Effect and mechanism of apelin on lipopolysaccharide induced acute pulmonary vascular endothelial barrier dysfunction.

Vascular endothelial barrier dysfunction is the most prominent manifestation and important cause of mortality in infectious acute lung injury (ALI). Exogenous apelin is effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory response in ALI lungs, reducing exudation of lung tissue and decreasing mortality. This study set out to investigate the association between apelin and Friend leukemia integration-1 (Fli-1) in the prevention and treatment of ALI, and to elucidate the molecular mechanism by which apelin protects the permeability of the vascular endothelial barrier. At the vivo functional level, lung wet/dry weight ratio was used to detect whole lung permeability, evans blue assay and dual fluorescent protein tracking assay were used to detect lung vascular endothelial permeability, HE staining to observe the inflammatory status of lung tissue, and immunofluorescence staining for VE-cadherin expression levels in blood vessels. The changes in inflammatory factors in bronchoalveolar lavage fluid (BALF) were detected by ELASA. Western blot was used to detect the expression level of proteins. qRT-PCR was performed to detect changes in mRNA expression of Fli-1 and adherent junction-related proteins. The correlation analysis of Fli-1 with vascular endothelial permeability and SRC showed that Fli-1 participated in the process of ALI. After preventive and therapeutic treatment of ALI mice with exogenous apelin, Fli-1, APJ, VE-cadherin, phosphorylated-VE-cadherin (p-VE-cadherin) and ß-catenin were up-regulated, while SRC, phosphorylated-SRC (p-SRC), VEGF and VEGF-R were down-regulated, which indicated that the stability of vascular endothelial barrier was enhanced. With the use of Fli-1 inhibitor irinotecan, the protective effect of apelin was weakened in various functional indexes, genes and proteins. The lung was maintained at the level of the injury. Our research shows that Fli-1 is involved in the LPS-induced ALI process. The molecular mechanism for apelin in preventing endothelial barrier dysfunction in ALI is through up-regulating Fli-1, thus regulating adherens junction-related proteins, and finally recovering the endothelial barrier function.

Critical role of VGLL4 in the regulation of chronic normobaric hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH), a rare but deadly cardiopulmonary disorder, is characterized by extensive remodeling of pulmonary arteries resulting from enhancement of pulmonary artery smooth muscle cell proliferation and suppressed apoptosis; however, the underlying pathophysiological mechanisms remain largely unknown. Recently, epigenetics has gained increasing prominence in the development of PH. We aimed to investigate the role of vestigial-like family member 4 (VGLL4) in chronic normobaric hypoxia (CNH)-induced PH and to address whether it is associated with epigenetic regulation. The rodent model of PH was established by CNH treatment (10% O2 , 23 hours/day). Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, immunoprecipitation, and adeno-associated virus tests were performed to explore the potential mechanisms involved in CNH-induced PH in mice. VGLL4 expression was upregulated and correlated with CNH in PH mouse lung tissues in a time-dependent manner. VGLL4 colocalized with α-smooth muscle actin in cultured pulmonary arterial smooth muscle cells (PASMCs), and VGLL4 immunoactivity was increased in PASMCs following hypoxia exposure in vitro. VGLL4 knockdown attenuated CNH-induced PH and pulmonary artery remodeling by blunting signal transducer and activator of transcription 3 (STAT3) signaling; conversely, VGLL4 overexpression exacerbated the development of PH. CNH enhanced the acetylation of VGLL4 and increased the interaction of ac-H3K9/VGLL4 and ac-H3K9/STAT3 in the lung tissues, and levels of ac-H3K9, p-STAT3/STAT3, and proliferation-associated protein levels were markedly up-regulated, whereas apoptosis-related protein levels were significantly downregulated, in the lung tissues of mice with CNH-induced PH. Notably, abrogation of VGLL4 acetylation reversed CNH-induced PH and pulmonary artery remodeling and suppressed STAT3 signaling. Finally, STAT3 knockdown alleviated CNH-induced PH. In conclusion, VGLL4 acetylation upregulation could contribute to CNH-induced PH and pulmonary artery remodeling via STAT3 signaling, and abrogation of VGLL4 acetylation reversed CNH-induced PH. Pharmacological or genetic deletion of VGLL4 might be a potential target for therapeutic interventions in CNH-induced PH.

