Pulmonary Artery Smooth Muscle Cell Senescence Promotes the Proliferation of PASMCs by Paracrine IL-6 in Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a critical and dangerous disease in cardiovascular system. Pulmonary vascular remodeling is an important pathophysiological mechanism for the development of pulmonary arterial hypertension. Pulmonary artery smooth muscle cell (PASMC) proliferation, hypertrophy, and enhancing secretory activity are the main causes of pulmonary vascular remodeling. Previous studies have proven that various active substances and inflammatory factors, such as interleukin 6 (IL-6), IL-8, chemotactic factor for monocyte 1, etc., are involved in pulmonary vascular remodeling in PH. However, the underlying mechanisms of these active substances to promote the PASMC proliferation remain to be elucidated. In our study, we demonstrated that PASMC senescence, as a physiopathologic mechanism, played an essential role in hypoxia-induced PASMC proliferation. In the progression of PH, senescence PASMCs could contribute to PASMC proliferation via increasing the expression of paracrine IL-6 (senescence-associated secretory phenotype). In addition, we found that activated mTOR/S6K1 pathway can promote PASMC senescence and elevate hypoxia-induced PASMC proliferation. Further study revealed that the activation of mTOR/S6K1 pathway was responsible for senescence PASMCs inducing PASMC proliferation via paracrine IL-6. Targeted inhibition of PASMC senescence could effectively suppress PASMC proliferation and relieve pulmonary vascular remodeling in PH, indicating a potential for the exploration of novel anti-PH strategies.

Role of the signal transducer and activator of transcription 3 protein in the proliferation of vascular smooth muscle cells.

OBJECTIVES: Cardiovascular disease (CVD) remains the primary cause of morbidity and mortality worldwide. The abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of CVD. The functional and phenotypic changes in vascular cells are mediated by complex signaling cascades that initiate and control genetic reprogramming. Many studies have demonstrated that signal transducer and activator of transcription 3 (STAT3) regulates a diverse array of functions relevant to atherosclerosis. METHODS: In this review, we summarize the studies on the STAT3-mediated proliferation of VSMCs and subsequent CVDs such as hypertension, atherosclerosis, stroke, coronary artery disease, and myocardial infarction. Furthermore, we describe the general background of STAT3, its structure, function and regulation as well as the STAT3 signaling pathway. Finally, we highlight some potential issues and propose some solutions to these issues.Results and conclusions: STAT3 activation promotes the proliferation of VSMCs by regulating the transcription of genes. Studying the mechanism of VSMC proliferation induced by the STAT3 pathway is valuable for finding therapeutic targets for CVD.

Rutaecarpine prevents hypertensive cardiac hypertrophy involving the inhibition of Nox4-ROS-ADAM17 pathway.

Rutaecarpine attenuates hypertensive cardiac hypertrophy in the rats with abdominal artery constriction (AAC); however, its mechanism of action remains largely unknown. Our previous study indicated that NADPH oxidase 4 (Nox4) promotes angiotensin II (Ang II)-induced cardiac hypertrophy through the pathway between reactive oxygen species (ROS) and a disintegrin and metalloproteinase-17 (ADAM17) in primary cardiomyocytes. This research aimed to determine whether the Nox4-ROS-ADAM17 pathway is involved in the protective action of rutaecarpine against hypertensive cardiac hypertrophy. AAC-induced hypertensive rats were adopted to evaluate the role of rutaecarpine in hypertensive cardiac hypertrophy. Western blotting and real-time PCR were used to detect gene expression. Rutaecarpine inhibited hypertensive cardiac hypertrophy in AAC-induced hypertensive rats. These findings were confirmed by the results of in vitro experiments that rutaecarpine significantly inhibited Ang II-induced cardiac hypertrophy in primary cardiomyocytes. Likewise, rutaecarpine significantly suppressed the Nox4-ROS-ADAM17 pathway and over-activation of extracellular signal-regulated kinase (ERK) 1/2 pathway in the left ventricle of AAC-induced hypertensive rats and primary cardiomyocytes stimulated with Ang II. The inhibition of Nox4-ROS-ADAM17 pathway and over-activation of ERK1/2 might be associated with the beneficial role of rutaecarpine in hypertensive cardiac hypertrophy, thus providing additional evidence for preventing hypertensive cardiac hypertrophy with rutaecarpine.

Protein Arginine Methyltransferase 2 Inhibits Angiotensin II-Induced Proliferation and Inflammation in Vascular Smooth Muscle Cells.

