LncRNA Chaer Prevents Cardiomyocyte Apoptosis From Acute Myocardial Infarction Through AMPK Activation.

Long non-coding RNA (lncRNA) is widely reported to be involved in cardiac (patho)physiology. Acute myocardial infarction, in which cardiomyocyte apoptosis plays an important role, is a life-threatening disease. Here, we report the lncRNA Chaer that is anti-apoptotic in cardiomyocytes during Acute myocardial infarction. Importantly, lncRNA Chaer is significantly downregulated in both oxygen-glucose deprivation (oxygen-glucose deprivation)-treated cardiomyocytes in vitro and AMI heart. In vitro, overexpression of lncRNA Chaer with adeno virus reduces cardiomyocyte apoptosis induced by OGD-treated while silencing of lncRNA Chaer increases cardiomyocyte apoptosis instead. In vivo, forced expression of lncRNA Chaer with AAV9 attenuates cardiac apoptosis, reduces infarction area and improves mice heart function in AMI. Interestingly, overexpression of lncRNA Chaer promotes the phosphorylation of AMPK, and AMPK inhibitor Compound C reverses the overexpression of lncRNA Chaer effect of reducing cardiomyocyte apoptosis under OGD-treatment. In summary, we identify the novel ability of lncRNA Chaer in regulating cardiomyocyte apoptosis by promoting phosphorylation of AMPK in AMI.

Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia.

OBJECTIVE: Regenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb. METHODS AND RESULTS: In this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism. CONCLUSION: In conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

MicroRNA expression profile and functional analysis reveal their roles in contact inhibition and its disruption switch of rat vascular smooth muscle cells.

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.

Impact of pneumonia and lung cancer on mortality of women with hypertension.

Essential hypertension is one of the most severe women's health problems. Modern life brings more chances of pulmonary diseases to human. The purpose of the study is to investigate weather pneumonia and lung cancer are associated with the mortality of women with hypertension in different age. A cross-sectional retrospective study was conducted in women with hypertension, who were admitted into our hospital in 2004-2013. 14219 women were enrolled and 68.8 ± 12.2 year old (y). Isolated hypertension was 14.7%. The age of death was 78.1 ± 9.8 y. The mortality was 4.4% for average and 0.2%, 1.1%, 2.4%, 4.8%, 10.4% and 15.8% in group age ≦49, 50-59, 60-69, 70-79, 80-89 and ≧90 y separately. This mortality increased with age was positively significantly correlated with the increased incidences of pneumonia (P < 0.05, r = 0.77). Pneumonia was a significant risk associated with the mortality in age 55-89 y (OR = 6.4-22.5; 95% CI = 3.06-51.12). While, lung cancer was the significant risk in 70-79 y. These observations indicate that pneumonia and lung cancer are significant risk factors associated with the mortality of certain age women with hypertension, and bring an alert that pneumonia and lung cancer should be prevented and treated intensively in modern life in order to reduce the mortality.

Downregulation of HDAC1 is involved in the cardiomyocyte differentiation from mesenchymal stem cells in a myocardial microenvironment.

Under myocardial microenvironment, bone marrow-derived mesenchymal stem cells (MSCs) can transdifferentiate into cardiomyocytes (CMs). However, the role of histone deacetylase 1 (HDAC1) in this directed differentiation process remains unclear. The current study is to determine the acetylation regulatory mechanisms that may be involved in the directed CM differentiation from MSCs. MSCs isolated from male Sprague-Dawley (SD) rats were marked with Ad-EGFP and co-cultured with CMs. Flow cytometry was used to sort EGFP-positive (EGFP+) MSCs from the co-culture system. Then, the expression of cardiac troponin T (cTnT) in these MSCs was detected by immunofluorescence assay. In addition, HDAC1 levels at different co-culture times were measured by quantitative real-time polymerase chain reaction (QT-PCR) and Western blotting. At 4 days after co-culture with CMs, the MSCs began to expression detectable levels of cTnT. The expression of HDAC1 in CMs was much lower than that in MSCs. After co-culture with CMs, the expression of HDAC1 in MSCs was significantly decreased in a time dependent manner. In addition, our recent study has also identified that knockdown of the HDAC1 could promote the directed differentiation of MSCs into CMs. The results suggest that HDAC1 has a negative correlation with cardiac cell differentiation from MSCs under a myocardial microenvironment. HDAC1 might play an important role in the directed differentiation of MSCs into CMs in heart.

Knockdown of the HDAC1 promotes the directed differentiation of bone mesenchymal stem cells into cardiomyocytes.