Upregulation of KDM6B contributes to lipopolysaccharide-induced anxiety-like behavior via modulation of VGLL4 in mice.

Histone H3K27me3 demethylase KDM6B (also known as Jumonji domain-containing protein D3, JMJD3) plays vital roles in the etiology of inflammatory responses; however, little is known about the role of KDM6B in neuroinflammation-induced anxiety-like behavior. The present study aimed to investigate the potential role of KDM6B in lipopolysaccharide (LPS)-induced anxiety-like behavior and to evaluate whether it is associated with the modulation of vestigial-like family member 4 (VGLL4). The elevated plus maze, light-dark box, and open-field test were performed to test the anxiety-like behavior induced by LPS in C57BL/6 J male mice. Levels of relative protein expression in the hippocampus were quantified by western blotting. KDM6B inhibitor GSK-J4 and microglia inhibitor minocycline as well as adeno-associated virus of Vgll4 shRNA were used to explore the underlying mechanisms. We found that KDM6B, VGLL4, interleukin-1ß (IL-1ß), and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) protein levels were increased in LPS-dose dependent manner in the hippocampus but not in prefrontal cortex. GSK-J4 treatment attenuated LPS-induced VGLL4, the signal transducer and activator of transcription 3 (STAT3), IL-1ß and Iba-1 upregulation and anxiety-like behavior. Knockdown VGLL4 with Vgll4 shRNA prevented the increase of anxiety-like behavior and levels of STAT3, IL-1ß, and Iba-1 expression in the hippocampus of LPS-treated mice. Moreover, minocycline, an inhibitor of microglia treatment blunted LPS-induced anxiety-like behavior. Collectively, these results demonstrate that the induction of neuroinflammation by LPS promotes KDM6B activation in the hippocampus, and LPS-induced anxiety-like behavior is associated with upregulation of VGLL4 by KDM6B in the hippocampus.

YAP activity protects against endotoxemic acute lung injury by activating multiple mechanisms.

The roles of the Hippo­Yes­associated protein (YAP) pathway in lung injury and repair remain elusive. The present study examined the effects of systemic inhibition or stimulation of YAP activity on lung injury, repair and inflammation in a mouse model of lipopolysaccharide (LPS)­induced lung injury. Mice were treated with or without YAP inhibitor, verteporfin, or with or without YAP stimulator, XMU­MP­1, and intraperitoneally injected with LPS (7.5 mg/kg). Lung injury and repair were evaluated by histological analysis and by testing for markers of lung injury. Lung inflammation was assessed by measuring tissue levels of inflammatory mediators. Lung injury was associated with a decreased, whereas lung repair was associated with an increased YAP activity evidenced by nuclear translocation. Lung injury was associated with a high level of lung inflammation and epithelial adherens junction disassembly, but not with cell proliferation or epithelial cell regeneration. The injury phase was defined as 0­48 h post­LPS injection, and the 48­168 h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU­MP­1, alleviated lung injury, lung inflammation and epithelial adherens junction disassembly. Inhibition or stimulation of YAP activity at the injury phase had no effects on cell proliferation or epithelial regeneration. By contrast, lung repair was associated with inflammation resolution, increased cell proliferation, epithelial regeneration and reassembly of epithelial adherens junctions. Inhibition of YAP activity at the repair phase delayed inflammation resolution, impeded lung recovery, inhibited cell proliferation and epithelial regeneration, and inhibited epithelial adherens junction reassembly. Stimulation of YAP activity at the repair phase reversed all these processes. The results of the current study demonstrated that the Hippo­YAP activity serves a protective role against endotoxemic lung injury. The Hippo­YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

YAP promotes endothelial barrier repair by repressing STAT3/VEGF signaling.

AIMS: Endothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. MAIN METHODS: A trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. KEY FINDINGS: YAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SIGNIFICANCE: Elucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.

Upregulation of miR-335-3p by NF-κB Transcriptional Regulation Contributes to the Induction of Pulmonary Arterial Hypertension via APJ during Hypoxia.

