| BACKGROUND&OBJECTIVEErectile dysfunction (ED) is a kind of common disease of Andrology, and men who are over45years old suffer from significantly higher risk of ED. Diabetes and ED have been proved to be highly related. Diabetes has been recognized as one of the most important risk factors for ED, while ED has been a common complication of diabetes. The pathogenesis of how does diabetes lead to ED may be involved in many aspects, Thus, exploring some related factors basing on the abnormal pathophysiology will be a new idea for treatment of ED. Corpus cavernosum smooth muscle (CCSM) is the structural basis of cavernous space relaxing and penile erection, which plays a key role in the change of hemodynamics when the penis erects. According to structure and function, vascular smooth muscle cells (SMC) can be further divided into two types:the contraction type and synthetic type. The major function of the former one is contraction, and that of the latter one is proliferation, migration, secretion and regulation of extracellular proteins, etc. The contraction type vascular SMC can transform into synthetic type ones and get to be proliferation when the vascular is injured or the vascular SMC cultured in vitro are stimulated by the growth factors. There is a structure of sinusoid in the corpus cavernosum, which is very similar with the vascular systems. Therefore, the corpus cavernosum is considered as a special kind of vascular tissue as well. However, to the best of our knowledge, the characteristics of the phenotypic modulation within CCSM cells under hyperglycemia condition in diabetic rats have not been well elucidated. Our previous study has demonstrated that the expression of SMA decreased in CCSM cells of diabetic rat with ED. Considering more genes having recently been proposed to be contractility-associated markers, we are prompted to further explore the possibility that phenotypic modulation of smooth muscle cells are involved in the development of diabetic ED.Myocardin belongs to the SAP (SAF-A/B, Acinus, PIAS) domain family of nuclear proteins, it is a remarkably potent transcriptional coactivator and the founding member of a class of muscle transcription factors expressed exclusively in cardiomyocytes and SMCs. It is well documented that the establishment of a differentiated phenotype in SMC is dependent on the activation of myocardin. It was reported that forced expression of myocardin transactivates multiple SMC-restricted transcriptional regulatory elements, remarkably, forced expression of myocardin in embryonic stem cells induces expression of multiple endogenous SMC genes including SM22a, smooth muscle myosin heavy chain (SMMHC), and SMa-actin. Mice harboring a null mutation in the myocardin gene survive only to embryonic day (E)10.5and exhibit obvious defects in the embryonic and extraembryonic vasculature including a block in SMC differentiation. Taken together, these data demonstrate that myocardin promotes SMC differentiation and the differentiated SMC phenotype.Considerable work had demonstrated that the establishment of a differentiated phenotype in SMC is dependent on the activation of myocardin. However, up to now, few reports were found about function and mechanism of myocardin in modulating of CCSM cell phenotype and improving erectile function in diabetic rats. In this study, we mainly focus on the functional alteration of myocardin in CCSM cell of diabetic rats by gene transfection. the aim is to insight into the role of myocardin gene in modulating of CCSM cell phenotype and improving erectile function in diabetic rats, and providing a molecular marker for ED treatment.METHODS1. Characterization of CCSM cell phenotype in diabetic rats with erectile dysfunctionThe cavernous tissue segments were subjected to qRT-PCR to determine the expressions of SMa-actin, SMMHC, smoothelin and calponin. Cell contractility in vitro and western blot analysis of SMa-actin and SMMHC in the cavernous tissues and cells were determined.2. Expression of myocardin and SRF in the cavernous tissue of diabetic ratsWestern blot and qRT-PCR were used to determine the protein and mRNA expression levels of myocardin and SRF.3. Effect of myocardin-overexpressed in modulating of CCSM cell phenotype and improving erectile function in diabetic ratsReplication-deficient recombinant adenovirus carrying myocardin gene under the control of cytomegalovirus promoter were generated. Erectile function was assessed by measuring intracavernous pressure (ICP) following electrostimulation of the cavernous nerves5-7days after vehicle or virus administration. After measurement of the erectile response, rats were killed and the serum was used to test testosterone levels, and the penile shaft was removed for qRT-PCR and histochemical analysis. qRT-PCR were used to determine the mRNA expression levels of myocardin, calponin, SMA, SMMHC, eNOS, nNOS and cGMP. Masson trichrom staining were used to assess the content of CCSM.RESULTS1. Characterization of CCSM cell phenotype in diabetic rats with erectile dysfunctionWe examined the expressions of contractility-associated genes at the mRNA level in the cavernous tissue from three groups by qRT-PCR. Compared with the control group and the DM group, the levels of SMA, calponin, SMMHC and smoothelin were significantly decreased in rats of