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Aim: To investigate the effects of Sonic hedgehog (shh) protein on bone marrow‐derived endothelial progenitor cells (BM‐EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. Methods: Bone marrow‐derived Flk‐1+ cells were enriched using the MACS system from adult Kunming mice and then BM‐EPC was cultured in gelatin‐coated culture dishes. The effects of shh N‐terminal pep‐tide on BM‐EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boy den chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. Results: The proliferation, migration and VEGF production in BM‐EPC could be promoted by endogenous shh N‐terminal peptide at concentrations of 0.1 ug/mL to 10 ug/mL, and could be inhibited by anti‐shh antibodies. Shh‐mediated proliferation and migration in BM‐EPC could be partly attenuated by anti‐VEGF. Phospho‐PI3 ‐kinase expression in newly separated BM‐EPC was low, and it increased significantly when exogenous shh N‐terminal peptide was added, but could be attenuated by anti‐human/mouse shh N‐terminal peptide antibody. Moreover, the inhibitor of the PI3‐kinase, but not the inhibitor of the PKC, significantly inhibited the shh‐mediated proliferation, migration and VEGF production. Conclusion: Shh protein can stimulate bone marrow‐derived BM‐EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3‐kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

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Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6−/−) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.

The present study investigated the association between Snail1 and atrial fibrosis in patients with atrial fibrillation (AF) and rheumatic heart disease (RHD) and to determine the possible mechanism underlying this interrelation. A total of 19 patients were included in the current study and were divided into two groups: A sinus rhythm (SR) group (n=9) and an AF group (n=10). All patients underwent heart valve replacement surgery, during which ~200 mg right atrium tissue was obtained. Hematoxylin and eosin and Masson's trichrome-stained sections were used to evaluate the morphological changes of cardiomyocytes and the level of fibrosis. Immunohistochemistry was applied to observe the location and expression of Snail1. Reverse transcription-quantitative polymerase chain reaction was used to measure Snail1 mRNA levels. Western blotting was used to determine changes in the expression of Snail1, as well as in the expression of proteins involved in the Wnt pathway, including Wnt1, Wnt 3a, Wnt8a, Wnt5a and Wnt11. Compared with the SR group, expanded cardiomyocytes and higher collagen deposition was detected in the atrial tissue of the AF group. The expression of Snail1 mRNA and protein was significantly higher in the AF group than in the SR group (P<0.05). Additionally, the expression of Wnt1, 3a and 8a in the canonical Wnt signaling pathway, and Wnt5a and 11 in the noncanonical Wnt signaling pathway were significantly increased in the AF group. Furthermore, the phosphorylation level of glycogen synthase kinase 3β (GSK3β) and the levels of β-catenin and GSK3β were significantly increased in the AF group compared with the SR group (P<0.05). Snail1 may be involved in the development and maintenance of atrial fibrosis in patients with atrial fibrillation and rheumatic heart disease and may be developed as a novel biomarker to evaluate myocardial fibrosis in the future. Additionally, the current study suggests that the Wnt signaling pathway may participate in the process of increased Snail1 expression and atrial fibrosis in patients with AF and RHD.

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Reactive oxygen species (ROS) impair neovascularization and perfusion recovery following limb ischemia in patients with peripheral arterial disease (PAD). Hydrogen molecules (H2) comprise an antioxidant gas that has been reported to neutralize cytotoxic ROS. The present study investigated whether H2 may serve as a novel therapeutic strategy for PAD. H2-saturated water or dehydrogenized water was supplied to mice with experimental PAD. Laser Doppler perfusion imaging demonstrated that H2-saturated water improved perfusion recovery, decreased the rate of necrosis, increased the capillary density in the gastrocnemius muscle and increased the artery density in the abductor muscle in the ischemic limbs, at 14 and 21 days post-hindlimb ischemia. Ischemic muscle tissue was harvested 7 days after experimental PAD for biochemical testing and H2 was observed to reduce the levels of malondialdehyde and increase the levels of cyclic guanine monophosphate (cGMP). In cultured endothelial cells, H2-saturated culture medium resulted in reduced ROS levels, increased tube formation and increased cGMP levels. In macrophages, H2 decreased cellular ROS levels and promoted M2 polarization. H2-saturated water increases angiogenesis and arteriogenesis and subsequently improves perfusion recovery in a mouse PAD model via reduction of ROS levels.