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease that can lead to heart failure and eventually death. MicroRNAs (miRs) play essential roles during PAH progression; however, their exact mechanism of action remains unclear. Apelin is a small bioactive peptide with a key protective function in the pathogenesis of PAH mediated by binding to the APJ gene. The aim of the present study was to investigate the role of miR-335-3p in chronic normobaric hypoxia (CNH)-induced PAH in mice and the potential underlying regulatory mechanism. Adult male C57BL/6 mice were exposed to normoxia (~21% O2) or CNH (~10% O2, 23 h/d) for 5 weeks. MiR-335-3p was significantly increased in lung tissue of CNH-induced PAH mice. Blocking miR-335-3p attenuated CNH-induced PAH and alleviated pulmonary vascular remodeling. Bioinformatics analysis and luciferase reporter assay indicated that nuclear factor-kappa beta (NF-κB) acted as a transcriptional regulator upstream of miR-335-3p. Pyrrolidine dithiocarbamate treatment reversed the CNH-induced increase in miR-335-3p expression and diminished CNH-induced PAH. Moreover, p50-/- mice were resistant to CNH-induced PAH. Finally, APJ was identified as a direct targeting gene downstream of miR-335-3p, and pharmacological activation of APJ by its ligand apelin-13 reduced CNH-induced PAH and improved pulmonary vascular remodeling. Our results indicate that NF-κB-mediated transcriptional upregulation of miR-335-3p contributes to the inhibition of APJ and induction of PAH during hypoxia; hence, miR-335-3p could be a potential therapeutic target for hypoxic PAH.

Epigallocatechin-3-gallate (EGCG) inhibits aggregation of pulmonary fibrosis associated mutant surfactant protein A2 via a proteasomal degradation pathway.

BACKGROUND/AIMS: Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, plays an important anti-tumor role and is involved in various other biological processes, such as, neuroprotection by prevention of aggregation of misfolded proteins generated because of genetic defects. Surfactant protein A2 mutations (G231V and F198S) have been identified to be associated with pulmonary fibrosis and lung cancer, and these mutations cause protein aggregation, instability as well as secretion deficiency. The present study focused on investigating the inhibitory effects of EGCG on aggregation of mutant SP-A2 and elucidating the potential mechanisms underlying this action. METHODS: Wild-type and mutant SP-A2 were transiently expressed in CHO-K1 cells. The aggregated and soluble proteins were separated into NP-40-insoluble and NP-40-soluble fractions. Protein stability was validated by chymotrypsin limited proteolysis assay. Western blot and RT-PCR were used to determine the protein and mRNA expression level, respectively. RESULTS: Mutant SP-A2 alone or wild-type SP-A2 co-expressed with G231V formed NP-40-insoluble aggregates in CHO-K1 cells. EGCG significantly suppressed this aggregation and alleviated mutant SP-A2 accumulation in the ER. When combined with 4-PBA, EGCG treatment completely blocked mutant SP-A2 aggregate formation. Though secretion of mutant protein was not affected, EGCG facilitated protein instability in both wild-type and mutant protein. Importantly, MG132, a proteasome inhibitor, reversed EGCG-induced aggregate reduction. CONCLUSIONS: EGCG inhibits aggregation of misfolded SP-A2 via induction of protein instability and activation of proteasomal pathway for aggregate degradation.

[Effects of apolipoprotein E on proliferation of mouse pulmonary arterial smooth muscle cells induced by hypoxia].

OBJECTIVE: To investigate the effects of apolipoprotein E (apoE) on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. METHODS: Primary culture of mouse PASMCs was prepared from male C57BL/6 mouse pulmonary artery by the method of tissue block anchorage. PASMCs were divided into four groups: normoxia group, normoxia with apoE administration group, hypoxia group and hypoxia with apoE administration group. The proliferation of PASMCs was observed by EdU incorporation. The protein levels of apoE, proliferating cell nuclear antigen (PCNA), protein kinase C (PKC) and phosphorylated protein kinase C (p-PKC) were analyzed by Western blot. RESULTS: The percentage of PASMCs proliferation of hypoxia group was significantly higher than that of normoxia group by 64.7% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia group were up-regulated than those of normoxia group by 69.0% and 120.0%, while the protein expression of apoE was down-regulated by 51.0% (P＜0.05), respectively. The percentage of PASMCs proliferation of hypoxia with apoE administration group was significantly lower than that of hypoxia group by 19.6% (P＜0.05), and the protein expression levels of PCNA and p-PKC of hypoxia with apoE administration group were down-regulated than those of hypoxia group by 19.8% and 103.2% (P＜0.05), respectively. There was no significant difference among each group in the protein expression of PKC, nor do there any significant difference between normoxia group and hypoxia group in the protein expression of p-PKC (P＞0.05). CONCLUSION: ApoE can inhibit the proliferation of PASMCs induced by hypoxia, and the mechanism of its effect may be attributed to blocking PKC pathway.

Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78.

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.

SIRT1 Mediates Apelin-13 in Ameliorating Chronic Normobaric Hypoxia-induced Anxiety-like Behavior by Suppressing NF-κB Pathway in Mice Hippocampus.

We previously showed that apelin-13 ameliorates chronic normobaric hypoxia (CNH)-induced anxiety-like behavior in mice, the mechanism, however, is not well known. This study aims to investigate whether SIRT1 is involved in the anxiolytic effect of apelin-13 in CNH-treated mice, and to illustrate the potential underlying mechanism. We showed that apelin-13 treatment reversed a decrease in SIRT1 and an increase in acetylated p65 (lysine 310) proteins' expression in hippocampus of CNH-treated mice, indicating that apelin-13 inhibited NF-κB signaling pathway by activating SIRT1. Behaviorally, apelin-13 ameliorated CNH-induced anxiety-like behavior, EX-527 blocked the beneficial effect of apelin-13, and the anxiogenic effect of CNH was attenuated by resveratrol pretreatment, suggesting that SIRT1 was involved in the effect of apelin-13 against CNH-induced anxiety-like behavior in mice. We also showed that resveratrol treatment decreased IL-1ß, IL-6, TNF-ɑ, PCNA, Bcl-2, and acetyl-p65 levels, but increased Bax and caspase 3 levels in hippocampus, suggesting a suppressive effect of resveratrol on cellular neuroinflammation and proliferation while a promotive effect on apoptosis of microglia in hippocampus. Finally, blockade of NF-κB activity by PDTC diminished CNH-induced anxiety-like behavior, indicating that NF-κB was involved in CNH-induced anxiety-like behavior in mice. In conclusion, this study provides the first evidence that SIRT1 mediates the anxiolytic effect of apelin-13 in CNH-treated mice through the inhibition of NF-κB pathway. These results imply that dysfunction of the apelin-SIRT1-NF-κB axis in hippocampus represents a potential mechanism that results in the induction of neuroinflammation and reduction in neuroprotection, thus induces anxiety-like behavior in CNH-treated mice.

Selective inhibition of endothelial NF-κB signaling attenuates chronic intermittent hypoxia-induced atherosclerosis in mice.

BACKGROUND AND AIMS: Chronic intermittent hypoxia (CIH) exposure causes atherosclerosis, although the underlying mechanisms are poorly understood. This study defines the role of endothelial intrinsic NF-κB signaling in the atherogenic response to CIH. METHODS: We created ApoE-ECI-κBmt mice that are deficient in the apolipoprotein E gene (ApoE-/-) and overexpress an I-κBα mutant (I-κBmt) selectively in endothelial cells. ApoE-/- and ApoE-ECI-κBmt mice were fed a normal chow diet (NCD) or high cholesterol diet (HCD) and exposed to sham or CIH, and atherosclerotic lesions were quantified. RESULTS: CIH exposure activated NF-κB in aortas, and induced the expression of endothelial-specific and NF-κB-dependent genes, E-selectin and vascular cell adhesion molecule (VCAM)-1, in the aortas and hearts. Endothelial I-κBmt overexpression in ApoE-ECI-κBmt mice significantly inhibited CIH-induced NF-κB activity, and suppressed E-selectin and VCAM-1 expressions, confirming endothelial NF-κB inhibition in ApoE-ECI-κBmt mice. ApoE-/- mice, on NCD, developed mild atherosclerotic lesions spontaneously, and developed advanced and larger areas of atherosclerotic plaques when exposed to CIH. ApoE-/- mice also developed advanced atherosclerotic lesions when fed an HCD alone. The HCD-induced atherosclerotic plaques became more advanced, and plaque area was doubled in mice exposed to HCD + CIH. Endothelial I-κBmt overexpression in ApoE-ECI-κBmt mice attenuated spontaneously developed atherosclerotic lesions, abrogated CIH-induced atherosclerosis and mitigated CIH-mediated facilitation of HCD-induced atherosclerosis. CONCLUSIONS: These results suggest that endothelial intrinsic NF-kB signaling may play a pivotal role in CIH-induced atherosclerosis.

