Proliferation Of Pulmonary Microvascular Endothelial Cells Research Articles

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

MiR144-3p inhibits PMVECs excessive proliferation in angiogenesis of hepatopulmonary syndrome via Tie2

Background/aimIncreasing evidence show microRNAs (miRNAs) are associated with hepatopulmonary syndrome (HPS). The aim of this study was to investigate the role of miR-144 in the angiogenesis of HPS, as well as to identify its underlying mechanism. MethodsThe expression levels of miR-144-3p were assessed in pulmonary micro-vascular endothelial cells (PMVECs), as well as in lung tissues from rats with HPS. We predicted the potential target of miR-144-3p. Tyrosine kinase 2(Tie2) was identified as a target gene of miR144-3p, which has an essential role in the angiogenesis of lung vessel. In addition, the effects of miR-144-3p regulated on Tie2 was examined. The upregulation and down-regulation of miR-144-3p can affect the proliferation of PMVECs. ResultsWe found that the levels of miR-144-3p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-144-3p dramatically inhibited PMVECs proliferation and cell cycle. We further verified the Tie2 as a novel and direct target of miR-144-3p in HPS. ConclusionmiR-144-3p can negatively regulate PMVECs proliferation by Tie2 expression. In addition, overexpression of miR-144-3p may prove beneficial as a therapeutic strategy for HPS treatment.

Cdc42 regulates LPS-induced proliferation of primary pulmonary microvascular endothelial cells via ERK pathway

BackgroundAfter stimulation due to injury, cell division cycle protein 42 (Cdc42) restores and enhances barrier functions by strengthening intercellular adherens junctions; however, its influence on cell proliferation after injury remains unknown. ObjectiveIn this study, we sought to investigate the effect of stimulation using small doses of lipopolysaccharide (LPS) on the proliferation of pulmonary microvascular endothelial cells (PMVECs). MethodsWe stimulated PMVECs with different doses of LPS and evaluated the effects on cell proliferation. We also constructed a primary gene-knockout cell line lacking Cdc42 to verify the role of Cdc42 in regulating the proliferation of PMVECs that were stimulated using LPS and to explore related signaling pathways. ResultsStimulating PMVECs with small doses of LPS increased proliferation. Cdc42 is involved in regulating this process, which was mediated by the extracellular regulated protein kinase (ERK) pathway. ConclusionsCdc42 plays a role in regulating the proliferation of PMVECs stimulated with small doses of LPS, and this regulation involves the ERK pathway.

Inhibition of autophagy ameliorates pulmonary microvascular dilation and PMVECs excessive proliferation in rat experimental hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is a defective liver-induced pulmonary vascular disorder with massive pulmonary microvascular dilation and excessive proliferation of pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that autophagy is involved in pulmonary diseases, protectively or detrimentally. Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS. In the present study, we report that autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary PMVECs induced by CBDL rat serum, two accepted in vivo and in vitro experimental models of HPS. Furthermore, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) significantly alleviated pathological alterations and typical symptom of HPS in CBDL rats in vivo, and consistently 3-MA significantly attenuated the CBDL rat serum-induced excessive proliferation of PMVECs in vitro. All these changes mediated by 3-MA might explain the observed prominent improvement of pulmonary appearance, edema, microvascular dilatation and arterial oxygenation in vivo. Collectively, these results suggest that autophagy activation may play a critical role in the pathogenesis of HPS, and autophagy inhibition may have a therapeutic potential for this disease.

The mechanism underlying alpinetin-mediated alleviation of pancreatitis-associated lung injury through upregulating aquaporin-1.

