Qian-Hui Shang

Role of TGF-β1/Smads pathway in carotid artery remodeling in renovascular hypertensive rats and prevention by Enalapril and Amlodipine.

Objective To investigate the role of transforming growth factor-β1 (TGF-β1), Smad2/3 and Smad7 expressions in carotid artery remodeling in renovascular hypertensive rats, and also the therapeutic effect of Enalapril and Amlodipine. Methods The renovascular hypertensive rat (RHR) models with “two-kidney and one-clip” were established, including model group (n = 6), sham-operated group (n = 6), Enalapril group (10 mg/kg per day, n = 6), Amlodipine group (5 mg/kg per day, n = 6) and combination group (Amlodipine 2.5 mg/kg per day + Enalapril 5mg/kg per day, n = 6). The medication were continuous administrated for six weeks. Carotid artery morphological and structural changes in the media were observed by HE staining, Masson staining and immuno histochemical staining. Media thickness (MT), MT and lumen diameter ratio (MT/LD), and the expression levels of media α-smooth muscle actin (α-actin), proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in carotid arteries were measured. Results The media of carotid arteries in RHR model group was significantly thickened, the volume of smooth muscle cell was increased, and the array was in disorder; MT, MT/LD, the proliferation index of smooth muscle cell and collagen fiber area percentage of carotid arteries in the model group were significantly higher than those in the sham-operated group (P < 0.01). Compared to sham-operated group, the model group had significantly higher expressions of TGF-β1 and p-Smad2/3 (P < 0.05) and lower Smad7 expression. Both Enalapril and Amlodipine improved smooth muscle hypertrophy and collagen deposition, reduced RHR carotid MT, MT/LD, proliferation index of smooth muscle cell, collagen fiber area percentage and the expressions of TGF-β1 and p-Smad2/3 (P < 0.05), increased Smad7 expression (P < 0.05). Moreover, the combination treatment of Enalapril and Amlodipine had significantly better effects than single Amlodipine group (P < 0.05), but not single Enalapril group. Conclusions TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodeling in RHR; the role of Amlodipine and Enalapril in inversing carotid artery remodeling may be related to the change of TGF-β1/Smads pathway, the combination treatment of Amlodipine and Enalapril had better effects than single administration of Amlodipine.

e0039 Effects of Lisinopril on the activities and mRNA expression of ion pumps in aortic smooth muscle cells from spontaneously hypertensive rats

Objectives To explore the effects of Lisinopril upon the activities of Na+, K+-ATPase and Ca2+-ATPase and mRNA expression levels of Na+, K+-ATPase a1-subunit and plasma membrane Ca2+-ATPase isoform 1 (PMCA1) in cultured thoracic aorta vascular smooth muscle cells (ASMCs) isolated from spontaneously hypertensive rats (SHR). Methods ASMCs were divided into four groups: Wistar-Kyoto (WKY) control, SHR control, Lisinopril (1×10−5) intervened SHR group and Lisinopril (1×10−6) intervened SHR group. The activities of ion pumps were detected by spectrophotography and mRNA expressions were measured by real time PCR. The content of Angiotensin II (Ang II) in cells-cultured medium were detected by radioimmunoassay. Results The activities of Na+, K+-ATPase, Ca2+-ATPase and the mRNA expression levels of Na+, K+-ATPase a1-subunit and PMCA1 in ASMCs from SHR were significantly lower than those from WKY control (p<0.01). Lisinopril significantly increased the activities of Na+, K+-ATPase and Ca2+-ATPase and mRNA expression levels of Na+, K+-ATPase a1-subunit and PMCA1 in ASMCs from SHR (p<0.01). Ang II content of culture medium in ASMCs from SHR was significantly more than those from WKY control (p<0.05), Lisinopril attenuated Ang II content of ASMCs culture medium from SHR (p<0.05). Conclusions The decreased activities of Na+, K+-ATPase and Ca2+-ATPase may be related to their lower expression of the mRNA in ASMCs from SHR. The Lisinopril may increase the activities of two ion pumps and upregrulae the mRNA expression of Na+, K+-ATPase a1-subunit and PMCA1 in ASMCs from SHR through blocking the generation of Ang II.

