Proliferation Of Cardiac Microvascular Endothelial Cells Research Articles

Salvianic acid A sodium facilitates cardiac microvascular endothelial cell proliferation by enhancing the hypoxia-inducible factor-1 alpha/vascular endothelial growth factor signalling pathway post-myocardial infarction.

Cardiac microvascular endothelial cells (CMECs) are important cells surrounding the cardiomyocytes in the heart that maintain microenvironment homeostasis. Salvianic acid A sodium (SAAS) has been reported to prevent myocardial infarction (MI) injury. However, the role of SAAS on CMEC proliferation remains unclear. CEMCs exposed to oxygen glucose deprivation (OGD) were used to explore the angiogenic abilities of SAAS. In vivo, C57BL/6 mice were divided into three groups: sham, MI and SAAS + MI groups. Compared to OGD group, SAAS led to a reduction in the apoptotic rate and an increase of the proliferation in vitro. Additionally, SAAS increased the protein levels of Bcl2, HIF-1α and vascular endothelial growth factor (VEGF) with the reduction of Bax. In terms of the specific mechanisms, SAAS might inhibit HIF-1α ubiquitination and enhance the HIF-1α/VEGF signalling pathway to increase CMEC proliferation. Furthermore, SAAS increased the density of vessels, inhibited myocardial fibrosis and improved cardiac dysfunction in vivo. The present study has revealed that SAAS could potentially be used as an active substance to facilitate CMEC proliferation post-MI.

Adipsin alleviates cardiac microvascular injury in diabetic cardiomyopathy through Csk-dependent signaling mechanism

BackgroundMicrovascular complications are associated with an overtly increased risk of adverse outcomes in patients with diabetes including coronary microvascular injury which manifested as disruption of adherens junctions between cardiac microvascular endothelial cells (CMECs). However, particular mechanism leading to diabetic coronary microvascular hyperpermeability remains elusive.MethodsExperimental diabetes was induced in mice with adipose tissue-specific Adipsin overexpression (AdipsinLSL/LSL-Cre) and their respective control (AdipsinLSL/LSL). In addition, cultured CMECs were subjected to high glucose/palmitic acid (HG + PA) treatment to simulate diabetes for a mechanistic approach.ResultsThe results showed that Adipsin overexpression significantly reduced cardiac microvascular permeability, preserved coronary microvascular integrity, and increased coronary microvascular density. Adipsin overexpression also attenuated cardiac dysfunction in diabetic mice. E/A ratio, an indicator of cardiac diastolic function, was improved by Adipsin. Adipsin overexpression retarded left ventricular adverse remodeling, enhanced LVEF, and improved cardiac systolic function. Adipsin-enriched exosomes were taken up by CMECs, inhibited CMECs apoptosis, and increased CMECs proliferation under HG + PA treatment. Adipsin-enriched exosomes also accelerated wound healing, rescued cell migration defects, and promoted tube formation in response to HG + PA challenge. Furthermore, Adipsin-enriched exosomes maintained adherens junctions at endothelial cell borders and reversed endothelial hyperpermeability disrupted by HG + PA insult. Mechanistically, Adipsin blocked HG + PA-induced Src phosphorylation (Tyr416), VE-cadherin phosphorylation (Tyr685 and Tyr731), and VE-cadherin internalization, thus maintaining CMECs adherens junctions integrity. LC-MS/MS analysis and co-immunoprecipitation analysis (Co-IP) unveiled Csk as a direct downstream regulator of Adipsin. Csk knockdown increased Src phosphorylation (Tyr416) and VE-cadherin phosphorylation (Tyr685 and Tyr731), while abolishing Adipsin-induced inhibition of VE-cadherin internalization. Furthermore, Csk knockdown counteracted Adipsin-induced protective effects on endothelial hyperpermeability in vitro and endothelial barrier integrity of coronary microvessels in vivo.ConclusionsTogether, these findings favor the vital role of Adipsin in the regulation of CMECs adherens junctions integrity, revealing its promises as a treatment target against diabetic coronary microvascular dysfunction.Graphical Graphical abstract depicting the mechanisms of action behind Adipsin-induced regulation of diabetic coronary microvascular dysfunction.