Characterized by its acute onset, critical condition, poor prognosis, and high mortality rate, severe acute pancreatitis (SAP) can cause multiple organ failure at its early stage, particularly acute lung injury (ALI). The pathogenesis of ALI is diffuse alveolar damage, including an increase in pulmonary microvascular permeability, a decrease in compliance, and invasion of many inflammatory cells. Corticosteroids are the main treatment method for ALI; however, the associated high toxicity and side effects induce pain in patients. Recent studies show that the effective components in many traditional Chinese medicines can effectively inhibit inflammation with few side effects, which can decrease the complications caused by steroid consumption. Based on these observations, the main objective of the current study is to investigate the effect of alpinetin, which is a flavonoid extracted from Alpinia katsumadai Hayata, on treating lung injury induced by SAP and to explore the mechanism underlying the alpinetin-mediated decrease in the extent of ALI. In this study, we have shown through in vitro experiments that a therapeutic dose of alpinetin can promote human pulmonary microvascular endothelial cell proliferation. We have also shown via in vitro and in vivo experiments that alpinetin upregulates aquaporin-1 and, thereby, inhibits tumor necrosis factor-α expression as well as reduces the degree of lung injury. Overall, our study shows that alpinetin alleviates SAP-induced ALI. The likely molecular mechanism includes upregulated aquaporin expression, which inhibits tumor necrosis factor-α and, thus, alleviates SAP-induced ALI.

MicroRNA-199a-5p Regulates the Proliferation of Pulmonary Microvascular Endothelial Cells in Hepatopulmonary Syndrome

Background/Aims: Pulmonary microvascular endothelial cell (PMVEC) proliferation and angiogenesis contribute to the development of hepatopulmonary syndrome (HPS). MicroRNA-199a-5p (miR-199a-5p) has emerged as a potent regulator of angiogenesis, and its expression levels significantly decrease in the serum of patients with hepatopathy. However, it has not been reported about whether miR-199a-5p might control PMVEC proliferation. Here, we described the miR-199a-5p governing PMVEC proliferation in HPS. Methods: PMVECs were treated with rat serum from common bile duct ligation (CBDL) or sham. MiR-199a-5p mimic or inhibitor was used to change the miR-199a-5p expression. Knockdown of caveolin-1 (Cav-1) was performed using siRNA. NSC-23766 was used to inhibit Rac1 activity. Gene and protein expressions were quantified by qRT-PCR and western blot. Cell proliferation was analyzed by ³H-TdR incorporation and CCK-8 assays. Stress fibers were detected by immunofluorescence. Results: CBDL rat serum induced the down-regulation of miR-199a-5p. Delivery of miR-199a-5p suppressed the CBDL rat serum-induced PMVEC proliferation whereas knockdown of miR-199a-5p promoted PMVEC proliferation. This was accompanied by a decrease and an increase in Cav-1 expression, respectively. Cav-1 siRNA abolished the enhancement of PMVEC proliferation induced by the miR-199a-5p inhibition. Although stress fibers were disrupted in Cav-1 deficient cells, NSC-23766 increased stress fibers and contributed to cell proliferation. Conclusions: CBDL rat serum induced down-regulation of miR-199a-5p in PMVECs, which led to an increase of Cav-1 gene expression. Increased Cav-1 expression, by inhibiting Rac1 activity, led to the formation of stress fibers, which contribute to PMVEC proliferation and thus the pathogenesis of HPS.

Proliferative phenotype of pulmonary microvascular endothelial cells plays a critical role in the overexpression of CTGF in the bleomycin-injured rat

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is not very clear, with evidence for the involvement of both inflammation and aberrant vascular remodeling (associated with angiogenesis). Pulmonary microvascular endothelial cells (PMVECs), which play a major role in inflammation, secrete cytokines that promote the transformation and collagen synthesis of fibroblasts. Moreover, angiogenesis is characterized by PMVEC proliferation. The main aim of this study was to confirm the role of PMVECs in pulmonary fibrosis. Accordingly, we observed the functional changes in PMVECs in bleomycin (BLM)-treated rats (pulmonary fibrosis model) in vivo, and compared them with those of rats with pneumonia. The proliferation phenotype and intracellular ionized calcium concentration ([Ca2+]i) of PMVECs from BLM-treated rats were also investigated. The functioning of PMVECs was abnormal in BLM-injured rats, particularly with regard to their proliferation and secretion of connective tissue growth factor (CTGF). [Ca2+]i was increased in the proliferated PMVECs from BLM-treated rats. The findings suggest that dysfunction of PMVECs characterized by overexpression of CTGF is critical in rat pulmonary injury induced by BLM, and is probably related with the proliferative phenotype and [Ca2+]i overload. It can be concluded from the results that proliferation of PMVECs plays an important role in the pathogenesis of BLM-induced PF.