EFFECTS OF HIGH-SALT DIET ON THE REMODELLING OF CAROTID ARTERIES AND THE INTERVENTION OF TELMISARTAN IN WISTAR RATS

Objectives To study the influence of high salt diet on blood pressure and carotid artery remodelling and the intervention of telmisartan in Wiatar rats. Methods 60 Wistar rats were fed a normal salt diet (Control group, 0.5%NaCl), high salt diet (M group: 8% NaCl), and high salt diet+Telmisartan (Tel group, 8% NaCl+ Telmisartan) until 24 weeks. After the end of experiment, M group was divided into hypertension group (MH) and normal blood pressure group (MN) according of the tail-cuff blood pressure. The structural changes and proliferation in the media of carotid artery were observed by HE staining, Masson staining and immunohistochemical. Expression of TGF-β1, smad2/3, smad7, AngII, AT1 and AT2 in media of carotid arteries were measured with immunohistochemistry method. Aldosterone in vessel was measured by radioimmunoassay. Results (1) Media thickness (MT), ratio of media to lumen (MT/LD), proliferation index (PI)、collagen fibre area percentage of carotid arteries in MH and MN groups were increased compared with that of the control group p<0.01), But MT, MT/LD, PI, the collagen volume fraction in Tel group decreased significantly p<0.01). (2) compared with the control group, the TGF-β1, smad2/3 in MH and MN groups were higher p<0.01), and in Tel group was decreased significantly p<0.01). smad7 of carotid arteries media in control group was increased than in other three groups p<0.01), Tel group was increased significantly compared with MH and MN groups p<0.01). (3) AngII of carotid artery was no difference in each group (p>0.05). The AT1 expression in MH and MN groups were higher than in the control group p<0.01), and were much lower in telmisartan group p<0.01). The AT2 expression in MH was increased significantly compared with that of other three groups p<0.01). The AT2 of expression in MN and Tel group were increased compared with that of the control group p<0.01). The aldosterone level in carotid arteries media was increased in MH groups compared with that of the control group p<0.05). Conclusions Long-term high-salt diet can cause the carotid artery remodelling directly or through high blood pressure, it may be related to positive and negative regulation of signal transduction in TGF-β1/smads and the RAAS components in local tissues. Telmisartan can prevent high salt-induced hypertension and remodelling of carotid artery.

EFFECTS OF HIGH-SALT DIET ON MYOCARDIAL REMODELLING AND THE INTERVENTION OF TELMISARTAN

Objectives To study the effects of high-salt diet on myocardial remodelling, and investigate the relevant mechanisms of telmisartan on the reverse of myocardial remodelling in Wistar rats. Methods Twenty-four Wistar rats fed by high-salt diet for 23 weeks which were divided into two groups: high-salt hypertension group (HSH n=12) and high-salt normal blood pressure group (HSN n=12) according to the level of systolic blood pressure (SBP). The rats of telmisartan group (T n=12) were fed high-salt and telmisartan for 23 weeks too. Thirteen age-matched rats fed by normal-salt were used as controls (NS n=13). Myocardial morphology and structural changes were observed by HE staining and Masson staining. The content of superoxide dismutase (SOD) and malondialdehyde (MDA) in blood and left ventricle (LV) were measured by biochemistry and enzymology. Radioimmunoassay and enzyme linked immunosorbent assay (ELISA) were employed to determine the content of tumour necrosis factor-a (TNF-a) and C-reactive protein (CRP). The protein levels of nuclear factor-κb p65 (NF-κb p65) were evaluated by western blot. Results SBP in HSH was higher than other groups. In the high-salt groups, the ratio of left ventricular mass and body mass (LVMI), the myocardial cell diameter (CMD), the fibrosis area of myocardial interstitial (MIFI), the content of CRP, TNF-a (HSH (48.86±8.25), HSN (56.67±9.67) vs NS (40.89±4.37) ng/g, p<0.05), NF-κb p65 protein (HSH (87.77±10.3), HSN (75.18±16.67) vs NS (57.13±10.00), p<0.05) and SOD in the blood were significantly increased, while the level of SOD (HSH (58.34±5.78), HSN (54.59±6.65) vs NS (68.14±9.98) U/mgprot, p<0.05) in LV decreased. LVMI, CMD and MIFI were negatively correlated with SOD activity in LV respectively, and positively correlated with the protein levels of NF-κb p65. Telmisartan partly reversed myocardial remodelling decreased the protein levels of NF-κb p65 and TNF-a, and increased the SOD activity in LV. Conclusions The myocardial remodelling caused by high salt diet may be related to decreased SOD activity and inflammatory mechanism. Telmisartan prevents the salt-induced myocardial remodelling at least in part through inhibiting oxidative stress and inflammation.