Irisin rescues diabetic cardiac microvascular injury via ERK1/2/Nrf2/HO-1 mediated inhibition of oxidative stress

AimsCardiac microvascular dysfunction is a common feature across cardiovascular complications in diabetes, while effective therapy remains elusive. This study was designed to evaluate the effect of irisin on cardiac microvascular injury in type 2 diabetes mellitus (T2DM). MethodsT2DM was induced in C57BL/6J mice. A cohort diabetic mice received a 12-week treatment of irisin. Cardiac function and microvessel density were evaluated. Whether irisin directly regulates cardiac microvascular endothelial cells (CMECs) function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms. ResultsIrisin improved cardiac function in diabetic mice, and increased microvessel density. In vitro study revealed that irisin promoted CMECs proliferation and reduced high glucose and high lipid (HGHL)-induced apoptosis. Mechanistically, irisin increased mRNA and protein levels of heme oxygenase 1 (HO-1), superoxide dismutase 1 and superoxide dismutase 2, among which HO-1 ranked top. Irisin stimulated the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and nuclear factor erythroid-derived 2—like 2 (Nrf2) nuclear translocation, while U0126 (the inhibitor of ERK1/2) inhibited irisin-induced Nrf2 nuclear translocation and HO-1 expression. Nrf2 siRNA inhibited irisin’s antioxidative effects. ConclusionIrisin could rescue cardiac microvessels against oxidative stress and apoptosis in diabetes via ERK1/2/Nrf2/HO-1 pathway.

MiR-543 in human mesenchymal stem cell-derived exosomes promotes cardiac microvascular endothelial cell angiogenesis after myocardial infarction through COL4A1.

To explore the impact and mechanism of human mesenchymal stem cells (hMSCs) on the angiogenesis of cardiac microvascular endothelial cells (CMECs) after ischemia insult. Exosomes derived from hMSCs (hMSCs-Exo) were identified by Western blotting and labeled by PHK-67. CMECs were isolated from rat myocardial tissues. After hypoxic treatment, CMECs were cultured with hMSCs and exosome inhibitor (GW4869) or transfected with si-COL4A1 + miR-543 inhibitor. CMEC proliferation, migration, invasion, and angiogenesis were examined. Target genes of miR-543 were predicted and then were identified by dual luciferase assay. Myocardial infarction (MI) rat model established by suture occlusion was intravenously injected with hMSCs-Exo. Fluorescence microscope was applied to visualize exosomes in myocardial tissues. Infarction volume and pathologies of myocardial tissues were observed. Ki-67 and miR-543 expressions were detected. The isolated hMSC-Exo expressed TSG101, HSP70, and CD63. Hypoxia-treated CMECs cultured with hMSCs exhibited high proliferation, migration, invasion, and angiogenesis ability, while incubation with exosome inhibitor GW4969 offset the promoting effects of hMSCs on the proliferation, migration, invasion, and angiogenesis of CMECs. hMSCs transfected with miR-543 inhibitor brought CMECs weak viability and angiogenesis ability. CMECs transfected with si-COL4A1 and miR-543 inhibitor showed low proliferation, migration, invasion, and angiogenesis compared to those transfected with si-COL4A1 alone. hMSCs-Exo entered the myocardial tissues of MI rats. Injection of hMSCs-Exo in MI rats diminished infarction size, attenuated MI-induced injuries, and increased Ki-67 expression. hMSCs-Exo facilitates the proliferation, migration, invasion, and angiogenesis of CMECs through transferring miR-543 and downregulating COL4A1 expression.

The lncRNA Malat1 regulates microvascular function after myocardial infarction in mice via miR-26b-5p/Mfn1 axis-mediated mitochondrial dynamics

