Xiuzhen Sun

Statins suppress MMP2 secretion via inactivation of RhoA/ROCK pathway in pulmonary vascular smooth muscles cells.

Recent studies have shown that statins ameliorate hypoxia-induced pulmonary hypertension in animal models. Yet, the underlying molecular mechanisms have not been completely understood. Here, we investigated the hypothesis that statins regulate vascular remodeling by modulation of matrix metalloproteinases (MMP) secretion in primary cultured pulmonary artery smooth muscle cells (PASMCs). Angiotensin II induced a concentration-dependent MMP2 secretion. A 2.75 fold increase was achieved by 100 nM angiotensin II stimulation for 24 h in primary cultured PASMCs (P<0.01 versus control), which was reversed by silencing Ras homolog gene family member A (RhoA) or inhibition of its downstream Rho-associated kinase (ROCK). There was no effect of angiotensin II on the expression of tissue inhibitors of matrix metalloproteinases (TIMPs). Pre-exposure of cells to simvastatin concentration-dependently blocked angiotensin II-stimulated MMP2 release. MMP2 release was reduced to1.34 fold increase in the presence of 10 microM simvastatin (P<0.01 versus angiotensin II-stimulated cells). We further revealed that simvastatin suppression of RhoA activation mediated its inhibitory effect on angiotensin II-triggered MMP2 release. Similarly, simvastatin suppressed endothelin-1-induced MMP2 release through RhoA/ROCK pathway. These results indicated a novel statins-regulation of RhoA/ROCK signaling against pulmonary vascular remodeling, and suggest that statins could prove useful in targeting this pathway in pulmonary hypertension and other disease conditions.

Heme oxygenase‐1/p21WAF1 mediates peroxisome proliferator‐activated receptor‐γ signaling inhibition of proliferation of rat pulmonary artery smooth muscle cells

Activation of peroxisome proliferator‐activated receptor (PPAR)‐γ suppresses proliferation of rat pulmonary artery smooth muscle cells (PASMCs), and therefore ameliorates the development of pulmonary hypertension in animal models. However, the molecular mechanisms underlying this effect remain largely unknown. This study addressed this issue. The PPARγ agonist rosiglitazone dose‐dependently stimulated heme oxygenase (HO)‐1 expression in PASMCs, 5 μm rosiglitazone inducing a 12.1‐fold increase in the HO‐1 protein level. Cells pre‐exposed to rosiglitazone showed a dose‐dependent reduction in proliferation in response to serotonin; this was abolished by pretransfection of cells with sequence‐specific small interfering RNA against HO‐1. In addition, rosiglitazone stimulated p21WAF1 expression in PASMCs, a 2.34‐fold increase in the p21WAF1 protein level being achieved with 5 μm rosiglitazone; again, this effect was blocked by knockdown of HO‐1. Like loss of HO‐1, loss of p21WAF1 through siRNA transfection also reversed the inhibitory effect of rosiglitazone on PASMC proliferation triggered by serotonin. Taken together, our findings suggest that activation of PPARγ induces HO‐1 expression, and that this in turn stimulates p21WAF1 expression to suppress PASMC proliferation. Our study also indicates that rosiglitazone, a medicine widely used in the treatment of type 2 diabetes mellitus, has potential benefits for patients with pulmonary hypertension.

Endothelin-1 induces hypoxia inducible factor 1α expression in pulmonary artery smooth muscle cells

Endothelin‐1 (ET‐1) dose‐dependently increased HIF1α expression in pulmonary artery smooth muscle cells (PASMCs). Inhibition of protein synthesis did not affect ET‐1‐induced HIF1α expression. The maximum effect of ET‐1 was similar to that caused by proteasome inhibitor MG132. Further study indicates that ET‐1 also dose‐dependently stimulated calcineurin activation, specific calcineurin inhibitor cyclosporine A (CsA), abolished ET‐1‐induced HIF1α elevation, and reversed ET‐1‐induced RACK1 (receptor of activated protein kinase C 1) de‐phosphorylation. Endothelin receptor A was found to specifically mediate the effects of ET‐1. To examine whether RACK1 is particularly involved in proteasome‐dependent HIF1α degradation, RACK1 was silenced by siRNA transfection. Cells lacking RACK1 exhibited significant elevation of HIF1α protein level. Taken together, our study suggests that ET‐1 suppressed proteasome‐dependent HIF1α degradation by calcineurin‐dependent RACK1 de‐phosphorylation.

