| Abnormal changes of proliferation and apoptosis in pulmonary artery smooth muscle cells (PASMCs) play a key role on vascular remodeling that occurs in chronic hypoxic pulmonary hypertension (HPH). Different growth factors, cytokine and proinflammatory mediators convey extracelluar signal to the nucleus by interacting with receptors on the cell surface, then regulate expression of target genes and contribute to cell stress reaction. However, the cellular signal mechanism of PASMC involved in hypoxic pulmonary vascular remodeling are not fully understood.The Hedgehog(Hh) signaling pathway has been well investigated in the studies of development. To date, three members of the hedgehog family have been identified in vertebrates: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). The stimuli of secreted hedgehog family peptides are mediated by a receptor system on the cell surface composed with two proteins: Patched (Ptc) and Smoothened (Smo). The hedgehog receptor Ptc represses the 7-transmembrane protein Smo, inhibiting the pathway in the absence of hedgehog ligands. Upon hedgehog ligand binding, Ptc is inactivated, and then Smo is de-repressed from the suppression of Ptc and is able to transduce the intracellular signals to transcriptor Gli. Gli transfers the signals into the nucleus and then activates and represses many target gene transcription in response to Hh signaling.The Hedgehog signaling pathway plays a fundamental role in the development of various embryonic tissues development. The pathway has been shown to have effects on cell proliferation, cell survival and cell fate determination in embryonic development. Mutations in the pathway often result in gross defects across species. Its regulatory roles in signal transduction in mature/adult cells and their implications in disease are now well-recognized recent years. Several observations highlight the involvement of Hh in the development of embryonic vascular tissues, including hypervascularization of neuroectoderm following overexpression of Shh ,and poor vascularization of the developing lung in Shh-deficient mice. Recently a study showed that Hh signaling is present in adult vascular smooth muscle cells and can be diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling. According to these, we hypothesized that Hh pathway may potentially have regulatory effects on VSMC's response in hypoxic vascular remolding .This study was therefore undertaken to observe the expression of Hh signaling pathway and its role on human PASMCs under hypoxia conditions.Methods1. After HPASMC were purchased and cultured, expressions of Shh, Ptc-1 and Gli-1 mRNA and protein were detected by RT-PCR and Western blot, locations of Ptc-1 and Gli-1 in static HPASMC were detected by immunofluorescence. Shh was used to investigate the role of Hh signal pathway on HPASMC growth.2. The quantitative assessment of Shh and Ptc-1 mRNA expression under hypoxia by Real-time RT-PCR and the protein expression by Western blot analysis. Gli-1 nuclear accumulation induced by hypoxia was detected by immunofluorescence.3. After various hours hypoxia treatment, HPASMC proliferation was detected by Alamar Blue assay, cell cycle distribution and apoptosis was measured by flow cytometry analysis.4. After hypoxia treatment, with or without cyclopamine block, the activities of both cytokine The activities of IL-6 and MCP-1 in HPASMC cell supernatant was detected by ELISA.5. HPASMC was cultured in hypoxia with or without Vitamin D3 treatment, the proliferation was measured by Alamar Blue assay,the expression of Gli-1 mRNA was measured by real-time RT-PCR, and the location of Gli-1 protein was detected by immunofluorescence。Results1.RT-PCR revealed that Shh, Ptc-1, and Gli-1 mRNA are expressed in HPASMC. Immunofluorescence showed that Ptc-1 protein was present on the plasma membranes of HPASMC, and Gli-1 protein was present on the cytoplasm but not nucleus of static HPASMC. Western blot analysis showed that Shh, Ptc-1,and Gli-1 protein were expressed in HPASMC. Shh enhanced proliferation of HPASMC in a dose-dependent manner. The enhancement can be inhibited by cyclopamine(a Hh signaling inhibitor).2.The expressions of Shh and Ptc-1 mRNA level in HPASMC measured by real-time RT-PCR were promoted after 4h hypoxic exposure, peaked after 8h and decreased after 12h. And the protein expression level of Shh, Ptc-1 measured by Western blot changed as the same mode, respectively. Gli-1 nuclear accumulation induced by hypoxia and can be blocked by cyclopamine.3. HPASMC were serum starved at least 24h and then incubated for various periods in normoxic or hypoxic conditions. Hypoxia significantly promoted HPASMC proliferation at the phase of 4h, 8h, and 12h. Cyclopamine inhibited the proliferation, increased G0/G1 phrase, decreased S and G2/M phrase cell cycles significantly. Effect of Hh signaling pathway on HPASMC apoptosis in 8h hypoxic exposure was assessed by annexin V staining. Compared with normoxia control, the rate of HPASMC apoptosis in hypoxia group was lower, which can be increased by cyclopamine significantly.4.The IL-6 activity in normoxia HPASMC cell supernatant was detectable by ELISA, but MCP-1 not. After hypoxia exposure, the activities of IL-6 and MCP-1 both increased significantly, and can be inhibited remarkably by cyclopamine.5.After hypoxic treatment, vitamin D3 can inhibit the Gli-1mRNA expression level and nuclear accumulation of HPASMC and decrease the proliferation induced by hypoxia.Conclusions1. Hh signal pathway main components are expressed in normal adult HPASMC and involve in the growth regulation.2. Hh signal pathway can be activated by hypoxia, which involes in the abnormal changes of proliferation, apoptosis, and proinflammation cytokine secretion of HPASMC induced by hypoxia.3. Vitamin D3 can inhibit the activation of Hh signal pathway and decrease HPASMC proliferation induced by hypoxia, which indicate its potential value for the treatment of hypoxic pulmonary vascular remodeling.
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