RationaleMyocardial infarction (MI) is a leading cause of cardiovascular mortality globally. The improvement of microvascular function is critical for cardiac repair after MI. Evidence now points to long non-coding RNAs (lncRNAs) as key regulators of cardiac remodelling processes. The lncRNA Malat1 is involved in the development and progression of multiple cardiac diseases. Studies have shown that Malat1 is closely related to the regulation of endothelial cell regeneration. However, the potential molecular mechanisms of Malat1 in repairing cardiac microvascular dysfunction after MI remain unreported. Methods and resultsThe present study found that Malat1 is upregulated in the border zone of infarction in mouse hearts, as well as in isolated cardiac microvascular endothelial cells (CMECs). Targeted knockdown of Malat1 in endothelial cells exacerbated oxidative stress, attenuated angiogenesis and microvascular perfusion, and as a result decreased cardiac function in MI mice. Further studies showed that silencing Malat1 obviously inhibited CMEC proliferation, migration and tube formation, which was at least in part attributed to disturbed mitochondrial dynamics and activation of the mitochondrial apoptosis pathway. Moreover, bioinformatic analyses, luciferase assays and pull-down assays indicated that Malat1 acted as a competing endogenous RNA (ceRNA) for miR-26b-5p and formed a signalling axis with Mfn1 to regulate mitochondrial dynamics and endothelial functions. Overexpression of Mfn1 markedly reversed the microvascular dysfunction and CMEC injuries that were aggravated by silencing Malat1 via inhibition of excessive mitochondrial fragments and mitochondria-dependent apoptosis. ConclusionsThe present study elucidated the functions and mechanisms of Malat1 in cardiac microcirculation repair after MI. The underlying mechanisms of the effects of Malat1 could be attributed to its blocking effects on miR-26b-5p/Mfn1 pathway-mediated mitochondrial dynamics and apoptosis.

Qiliqiangxin Prescription Promotes Angiogenesis of Hypoxic Primary Rat Cardiac Microvascular Endothelial Cells via Regulating miR-21 Signaling.

Angiogenesis is the most important repair process of tissues subjected to ischemic injury. The present study aims to investigate whether the pro-angiogenic effect of Qiliqiangxin prescription (QL) is mediated through miR-21 signaling. Cardiac microvascular endothelial cells (CMECs) were isolated and cultured from 2-3 weeks old SD rats by the method of planting myocardium tissues. The purity was identified by CD31 immunofluorescence staining. CMECs were then cultured under 1% O2 hypoxia or normoxia condition for 24h in the presence or absence of QL pretreatment (QL, 0.5mg/ml, 24h). The mimics and inhibitors of miR-21 were transfected into CMECs. miR-21, HIF-1α, and VEGF expressions of CMECs were then detected by qRT-PCR and/or Western blot. The proliferation, migration, and tube formation functions of CMECs were assessed using the BrdU assay, wound healing test, and tube formation assay, respectively. The results showed that compared with the control group, hypoxia significantly upregulated the expression of miR-21 and impaired CMECs proliferation, migration, and tube formation functions. Compared with the hypoxia group, QL further upregulated miR-21, HIF-1α, and VEGF expressions, and improved cell proliferation, migration, and tube formation of hypoxic CMECs. These effects of QL were abolished by a knockdown of miR-21. Conversely, treatment with miR-21 mimics further enhanced QL induced changes in hypoxic CMECs. Results indicate that the pro-angiogenesis effects of QL on hypoxic CMECs are mediated by activating miR-21 and its downstream HIF-1α/VEGF pathway possibly.

Methylation‐dependent transcriptional repression of RUNX3 by KCNQ1OT1 regulates mouse cardiac microvascular endothelial cell viability and inflammatory response following myocardial infarction

Myocardial infarction (MI) is a major contributor to death and disability throughout the world. Increasing evidence shows that long noncoding RNAs (lncRNAs) are involved in the progression of MI. Here, we hypothesized that lncRNA potassium voltage-gated channel subfamily q member 1 overlapping transcript 1 (KCNQ1OT1) could affect the development of MI via regulation of Runt-related transcription factor (RUNX)3 by methylation. Initially, by ligation of the left anterior descending coronary artery, an acute MI (AMI) mouse model was established to collect the cardiac microvascular endothelial cells (CMECs), which revealed a high KCNQ1OT1 expression and a low RUNX3 expression with its high methylation. After that, KCNQ1OT1 knockdown or RUNX3 overexpression were transduced into the CMECs in order to detect their role in CMEC proliferation, apoptosis, and inflammatory response. Moreover, we assessed their interaction with the inflammatory Notch pathway, by determining the expression of Jagged 1, Hey1, Hes1, Notch intracellular domain, and Notch1. It was observed that after KCNQ1OT1 knockdown, the proliferation of AMI-CMECs was promoted, whereas their apoptosis was inhibited, accompanied by reduced level of inflammatory factors. These trends could also be achieved by RUNX3 overexpression via the Notch pathway. Finally, the regulation of DNA methyltransferase (DNMT)1-dependent methylation in RUNX3 by KCNQ1OT1 was determined, suggesting that KCNQ1OT1 could result in down-regulated RUNX3 expression through promoted RUNX3 methylation caused by recruiting DNMT1. Overall, this study demonstrates that KCNQ1OT1 silencing inhibits RUNX3 methylation, thereby offering protection against CMEC injury and inflammatory response in AMI, which may serve as a promising target for the disease treatment. -Wang, Y., Yang, X., Jiang, A., Wang, W., Li, J., Wen, J. Methylation-dependent transcriptional repression of RUNX3 by KCNQ1OT1 regulates mouse cardiac microvascular endothelial cell viability and inflammatory response following myocardial infarction.