Inhibition of cGMP phosphodiesterase 5 suppresses matrix metalloproteinase-2 production in pulmonary artery smooth muscles cells

1. It has been shown that the beneficial effects of phosphodiesterase (PDE) 5 inhibition on pulmonary hypertension (PH) are associated with the induction of vascular relaxation and suppression of the proliferation of pulmonary artery smooth muscle cells (PASMC). In the present study, we investigated whether PDE5 inhibition affects the production and/or secretion of matrix metalloproteinases (MMPs) in PASMC, resulting in extracellular matrix remodelling in the pulmonary vasculature and, thus, the development of PH.

The PPARγ agonist, rosiglitazone, attenuates airway inflammation and remodeling via heme oxygenase-1 in murine model of asthma

Aim:Rosiglitazone is one of the specific PPARγ agonists showing potential therapeutic effects in asthma. Though PPARγ activation was considered protective in inhibiting airway inflammation and remodeling in asthma, the specific mechanisms are still unclear. This study was aimed to investigate whether heme oxygenase-1 (HO-1) related pathways were involved in rosiglitazone-activated PPARγ signaling in asthma treatment.Methods:Asthma was induced in mice by multiple exposures to ovalbumin (OVA) in 8 weeks. Prior to every OVA challenge, the mice received rosiglitazone (5 mg/kg, po). After the mice were sacrificed, the bronchoalveolar lavage fluid (BALF), blood samples and lungs were collected for analyses. The activities of HO-1, MMP-2 and MMP-9 in airway tissue were assessed, and the expression of PPARγ, HO-1 and p21 proteins was also examined.Results:Rosiglitazone administration significantly attenuated airway inflammation and remodeling in mice with OVA-induced asthma, which were evidenced by decreased counts of total cells, eosinophils and neutrophils, and decreased levels of IL-5 and IL-13 in BALF, and by decreased airway smooth muscle layer thickness and reduced airway collagen deposition. Furthermore, rosiglitazone administration significantly increased PPARγ, HO-1 and p21 expression and HO-1 activity, decreased MMP-2 and MMP-9 activities in airway tissue. All the therapeutic effects of rosiglitazone were significantly impaired by co-administration of the HO-1 inhibitor ZnPP.Conclusion:Rosiglitazone effectively attenuates airway inflammation and remodeling in OVA- induced asthma of mice by activating PPARγ/HO-1 signaling pathway.

Inhibition of cGMP phosphodiesterase 5 suppresses serotonin signalling in pulmonary artery smooth muscles cells.

cGMP-specific phosphodiesterase 5 (PDE5) inhibition has been shown to be effective in improving pulmonary haemodynamics in both animal models and clinic patients with pulmonary hypertension. Here, we reveal a novel mechanism whereby PDE5 inhibition suppresses serotonin signalling and consequent cellular proliferation in primary cultured pulmonary artery smooth muscle cells (PASMCs). 1 microM serotonin induced 4.15-fold increases in DNA synthesis compared with control; this was accompanied by significant Ras homolog gene family member A (RhoA) activation and ERK1/2 MAP kinase phosphorylation/nucleus translocation. Furthermore, inhibition of Rho-associated kinase (ROCK) abolished serotonin-triggered DNA synthesis and ERK1/2 nucleus translocation without changing ERK1/2 phosphorylation, indicating that ERK1/2 phosphorylation and nucleus translocation are coupled to different upstream effectors. Pre-exposure of cells to sildenafil dose-dependently blocked the activation of these signalling pathways and elevated DNA synthesis. The inhibitive effects of sildenafil, however, were fully reversed by concomitant presence of PKG inhibitor in cells. These data suggest that RhoA and ERK1/2 pathways are partially cross-talked and concordantly mediate serotonin-stimulated PASMCs proliferation thereby vascular remodelling leading to the occurrence of pulmonary hypertension. Meanwhile, these two pathways are also separately modulated by enhanced cGMP-PKG signalling derived from inhibition of PDE5 with sildenafil to confer the overall roles of sildenafil against pulmonary hypertension.

MiR-138 suppresses airway smooth muscle cell proliferation through the PI3K/AKT signaling pathway by targeting PDK1

ABSTRACT Background: Airway smooth muscle cells (ASMCs) play important physiological roles in the lung, and their abnormal proliferation directly contributes to airway remodeling during development of lung diseases such as asthma. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation, but little is known about the precise role of microRNAs in the proliferation of ASMCs. Methods: In this study, human ASMCs from asthmatic and non-asthmatic donors were used. MicroRNA and mRNA expression were measured by quantitative real-time PCR. Dual-luciferase reporter assays were performed to determine whether microRNA-138 (miR-138) binds directly to 3-phosphoinositide-dependent protein kinase-1(PDK1) 3′ untranslated region (3′-UTR) to alter gene expression. Results: The results showed that overexpression of miR-138 reduced proliferation of human ASMCs, whereas inhibition of miR-138 increased proliferation of ASMCs. MiR-138 directly suppressed PDK1 expression by targeting the 3′-UTR of the gene. MiR-138 controls ASMC proliferation through directly inhibiting the phosphoinositide 3-kinase (PI3K) pathway. Conclusions: Our study indicated that miR-138 regulation of PI3K signaling in ASMCs by altering the expression of PDK1 can have a profound impact on cell proliferation.

