The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension.

BACKGROUND: Recent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. MATERIAL AND METHODS: Our research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. RESULTS: Chronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. CONCLUSION: Our research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

[Monocrotaline pyrrole induces A549 cells and activates TGF-ß1/SMAD2/SMAD3 pathway to promote proliferation and migration of human pulmonary artery smooth muscle cells].

Objective To explore the effects of A549 cells on the proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) and its mechanism. Methods A549 cells and HPASMCs were cultured in vitro. The A549 cells were randomly divided into four groups: control group, dimethylformamide (DMF) solvent group, monocrotaline pyrrole (MCTP) group, MCTP combined with SB431542 group. The cells were assigned into four groups: HPASMC group, A549 and HPASMC co-culture group, MCTP-stimulated A549 and HPASMC co-culture group, MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and IL-6-stimulated HPASMC group. A549 cell viability was detected by CCK-8 assay. The level of IL-6 in the A549 cell culture supernatant was tested by ELISA. The mRNA levels of SMAD2 and SMAD3 in the A549 cells were detected by real-time PCR. The protein levels of TGF-ß1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were detected by Western blot analysis. The protein levels of TGF-ß1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were examined by Western blot analysis. The protein levels of osteopontin (OPN) and proliferating nuclear antigen (PCNA) in the HPASMCs were determined by Western blot analysis. The migration ability of HPASMCs was measured by wound healing and TranswellTM assay. Results In the A549 cells, compared with the control group, the cell proliferation ability decreased, the production of IL-6 increased, the mRNA levels of SMAD2 and SMAD3, and the expression of TGF-ß1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly increased in the MCTP group. Compared with the MCTP group, the cell proliferation ability increased, the production of IL-6 decreased, the mRNA levels of SMAD 2 and SMAD3, and the expression of TGF-ß1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly decreased in the MCTP and SB431542-stimulated group. In the co-culture system, compared with the HPASMC group, the expression of PCNA and OPN proteins and migration ability did not change significantly in the A549 and HPASMC co-cultured group. The expression of PCNA and OPN proteins significantly increased in the MCTP-stimulated A549 and HPASMC co-culture group, and the cell migration ability increased. Compared with the MCTP-stimulated A549 and HPASMC co-culture group, the expression of PCNA and OPN proteins significantly decreased in MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and the cell migration ability decreased. Compared with the HPASMC group, the migration ability of HPASMCs increased and the expression of PCNA and OPN proteins increased in the IL-6 control group. Conclusion Activation of the TGF-ß1/SMAD2/SMAD3 signaling pathway in A549 cells induced by MCTP increases IL-6 secretion, thus promoting the proliferation and migration of HPASMCs.

[Up-regulation of connexin 43 (Cx43) by angiotensin II promotes the proliferation and migration of human pulmonary artery smooth muscle cells].

Objective To explore the role of Cx43 in the proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) induced by angiotensin II (Ang II). Methods HPASMCs were cultured in vitro and randomly divided into four groups: control group, Ang II group, Ang II combined with DMSO group, and Ang II combined with candesartan group, and cells were collected in logarithmic growth phase. Cell viability was detected by CCK-8 assay; the migration ability of HPASMCs were measured by wound-healing and TranswellTM assay. The protein levels of Cx43, osteopontin (OPN), proliferating cell nuclear antigen (PCNA), SMAD2 and SMAD3 in HPASMCs were detected by Western blot analysis. Results Compared with the control group, the expression of OPN and PCNA proteins significantly went up in Ang II group, and the cell proliferation and migration ability increased. The cell proliferation and migration ability of the Ang II combined with candesartan group were significantly lower than that in the Ang II group. Compared with the control group, the Cx43 protein and its phosphorylation level increased significantly in the Ang II group, and the protein expression of SMAD2 and SMAD3 increased, while the expression of each protein in the Ang II combined with candesartan group was significantly lower than those in the Ang II group. Conclusion Ang II up-regulates the expression of Cx43 protein to promote the proliferation and migration of HPASMCs, which may be related to the activation of SMAD2/3 signaling pathway.

[Effects of Ramipril on the expression of connexin 43 in cerebral arteries of spontaneously hypertensive rats].

The present study was designed to examine whether Ramipril (an inhibitor of angiotensin-converting enzyme) affected spontaneous hypertension-induced injury of cerebral artery by regulating connexin 43 (Cx43) expression. Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were randomly divided into WKY, WKY + Ramipril, SHR, and SHR + Ramipril groups (n = 8). The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin-eosin (HE) staining was used to show vascular remodeling. The expression and distribution of Cx43 was determined by using immunofluorescence and immunohistochemistry analysis. The protein and mRNA levels of Cx43 were examined by Western blot and real-time PCR analysis, respectively. The results showed that chronic Ramipril treatment significantly attenuated blood pressure elevation (P < 0.01, n = 8) and blood vessel wall thickness in SHR (P < 0.01, n = 8). The cerebral artery contraction rate in the SHR group was higher than that in the WKY group (P < 0.05, n = 8). The cerebral artery contraction rate in the SHR + Ramipril group was lower than that in the SHR group (P < 0.05, n = 8). Pretreatment with 2-APB (Cx43 non-specific blocker) or Gap26 (Cx43 specific blocker) significantly decreased the vasoconstriction rate, while pretreatment with AAP10 (Cx43 non-specific agonist) significantly increased the vasoconstriction in the SHR + Ramipril group (P < 0.05, n = 8). In addition, the expression of Cx43 mRNA and protein in cerebral arteries of SHR group was higher than that of WKY group (P < 0.05, n = 8). The mRNA and protein expression of Cx43 in cerebral arteries of SHR + Ramipril group was significantly lower than that of SHR group (P < 0.05, n = 8). These results suggest that Ramipril can down-regulate the expression of Cx43 mRNA and protein in cerebral arterial cells of SHR, lower blood pressure, promote vasodilation, and improve arterial damage and vascular dysfunction caused by hypertension.

[Candesartan inhibits the angiotensin II-induced proliferation and migration of rat thoracic aortic smooth muscle A7r5 cells and its mechanism].

Objective To investigate the effect of candesartan on angiotensin II (Ang II)-induced proliferation and migration of vascular smooth muscle cells and its effect on connexin 43 (Cx43). Methods A7r5 cells were cultured in vitro and randomly divided into control group, AngII group and AngII combined with candesartan group. Cell viability was detected by CCK-8 assay; the migration and invasion ability of A7r5 cells were measured by wound-healing and TranswellTM assay; the expression and distribution of Cx43 on A7r5 cells were detected by immunofluorescence assay. Cx43, osteopontin (OPN), proliferating cell nuclear antigen (PCNA), p-MEK1/2 and p-ERK1/2 protein levels of A7r5 cells were detected by Western blot analysis. Results Compared with the control group, the AngII group had higher cell proliferation and migration ability, and the AngII combined with candesartan group had a lower concentration than the AngII group. Cx43 was expressed in the A7r5 cell membrane and nuclear membrane. The expression of Cx43 were enhanced in the AngII group than in the control group, and the expressions of Cx43, OPN, PCNA, p-MEK1/2 and p-ERK1/2 significantly increased compared with AngII. The expression of protein significantly decreased in AngII combined with candesartan group. Conclusion Candesartan can reduce the proliferation and migration of smooth muscle cells induced by AngII, and its mechanism may be related to Cx43 and MEK/ERK signaling pathways.