Over-expression of JAZF1 promotes cardiac microvascular endothelial cell proliferation and angiogenesis via activation of the Akt signaling pathway in rats with myocardial ischemia-reperfusion

ABSTRACT Myocardial ischemia-reperfusion (I/R) injury is caused by endothelial dysfunction and enhanced oxidative stress. The overexpression of JAZF1, a zinc finger protein, has been reported to promote cell proliferation and suppress myogenic differentiation in type 2 diabetes. However, the involvement of JAZF1 in myocardial I/R injury remains to be unclear. The current study aims to investigate the role by which JAZF1 influences cardiac microvascular endothelial cells (CMECs) in a rat model of myocardial I/R injury. A total of 50 rats were established as a myocardial I/R model to isolate CMECs, with alterations in JAZF1 expression. After that, the gain- or loss-function of JAZF1 on the proliferation, apoptosis and tube formation ability of CMECs were evaluated by a series of in vitro experiments. Results indicated that JAZF1 was down-regulated in CMECs of rats with myocardial I/R injury. After treatment with JAZF1, the levels of VEGF, Bcl-2, PDGF and p-Akt/Akt were all increased; however, the expression of Bax, caspase-3, caspase-9, p-Bad/Bad, c-caspase-3/caspase-3, c-caspase-9/caspase-9, and p-FKHR/FKHR exhibited decreased levels; CMEC proliferation and angiogenesis were increased, while cell apoptosis was attenuated. CMECs transfected with JAZF1 shRNA exhibited the contrary tendencies. The key findings of this study suggest that the over-expression of JAZF1 alleviates myocardial I/R injury by enhancing proliferation and angiogenesis of CMECs and in turn inhibiting apoptosis of CMECs via the activation of the Akt signaling pathway.

Advanced glycation end products facilitate the proliferation and reduce early apoptosis of cardiac microvascular endothelial cells via PKCβ signaling pathway: Insight from diabetic cardiomyopathy.

Objective:To investigate the effects of advanced glycation end products (AGEs) on the proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs) in rats and their underlying signaling pathway.Methods:CMECs were isolated from Sprague–Dawley rats. We first examined the effects of AGEs on the proliferation and apoptosis of CMECs and then tested whether protein kinase C (PKC) β blockers could counteract the effects of AGEs. The PKC agonists phorbol 12-myristate 13-acetate (PMA) and PKCβ blockers were also used to verify whether PKC could act independently on CMECs. The receptor for AGEs (RAGE)–small interfering RNA (siRNA) transfection was used to verify the effect of AGEs on PKC. Following the above steps, we explained whether AGEs regulated the CMEC proliferation and early apoptosis through the PKCβ signaling pathway. Proliferation of CMECs was detected using the Cell Counting Kit-8 (CCK-8) assay, and early apoptosis was determined using the Annexin V- Fluorescein Isothiocyanate (FITC)/propidium iodide (PI) double staining. Expression of proliferation and apoptosis-related proteins and PKC phosphorylation were determined by western blotting analysis. Cell cycle distributions were assayed using a BD FACSCalibur cell-sorting system.Results:AGEs facilitated the proliferation of CMECs, upregulated phosphorylated extracellular signal regulated kinase (p-ERK), and accelerated the entry of cells from G1 phase to the S+G2/M phase, which was consistent with the upregulated cyclin D1 by AGEs. AGEs inhibited early apoptosis of CMECs by increasing the expression of survivin and decreasing the expression of cleaved-caspase3. All these effects can be reversed by PKCβ1/2inhibitors. In addition, AGE upregulated the RAGE expression and phosphorylation of PKCβ1/2 in CMECs, while the inhibition of RAGE reversed the phosphorylation, as well as the effects of AGEs on proliferation and apoptosis in CMECs.Conclusion:The study indicated that AGEs facilitated the proliferation and reduced early apoptosis of CMECs via the PKCβ signaling pathway.

