Role Of Connective Tissue Growth Factor In The Proliferation And Extracellular Matrix Accumulation Of Pulmonary Artery Smooth Muscle Cells

PARTⅠThe relationship between CTGF expression of pulmonary arteries and smoke-induced pulmonary arteries remodelingAIM: To study the relationship between smoke-induced pulmonary arteries remodeling and CTGF expression of pulmonary arteries. METHODS: Twenty-four male Wistar rats were randomly assigned into control group and smoke exposure group, and the latter was further divided into three time points of 1, 2, 3 months. Pulmonary arteries remodeling was observed by Hematoxylin-Eosin staining, and collagen contents were investigated by sirius red staining. Immunohistochemistry methods were used to determine Proliferating Cellular Nuclear Antigen (PCNA) expression and Western Blot were performed to determine CTGF protein expression of pulmonary artery smooth muscle. The CTGF mRNA expressions of pulmonary arteries were evaluated by RT-PCR. The difference between the groups was analyzed. RESULTS: (1) The pulmonary artery WA% of four groups were (30.5±1.6)%, (43.3±2.0)%, (49.3±1.2)%, (60.3±2.9)%. Compared to control group, there were significant increases in smoke exposure groups (P<0.01). (2) The collagen thickness of pulmonary artery wall were respectively (4.5±0.7)μm, (8.8±1.2)μm, (11.8±2.7)μm, (15.3±2.9)μm. Collagen especially typeⅠcollagen was abnormally deposited in pulmonary artery walls of smoke exposure groups. (3) The PCNA protein expressions of pulmonary artery walls were respectively (0.082±0.022), (0.147±0.032), (0.286±0.022), (0.297±0.041). Compared to control group, there were significant increases in smoke exposure groups (P<0.01). (4) The CTGF mRNA expressions of pulmonary artery walls were respectively (0.032±0.012), (0.644±0.351), (2.749±0.458), (2.337±0.149). There were markedly differences among four groups (P<0.01). (5) The CTGF protein expressions of pulmonary artery walls were respectively (0.218±0.037),(0.754±0.142),(1.439±0.348),(2.267±0.519). There were markedly differences among four groups (P<0.01). (6) Both CTGF mRNA and protein were positively correlated with WA% (r=0.841, P<0.01; r=0.862, P<0.01). Similarly, both CTGF mRNA and protein were positively correlated with PCNA expression (r=0.869, P<0.01; r=0.887, P<0.01). CONCLUSION: Pulmonary arteries remodeling happened in early period after smoke exposure, and became worse and worse. CTGF expressions of pulmonary arteries in smoke exposure groups were obviously increased, and may play an important role in pulmonary arteries remodeling.PARTⅡRole of Connective Tissue Growth Factor on Rat Pulmonary Arteries Remodeling induced by smoke exposureObjective To elucidate the role of connective tissue growth factor (CTGF) in rat pulmonary arteries remodeling induced by smoke exposure. Methods Thirty-five male Wistar rats were randomly assigned into normal control group(A), smoke exposure one month group(B), smoke exposure and large dose CTGF antisense oligonucleotide (ASON) one month group(C), smoke exposure and low dose CTGF ASON one month group(D), smoke exposure two month group(E), smoke exposure and large dose CTGF ASON two month group(F), smoke exposure and low dose CTGF ASON two month group(G). Pulmonary arteries remodeling was observed by Hematoxylin-Eosin staining, and the CTGF mRNA expressions of pulmonary arteries were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry methods were performed to determine proliferating cellular nuclear antigen (PCNA) and CTGF protein expression of pulmonary artery smooth muscle. The difference between the groups was analyzed. Results (1) The pulmonary artery WA% of seven groups were respectively (28.6±1.2)%, (42.5±2.3)%, (33.7±1.8)%, (42.1±2.4)%, (49.6±2.1)%, (34.3±1.9)% and (38.4±2.0)%. The pulmonary artery walls were much thinner in CTGF ASON treatment groups than those of smoke exposure groups. (2) The PCNA protein expressions (A value) of pulmonary artery smooth muscle were respectively (0.102±0.026), (0.186±0.033), (0.146±0.022), (0.179±0.032), (0.348±0.041), (0.149±0.030) and (0.156±0.034). The expressions of CTGF ASON treatment groups were significant decreased than those of smoke exposure