Dong Kuang

Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK.

ObjectiveIt was reported that there are cardiac stem cells (CSCs) in the rat heart, and they could reconstitute well-differentiated myocardium that are formed by blood-carrying new vessels and myocytes. However, how do the CSCs migrate into the peri-infarcted areas after myocardial infarction (MI)? It remains entirely unknown about the signal transduction involved in the migration of CSCs.Methods and resultsRat heart MI was induced by left coronary artery ligation. Both immunohistochemical staining and Western blotting analysis was performed to detect the expression of SCF protein, and RT-PCR was conducted for the expression of SCF mRNA. Cardiac stem cells were isolated from rat hearts, and a cardiac stem cell migration assay was performed using a 48-well chemotaxis chamber system. On day 5 after MI in rats, the expression of stem cell factor (SCF) mRNA and protein was significantly increased in the peri-infarcted area, which was matched with more accumulation of CSCs in the region and improvement of cardiac function, which was blocked by p38 MAPK selective inhibitor SB203580. In in vitro experiments, SCF induced CSC migration in a concentration-dependent manner, and the antibody against SCF receptor (c-kit) blocked the SCF-induced CSC migration. Western blot analysis showed that the phosphorylated p38 MAPK (Phospho-p38 MAPK) was highly increased in the SCF-treated CSCs, and the inhibition of p38 MAPK activity significantly attenuated SCF-induced the migration of CSCs.ConclusionIt demonstrated that SCF/c-kit signaling may mediate the migration of CSCs via activation of p38 MAPK.

SDF-1α/CXCR4 axis is involved in glucose-potentiated proliferation and chemotaxis in rat vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs), which migrate from the tunica media to the subendothelial region, is one of the primary lesions involved in atherogenesis in diabetes. Here, we investigated whether high glucose potentiated the proliferation and chemotaxis of VSMCs by activating SDF‐1α/CXCR4/PI‐3K/Akt signalling. The expression of SDF‐1α, CXCR4 and PCNA was up‐regulated in tunica media of thoracic aortas by streptozotocin‐induced hyperglycaemic Sprague–Dawley rats. Exposure of primary VSMCs to high glucose (25 mM) led to the up‐regulated expression of SDF‐1α and CXCR4, activated PI‐3K/Akt signalling, and consequently promoted the proliferation and chemotaxis of VSMCs. Interestingly, the administration of SDF‐1 siRNA or neutralizing antibody against SDF‐1α abolished high glucose‐induced up‐regulation of CXCR4. Moreover, pretreatment with SDF‐1α neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or PI‐3K inhibitor (LY294002) attenuated the high glucose‐potentiated proliferation and chemotaxis in VSMCs. These results suggested that high glucose activated the SDF‐1α/CXCR4/PI‐3K/Akt signalling pathway in VSMCs in an autocrine manner, which enhanced the proliferation and chemotaxis of VSMCs.

Ischaemia/reperfusion induced cardiac stem cell homing to the injured myocardium by stimulating stem cell factor expression via NF-κB pathway

Ischaemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells (CSCs) were proposed as the most appropriate cell type for heart disease therapy. However, it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium. Male Sprague–Dawley rats were subjected to a 30‐min ischaemia followed by reperfusion of different intervals. RT‐PCR, western blotting and immunohistochemistry were performed to detect stem cell factor (SCF) expression at mRNA and protein levels respectively. Activation of nuclear factor‐κB (NF‐κB) was determined by electrophoretic mobility shift assay. To assess the homing of CSCs in vivo, BrdU‐labelled CSCs were injected into AV‐groove before induction of ischaemia and examined by immunofluorescent staining in the injured myocardium after I/R. From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevated in the injured area, which was matched with the increased SCF expression during I/R. Pretreatment of rats with NF‐κB inhibitor, N‐acetyl‐l‐cysteine (NAC) not only suppressed NF‐κB activation induced by I/R but also attenuated SCF expression. Further analysis revealed that I/R induced phosphorylation of IκBα after 15 min of reperfusion, and the raised phosphor‐IκBα returned to the basal level at 2 h of reperfusion. In simulated I/R(SI/R) in vitro, it enhanced NF‐κB activation and SCF expression in cultured neonatal rat cardiomyocytes, which was markedly inhibited by NF‐κB decoy oligodeoxynucleotide or NAC. Taken together, our results demonstrated that I/R induced CSCs homing to the injured myocardium by stimulating myocardial SCF expression via activation of NF‐κB.

Crosstalk between SDF-1/CXCR4 and SDF-1/CXCR7 in cardiac stem cell migration.

