scholarly journals Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation

2014 ◽  
Vol 307 (7) ◽  
pp. L576-L585 ◽  
Author(s):  
Krishna C. Penumatsa ◽  
Deniz Toksoz ◽  
Rod R. Warburton ◽  
Andrew J. Hilmer ◽  
Tiegang Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Vascular Remodeling ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transglutaminase 2 ◽  
Transient Receptor Potential Channel ◽  
Pulmonary Arterial Hypertension Patient ◽  
Pulmonary Artery Smooth Muscle

We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [3H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.

Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation

2002 ◽  
Vol 283 (1) ◽  
pp. L144-L155 ◽  
Author(s):  
Michele Sweeney ◽  
Ying Yu ◽  
Oleksandr Platoshyn ◽  
Shen Zhang ◽  
Sharon S. McDaniel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Cell Growth ◽  
Pulmonary Artery ◽  
Transient Receptor Potential ◽  
Critical Role ◽  
Cyclopiazonic Acid ◽  
Receptor Potential ◽  
Normal Growth ◽  
Pulmonary Artery Smooth Muscle

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents ( I SOC) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I SOC, was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I SOC and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I SOC and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+]cyt.

scholarly journals Long Non-Coding RNA MEG3 Downregulation Triggers Human Pulmonary Artery Smooth Muscle Cell Proliferation and Migration via the p53 Signaling Pathway

2017 ◽  
Vol 42 (6) ◽  
pp. 2569-2581 ◽  
Author(s):  
Zengxian Sun ◽  
Xiaowei Nie ◽  
Shuyang Sun ◽  
Shumin Dong ◽  
Chunluan Yuan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Remodeling ◽  
Pulmonary Artery Smooth Muscle ◽  
And Migration ◽  
Proliferation And Migration

Background/Aims: Increasing evidence has demonstrated a significant role of long non-coding RNAs (lncRNAs) in diverse biological processes, and many of which are likely to have functional roles in vascular remodeling. However, their functions in pulmonary arterial hypertension (PAH) remain largely unknown. Pulmonary vascular remodeling is an important pathological feature of PAH, leading to increased vascular resistance and reduced compliance. Pulmonary artery smooth muscle cells (PASMCs) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of PASMCs function. Herein, we determined whether long noncoding RNA–maternally expressed gene 3 (MEG3) was involved in PAH-related vascular remodeling. Methods: The arterial wall thickness was examined by hematoxylin and eosin (H&E) staining in distal pulmonary arteries (PAs) isolated from lungs of healthy volunteers and PAH patients. The expression level of MEG3 was analyzed by qPCR. The effects of MEG3 on human PASMCs were assessed by cell counting Kit-8 assay, BrdU incorporation assay, flow cytometry, scratch-wound assay, immunofluorescence, and western blotting in human PASMCs. Results: We revealed that the expression of MEG3 was significantly downregulated in lung and PAs of patients with PAH. MEG3 knockdown affected PASMCs proliferation and migration in vitro. Moreover, inhibition of MEG3 regulated the cell cycle progression and made more smooth muscle cells from the G0/G1 phase to the G2/M+S phase and the process could stimulate the expression of PCNA, Cyclin A and Cyclin E. In addition, we found that the p53 pathway was involved in MEG3–induced smooth muscle cell proliferation. Conclusions: This study identified MEG3 as a critical regulator in PAH and demonstrated the potential of gene therapy and drug development for treating PAH.

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

2007 ◽  
Vol 292 (5) ◽  
pp. L1202-L1210 ◽  
Author(s):  
Shen Zhang ◽  
Hemal H. Patel ◽  
Fiona Murray ◽  
Carmelle V. Remillard ◽  
Christian Schach ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Phosphodiesterase Inhibitor ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Normal Subjects ◽  
Pulmonary Artery Smooth Muscle ◽  
The Impact

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca2+ concentration, resulting from enhanced capacitative Ca2+ entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 μM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 μM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.

scholarly journals Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells

2016 ◽  
Vol 311 (1) ◽  
pp. C24-C34 ◽  
Author(s):  
Prasanna Abeyrathna ◽  
Laszlo Kovacs ◽  
Weihong Han ◽  
Yunchao Su
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Vascular Remodeling ◽  
Collagen Synthesis ◽  
Pulmonary Vascular Remodeling ◽  
Calpain 2 ◽  
Pulmonary Artery Smooth Muscle

Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism.

Mibefradil suppresses the proliferation of pulmonary artery smooth muscle cells

2016 ◽  
Vol 64 (1) ◽  
pp. 45-49 ◽  
Author(s):  
Hong-Hong Li ◽  
Li-Jian Xie ◽  
Ting-Ting Xiao ◽  
Min Huang ◽  
Jie Shen
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Transient Receptor Potential ◽  
Critical Role ◽  
Receptor Potential ◽  
Channel Expression ◽  
Intracellular Ca ◽  
Pulmonary Arterial ◽  
Pulmonary Artery Smooth Muscle ◽  
Transient Receptor

Intracellular Ca2+ levels play a critical role in the regulation of vasodilation and vasoconstriction by stimulating pulmonary artery smooth muscle cell (PASMC) proliferation, which is important in the pathogenesis of pulmonary arterial hypertension (PAH); however, L-type Ca2+ channel antagonists are useful in only few patients with PAH. The present study sought to assess the effect of mibefradil, which blocks T-type Ca2+ channels, on PASMC proliferation and Ca2+ channel profile. Human PASMCs were stimulated with 25 ng/mL platelet-derived growth factor-BB (PDGF-BB) with and without 10 µM mibefradil or 100 nM sildenafil. After 48 or 72 h, PASMC proliferation and Ca2+ channel expression were assessed by MTT assays and western blot analysis, respectively. PDGF-BB-induced PASMC proliferation at 72 h (p<0.01), which was inhibited by both sildenafil and mibefradil (p<0.01). Transient receptor potential Ca2+ channel 6 (TRPC6) expression was significantly increased with PDGF-BB stimulation (p=0.009); however, no changes in TRPC1, TRPC3, CAV1.2, and CAV3.2 levels were observed. Although both TRPC1 and CAV1.2 expression levels were increased in PDGF-stimulated PASMCs on mibefradil and sildenafil treatment, it was not statistically significant (p=0.086 and 1.000, respectively). Mibefradil inhibits PDGF-BB-stimulated PASMC proliferation; however, the mechanism through which it functions remains to be determined. Further studies are required to elucidate the full therapeutic value of mibefradil for PAH.

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

2005 ◽  
Vol 288 (2) ◽  
pp. C245-C252 ◽  
Author(s):  
Shen Zhang ◽  
Jason X.-J. Yuan ◽  
Kim E. Barrett ◽  
Hui Dong
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Reverse Mode ◽  
Store Depletion ◽  
Pulmonary Artery Smooth Muscle ◽  
Transient Receptor

A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+ stores opens store-operated Ca2+ channels (SOC) and causes Ca2+ entry. Transient receptor potential (TRP) cation channels that are permeable to Na+ and Ca2+ are believed to form functional SOC. Because sarcolemmal Na+/Ca2+ exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cyt by contributing to store depletion-mediated Ca2+ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+ exchanger from the forward (Ca2+ exit) to reverse (Ca2+ entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+ exchanger with KB-R7943 (10 μM) markedly attenuated the increase in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Store depletion also induced a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Removal of extracellular Na+ or inhibition of the Na+/Ca2+ exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension.

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