[Changes of apoE protein expression in lung of mice with hypoxic pulmonary arterial hypertension].

OBJECTIVE: To observe the changes of apolipoprotein E (apoE) protein expression of pulmonary tissue in mice with pulmonary hypertension induced by hypoxia. METHODS: The animal model of hypoxic pulmonary hypertension was established by exposing the mice to isobaric hypoxic chamber for 3 weeks (23 h/d, regular chow feed).Twenty male wild type (WT) C57BL/6 mice and twenty apoE gene knockout (apoE-KO) mice were randomly divided into normoxia group and hypoxia group. The plasma concentrations of low density lipoprotein (LDL), high density lipoprotein (HDL) and total cholesterol were detected by ELISA method. The protein expression of apoE in lung and liver, and peroxisome proliferators-activated receptor gamma (PPARγ) in lung were measured by Western blot. RESULTS: ①In WT mice, the right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) of hypoxia group were significantly higher than those of normoxia group by 68% and 59% (P<0.05), respectively. The plasma concentration of HDL and HDL/LDL of hypoxia group were significantly lower than those of normoxia group by 17% and 40% (P<0.05), respectively.The protein expression of apoE in lung and in liver of hypoxia group were significantly down-regulated than those of normoxia group by 48% and 52% (P<0.05), respectively.The protein expression of PPARγ in lung was significantly down-regulated than that of normoxia group by 37%(P<0.05).RVSP were significantly negative correlated with the protein levels of apoE and PPARγ in lung (P<0.01).② In apoE-KO mice, RVSP and the weight ratio of RV to LV+S of hypoxia group were significantly higher than those of normoxia group by 96% and 86% (P<0.05), respectively.RVSP and RV to (LV+S) of hypoxia group in apoE-KO mice were significantly higher than those of hypoxia group in WT mice by 29% and 24% (P<0.05), respectively. CONCLUSIONS: Down-regulated expression of apoE in lung tissue participates in the pathological proceeding of pulmonary hypertension induced by hypoxia.

Amelioration of apelin-13 in chronic normobaric hypoxia-induced anxiety-like behavior is associated with an inhibition of NF-κB in the hippocampus.

Apelin, a small bioactive peptide, plays an important role in the pathogenesis of mood disorders through the endogenous ligand APJ. Although the anxiolytic effect of apelin is well established, the mechanisms are poorly understood. In this study, we hypothesized that apelin played an anxiolytic role in chronic normobaric hypoxia (CNH)-induced anxiety like behavior in mice, which might be associated with an inhibition of nuclear factor-κB (NF-κB) activation in the hippocampus. To this end, mice were exposed in a normobaric hypoxic chamber with a fraction of inspired oxygen (FIO2, ∼10%, 23h/d) with or without apelin-13 application (20 nmolkg-1d-1, i.p.), for 4 weeks. The anxiety-like behavior was tested by elevated plus maze and open field. Activities of NF-κB, microglial, and related signaling pathways in the hippocampus during this pathological process were examined. We found that CNH treatment decreased APJ but increased Iba-1 proteins expression, as well as nucleus translocation of p50 and p65 in the hippocampus, which were reversed by apelin-13 treatment. In addition, apelin-13 treatment ameliorated CNH-induced anxiety-like behavior in mice, suggesting anxiogenic effect of apelin-13 might be mediated by an inhibition of NF-κB activation in microglial of the hippocampus. Furthermore, apelin-13 treatment reversed p-CAMKII decrease in the hippocampus under CNH treatment. Apelin-13 treatment did not affect anxiety-like behavior and relative proteins expression in normoxia control mice. Finally, we found that rats with CNH treatment decreased APJ expression while enhanced NF-κB activation in the hippocampus, providing additional evidences that NF-κB activation in hippocampus in CNH-induced anxiety-like behavior in rats we reported previously might be associated with an inhibition of APJ activity. In conclusion, the present results illustrated that inhibition of APJ and promotion of NF-κB activation in the microglial of hippocampus might be involved in anxiogenic effect in CNH-exposed mice, and apelin-13 ameliorates CNH-induced anxiety-like behavior might be associated with an inhibition of NF-κB activation.