Stromal cell-derived factor 1 (SDF-1) is a chemokine that can be expressed in injured cardiomyocytes after myocardial infarction (MI). By combining with its receptor CXCR4, SDF-1 induced stem and progenitor cells migration. CXCR7, a novel receptor for SDF-1, has been identified recently. We aimed to explore the roles of SDF-1/CXCR4 and SDF-1/CXCR7 pathway and their crosstalk in CSCs migration. In the present study, CXCR4 and CXCR7 expression were identified in CSCs. Transwell assay showed that SDF-1 caused CSCs migration in a dose- and time-dependent manner, which could be significantly suppressed by CXCR4 or CXCR7 siRNA. Phospho-ERK, phospho-Akt and Raf-1 significantly elevated in CSCs with SDF-1 stimulation. Knockdown of CXCR4 or CXCR7 significantly decreased phospho-ERK or phospho-Akt, respectively, and eventually resulted in the inhibition of CSCs migration. Moreover, western blot showed that MK2206 (Akt inhibitor) increased the expression of phospho-ERK and Raf-1, whereas PD98059 (ERK inhibitor) had no effect on phospho-Akt and Raf-1. GW5074 (Raf-1 inhibitor) upregulated the expression of phospho-ERK, but had no effect on phospho-Akt. The present study indicated that SDF-1/CXCR7/Akt and SDF-1/CXCR4/ERK pathway played important roles in CSCs migration. Akt phosphorylation inhibited Raf-1 activity, which in turn dephosphorylated ERK and negatively regulated CSCs migration.

Hyperhomocysteinemia inhibited cardiac stem cell homing into the peri-infarcted area post myocardial infarction in rats.

BACKGROUND Hyperhomocysteinemia (HHcy) has been reported as an independent risk factor for coronary artery disease; however it is not clear regarding the action of HHcy on the homing of cardiac stem cells (CSCs) to the damaged myocardium and the consequent CSCs-mediated cardiac repair post myocardial infarction. METHODS Sprague-Dawley (SD) rats were divided into 4 groups. HHcy was induced in the rats by a 6-week high-methionine diet. Rat heart MI model was developed by left coronary artery ligation. Immunofluorescence was used to examine the CSCs migration in vivo via injecting BrdU-labeled CSCs into AV-groove followed by a coronary ligation. Immunohistochemistry, western blot and ELISA analysis were carried out to detect the expression of stem cell factor (SCF) protein, and RT-PCR was conducted for the expression of SCF mRNA. RESULTS On day 5 of MI model creation, accumulation of CSCs was significantly increased in the peri-infarcted area by the non-hyperhomocysteinemic rats, which led to an improvement of cardiac function at 3 weeks after MI. however, the accumulation of CSCs was markedly decreased by the hyperhomocysteinemic rats followed with the decline of cardiac function. SCF expression was also significantly decreased in the peri-infarcted area by the hyperhomocysteinemic rats compared to the non-hyperhomocysteinemic rats. The experiments in vitro confirmed that homocysteine (Hcy) decreased SCF expression via inhibition of TNF-α-induced activity of NF-κB, further reduced the migration of CSCs. CONCLUSION It demonstrated that hyperhomocysteinemia may significantly contribute to restrain CSCs-mediated cardiac repair by reducing SCF-induced homing of CSCs.

SCF/c-kit transactivates CXCR4-serine 339 phosphorylation through G protein-coupled receptor kinase 6 and regulates cardiac stem cell migration

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

Hyperglycemia suppresses cardiac stem cell homing to peri-infarcted myocardium via regulation of ERK1/2 and p38 MAPK activities

Hyperglycemia in the acute phase of myocardial infarction (MI) is a marker of worse prognosis in both diabetic and non-diabetic patients; however, the role of hyperglycemia in the homing of cardiac stem cells (CSCs) to damaged myocardium post-MI and the possible mechanisms involved are not well understood. In this study, an MI model was induced in normoglycemic and hyperglycemic rats by left coronary artery ligation. Immunofluorescence was used to examine the migration of CSCs in vivo by injecting BrdU-labeled CSCs into the atrium-ventricle groove (AV-groove). Immunohistochemistry, western blot analysis and ELISA were carried out to detect the expression of stem cell factor (SCF) protein and RT-PCR was conducted for the expression of SCF mRNA. Phosphorylation of ERK1/2 and p38 MAPK was detected by western blot analysis. Afterwards, cardiac function was evaluated by hemodynamic measurement. On Day 5 post-MI, the accumulation of CSCs significantly increased in the peri-infarcted myocardium in normoglycemic rats, which led to an improvement in cardiac function 3 weeks after MI. However, the accumulation of CSCs markedly decreased in hyperglycemic rats, followed by the decline of cardiac function. SCF expression, followed with phosphorylation of ERK1/2 and p38 MAPK, were also significantly downregulated in the peri-infarcted myocardium in hyperglycemic rats compared to normoglycemic rats. Moreover, SCF expression and the migration of CSCs were blocked by either the MEK-specific inhibitor PD98059 or the p38 MAPK-selective inhibitor SB203580. The experiments in vitro confirmed that hyperglycemia decreased SCF expression via reduction in ERK1/2 and p38 MAPK activities and further inhibited the migration of CSCs. The results suggest that hyperglycemia suppresses CSC migration towards the ischemic area post-MI. This is possibly due to decreased myocardial SCF expression via reduction of ERK1/2 and p38 MAPK activities in hyperglycemic rats.

