| BACKGROUND & OBJECTIVEErectile dysfunction (ED) is a kind of common disease of Andrology, especially men who are over 45 years old suffer from significantly higher risk of ED. The causes of ED include many aspects, such as blood vessels, nerves, psychological, endocrine. In recent years, vascular factors have drew more and more attention of doctors and patients. The corpus cavernosum is very similar with the vascular systems. Therefore, it is considered as a rather exceptional vascular bed. Corpus cavernosum smooth muscle (CCSM) is the structural basis of cavernoussinus space relaxing and penile erection, which plays a critical role in the change of hemodynamic when the penis erects. Simultaneously CCSM is at the core position because it is the target of various factors. Any factor caused CCSM tissue or cells damage may lead to the occurrence of ED.The phenotypic modulation of CCSM cells can alter the normal structure and function of the corpus cavernosum, which is closely related to the occurrence of ED.The smooth muscle cell phenotypic modulation means:the morphological, structural and functional changes of smooth muscle cells in body’s various stages of development or different diseases. The CCSM cell divides into two kinds of types: the contractile and synthetic phenotype, similar to the vascular smooth muscle cell (VSMC), and can change its phenotype according to the local environmental variations because of its bi-directional differentiation function. The phenotypic modulation of CCSM cells can reduce the contraction and relaxation capacity of cells, increase synthesis of extracellular matrix, and even cause cavernous fibrosis and hemodynamic changes of the penis, which plays a very important role in the development of ED.Among many factors that can cause the structural and functional changes of the corpus cavernosum, the hypoxia may easily be overlooked. Hypoxia refers to the reduce of oxygen supply with the ratio imbalance of tissue oxygen supply and demand, leading to insufficient to sustain metabolism and maintain the normal function and structure of the organization. Hypoxia is prevalent in many diseases causing to organic ED, such as diabetes, sleep apnea hypopnea syndrome (SAHS), the neurovascular bundle injury after radical prostate cancer surgery, plateau hypoxia and so on. The corpus cavernosum tissue is in a hypo-oxygenated at flaccidity, only during sleep-or sexual-related erections does the increased arterial blood flow allow corpus cavernosum oxygen tension to achieve arterial values. The periodic oxygenation is important for the maintenance of normal erectile function and avoiding prolonged hypoxia which can alter the normal structure and function of the corpus cavernosum, even cause cavernous fibrosis. Thus, chronic hypoxia plays a very important role in the occurrence of organic ED.However, whether does hypoxia causes the phenotypic modulation of CCSM cell and underlying molecular mechanisms are still unclear at present.Platelet-derived growth factor-BB (PDGFBB) belonging to the PDGF family, is the main type of mitotic agents in vivo. In many pathological and physiological conditions, such as local tissue ischemia and hypoxia, inflammation, many kinds of cells including endothelial cell, smooth muscle cell, macrophage, many tumor cells and so on could secrete it. PDGFBB combined with PDGFR and make its receptor phosphorylation, activation of downstream important signaling molecules, which is involved in many signal transduction pathways such as ras/GTP, PI3K, Src, MAPK, etc, and play a important role in many cardiovascular diseases such as atherosclerosis and arterial restenosis and so on. PDGFBB can down regulate the expression of myocardin in VSMCs and lead to phenotypic modulation. Our previous studies have shown that myocardin also plays a key role in the phenotypic modulation of CCSM cells, myocardin significantly decreased in corpus cavernosums of rats with diabetic ED, in turn over-expression of myocardin in corpus cavernosum can reverse CCSM cells phenotypic modulation and alleviate erectile dysfunction of rats with diabetes.In summary, we suppose that PDGFBB down-regulating myocardin may play an important role in hypoxia-induced phenotypic modulation of rat CCSM cells. In recent years, PDGFBB is thoroughly studied in the cardiovascular system disease, however, whether does hypoxia can promote the phenotypic modulation of CCSM cellsand PDGFBB secretion, and whether does the latter down-regulates myocardin in CCSM cells and induces its phenotypic modulation is still unclear at present. In this study, we cultured CCSM cells under hypoxia condition or treated CCSM cells with restructuring PDGFBB, and observed the effect of hypoxia on the phenotypic modulation of CCSM cells and the secretion of PDGFBB in cell culture supernatant, and the effect of PDGFBB on the phenotypic modulation of CCSM cells and the levels of myocardin in CCSM cells, to understand the important molecular mechanism of hypoxia-induced phenotypic modulation of rat CCSM cells, which may provide a basic theory for the treatment of ED.METHODS1. Isolation, culture and identification of rat CCSM cells.We establishment of the original generation of CCSM cells through the modified tissue sticking method, further by the method of differential velocity adherent duringcell subculture, we could obtain high-purity CCSM cells.2. Effect of hypoxia on the phenotypic modulation of CCSM cells and PDGFBB secretion in cell culture supernatant.Taking the second generation CCSM cells with stable biology feature, we had cultured CCSM cells for 24h and 48h in low oxygen training box with 2.5% oxygen concentration, whereas the cells in control group were in 5%CO2 training box withnormoloxygen concentration. The mRNA expressions of transcription factor myocardin and contractile phenotype markers α-SMA, SMMHC and smoothelin in CCSM cells were determined by real-time fluorescence quantitative PCR (qRT-PCR), the protein expression of myocardin in CCSM cells was examined by western blotting, and the expression of PDGFBB at protein