Hypoxia promotes rabbit pulmonary artery smooth muscle cells proliferation through a 15-LOX-2 product 15(S)-hydroxyeicosatetraenoic acid

The initial event of hypoxic pulmonary hypertension is acute hypoxic pulmonary vasoconstriction followed by remodeling of pulmonary arteries. Although 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] is found to be able to induce hypoxic pulmonary vasoconstriction, role of 15(S)-HETE in pulmonary artery smooth muscle cells (PASMCs) proliferation has been studied less. We sought evidence for a role of 15(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that hypoxia enhances 15-lipoxygenase-2 (15-LOX-2) expression and stimulates cultured rabbit PASMCs proliferation. 15(S)-HETE at concentration 0.1 μM stimulated proliferation of PASMCs and induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 15(S)-HETE-stimulated PASMC proliferation was blocked by the MEK inhibitors PD-98059. Hypoxia (3% O(2))-stimulated PASMC proliferation was blocked by U0126, a MEK inhibitor, as well as by NDGA and CDC, inhibitors of 15-LOX, but not by the p38 MAPK inhibitor SB-202190. We conclude that 15-LOX-2 and its product, 15(S)-HETE, are important intermediates in hypoxia-induced rabbit PASMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

Silencing of STIM1 attenuates hypoxia-induced PASMCs proliferation via inhibition of the SOC/Ca2+/NFAT pathway

Background

Stromal interaction molecule 1 (STIM1) is a newly discovered Ca²⁺ sensor on the endoplasmic reticulum which is an indispensable part in the activation of store-operated Ca²⁺ channels (SOC). Recent studies demonstrate that SOC of pulmonary smooth muscle cells (PASMCs) were upregulated by chronic hypoxia which contribute to the enhanced pulmonary vasoconstriction and vascular remodeling. However, the exact role of STIM1 in the development of chronic hypoxic pulmonary hypertension(HPH) remains unclear.

Methods

In this study we investigated the cellular distribution and expression of STIM1 by immunofluorescence, qRTPCR and Western blotting methods in Wistar rat distal intrapulmonary arteries under normal and chronic hypobaric hypoxic conditions. In vitro, Wistar rat PASMCs were isolated and cultured. PASMCs were transfected with siRNA targeting STIM1 gene by liposome. The expression of STIM1 protein was detected by Western blotting. [³H]-thymidine ([³H]-TdR) incorporation were performed to detect PASMCs proliferation. The cell cycle was analyzed by flow cytometry. The SOC-mediated Ca²⁺ influx was calculated by Ca²⁺ fluorescence imaging and the nuclear translocation of NFATc3 was determined by immunofluorescence and Western blot analysis of nuclear extracts.

Results

We found that during the development of HPH and the initiation of vascular remodeling, the mRNA and protein expression levels of STIM1 significantly increased in the distal intrapulmonary arteries. Moderate hypoxia significantly promotes PASMCs proliferation and cell cycle progression. Silencing of STIM1 significantly decreased cellular proliferation and delayed the cell cycle progression induced by hypoxia. Silencing of STIM1 also significantly decreased SOC-mediated Ca²⁺ influx and inhibited the nuclear translocation of NFATc3 in hypoxic PASMCs.

Conclusion

Our findings suggest that chronic hypobaric hypoxia upregulates the expression of STIM1 in the distal intrapulmonary arteries which plays an important role in the hypoxia-induced PASMCs proliferation via SOC/Ca²⁺/NFAT pathway and may represent a novel therapeutic target for the prevention of hypoxia pulmonary hypertension.     

Platelet-derived growth factor (PDGF) induces pulmonary vascular remodeling through 15-LO/15-HETE pathway under hypoxic condition