Annexin A1 protein regulates the expression of PMVEC cytoskeletal proteins in CBDL rat serum-induced pulmonary microvascular remodeling

BackgroundHepatopulmonary syndrome (HPS) is characterized by advanced liver disease, hypoxemia and intrapulmonary vascular dilatation (IPVD). The pathogenesis of HPS is not completely understood. Recent findings have established the role of proliferation and phenotype differentiation of pulmonary microvascular endothelial cells (PMVECs) in IPVD of HPS; the change in cytoskeletal proteins and their molecular mechanism play an essential role in the proliferation, phenotype modulation and differentiation of PMVECs. However, little is known about the relevance of cytoskeletal protein expression and its molecular mechanism in IPVD of HPS. In addition, ANX A1 protein has been identified as a key regulator in some important signaling pathways, which influences cytoskeletal remodeling in many diseases, such as lung cancer, liver cancer, etc.MethodsPMVECs were cultured from the normal rats and then divided into three groups(Ad-ANXA1-transfected group, a non-transfected group, and an adenovirus empty vector group) and incubated by nomal rat serum or hepatopulmonary syndrome rat serum respectively. mRNA level was evaluated by real time reverse transcription polymerase chain reaction, and protein expression was detected by western blot. Cell proliferation was determined by the MTT and thymidine incorporation assay.ResultsIn this study, we found that the serum from a common bile duct ligation(CBDL) Rat model decreased the expression levels of the ANX A1 mRNA and protein by at least two-fold in human PMVECs. We also found the expression of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) in PMVECs significantly increased. After stimulating ANX A1 over-expression in PMVECs by adenovirus-mediated ANX A1 (Ad-ANXA1) transfection, we found the expression levels of cytoskeletal proteins were significantly suppressed in PMVECs at all time points. Additionally, we report here that serum from a CBDL Rat model increases the proliferation of PMVECs by nearly two-fold and that over-expression of Ad-ANXA1 significantly inhibits HPS-rat-serum-induced PMVEC proliferation (p <0.05). These findings suggest that the ANX A1 down-regulation of PMVEC proliferation in the presence of HPS-rat-serum may be the major cause of aberrant dysregulation of cytoskeletal proteins (Destrin, a1-actin, and a1-tubulin) and may, therefore, play a fundamental role in the proliferation and phenotype differentiation of PMVECs in the PVD of HPS.ConclusionFinally, the fact that transfection with Ad-ANXA1 results in inhibition of the aberrant dysregulation of cytoskeletal proteins and proliferation of PMVECs suggests a potential therapeutic effect on PVD of HPS.

Endostatin limits pulmonary endothelial repair via RhoA inactivation

Endothelial repair following vascular injury involves cell proliferation and migration. Endostatin regulates cell proliferation, and is essential for maintenance of a functional vascular barrier. However, elevated endostatin levels correlate with severity of inflammatory lung damage. The source of endostatin or its role in progression of vascular disease is not known. We reasoned endostatin release by lung endothelium during an injury event contributes to pulmonary vascular damage by limiting endothelial proliferation and migration; thus slowing barrier restoration. LPS increased endostatin expression in lung tissue in vivo and in pulmonary microvascular endothelial cells (PMVECs) in vitro. LPS caused translocation of endostatin from peri‐nuclear regions to cell membranes in PMVECs consistent with release of endogenously synthesized endostatin. LPS inhibits PMVEC proliferation and wound healing, an effect exacerbated by exogenous application of endostatin. Endostatin decreased RhoA activity in PMVECs implicating the RhoA pathway in endostatin signaling.Conclusioninflammation induces endostatin release from endothelium sufficient to limit lung vascular repair. Supported by HL102296 and HL 110803.

