Performance of Subcutaneous Implantable Cardioverter Defibrillator (S-ICD) in Chinese Population with Primary Prevention Indications: A Prospective Observational Cohort Study.

BACKGROUND International studies have shown that use of a subcutaneous implantable cardioverter defibrillator (S-ICD) could reduce lead-related complications while maintaining adequate defibrillation performance; however, data from the Chinese population or other Asian groups are limited. MATERIAL AND METHODS SCOPE is a prospective, multicenter, observational cohort study. Two hundred patients with primary prevention indication for sudden cardiac death (SCD), who are candidates for S-ICD, will be enrolled. From the same population, another 200 patients who are candidates for transvenous implantable cardioverter defibrillator (TV-ICD) will be enrolled after being matched for age, sex, SCD high-risk etiology (ischemic cardiomyopathy, and non-ischemic cardiomyopathy, ion channel disease, and other) and atrial fibrillation in a 1: 1 ratio with enrolled S-ICD patients. All the patients will be followed for 18 months under standard of care. RESULTS The primary endpoint is proportion of patients free from inappropriate shock (IAS) at 18 months in the S-ICD group. The lower 95% confidence bound of the proportion will be compared with a performance goal of 90.3%, which was derived from the previous meta-analysis. The comparisons between S-ICD and TV-ICD on IAS, appropriate shock, and complications will be used as secondary endpoints without formal assumptions. CONCLUSIONS This is the first prospective multicenter study focusing on the long-term performance of S-ICD in a Chinese population. By comparing with the data derived from international historical studies and a matched TV-ICD group, data from SCOPE will allow for the assessment of S-ICD in the Chinese population in a contemporary real-world implantation level and programming techniques, which will help us to further modify the device implantation and programming protocol in this specific population in the future.

Myocardial fibrosis induced by nonylphenol and its regulatory effect on the TGF-ß1/LIMK1 signaling pathway.

OBJECTIVE: We here explored whether perinatal nonylphenol (NP) exposure causes myocardial fibrosis (MF) during adulthood in offspring rats and determined the role of the TGF-ß1/LIMK1 signaling pathway in NP-induced fibrosis in cardiac fibroblasts (CFs). METHODS AND RESULTS: Histopathology revealed increased collagen deposition and altered fiber arrangement in the NP and isoproterenol hydrochloride (ISO) groups compared with the blank group. Systolic and diastolic functions were impaired. Western blotting and qRT-PCR demonstrated that the expression of central myofibrosis-related proteins (collagens Ι and ΙΙΙ, MMP2, MMP9, TGF-ß1, α-SMA, IL-1ß, and TGF-ß1) and genes (Collagen Ι, Collagen ΙΙΙ, TGF-ß1, and α-SMA mRNA) was upregulated in the NP and ISO groups compared with the blank group. The mRNA-seq analysis indicated differential expression of TGF-ß1 signaling pathway-associated genes and proteins. Fibrosis-related protein and gene expression increased in the CFs stimulated with the recombinant human TGF-ß1 and NP, which was consistent with the results of animal experiments. According to the immunofluorescence analysis and western blotting, NP exposure activated the TGF-ß1/LIMK1 signaling pathway whose action mechanism in NP-induced CFs was further validated using the LIMK1 inhibitor (BMS-5). The inhibitor modulated the TGF-ß1/LIMK1 signaling pathway and suppressed the NP-induced increase in fibrosis-related protein expression in the CFs. Thus, the aforementioned pathway is involved in NP-induced fibrosis. CONCLUSION: We here provide the first evidence that perinatal NP exposure causes myocardial fibrosis in growing male rat pups and reveal the molecular mechanism and functional role of the TGF-ß1/LIMK1 signaling pathway in this process.

ANNEXIN A2 FACILITATES NEOVASCULARIZATION TO PROTECT AGAINST MYOCARDIAL INFARCTION INJURY VIA INTERACTING WITH MACROPHAGE YAP AND ENDOTHELIAL INTEGRIN Β3.