[Apelin attenuates hypoxia induced pulmonary hypertension of mice through regulation of lipid metabolism].

OBJECTIVE: To observe the role of apelin in the prevention of pulmonary hypertension induced by hypoxia in mice. METHODS: Adult male apoE gene knockout (apoE-KO) mice were exposed to isobaric hypoxic chamber (9%~11% O2, regular chow feed, 23 h/d)for 3 weeks to establish hypoxia-induced pulmonary hypertension. Thirty apoE-KO mice were randomly divided into normoxia group, hypoxia group and hypoxic with apelin (10 nmol/(kg·d), ip) group. The concentrations of high density lipoprotein (HDL), low density lipoprotein (LDL)and total cholesterol in plasma were detected by Elisa method. The mRNA levels of ATP-binding cassette transporter A1(ABCA1), low density lipoprotein receptor (LDLR), scavenger receptor class B1 (SR-B1), and HMG-CoA reductase (HMGCR)in liver were measured by real-time PCR. The protein level of peroxisome proliferators-activated receptor gamma (PPARγ) in lung was measured by Western blot. RESULTS: ①The right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) of hypoxia group were significantly higher than those of normoxia group by 87% and 85% (P<0.05), respectively. RVSP and RV/(LV+S) of apelin group were significantly lower than those of hypoxia group by 39% and 33%(P<0.05), respectively. ②The plasma concentration of HDL and HDL/LDL of apelin group were significantly higher than those of hypoxia group by 21% and 20%(P<0.05), respectively. ③The mRNA levels of LDLR, SR-B1 and ABCA1 in liver of apelin group were significantly up-regulated than those of hypoxia group by 241%, 112% and 69% (P<0.05), respectively, while the mRNA level of HMGCR was down-regulated by 45% (P<0.05). ④The protein level of PPARγ in lung of apelin group was significantly up-regulated than that of hypoxia group by 47% (P<0.05). CONCLUSIONS: Apelin attenuates hypoxia-induced pulmonary hypertension of mice through regulation of lipid metabolism.

[Changes of lipid levels in mice with hypoxic pulmonary arterial hypertension].

OBJECTIVE: To observe the changes of lipid levels in mice with pulmonary hypertension induced by hypoxia. METHODS: The animal model of hypoxic pulmonary hypertension was established by exposing the mice to isobaric hypoxic chamberfor 3 weeks (23 h/d, regular chow feed). Twenty male C57BL/6 mice were randomlydivided into normoxia group and hypoxia group (n=10). The concentrations of total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma were detected by Elisa method.The mRNA levels of HMG-CoAreductase (HMGCR), low density lipoprotein receptor (LDLR), scavenger receptor class B1 (SR-B1), and sterol regulatory element-binding factor-2 (SREBF2) in liverwere measured by real-time PCR. RESULTS: ① The right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) of hypoxia group were significantly higher than those of normoxia group (P<0.05).② The concentrations of HDL and HDL/LDL in plasma were significantly higher in hypoxia group, compared with normoxia group (P<0.05).③The mRNA levels of LDLR and SR-B1in liver were significantly down-regulated in hypoxia group(P<0.05).④RVSP were significantly negative correlated with HDL/LDL, the gene expression of LDLR and SR-B1 (P<0.05). CONCLUSIONS: Abnormal lipid metabolism participates in the pathological proceeding of pulmonary hypertension induced by hypoxia.

Resident Endothelial Cells and Endothelial Progenitor Cells Restore Endothelial Barrier Function After Inflammatory Lung Injury.

