Angiogenesis, the process by which new blood vessels are formed, is a critical phenomenon that is activated during various stages of mammalian development. MicroRNAs (miRNAs), a class of short, ...single stranded, non-coding RNAs, are recognized as important regulators of angiogenesis, and the role of intracellular miRNAs in modulating angiogenesis signaling has been identified. The recent discovery of extracellular and circulating miRNAs has sparked new questions regarding their potential in modulating angiogenesis signaling not only within cells but also between cells. In this review, we discuss the characteristics of intracellular and extracellular miRNAs and decipher the potential functional roles for these molecules in regard to the angiogenic process. We summarize what is currently known about circulating miRNAs in distinct clinical populations and discuss evidence that implicates extracellular miRNAs as novel mediators of angiogenesis-associated intercellular signaling. Lastly, we offer a new perspective on the functional role of vesicle-encapsulated circulating miRNA in modulating angiogenesis signaling pathways.
Abstract only Introduction: Previously, we used plasma metabolomics profiling to identify metabolites underlying cardiovascular health (CVH) in participants of the Morehouse-Emory Cardiovascular ...(MECA) Center for Health Equity study, a cohort of Black adults living in the greater Atlanta metropolitan area. To determine whether a health intervention could alter expression of metabolites associated with CVH, metabolomics profiles were assessed pre- and post-intervention in participants with poor CVH. Hypothesis: Metabolites previously shown to be associated with CVH in MECA participants would be altered after the health intervention. Methods: Seventy-one Black adults without known cardiovascular disease and with poor CVH (AHA Life’s Simple 7 LS7 score <8), used a technology-enabled behavioral intervention platform for 6 months with or without a health coach. Metabolomics profiles were assessed pre- and post-intervention by high-resolution metabolomics profiling. A metabolome wide association study (MWAS) identified differentially expressed metabolites and enriched metabolic pathways. Metabolites were annotated by matching to an in-house library of confirmed metabolites. Changes in clinical metrics after intervention were also explored. Results: Mean age was 55 (standard deviation SD 9.0) years, 69% female. Total LS7 scores improved from 6.2 (SD 1.49) to 6.5 (SD 1.74) after the intervention. While total and subcomponent LS7 scores and clinical metrics (BMI, blood pressure, glucose and cholesterol levels) trended toward improvement, the changes were not statistically significant. MWAS identified 18 confirmed metabolites that were significantly changed after intervention, including glutamine and glutamate. Pathway analysis identified 29 enriched metabolic pathways, including those for glutamate, aspartate, asparagine, arginine and proline metabolism. Conclusions: A six-month lifestyle intervention significantly altered activity of select plasm metabolites while only modestly altering clinical metrics. These metabolites appear to be sensitive indicators of a healthier lifestyle, potentially supporting their use as markers of CVH and therapeutic targets.
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Intro:
Integrative analysis of extracellular microRNA (miRNA) and metabolomics is a novel approach to identifying molecular pathways underlying cardiovascular health (CVH). Levels of ...miRNAs are linked to cardiovascular disease (CVD), while cardiometabolic risk factors are known to alter activity of select metabolic pathways. We identified miRNAs and metabolites underlying CVH in a cohort of Black adults through an integrative analysis of extracellular miRNA levels, metabolite concentrations and clinical factors associated with CVH.
Methods:
The MECA cohort consisted of 371 Black adults (age 53+10, 38.5% male) without known CVD. CVH was determined by Life’s Simple 7 (LS7) score, calculated from blood pressure, fasting glucose, cholesterol, BMI, exercise, diet, and smoking. Metabolite concentrations in plasma was assessed by high-resolution metabolomics profiling, and expression of miRs-122-5p, -150-5p, and -30c-5p was assessed by RT-qPCR. Metabolome wide association study (MWAS) identified metabolites associated with LS7. Association of miRNA levels with CVH clinical domains was assessed using Spearman correlation. Integrative analysis of select miRNAs, metabolites, and CVH clinical domains was performed using the program xMWAS.