ABSTRACT: Cardiac macrophages with different polarization phenotypes regulate ventricular remodeling and neovascularization after myocardial infarction (MI). Annexin A2 (ANXA2) promotes macrophage polarization to the repair phenotype and regulates neovascularization. However, whether ANXA2 plays any role in post-MI remodeling and its underlying mechanism remains obscure. In this study, we observed that expression levels of ANXA2 were dynamically altered in mouse hearts upon MI and peaked on the second day post-MI. Using adeno-associated virus vector-mediated overexpression or silencing of ANXA2 in the heart, we also found that elevation of ANXA2 in the infarcted myocardium significantly improved cardiac function, reduced cardiac fibrosis, and promoted peri-infarct angiogenesis, compared with controls. By contrast, reduction of cardiac ANXA2 exhibited opposite effects. Furthermore, using in vitro coculture system, we found that ANXA2-engineered macrophages promoted cardiac microvascular endothelial cell (CMEC) proliferation, migration, and neovascularization. Mechanistically, we identified that ANXA2 interacted with yes-associated protein (YAP) in macrophages and skewed them toward pro-angiogenic phenotype by inhibiting YAP activity. In addition, ANXA2 directly interacted with integrin ß3 in CMECs and enhanced their growth, migration, and tubule formation. Our results indicate that increased expression of ANXA2 could confer protection against MI-induced injury by promoting neovascularization in the infarcted area, partly through the inhibition of YAP in macrophages and activation of integrin ß3 in endothelial cells. Our study provides new therapeutic strategies for the treatment of MI injury.

The relationship between perfluoroalkyl substances and hypertension: A systematic review and meta-analysis.

Hypertension is an important risk factor for cardiovascular diseases (CVDs) and a leading cause of premature death. Epidemiological studies have found that perfluoroalkyl substances (PFASs) are associated with hypertension. However, the correlation between PFASs and hypertension has not been systematically reported. Based on evidence from population epidemiological surveys, we conducted a meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to assess the correlation between PFASs exposure and hypertension. In this study, three databases of PubMed, Web of science, Embase were searched and 13 literatures with 81,096 participants were included. Literature heterogeneity was evaluated by I2 statistic, and the random effect model (I2 > 50%) and fixed effect model (I2 < 50%) were used to combine the studies in meta-analysis. The results showed that PFNA (OR = 1.11, 95% CI: 1.04-1.19), PFOA (OR = 1.12, 95% CI: 1.02-1.23), PFOS (OR = 1.19, 95% CI: 1.06-1.34) and PFHxS (OR = 1.03, 95% CI: 1.00-1.06) were significantly associated with hypertension, while other types of PFASs (∑PFAS, PFDA, PFUnDA) had no statistical significance. In addition, PFNA (OR = 1.12, 95% CI: 1.03-1.22), PFOA (OR = 1.12, 95% CI: 1.01-1.25) and PFOS (OR = 1.12, 95% CI: 1.00-1.25) exposure were positively correlated with the risk of hypertension in men, but not in women. Our study reveals that PFASs are risk factors for hypertension, with notable gender differences observed in PFASs-exposed populations. Specifically, males exposed to PFNA, PFOA, and PFOS exhibit a higher risk of hypertension compared to females. However, further investigations are needed to delve into the precise mechanism through which PFASs contribute to the development of hypertension.

Effects and mechanism of perinatal nonylphenol exposure on cardiac function and myocardial mitochondria in neonatal rats.

BACKGROUND: Nonylphenol (NP) is a common environmental endocrine disruptor that is associated with the development of cardiovascular disease. However, the toxic effect of NP on mitochondria in the heart of offspring to exposed individuals remains exclusive. OBJECTIVE: To investigate whether perinatal NP exposure causes mitochondrial damage in the hearts of offspring of exposed individuals and determine its mechanism of action through both animal and cell experiments. METHODS AND RESULTS: For the in vivo experiment, pregnant rats were randomly divided into four groups: the control group (corn oil, C), low dose group (2.5 mg/kg/day, L-NP group), medium dose group (50 mg/kg/day, M-NP group), and high dose group (100 mg/kg/day, H-NP group), with 12 rats in each group. The NP concentration in the hearts of offspring at PND21 and PND90 increased with the increase of the NP dose. Perinatal NP exposure induced a gradual increase in systolic blood pressure in offspring at PND90. In the H-NP group, there was a high degree of inflammatory cell infiltration, myofibril breaks, inconspicuous or absent nuclei, and pink collagen deposition. At PND90, the membrane integrity of mitochondria in the H-NP group was disrupted, the cristae disorder was aggravated, and there was internal lysis with vacuolation. Compared to the control group, the mitochondrial membrane potential of offspring at PND21 and PND90 was decreased in each of the NP exposure groups. NP exposure decreased the activity of mitochondrial respiratory enzyme complex I (CI) and increased the activity of mitochondrial respiratory enzyme complex IV (CIV) in the offspring. At PND21 and PND90, the mRNA and protein expression levels of cardiac mitochondrial PGC-1α, NRF-1, and TFAM decreased with increasing NP dose in a dose-dependent manner. In the in vitro experiment, H9C2 cells were divided into the following four groups: the blank group, RSV group (15 µg/ml), RSV + NP group (15 µg/ml RSV + 120 mmol/L NP), and NP group (120 mmol/L). With increasing NP concentration, the cell survival rate gradually decreased. Compared to the control, the membrane potential was significantly decreased in the NP group; the protein expression levels of SIRT1, PGC-1α, NRF-1, and TFAM in the NP group were significantly lower. CONCLUSION: Perinatal NP exposure caused mitochondrial damage and dysfunction in the offspring of exposed individuals in a dose-dependent manner. This toxic effect may be related to NP-induced mitochondrial pathology in the offspring and the inhibition of both gene and protein expression involved in the PGC-1α/NRF-1/TFAM mitochondrial biogenesis signaling pathway following NP exposure.

