Primary Pulmonary Arterial Smooth Muscle Cells Research Articles

Losartan attenuates chronic cigarette smoke exposure-induced pulmonary arterial hypertension in rats: Possible involvement of angiotensin-converting enzyme-2

Chronic cigarette smoking induces pulmonary arterial hypertension (PAH) by largely unknown mechanisms. Renin–angiotensin system (RAS) is known to function in the development of PAH. Losartan, a specific angiotensin II receptor antagonist, is a well-known antihypertensive drug with a potential role in regulating angiotensin-converting enzyme-2 (ACE2), a recently found regulator of RAS. To determine the effect of losartan on smoke-induced PAH and its possible mechanism, rats were daily exposed to cigarette smoke for 6 months in the absence and in the presence of losartan. Elevated right ventricular systolic pressure (RVSP), thickened wall of pulmonary arteries with apparent medial hypertrophy along with increased angiotensin II (Ang II) and decreased ACE2 levels were observed in smoke-exposed-only rats. Losartan administration ameliorated pulmonary vascular remodeling, inhibited the smoke-induced RVSP and Ang II elevation and partially reversed the ACE2 decrease in rat lungs. In cultured primary pulmonary artery smooth muscle cells (PASMCs) from 3- and 6-month smoke-exposed rats, ACE2 levels were significantly lower than in those from the control rats. Moreover, PASMCs from 6-month exposed rats proliferated more rapidly than those from 3-month exposed or control rats, and cells grew even more rapidly in the presence of DX600, an ACE2 inhibitor. Consistent with the in vivo study, in vitro losartan pretreatment also inhibited cigarette smoke extract (CSE)-induced cell proliferation and ACE2 reduction in rat PASMCs. The results suggest that losartan may be therapeutically useful in the chronic smoking-induced pulmonary vascular remodeling and PAH and ACE2 may be involved as part of its mechanism. Our study might provide insight into the development of new therapeutic interventions for PAH smokers.

BFGF induces S1P1 receptor expression and functionality in human pulmonary artery smooth muscle cells

Sphingosine-1-phosphate (S1P), acting through five closely related G-protein coupled receptors termed S1P1-5, has recently emerged as a possible regulator of smooth muscle cell (SMC) physiology with the potential to induce contraction, proliferation and stress fiber formation. In the present study, real-time quantitative PCR was used to determine the expression patterns of S1P receptor subtypes in human primary pulmonary artery smooth muscle cells (PASMC). We report here that subconfluent PASMC express predominantly S1P2 and S1P3 receptors and we show that S1P1 receptor mRNA levels are significantly up-regulated following basic fibroblast growth factor (bFGF) treatment. As a consequence, increased responsiveness, as measured by impedance and ERK1/2 phosphorylation, was observed upon stimulation with a specific S1P1 receptor agonist SEW2871. We therefore demonstrate, for the first time, that a growth factor that was previously shown to be involved in physiological and pathological changes of SMC function induced S1P1 receptor expression and we propose that S1P1 receptor up-regulation could contribute to vascular remodeling.

Increased hyaluronic acid content in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease characterised by elevated blood pressure in the pulmonary circulation. Initial vasoconstriction, proliferation of pulmonary arterial smooth muscle cells (PASMC) and increased deposition of extracellular matrix (ECM) contribute to pathological remodelling of pulmonary arterioles in IPAH. Glycosaminoglycans (GAGs), components of the ECM, control cellular proliferation and differentiation, but their expression in IPAH remains elusive. In the present study, GAG expression was investigated in the lungs of patients with IPAH or control transplant donors, and expression and localisation of GAG-metabolising enzymes were analysed in vivo and in vitro. A significant increase in the expression of hyaluronic acid (HA) was detected in IPAH lungs, associated with increased hyaluronan synthase (Has)1 and decreased hyaluronoglucosaminidase 1 gene expression, as assessed by quantitative RT-PCR and Western blotting. HAS1 protein localised to PASMC in vivo and increased HA deposition was observed in remodelled pulmonary arteries in IPAH. Transforming growth factor-beta1, a profibrotic growth factor, led to increased HA secretion and HAS1 expression in primary PASMC. The results demonstrate an increased hyaluronic acid content in idiopathic pulmonary arterial hypertension lungs, associated with increased hyaluronan synthase 1 and decreased hyaluronoglucosaminidase 1 gene expression. Synergistic regulation of glycosaminoglycan-metabolising enzymes in favour of accumulation may, thus, regulate pathological vascular remodelling in idiopathic pulmonary arterial hypertension lungs.

