Anastasia Familtseva

Cardiosome mediated regulation of MMP9 in diabetic heart: role of mir29b and mir455 in exercise

‘Cardiosomes’ (exosomes from cardiomyocytes) have recently emerged as nanovesicles (30–100 nm) released in the cardiosphere by myocytes and cardiac progenitor cells, though their role in diabetes remains elusive. Diabetic cardiovascular complications are unequivocally benefitted from exercise; however, the molecular mechanisms need exploration. This novel study is based on our observation that exercise brings down the levels of activated (Matrix Metalloprotease 9) in db/db mice in a model of type 2 diabetes. We hypothesize that exosomes that are released during exercise contain microRNAs (mir455, mir29b, mir323‐5p and mir466) that bind to the 3′ region of MMP9 and downregulate its expression, hence mitigating the deleterious downstream effects of MMP9, which causes extracellular matrix remodeling. First, we confirmed the presence of exosomes in the heart tissue and serum by electron microscopy and flow cytometry, respectively, in the four treatment groups: (i) db/control, (ii) db/control+exercise, (iii) db/db and (iv) db/db+exercise. Use of exosomal markers CD81, Flottilin 1, and acetylcholinesterase activity in the isolated exosomes confirmed enhanced exosomal release in the exercise group. The microRNAs isolated from the exosomes contained mir455, mir29b, mir323‐5p and mir466 as quantified by qRTPCR, however, mir29b and mir455 showed highest upregulation. We performed 2D zymography which revealed significantly lowered activity of MMP9 in the db/db exercise group as compared to non‐exercise group. The immunohistochemical analysis further confirmed the downregulated expression of MMP9 after exercise. Since MMP9 is involved in matrix degradation and leads to fibrosis and myocyte uncoupling, the present study provides a strong evidence how exercise can mitigate these conditions in diabetic patients.

Role of microRNA29b in blood–brain barrier dysfunction during hyperhomocysteinemia: an epigenetic mechanism

Although blood–brain barrier (BBB) integrity is maintained by the cross-talk of endothelial cells, junction proteins, and neurogliovascular network, the epigenetic mechanisms behind BBB permeability are largely unknown. We are reporting for the first time miR29b-mediated regulation of BBB, which is a novel mechanism underlying BBB integrity. We hypothesize that miR29b regulates BBB dysfunction by regulating DNMT3b, which consequently regulates the levels of metalloproteinases, that can eat up the membrane and junction proteins leading to leaky vasculature. In addition, 5′-azacytidine (5′-aza) was used to test its efficacy on BBB permeability. Blood–brain barrier disruption model was created by using homocysteine, and in the models miR29b was identified to be most affected, by using microRNA RT 2 -qPCR array. MiR29b mimics and inhibitors also confirmed that miR29b regulates the levels DNMT3b and MMP9. In hyperhomocysteinemic cystathionine-β-synthase deficient (CBS+/−) mice with high brain vessel permeability, miR29b levels were also high as compared with wild-type (WT) mice. Interestingly, 5′-aza improved BBB permeability by decreasing the expression of miR29b. In conclusion, our data suggested miR29b-mediated regulation of BBB dysfunction through DNMT3b and MMP9. It also potentiates the use of microRNAs as candidates for future epigenetic therapies in the improvement of BBB integrity.

Mitochondrial mitophagy in mesenteric artery remodeling in hyperhomocysteinemia.

Although high levels of homocysteine also termed as hyperhomocysteinemia (HHcy) has been associated with inflammatory bowel disease and mesenteric artery occlusion, the mitochondrial mechanisms behind endothelial dysfunction that lead to mesenteric artery remodeling are largely unknown. We hypothesize that in HHcy there is increased mitochondrial fission due to altered Mfn‐2/Drp‐1 ratio, which leads to endothelial dysfunction and collagen deposition in the mesenteric artery inducing vascular remodeling. To test this hypothesis, we used four groups of mice: (i) WT (C57BL/6J); (ii) mice with HHcy (CBS+/−); (iii) oxidative stress resistant mice (C3H) and (iv) mice with HHcy and oxidative stress resistance (CBS+/−/C3H). For mitochondrial dynamics, we studied the expression of Mfn‐2 which is a mitochondrial fusion protein and Drp‐1 which is a mitochondrial fission protein by western blots, real‐time PCR and immunohistochemistry. We also examined oxidative stress markers, endothelial cell, and gap junction proteins that play an important role in endothelial dysfunction. Our data showed increase in oxidative stress, mitochondrial fission (Drp‐1), and collagen deposition in CBS+/− compared to WT and C3H mice. We also observed significant down regulation of Mfn‐2 (mitochondrial fusion marker), CD31, eNOS and connexin 40 (gap junction protein) in CBS+/− mice as compared to WT and C3H mice. In conclusion, our data suggested that HHcy increased mitochondrial fission (i.e., decreased Mfn‐2/Drp‐1 ratio, causing mitophagy) that leads to endothelial cell damage and collagen deposition in the mesenteric artery. This is a novel report on the role of mitochondrial dynamics alteration defining mesenteric artery remodeling.