Curative effect of zinc-selenium tea on rat's cardiotoxicity induced by long-term exposure to nonylphenol.

This study aimed to explore whether zinc-selenium tea has an curative effect on the cardiotoxicity induced by nonylphenol (NP), and to compare the effect of zinc-selenium tea and green tea. After drinking of zinc-selenium tea or green tea, compared with the control group, the left ventricular anterior wall became thinner, and the left ventricular end-diastolic diameter increased, the anterior wall of the left ventricle became thin at the end of diastole in the NP group. The serum myocardial enzymes aspartate aminotransferase, creatine kinase, creatine kinase isoenzyme, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase in the NP group were significantly increased, and the serum myocardial enzymes were significantly decreased after the intervention of zinc-selenium tea. Proteins and mRNA expressions of Collagen I and Collagen III in the tea groups were lower than those in the NP group. In the green tea and zinc-selenium tea intervention groups, the disorder and degree of myocardial fiber were alleviated to varying degrees. The disturbance, breakage, and inflammatory cell infiltration of myocardial fibers in zinc-selenium tea and green tea groups were less than that of NP group. After tea intervention, collagen I and collagen III in the myocardium decreased. The intervention effect of zinc-selenium tea was better than that of green tea. Zinc-selenium tea and green tea could interfere with the cardiotoxicity indued by NP, which would alleviate the myocardial fibrosis by reducing expressions of collagen I and collagen III. Moreover, the curative effect of zinc-selenium tea was better than that of green tea.

Methyltransferase like 3-mediated N6-methylatidin methylation inhibits vascular smooth muscle cells phenotype switching via promoting phosphatidylinositol 3-kinase mRNA decay.

N6-methylatidine (m6A) is involved in post-transcriptional metabolism and a variety of pathological processes. However, little is known about the role of m6A in vascular proliferative diseases, particularly in vascular smooth muscle cells (VSMCs) phenotype switching-induced neointimal hyperplasia. In the current study, we discovered that methyltransferase like 3 (METTL3) is a critical candidate for catalyzing a global increase in m6A in response to carotid artery injury and various VSMCs phenotype switching. The inhibited neointimal hyperplasia was obtained after in vivo gene transfer to knock-down Mettl3. In vitro overexpression of Mettl3 resulted in increased VSMC proliferation, migration, and reduced contractile gene expression with a global elevation of m6A modification. In contrast, Mettl3 knockdown reversed this facilitated phenotypic switch in VSMCs, as demonstrated by downregulated m6A, decreased proliferation, migration, and increased expression of contractile genes. Mechanistically, Mettl3 knock-down was found to promote higher phosphatidylinositol 3-kinase (Pi3k) mRNA decay thus inactivating the PI3K/AKT signal to inhibit VSMCs phenotype switching. Overall, our findings highlight the importance of METTL3-mediated m6A in VSMCs phenotype switching and offer a novel perspective on targeting METTL3 as a therapeutic option for VSMCs phenotype switching modulated pathogenesis, including atherosclerosis and restenosis.

Role of Mydgf in the regulation of hypoxia/reoxygenation-induced apoptosis in cardiac microvascular endothelial cells.

