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Principal Investigator: Edward Lakatta
Organization: NATIONAL INSTITUTE ON AGING
Fiscal Year: 2022
Award: $307,915
Funding agency: National Institute on Aging
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.
Terms: <66-kDa Shc protein><ACE2><Ablation><Active Oxygen><Adult-Onset Diabetes Mellitus><Age><Aging><AngII><Angiotensin II><Angiotensin II Receptor><Angiotensin-Forming Enzyme><Angiotensinogen><Angiotensinogenase><Angiotensins><Animal Model><Animal Models and Related Studies><Animals><Anti-Inflammatories><Anti-Inflammatory Agents><Anti-inflammatory><Antiinflammatories><Antiinflammatory Agents><Antioncogene Protein p53><Antioxidants><Apoptosis><Apoptosis Pathway><Assay><Atheroscleroses><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Basal Transcription Factor><Basal transcription factor genes><Beta Cell><Binding><Bioassay><Biologic Assays><Biological Assay><Blood Vessels><Bone Marrow><Bone Marrow Reticuloendothelial System><CCR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cardiac><Cardiac Muscle Cells><Cardiac Myocytes><Cardiac artery><Cardiocyte><Cardiology><Cardiovascular><Cardiovascular Body System><Cardiovascular Organ System><Cardiovascular system><Carotid Artery Plaque><Carotid Artery Plaques><Cas nuclease technology><Cell Aging><Cell Body><Cell Communication and Signaling><Cell Death><Cell Senescence><Cell Signaling><Cells><Cellular Aging><Cellular Senescence><Cellular Tumor Antigen P53><Central Artery><Chymase><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Collaborations><Common Rat Strains><Common carotid artery><Conceptions><Coronary artery><Coupling><D-Glucose><Defense Mechanisms><Dextrose><Diabetes Mellitus><Diabetic Foot Ulcer><Diabetic mouse><Disease><Disorder><Doctor of Medicine><Dose><Dysfunction><Echocardiogram><Echocardiography><Endothelial Cells><Enzyme Gene><Enzymes><Exhibits><FKHR><FOXO1><FOXO1A><FOXO1A gene><Family><Family member><Feedback><Fibroblasts><Forkhead Box O1A><Forkhead in Rhabdomyosarcoma><Functional RNA><Functional disorder><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Genomics><Genotype><Glucose><Growth><Guide RNA><Heart><Heart Muscle Cells><Heart Vascular><Heart artery><Heart failure><Heart myocyte><High Fat Diet><Human><Hypertensinogen><Hypertension><IP injection><IPO-B><Impairment><In Vitro><Indophenol Oxidase B><Induction of Apoptosis><Insulin Cell><Insulin Resistance><Insulin Secreting Cell><Integral Membrane Protein><Intracellular Communication and Signaling><Intraperitoneal Injections><Intrinsic Membrane Protein><Italy><KO mice><Ketosis-Resistant Diabetes Mellitus><Knock-out Mice><Knockout Mice><L-Serine><Lead><Leiomyocyte><Link><Liver><Luciferase Immunologic><Luciferases><M.D.><MMCP-1><MNSOD><Manganese Superoxide Dismutase><Manuscripts><Maturity-Onset Diabetes Mellitus><Mediating><Mice><Mice Mammals><Micro RNA><MicroRNAs><Mitochondrial Superoxide Dismutase><Mn Superoxide Dismutase><Mn-SOD><Modeling><Modern Man><Molecular Interaction><Monkeys><Murine><Mus><Muscle function><Myocardial><Myocardial depression><Myocardial dysfunction><Myocardium><NIDDM><Non-Coding><Non-Coding RNA><Non-Insulin Dependent Diabetes><Non-Insulin-Dependent Diabetes Mellitus><Non-translated RNA><Noncoding RNA><Noninsulin Dependent Diabetes><Noninsulin Dependent Diabetes Mellitus><Nontranslated RNA><Null Mouse><Obesity><Oligo><Oligonucleotides><Oncoprotein p53><Oxidative Stress><Oxygen Radicals><P53><Pathway interactions><Patients><Pb element><Phosphoprotein P53><Phosphoprotein pp53><Phosphorylation><Physiopathology><Play><Preparation><Pro-Oxidants><Proangiotensin><Process><Production><Programmed