Effects of Mycophenolic Acid on the Proliferation and Endothelin-1 and Interleukin-6 Secretion of Rat Pulmonary Microvascular Endothelial Cells

Objective: To investigate the effect of mycophenolic acid (MPA) on the proliferation of rat pulmonary microvascular endothelial cells (PMVECs) and on the secretion of endothelin-1 (ET-1) and interleukin-6 (IL-6) by these cells. Methods: Rat PMVECs were treated with five different final concentrations of MPA (0, 0.1, 1, 10 and 100 μM). The fetal bovine serum-induced proliferation of the PMVECs was detected using the Cell Counting Kit-8 (CCK-8). The levels of ET-1 and IL-6 secretion were assessed by radioimmunoassays. Results: At the 24 h time point, the optical density (OD) values at 450 nm for the five groups showed that there were no significant differences between the groups treated with 0 and 0.1 μM MPA (P=0.388) or between the groups treated with 10 and 100 μM MPA (P=0.292), but the OD values were significantly different between all other pairs of groups (P<0.001). At the 48 h time point, the OD values at 450 nm for the five groups showed that there was no significant difference between the groups treated with 0 and 0.1 μM MPA (P=0.094), but the OD values were significantly different between all the other groups (P<0.001). At the 72 h time point, the OD values at 450 nm for the five groups showed that there was no significant difference between the groups treated with the final concentrations of 0 and 0.1 μM MPA (P=0.931), but the OD values were significantly different between all other pairs of groups (10 μM MPA vs 100 μM MPA: P=0.037; 0 μM MPA vs 1 μM MPA: P=0.006; 1 μM MPA vs 10 μM MPA: P=0.005; all other comparisons: P<0.001). There were no significant differences in ET-1 concentration between the groups treated with 0 and 0.1 μM MPA (P=0.156) or the groups treated with 10 and 100 μM MPA (P=0.262), but there were significant differences between all other pairs of groups (P<0.001). By contrast, there were no significant differences in IL-6 concentrations between any pair of groups (P>0.05). Conclusions: MPA inhibits fetal bovine serum-induced rat PMVEC proliferation and ET-1 secretion in a dose-dependent manner.

MicroRNA-101 inhibits proliferation of pulmonary microvascular endothelial cells in a rat model of hepatopulmonary syndrome by targeting the JAK2/STAT3 signaling pathway

Hepatopulmonary syndrome (HPS) is one of the reasons for the mortality of patients with perioperative liver disease. Intrapulmonary vascular dilatation is the most important mechanism underlying HPS, and it primarily occurs due to cell proliferation. Inhibiting the proliferation of pulmonary microvascular endothelial cells (PMVECs) may provide a novel strategy to prevent HPS. MicroRNAs (miRNAs) regulate gene expression and are crucial in cell proliferation. To investigate the mechanism underlying the proliferation of PMVECs in HPS, PMVECs were isolated from rat models of HPS. It was demonstrated that interleukin (IL)‑6 could stimulate the janus kinase 2 (JAK2)/ signal transducer and activator of transcription (STAT3) signaling pathway, which promotes the cell proliferation of PMVECs. JAK2 is a novel target gene of miR-101 and it was shown that miR-101 could inhibit cell proliferation by targeting the IL-6/JAK2/STAT3 signal pathway. In conclusion, the present study demonstrated that miR-101 could inhibit the proliferation of PMVECs by targeting the IL-6/JAK2/STAT3 signaling pathway. This clarifies the role of miR-101 in HPS and provides the theoretical basis of the pathogenesis of HPS.

