Role of angiotensin Ⅱ in the regulation of platelet-derived growth factor receptor β subunit of vascular smooth muscle

AIM:To investigate the crosstalk between angiotensin Ⅱ (AngⅡ)-mediated and platelet-derived growth factor (PDGF)-mediated signal transduction in vascular smooth muscle proliferation.METHODS:A model of renal hypertension was made by two kidney/one-clip operation. Level of PDGF receptor β subunit of aorta was measured by Western Blot analysis. The effect of Ang Ⅱ on PDGF receptor β subunit expression was investigated in culture rat aortic vascular smooth muscle cells (VSMC).RESULTS:Systolic blood pressure obviously increased at 8th week after operation, whereas the level of PDGF receptor β subunit of aorta significantly increased by 126.6% (P＜0.05) in 2K1C rats compared with control group. The expression of PDGF receptor β subunit in cultured VSMC stimulated by AngⅡ was higher than that of control by 192.74%(P＜0.01). The effect of AngⅡ was inhibited remarkably by pretreated with losartan, a kind of specific AngⅡ receptor 1 (AT₁) subtype antagonist and U73122, a kind of phospholipase C inhibitor. The effect was partly blocked by PD98059, which inhibit the activity of mitogen-activated, ERK-activating kinase (MEK).CONCLUSION:AngⅡ-induced PDGF receptor β subunit expression is regulated by the AT₁ and its downstream signal molecule-PLC and ERK, might participate in the intracellular signal transduction pathway.     

Inhibitory effect of Salidroside on the proliferation of rabbit pulmonary artery smooth muscle cells under hypoxia

AIM: To investigate the effect of Salidroside on the proliferation, DNA synthesis, intracellular Ca²⁺ content of rabbit PASMC (pulmonary artery smooth muscle cells) under hypoxia. METHODS: Techniques of cell culture, MTT test, [³H][³H][³H]-TdR incorporation, fluo-3 and confocal laser scanning microscopy were used. RESULTS: The A value of MTT and [³H][³H]-TdR incorporation of PASMC increased significantly by 62% (P＜0.05) and 138% (P＜0.01) after 24 h hypoxia. Salidroside (32×10^-5 mol/L) inhibited the action of hypoxia on the proliferation of PASMC, the A value of MTT and [³H][³H]-TdR incorporation declined significantly by 29% (P＜0.05) and 37% (P＜0.01) compared with hypoxia group. A calcium channel blocker, verapamil could also inhibit the accelerative effect of hypoxia on the proliferation of PASMC. The intracelluler Ca²⁺ content of PASMC raised markedly under hypoxia, but the effect of hypoxia on the intracelluler Ca²⁺ content could be inhibited by Salidroside. CONCLUSION: Salidroside inhibited the proliferation, DNA synthesis of PASMC induced by hypoxia. The inhibitory action of Salidroside on the increase in intracellular Ca²⁺ concentration under hypoxia might be one of the mechenisms.     

Role of cyclic nucleotides in the acute hypoxic responses of hypoxic subcultured porcine pulmonary arterial smooth muscle and endothelial cells

AIM: To detect the role of cyclic nucleotides in the alleviation of hypoxic pulmonary vasoconstriction (HPV) in chronic hypoxic animals. METHODS: The intracellular cAMP and cGMP of the cultured porcine pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were assayed by RIA. The length of single PASMC during acute hypoxia was measured by imaging analysis system. RESULTS: The basal levels of cAMP and cGMP in PASMC and cGMP in PAEC of Chronic hypoxic groups decreased remarkably compared with normoxic groups (P＜0.01). Under acute hypoxia, the contents of cAMP and cGMP in PASMC of chronic hypoxic groups increased significantly (P＜0.01). Meanwhile, the percentage of PASMC with weak constrictive response in chronic hypoxic group was higher than that of control group. CONCLUSION:It's suggested that the changes of cAMP and cGMP in chronic hypoxic PASMC and PAEC might contribute to the increase in the basic tension of pulmonary artery and the alleviation of HPV in chronic hypoxic animals.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

Role of α1 adrenoceptor in proliferation of hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the role of α₁ and β₂ adrenoceptors(α₁AR and β₂AR) in the proliferation of hypoxic pulmonary artery smooth muscle cells (PASMCs).METHODS: PASMCs were isolated by an explant method from neonatal bovine pulmonary arteries. The cultured PASMCs were exposed to 6.6% O₂ for 6 h, 12 h and 24 h. The method of -TdR incorporation was used to measure the proliferation of PASMCs. _i was assayed with Fura-2/AM. The mRNA expression of α₁AR, β₂AR, c-fos and c-myc was determined by Northern blotting. The effects of activation of α₁AR and β₂AR, and inhibition of α₁AR on the above indexes were observed by treating PASMCs with different AR agonists and antagonists under hypoxic condition.RESULTS: Significant increase in TdR incorporation in hypoxic PASMCs with α₁AR activation was observed, and marked decrease in that was induced by α₁AR inhibition. However, no significant change was found after β₂AR activation. _i , the mRNA expression of c-fos, c-myc, α₁AR and β₂AR in PASMCs were increased after hypoxia.CONCLUSION: Hypoxia induces the increase in _i and mRNA expression of c-fos and c-myc, leading to the proliferation of PASMCs. The hypoxic proliferation of PASMCs is intervened by α₁AR, but not β₂AR. The remodeling of pulmonary arteriole and pulmonary hypertension may be involved in the processes of pulmonary arteriole constriction and proliferation induced by hypoxia through up-regulation of α₁AR.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Changes of nuclear factor-κB and inducible nitric oxide synthase in hypoxic pulmonary hypertension