We aimed to explore the effects of myeloid-derived growth factor (Mydgf) on the regulation of hypoxia/reoxygenation (HR)-induced apoptosis of cardiac microvascular endothelial cells (CMECs). CMECs were exposed to hypoxia for 24 h and reoxygenation for 6 h to establish an HR cell model. Subsequently, an adenovirus was used to overexpress Mydgf in CMECs. Flow cytometry and TUNEL staining were used to detect the extent of apoptosis, whereas qPCR was used to detect the relative expression of Mydgf mRNA. Western blotting was also performed to detect the expression of apoptosis-related proteins and endoplasmic reticulum stress (ERS)-related proteins, including C/EBP Homologous Protein (CHOP), glucose-regulated protein 78 (GRP 78), and cleaved Caspase-12. The endoplasmic reticulum stress agonist tunicamycin (TM) was used to stimulate CMECs for 24 h as a rescue experiment for Mydgf. Flow cytometry revealed that the HR model effectively induced endothelial cell apoptosis, whereas qPCR and western blotting showed that Mydgf mRNA and protein levels decreased significantly after HR treatment (P < 0.05). Overexpression of Mydgf in cells effectively reduced apoptosis after HR. Furthermore, western blotting showed that HR induced a significant upregulation of CHOP, GRP78, and cleaved-Caspase-12 expression in CMECs, whereas HR-treated cells downregulated the expression of CHOP, GRP78, and cleaved-Caspase-12 after Mydgf overexpression. Under HR conditions, TM significantly reversed the protective effect of Mydgf on CMECs. Mydgf may reduce CMEC apoptosis induced by HR by regulating oxidative stress in ERS.

CircERBB2IP promotes post-infarction revascularization via the miR-145a-5p/Smad5 axis.

Myocardial infarction is one of the leading diseases causing death and disability worldwide, and the revascularization of damaged tissues is essential for myocardial-injury repair. Circular RNAs (circRNAs) are widely involved in physiological and pathological processes in various systems throughout the body, and the role of circRNAs in cardiovascular disease is gaining attention. In this study, we determined that circERBB2IP is highly expressed in the hearts of newborn mice. Silencing or overexpression of circERBB2IP inhibited and promoted angiogenesis in vivo and in vitro, respectively. Mechanistically, the transcription factor GATA4 promotes the production of circERBB2IP. Furthermore, circERBB2IP functioned as an endogenous miR-145a-5p sponge and was able to sequester and repress miR-145a-5p activity, which led to an increased expression level of Smad5. In summary, circERBB2IP can promote angiogenesis after myocardial infarction through the miR-145a-5p/Smad5 axis. These data suggest that circERBB2IP may be a potential therapeutic target for the treatment of myocardial infarction.

Exosomal MiR-29a in Cardiomyocytes Induced by Angiotensin II Regulates Cardiac Microvascular Endothelial Cell Proliferation, Migration and Angiogenesis by Targeting VEGFA.

BACKGROUND: Exosomes released from cardiomyocytes (CMs) potentially play an important role in angiogenesis through microRNA (miR) delivery. Studies have reported an important role for miR-29a in regulating angiogenesis and pathological myocardial hypertrophy. However, whether CMderived exosomal miR-29a is involved in regulating cardiac microvascular endothelial cell (CMEC) homeostasis during myocardial hypertrophy has not been determined. METHODS: Angiotensin II (Ang II) was used to induce CM hypertrophy, and ultracentrifugation was then used to extract exosomes from a CM-conditioned medium. CMECs were cocultured with a conditioned medium in the presence or absence of exosomes derived from CMs (Nor-exos) or exosomes derived from angiotensin II-induced CMs (Ang II-exos). Moreover, a rescue experiment was performed using CMs or CMECs infected with miR-29a mimics or inhibitors. Tube formation assays, Transwell assays, and 5-ethynyl-20-deoxyuridine (EdU) assays were then performed to determine the changes in CMECs treated with exosomes. The miR-29a expression was measured by qRT-PCR, and Western blotting and flow cytometry assays were performed to evaluate the proliferation of CMECs. RESULTS: The results showed that Ang II-induced exosomal miR-29a inhibited the angiogenic ability, migratory function, and proliferation of CMECs. Subsequently, the downstream target gene of miR- 29a, namely, vascular endothelial growth factor (VEGFA), was detected by qRT-PCR and Western blotting, and the results verified that miR-29a targeted the inhibition of the VEGFA expression to subsequently inhibit the angiogenic ability of CMECs. CONCLUSION: Our results suggest that exosomes derived from Ang II-induced CMs are involved in regulating CMCE proliferation, migration, and angiogenesis by targeting VEGFA through the transfer of miR-29a to CMECs.