OBJECTIVE: Disruption of endothelial barrier integrity is a characteristic of many inflammatory conditions. However, the origin and function of endothelial cells (ECs) restoring endothelial barrier function remain unknown. This study defined the roles of resident ECs (RECs) and bone marrow-derived endothelial progenitor cells (BMDEPCs) in endothelial barrier restoration after endotoxemic lung injury. APPROACH AND RESULTS: We generated mice that enable to quantify proliferating RECs or BMDEPCs and also to study the causal link between REC or BMDEPC proliferation and endothelial barrier restoration. Using these mouse models, we showed that endothelial barrier restoration was associated with increased REC and BMDEPC proliferation. RECs and BMDEPCs participate in barrier repair. Immunofluorescence staining demonstrated that RECs proliferate in situ on endothelial layer and that BMDEPCs are engrafted into endothelial layer of lung microvessels at the active barrier repair phase. In lungs, 8 weeks after lipopolysaccharide-induced injury, the number of REC-derived ECs (CD45(-)/CD31(+)/BrdU(+)/rtTA(+)) or BMDEPC-derived ECs (CD45(-)/CD31(+)/eNOS(+)/GFP(+)) increased by 22- or 121-fold, respectively. The suppression of REC or BMDEPC proliferation by blocking REC or BMDEPC intrinsic nuclear factor-κB at the barrier repair phase was associated with an augmented endothelial permeability and impeded endothelial barrier recovery. RECs and BMDEPCs contributed differently to endothelial barrier repair. In lungs, 8 weeks after lipopolysaccharide-induced injury, REC-derived ECs constituted 22%, but BMDEPC-derived ECs constituted only 3.7% of the total new ECs. CONCLUSIONS: REC is a major and BMDEPC is a complementary source of new ECs in endothelial barrier restoration. RECs and BMDEPCs play important roles in endothelial barrier restoration after inflammatory lung injury.

The Apelin-APJ axis is an endogenous counterinjury mechanism in experimental acute lung injury.

BACKGROUND: Although the mechanisms and pathways mediating ARDS have been studied extensively, less attention has been given to the mechanisms and pathways that counteract injury responses. This study found that the apelin-APJ pathway is an endogenous counterinjury mechanism that protects against ARDS. METHODS: Using a rat model of oleic acid (OA)-induced ARDS, the effects of ARDS on apelin and APJ receptor expressions and on APJ receptor binding capacity were examined. The protective effect of activating the apelin-APJ pathway against OA- or lipopolysaccharide (LPS)-induced ARDS was evaluated. RESULTS: ARDS was coupled to upregulations of the apelin and APJ receptor. Rats with OA-induced ARDS had higher lung tissue levels of apelin proprotein and APJ receptor expressions; elevated plasma, BAL fluid (BALF), and lung tissue levels of apelin-36 and apelin-12/13; and an increased apelin-APJ receptor binding capacity. Upregulation of the apelin-APJ system has important pathophysiologic function. Stimulation of the apelin-APJ signaling using receptor agonist apelin-13 alleviated, whereas inhibition of the apelin-APJ signaling using receptor antagonist [Ala]-apelin-13 exacerbated, OA-induced lung pathologies, extravascular lung water accumulation, capillary-alveolar leakage, and hypoxemia. The APJ receptor agonist inhibited, and the APJ receptor antagonist augmented, OA-induced lung tissue and BALF levels of tumor necrosis factor-α and monocyte chemoattractant protein-1, and plasma and lung tissue levels of malondialdehyde. Postinjury treatment with apelin-13 alleviated lung inflammation and injury and improved oxygenation in OA- and LPS-induced lung injury. CONCLUSIONS: The apelin-APJ signaling pathway is an endogenous anti-injury and organ-protective mechanism that is activated during ARDS to counteract the injury response and to prevent uncontrolled lung injury.

An obligatory role of NF-κB in mediating bone marrow derived endothelial progenitor cell recruitment and proliferation following endotoxemic multiple organ injury in mice.

