Yanbo Zhao

Thymosin β4 Induces Endothelial Progenitor Cell Migration via PI3K/Akt/eNOS Signal Transduction Pathway

Thymosin β4, a G-actin-sequestering peptide, has been shown to play an important role in cell migration. However, little is known about the effect of thymosin β4 on circulating endothelial progenitor cell (EPC) directional migration, which is essential for EPC-mediated reendothelialization and neovascularization. In our study, using a transwell migration assay, we showed that thymosin β4 induced EPC migration in a concentration-dependent manner. Western blot analysis revealed that treatment of EPCs with thymosin β4 resulted in a time and concentration-dependent phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK)1/2. Functional analysis showed that thymosin β4-induced EPC migration was blocked by phosphatidylinositol 3-kinase inhibitors (LY294002 or wortmannin) or eNOS inhibitor (Nω-nitro-L-arginine methyl ester) but was not significantly attenuated by mitogen-activated protein kinase (MAPK)/ERK inhibitor (PD98059). These findings suggest that thymosin β4 stimulates EPC directional migration via phosphatidylinositol 3-kinase/Akt/eNOS, rather than via MAPK/ERK signal transduction pathway.

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.

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.

P38 MAPK/miR-1 are involved in the protective effect of EGCG in high glucose-induced Cx43 downregulation in neonatal rat cardiomyocytes.

The remodeling of cardiac gap junctions contributes to various arrhythmias in a diabetic heart. We previously reported that Epigallocatechin‐3‐gallate (EGCG) attenuated connexin43 (Cx43) protein downregulation induced by high glucose (HG) in neonatal rat cardiomyocytes, but Cx43 mRNA expression was not affected. It indicated the possible mechanisms of post‐transcriptional regulation, which still remains unclear. As microRNAs (miRNAs) regulate gene expression widely at post‐transcriptional level, we measured miR‐1/206 in cardiomyocytes treated with HG and EGCG by quantitative RT‐PCR and investigated their relationship with signal transduction pathways. The results showed that HG induced miR‐1/206 elevation by PKC MAPK pathway. Moreover, we tested the negative regulation effect of miR‐1/206 on Cx43 protein by miRNAs transfection. EGCG, however, nearly abolished the HG‐induced miR‐1 augmentation via P38 MAPK pathway. Therefore, our study suggested that PKC‐activated miR‐1/206 expression might contribute to Cx43 downregulation in HG‐treated cardiomyocytes, and EGCG conferred protective effect by inhibiting miR‐1 elevation via P38 MAPK pathway.

Pioglitazone restores the homocysteine‑impaired function of endothelial progenitor cells via the inhibition of the protein kinase C/NADPH oxidase pathway

Homocysteine (Hcy) has been shown to impair the migratory and adhesive activity of endothelial progenitor cells (EPCs). As a peroxisome proliferator-activated receptor γ agonist, pioglitazone (PIO) has been predicted to regulate angiogenesis, and cell adhesion, migration and survival. The aim of the present study was to determine whether PIO could inhibit Hcy-induced EPC dysfunctions such as impairments of cell migration and adhesion. EPC migration and adhesion were assayed using 8.0-µm pore size Transwell membranes and fibronectin-coated culture dishes, respectively. Hcy at a concentration of 200 µM was observed to markedly impair cell migration and adhesiveness, and PIO at a concentration of 10 µM attenuated the Hcy-mediated inhibition of EPC migration and adhesion. The mechanism of these effects may be through the inhibition of protein kinase C (PKC) and reactive oxygen species production. The expression levels of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, NADPH oxidase 2 (Nox2) and p67phox, were upregulated by Hcy, with a peak in levels following treatment with a concentration of 200 µM. PIO downregulated the expression levels of Nox2 and p67phox via the PKC signaling pathway. Furthermore, the mechanism of PIO associated with downregulating the p67phox and Nox2 subunits of NADPH oxidase was verified. Thus, PKC and NADPH oxidase may serve a major role in the protective effects of PIO in EPCs under conditions of high Hcy concentrations.

Thymosin β4 promotes glucose‑impaired endothelial progenitor cell function via Akt/endothelial nitric oxide synthesis signaling pathway

Circulating endothelial progenitor cells (EPCs) are a subtype of hematopoietic stem cells, which can differentiate into endothelial cells and restore endothelial function. However, high glucose decreases the number and impairs the function of EPCs. A previous study showed that thymosin β4 (Tβ4), a pleiotropic peptide beneficial for multiple functions of various types of cells, could promote EPC migration and dose-dependently upregulate the phosphorylation of Akt and endothelial nitric oxide synthesis signaling (eNOS). In present study, the hypothesis that Tβ4 can improve glucose-suppressed EPC functions via the Akt/eNOS signaling pathway and restores the production of nitric oxide (NO) is investigated. EPCs were isolated from the peripheral blood of healthy volunteers and formed a cobblestone shape after 3–4 weeks of cultivation. Then, EPCs were treated with high concentrations of glucose (25 mM) for 4 days and administrated with Tβ4 for further study. Transwell migration and tube formation assays were performed to access the migratory and angiogenic ability of EPCs. In addition, the quantity of Akt, eNOS and the concentration of nitric oxide (NO) was investigated. Functional studies showed that high concentrations of glucose significantly suppressed EPC function, while this adverse effect was reversed by the administration of Tβ4. In addition, Akt small interfering (si)RNA and eNOS siRNA were demonstrated to reduce the protective effect of Tβ4 against glucose-impaired EPC functions. These findings suggest that Tβ4 improves glucose-impaired EPC functions via the Akt/eNOS signaling pathway.