Corpus Cavernous Smooth Muscle Cell Phenotypic Modulation Induced By Hypoxia And The Effect Of Salidroside Intervent In Rats

It is well known that Corpus cavernous smooth muscle cell (CCSMC) is the main effector cells in the progress of regulation and maintain in male erection. It has been observed that the changes of CCSMCs content and hypoxic condition in penile tissue is the common pathophysiology progress of erectile dysfunction (ED) induced by diabetes, hypertension, hyperlipidemia, and prostate cancer radical prostatectomy and cardiovascular disease. There are various studies showed that smooth muscle cells participate in Multiple organ fibrosis through the modulation of phenotypic stimulated by various localization cues including hypoxia. However, there is no report about the relationship between CCSMCs and fibrosis.Purpose:To investigate the changes of CCSMCs induced by hypoxic condition and the effect of salidroside intervention in vitro and in vivo.Materials and Methods:1. Part of the in vitro:The isolation and culture of primary CCSMCs was prepared by tissue explant adherent method and trypsin digestion method and identified by immunofluorescence (IF) and transmission electron microscopy (TEM). Hypoxia cell model was established using a hypoxia chamber and assess CCSMC morphology and ultrastructure changes after hypoxia treatment by using light microcopy and TEM. CCSMCs proliferation and apoptosis were assessed with MTT analysis and flow cytometry analysis. Assess the changes of hypoxia inducible factors-la (HIF-la), phenotypic modulation related transcription factor, such as Myocd, Elk-1and KLF, and phenotypic modulation marker proteins (ACTA2> Vimentin、CNN1). Moreover, we knocked down and overexpressed the level of HIF-la with virus transfection to verify the progress of phenotypic modulation induced by hypoxic condition.2. Part of in vivo:Hypoxia in the rat ED model was established by bilateral cavernous neurotomy (BCN). All rats were treated with sham (n=10) or BCN (n=10) surgery. At12weeks flowing BCN, Intracavernosal pressure (ICP) was measured through multifunctional biological information collector and harvesting the penile tissues for further assessment. The penile tissues were assessed for the level of fibrosis by Masson trichrome staining and CCSMC phenotype markers (a-SMA, SMMHC and Vimentin) and HIF-al by immunohistochemistry (IHC) and western blot. Morphological structure of CCSMCs was evaluated by hematoxylin-eosin (H&E) staining and (TEM).3. The effect of salidroside intervention:1). In vitro:CCSMC were pretreated with salidroside (3ug/mL and30ug/ml) and then treated with hypoxia for48h. Morphological structure of CCSMCs was evaluated by light microscope and TEM. The effect of salidroside intervention on CCSMCs proliferation and apoptosis were assessed with MTT analysis and flow cytometry technic. The level of HIF-la, CCSMC phenotype markers and the degree of synthetic collagen-III were assessed by western blotting.2). In vivo:Hypoxia rat model was established as previous described, all rats were treated with sham (n=10), BCN (n=10) surgery and salidroside (6mg/kg) for12weeks. The assessments of ICP, HIF-la and CCSMC phenotype markers were the same as previous described.Results:1. The primary CCSMCs were spindle, high expression of a-SMA、desmin and with a lot of myofilament bundle. After hypoxia treatment, CCSMCs became hypertrophic with loss of myofilamentbundles and formation of an extensive rough endoplasmic reticulum (RER) with inhibited cell proliferation and enhanced cell apoptosis. This was accompanied with the increased synthesis of TGF-β1and types I and III collagen. Moreover, smooth muscle cell phenotypic markers were also attected by hypoxic conditions, as indicated by the decrease in α-SMA, desmin and CNN1expression and increase in vimentin expression. These changes corresponded to changes in associated transcriptional factors, such as the increase in Elk-1and KLF-4expression and decrease in Myocd expression. In addition, a HIF-la knockdown effectively reversed the hypoxia-induced CCSMC phenotype, whereas its overexpression induced the dedifferentiation phenotype.2. Erectile function in BCN rats was significantly reduced compared to sham rats. BCN increased hypoxia-inducible factor (HIF)-la and collagen protein expression in corpora cavernous tissue. The H&E-staining and TEM showed CCMSCs in BCN rats undergo a process of hypertrophic and formation of rough endoplasmic reticulum (RER). Compared with the sham rats, the expression of CCSMC phenotypic markers, such as a-SMA and desmin, were markedly decreased in BCN rats, while vimentin protein was significantly increased.3. Salidroside effectively inhibit the hypoxia-induced CCSMCs apoptosis and reverse CCSMCs phenotype modulation in vitro. Salidroside inhibit the expression of HIF-1α and the extent of fibrosis induced by hypoxia. Moreover, salidroside increased the expression of contractile protein (a-SMA and desmin) as well as reduced the expression of synthetic protein (vimentin).Conclusion:1. Successfully established the method of isolating and culturing primary CCSMCs in vitro.2. Successfully established the hypoxic model of CCSMCs in vitro.3. CCSMCs undergo a modulation of phenotype under hypoxic condition in vitro, HIF-α1involed in these processes.4. Successfully established the hypoxia model in ED rat induced by BCN.5. BCN-mediated ED rats occured in corpus cavernous hypoxia, fibrosis and CCSMCs phenotypic modulation.6. Salidroside had an effect of anti-hypoxia and reversed CCSMCs phenotypic modulation in vitro and in vivo.