RP105 ameliorates hypoxia̸reoxygenation injury in cardiac microvascular endothelial cells by suppressing TLR4̸MAPKs̸NF-κB signaling.

The radioprotective 105kDa protein (RP105) has been implicated in the pathological process of multiple cardiovascular diseases through its functional and physical interactions with Toll‑like receptor4 (TLR4). However, the effects of RP105 on cardiac microvascular endothelial cells (CMECs) in response to hypoxia̸reoxygenation(H̸R) injury have not been extensively investigated. The aim of the present study was to elucidate the potential roles of RP105 in the protection of CMECs against H̸R injury, and investigate the underlying mechanisms. CMECs isolated from Sprague‑Dawley rats were transduced with adenoviral vectors encoding RP105 or green fluorescent protein (GFP). At 48h post‑transfection, CMECs were subjected to hypoxia for 4h and reoxygenation for 2h (H̸R) to simulate the invivo ischemia̸reperfusion model. The mRNA and protein levels of RP105 were detected by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The effects of RP105 on CMEC proliferation, migration and apoptosis were measured by GFP‑8, Transwell chamber and flow cytometry assays, respectively. The secretion of interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α in the culture medium was measured by ELISA. Moreover, the expression level of TLR4, p38mitogen‑activated protein kinase (MAPK), extracellular-signal-regulated kinase1̸2, c-Jun N-terminal kinase, nuclear factor (NF)‑κB̸p65, IL‑6, TNF‑α and intercellular adhesion melecule‑1 was evaluated by western blot analysis. The results demonstrated that RP105 was minimally expressed in CMECs subjected to H̸R injury. Overexpression of RP105 via adenoviral vectors was able to significantly protect CMECs against H̸R injury, as evidenced by the promotion of cell proliferation and migration, as well as the amelioration of inflammation and apoptosis. These beneficial effects were at least partly mediated through inhibition of TLR4̸MAPKs̸NF‑κB signaling. Therefore, RP105 may be a promising candidate for prevention against CMECs‑associated H̸R injury.

The Effect of Velvet Antler Proteins on Cardiac Microvascular Endothelial Cells Challenged with Ischemia-Hypoxia.

Velvet antler (VA) is a precious traditional Chinese medicine that is capable of repeated regeneration. Based on the Chinese medicine theory of coordination the heart and kidneys, VA has been employed to treat heart diseases, including ischemic heart disease, heart failure, and arrhythmia. We examined the effects of VA proteins on primary cardiac microvascular endothelial cells (CMECs) that were subjected to ischemia-hypoxia (IH) to investigate their effects on and mechanism of action in the treatment of ischemic heart disease. Velvet antler proteins (VA-pro) were extracted with water as the solvent, the ultrasonic wave method, and freeze-drying technology; then it was analyzed by Nano LC-MS/MS. In addition, the role of VA-pro in cell viability, proliferation, apoptosis, and mitochondrial membrane potential (MMP) were evaluated by the MTS assay, the EdU assay, the Annexin V-FITC/PI double-staining assay, and the JC-1 assay, respectively. Cell migration were evaluated by the scratch assay and the Transwell assay. The expression of apoptosis-associate proteins, Akt and p-Akt, and tube formation in Matrigel of CMECs were also detected. In total, 386 VA-pro were identified. Our results showed that IH significantly reduced the viability of the CMECs (P < 0.001) and suppressed copies of DNA to hold back CMEC proliferation (P < 0.001). The OD of control group was 1.81 ± 0.08 and IH group OD was 1.25 ± 0.03. After suffering with IH for 46 h, CMECs were 75% less likely to migrate (P < 0.001), and its tubule formation ability and MMP were also decreased (P < 0.001). VA-pro treatment resulted in an improvement in CMECs’ viability and proliferation (P < 0.001). Such as, the OD of 0.5, 1, and 2 mg/ml rose to 1.56 ± 0.5, 1.74 ± 0.1 and 1.65 ± 0.1, respectively. Similarly, CMECs’ migration (for the scratch assay P < 0.001, for the Transwell assay P < 0.05) and tubule formation (P < 0.05) ability were better after treated with VA-pro. At the same time, the stability of MMP was retained preferably (P < 0.001). 50% apoptosis was induced after CMECs were cultured in IH conditions (P < 0.001), while VA-pro decreased the number of apoptotic cells (P < 0.001). All above results showed that 1 mg/ml VA-pro produced maximum results. Furthermore, the expression of pro-apoptosis proteins was higher, but the expression of anti-apoptosis proteins was lower in the IH group (P < 0.05); VA-pro reversed these changes (P < 0.001). These findings suggest that VA-pro ameliorate CMEC injuries induced by IH via regulating the PI3K/Akt signaling pathway.