Copper Dependence of Angioproliferation in Pulmonary Arterial Hypertension in Rats and Humans

Obliteration of the vascular lumen by endothelial cell growth is a hallmark of many forms of severe pulmonary arterial hypertension. Copper plays a significant role in the control of endothelial cell proliferation in cancer and wound-healing. We sought to determine whether angioproliferation in rats with experimental pulmonary arterial hypertension and pulmonary microvascular endothelial cell proliferation in humans depend on the proangiogenic action of copper. A copper-depleted diet prevented, and copper chelation with tetrathiomolybdate reversed, the development of severe experimental pulmonary arterial hypertension. The copper chelation-induced reopening of obliterated vessels was caused by caspase-independent apoptosis, reduced vessel wall cell proliferation, and a normalization of vessel wall structure. No evidence was found for a role of super oxide-1 inhibition or lysyl-oxidase-1 inhibition in the reversal of angioproliferation. Tetrathiomolybdate inhibited the proliferation of human pulmonary microvascular endothelial cells, isolated from explanted lungs from control subjects and patients with pulmonary arterial hypertension. These data suggest that the inhibition of endothelial cell proliferation by a copper-restricting strategy could be explored as a new therapeutic approach in pulmonary arterial hypertension. It remains to be determined, however, whether potential toxicity to the right ventricle is offset by the beneficial pulmonary vascular effects of antiangiogenic treatment in patients with pulmonary arterial hypertension.

Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.

Methylnaltrexone potentiates the antiangiogenic effects of the mTOR inhibitor, temsirolimus

e14636 Background: Angiogenesis is important in cancer growth and metastasis. Recent therapeutic interventions cancer, include drugs such as temsirolimus that target both tumor growth and angiogenesis including the mammalian target of rapamycin (mTOR) inhibitor. Previously, we have demonstrated that the peripheral opioid antagonist methylnaltrexone (MNTX) potentiates the effects of bevacizumab and 5FU on endothelial cell (EC) migration and proliferation. Since mTOR inhibitor therapy is also associated with significant side effects, we examined potential adjunctive agents that could reduce the therapeutic dose and improve the therapeutic window for this approach. Methods: We determined the effect(s) of MNTX, naloxone and/or temsirolimus on VEGF-induced human pulmonary microvascular EC proliferation and migration assays. We used siRNA and specific inhibitors to analyze Src, Akt and mTOR regulation. Results: MNTX inhibited EC VEGF-induced proliferation and migration with an IC50 of ∼100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation from ∼10 nM to ∼1 nM. Further, adding 10 nM MNTX shifted the IC50 of temsirolimus on inhibition of EC migration from ∼50 nM to ∼10 nM. These synergistic effects were not observed with naltrexone, a tertiary mu opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased protein tyrosine phosphatase (PTP) activity which was independent of mu opioid receptor expression. Treatment of human EC with the PTP inhibitor, 3,4-Dephostatin, inhibited both the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR complex 1 (temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events. Conclusions: Synergy of MNTX with mTOR inhibitors may have therapeutic implications which await clinical proof-of-concept. [Table: see text]

Essential role of lactate in controlling the rapid proliferation of pulmonary microvascular endothelial cells

Pulmonary microvascular endothelial cells (PMVECs) constitutively up‐regulate glycolytic enzymes necessary to generate ATP. One of these enzymes is lactate dehydrogenase (LDH), which converts pyruvate to lactate, causing lactic acidosis in proliferating cells. Since the role of LDH and lactate in endothelial pro‐proliferation is unknown, we tested whether lactate is a key contributor of rapid PMVEC proliferation. PMVEC growth curves were generated using standard culture conditions (10% serum with 22 mM glucose) in the presence of the LDH inhibitor, oxamate (4, 8 and 16 mM). Oxamate inhibited growth, glucose consumption and lactate production in a dose‐dependent manner, suggesting that conversion of pyruvate to lactate is essential for rapid proliferation. To determine whether glycolytic flux is necessary to sustain proliferation, galactose was substituted for glucose, since galactose is an ineffective glycolysis substrate. Galactose treatment reduced cellular ATP concentrations ?2‐fold, and inhibited growth, glucose consumption and lactate production. To examine whether lactate results in glycolytic‐independent cell growth, lactate was added to galactose‐rich media. Addition of exogenous lactate restored cellular ATP concentrations and partially rescued PMVEC proliferation. Thus, LDH contributes to the rapid PMVEC proliferation by controlling the rate of pyruvate conversion to lactate.