ROLE OF TGF-β1/SMADS PATHWAY IN CAROTID ARTERY REMODELLING AND PREVENTION OF ENALAPRIL AND AMLODIPINE IN RENOVASCULAR HYPERTENSIVE RATS

Objectives To investigate the role of TGF-β1/Smads pathway in carotid artery remodelling in renovascular hypertensive rats and the prevention of enalapril and amlodipine. Methods The renovascular hypertensive rats (RHR) developed by ‘two-kidney and one-clip’ method were treated consecutively with distilled water (model group, n=6), enalapril (10 mg/(kg/d), n=6), amlodipine (50 mg/(kg/d), n=6) for 6 weeks. Six sham-operated rats were used as controls. Carotid artery morphology and structural changes in the media were observed by HE staining, immunohistochemical staining and Masson staining. Media thickness (MT), lumen diameter (LD), media thickness and lumen diameter ratio (MT/LD) and collagen fibre area percentage of carotid arteries were measured. In addition, the immunohistochemical staining was applied to detect the expression of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), TGF-β1, p-Smad2/3 and Smad7. Results MT, LD, MT/LD, α-SMA, PCNA and collagen fibre area percentage of carotid arteries in the model group were higher than those in the sham-operated group (p<0.01), and TGF-β1 and p-smad2/3 were significantly increased compared to sham-operated group, Smad7 was much lower in the model group (p<0.01). Single therapy of enalapril or amlodipine decreased MT, MT/LD and the protein expression of TGF-β1, p-Smad2/3, and increased the expression of Smad7. The combination treatment of enalapril and amlodipine was significantly better than that in single amlodipine group (p<0.05), but not in single enalapril group. Conclusions In RHR, TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodelling. The enalapril or amlodipine could attenuate carotid remodelling of RHR through the intervention in TGF-β1/Smads pathway. The combination of enalapril and amlodipine is better than amlodipine therapy.

EFFECTS OF ENALAPRIL AND IRBESARTAN ON CAROTID ARTERY REMODELLING AND TGF-β1/SMADS PATHWAY IN RENOVASCULAR HYPERTENSIVE RATS

Objectives To investigate the effects of single-drug or combination therapy of enalapril and irbesartan on carotid artery remodelling and TGF-β1/Smads signal pathway. Methods Renovascular hypertensive rats (RHR) developed by ‘two-kidney and one-clip’ method were treated respectively with distilled water (model group, n=6), enalapril (10 mg/(kg d), n=6), irbesartan (50 mg/(kg/d), n=6) and enalapril plus irbesartan (5 mg/kg/d+25 mg/(kg d), n=6) for 6 weeks. Six sham-operated rats were used as controls. Carotid artery morphology and structural changes were observed through HE staining, immunohistochemical staining and Masson staining. Media thickness (MT), lumen diameter (LD), media thickness and lumen diameter ratio (MT/LD) and collagen fibre area percentage of carotid arteries were measured. Moreover, the immunohistochemical staining was applied to detect the expression of alpha-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), TGF-β1, p-Smad2/3 and Smad7. Results In the model group, the media thickness was significantly increased, and the volume of vascular smooth muscle cell (VSMC) increased and disarranged. MT, LD, MT/LD, α-SMA, PCNA and collagen fibre area percentage of carotid arteries in the model group were higher than those in the sham-operated group (p<0.01), and TGF-β1 and p-smad2/3 were increased whereas Smad7 was decreased in the model group (p<0.01). Single enalapril or irbesartan therapy decreased MT, MT/LD and the protein expression of TGF-β1, p-Smad2/3, and increased the expression of Smad7. Combined enalapril and irbesartan treatment showed significant reductions in above experimental indices than single drug interventions (all p<0.05). Conclusions The TGFβ1/Smads signalling pathway may be involved in carotid remodelling of RHR. Enalapril or irbesartan can attenuate carotid remodelling of RHR through regulating TGF-β1/Smads pathway and both combination treatment seems to have interaction.