15-lipoxygenase (15-LO) is known to play an important role in chronic pulmonary hypertension. Accumulating evidence for its down-stream participants in the vasoconstriction and remodeling processes of pulmonary arteries, while how hypoxia regulates 15-LO/15-hydroxyeicosatetraenoic acid (15-HETE) to mediate hypoxic pulmonary hypertension is still unknown. Platelet-derived growth factor (PDGF) is an important vascular regulator whose concentration increases under hypoxic condition in the lungs of both humans and mice with pulmonary hypertension. The present study was carried out to determine whether hypoxia advances the pulmonary vascular remodeling through the PDGF/15-LO/15-HETE pathway. We found that pulmonary arterial medial thickening caused by hypoxia was alleviated after a treatment of the hypoxic rats with imatinib, which was associated with down-regulations of 15-LO-2 expression and 15-HETE production. Moreover, the increases in cell proliferation and endogenous 15-HETE content by hypoxia were attenuated by the inhibitors of PDGF-β receptor in pulmonary artery smooth muscle cells (PASMCs). The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of 15-LO inhibitors or 15-LO RNA interference. These results suggest that hypoxia promotes PASMCs proliferation and survival, contributing to pulmonary vascular medial hypertrophy, which is likely to be mediated via the PDGF-BB/15-LO-2/15-HETE pathway.     

Kv3.4通道参与15-羟二十碳四烯酸诱导的大鼠肺动脉收缩

通过组织浴槽血管环方法观察Kv3．4通道特异阻断剂BDS-Ⅰ对15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-FETE）收缩肺动脉血管的影响;通过酶法分离、培养Wistar大鼠肺动脉血管平滑肌细胞（pulmonary artery smooth musclecells,PASMCs）,RT-PCR和Western blot技术观察15-HETE对大鼠PASMCs上Kv3．4通道表达的影响,以探讨Kv3．4通道在15-HETE收缩肺动脉过程中的作用。结果如下：（1）15-HETE以浓度依赖方式使肺动脉环张力增加,对缺氧组大鼠肺动脉环张力作用更为明显,与正常对照组相比差异显著;（2）除去肺动脉内皮后,15-HETE引起血管收缩的强度较内皮完整时增强,呈剂量依赖性收缩反应;（3）阻断Kv3．4通道可抑制15-HETE收缩肺动脉;（4）15-HETE下调PASMCs膜上Kv3．4通道mRNA及蛋白质表达。上述观察结果提示Kv3．4通道参与由15-HETE引起的缺氧肺动脉血管收缩（hypoxic pulmonary vasoconstriction,HPV）。     

Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca²⁺ released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30?min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca²⁺ and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.     

Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels

Activity of voltage-gated potassium (Kv) channels controls membrane potential, which subsequently regulates cytoplasmic free calcium concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMCs). Acute hypoxia inhibits Kv channel function in PASMCs, inducing membrane depolarization and a rise in [Ca²⁺ ]_cyt that triggers vasoconstriction. Prolonged hypoxia inhibits expression of Kv channels and reduces Kv channel currents in PASMCs. The consequent membrane depolarization raises [Ca²⁺]_cyt, thus stimulating PASMC proliferation. The present review discusses recent evidence for the involvement of Kv channels in initiation of hypoxic pulmonary vasoconstriction and in chronic hypoxia-induced pulmonary hypertension.     

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）在低氧性肺血管收缩中起着重要作用,低氧肺动脉高压下调内皮型。氧化氮合酶（endothelial nitric oxide synthase,eNOS）,使一氧化氮（nitric oxide,NO）的产量下降,但目前尚无关于15-HETE与eNOS／NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS／NO的相互作用进行研究。首先分离人鼠肺动脉,分为eNOS抑制剂L-NAME组（0．1mmol／L）、去缸管内皮组与内皮完整组,用15-HETE作用夫鼠离体肺动脉环,测定肺动脉张力。结果表明,L-NAME组、去除内皮组与内皮完整组分别比较,15-HETE对血管的收缩作用增强,且都有统计学意义（P〈0．05）。培养牛肺动脉内皮细胞,分别用15-HETE、15-脂氧酶（15-lipoxygenase,15-LO）抑制剂[（cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate,CDC）和（nordihydroguiairetic acid,YDGA）]处理细胞,通过Greiss方法检测亚硝酸盐含量,间接测定NO产量,与对照组比较,1μmol／L 15-HETE明显降低肺动脉内皮细胞NO水平（P〈0．05）,10μmol／L CDC和0．1mmol／L NDGA显著增加NO水平（分别是P〈0．05,P〈0．01）;通过Western blot检测不同时间（5,10,15,20,30,60min）eNOS的表达情况,结果显示,15-HETE的不同作用时间,没有引起eNOS表达的明显不同;用苏氨酸495位点磷酸化eNOS（Thr495）抗体进行免疫沉淀,再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量,问接测定eNOS活性,结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点,因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS／NO参与,15-HETE可以通过磷酸化Thr495位点降低eNOS活性,并且首次发现磷酸化eNOS（Thr495）和15-LO之间存在蛋白质相互作用。     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