Cell Death><Protein Phosphorylation><Protein TP53><Proteins><Rat><Rats Mammals><Rattus><Reactive Oxygen Species><Receptor Protein><Renin><Renin-Angiotensin-Aldosterone System><Renin-Substrate><Replicative Senescence><Reporting><Research><Rodent><Rodentia><Rodents Mammals><Role><SIRT1><SIRT1 gene><SOD2><SOD2 gene><STZ><Sequence Homology><Serine><Signal Transduction><Signal Transduction Systems><Signaling><Single-Stranded DNA><Sirtuin 1><Site><Skeletal Muscle><Skin><Slow-Onset Diabetes Mellitus><Smooth Muscle Cells><Smooth Muscle Myocytes><Smooth Muscle Tissue Cell><Src homology 2 domain-containing, transforming protein 1, 66 kDa isoform><Stable Diabetes Mellitus><Streptozocin><Streptozotocin><Superoxide Dismutase 2><T2 DM><T2D><T2DM><TP53><TP53 gene><TRP53><Tamoxifen><Technology><Tissue Growth><Transcription Factor Proto-Oncogene><Transcription factor genes><Translational Research><Translational Science><Transmembrane Protein><Transmembrane Protein Gene><Transthoracic Echocardiography><Tumor Protein p53><Tumor Protein p53 Gene><Type 2 Diabetes Mellitus><Type 2 diabetes><Type II Diabetes Mellitus><Type II diabetes><Umbilical vein><Untranslated RNA><Up-Regulation><Upregulation><Vascular Diseases><Vascular Disorder><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><Vascular Smooth Muscle><Vasodilatation><Vasodilation><Vasorelaxation><Voluntary Muscle><Zanosar><adaptor protein p66shc><adiposity><adult onset diabetes><age dependent><age related><ages><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-oxidant><antiinflammatory><arteriole><atheromatosis><atherosclerotic disease><atherosclerotic vascular disease><biological signal transduction><blood pressure elevation><blood vessel disorder><cancer genetics><cardiac dysfunction><cardiac failure><cardiac fibrosis><cardiac function><cardiac muscle><cardiomyocyte><carotid plaque><catalase><chymase-1><chymotrypsin-like protease><circulatory system><clinical relevance><clinically relevant><co-morbid><co-morbidity><comorbidity><conference><convention><coronary fibrosis><corpulence><design><designing><diabetes><diabetes mouse model><diabetic><diabetic foot wound><elevated blood pressure><endothelial progenitor cell><endothelial stem cell><femoral artery><function of the heart><gRNA><gene locus><genetic locus><genome editing><genomic editing><genomic locus><heart dysfunction><heart function><heart muscle><heart sonography><heavy metal Pb><heavy metal lead><hepatic body system><hepatic organ system><high blood pressure><homologous recombination><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><in vivo><increase in blood pressure><increased blood pressure><inhibitor><insulin resistant><ketosis resistant diabetes><mRNA Expression><mast cell protease><mast cell protease 1><mast cell protease I><mast cell proteinase-1><maturity onset diabetes><miRNA><miRNAs><model of animal><model organism><mouse model><murine model><myocardial fibrosis><necrocytosis><non-human primate><noncoding><nonhuman primate><oligos><ontogeny><overexpress><overexpression><p53 Antigen><p53 Genes><p53 Tumor Suppressor><p66(ShcA) protein><p66shc><pathophysiology><pathway><peroxiredoxin 2><peroxiredoxin II><posters><prevent><preventing><programs><protein expression><protein function><protein p53><psychological defense mechanism><receptor><response><senescence><senescent><skeletal muscle protease><social role><summit><symposia><symposium><transcription factor><translation research><type 2 DM><type II DM><type two diabetes><vascular><vascular dysfunction><vasculopathy><β-cell><β-cells><βCell>