miR-497-5p inhibits cell proliferation and invasion by targeting KCa3.1 in angiosarcoma

Angiosarcoma is a rare malignant mesenchymal tumor with poor prognosis. We aimed to identify malignancy-associated miRNAs and their target genes, and explore biological functions of miRNA and its target in angiosarcoma. By miRNA microarrays and reverse transcription polymerase chain reaction, we identified 1 up-regulated miRNA (miR-222-3p) and 3 down-regulated miRNAs (miR-497-5p, miR-378-3p and miR-483-5p) in human angiosarcomas compared with human capillary hemangiomas. The intermediate-conductance calcium activated potassium channel KCa3.1 was one of the putative target genes of miR-497-5p, and marked up-regulation of KCa3.1 was detected in angiosarcoma biopsy specimens by immunohistochemistry. The inverse correlation of miR-497-5p and KCa3.1 also was observed in the ISO-HAS angiosarcoma cell line at the mRNA and protein levels. The direct targeting of KCa3.1 by miR-497-5p was evidenced by reduced luciferase activity due to complementary binding of miR-497-5p to KCa3.1 mRNA 3′ untranslated region. For the functional role of miR-497-5p/KCa3.1 pair, we showed that application of TRAM-34, a specific KCa3.1 channel blocker, or transfection of ISO-HAS cells with KCa3.1 siRNA or miR-497-5p mimics inhibited cell proliferation, cell cycle progression, and invasion by down-regulating cell-cycle related proteins including cyclin D1, surviving and P53 and down-regulating matrix metallopeptidase 9. In an in vivo angiosarcoma xenograft model, TRAM-34 or miR-497-5p mimics both inhibited tumor growth. In conclusion, the tumor suppressor miR-497-5p down-regulates KCa3.1 expression and contributes to the inhibition of angiosarcoma malignancy development. The miR-497-5p or KCa3.1 might be potential new targets for angiosarcoma treatment.

Contribution of CXCR4+/PDGFRβ+ progenitor cells in hypoxic alveolar arterioles muscularization: role of myocardin

AIMS Bone marrow (BM) progenitor cells may contribute to vascular remodelling. The present study aimed to investigate the contribution of BM-derived CXCR4(+) (a CXC chemokine receptor) and PDGFRbeta(+) (platelet-derived growth factor receptor beta) progenitor cells in hypoxia-induced muscularization of alveolar arterioles. METHODS AND RESULTS Accumulation of GFP(+) (green fluorescent protein) cells was markedly increased in the pulmonary vasculature by the hypoxic (10% O(2,) 4 weeks) chimeric mice with transgenic GFP-tagged BM. After injection of BM-derived CXCR4(+)/PDGFRbeta(+) progenitor cells into C57BL/6J mice, followed by 6-week hypoxia, the cells were found to home to the alveolar arterioles and readily differentiated into smooth muscle cells (SMCs). Under the same hypoxic conditions, mice infused with myocardin lentiviral RNAi vector-transduced progenitor cells displayed lower myocardin expression in the muscularized alveolar arterioles, correlating with decreased pulmonary artery pressure and arteriole muscularization. In vitro experiments further confirmed that the differentiation of the progenitor cells into SMCs occurred under hypoxia (1% O(2)), and SMC differentiation could be suppressed when myocardin RNAi was administered. CONCLUSION Theses results suggest that myocardin may contribute to the differentiation of CXCR4(+)/PDGFRbeta(+) progenitor cells into SMCs induced by hypoxia, which leads to the muscularization of alveolar arterioles.

Co-targeting of IGF1R/mTOR pathway by miR-497 and miR-99a impairs hepatocellular carcinoma development

Persistent activation of IGF1R/mTOR signaling pathway plays crucial role in the development of hepatocellular carcinoma (HCC). Therefore, our goal was to elucidate microRNAs (miRNAs) targeting IGF1R/mTOR and the therapeutic potential of single or dual miRNA on HCC development. In this study, we found that miR-497 and miR-99a that target the 3′-UTR of both IGF1R and mTOR were down-regulated in HCC human tissues and cell lines. Functional assay revealed that ectopic expression of miR-497 or miR-99a in HCC cells resulted in a significant inhibition on tumor growth and invasiveness in vitro and tumor development in vivo via repressing the expression of IGF1R and mTOR. Such inhibitory effect on tumor growth is reversed by application of IGF1 ((IGF1R ligand) or MHY1485 (mTOR agonist) in vitro. Furthermore, we found that simultaneous over-expression of both miR-497 and miR-99a exhibited much stronger inhibitory effects on tumor growth than their individual effect, which is still correlated with significantly stronger repression of IGF1R and mTOR. Overall, our results suggest that miR-497 and miR-99a both function as tumor-suppressive miRNAs by suppressing IGF1R/mTOR signaling pathway. The synergistic actions of these two miRNAs partly correlated with IGF1R and mTOR levels, which may represent new strategies for the molecular treatment of HCC.