AIM: To observe the changes of nuclear factor-κB (NF-κB) activity and inducible nitric oxide synthase (iNOS) expression in hypoxic pulmonary hypertension (HPH). METHODS :The rat model of HPH was used. The NF-κB activity and iNOS expression in lung tissue were determined by immunohistochemistry (IHC), in situ hybridization (ISH), RT-PCR and Western blot. RESULTS: ISH showed that iNOS mRNA expression in intraacinar pulmonary arteriole (IAPA) in H28d group (hypoxic treatment for 28 days) was stronger than that in normal group, H5d group and H14d group. RT-PCR showed that the iNOS mRNA in H28 group was 2.1 times, 1.9 times and 1.8 times higher than that in normal group, H5d group and H14d group, respectively. The nucleic anti-NF-κB stain was observed in H28d group, which was significantly stronger, but the I-κB amount was 2.8 times, 2.7 times and 2.5 times lower than that in normal group, H5d group and H14d group, respectively. CONCLUSION: The activity of NF-κB was correlated with the hypoxic pulmonary vessel structural remodeling and iNOS expression.     

Effect of urotensin II on cultured pulmonary arterial smooth muscle cells of rabbits

AIM: The effect of urotensin II (U-II) on proliferation of cultured pulmonary arterial smooth muscle cells (PASMCs) of rabbits and its mechanism are investigated. METHODS: PASMCs were isolated using explant technique. RPASMCs were incubated in serum-free medium with different concentrations of nicardipine, calcimodulin antagonist W₇, PKC inhibitor H₇ or MAPK inhibitor (PD₉₈₀₅₉), with or without U-II. RPASMC proliferation was examined by MTT assay and by the increase in [³H]-thymidine incorporation into DNA. RESULTS: U-II (10^-9 mol/L-10^-7 mol/L) increased A value of PASMCs by MTT assay and [³H]-thymidine incorporation in PASMCs in a dose-dependent manner. U-II induced a maximal effect at a concentration of 10^-7 mol/L. A value and [³H]-thymidine incorporation rose 42.9% and 68.5% (P<0.05), respectively. U-II had no effect at a concentration of 10^-10 mol/L. Nicardipine, W₇, H₇, PD₉₈₀₅₉ (10^-7 mol/L-10^-5 mol/L) inhibited the effect of U-II in inducing increase of A value and -thymidine incorporation in a dose-dependent manner, with the maximal inhibitory rate of 42.3%, 19.6%, 23.2%, 10.5% (P<0.05) in A value and 46.6%, 9.8%, 21.7%, 14.7% (P<0.05) in [³H]-thymidine incorporation at concentration of 10^-5 mol/L, respectively. CONCLUSION: Our results suggest that U-II may induce proliferation of PASMCs of rabbits by Ca²⁺, CaM, PKC and MAPK signal transduction pathway.     

Change of c-fos in CCK-8 against the pulmonary artery smooth muscle cell injury induced by LPS

AIM: To observe the role of c-fos in the protection by cholecystokinin-octapeptide (CCK-8) against pulmonary artery smooth muscle cell (PASMC) injury induced by lipopolysaccharide (LPS). METHODS: The ultrastructure of PASMC was observed under transmission electron microscope (TEM). MDA content, LDH release and the rate of trypan blue in PASMC were measured, and immunocytochemistry technique was adopted to observe the c-fos protein expression. RESULTS: The TEM results showed significant PASMC structural injury in LPS group and alleviated structural changes in LPS+CCK-8 group. CCK-8 reduced the increase in the rate of trypan blue uptake, MDA content and LDH release in PASMC induced by LPS. LPS lightly increased c-fos protein expression, which was enhanced by CCK-8. CONCLUSION: CCK-8 attenuated the injury of PASMC induced by LPS, which may be concerned with the increase in c-fos protein expression.     

Effects of β3 integrin on adhesion and migration of vascular smooth muscle cells induced by growth factor

AIM: To investigate the effect of platelet-derived growth factor (PDGF) on β₃ integrin gene expression and the role of β₃ integrin on adhesion, migration and proliferation of vascular smooth muscle cells (VSMC) induced by PDGF. METHODS: β₃ integxin gene expression was detected by RT-PCr. After β₃ integrin extracellular do-main was blocks, VSMC adhesio, migration and proliferation were measured by adhesion assay awound-culture model an [³H]-TSR incorporation respectively.RESULTS: After the interaction between β₃ integrin and extracellular matrix was blocked, VSMC proliferation was inhibited in some degree and the rate of [³H]-TdR incorporation into VSMC decreased 39%. The cell adhesion and migration were significantly inhibited when 10 mg/L anti-β₃ integrin antibody was added (P＜0.05). When VSMC were treated by PDGF for 6 hours, the expression of β₃ integrin gene was 87% higher than that of control. CONCLUSION: PDGF significantly induces expression of β₃ integrin gene in VSMC, and the interaction between β₃ integrin and ECM protein may play an important role in VSMC adhesion and migration.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.     

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

ATM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were cultured in vitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05﹚,while no difference in the expression of Ang-2 between the 2 groups was found.Compared with hypoxia and serum deprivation group,the expression of VEGF and Ang-1 in the H CASM Cs treated with NRG-1 was furtherincreased﹙P<0.05﹚,and no difference in the expression of Ang-2 between the 2 groups was observed.CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.