the DM&ED group (P=0.001and0.020, P=0.014and0.012, both P<0.001, P=0.005and<0.001respectively). Expressions of contractility-associated genes were down-regulated in the DM group when compared with the control group, however, the difference did not show statistical significance (all P>0.05). To confirm whether decreased mRNA of contractility-associated genes was associated with a decrease at the protein level, western blot was performed. In accordance with expressions at the mRNA level, the levels of SMA and SMMHC at the protein level in the cavernous tissues were decreased in rats of the DM&ED group(all P<0.001). In order to get better understanding of the characterization of CCSM cell, we established and passaged primary cultures of cells from rat penile cavernous tissue fragments of three groups. No distinct morphological features were noted in the three types of cells. They had uniformly spindle-shaped morphology, and ’hill and valley’ appearance in the confluent state. Subsequently, the levels of SMA and SMMHC at the protein level in cultured CCSM cells were examined. We firstly found that when CCSM cells were placed in culture, they gradually lost their SMA and SMMHC proteins. Then, we chose CCSM cells from the steady early stage (the2nd passage), and decreased expressions of SMA and SMMHC proteins were found in rats of DM&ED group when compared with those of the other groups. To confirm whether the contractile function of CCSM cell can be affected by the decreased levels of contractility-associated genes, the lattice contractility assay was performed. Consequently, CCSM cells of the DM&ED group exhibited significantly less contractile than those of other groups in response to10%serum and growth factor calcium-ionophore (all P<0.001).2. Expression of myocardin and SRF in the cavernous tissue of diabetic ratsCompared with the control group (23.226±9.712), the myocardin mRNA levels in the cavernous tissue of diabetic rats (4.931±3.987) were significantly decreased (P<0.001), the SRF mRNA levels in diabetic rats (7.524±4.767) were also significantly decreased (P<0.001) compared with the control group (19.054±5.236). Concomitant with expressions at the mRNA level, the expressions of myocardin and SRF at the protein level in the cavernous tissue were less in diabetic rats (P<0.001). The expressions at mRNA level demonstrated positive correlation with these at protein level for myocardin (r=0.551, P<0.001) and SRF (r=0.689, P<0.001).3. Effect of myocardin-overexpressed in modulating of CCSM cell phenotype and improving erectile function in diabetic ratsErectile function was assessed by measuring intracavernous pressure (ICP) following electrostimulation of the cavernous nerves and mean arterial pressure (MAP)5-7days after vehicle or virus administration. MAP in adCMV-myocardin-transfected rats (A) were compared with vehicle-treated rats (B), and PBS-treated rats (C), and sham operation rats (D), and control rats (E), however, the difference did not show statistical significance(F=0.721, P=0.587). Compared with B group, C group and D group, the ICP were significantly increased in rats of A group (all P<0.001), The difference of ICP in A group did not show statistical significance when compared with E group (P>0.05). Compared with B group, C group and D group, ICP/MAP were significantly increased in rats of A group (P=0.033,0.004,0.007), The difference of ICP/MAP in A group did not show statistical significance when compared with E group (P>0.05). We examined the expressions of myocardin, calponin, SMA, SMMHC, eNOS, nNOS and cGMP at the mRNA level in the cavernous tissue by qRT-PCR. Compared with B group, C group D group and E group, the expressions of myocardin were significantly increased in rats of A group (F=14.017, P<0.001). Compared with B group, C group and D group, the expressions of SMA(F=17.177, P<0.001), SMMHC(F=8.459, P<0.001) and calponin(F=29.938, P<0.001) were significantly increased in rats of A group and E group. The difference of the expressions of SMA, SMMHC and calponin in A group did not show statistical significance when compared with E group. Compared with A group, B group, C group and D group, the expressions of eNOS(F=7.500, P<0.001), nNOS(F=25.923, P<0.001) and cGMP(F=39.666, P<0.001) were significantly increased in rats E group. The difference did not show statistical significance among A group, B group, C group and D group. The difference of serum testosterone levels did not show statistical significance among A group, B group, C group D, group and E group (F=0.033, P=0.998). Compared with A group, B group, C group and D group, the smooth muscle/collagen ratio were significantly increased in rats E group (P<0.001, P<0.001, P=0.001and P<0.001).CONCLUSION1. CCSM cell possesses the ability to modulate phenotype under hyperglycemia condition which could play a key role in the pathogenesis of diabetic erectile dysfunction.2. Diabetes can inhibit mRNA and protein expression of myocardin and SRF in penile corpus cavernosum tissue of rats, It is well documented that myocardin and SRF have a key role in maintaining differentiated phenotype in CCSM cells.3. Myocardin gene therapy can improve erectile function in diabetic rats, which has the role in modulating of phenotype in CCSM cells. |
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