Guosheng Fu

MicroRNA‐21 mediates the rapamycin‐induced suppression of endothelial proliferation and migration

Rapamycin suppresses endothelial proliferation and migration, which leads to delayed re‐endothelialization in the rapamycin‐eluted stents that are used in coronary heart disease patients. Because microRNAs (miRs) play important roles in endothelial angiogenesis, we tested the hypothesis that rapamycin induces endothelial suppression, partly through pathways that involve miRs. Rapamycin treatment increased the expression of miR‐21 in HUVECs. The downregulation of miR‐21 by inhibitors abolished the negative effects of rapamycin on endothelial cell growth and mobility. RhoB was confirmed as a direct target gene of miR‐21. Knockdown of Raptor by siRNA mimicked the effects of rapamycin on miR‐21 expression. Our study provides a new explanation of the mechanism of rapamycin‐mediated inhibition of endothelial proliferation and migration.

Thymosin β4 activates integrin-linked kinase and decreases endothelial progenitor cells apoptosis under serum deprivation.

Thymosin β4 (Tβ4) has been suggested to regulate multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival, and angiogenesis. Here, we investigated the effect of Tβ4 on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the corresponding signal transduction pathways involved in this process. Circulating EPCs, isolated from healthy volunteers, were cultured in the absence or presence of Tβ4 and various signal cascade inhibitors. Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Incubation of EPCs with Tβ4 caused a concentration dependent increase in cell viability and proliferation activity. It also caused an inhibitory effect on EPCs apoptosis, which was abolished by PI3K inhibitors (either LY294002 or Wortmannin) or JNK MAPK inhibitor SP600125. In addition, the expression and activity of caspase‐3 and ‐9 were decreased by Tβ4, which markedly increased the Bcl‐2/Bax ratio within EPCs. Furthermore, Tβ4 was immunoprecipitated with integrin‐linked kinase (ILK), accompanied by augmentation of ILK activity. Transfection of EPCs with ILK‐siRNA resulted in abolishment of the activation of ILK‐Akt and the ameliorative effect on apoptosis by Tβ4. Together, Tβ4 mediated inhibitory effect on EPCs apoptosis under serum deprivation can be attributed, at least in part, to ILK‐Akt activation. The activation of JNK MAPK might also be involved in this process. J. Cell. Physiol. 226: 2798–2806, 2011.

Interleukin-8 prevents oxidative stress-induced human endothelial cell senescence via telomerase activation

Senescence is an irreversible growth arrest which can be triggered by stresses such as oxidative reaction, telomere shortening, DNA damage, or oncogene signaling. Oxidative stress accelerates vascular endothelial cell senescence, and may promote atherosclerosis in humans. Interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis, and metastasis, and has close relationship with oxidative stress. The objective of this study was to determine if IL-8 might be able to prevent oxidative stress-induced senescence of endothelial cells and the mechanisms. Human umbilical vein endothelial cells (HUVECs) were cultured and stimulated with hydrogen peroxide in the absence or presence of IL-8. After ex vivo cultivation, HUVECs became senescent as determined by acidic beta-galactosidase staining. IL-8 dose-dependently inhibited the onset of HUVEC senescence. Western blots indicated that IL-8 attenuated the oxidative stress induced high-expression of cell cycle regulation protein and inhibited the activation of p38 and NF-κB pathway. IL-8 also increased telomerase activity which was accompanied with upregulation of the catalytic subunit, telomerase reverse transcriptase (TERT), whereas these effects were significantly attenuated by SB 225002 (selective non-peptide CXCR2 antagonist). In conclusion, IL-8 exerted protective effects against endothelial senescence, which may be related to the activation of telomerase.

Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (-)-Epigallocatechin gallate via ERK MAP kinase pathway.