groups. (3) The CTGF protein expressions (A value) of pulmonary artery smooth muscle were respectively (0.098±0.015), (0.159±0.023), (0.118±0.017), (0.153±0.022), (0.406±0.036), (0.109±0.012) and (0.146±0.024). The expressions of CTGF ASON treatment groups were significant decreased than those of smoke exposure groups. (4) The CTGF mRNA expressions (ACTGF/Aβ-actin) of pulmonary artery were respectively (0.051±0.010), (0.823±0.096), (0.216±0.056), (0.810±0.085), (2.452±0.267), (0.207±0.062) and (0.509±0.067). Compared to smoke exposure groups, there were significant decreases in CTGF ASON treatment groups. (5) Both CTGF mRNA and protein were positively correlated with WA% (r=0.841, P<0.01; r=0.902, P<0.01). Similarly, both CTGF mRNA and protein were positively correlated with PCNA expression (r=0.929, P<0.01; r=0.887, P<0.01). Conclusion Administration of CTGF ASON in vivo could significant reduce the rat pulmonary arteries remodeling induced by smoke exposure, and CTGF may play an important role in pulmonary hypertension.PARTⅢEffects of cigarette smoke extract on proliferation and CTGF expression of rat pulmonary artery smooth muscle cellsObjective To investigate the effects of cigarette smoke extract (CSE) on proliferation and connective tissue growth factor (CTGF) expression of rat pulmonary artery smooth muscle cells (rPASMC). Methods Cultured rPASMCs were respectively divided into 6 groups randomly, group A (normal control group), group B (CSE group), group C (hypoxia control group), group D (CSE and rhTGF-β1 group), group E (CSE and CTGF sense oligonucleotide group), group F (CSE and CTGF antisense oligonucleotide group). Then, cell proliferation was measured by MTT colorimetry and proliferating cellular nuclear antigen (PCNA) immunocytochemical staining, and expression of CTGF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemisty respectively. Enzyme-linked immunosorbent assay (ELISA) method was performed to determine collagen type ? contents of supernatant. The difference between the groups was analyzed. Results In rPASMCs, the MTT result in group B (0.311±0.009) was significant higher than those in group A (0.230±0.010), group F (0.260±0.004), and lower than those in group D (0.351±0.014). There was no significant difference between group B and group E (0.303±0.011). The tendency of PCNA protein expression was similar with MTT. The CTGF protein expression of group B (0.314±0.005) was significant higher than those of group A (0.199±0.005), group F (0.202±0.009), and lower than those of group D (0.392±0.008). There was no significant difference between group B and group E(0.323±0.007). The tendency of CTGF mRNA expression was similar with protein expression. The type I collagen content of supernatant of cultured rPASMCs was measured by ELISA. The content of group B [(78.7±1.3)ng/ml] was significant higher than those of group A [(50.9±1.0)ng/ml], group F [(51.4±0.6)ng/ml], and lower than those of group D [(104.9±2.0)ng/ml]. There was no significant difference between group B and group E [(81.7±1.8)ng/ml]. Conclusion CSE could induce rPASMCs proliferation, and CTGF overexpression may be involved in the process. CTGF ASON showed high inhibitory activity against the PASMC proliferation of pulmonary arterial hypertension induced by CSE.PARTⅣThe relationship between CTGF expression of pulmonary arteries and pulmonary arteries remodeling of smokers and COPD patientsObjective To study the relation between Connective Tissue Growth Factor (CTGF) expression of pulmonary arteries and pulmonary arteries remodeling of smokers and Chronic Obstructive Pulmonary Disease (COPD) patients. Methods Twenty-four pulmonary lobes after operation because of lung cancer were assigned into non-smoke control group (N), smoker group (S), smoker and COPD group (S+C). The specimen were obtained far from the cancer focus (>5cm). Pulmonary arteries remodeling was observed by Hematoxylin-Eosin staining, and collagen contents of pulmonary artery wall were investigated by sirius red staining and picro-polarization method. Immunohistochemistry methods were performed to determine the Proliferating Cell Nuclear Antigen (PCNA) expression of pulmonary artery smooth