Results:
Expression of 30 metabolites was associated with improved CVH (FDR<0.2) where glutamine was higher (p=0.005), and glutamate was lower (p<0.001). Integrative analysis showed association between miR-122-5p, glutamine and fasting glucose. miR-122-5p expression independently correlated with fasting glucose (p=0.03) and glutamine/glutamate is known to be associated with glucose metabolism. In silico and in vitro analyses revealed glutamine synthetase as a potential target of miR-122-5p (Fig).
Conclusions:
We demonstrate novel associations between glutamine, miR-122-5p and fasting glucose in CVH, suggesting that select metabolites and miRNAs are markers of CVH and maybe potential therapeutic targets.
Abstract only
Introduction:
Previously, we used plasma metabolomics profiling to identify metabolites underlying cardiovascular health (CVH) in participants of the Morehouse-Emory Cardiovascular ...(MECA) Center for Health Equity Study. Herein, we explored whether a health intervention could alter plasma concentrations of metabolites associated with CVH in MECA participants with poor CVH.
Methods:
71 Black adults without known cardiovascular disease and with poor CVH, defined as AHA Life’s Simple 7 (LS7) score <8, were given a technology-enabled behavioral intervention platform (Health360x) for 6 months with or without access to a health coach. Metabolites were assessed pre- and post-intervention by untargeted high-resolution metabolomics profiling. Metabolome wide association study (MWAS) was conducted to identify differentially expressed metabolites and Mummichog was used to identify metabolic pathways that were differentially enriched after the intervention. Annotations were performed by matching to an in-house library of confirmed metabolites. Paired t-test and Wilcoxon signed-rank test were used to compare clinical metrics.
Results:
Mean age was 55
+
9.0 years, 69% female. Total LS7 scores improved from 6.2 (
+
1.49) to 6.5 (
+
1.74) with the intervention. While total and subcomponent LS7 scores and clinical metrics (BMI, blood pressure, glucose and cholesterol levels) trended toward improvement, the changes were not statistically significant. MWAS identified 18 metabolites that were significantly changed after intervention, including glutamine and glutamate. Pathway analysis demonstrated 30 metabolic pathways that were significantly changed with intervention, including glutamate, aspartate, asparagine, arginine and proline metabolism.
Conclusions:
Six-months of lifestyle intervention altered activity of 30 metabolic pathways without significant alterations in the clinical metrics. These metabolites and pathways appear to be indicators of a healthier lifestyle in the Black population, potentially supporting their use as markers of CVH and possible therapeutic targets.
Abstract only Introduction: Shear stress forces play an integral role in dictating the vascular wall response to changes in blood flow, induction of pro-inflammatory response and hence development of ...atherosclerosis. Previously, our group and others have identified an inverse relationship between microRNA-155 (miR-155) and AT1R expression and/or activity in atheroprone areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and in-vitro. Hypothesis: we hypothesized that acute induction of OSS mediates vascular inflammation and dysfunction, via activation of the AT1R-ETS1 pathway and dysregulation of miR-155. Methods: 12-week old C57B/6J mice were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of acute OSS, for 3 days. Downstream segments of acute OSS were compared to upstream unidirectional shear stress (USS) segments of the thoracic aorta using RT-PCR, western blot analysis and unpaired student t-test. Results: Acute OSS resulted in vascular inflammation evidenced by upregulation of the AT1R-ETS1 pathway and several of its downstream targets including phosphorylated ERK2, MCP-1 and VCAM-1 in OSS segments compared with USS. This was associated with loss of EC barrier function as evaluated by extravasation of Evans-blue dye assay along with increased expression of MMP3 and MMP9 in in OSS segments compared with USS. However, vascular miR-155 expression was also higher in OSS segments compared with USS (n=6-12, P<0.05). Nevertheless, tail vein injections of miR-155 overexpressing lentivirus particles after AAC resulted in further upregulation of miR-155 expression and inverse downregulation of the AT1R-ETS1 pro-inflammatory pathway and MMPs 3 and 9 expression in OSS segments compared with USS versus scramble control (n=5-6, P<0.05). Conclusions: Despite the early upregulation of the shear-sensitive miR-155, our data suggest that it could serve as a negative feedback regulator to acute OSS-induced upregulation of the pro-inflammatory AT1R-ETS1 pathway. Further studies are in progress to evaluate the effect of exogenous miR-155 on OSS-induced oxidative stress and vascular dysfunction, which can serve as the basis for developing novel miRNA-based therapeutic modalities.
