Non-coding RNAs (ncRNAs) in Cardiovascular Aging

National Institute On Aging

Investigators

Linked publications, trials & patents

Abstract

Some projects that require expertise in microRNAs and, specifically, in miR-200 family and miR-34a, have been initiated. It has been shown that miR-200 family members (co-transcribed miR-200c, -141; co-transcribed miR-200b, -200a, -429) and miR-34a increase in response to oxidative stress suggesting that these miRNAs may play a functional role in conditions associated with enhanced production of reactive oxygen species (ROS) such as aging and diabetes. Some miR-200 family members exhibit an age-associated increase in human skin fibroblasts and liver, and in non-human primates skeletal muscle. Further, miR-200c increases within the myocardium of diabetic rodents, in femoral arteries of diabetic rodents and in skin cells of patients with type 2 diabetes and diabetic foot ulcers. Dr Capogrossi directs the conception and implementation of the following ongoing projects: 1. Establish the role of miR-200 family members in myocardial function and its potential link to systolic and diastolic dysfunction in aging and diabetes. 2. Establish the role of miR-200 family members and miR-34a in vascular dysfunction in aging and diabetes; 3. Establish the expression level of circulating miR-200 family members in human aging, diabetes, obesity; 4. Establish the role of miR-200 family members in skeletal muscle function. REPORTS 2021 1. miR-200 family members in cardiac function. Oxidative stress is defined as a dysregulation between the production of ROS and endogenous antioxidant defense mechanisms. Reactive oxygen species (ROS) upregulate miR-200c via a p53-dependent mechanism and p53 is implicated in the induction of apoptosis and senescence. miR-200c upregulation induces growth arrest, senescence, and apoptosis through the inhibition of ZEB1 and SIRT1/ eNOS/FOXO1 regulatory loop by directly targeting all of them. Diminished SIRT1 expression associates with cell senescence. Moreover, miR-200c increases ROS production by 2 mechanisms: (1) it decreases ROS scavengers by targeting peroxiredoxin 2 (PRDX2), and FOXO1, a transcription factor required for catalase (CAT) and manganese superoxide dismutase (MnSOD) expression; (2) it induces ROS production via p66Shc phosphorylation in Serine 36. Therefore, a positive feedback loop occurs between enhanced oxidative stress increasing miR-200c expression and miR-200c further increasing oxidative stress. Conditions such as aging, diabetes, obesity, and hypertension are associated with increased oxidative stress in the heart and lead to the impairment of cellular differentiation and proliferation, alterations in excitation-contraction coupling, cardiac fibrosis and heart failure. The potential role of miR-200 family in these clinically relevant conditions remains to be elucidated. The expression of miR-200 family members was evaluated in diabetic mice myocardial cells. Diabetes was induced with the intraperitoneal injection of 50 mg/kg streptozotocin (STZ). Co-transcribed miR-141 and miR-200c significantly increased in cardiomyocytes isolated form diabetic mice; in contrast, co-transcribed miR-200a, -200b and -429 expression did not increase. Moreover, miR-200c and miR-141 are upregulated in vitro, in human umbilical vein endothelial cells grown in high glucose, and in vivo, in mouse endothelial cells, bone marrow-derived endothelial progenitor cells, human skin fibroblasts, and human skin. To establish the role of miR-200 family members in myocardial function and its potential link to systolic and diastolic dysfunction in aging and diabetes, we used a mouse model where the locus with miR-141 and -200c is deleted. Cardiac fractional shortening evaluated by echocardiography increased in KO mice when compared to WT animals. However, it was not possible to induce diabetes in this model since the ablation of miR-141/-200c protects against STZ-induced beta cell death and the consequent diabetes induction. To better evaluate the role of miR-200c, we created in collaboration with Mouse Cancer Genetics Program at the NCI/CCR a mouse model where only miR-200c is deleted. The CRISPR/Cas9 genome editing technology was used to introduce loxP sites flanking the miR-200c genomic sequence in mouse. Specifically, two guide RNAs were designed to target the region upstream and downstream of the miR-200c genomic locus. A single strand DNA oligonucleotide (ssDNA oligo; 907 nt) containing the loxP sites flanking the specific miR-200c sequence and homology regions was used as donor template for the homologous recombination process. The cardiac function was evaluated in vivo but no differences were detected in the two genotypes. The animals have been then treated with STZ, but the KO animals did not develop diabetes. Some preliminary results have been presented as a poster presentation at the Cardiac Regulatory mechanisms Gordon Research Seminar and Conference in June 2022. We plan to create two new tamoxifen - inducible mouse models, where miR-200c is KO only in cardiomyocytes or in fibroblasts, to better assess its role in cardiac function and cardiac fibrosis. Specifically, we will evaluate whether the deletion of miR-200c can prevent cardiac dysfunction and cardiac fibrosis, respectively, in the diabetic mouse. In fact, it has been shown that miR-200c targets ACE2, responsible for the conversion of Angiotensin II into Angiotensin 1,7; the decreased ACE2 expression is expected to enhance proinflammatory AT1 receptor activity and decrease anti-inflammatory MAS receptor activity. Further, we are planning to evaluate another approach to induce diabetes in miR-200c KO mice. The combination of high fat diet and a single high dose of STZ (as reported by Bengt-Frederik Belgardt et al, Nat Med 2015) is expected to induce diabetes in miR-200c KO mice; this approach, if successful in our hands, will allow us to investigate the role of miR-200c in cardiac dysfunction and cardiac fibrosis associated with diabetes, obesity and insulin resistance. 2. miR-34a in vascular dysfunction in cardiovascular aging. The Renin-angiotensin-aldosterone system (RAAS) plays a key role in cardiovascular aging and disease. Renin secretion is the first step in the activation of the RAAS pathway. Renin cleaves angiotensinogen to form Ang I, which is then transformed into Ang II by ACE and chymase enzyme. Specific receptors, AT1R and AT2R, can then bind Ang II. AT1R increases blood pressure, promotes cardiac remodeling and atherosclerosis, whereas AT2R activation has opposite effects. ACE2 cleaves Ang I and Ang II to form Ang(19) to Ang(17), respectively. Ang(17) induces vasodilation, anti-inflammatory, antifibrotic, and anti-remodeling effects, through MasR. Ang II effects are mediated by combining with Ang II receptor type 1 (AT1R). AT1R is Angiotensin II receptor-associated protein (AGTRAP) is a transmembrane protein that functions as an inhibitor of AT1R signaling. Presently is still unknown if AGTRAP expression in central arteries is modulated by aging. We demonstrated that miR-34a increases in monkey common carotid artery (CCA), in rat aortic wall, and CCA in an age dependent manner. BY the luciferase activity assay, we demonstrated that AGTRAP is directly targeted by miR-34a. AGTRAP mRNA and protein expression are decreased in rat vascular smooth muscle cells (VSMCs) isolated from old rats and human VSMCs overexpressing miR-34a. Moreover, miR-34a increased in carotid plaques compared to arterioles of healthy patients and negatively correlated with SIRT1 and AGTRAP. In collaboration with the Centro Cardiologico Monzino in Milan (Italy) we are investigating age-dependent AGRAP expression in miR-34a KO mice. A manuscript is currently in preparation.