Smad-Dependent MicroRNA Processing

Signaling initiated by binding of ligands of the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) families to their cognate receptors stimulates the activity of Smad proteins (see Connections Map by Wrana and Attisano). In the canonical pathway, receptor Smads (R-Smads) are phosphorylated by the ligand-bound receptor complex and then interact with the common Smad, Smad4, which leads to nuclear translocation of the R-Smad/Smad4 complex and regulation of specific genes. Davis et al . show that a Smad4-independent pathway for TGF-β and BMP signaling is mediated by the interaction of R-Smads with specific primary microRNA (miRNA) transcripts and the miRNA processing complex called DROSHA, which contains the RNase III DROSHA, the DiGeorge syndrome critical region gene 8 (DGCR8), and RNA helicases p68 and p72, and that this interaction allows TGF-β and BMP to stimulate the production of the mature miRNAs, which then repress the expression of specific genes. In vascular smooth muscle cells, BMP or TGF-β signaling leads to the increased abundance of smooth muscle cell markers, such as smooth muscle α-actin (SMA). Davis et al . show that BMP2, 4, or 7 stimulated the abundance of mature miR-21 and miR-199a through a nontranscriptional mechanism in human primary pulmonary artery smooth muscle cells (PASMCs). The abundance of the primary miR-21 transcripts did not increase, only the abundance of the precursor and mature miR-21 increased in response to BMP or TGF-β. Expression of the miR-21 target PDCD4 (encoding programmed cell death 4) was decreased in response to BMP. Forced expression of PDCD4 prevented the induction of smooth muscle cell markers by BMP, and knockdown of PDCD4 with small-interfering RNA (siRNA) prevented BMP from stimulating SMA abundance. Knockdown of both BMP-specific R-Smads, Smad1 and Smad5, which are present in PASMCs, prevented BMP-induced increase in the precursor and mature miR-21 ; however, knockdown of Smad4 had no effect. In vitro assays suggested that Smad1, Smad5, and the TGF-β-specific Smad3 interacted with p68 through an RNA-independent mechanism and that BMP stimulated the coimmunoprecipitation of DROSHA and p68 with Smad1 or 5 from PASMCs. RNA chromatin immunoprecipitation experiments revealed that BMP4 stimulated the association of Smad1, but not Smad2 or Smad3, with primary miR-21 or miR-199a , but not miR-214 , and that TGF-β stimulated the association of Smad2 or Smad3, but not Smad1, with primary miR-21 or miR-199a , but not miR-214 . Thus, the interaction between the Smads and the microRNA precursor is ligand specific and microRNA specific. Nuclear extracts from Cos7 cells treated with BMP4 or TGF-β exhibited enhanced primary miR-21 processing activity. Promotion of microRNA processing may be a mechanism by which TGF-β signaling contributes to oncogenesis. In the Smad4-deficient breast cancer cell line MDA-MB-468, TGF-β stimulation increased precursor and mature miR-21 abundance, and expression of a dominant-negative TGF-β receptor subunit in these cells decreased the abundance of the precursor miR-21 , suggesting that autocrine signaling by TGF-β through the Smad4-independent miRNA processing pathway may contribute to the cancerous phenotype. B. N. Davis, A. C. Hilyard, G. Lagna, A. Hata, SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454 , 56-61 (2008). [PubMed]

Shroom expression is attenuated in pulmonary arterial hypertension

Shroom is a PDZ-domain protein involved in the regulation and maintenance of cytoskeletal architecture by binding to actin. Hypertrophy and altered actin organisation of pulmonary arterial smooth muscle cells (PASMC) is a hallmark of pulmonary arterial hypertension (PAH). The aim of the present study was to localise and characterise Shroom expression in the lung in experimental and idiopathic PAH (IPAH). Shroom expression and localisation in hypoxia-induced PAH in mice and IPAH in humans, in vivo, as well as in primary PASMC, in vitro, was assessed by quantitative RT-PCR, immunofluorescence, laser-assisted microdissection and immunohistochemistry. Shroom localised exclusively to PASMC (both bronchial and vascular) in mouse and human lungs. Both in vivo and in primary PASMC, in vitro, Shroom exhibited spatially similar expression with alpha-smooth muscle actin (alpha-SMA). Shroom expression was significantly reduced in the mouse model of PAH, in primary murine PASMC exposed to hypoxia, and in primary PASMC isolated from patients with IPAH. The ratio between Shroom and alpha-SMA RNA expression further confirmed Shroom downregulation in both mouse and human PASMC. In summary, Shroom localises exclusively to pulmonary smooth muscle cells. Shroom downregulation in pulmonary arterial hypertension suggests a link between Shroom expression and pulmonary arterial smooth muscle cell hypertrophy in pulmonary arterial hypertension.