Chronic heart failure is associated with pathophysiologic alterations in myocardial and vascular function. Accompanying these changes are increased oxidative stress and modulation of the nitric oxide ...pathway. The role of the nitric oxide pathway in heart failure and the effect of its interaction with reactive oxygen species are complex, with diverse pathophysiologic implications in both the heart and the peripheral vasculature. This review discusses current information regarding the nitric oxide˙ pathway in heart failure and its relationship with increased oxidative stress.
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MicroRNAs(miRNAs) are small, non‐coding RNAs that are post‐transcriptional regulators of gene expression. They are recognized for their roles both as modulators of disease progression ...and as biomarkers of disease activity in diseases such as neurological diseases, cancers, and cardiovascular diseases (CVD). CVD is a chronic inflammatory process that is associated with changes in intra‐ and extracellular miRNA abundance, and these circulating miRNA profiles are promising novel biomarkers for CVD severity. Many extracellular miRNAs are enriched in microvesicles (MV) and could potentially be therapeutic targets. We hypothesized that CVD‐associated variations in the circulating miRNA profile are related to: 1) changes in the abundance of miRNA isoforms (isomiRs),2) shifts in the isomiRs’ extracellular transport modality, and 3) alterations inMV counts based on CVD severity. IsomiR sequences are typically 1–3 nucleotides different from the consensus miRNA sequences but little is known about their biological function. Using RNA‐deep sequencing, the abundance/frequency of consensus miRNAs and isomiRs were assessed in 18 human blood samples: nine samples were from patients with only clinical risk factors (RFs) for CVD and nine samples were from patients diagnosed with significant CVD. From these analyses, 46 different miRNAs were identified as having both high frequency and abundance in these human blood samples; for 22 of these miRNAs, the expression of isomiRs were higher than that of the consensus miRNA. Among the 22 miRNAs that were isomiR‐dominant, 90% had a deletion orinsertion in the 3′ region with no changes in the seed sequence region. Using qRT‐PCRprimers that were custom‐designed for quantitation of each of the 22 dominant isomiRs, three isomiRs (isomiR‐10b, ‐93, and ‐181a) were determined to demonstrate statistical differences among patients (20 RFs; 24 CVD) with varying CVD severity and contained the consensus miRNA seed sequences. The abundance of isomiR‐10b was 10‐fold higher in the plasma of patients with significant CVD compared to those with RFs for CVD, whereas the abundance of isomiR‐181a was 10‐fold higher only in the MV fraction of plasma from patients with CVD compared to patients with only RFs. Lastly, isomiR‐93 was 1,000‐fold higher in the plasma of patients with CVD compared to RFs and a 100,000‐fold higher in the MVs of patients with CVD. Additionally, plasma MV counts were quantitated by flow cytometry. There was a significant increase in the number of MVs from patients with RFs for CVD compared to those with severe CVD. IsomiR abundance and frequency within certain extracellular transport modalities coupled with circulating MV counts could be used as biomarkers to predict CVD severity. Furthermore, these data give insight into why understanding the function of these isomiRs is essential; once the function is completely understood, these 3 isomiRs could be used as therapeutic targets for patients with CVD.