Endothelial progenitor cells may inhibit apoptosis of pulmonary microvascular endothelial cells: new insights into cell therapy for pulmonary arterial hypertension

Endothelial apoptosis underlies the pathophysiology of pulmonary arterial hypertension (PAH). Some factors/cytokines released by endothelial progenitor cells (EPC) have been revealed as potent inhibitors of apoptosis. The aim of this study was to investigate the effects of EPC on pulmonary microvascular endothelial cell (PMVEC) survival with the PAH condition. PMVEC apoptosis was induced by high shear stress (HSS) with serum starvation or pro-inflammatory factors in an artificial capillary system. EPC were delivered into monocrotaline-induced PAH nude rats. PMVEC apoptosis under HSS and serum starvation conditions was significantly inhibited by EPC conditioned medium (CM). It was attenuated by vascular endothelial growth factor (VEGF)-A or -B blocking. EPC CM promoted PMVEC proliferation, which was weakened by VEGF-A or interleukin (IL)-8 blocking. The EPC CM caused less apoptosis of PMVEC induced by HSS plus pro-inflammatory factors. The anti-apoptotic effect of EPC CM was attenuated by blockade of either vascular endothelial growth factor receptor (VEGFR)-1 or -2. However, the pro-proliferating effect appeared to be weakened only by VEGFR-2 blocking. Both Erk1/2 and Akt phosphorylation were enhanced by EPC CM. VEGFR-2 blockage led to significant inhibition of Erk1/2 and Akt activation; VEGFR-1 blockage only of Erk1/2 activation. Human-origin VEGF co-localized with incorporated EPC in small pulmonary arterioles, and EPC transplantation resulted in down-regulation of caspase-3 expression. The VEGF-A/B-VEGFR-1/2-Erk1/2 signal pathway took major responsibility for the anti-apoptotic effects of EPC on PMVEC, and VEGF-A-VEGFR-2-Akt for pro-proliferating effects. Growth factors, secreted in a paracrine manner by transplanted EPC, inhibited cell apoptosis in PAH lung.

Glucose restriction inhibits glycolytic flux and limits proliferation in lung microvascular endothelial cells

Pulmonary microvascular endothelial cells (PMVECs) exhibit increased glycolytic flux, which may contribute to their highly proliferative nature. We therefore sought to determine whether PMVECs rely on extracellular glucose to sustain glycolysis and growth under normoxic conditions. We performed growth curves and pH measurements using PMVECs and pulmonary artery endothelial cells (PAECs) in different glucose concentrations: High (22 mM), normal (11 mM) and low (5 mM). PMVEC proliferation was greatest in high glucose, and decreased progressively with lower glucose concentrations. In contrast, glucose concentration did not affect PAEC growth. Whereas PMVECs acidified the media in high glucose, they did not acidify the media at low glucose concentrations; PAECs did not acidify the media at any glucose concentration. QRT‐PCR analysis for glucose transporter‐1 (GLUT‐1), hexokinase‐2 (HK‐2), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) revealed that media glucose did not influence upon the expression of these key glycolytic enzymes, suggesting genes responsible for increased glycolysis are constitutively upregulated in PMVECs. The constitutive upregulation of GLUT‐1, HK‐2, PFK, and LDH in PMVECs illustrate the intrinsic preference of these cells to use aerobic glycolysis during proliferation. Thus, glucose availability is a key determinant of growth and acidosis in PMVECs. HL66299.