OBJECTIVES: Protein arginine methyltransferase 2 (PRMT2) protects against vascular injury-induced intimal hyperplasia; however, little is known about the role of PRMT2 in angiotensin II (Ang II)-induced VSMCs proliferation and inflammation. This research aims to determine whether PRMT2 inhibits Ang II-induced proliferation and inflammation of vascular smooth muscle cells (VSMCs). MATERIALS AND METHODS: PRMT2 overexpression was used to elucidate the role of PRMT2 in Ang II-induced VSMCs proliferation and inflammation. Western blotting and reverse transcriptional PCR were adopted to detect protein and mRNA expression severally. Cell viability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and cell cycle distribution by flow cytometry. RESULTS: Ang II significantly reduced mRNA and protein levels of PRMT2 in VSMCs in time-dependent and dose-dependent manner. Results of PRMT2 overexpression indicated that PRMT2 inhibited proliferation of VSMCs stimulated with 100 nmol/L Ang II for 24 hours. Furthermore, overexpression of PRMT2 reduced Ang II-induced production of proinflammatory cytokines such as interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in VSMCs. CONCLUSIONS: These findings suggest that PRMT2 alleviates Ang II-induced VSMCs proliferation and inflammation, providing a new mechanism about how Ang II mediated VSMCs proliferation and inflammation.

Evidence That ADAM17 Mediates the Protective Action of CGRP against Angiotensin II-Induced Inflammation in Vascular Smooth Muscle Cells.

Calcitonin gene-related peptide (CGRP) has a potent protective action on the cardiovascular system; however, little is known about the role of CGRP in angiotensin II- (Ang II-) induced inflammation of vascular smooth muscle cells (VSMCs). This study is aimed at determining the anti-inflammatory effect of CGRP in Ang II-treated VSMCs and whether a disintegrin and metalloproteinase 17 (ADAM17) modulates this protective action. Small interference RNA (siRNA) and inhibitors of CGRP, epidermal growth factor receptor (EGFR), and extracellular signal-regulated kinase 1/2 (ERK1/2) were adopted to investigate their effect on Ang II-induced inflammation in VSMCs. Here, we found that CGRP could inhibit inflammation and decrease ADAM17 expression and activation of EGFR and ERK1/2 in VSMCs stimulated with Ang II. Results of siRNA demonstrated that ADAM17 siRNA attenuated Ang II-induced inflammation and up-regulation of activities of EGFR and ERK1/2 in VSMCs. Furthermore, the EGFR-ERK1/2 pathway promoted Ang II-induced VSMC inflammation. In summary, these findings identify the anti-inflammatory effect of CGRP in VSMCs stimulated by Ang II and suggest that ADAM17 is involved in the protective effect of CGRP against Ang II-induced inflammation via the EGFR-ERK1/2 pathway in VSMCs.

The ERK1/2 pathway participates in the upregulation of the expression of mesenteric artery α1 receptors by intravenous tail injections of mmLDL in mice.

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular diseases. However, no studies examining the effect of mmLDL on vascular smooth muscle receptors have been released. The current study investigated the effect of mmLDL on the mesenteric artery α1 adrenoceptor and the molecular mechanisms. Mice were divided into the normal saline (NS), mmLDL, and mmLDL+U0126 groups. In the mmLDL+U0126 group, the animals were subjected to an intravenous tail injection of mmLDL and an intraperitoneal injection of U0126. Vascular tension caused by noradrenaline (NA) in mesenteric arteries was measured with a sensitive myograph system. The serum levels of oxLDL, TNF-α, and IL-1ß were detected using enzyme-linked immunosorbent assays. The expressions of the α1 adrenoceptor, the α2 adrenoceptor, TNF-α, IL-1ß, and pERK1/2 were detected using real-time polymerase chain reactions and Western blot analysis. Compared with the NS group, the mmLDL group exhibited a noticeably enhanced NA shrinkage dose-response curve and a significantly increased Emax value (P<0.01). Prazosin (α1 adrenoceptor antagonist) caused a noticeable right shift of the dose-response curve. U0126 inhibited the increases in the serum levels and vessel wall expression of IL-1ß and TNF-α and enhanced the NA shrinkage dose-response curve caused by mmLDL, as observed by a significantly decreased Emax value (P<0.01). It inhibited the increased α1 adrenoceptor expression caused by mmLDL. The serum levels of IL-1ß and TNF-α demonstrated a positive correlation with the NA-induced maximum shrinkage percentage. U0126 inhibited the mmLDL-induced increase in the pERK1/2 protein level in the vessel wall. In conclusion, mmLDL increased the serum levels of IL-1ß and TNF-α in vivo by activating the ERK1/2 pathway, which resulted in α1 receptor-mediated vasoconstriction and an increase in the expression of α1 adrenoceptor. The results of this study may provide new ideas for the prevention and cure of cardiovascular diseases in the future.