15-PGDH/15-KETE plays a role in hypoxia-induced pulmonary vascular remodeling through ERK1/2-dependent PAR-2 pathway

We have established that 15-hydroxyeicosatetraenoic acid is an important factor in regulation of pulmonary vascular remodeling (PVR) associated with hypoxia-induced pulmonary hypertension (PH), which is further metabolized by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to form 15-ketoeicosatetraenoic acid (15-KETE). However, the role of 15-PGDH and 15-KETE on PH has not been identified. The purpose of this study was to investigate whether 15-PGDH/15-KETE pathway regulates hypoxia-induced PVR in PH and to characterize the underlying mechanisms. To accomplish this, Immunohistochemistry, Ultra Performance Liquid Chromatography, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed. Our results showed that the levels of 15-PGDH expression and endogenous 15-KETE were drastically elevated in the lungs of humans with PH and hypoxic PH rats. Hypoxia stimulated pulmonary arterial smooth muscle cell (PASMC) proliferation, which seemed to be due to the increased 15-PGDH/15-KETE. 15-PGDH/15-KETE pathway was also capable of stimulating the cell cycle progression and promoting the cell cycle-related protein expression. Furthermore, 15-KETE-promoted cell cycle progression and proliferation in PASMCs depended on protease-activated receptor 2 (PAR-2). ERK1/2 signaling was likely required for 15-PGDH/15-KETE-induced PAR-2 expression under hypoxia. Our study indicates that 15-PGDH/15-KETE stimulates the cell cycle progression and proliferation of PASMCs involving ERK1/2-mediated PAR-2 expression, and contributes to hypoxia-induced PVR.     

Isolation,culture and identification of pulmonary arterial smooth muscle cells from rat distal pulmonary arteries

The culture of pulmonary arterial smooth muscle cells (PASMCs) is one of the most powerful tools for exploring the mechanisms of pulmonary hypertension (PH). Both pulmonary vasoconstriction and remodeling occur predominantly in distal pulmonary arteries (PA). In this study, we provide our detailed and standardized protocol for easy isolation and culture of PASMCs from rat distal PA to supply every investigator with a simple, economical and useful method in studying PH. The protocol can be divided into four stages: isolation of distal PA, isolation of cells, growth in culture and passage of cells. Rat distal PASMCs were characterized by morphological activity and by immunostaining for smooth muscle α-actin and smooth muscle myosin heavy chain, but not for CD90/Thy-1 or von Willebrand factor. Furthermore, functional assessments were performed, confirming the presence of voltage-dependent Ca²⁺ channels and physiological characteristic of response to hypoxia. In conclusion, we have developed a detailed and simple protocol for obtaining rat distal PASMCs. These PASMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to further explore the pathophysiological mechanisms of PH.     

15-HETE suppresses K+ channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K⁺ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K⁺ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K⁺ channels. Yumei Li and Qian Li contributed equally to this work.     

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.     

ATP Antagonizes Thrombin-Induced Signal Transduction through 12(S)-HETE and cAMP

In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y₁ and P2Y₁₂ receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca²⁺]_i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.     

Role of 15-hydroxyeicosatetraenoic acid in phosphorylation of ERK1/2 and caldesmon in pulmonary arterial smooth muscle cells