THE EFFECTS OF LONG-TERM HIGH-SALT DIET ON BLOOD PRESSURE AND KIDNEY IN WISTAR RATS AND THE INTERVENTION OF TELMISARTAN

Objectives To investigate the effects of long-term high-salt diet on blood pressure and kidney and the intervention of telmisartan. Methods Wistar rats were randomly divided into three groups: Control group (NS group: given 0.5% NaCl), High-salt group (given 8% NaCl), and Intervention group (GY group: given 8% NaCl+telmisartan). Systolic blood pressure was assessed by the tail-cuff artery pressure. The urine was collected to measure the concentration of Na+, K+, microalbumin, total protein, and creatinine. At 24 weeks, the renal hypertrophy index was calculated. HE, Masson staining were used to observe the kidney morphology. Results Compared with NS group, systolic blood pressure was significantly increased and continued until the end of the experiment in one part of rats fed high-salt diet, whereas the other part of rats fed high-salt diet developed transient increase in blood pressure only from 8 weeks to 10 weeks of the experiment. So high-salt group rats were finally divided into High-salt hypertension group (HH group) and High-salt normal blood pressure group (HN group). In high-salt group rats, the renal hypertrophy index, microalbumin, total protein, and the ratio of Na+/K+ were increased p<0.01), creatinine clearance rate was decreased p<0.01), but the real damage in HN group was lighter than that in HH group. In GY group, systolic blood pressure was decreased, and the content of microalbumin, and total protein was reduced p<0.01), renal damage was ameliorated, but the renal hypertrophy index, creatinine clearance, and the ratio of Na+/K+ did not change (p>0.05). Conclusions Long-term high-salt diet may induce high blood pressure in part of the Wistar rats and cause renal damage independent of high blood pressure. Telmisartan can prevent high blood pressure and renal damage induced by high-salt diet in Wistar rats.

EFFECTS OF HIGH SALT DIET ON ARTERIAL REMODELLING AND THE INTERVENTION OF TELMISARTAN IN WISTAR RATS

Objectives To investigate the effects of dietary salt on aorta and mesenteric artery remodelling in Wistar Rats and explore the possible mechanism of salt-induced arterial remodelling and AngiotensinII receptor blockers telmisartan intervention. Methods 60 Wistar rats were randomly divided into normal control group, high salt (8%) model group and high salt+telmisartan group. The tail artery pressure was determined every 2 weeks. After 24 weeks, high salt model group was divided into model hypertension (MH) group and model normal pressure (MN) group. The structural changes and proliferation in the media of aorta and mesenteric arteries were observed by HE staining, Masson staining and immunohistochemical staining. The activities and mRNA levels of Na+ pump and Ca2+ pump in aortic media were determined by enzyme colorimetry and real-time PCR respectively. Results Compared with control group, the blood pressure was significantly increased in MH Group, Media thickness (MT), lumen diameter (LD), ratio of media to lumen (MT/LD), the collagen volume fraction and PCNA positive expressive percentage of arteries in high-salt group were increased (p<0.05), the activities of Na+-K+-ATPase and Ca2+-ATPase in MH group were decreased (p<0.05).The mRNA expression of Na+-K+-ATPase α1 subunit in MH and MN groups was decreased (P<0.05), and PMCA1 expression raised in MH group, Correlation analysis showed that two ATPase activities and vascular remodelling indicators have a negative correlation (p<0.05).Compared with high-salt group, blood pressure, media thickness, ratio of media to lumen, the collagen volume and PCNA positive expressive percentage were lower in telmisartan group (p<0.05). Conclusions High-salt diet could lead to arterial remodelling directly or indirectly (elevated blood pressure), The decreased ion pump activity and abnormal gene expression may be one of the mechanisms of high-salt induced arterial remodelling. Telmisartan may inhibit the proliferation of vascular smooth muscle and collagen accumulation, and prevent salt-induced hypertension and arterial remodelling.