CircHIPK3 regulates the autophagy and apoptosis of hypoxia/reoxygenation-stimulated cardiomyocytes via the miR-20b-5p/ATG7 axis.

Autophagy and apoptosis are involved in myocardial ischemia/reperfusion (I/R) injury. Research indicates that circular RNA HIPK3 (circHIPK3) is crucial to cell autophagy and apoptosis in various cancer types. However, the role of circHIPK3 in the regulation of cardiomyocyte autophagy and apoptosis during I/R remains unknown. Our study aimed to examine the regulatory effect of circHIPK3 during myocardial I/R and investigate its mechanism in cardiomyocyte autophagy and apoptosis. Methods and results. The expression of circHIPK3 was upregulated during myocardial I/R injury and hypoxia/reoxygenation (H/R) injury of cardiomyocytes. To study the potential role of circHIPK3 in myocardial H/R injury, we performed gain-of-function and loss-of-function analyses of circHIPK3 in cardiomyocytes. Overexpression of circHIPK3 significantly promoted H/R-induced cardiomyocyte autophagy and cell injury (increased intracellular reactive oxygen species (ROS) and apoptosis) compared to those in the control group, while silencing of circHIPK3 showed the opposite effect. Further research found that circHIPK3 acted as an endogenous miR-20b-5p sponge to sequester and inhibit miR-20b-5p activity, resulting in increased ATG7 expression. In addition, miR-20b-5p inhibitors reversed the decrease in ATG7 induced by silencing circHIPK3. Conclusions. CircHIPK3 can accelerate cardiomyocyte autophagy and apoptosis during myocardial I/R injury through the miR-20b-5p/ATG7 axis. These data suggest that circHIPK3 may serve as a potential therapeutic target for I/R.

Exosomal circHIPK3 Released from Hypoxia-Pretreated Cardiomyocytes Regulates Oxidative Damage in Cardiac Microvascular Endothelial Cells via the miR-29a/IGF-1 Pathway.

BACKGROUND/AIMS: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. Recently, exosomes from cardiomyocytes (CMs) have been found to facilitate cell proliferation and survival by transporting various bioactive molecules, including circRNA. However, the functions of exosomal circRNAs are not clear. The present research is aimed at determining whether circHIPK3 released from hypoxia-pretreated CMs is transferred into cardiac microvascular endothelial cells (CMVECs) by exosomes and becomes functionally active in the CMVECs under oxidative stress conditions. METHODS: Quantitative polymerase chain reactions were conducted to detect the expression pattern of circHIPK3 in CMVECs under oxidative stress. Annexin V-FITC/propidium iodide (PI) staining assays, TUNEL assays, ROS assays, and Western blot analysis were conducted to detect the role of exosomal circHIPK3 in CMVEC function in vitro. Luciferase activity assays and RNA immunoprecipitation studies were conducted in vitro to reveal the mechanism of circHIPK3-mediated CMVEC function. RESULTS: circHIPK3 expression was significantly upregulated in hypoxic exosomes (HPC-exos) compared with normoxic exosomes (Nor-exos). Moreover, HPC-exos induced stronger antioxidant effects than Nor-exos. The silencing or overexpression of circHIPK3 changed CMVEC survival under oxidative conditions in vitro. Furthermore, circHIPK3 silencing in HPC-exos abrogated the protective effects of HPC-exos in CMVECs, as shown by increased levels of apoptosis, ROS, MDA, and proapoptotic proteins. circHIPK3 acted as an endogenous miR-29a sponge to sequester and inhibit miR-29a activity, which led to increased IGF-1 expression. The ectopic expression of miR-29a mimicked the effect of circHIPK3 silencing in CMVECs in vitro. CONCLUSIONS: circHIPK3 in HPC-exos plays a role in CMVECs under oxidative conditions through miR-29a-mediated IGF-1 expression, leading to a decrease in oxidative stress-induced CMVECs dysfunction. These data suggest that the exosomal circRNA in CMs is a potential target to control CMVECs dysfunction under oxidative conditions.