High Methionine Diet Poses Cardiac Threat: A Molecular Insight

High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress; (2) by inflammatory manifestations; and (3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice: (1) WT; (2) WT + methionine; (3) CBS+/−; (4) CBS+/−+methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS+/− and CBS+/−+methionine. Higher oxidative stress was indicated by high levels of SOD‐1 in methionine fed mouse hearts whereas IL‐1β, IL‐6, TNFα, and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion, our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling, and decreased cardiac function. J. Cell. Physiol. 231: 1554–1561, 2016.

Toll-like receptor 4 mutation suppresses hyperhomocysteinemia-induced hypertension.

Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll-like receptor 4 (TLR-4) is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. TLR-4 activation has been known to promote inflammation that has been associated with the pathogenesis of hypertension. In this study we hypothesize that HHcy induces hypertension by TLR-4 activation, which promotes inflammatory cytokine (IL-1β, IL-6, and TNF-α) upregulation and initiation of mitochondria-dependent apoptosis, leading to cell death and chronic vascular inflammation. To test this hypothesis, we used C57BL/6J (WT) mice, cystathionine β-synthase (CBS)-deficient (CBS+/-) mice with genetic mild HHcy, C3H/HeJ (C3H) mice with TLR-4 mutation, and mice with combined genetic HHcy and TLR-4 mutation (CBS+/-/C3H). Ultrasonography of the superior mesenteric artery (SMA) detected an increase in wall-to-lumen ratio, resistive index (RI), and pulsatility index (PI). Tail cuff blood pressure (BP) measurement revealed elevated BP in CBS+/- mice. RI, PI, and wall-to-lumen ratio of the SMA in CBS+/-/C3H mice were similar to the control group, and BP was significantly alleviated. TLR-4, IL-1β, IL-6, and TNF-α expression were upregulated in the SMA of CBS+/- mice and reduced in the SMA of CBS+/-/C3H mice. Molecules involved in the mitochondria-mediated cell death pathway (BAX, caspase-9, and caspase-3) were upregulated in CBS+/- mice and attenuated in CBS+/-/C3H mice. We conclude that HHcy promotes TLR-4-driven chronic vascular inflammation and mitochondria-mediated cell death, inducing hypertension. TLR-4 mutation attenuates vascular inflammation and cell death, which suppress hypertension.

Cardiac tissue inhibitor of matrix metalloprotease 4 dictates cardiomyocyte contractility and differentiation of embryonic stem cells into cardiomyocytes: Road to therapy☆

BACKGROUND TIMP4 (Tissue Inhibitors of Matrix Metalloprotease 4), goes down in failing hearts and mice lacking TIMP4 show poor regeneration capacity after myocardial infarction (MI). This study is based on our previous observation that administration of cardiac inhibitor of metalloproteinase (~TIMP4) attenuates oxidative stress and remodeling in failing hearts. Therefore, we hypothesize that TIMP4 helps in cardiac regeneration by augmenting contractility and inducing the differentiation of cardiac progenitor cells into cardiomyocytes. METHODS To validate this hypothesis, we transfected mouse cardiomyocytes with TIMP4 and TIMP4-siRNA and performed contractility studies in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. We evaluated the calcium channel gene serca2a (sarcoplasmic reticulum calcium ATPase2a) and mir122a which tightly regulates serca2a to explain the changes in contractility. We treated mouse embryonic stem cells with cardiac extract and cardiac extract minus TIMP4 (using TIMP4 monoclonal antibody) to examine the effect of TIMP4 on differentiation of cardiac progenitor cells. RESULTS Contractility was augmented in the TIMP4 transfected cardiomyocytes as compared to siRNA-TIMP4 transfected cardiomyocytes. There was elevated expression of serca2a in the TIMP4 transformed myocytes and down regulation of mir122a. The cells treated with cardiac extract containing TIMP4 showed cardiac phenotype in terms of Ckit+, GATA4+ and Nkx2.5 expression. CONCLUSION This is a novel report suggesting that TIMP4 augments contractility and induces differentiation of progenitor cells into cardiac phenotype. In view of the failure of MMP9 inhibitors for cardiac therapy, TIMP4 provides an alternative approach, being an indigenous molecule and a natural inhibitor of MMP9.

Toll-like receptor 4 mediates vascular remodeling in hyperhomocysteinemia

Although hyperhomocysteinemia (HHcy) is known to promote downstream pro-inflammatory cytokine elevation, the precise mechanism is still unknown. One of the possible receptors that could have significant attention in the field of hypertension is toll-like receptor 4 (TLR-4). TLR-4 is a cellular membrane protein that is ubiquitously expressed in all cell types of the vasculature. Its mutation can attenuate the effects of HHcy-mediated vascular inflammation and mitochondria- dependent cell death that suppresses hypertension. In this review, we observed that HHcy induces vascular remodeling through immunological adaptation, promoting inflammatory cytokine up-regulation (IL-1β, IL-6, TNF-α) and initiation of mitochondrial dysfunction leading to cell death and chronic vascular inflammation. The literature suggests that HHcy promotes TLR-4-driven chronic vascular inflammation and mitochondria-mediated cell death inducing peripheral vascular remodeling. In the previous studies, we have characterized the role of TLR-4 mutation in attenuating vascular remodeling in hyperhomocysteinemia. This review includes, but is not limited to, the physiological synergistic aspects of the downstream elevation of cytokines found within the vascular inflammatory cascade. These events subsequently induce mitochondrial dysfunction defined by excessive mitochondrial fission and mitochondrial apoptosis contributing to vascular remodeling followed by hypertension.