We have reported that 15-hydroxyeicosatetraenoic acid (15-HETE) induces pulmonary artery (PA) contraction in rats exposed to hypoxia by activating extracellular signal-regulated kinase 1/2 (ERK1/2). In this study, we investigated the characteristics of 15-HETE mediating phosphorylation of ERK1/2 and caldesmon in rat pulmonary arterial smooth muscle cells (PASMCs). Our data showed that 15-HETE upregulated ERK1/2 phosphorylation in a dose-dependent manner, which could be blocked by ERK pathway inhibitors U0126 and PD98059. ERK1/2 phosphorylation was attenuated by inhibiting endogenous 15-HETE formation with lipoxygenase inhibitor, cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate (CDC), in both normoxic and hypoxic PASMCs. ERK1/2 phosphorylation in response to 15-HETE was detected in cytosol as well as in nucleus and phosphorylatd ERK1/2 partly translocated into nucleus, which could be blocked by PD98059. In addition, caldesmon was phosphorylated in 15-HETE-stimulated cells; this could be inhibited by PD98059. These data demonstrated that 15-HETE is associated with ERK1/2 activation and caldesmon phosphorylation in PASMCs and that 15-HETE is at least partly involved in mediating activation of hypoxia-initiated ERK pathway, possibly leading to hypoxic pulmonary vasoconstriction.     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca²⁺] _i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca²⁺]_i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca²⁺]_i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca²⁺]_i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.     

Hypoxia suppresses KV1.5 channel expression through endogenous 15-HETE in rat pulmonary artery

Hypoxia initiated pulmonary vasoconstriction is due to the inhibition of voltage-gated K(+) (K(V)) channels. But the mechanism is unclear. We have evidence that hypoxia activates 15-lipoxygenase (15-LOX) in distal pulmonary arteries and increases the formation of 15-hydroxyeicosatetraenoate (15-HETE). 15-HETE-induced pulmonary artery constriction to be through the inhibition of K(V) channels (K(V)1.5, K(V)2.1 and K(V)3.4). However, no direct link among hypoxia, 15-HETE and inhibition of K(V) subtypes is established. Therefore, we investigated whether 15-LOX/15-HETE pathway contributes to the hypoxia-induced down-regulation of K(V) channels. As K(V)1.5 channel is O(2)-sensitive, it was chosen in the initial study. We found that inhibition of 15-LOX suppressed the response of hypoxic pulmonary artery rings to phenylephrine. The expressions of K(V)1.5 channel mRNA and protein was robustly up-regulated in cultured PASMC and pulmonary artery after blocking of 15-LOX by lipoxygenase inhibitors in hypoxia. The 15-LOX blockade also partly rescued the voltage-gated K(+) current (I(K(V))). 15-HETE contributes to the down-regulation of K(V)1.5 channel, inhibition of I(K(V)) and increase of native pulmonary artery tension after hypoxia. Hypoxia inhibits K(V)1.5 channel through 15-LOX/15-HETE pathway.     

The key role of transforming growth factor-beta receptor I and 15-lipoxygenase in hypoxia-induced proliferation of pulmonary artery smooth muscle cells

Our laboratory has proved that 15-hydroxyeicosatetraenoic acid, a product of arachidonic acid catalyzed by 15-lipoxygenase (15-LO), plays a pivotal role in hypoxic pulmonary arterial hypertension. However, the mechanisms of how hypoxia regulates 15-LO expression are still unclear. As the formation of endogenous transforming growth factor-beta1 (TGF-β1), implicated in pulmonary arterial hypertension pathogenesis, was promoted by hypoxia, we suspect whether hypoxia-induced the expression of 15-LO is via the TGF-β1 pathway. We found that treatment of pulmonary artery smooth muscle cells with TGF-β1 significantly increased the expression of 15-LO and levels of 15-hydroxyeicosatetraenoic acid, product of 15-LO, which were inhibited by transforming growth factor-beta receptor I (TGFβRI) inhibitor, SD-208 and siRNA targeted to knockdown rat TGFβRI. Moreover, our results showed that TGF-β1 regulated the cell cycle progression and made more cells from the G(0)/G(1) phase to the G(2)/M+S phase and enhanced the microtubule formation in cell nucleus. Additionally, we found that the 15-LO pathway was involved in TGFβ-1-mediated cell viability, DNA synthesis and the cell cycle progression. Our data provide novel evidence that hypoxia induced 15-LO expression is through TGF-β1, and 15-LO pathway plays a critical role in TGFβRI mediated the proliferation of pulmonary artery smooth muscle cells induced by hypoxia. Thus, new strategies aimed at combined blockade of TGFβRI as well as 15-LO may yield optimal therapeutic benefits.