Exosomes Derived from miR-214-Enriched Bone Marrow-Derived Mesenchymal Stem Cells Regulate Oxidative Damage in Cardiac Stem Cells by Targeting CaMKII.

Cardiac stem cells (CSCs) have emerged as one of the most promising stem cells for cardiac protection. Recently, exosomes from bone marrow-derived mesenchymal stem cells (BMSCs) have been found to facilitate cell proliferation and survival by transporting various bioactive molecules, including microRNAs (miRs). In this study, we found that BMSC-derived exosomes (BMSC-exos) significantly decreased apoptosis rates and reactive oxygen species (ROS) production in CSCs after oxidative stress injury. Moreover, a stronger effect was induced by exosomes collected from BMSCs cultured under hypoxic conditions (Hypoxic-exos) than those collected from BMSCs cultured under normal conditions (Nor-exos). We also observed greater miR-214 enrichment in Hypoxic-exos than in Nor-exos. In addition, a miR-214 inhibitor or mimics added to modulate miR-214 levels in BMSC-exos revealed that exosomes from miR-214-depleted BMSCs partially reversed the effects of hypoxia-induced exosomes on oxidative damage in CSCs. These data further confirmed that miR-214 is the main effector molecule in BMSC-exos that protects CSCs from oxidative damage. miR-214 mimic and inhibitor transfection assays verified that CaMKII is a target gene of miR-214 in CSCs, with exosome-pretreated CSCs exhibiting increased miR-214 levels but decreased CaMKII levels. Therefore, the miR-214/CaMKII axis regulates oxidative stress-related injury in CSCs, such as apoptosis, calcium homeostasis disequilibrium, and excessive ROS accumulation. Collectively, these findings suggest that BMSCs release miR-214-containing exosomes to suppress oxidative stress injury in CSCs through CaMKII silencing.

Bone marrow mesenchymal stem cell-derived exosomal miR-21 protects C-kit+ cardiac stem cells from oxidative injury through the PTEN/PI3K/Akt axis.

Stem cell (SC) therapy for ischemic cardiomyopathy is hampered by poor survival of the implanted cells. Recently, SC-derived exosomes have been shown to facilitate cell proliferation and survival by transporting various proteins and non-coding RNAs (such as microRNAs and lncRNAs). In this study, miR-21 was highly enriched in exosomes derived from bone marrow mesenchymal stem cells (MSCs). Interestingly, exosomes collected from hydrogen peroxide (H2O2)-treated MSCs (H-Exo) contained higher levels of miR-21 than exosomes released from MSCs under normal conditions (N-Exo). The pre-treatment of C-kit+ cardiac stem cells (CSCs) with H-Exos resulted in significantly increased levels of miR-21 and phosphor-Akt (pAkt) and decreased levels of PTEN, which is a known target of miR-21. AnnexinV-FITC/PI analysis further demonstrated that the degree of oxidative stress-induced apoptosis was markedly lower in H-Exo-treated C-kit+ CSCs than that in N-Exo-treated cells. These protective effects could be blocked by both a miR-21 inhibitor and the PI3K/Akt inhibitor LY294002. Therefore, exosomal miR-21 derived from H2O2-treated MSCs could be transported to C-kit+ cardiac stem cells to functionally inhibit PTEN expression, thereby activating PI3K/AKT signaling and leading to protection against oxidative stress-triggered cell death. Thus, exosomes derived from MSCs could be used as a new therapeutic vehicle to facilitate C-kit+ CSC therapies in the ischemic myocardium.

miR-21 increases c-kit+ cardiac stem cell proliferation in vitro through PTEN/PI3K/Akt signaling.

