Dataset Information

Mechanism of anti-remodelling action of treprostinil in human pulmonary arterial smooth muscle cells.

ABSTRACT: Treprostinil is applied for pulmonary arterial hypertension (PAH) therapy. However, the mechanism by which the drug achieves its beneficial effects in PAH vessels is not fully understood. This study investigated the effects of treprostinil on PDGF-BB induced remodelling parameters in isolated human pulmonary arterial smooth muscle cells (PASMC) of four PAH patients. The production of TGF-?1, CTGF, collagen type-I and -IV, and of fibronectin were determined by ELISA and PCR. The role of cAMP was determined by ELISA and di-deoxyadenosine treatment. Proliferation was determined by direct cell count. Treprostinil increased cAMP levels dose and time dependently, which was not affected by PDGF-BB. Treprostinil significantly reduced PDGF-BB induced secretion of TGF-?1 and CTGF, both was counteracted when cAMP generation was blocked. Similarly, the PDGF-BB induced proliferation of PASMC was dose dependently reduced by treprostinil through signalling via cAMP-C/EBP-? p42 -p21(WAf1/Cip1). In regards to extracellular matrix remodelling, treprostinil significantly reduced PDGF-BB-TGF-?1-CTGF induced synthesis and deposition of collagen type I and fibronectin, in a cAMP sensitive manner. In contrast, the deposition of collagen IV was not affected. The data suggest that this action of treprostinil in vessel wall remodelling may benefit patients with PAH and may reduce arterial wall remodelling.

SUBMITTER: Lambers C

PROVIDER: S-EPMC6211661 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Mechanism of anti-remodelling action of treprostinil in human pulmonary arterial smooth muscle cells.

Lambers Christopher C Kornauth Christoph C Oberndorfer Felicitas F Boehm Panja M PM Tamm Michael M Klepetko Walter W Roth Michael M

PloS one 20181101 11

Treprostinil is applied for pulmonary arterial hypertension (PAH) therapy. However, the mechanism by which the drug achieves its beneficial effects in PAH vessels is not fully understood. This study investigated the effects of treprostinil on PDGF-BB induced remodelling parameters in isolated human pulmonary arterial smooth muscle cells (PASMC) of four PAH patients. The production of TGF-β1, CTGF, collagen type-I and -IV, and of fibronectin were determined by ELISA and PCR. The role of cAMP was ...[more]

PMID: 30383775

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Project description:Smooth muscle cell (SMC) phenotypic switch from a quiescent 'contractile' phenotype to a dedifferentiated and proliferative state underlies the development of cardiovascular diseases (CVDs); however, our understanding of the mechanism is still incomplete. In the present study, we explored the potential role of ferroptosis, a novel nonapoptotic form of cell death, in SMC phenotypic switch and related neointimal formation. We found that ferroptotic stress was triggered in cultured dedifferentiated SMCs and arterial neointimal tissue of wire-injured mice. Moreover, pro-ferroptosis stress was activated in arterial neointimal tissue of clinical patients who underwent carotid endarterectomy. Blockade of ferroptotic stress via administration of a pharmacological inhibitor or by global genetic overexpression of glutathione peroxidase-4 (GPX4), a well-established anti-ferroptosis molecule, delayed SMC phenotype switch and arterial remodelling. Conditional SMC-specific gene delivery of GPX4 using adreno-associated virus in the carotid artery inhibited ferroptosis and prevented neointimal formation. Conversely, ferroptosis stress directly triggered dedifferentiation of SMCs. Transcriptomics analysis demonstrated that inhibition of ferroptotic stress mainly targets the mitochondrial respiratory chain and oxidative phosphorylation. Mechanistically, ferroptosis inhibition corrected the disrupted mitochondrial homeostasis in dedifferentiated SMCs, including enhanced mitochondrial ROS production, dysregulated mitochondrial dynamics, and mitochondrial hyperpolarization, and ultimately inhibited SMC phenotypic switch and growth. Copper-diacetyl-bisN4-methylthiosemicarbazone (CuATSM), an agent used for clinical molecular imaging and that potently inhibits ferroptosis, prevented SMC phenotypic switch, neointimal formation and arterial inflammation in mice. These results indicate that pro-ferroptosis stress is likely to promote SMC phenotypic switch during neointimal formation and imply that inhibition of ferroptotic stress may be a promising translational approach to treat CVDs with SMC phenotype switch.

Project description:Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.

Dataset Information

Mechanism of anti-remodelling action of treprostinil in human pulmonary arterial smooth muscle cells.

Publications

Mechanism of anti-remodelling action of treprostinil in human pulmonary arterial smooth muscle cells.

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