Intercellular communication through gap junctions (GJIC) plays an essential role in maintaining the functional integrity of vascular endothelium. Despite emerging evidence suggests that (-)-Epigallocatechin gallate (EGCG) may improve endothelial function. However, its effect on Cx43 gap junction in endothelial cells remains unexplored. Here we investigated the effect of EGCG on connexin43 (Cx43) gap junction in endothelial cells. The levels of Cx43 protein in human umbilical vein endothelial cells (HUVECs) cultured under serum-deprivation 48 h decreased about 50%, accompanied by decreased GJIC. This reduction can be reversed by treatments with EGCG. In addition, EGCG activated ERK, P38, and JNK mitogen-activated protein kinases (MAPKs), which were supposed to participate in the regulation of Cx43. A MEK inhibitor PD98059, but not SB203580 (a p38 kinase inhibitor) or SP600125 (a JNK kinase inhibitor), abolished the effects of EGCG on Cx43 expression and GJIC. Moreover, although both Akt and eNOS phosphorylation were time-dependently augmented by EGCG, neither PI3K inhibitor LY294002 nor eNOS inhibitor L-NAME blocked the effects of EGCG on Cx43 gap junctions. Thus, EGCG attenuated Cx43 down-regulation and impaired GJIC induced by serum deprivation, ERK MAPK Signal transduction pathway appears to be involved in these processes.

Stromal cell-derived factor 1α reduces senescence of endothelial progenitor subpopulation in lectin-binding and DiLDL-uptaking cell through telomerase activation and telomere elongation

Recent studies have suggested that reduced endothelial progenitor subpopulation in lectin‐binding and DiLDL‐uptaking cell (EPC subpopulation) number and activity was associated with EPC subpopulation senescence that involved telomerase activity and telomere length. Stromal cell‐derived factor‐1α (SDF‐1α) has been shown to augment a variety of cellular functions of EPC subpopulation and subsequently contribute to ischemic neovascularization. Therefore, we investigated whether SDF‐1α might be able to prevent senescence of EPC subpopulation and also investigated the effects of SDF‐1α on the telomerase activity and telomere length. EPC subpopulation were isolated from peripheral blood and characterized. After ex vivo prolonged cultivation, EPC subpopulation became senescent as determined by acidic β‐galactosidase staining. SDF‐1α dose‐dependently inhibited the onset of EPC subpopulation senescence. Moreover, SDF‐1α increased proliferation and colony‐forming activity of EPC subpopulation. SDF‐1α also increased telomerase activity and telomere length, which was accompanied with upregulation of the catalytic subunit, telomerase reverse transcriptase (TERT). Whereas these effects of SDF‐1α on telomerase activity and expression of hTERT mRNA were significantly attenuated by CXCR4‐specific peptide antagonist (AMD3100) and phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002). In conclusions, SDF‐1α delays the onset of EPC subpopulation senescence, which may be related to the activation of telomerase and elongation of telomere length. The inhibition of EPC subpopulation senescence and induction of EPC subpopulation proliferation by SDF‐1α in vitro may importantly improve the functional activity of EPC subpopulation for potential cell therapy. J. Cell. Physiol. 223:757–763, 2010.

Advanced Glycation End Product (AGE)-AGE Receptor (RAGE) System Upregulated Connexin43 Expression in Rat Cardiomyocytes via PKC and Erk MAPK Pathways

The remodeling of cardiac gap junction contributes to the arrhythmias in a diabetic heart. We previously reported that high glucose reduced Cx43 protein level in neonatal rat cardiomyocytes. But, the effect and mechanisms of advanced glycation end product (AGE) on Cx43 expression still remain unclear. In this study, we measured the AGE receptor (RAGE) and Cx43 expression by immunohistochemisty in AGE-infused Sprague-Dawley (SD) rats. In vitro, the Cx43 and RAGE levels were detected in AGE-treated cardiomyocytes by Western blot and real-time RT-PCR. The function of cells coupling was measured by Scrap loading dye transfer assay. Our results showed that the AGE-infused rat hearts exhibited increased cardiac RAGE and Cx43, as well as Cx43 redistribution. In cultured cardiomyocytes, AGE elevated RAGE expression in a time- and dose-dependent manner. Cx43 protein and mRNA levels were upregulated by AGE (200 mg/L, 24 h), but the gap junction function was not enhanced. RAGE-targeted knock-down or the addition of PKC, and Erk inhibitors abolished the effect of AGE on Cx43. Therefore, AGE-RAGE system might elevate Cx43 expression in rat cardiomyocytes by activating PKC and Erk MAPK pathways, and it also enhanced Cx43 redistribution in vivo, which might contribute to the arrhythmias in diabetes.