[Involvement of the receptor component protein in the regulation of vascular peroxidase-1 expression induced by calcitonin gene-related peptide and angiotensin II in vascular smooth muscle cell].

Angiotensin II (Ang II) and calcitonin gene-related peptide (CGRP) play important roles in vascular injury and protection. In order to determine the role of CGRP receptor component protein (RCP) in signal transduction whereby CGRP and Ang II mediate the expression of vascular peroxidase-1 (VPO1) in vascular smooth muscle cell (VSMC), mouse derived A10 vascular smooth muscle cell line (A10VSMC) was cultured with CGRP or/and Ang II in vitro. RCP-specific small interference RNA (siRNA-RCP) was used to silence oligonucleotide sequence. Western blot and RT-PCR were used to determine the protein and mRNA expressions of RCP and VPO1, respectively. The results showed that the expressions of RCP and VPO1 were increased in the presence of CGRP or Ang II in the quiescent A10VSMC. But the protein expressions of RCP and VPO1 induced by Ang II were decreased by pretreatment of CGRP (P < 0.05). The expressions of VPO1 were decreased in all the groups treated with siRNA-RCP, compared with those of wide-type counterparts. Meanwhile, the expression of VPO1 was significantly induced by CGRP but not Ang II in the siRNA-RCP treated A10VSMCs. Ang II in combination with CGRP increased the protein expression of VPO1 in the siRNA-RCP-transfected cells, compared with Ang II alone, and this effect could be abolished by catalase. The results suggest that RCP may play an important role in the integration of signal transduction whereby CGRP and Ang II receptors jointly regulate VPO1 expression in VSMC.

The role of caveolae in regulating calcitonin receptor-like receptor subcellular distribution in vascular smooth muscle cells.

To determine whether caveolae and caveolin-1 affect the distribution of calcitonin receptor-like receptors (CLR) in vascular smooth muscle cell (VSMC) membranes, we have used VSMCs cell line A10. We found that calcitonin gene-related peptide (CGRP) reduced CLR protein in the VSMC membrane in a time-dependent manner, which was dramatically decreased after 4 h CGRP treatment, and remained at a low level after 16 h. CGRP8-37 or ß-cyclodextrin (ß-CD) blocked this effect, without changing the total levels of CLR protein and mRNA in the cells. Co-immunoprecipitation experiments showed that CLR bound to caveolin-1 in cell membrane fractions. Confocal laser microscopic studies confirmed this co-localization relationship at the cell plasma membrane. Thus, our data indicate that the structural integrity of caveolae plays an important role in regulating subcellular distribution of CLR.

[Effect of losartan on the protection of the kidney and PRCP-kallikrein axis of the two-kidney, one-clipped renovascular hypertensive rats].

To investigate the effect of losartan on the axis of prolylcarboxypeptidase (PRCP)--kallikrein of the two-kidney, one-clipped (2K1C) hypertensives rats, and explore the novel protection mechanism of losartan on the kidney. Sprague-Dawley (SD) rats were used to develop the 2K1C hypertensive rats. Then, the rats were treated with prazosin (5 mg x kg(-1) x d(-1)) or losartan (5, 15 and 45 mg x kg(-1) x d(-1)) or vehicle, separately. At the same time, the blood pressures were observed. After treated for four weeks, the ratio of right kidney weight and body weight, the change of glomerular morphology, and K+, Na+, creatinine and blood urea nitrogen (BUN) of the serum were used for evaluation of kidney. The expressions of PRCP mRNA in the kidneys were determined by RT-PCR. The protein levels of PRCP, tissue kallikrein, plasma kallikrein, TGF-beta1 in kidney or plasma were measured by Western blotting. Results showed that the changes of body weight and kidney weight ratio, glomerular fibrosis degree and the biochemistrical index of serum induced by hypertension were relieved when the hypertensive rats treated with losartan for four weeks. Meanwhile, treatment of losartan also significantly decreased expression of TGF-beta1 and increased expressions of PRCP, plasma kallikrein and tissue kallikrein. The protective effects of losartan on the kidney of 2K1C hypertensive rats are activation of the axis of PRCP-kallikrein and reducing the expression of TGF-beta1.