The low survival rate of cardiac stem cells (CSCs) in the ischemic myocardium is one of the obstacles in ischemic cardiomyopathy cell therapy. The MicroRNA (miR)-21 and one of its target protein, the tensin homolog deleted on chromosome ten (PTEN), contributes to the proliferation of many kinds of tissues and cell types. It is reported that miR-21 promotes proliferation through PTEN/PI3K/Akt pathway, but its effects on c-kit+ CSC remain unclear. The authors hypothesized that miR-21 promotes the proliferation in c-kit + CSC, and evaluated the involvement of PTEN/PI3K/Akt pathway in vitro. miR-21 up-regulation with miR-21 efficiently mimics accelerated cell viability and proliferation in c-kit + CSC, which was evidenced by the CCK-8, EdU and cell cycle analyses. In addition, the over-expression of miR-21 in c-kit + CSCs notably down-regulated the protein expression of PTEN although the mRNA level of PTEN showed little change. Gain-of-function of miR-21 also increased the phosphor-Akt (p-Akt) level. Phen, the selective inhibitor of PTEN, reproduced the pro-proliferation effects of miR-21, while PI3K inhibitor, LY294002, totally attenuated the pro-survival effect of miR-21. These results indicate that miR-21 is efficient in promoting proliferation in c-kit+ CSCs, which is contributed by the PTEN/PI3K/Akt pathway. miR-21 holds the potential to facilitate CSC therapy in ischemic myocardium.

miR-21 Reduces Hydrogen Peroxide-Induced Apoptosis in c-kit+ Cardiac Stem Cells In Vitro through PTEN/PI3K/Akt Signaling.

The low survival rate of cardiac stem cells (CSCs) in the infarcted myocardium hampers cell therapy for ischemic cardiomyopathy. MicroRNA-21 (miR-21) and one of its target proteins, PTEN, contribute to the survival and proliferation of many cell types, but their prosurvival effects in c-kit+ CSC remain unclear. Thus, we hypothesized that miR-21 reduces hydrogen peroxide- (H2O2-) induced apoptosis in c-kit+ CSC and estimated the contribution of PTEN/PI3K/Akt signaling to this oxidative circumstance. miR-21 mimics efficiently reduced H2O2-induced apoptosis in c-kit+ CSC, as evidenced by the downregulation of the proapoptosis proteins caspase-3 and Bax and upregulation of the antiapoptotic Bcl-2. In addition, the gain of function of miR-21 in c-kit+ CSC downregulated the protein level of PTEN although its mRNA level changed slightly; in the meantime, miR-21 overexpression also increased phospho-Akt (p-Akt). The antiapoptotic effects of miR-21 were comparable with Phen (bpV), the selective inhibitor of PTEN, while miR-21 inhibitor or PI3K's inhibitor LY294002 efficiently attenuated the antiapoptotic effect of miR-21. Taken together, these results indicate that the anti-H2O2-induced apoptosis effect of miR-21 in c-kit+ CSC is contributed by PTEN/PI3K/Akt signaling. miR-21 could be a potential molecule to facilitate the c-kit+ CSC therapy in ischemic myocardium.

[Impact and related mechanism of exogenous receptor activity modifying protein 1 on calcitonin gene-related peptide modified bone marrow mesenchymal stem cells on the migration of vascular smooth muscle cells in vitro].

OBJECTIVE: To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms. METHODS: The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups. RESULTS: VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05). CONCLUSIONS: CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.

[Role of CGRP modified mesenchymal stem cells in restenosis in rat model with carotid angioplasty].

OBJECTIVE: To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis. METHODS: The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed. And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP, Lenti-GFP and phosphate buffer saline (PBS). Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively. Then MSCs were co-cultured with VSMCs. Experimental groups were MSCs(CGRP+/+)+VSMCs, MSCs(CGRP-/-)+VSMCs and MSCs(PBS)+VSMCs groups. The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells. Sacculus damaged atherosclerotic carotid was prepared according to the previous study. MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model. At Day 7 post-transplantation, injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence. At Day 28 post-transplantation, injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain. RESULTS: As compared with control and Lenti-GFP groups, the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P < 0.05). After 72-hour co-culturing with VSMCs, the proliferation ability of VSMCs was the lowest in MSCs(CGRP+/+)+VSMCs group versus other three groups (P < 0.05). At Day 7 post-transplantation, as compared with control and Lenti-GFP groups, the expression of CGRP proteins increased significantly in MSCs-CGRP group (P < 0.05). A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group, but not in control group. At Day 28 post-transplantation, the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups. Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P < 0.05). CONCLUSIONS: CGRP protein and CGRP mRNA are expressed after CGRP transfection. And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.

Transplantation of mesenchymal stem cells carrying the human receptor activity-modifying protein 1 gene improves cardiac function and inhibits neointimal proliferation in the carotid angioplasty and myocardial infarction rabbit model.