level in cell culture supernatant from each group wasdetermined by Elisa.3. Effect of PDGFBB on the proliferation, migration and the phenotypic modulation of CCSM cells and the expression of myocardinin CCSM cells.Using the second generation cells for experiments, we observed the influence of PDGFBB in different concentrations on the proliferation of rat CCSM cells by CCK-8, and filtered out the optimum PDGFBB concentration. Then, we observed the effect of PDGFBB in the optimum concentration on the migration of CCSM cells in vitro by the scratch assay. After treated with PDGFBB in the optimum concentration or without PDGFBB for 24h and 48h, the mRNA expressions of transcription factor myocardin and contractile phenotype markers a-SMA, SMMHC in CCSM cells were determined by real-time fluorescence quantitative PCR (qRT-PCR).After CCSM cells had treated with PDGFBB in the optimum concentration for 0,24 and 48h, the expression of myocardin at protein level was examined by western blotting.RESULTS1. Results of isolation, culture and identification of rat CCSM cellsObserved under the inverted phase contrast microscope, there were few spindle cells climbing out from the edge of tissue blocks at the 4th or 5th day of primary culture and gradually forming cell halo, to 22th day cells approached confluence approximately. The cells filtered by the method of differential velocity adherent were mostly spindle shaped with directional arrangement, and a small part of the cells were star-shaped. A typical swirling or "peak-valley" like growth of CCSM cells were displayed when the cells grow crowdedly.Immunofluorescence staining showed that under the fluorescence microscope, the cells were spindle shaped or star-shaped, and all cell nuclei were showed blue fluorescence. About 97% of cells cytoplasm showed green fluorescence were positive fora-SMA, about 96.5% of cells cytoplasm showed red fluorescence were positive for smoothelin.2. Hypoxia down-regulate myocardin and contractile phenotype markers a-SMA, SMMHC and smoothelin in CCSM cells.The resuts of qRT-PCR showed that compared with the control group, the mRNA levels of myocardin in CCSM cells were significantly decreased in hypoxia group both at 24h and 48h(both P<0.001), Similarly, at the mRNA level, the expressions of a-SMA in CCSM cells were significantly decreased(both P<0.001), the expressions of SMMHC were also significantly decreased(24h P< 0.001,48h P<0.05, respectively), and the expressions of smoothelin were significantly decreased(24h P< 0.001,48h P<0.05, respectively). The resuts of western blotting showed that compared with the control group, the protein levels of myocardin were significantly decreased in hypoxia group both at 24h and 48h(both P<0.05). Obviously, hypoxia reduced the expressions of myocardin and contractile phenotype markers a-SMA, SMMHC and smoothelin in CCSM cells, which shows that the phenotypic modulation of CCSM cells could be induced by hypoxia.3. Hypoxia promotes the PDGFBB secretion of CCSM cells.We examined the expressions of PDGFBB at the protein level in cell culture supernatant from each group by the Elisa assay. Compared with the control group, the levels of PDGFBB were significantly increased in hypoxia group both at 24h and 48h (both P<0.05).4. PDGFBB markedly promotes the proliferation and migration of CCSM cells, and the optimum concentration of PDGFBB on CCSM cells is 12.5ng/ml.Effect of PDGFBB on the proliferation of CCSM cells:CCSM cells were treated with PDGFBB in different concentrations (0,2.5,5.0,12.5and 25.0ng/ml), the proliferation of cells in each group was measured with the method of CCK-8, and the results were recorded in terms of OD value. Compared with the control group,The proliferation rates of CCSM cells in different concentrations PDGFBB were significantly higher than that of control group both at 24h and 48h(all P<0.001).The most obvious effect on the proliferation of CCSM cells was observed when they were treated with 12.5ng/m PDGFBB, thus, we considered that the optimum concentration of PDGFBB on CCSM cells is 12.5ng/ml.Effect of PDGFBB on the migration of CCSM cells:The result of the scratch assay showed that compared with the control group, the migration rates of CCSM cells in 12.5ng/ml PDGFBB were significantly higher than that of control group both at 12h and 24h.5. PDGFBB significantly down-regulate myocardin and contractile phenotype markers a-SMA and SMMHC in CCSM cells.The resuts of qRT-PCR showed that compared with the control group, the mRNA levels of myocardin in CCSM cells were decreased by 45% and 66% in PDGFBB group both at 24h and 48h, respectively, Similarly, at the mRNA level, the expressions of a-SMA in CCSM cells were decreased by 28% and 50%, respectively, and the expressions of SMMHC were decreased by 44% and 58%, respectively. That means PDGFBB can promote the phenotypic transition of CCSM cells. The results of western blotting showed that compared with the control group (at Oh), the protein level of myocardin was decreased at 24h (P<0.001). Similarly, compared with the control group (at Oh), the protein level of myocardin was significantly decreased at 48h (P<0.001). Obviously, the protein level of myocardin wasgradually decreased within 48h when CCSM cells were treated with PDGFBB. To sum up, PDGFBB promotes the phenotypic transition of CCSM cells, maybe through down-regulating myocardin.CONCLUSION1. High purity CCSM cells can be obtained bythe modified tissue sticking method and the method of differential velocity adherent.2. The phenotypic modulation of CCSM cells can be induced by hypoxia.3. Hypoxia promotes the PDGFBB secretion of CCSM cells.4. PDGFBB markedly promotes the proliferation and migration ofCCSM cells, and the optimum concentration of PDGFBB on CCSM cells is 12.5ng/ml.5. PDGFBB promotes the phenotypic transition of CCSM cells, maybe through down-regulating myocardin.6. In summary, PDGFBB down-regulating myocardin may play an important role in hypoxia-induced phenotypic modulation of rat CCSM cells. |
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