Effect Of Calcitonin Gene-related Peptide On Proliferation And Phenotype Modulation Of Corpus Cavernosum Smooth Muscle Cells In Diabetic Rats With Erectile Dysfunction

Research BackgroundErectile dysfunction (ED) is a kind of common disease of Andrology. Epidemiological investigations reveal that there are about 152,000,000 men being troubled by ED currently, and men who are over 45 years old suffer from significantly higher risk of ED. It is supposed that there will be 322,000,000 men suffered from ED all over the world until the year of 2025. Diabetes and ED have been proved to be highly related. Diabetes has been recognized as one of the most important risk factors for ED by most researchers, while ED has been a common complication of diabetes. The reports from WHO point out that 50% to 75% of the diabetic patients are accompanied with different degrees of ED, and the incidence of ED in diabetes group is three times higher than that in normal group.The pathogenesis of how does diabetes lead to ED may be involved in many aspects, however, vascular factors is an important aspect. 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. Corpus cavernosum smooth muscle 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 (VSMC) can be further divided into two types:the contraction type (differentiated) and synthetic type (de-differentiated). The major fuction 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 VSMC can transform into synthetic type ones and get to be proliferation when the vascular is injured or the VSMC cultured in vitro are stimulated by the growth factors. That changing of VSMC in mophorlogy, structure and fuction is defined as phenotype modulation. The proliferation and migration of VSMC can be the important pathological changes in the formation of atherosclerosis, and the vascular remodeling caused by phenotype modulation of VSMC is the basis for vascular restenosis, atherosclerosis and other vascular lesions. Because of the similarity between the sinusoid of corpus cavernosum and the vascular systems, our preliminary study has confirmed that corpus cavernosum smooth muscle cells (CCSM) derived from animal methods with ED caused by diabetes or hypertension also exist the phenomenon of phenotype modulation, which is associated with the occurrence of ED.Therefore, exploring some related factors basing on the abnormal proliferation and the phenotype modulation of smooth muscle cells that could have an effect on the proliferation and phenotype modulation of CCSM will be a new idea for the pathogenesis and treatment of ED. Calcitonin gene-related peptide (CGRP) is a kind of bioactive peptides which widely distributes in the central and peripheral nervous system and cardiovascular system, and CGRP has many functions such as strong expansion of peripheral vascular, lowering blood pressure, protecting the ischemic myocardium, inhibiting proliferation of smooth muscle cells, etc. Especially, recent researchs have found that CGRP could inhibit the proliferation of vascular smooth muscle cells, and it could make synthetic type VSMC transform into contraction type ones. However, it has not been reported whether CGRP could have the same effect on CCSM which are similar to VSMC as that on VSMC, especially when the CCSM are in diabetic state.It is important to choose the right markers of cell phenotype, which are used to judge whether there has been transformation of cell phenotype. Calponinl (Cnnl) is a smooth muscle-specific regulatory protein, which is discovered as a new specific marker for the phenotype of smooth muscle in recent years. Because of its only high expression in the contraction type VSMC, it is more specific for the differentiation of phenotype. Osteopontin (OPN) is a secreted acidic glycoprotein, which is related to bone formation, bone absorption, calcium regulation, some tumors, migration and proliferation of VSMC etc. OPN is considered as a marker of synthetic type smooth muscle cells. Increase in the expression of calponin 1 or decrease in OPN demonstrate that the phenotype of smooth muscle cells modulated from the synthetic type to the contraction type.Basing on the studies mentioned above, our research project will Establish a cell culture model in vitro of corpus cavernosum smooth muscle cells derived from experimental diabetic rats with erectile dysfunction at first. Then we add CGRP to the cell model cultured in vitro, and compare the proliferation of CCSM and the expression of Cnnl mRNA and OPN mRNA between CGRP group and no-CGRP