Resveratrol prevents endothelial progenitor cells from senescence and reduces the oxidative reaction via PPAR‑γ/HO‑1 pathways

Increasing evidence suggests endothelial progenitor cells (EPCs) improve neovascularization and endothelium regeneration. Resveratrol (RSV) is a natural polyphenolic compound, which has been demonstrated to exert multiple protective effects on the cardiovascular system, including inhibition of platelet adhesion and aggregation, reduction of myocardial ischemia-reperfusion injury, and suppression of neointimal hyperplasia of injured vascular tissue. The present study investigated the role of RSV on levels of oxidative stress and senescence of EPCs, and the effects of RSV on vascular-promoting and/or vascular-healing capacity of EPCs. It was demonstrated that EPCs could promote the repair of endothelium of the injured artery. RSV reduced the oxidative reaction of EPCs and inhibited EPC senescence, and these effects may occur via the peroxisome proliferator-activated receptor-γ/heme oxygenase-1 signaling pathways.

MicroRNAs as potential novel therapeutic targets and tools for regulating paracrine function of endothelial progenitor cells

Summary Endothelial progenitor cells (EPCs) play a protective role in the cardiovascular system by enhancing the maintenance of endothelium homeostasis and the process of new vessel formation. Recent studies show that EPCs may induce vascular regeneration and neovascularization mainly through paracrine signaling, that is, through the secretion of growth factors and pro-angiogenic cytokines [1]. However, multiple factors might function synergistically and therefore make it difficult to manipulate EPC paracrine effects. MicroRNAs, a family of small, non-coding RNAs, are characterized by post-transcriptionally regulating multiple functionally related genes, which renders them potentially powerful therapeutic targets or tools. In this paper we propose the hypothesis that microRNAs can be utilized as a novel therapeutic strategy for regulating EPC paracrine secretion.

Alendronate prevents angiotensin II-induced collagen I production through geranylgeranylation-dependent RhoA/Rho kinase activation in cardiac fibroblasts.

Collagen I is the main component of extracellular matrix in cardiac fibrosis. Our previous studies have reported inhibition of farnesylpyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis, while the exact molecular mechanism was still unclear. This paper was designed to investigate the effect of alendronate, a farnesylpyrophosphate synthase inhibitor, on regulating angiotensin II-induced collagen I expression in cultured cardiac fibroblasts and to explore the underlying mechanism. By measuring the mRNA and protein levels of collagen I, we found that alendronate prevented angiotensin II-induced collagen I production in a dose-dependent manner. The inhibitory effect on collagen I expression was reversed by geranylgeraniol, and mimicked by inhibitors of RhoA/Rho kinase pathway including C3 exoenzyme and GGTI-286. Thus we suggested geranylgeranylation-dependent RhoA/Rho kinase activation was involved in alendronate-mediated anti-collagen I synthetic effect. Furthermore, we accessed the activation status of RhoA in alendronate-, geranylgeraniol- and GGTI-286-treated cardiac fibroblasts and gave an indirect evidence for RhoA activation via geranylgeranylation. Then we came to the conclusion that in cardiac fibroblasts, alendronate could protect against angiotensin II-induced collagen I synthesis through inhibition of geranylgeranylation and inactivation of RhoA/Rho kinase signaling. Targeting geranylgeranylation and RhoA/Rho kinase signaling will hopefully serve as therapeutic strategies to reduce fibrosis in heart remodeling.

Stromal cell-derived factor-1α prevents endothelial progenitor cells senescence and enhances re-endothelialization of injured arteries via human telomerase reverse transcriptase

Recent studies have suggested that endothelial progenitor subpopulation (EPCs) number and activity were associated with EPCs senescence. Our previous study had shown that stromal cell‐derived factor‐1alpha (SDF‐1α) could prevent EPCs senescence, which may be via telomerase. In this study, we further investigated the role of human telomerase reverse transcriptase (h‐TERT) on the protective effect of SDF‐1α against senescence. Knockdown h‐TERT abrogated the protective effect of SDF‐1α and abolished the effects of SDF‐1α on migration and proliferation. Moreover, it inhibited EPCs recruitment. In conclusion, h‐TERT served a critical role in the progress that SDF‐1α prevented EPCs senescence and enhanced re‐endothelialization of the injured arteries.