[Minimally modified LDL induced impairment of endothelium-dependent relaxation in mesenteric arteries of mice].

This study is to investigate the impairment and possible mechanism of endothelium-dependent relaxation of mice mesenteric arteries induced by mmLDL. Wire myography was employed to examine endothelial function of mesenteric arteries. Ultramicrostructure of mesenteric vascular beds were detected by transmission electron microscope. The results showed that endothelium cell edema and peeling, vascular elastic membrane fracture traces in mmLDL group. Endothelium-dependent relaxation was decreased in a time-dependent and dose-dependent manner by using mmLDL, compared with normal arteries. In endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation, the Rmax and pIC50 were decreased from (63 +/- 5) % and 6.42 +/- 0.09 of normal saline control to (31 +/- 3) % and 5.67 +/- 0.07 in mmLDL group (P < 0.001, P < 0.001), respectively. In nitric oxide (NO)-mediated relaxation, the Rmax and pIC50 were decreased from (45 +/- 4) % and 5.93 +/- 0.08 in normal saline control to (32 +/- 4) % and 5.43 +/- 0.11 in mmLDL group (P < 0.05, P < 0.01), respectively. There is no significant alteration of prostacyclin I2 (PGI2) pathway between these two groups. In conclusion, mmLDL induced the impairment of the ultramicrostructure of mesenteric vascular endothelium cell as well as the endothelium-dependent relaxation. The latter includes the dysfunction of NO- and EDHF pathway mediated endothelium-dependent relaxation.

Involvement of prolylcarboxypeptidase in the effect of rutaecarpine on the regression of mesenteric artery hypertrophy in renovascular hypertensive rats.

1. Previous studies indicate that rutaecarpine blocks increases in blood pressure and inhibits vascular hypertrophy in experimentally hypertensive rats. The aim of the present study was to determine whether the effects of rutaecarpine are related to activation of prolylcarboxypeptidase (PRCP). 2. Renovascular hypertensive rats (Goldblatt two-kidney, one-clip (2K1C)) were developed using male Sprague-Dawley rats. Chronic treatment with rutaecarpine (10 or 40 mg/kg per day) or losartan (20 mg/kg per day) for 4 weeks to the hypertensive rats caused a sustained dose-dependent attenuation of increases in blood pressure, increased lumen diameter and decreased media thickness, which was accompanied by a similar reduction in the media cross-sectional area : lumen area ratio in mesenteric arteries compared with untreated hypertensive rats. 3. Angiotensin (Ang) II expression was significantly increased in mesenteric arteries of hypertensive rats compared with sham-operated rats. No significant differences in plasma AngII levels were observed between untreated hypertensive and sham-operated rats. Hypertensive rats treated with high-dose rutaecarpine had significantly decreased Ang II levels in both the plasma and mesenteric arteries. 4. Expression of PRCP protein or kallikrein mRNA was significantly inhibited in the right kidneys and mesenteric arteries of hypertensive rats. However, expression of PRCP protein and kallikrein mRNA was significantly increased after treatment with rutaecarpine or losartan (20 mg/kg per day). 5. The data suggest that the repression of increases in systolic blood pressure and reversal of mesenteric artery remodelling by rutaecarpine may be related to increased expression of PRCP in the circulation and small arteries in 2K1C hypertensive rats.

Calcitonin gene-related Peptide-mediated depressor effect and inhibiting vascular hypertrophy of rutaecarpine in renovascular hypertensive rats.

Calcitonin gene-related peptide (CGRP), the predominant neurotransmitter in capsaicin-sensitive sensory nerves, is a potent vasodilator and inhibits proliferation of vascular smooth muscle cells. Previous investigations have demonstrated that the hypotensive effect of rutaecarpine (Rut) is associated to stimulation of CGRP synthesis and release via activation of the vanilloid receptor subtype 1 (VR1) in the phenol-induced hypertensive rat. This study tested whether the depressor effect and inhibiting vascular hypertrophy of Rut is mediated by endogenous CGRP in 2-kidney, 1-clip (2K1C) hypertensive rats. Systolic blood pressure (SBP) was measured by tail-cuff method in conscious. Mesenteric arteries were isolated for examination of morphological changes. The concentration of CGRP in the plasma and the expression of CGRP mRNA in dorsal root ganglia (DRG) were measured. Chronic administration of Rut (10, 20, or 40 mg/kg/day, respectively) for 4 weeks caused a depressor effect and significantly regressed the lumen diameter and decreased the medium thickness of mesenteric arteries in hypertensive rats concomitantly with an increase in the plasma concentration of CGRP and the expression of CGRP mRNA in DRG. In conclusion, chronic administration of Rut can reduce blood pressure and relieve mesenteric artery hypertrophy in the 2K1C hypertensive rats, and the effects of Rut may be related to stimulation of CGRP synthesis and release.