Pulsed magnetic field accelerate proliferation and migration of cardiac microvascular endothelial cells.

Heart failure is a disease with multifactorial causes. Recently it was established that reduction in vascular density promoted the progression from adaptive cardiac hypertrophy to heart failure, therefore, therapeutic angiogenesis may be a promising method for treating heart failure. Cardiac microvascular endothelial cells (CMECs) play a major role in cardiac angiogenesis. In the present study, we investigated the direct and indirect effect of pulsed magnetic field (PMF) on the proliferation and migration of CMECs. CMECs were isolated from adult Sprague-Dawley (SD) rat hearts. We found PMF with a frequency of 15 Hz and an intensity of 1.8 mT accelerated the proliferation and migration of CMECs and cardiac myocytes (CMs). Moreover, CMECs treated with PMF released 1.5-fold higher vascular endothelial growth factor (VEGF) and 2-fold higher fibroblast growth factor-2 (FGF-2) when compared with PMF-free cells. In addition, CMs treated with PMF released twofold higher FGF-2 compared with PMF-free cells, but there was no change in VEGF levels. Those results suggested PMF has both a direct autocrine mitogenic and an indirect paracrine effect on CMECs proliferation and migration, and the effect of PMF on intercellular communication between CMECs and CMs was partially dependent on FGF-2, but independent on VEGF.

Effect of glutahione S-transferases on cardiac microvascular endothelial cells

Objective To detect the effect of glutahione S-transferases (GSTP1) expression on the migration,proliferation and apoptosis of cardiac microvascular endothelial cells (CMECs).Methods After obtaining CMECs from SD rats by enzymatic digestion method,the special plasmid of GSTP was transfected.The ability of migration,proliferation and apoptosis of CMECs were examined by using Transwell assay,methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results The ability of migration of CMECs was significantly reduced in GSTP1 over-expression group as compared with control group (9.0 ±2.7 vs.12.8 ±2.5,P ＜0.05),and the ability of proliferation of CMECs was also obviously reduced in comparison with control group (0.26 ± 0.02 vs.0.05 ± 0.01,P ＜ 0.05).But the apoptotic index was increased significantly (P ＜ 0.05).Conclusion GSTP1 expression can effectively inhibit migration and proliferation,and promote apoptosis of CMECs. Key words: Glutahione S-transferases; Cardiac microvascular endothelial cells; Migration; Proliferation

Simvastatin promotes cardiac microvascular endothelial cells proliferation, migration and survival by phosphorylation of p70 S6K and FoxO3a

Restenosis severely limits the overall efficacy of interventions. One of the reasons is the lack of reendothelialization related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface. Statins can promote angiogenic processes by improving endothelial function, proliferation and migration in cardiac microvascular endothelial cells (CMECs). This study clarified the effect of simvastatin on Akt/mTOR/p70 S6K and FoxO3a signalling pathways in rat CMECs following pretreated with rapamycin. Rapamycin treatment for 24 h inhibited CMECs' proliferation, migration and NO (nitric oxide) secretion, but with increased cell apoptosis and reactive oxygen species (ROS) production. In contrast, simvastatin pretreatment significantly improved proliferation, migration and NO secretion, and inhibited CMECs' apoptosis and ROS production in rapamycin-induced CMECs. Western blot assay showed that, after treatment with simvastatin, the phosphorylation of Akt/mTOR/p70 S6K and FoxO3a were up-regulated in rapamycin-induced CMECs, which was significantly reversed by pretreatment with LY294002. The data suggest that simvastatin inhibits rapamycin-induced CMECs dysfunction and apoptosis, probably through activation of PI3K/Akt/mTOR/p70 S6K and mTOR/FoxO3a signalling pathway in a sequential manner and this pathway may be important in some of the pleiotropic effects of statins.

Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059+LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.

The effects of low frequency pulsed magnetic fields with different waveforms on cardiac microvascular endothelial cells

Objective To investigate the effects of low frequency pulsed magnetic fields (LF-PMFs) on proliferation, the cell cycle, apoptosis, migration, cytoskeleton formation and nitric oxide (NO) secretion in cardiac microvascular endothelial cells (CMECs). Methods LF-PMFs with rectangular and triangular waveforms at 15 Hz were tested. The CMECs were randomly divided into a control group, 0.6 mT group, 1.2 mT group, 1.8 mT group and 2.4 mT group. Except for the control group, the CMECs were exposed to LF-PMFs 4 h/day for 5 days. Results After 5 days of intervention with rectangular wave LF-PMFs the proliferation of CMECs accelerated and NO secretion was enhanced significantly. The percentages of cells at ( S+G2 ) phase increased significantly, whereas apoptosis rates were significantly lower than in the control group. The migration of CMECs was facilitated, stress fibers increased and cytoskeleton components were reorganized. After 5 days of intervention with triangular wave LF-PMFs proliferation of CMECs accelerated and NO secretion was significantly enhanced. The percentages of cells at(S+G2)phase and migration increased, while apoptosis was inhibited. Cytoskeleton components were reorganized after exposure to 0.6 mT and 2.4 mT triangular waveform LF-PMFs. No significant change was detected with 1.2 mT and 1.8 mT triangular waveform LF-PMFs with regard to these variables. Conclusions The effects of LF-PMFs on proliferation, the cell cycle, apoptosis, migration, cytoskeleton formation and NO secretion function of CMECs were infiuenced by the waveform in addition to it's intensity and frequency. Key words: Low frequency pulsed magnetic fields; Cardiac microvascular endothelial cells; Proliferation; Cell cycle; Apoptosis; Migration; Cytoskeleton; Nitric oxide

E0002 Effect of toll-like receptor-4 signal pathway on the dysfunction of cardiac microvascular endothelial cells caused by hypoxia/reoxygenation injury

AimTLR-4 has been proved to take part in MIRI of heart. But the researches mostly focused on the relationship between TLR-4 and global heart dysfunction or cadiocyte apoptosis. The effect...

Mechanical stimulation regulates voltage-gated potassium currents in cardiac microvascular endothelial cells.

Vascular endothelial cells are constantly exposed to mechanical forces resulting from blood flow and transmural pressure. The goal of this study was to determine whether mechanical stimulation alters the properties of endothelial voltage-gated K+ channels. Cardiac microvascular endothelial cells (CMECs) were isolated from rat ventricular muscle and cultured on thin sheets of silastic membranes. Membrane currents were measured with the use of the whole-cell arrangement of the patch-clamp technique in endothelial cells subjected to static stretch for 24 hours and compared with measurements from control, nonstretched cells. Voltage steps positive to -30 mV resulted in the activation of a time-dependent, delayed rectifier K+current (IK) in the endothelial cells. Mechanically induced increases of 97%, 355%, and 106% at +30 mV were measured in the peak amplitude of IK in cells stretched for 24 hours by 5%, 10%, and 15%, respectively. In addition, the half-maximal voltage required for IK activation was shifted from +34 mV in the nonstretched cells to -5 mV in the stretched cells. Although IK in both groups of CMECs was blocked to a similar extent by tetraethylammonium, currents in the stretched endothelial cells displayed an enhanced sensitivity to inhibition by charybdotoxin. Preincubation of the CMECs with either pertussis toxin or phorbol 12-myristate 13-acetate during the 24 hours of cell stretch did not prevent the increase in IK. The application of phorbol 12-myristate 13-acetate and static stretch stimulated the proliferation of CMECs. Stretch-induced regulation of K+ channels may be important to control the resting potential of the endothelium and may contribute to capillary growth during periods of mechanical perturbation.