e0037 Effects of enalapril and irbesartan on aorta remodelling and ion pumps in renovascular hypertensive rats

Objective To investigate the effects of single-drug or combination therapy of enalapril and irbesartan on aorta remodelling and its mechanisms. Methods Renovascular hypertensive rats (RHD) induced by two-kidney one-clip method were treated with normal saline (model group, n=6), enalapril [10 mg/ (kg d), n=6], irbesartan [50 mg/ (kg·d), n=6] and enalapril+irbesartan [5 mg/ (kg d)+25 mg/ (kg d), n=6] for 6 weeks. Six sham-operated rats were used as controls. Aortic morphology and structural changes in the media were observed by HE staining, immunohistochemistry and Masson staining. The content of Angiotensin II (Ang II) was measured by radioimmunoassay. The activities and mRNA levels of Na+ pump and Ca2+ pump in aortic media were determined by enzyme assay and real-time PCR respectively. Results The media area of aorta and the Ang II content were significantly increased in model group, while the activities and the mRNA levels of Na+ pump and Ca2+ pump in aortic media were obviously decreased, and Na+ pump and Ca2+ activities were increased in enalapril group and irbesartan group (p<0.01). The Ang II content was obviously decreased in enalapril group, while increased in irbesartan group (p<0.01). The mRNA levels of sodium pump a1-subunit and plasma membrane calcium pump isoform 1 (PMCA1) in aorta smooth muscle tissue were significantly increased in enalapril group (p<0.01). The amelioration of blood pressure, Na+ pump and Ca2+ pump activities, media area and thickness in combination group was significantly better than single-drug intervened group (p<0.01). Conclusion The amelioration of aorta remodelling induced by enalapril and irbesartan may be associated with the increase of Na+ pump and Ca2+ pump activities. There may be some synergistic effects on ameliorating of Na+ pump and Ca2+ pump activities and aorta remodelling from combination of the two drugs. The effect of enalapril on Na+ pump and Ca2+ pump activities may be mediated by increasing their mRNA expression.

e0595 AngiotensinII modulates ion pumps of smooth muscle cells derived from umbilical artery of human neonates with hypertensive family history

Objective To investigate sodium pump and calcium pump activities and mRNA expression level and the changes after AngiotensinII (AngII) treatment in human umbilical artery smooth muscle cells (HUASMCs) isolated from neonates with positive hypertensive family history (FH+) or with negative hypertensive family history (FH-). Methods Ion pump activities in cultured HUASMCs were detected by spectrophotography. The mRNA expression of sodium pump α1-subunit and plasma membrane Ca2+-ATPase isoform 1 (PMCA1) in FH+ and FH- HUASMCs was measured by RT-PCR. Results Sodium pump, calcium pump activities in FH+ HUASMCs were higher than those in FH- group (p<0.05), but the mRNA expression of sodium pump α1subunit and PMCA1 showed no difference between two groups. In FH- group, after 24-h treatment, AngII (1×10−7 mol/L) elevated the activities of sodium pump (4.62±0.26 vs 3.52±0.33) and calcium pump (4.00±0.31 vs 3.01±0.32), and up-regulated sodium pump α1-subunit mRNA expression (0.946±0.099 vs 0.697±0.050, n=5, p<0.01), however higher concentration AngII (1×10−6 mol/L) suppressed the activities of sodium (2.47±0.27) and calcium pump (1.79±0.27), and down-regulated sodium pump mRNA expression (0.445±0.065). Whereas, in FH+ groups, both concentration (10−6 and 10−7 mol/l) of AngII suppressed the activities of sodium pump (3.49±0.34, 2.21±0.23 vs 4.70±0.44) and calcium pump (2.85±0.31, 1.87±0.16 vs 4.27±0.48), but only AngII (10−7 mol/l) down-regulated their mRNA expression (α1-subunit: 0.515±0.133 vs 0.885±0.097, PMCA1: 0.165±0.049 vs 0.397±0.046, n=5, p<0.01). Conclusions The activity of sodium pump and calcium pump is increased in FH+ HUASMCs. AngII inhibits both Na+ and Ca2+ ion pumps activities and mRNA expression in FH+ HUASMCs, and may have biphasic effects on ion pump activities and mRNA expression in FH- hUASMCs.