[Effect of pravastatin on cholesteryl esters in foam cells and the relation with caveolin-1].

The purpose of the present study was to investigate the effect of pravastatin on cholesteryl esters in foam cells of murine macrophages and the relation with caveolin-1. RAW 264.7 murine macrophages were coincubated with 80 mg/L oxidized low density lipoprotein (ox-LDL) and pravastatin (0~100 mumol/L) respectively for 24 h. When the best control concentration of pravastatin was confirmed, RAW 264.7 murine macrophages were coincubated with 80 mg/L ox-LDL and pravastatin of the best concentration respectively for 0, 6, 12, 24 h. Oil red O dyeing experiment was used to show the lipid droplets in foam cells. High performance liquid chromatography (HPLC) analysis was performed to determine the content of cellular cholesterol. The level of caveolin-1 was determined by Western blot analysis. The result showed that when macrophages were incubated with 80 mg/L ox-LDL, the ratio of cellular cholesteryl ester to total cholesterol (CE/TC) was beyond 50% through HPLC analysis, and a great deal of lipid droplets displayed in cells through Oil red O dyeing experiment, which manifested the formation of the foam cells. Pravastatin could decrease CE in foam cells in a concentration-dependent manner (1~100 mumol/L). At the concentration of 100 mumol/L, pravastatin decreased cellular CE more than 50%. The effects of pravastatin on the decrease of CE in murine macrophages also displayed a time-dependent manner (incubated with 100 mumol/L pravastatin from 6 to 24 h). Moreover, the expression of caveolin-1 was decreased when the macrophages were incubated with ox-LDL (80 mg/L), while treatment with pravastatin increased the level of caveolin-1 and displayed a concentration- and time-dependent manner. These results suggest that pravastatin could inhibit the development of foam cells through the decrease of cellular CE, which may be related to the upregulation of caveolin-1.

Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells.

The present study was designed to examine the effect of calcitonin gene-related peptide (CGRP) on angiotensin II-induced proliferation of cultured rat vascular smooth muscle cells. Vascular smooth muscle cells were grown from explants of Sprague-Dawley rat aorta. Vascular smooth muscle cells (between passages 5 and 10) were incubated with 0.1% neonatal calf serum for 48 h, and then treated with angiotensin II (100 nM) in the absence or presence of CGRP for 24 h. The viability, DNA synthesis and cell cycle of vascular smooth muscle cells were measured. Western blotting was used to determine the activity of intracellular extracellular regulated kinase (ERK1/2). Angiotensin II significantly decreased the viability and proliferation of vascular smooth muscle cells, decreased the proliferation index, and increased the activity of ERK1/2; the effects of angiotensin II were inhibited by CGRP (1-100 nM) in a concentration-dependent manner. In conclusion, CGRP significantly inhibits angiotensin II-induced proliferation of vascular smooth muscle cells, an effect related to a decrease in the activation of mitogen-activated protein kinase pathway.

Involvement of calcitonin gene-related peptide in the depressor effects of losartan and perindopril in rats.

Previous investigations have indicated that calcitonin gene-related peptide (CGRP) plays an important role in the regulation of cardiovascular function, and that the development of hypertension may be related to the reduction of sensory vasodilator nerve actions. In the present study, we examined the effect of perindopril, an angiotensin-converting enzyme inhibitor, and losartan, an angiotensin II receptor antagonist, on the plasma level and synthesis of CGRP in 2 kidneys, 1-clip hypertensive rats (2K1C, Goldblatt). In the hypertension group, systolic blood pressure and mean artery pressure were raised, and the level of CGRP in plasma was slightly raised compared with control groups. Chronic treatment with losartan or perindopril significantly increased the plasma concentration of CGRP and the expression of CGRP mRNA in dorsal root ganglia in the 2K1C, Goldblatt hypertensive rats. These results suggest that the 2K1C, Goldblatt hypertensive model has a compensatory increase of sensory nerve actions, and that the depressor effects of perindopril or losartan may be related to stimulation of the synthesis and release of CGRP in the 2K1C, Goldblatt hypertensive rats.