Although transplanting mesenchymal stem cells (MSCs) can improve cardiac function and contribute to endothelial recovery in a damaged artery, natural MSCs may induce neointimal hyperplasia by directly or indirectly acting on vascular smooth muscle cells (VSMCs). Receptor activity-modifying protein 1 (RAMP1) is the component and the determinant of ligand specificity of calcitonin gene-related peptide (CGRP). It is recently reported that CGRP and its receptor involve the proliferation and the apoptosis in vivo and in vitro, and the exogenous RAMP1 enhances the antiproliferation effect of CGRP in VSMCs. Here, we investigated the effects of MSCs overexpressing the human receptor activity-modifying protein 1 (hRAMP1) on heart function and artery repair in rabbit models of myocardial infarction (MI) reperfusion and carotid artery injury. MSCs transfected with a recombinant adenovirus containing the hRAMP1 gene (EGFP-hRAMP1-MSCs) were injected into the rabbit models via the ear vein at 24 h after carotid artery injury and MI 7 days post-EGFP-hRAMP1-MSC transplantation. The cells that expressed both enhance green fluorescent protein (EGFP) and CD31 were detected in the neointima of the damaged artery via immunofluorescence. EGFP-hRAMP1 expression was observed in the injured artery and infarcted myocardium by western blot analysis, confirming that the engineered MSCs targeted the injured artery and infarcted myocardium and expressed hRAMP1 protein. Compared with the EGFP-MSCs group, the EGFP-hRAMP1-MSCs group had a significantly smaller infarcted area and improved cardiac function by 28 days after cell transplantation, as detected by triphenyltetrazolium chloride staining and echocardiography. Additionally, arterial hematoxylin-eosin staining revealed that the area of the neointima and the area ratio of intima/media were significantly decreased in the EGFP-hRAMP1-MSCs group. An immunohistological study showed that the expression of α-smooth muscle antigen and proliferating cell nuclear antigen in the neointima cells of the carotid artery of the EGFP-hRAMP1-MSCs group was approximately 50% lower than that of the EGFP-MSCs group, suggesting that hRAMP1 expression may inhibit VSMCs proliferation within the neointima. Therefore, compared with natural MSCs, EGFP-hRAMP1-engineered MSCs improved infarcted heart function and endothelial recovery from artery injury more efficiently, which will provide valuable information for the development of MSC-based therapy.

[Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells].

OBJECTIVE: To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell. METHODS: Firstly (Lenti-GFP-CGRP, referred to CGRP (+/+)) MSCs were transfected with high expression lentivirus vector of CGRP (MSCs(CGRP+/+)). Protein secretion in the above-mentioned MSCs(CGRP+/+) supernatant was detected with enzyme-linked immunosorbent assay (ELISA). And then MSCs(CGRP+/+) was co-cultured with VSMCs(RAMP1)(+/+) and VSMCs(RAMP1-/-) respectively. Experimental groups were as follows: MSCs +VSMCs MSCs(CGRP+/+) +VSMCs, MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+) and MSCs(CGRP+/+) + VSMCs (RAMP1-/-). Flow cytometry was applied to detect the cycle variation of smooth muscle cells, thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively. RESULTS: After the transfection of CGRP(+/+), MSCs(CGRP+/+) secreted and expressed CGRP protein, the secretory volume of CGRP protein in MSCs(CGRP+/+) increased significantly compared with the control group (19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001, P < 0.05) . After a 72 h co-culturing with VSMCs, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+)group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070, P < 0.05) and the number of cells staying in G0 phase significantly increased (93.51% ± 0.38% vs 84.48% ± 0.31%, P < 0.05) , the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP(+/+) +VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ± 635, P < 0.05), OPN (26 026 ± 2595 vs 44 201 ± 2811, P < 0.05) }; but compared with MSCs(CGRP+/+)+VSMCs(RAMP1)(+/+)group, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)-/-group significantly increased (0.601 ± 0.04 vs 0.270 ± 0.263, P < 0.05) while the number of cells staying in G0 phase significantly declined (78.57% ± 0.68% vs 93.51% ± 0.38%, P < 0.05) . The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCs(CGRP+/+)+VSMCs(RAMP1)-/-group {α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847, P < 0.05), OPN (53 933 ± 1192 vs 26 026 ± 2595, P < 0.05)}. CONCLUSIONS: CGRP gene-modified MSCs may secrete target protein stably. And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation. It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.