muscle. The CTGF mRNA expressions of pulmonary arteries were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and CTGF protein expressions of pulmonary arteries were observed by Western Blot. The difference between the groups was analyzed. Results (1) The percent of pulmonary artery wall area to total area (WA%) of there groups were (28.4±4.7)%, (46.3±3.5)% and (55.5±3.9)%. Compared to non-smoke control group, there were significant increases in S and S+C groups (P<0.01). (2) The collagen thickness of pulmonary arteries were respectively (6.4±1.6)μm, (15.9±2.4)μm and (16.4±2.3)μm. Collagen was abnormally increased and deposited in pulmonary artery walls of S and S+C groups (P<0.01). (3) PCNA expressions of pulmonary artery walls were respectively (0.084±0.006), (0.178±0.018) and (0.226±0.134). The expressions of S and S+C groups were significant increased than which of non-smoke control group (P<0.01). (4) CTGF mRNA expressions of pulmonary artery walls were respectively (0.095±0.015), (0.396±0.167) and (0.501±0.177). Compared to non-smoke control group, there were significant increases in S and S+C groups (P<0.01). (5) CTGF protein expressions of pulmonary artery walls were respectively (0.158±0.021), (0.545±0.095) and (1.303±0.191). Compared to non-smoke control group, there were significant increases in S and S+C groups (P<0.01). (6) Both CTGF mRNA and protein expression of pulmonary arteries were positively correlated with WA% (r=0.815, P<0.01; r=0.799, P<0.01). Similarly, both CTGF mRNA and protein expression of pulmonary arteries were positively correlated with PCNA expression (r=0.838, P<0.01; r=0.821, P<0.01). Conclusion Pulmonary arteries remodeling were founded in merely smoker group, and the condition of COPD patients became worse. CTGF expressions of pulmonary arteries in S and S+C groups were obviously increased, and which may play an important role in pulmonary arteries remodeling.PARTⅤEffects of cigarette smoke extract on proliferation and CTGF expression of human pulmonary artery smooth muscle cellsObjective To investigate the effects of cigarette smoke extract (CSE) on cell proliferation and connective tissue growth factor (CTGF) expression of human pulmonary artery smooth muscle cells (hPASMC). Methods Cultured hPASMCs were respectively divided into 6 groups randomly, group A (normal control group), group B (hypoxia control group), group C (CSE group), group D (CSE and rhTGF-β1 group), group E (CSE and CTGF sense oligonucleotide group), group F (CSE and CTGF antisense oligonucleotide group). Then, cell proliferation was measured by MTT colorimetry and proliferating cellular nuclear antigen (PCNA) immunocytochemical staining, and expression of CTGF mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemisty respectively. Enzyme-linked immunosorbent assay (ELISA) method was performed to determine collagen type ? contents of cell culture supernatant. The difference between the groups was analyzed. Results In hPASMCs, the MTT result in group C (0.275±0.007) was significant higher than those in group A (0.212±0.007), group F (0.245±0.005), and lower than those in group D (0.322±0.009). There was no significant difference between group C and group E (0.283±0.010). The tendency of PCNA protein expression was similar with MTT. The CTGF protein expression of group C (0.368±0.006) was significant higher than those of group A (0.208±0.003), group F (0.218±0.004), and lower than those of group D (0.432±0.008). There was no significant difference between group C and group E(0.380±0.009). The tendency of CTGF mRNA expression was similar with protein expression. The type I collagen content of supernatant of cultured hPASMCs was measured by ELISA. The content of group C [(215.4±3.9)ng/ml] was significant higher than those of group A [(140.0±1.8)ng/ml], group F [(143.0±1.1)ng/ml], and lower than those of group D [(257.5±3.4)ng/ml]. There was no significant difference between group C and group E [(211.2±2.5)ng/ml]. Conclusion CSE could induce hPASMCs proliferation, and CTGF overexpression may be involved in the process. CTGF ASON showed high inhibitory activity against the PASMC proliferation of pulmonary arterial hypertension induced by CSE.