deep all-sky search for periodic gravitational waves from isolated neutron stars in data from the first Advanced LIGO observing run. This search investigates the low frequency range of Advanced LIGO data, between 20 and 100 Hz, much of which was not explored in initial LIGO. The search was made possible by the computing power provided by the volunteers of the Einstein@Home project. We find no significant signal candidate and set the most stringent upper limits to date on the amplitude of gravitational wave signals from the target population, corresponding to a sensitivity depth of 48.7  [1/Hz]. At the frequency of best strain sensitivity, near 100 Hz, we set 90% confidence upper limits of 1.8×10-25. At the low end of our frequency range, 20 Hz, we achieve upper limits of 3.9×10-24. At 55 Hz we can exclude sources with ellipticities greater than 10-5 within 100 pc of Earth with fiducial value of the principal moment of inertia of 1038  kg m2