MiR-21 modulates human airway smooth muscle cell proliferation and migration in asthma through regulation of PTEN expression.

ABSTRACT Background: Asthma is characterized by airway remodeling arising from an increase in airway smooth muscle (ASM) mass. This increase is regulated in part by ASM cell proliferation and migration. MicroRNA (miR)-21 also plays a role in asthma, but the molecular mechanisms underlying its effects are not completely understood. This study investigated the effects and mechanism of miR-21 on the human ASM (HASM) cell proliferation and migration. Materials and Methods: HASM cells were transduced with a miR-21 vector, and the expression of miR-21 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of the miR-21 on HASM cell proliferation and migration was analyzed by CCK8 and transwell assay. The expression level of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in HASM cells was assessed by qRT-PCR and Western blot analysis. Meanwhile, the activity of PTEN was measured by PTEN malachite green assay kit. Results: Lentivirus-mediated miR-21 overexpression markedly enhanced the proliferation and migration of HASM cells (P < .05), and ablation of miR-21 by anti-miR-21 inhibitor markedly reduced cell proliferation and migration. We demonstrated that miR-21 overexpression significantly reduced the expression of PTEN (P < .05), while PTEN knock-down markedly increased HASM cell proliferation and migration. Furthermore, we found that overexpression of PTEN led to a decrease of HASM cell proliferation and migration. MiR-21 mediated HASM cell proliferation and migration through activation of the phosphoinositide 3-kinase pathway. Conclusions: This study provides the first in vitro evidence that overexpression of miR-21 in HASM cells can trigger cell proliferation and migration, and the effects of miR-21 depend on the level of PTEN.

Benefits of adding fluticasone propionate/salmeterol to tiotropium in COPD: a meta-analysis.

OBJECTIVE This meta-analysis was performed to evaluate the efficacy and safety of adding fluticasone propionate/salmeterol (FSC) to tiotropium (Tio) in COPD patients. METHODS A systematic search was made of MEDLINE, Cochrane, ISI Web of Science and SCOPUS databases, and a hand search of leading respiratory journals. Randomized clinical trials on treatment of stable COPD with the addition of FSC, compared with tiotropium alone, were reviewed. Studies were pooled to odds ratio (OR) and weighted mean differences (WMD), with 95% confidence interval (CI). RESULTS Six trials met the inclusion criteria. Compared with tiotropium, addition of FSC presented significant effects on trough forced expiratory volume in 1s (FEV1) (WMD 54.64 mL; 95% CI 51.76 to 57.52 mL; P<0.001), COPD exacerbations (OR 0.73; 95% CI 0.55 to 0.96; p=0.03), and health-related quality of life (WMD 4.63; 95% CI 4.26 to 5.01; P<0.001). No significant increase was noticed in adverse events in the Tio+FSC group (OR 1.24; 95% CI 0.98 to 1.57; p=0.07). CONCLUSIONS The addition of FSC to subjects with COPD treated with tiotropium significantly improves lung function, quality of life and COPD exacerbations without increasing the risk of adverse events.

Effects of miRNA-145 on airway smooth muscle cells function

The pathological changes of airway smooth muscle (ASM) contribute to airway remodeling during asthma. Here, we investigated the effect of miR-145 on ASM function. We found that miR-145 was aberrantly more highly expressed in ASM cells exposed to cytokine stimulation that mimic the airway conditions of patients with asthma. Repression of miR-145 resulted in decreased ASM cell proliferation and migration in a dose-dependent manner and down-regulation of type I collagen and contractile protein MHC in ASM cells. qRT-PCR and Western blot analysis demonstrated that miR-145 negatively regulated the expression of downstream target Krüppel-like factor 4 (KLF4) protein, and overexpression of KLF4 attenuated the effects of miR-145 on ASM cells. Further studies showed that KLF4 significantly up-regulated the expression of p21 and down-regulated matrix metalloproteinase (MMP-2 and MMP-9). In conclusion, miR-145 overexpression in ASM cells significantly inhibited KLF4, and subsequently affected downstream p21, MMP-2, and MMP-9 expressions, eventually leading to enhanced proliferation and migration of ASM cells in vitro.