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Coronary artery disease (CAD) is a chronic inflammatory process that is associated with alterations in the intracellular expression and extracellular release of microRNAs (miRNAs) from ...different cell types, including endothelial cells (ECs), white blood cells, platelets and red blood cells. MiRNAs, in turn, are involved in modulating the expression of pro‐ and anti‐inflammatory genes that impact disease progression. It has been shown that variations in the circulating miRNA profile of CAD patients are associated with alterations in extracellular miRNA transport as well as a shift in the ratio between the consensus miRNA and isoforms of the miRNA (isomiRs). IsomiRs are relatively abundant variants of consensus miRNAs that differ by one to three nucleotides, but their biological function is not yet known. Previously, we demonstrated that TNF‐α alters the expression of select miRNAs in cultured human aortic endothelial cells (HAECs) and their release into microvesicles (MVs). MVs are major extracellular transport modalities for miRNAs. MV formation was altered by inhibitors that targeted pathways involved in MV release from ECs. Here, we hypothesized that TNF‐α and agents that target pathways involved in EC MV release would alter the abundance of select isomiRs in HAECs and in EC MVs. Using qRT‐PCR, the intracellular and MV abundance of seven isomiRs (isomiR‐10b, ‐ 30d,‐93, ‐143, ‐181a, ‐182, and ‐744) was quantitated in cultured HAECs treated with TNF‐α (100 ng/mL) plus or minus inhibitors of the caspase (Q‐VD‐OPH, 10 μM) and RhoA/ROCK pathways (Y‐27632, 10 μM). Overall, the intracellular abundance of isomiR‐10b, ‐143,‐181a, and ‐744 among all treatment groups were 10–10,000 fold higher in cells compared to their levels in EC MVs. In contrast, among all treatment groups the abundance of isomiR‐30d and ‐182 were 10–100 fold higher in EC MVs compared to their intracellular levels. TNF‐α treatment alone significantly increased the intracellular abundance of isomiR‐181a (P<0.05; 0.016) only. Treatment of HAECs with the caspase inhibitor alone increased intracellular levels (100–10,000‐fold)of isomiR‐10b, ‐93, and ‐181a compared to their levels in EC MVs while the ROCK inhibitor alone increased (100–10,000‐fold) intracellular levels of isomiR‐10band ‐143 versus their levels in EC MVs. In contrast, the ROCK inhibitor alone increased isomiR‐182 (100‐fold) in EC MVs compared to levels in HAECs. Co‐treatment of HAECs with TNF‐α and caspase inhibitor (P<0.05; 0.027) or TNF‐α and ROCK inhibitor (P<0.05; 0.017) significantly increased intracellular isomiR‐181a levels compared to isomiR‐181a levels in EC MVs. However, co‐treatment of HAECs with TNF‐α and ROCK inhibitor increased isomiR‐30dlevels in EC MVs compared to those in HAECs. Co‐treatment with TNF‐α and caspase inhibitor showed a similar trend for isomiR‐30d levels. These data provide insight into the relationship between the intracellular expression of isomiRs in ECs and their extracellular release in response to inflammation and modulation of pathways involved in MV release. These data also enhance our understanding of using MV‐encapsulated isomiR profiles as biomarkers for determining CAD severity.
Abstract only Introduction: Black Americans suffer from disparate levels of CVD and CVD risk factors, but metabolic pathways that underlie cardiometabolic risk in Blacks remain largely unknown. ...Objective: We defined metabolomic profiles and associated metabolic pathways underlying CVH in a Black community that is richly diverse in ethnic origin and socioeconomic status. Methods: The MECA study cohort consisted of 375 Black adults (age 53 + 10, 39% male) without known CVD. CVH was determined by the AHA Life’s Simple 7 (LS7) score, calculated from measured blood pressure, body mass index (BMI), fasting blood glucose, total cholesterol, and self-reported physical activity, diet, and smoking. Plasma metabolites were assessed by high-resolution metabolomics. Metabolome wide association study (MWAS) was used to identify metabolites associated with LS7 after adjusting for age and sex. Metabolic pathways significantly enriched in metabolites associated with LS7 were identified using Mummichog software. Concentrations of metabolites representative of these pathways were compared across clinical domains of LS7 score (ANOVA) and then a multivariable model was developed into a metabolomics risk score for prediction of CVH. Results: MWAS and pathway analysis identified 301 metabolites significantly associated with LS7 score (FDR<0.2) and enriched in pathways responsible for glutathione, alanine, tyrosine and glutamate metabolism. Plasma concentrations of six metabolites (glutamine, glutamate, serine, alanine, tyrosine and urate) were associated with blood pressure, fasting glucose, BMI, and physical activity (p<0.05). Assessed in conjunction, these metabolites were independent predictors of CVH - 1 SD increase in metabolomics risk score was associated with 0.93 increase in LS7 or 12.1% decrease in incident CVD. Conclusions: We identified a novel metabolomic signature of ideal CVH in Blacks without known CVD. This includes five non-essential amino acids and urate, which are known to be central elements of key metabolic processes (i.e. TCA cycle, urea cycle, nucleotide synthesis, one carbon metabolism). These data provide insight into metabolic pathways underlying cardiometabolic risk in Blacks and potential avenues for addressing health disparities.
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