Failure of the directed differentiation of the transplanted stem cells into cardiomyocytes is still a major challenge of cardiac regeneration therapy. Our recent study has demonstrated that the expression of histone deacetylase 1 (HDAC1) is decreased in bone mesenchymal stem cells (BMSCs) during their differentiation into cardiomyocytes. However, the potential roles of HDAC1 in cardiac cell differentiation of BMSCs, as well as the mechanisms involved are still unclear. In current study, the expression of HDAC1 in cultured rat BMSCs is knocked down by lentiviral vectors expressing HDAC1-RNAi. The directed differentiation of BMSCs into cardiomyocytes is evaluated by the expression levels of cardiomyocyte-related genes such as GATA-binding protein 4 (GATA-4), Nirenberg, Kim gene 2 homeobox 5 (Nkx2.5), cardiac troponin T (CTnT), myosin heavy chain (MHC), and connexin-43. Compared with that in control BMSCs, the expression of these cardiomyocyte-related genes is significantly increased in these HDAC1 deficient stem cells. The results suggest that HDAC1 is involved in the cardiomyocyte differentiation of BMSCs. Knockdown of the HDAC1 may promote the directed differentiation of BMSCs into cardiomyocytes.

[Effects of simvastatin on vasa vasorum and aortic endothelial function in rats].

OBJECTIVE: To investigate the effect of hyperlipidemia on vasa vasorum and vascular endothelial growth factor (VEGF) and study the role of vasa vasorum in arteriosclerosis. METHODS: Thirty SD rats were randomized into normal control, hyperlipidemic and simvastatin treatment groups (n=10). In simvastatin group, hyperlipidemia was induced by a 4-week administration of atherogenic diet followed by a 16-week treatment with simvastatin at the daily dose of 10 mg/kg, and the rats in hyperlipidemic rats received no treatment. The changes in the aorta and vasa vasorum were examined, and serum lipid concentration and VEGF and NO levels were measured. RESULTS: Compared with the control group, the hyperlipidemic rats showed significantly thickened intima and media aorta and increased vasa vasorum density with lowered NO level, but VEGF underwent no significant changes. Simvastatin treatment significantly reduced the thickness of the intima and media aorta and increased vasa vasorum density in comparison with those in hyperlipidemic group. Simvastatin treatment also significantly increased VEGF and NO levels and a positive correlation was noted between their levels. CONCLUSION: Hyperlipidemia can impair the vasa vasorum and aortic endothelial function. Simvastatin increases VEGF and NO and promotes neogenesis of the vasa vasorum for the benefit of the aortic function.

[Leptin promotes neointimal formation by stimulating vascular smooth muscle cell proliferation through leptin receptor].

OBJECTIVE: To evaluate the role of leptin in neointimal formation and related mechanisms. METHODS: Femoral arterial injury was induced in wild-type (Wt, n = 10), leptin-deficient (Lep(-)/-, n = 12), and leptin receptor-deficient (LepR(-)/-, n = 10) mice. Leptin treatment studies (tail vein injection of adenovirus expressing murine leptin on the RSV promoter, ad-leptin) were performed on Lep(-)/- (n = 5) and LepR(-)/- (n = 4) mice. Intimal (I) and medial (M) areas were measured and the ratio of I/M was calculated. Smooth muscle cells were detected by smooth muscle alpha-actin staining using an alpha-actin monoclonal antibody. Cellular proliferation was analyzed with BrdU Staining Kit and the number of BrdU-positive cells was counted manually. Plasma leptin level was measured by ELISA. RESULTS: The I/M ratio of Lep(-)/- and LepR(-)/- mice was significantly lower than that in Wt separately (Lep(-)/- vs. Wt = 0.80 +/- 0.14 vs. 1.50 +/- 0.22, P < 0.01; LepR(-)/- vs. Wt = 0.55 +/- 0.20 vs. 1.50 +/- 0.22, P < 0.05). Plasma leptin level was significantly increased in Lep(-)/- and LepR(-)/- mice post leptin treatment. I/M was significantly increased in Lep(-)/- mice receiving ad-leptin compared with untreated Lep(-)/- mice (P < 0.05), while I/M was similar between LepR(-)/- mice with and without ad-leptin treatment (P > 0.05). The changes on number of positive alpha-actin and BrdU stained smooth muscle cells were consistent with the neointimal formation findings in various groups. CONCLUSIONS: Mice lacking leptin or the leptin receptor were protected from neointimal formation following vascular injury. Leptin treatment increased neointimal formation in Lep(-)/- but not in LepR(-)/- mice, suggesting leptin receptor activation and vascular smooth muscle cell proliferation played a pivotal role on neointimal formation post-injury in this model, giving an evidence that high plasma leptin level is a risk factor for neointimal formation.