BACKGROUND: Recruitment of bone marrow derived endothelial progenitor cells (BMDEPCs) alleviates multiple organ injury (MOI) and improves outcomes. However, mechanisms mediating BMDEPC recruitment following septic MOI remain largely unknown. This study characterized the kinetics of BMDEPC recruitment and proliferation and defined the role of NF-κB in regulating BMDEPC recruitment and proliferation. METHODS AND MAIN FINDINGS: Chimeric mice with an intact or disrupted NF-κB p50 gene and BMDEPC-restricted expression of green fluorescent protein were created and injected with LPS (2 mg/kg, i.p.). BMDEPC recruitment and proliferation in multiple organs were quantified. BMDEPC recruitment and proliferation are highly organ-dependent. Lungs had the highest number of BMDEPC recruitment, whereas heart, liver and kidney had only a small fraction of the number of BMDEPCs in lungs. Number of proliferating BMDEPCs was several-fold higher in lungs than in other 3 organs. Kinetically, BMDEPC recruitment into different organs showed different time course profiles. NF-κB plays obligatory roles in mediating BMDEPC recruitment and proliferation. Universal deletion of NF-κB p50 gene inhibited LPS-induced BMDEPC recruitment and proliferation by 95% and 69% in heart. However, the contribution of NF-κB to these regulations varies significantly between organs. In liver, universal p50 gene deletion reduced LPS-induced BMDEPC recruitment and proliferation only by 49% and 35%. NF-κB activities in different tissue compartments play distinct roles. Selective p50 gene deletion either in stromal/parenchymal cells or in BM/blood cells inhibited BMDEPC recruitment by a similar extent. However, selective p50 gene deletion in BM/blood cells inhibited, but in stromal/parenchymal cells augmented BMDEPC proliferation. CONCLUSIONS: BMDEPC recruitment and proliferation display different kinetics in different organs following endotoxemic MOI. NF-κB plays obligatory and organ-dependent roles in regulating BMDEPC recruitment and proliferation. NF-κB activities in different tissue compartments play distinct roles in regulating BMDEPC proliferation.

Intermedin modulates hypoxic pulmonary vascular remodeling by inhibiting pulmonary artery smooth muscle cell proliferation.

BACKGROUND: Hypoxic pulmonary arterial hypertension (PAH) is a disabling disease with limited treatment options. Hypoxic pulmonary vascular remodeling is a major cause of hypoxic PAH. Pharmacological agents that can inhibit the remodeling process may have great therapeutic value. OBJECTIVE: To examine the effect of intermedin (IMD), a new calcitonin gene-related peptide family of peptide, on hypoxic pulmonary vascular remodeling. METHODS: Rats were exposed to normoxia or hypoxia (∼10% O(2)), or exposed to hypoxia and treated with IMD, administered by an implanted mini-osmotic pump (6.5 µg/rat/day), for 4 weeks. The effects of IMD infusion on the development of hypoxic PAH and right ventricle (RV) hypertrophy, on pulmonary vascular remodeling, on pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis, and on the activations of l-arginine nitric oxide (NO) pathway and endoplasmic reticulum stress apoptotic pathway were examined. RESULTS: Rats exposed to hypoxia developed PAH and RV hypertrophy. IMD treatment alleviated PAH and prevented RV hypertrophy. IMD inhibited hypoxic pulmonary vascular remodeling as indicated by reduced wall thickness and increased lumen diameter of pulmonary arterioles, and decreased muscularization of distal pulmonary vasculature in hypoxia-exposed rats. IMD treatment inhibited PASMC proliferation and promoted PASMC apoptosis. IMD treatment increased tissue level of constitutive NO synthase activity and tissue NO content in lungs, and enhanced l-arginine uptake into pulmonary vascular tissues. IMD treatment increased cellular levels of glucose-regulated protein (GRP) 78 and GRP94, two major markers of endoplasmic reticulum (ER) stress, and increased caspase-12 expression, the ER stress-specific caspase, in lungs and cultured PASMCs. CONCLUSIONS: These results demonstrate that IMD treatment attenuates hypoxic pulmonary vascular remodeling, and thereby hypoxic PAH mainly by inhibiting PASMC proliferation. Promotion of PASMC apoptosis may also contribute to the inhibitory effect of IMD. Activations l-arginine-NO pathway and of ER stress-specific apoptosis pathway could be the mechanisms mediating the anti-proliferative and pro-apoptotic effects of IMD.