group. At last, we could judge whether CGRP has an influence on the proliferation and phenotype modulation of CCSM derived from diabetic rats with ED.ObjectiveTo learn the biological characteristics of diabetic CCSM by establishing a cell culture model in vitro of CCSM derived from diabetic rats with ED. Add CGRP to the cell model cultured in vitro, and explore the effect of CGRP on the proliferation and phenotype modulation of CCSM by analyzing the proliferation of CCSM and the expression of markers of cell phenotype.Methods1.15 8-week-old SPF male Sprague Dawley (SD) rats were divided into two groups randomly:(1) Diabetic group:10 rats, induced by a single ip of streptozotocin (STZ) (60mg/kg); (2) Control group:5 rats, were injected with an equivalent volume of citrate buffer. Take the random blood glucose level which was higher than 16.7 mmol/L as the criterion for successful diabetic animal model by measuring blood glucose at 72 hours,1 week and 2 weeks. Subcutaneous injection of apomorphine (APO) (100μg/kg) was given, and the frequencies of penile erection in each rat within 30 Minutes were observed and counted after giving APO. The rats with ED were selected out according to not erection. After getting corpus cavernosum derived from diabetic and control groups, CCSM were cultured using the method of modified tissue sticking culture, and the morphology and proliferation of the cells were observed. The bred cells were identified by immunohistochemical staining for a-smooth muscle actin(a-SM-Actin) and desmin.2. The second-generation cells cultured were used for our research. The diabetic and normal cells were divided into four groups separately:Group A was normal cells exposed to CGRP; Group B was normal cells unexposed to CGRP; Group C was diabetic cells exposed to CGRP; Group D was diabetic cells unexposed to CGRP. The four groups of cells were exposed to CGRP (1,10, 100nmol/L) separately. PBS buffer was used as negative control group. Proliferation of cells in each group was measured with the method of MTT, and the results were recorded in terms of OD. Cell growth curve of each group was mapped and the percentage of inhibition for the cell proliferation was calculated. The percentage of inhibition for cell proliferation was derived from the following formula:inhibition(%) =(ODcontrol-ODCGRP)/ODcontrol×100%.3. Real-time fluorescence quantitative RT-PCR (qRT-PCR) was used to analyze the relative expression of Cnnl mRNA and OPN mRNA after adding CGRP(100nmol/L) to each group of cells for 24 hours.Results1. Animal model of diabetic rat with ED was established successfully. The primary culture of CCSM was performed successfully by the method mentioned above, the cells could be seen from the small tissue pieces 3 days later, and 16-18 days afterward the cells approached confluence approximately. A typical "hill-valley" growth pattern was displayed after cells were passaged. Immunohistochemica staining for a-smooth muscle actin(a-SM-Actin) and desmin were possitive.2. Values of OD derived from the groups which were exposed to CGRP at different time were all significantly lower than that from control group (P< 0.05)Inhibition rates of diabetic group were significantly higher than that of control group at 48 and 72 hours (P< 0.05)The inhibitory rates of CGRP in different concentration to diabetic CCSM showed that inhibition rates of the group with CGRP (100nmol/L) were significantly higher than that of the other two groups (F=12.930, P=0.001; F=20.216, P=0.000).3. There were no significant statistical difference of expression of Cnn1 mRNA or OPN mRNA between normal cells exposed to CGRP and that unexposed to CGRP. The expression of Cnn1 mRNA of diabetic cells exposed to CGRP were significantly higher than that of diabetic cells unexposed to CGRP (P= 0.000), when the expression of OPN mRNA got lower (P< 0.05)Conclusion1. Using the method of modified tissue sticking culture to establish the cell culture model in vitro of CCSM from diabetic rats with ED is convenient and stable.The CCSM from diabetic rats with ED proliferates faster than normal CCSM.The difference of morphology between diabetic and normal CCSM is unobvious by light microscope.2. Different concentrations of CGRP all have an effect of inhibition on the diabetic CCSM, and the degree of inhibition increases with the concentration of CGRP increasing.3. CGRP could make the phenotype of diabetic CCSM transform from synthetic type to contraction type.It means CGRP could reverse the phenotype modulation of diabetic CCSM.