A20 deficiency leads to angiogenesis of pulmonary artery endothelial cells through stronger NF‐κB activation under hypoxia

A20 is a zinc finger protein associated with hypoxia. As chronic hypoxia is responsible for intimal hyperplasia and disordered angiogenesis of pulmonary artery, which are histological hallmarks of pulmonary artery hypertension, we intended to explore the role of A20 in angiogenesis of pulmonary artery endothelial cells (ECs). Here, we found a transient elevation of A20 expression in the lung tissues from hypoxic rats compared with normoxic controls. This rapid enhancement was mainly detected in the endothelium, and similar results were reproduced in vitro. During early hypoxia, genetic inhibition of A20 increased proliferation in pulmonary artery ECs, linking to advanced cell cycle progression as well as microtubule polymerization, and aggravated angiogenic effects including tube formation, cell migration and adhesion molecules expression. In addition, a negative feedback loop between nuclear factor‐kappa B and A20 was confirmed. Our findings provide evidence for an adaptive role of A20 against pulmonary artery ECs angiogenesis via nuclear factor‐kappa B activation.     

MicroRNA let‐7g possesses a therapeutic potential for peripheral artery disease

Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis and conveys a significant health burden globally. Critical limb ischaemia encompasses the most severe consequence of PAD. Our previous studies indicate that microRNA let‐7g prevents atherosclerosis and improves endothelial functions. This study aimed to investigate whether and how let‐7g therapy may improve blood flow to ischaemic limbs. The present study shows that let‐7g has multiple pro‐angiogenic effects on mouse ischaemic limb model and could be a potential therapeutic agent for PAD. Mice receiving intramuscular injection of let‐7g had more neovascularization, better local perfusion and increased recruitment of endothelial progenitor cells after hindlimb ischaemia. The therapeutic effects of let‐7g's on angiogenesis are mediated by multiple regulatory machinery. First, let‐7g increased expression of vascular endothelial growth factor‐A (VEGF‐A) and VEGF receptor‐2 (VEGFR‐2) through targeting their upstream regulators HIF‐3α and TP53. In addition, let‐7g affected the splicing factor SC35 which subsequently enhanced the alternative splicing of VEGF‐A from the anti‐angiogenic isoform VEGF‐A_165b towards the pro‐angiogenic isoform VEGF‐A_164a. The pleiotropic effects of let‐7g on angiogenesis imply that let‐7g may possess a therapeutic potential in ischaemic diseases.     

Endothelial microparticles reduce ICAM‐1 expression in a microRNA‐222‐dependent mechanism

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced.     

Autophagy in pulmonary hypertension: Emerging roles and therapeutic implications

Autophagy is an important mechanism for cellular self-digestion and basal homeostasis. This gene- and modulator-regulated pathway is conserved in cells. Recently, several studies have shown that autophagic dysfunction is associated with pulmonary hypertension (PH). However, the relationship between autophagy and PH remains controversial. In this review, we mainly introduce the effects of autophagy-related genes and some regulatory molecules on PH and the relationship between autophagy and PH under the conditions of hypoxia, monocrotaline injection, thromboembolic stress, oxidative stress, and other drugs and toxins. The effects of other autophagy-related drugs, such as chloroquine, 3-methyladenine, rapamycin, and other potential therapeutic drugs and targets, in PH are also described.     

Chrysin ameliorates ANTU‐induced pulmonary edema and pulmonary arterial hypertension via modulation of VEGF and eNOs

Alpha‐naphthylthiourea (ANTU), a rodenticide induces lung toxicity. Chrysin a flavonoid possesses antioxidant, anti‐inflammatory, and antihypertensive potential. The aim of this study was to evaluate the efficacy of chrysin against ANTU‐induced pulmonary edema (PE) and pulmonary arterial hypertension (PAH) in laboratory rats. Sprague‐Dawley rats were used to induce PE (ANTU, 10 mg/kg, ip) and PAH (ANTU, 5 mg/kg, ip, 4 weeks). Animals were treated with chrysin (10, 20, and 40 mg/kg) and various biochemical, molecular, and histological parameters were evaluated. Acute administration of ANTU induces PE revealed by significant (P < 0.05) increase in relative lung weight, pleural effusion volume, lung edema, bronchoalveolar lavage fluid cell counts, total protein, 5‐hydroxytryptamine (5‐HT), lactate dehydrogenase (LDH), and γ‐glutamyl transferase (GGT), whereas pretreatment with chrysin (20 and 40 mg/kg, ip) significantly (P < 0.05) attenuated these ANTU‐induced biochemical and histological alterations. Repeated administration of ANTU caused induction of PAH evaluated by significant (P < 0.05) alterations in electrocardiographic, hemodynamic changes, and left ventricular function, whereas chrysin (20 and 40 mg/kg, p.o.) treatment significantly (P < 0.05) attenuated these alterations. ANTU‐induced hematological and serum biochemical (aspartate transaminase, alanine transaminase, LDH, and creatinine kinase MB) alterations were significantly (P < 0.05) inhibited by chrysin. It also significantly (P < 0.05) decreased elevated levels of oxido‐nitrosative stress in the right ventricle (RV) and lung. Chrysin significantly (P < 0.05) attenuated downregulated endothelial nitric oxide synthase and upregulated vascular endothelial growth factor messenger RNA and protein expressions both in the RV and pulmonary artery. Chrysin inhibited ANTU‐induced PE and PAH via modulation of inflammatory responses (5‐HT, LDH, and GGT), oxido‐nitrosative stress, and VEGF and eNOs levels.     

Yin Yang‐1 suppresses CD40 ligand‐CD40 signaling‐mediated anti‐inflammatory cytokine interleukin‐10 expression in pulmonary adventitial fibroblasts by promoting histone H3 tri‐methylation at lysine 27 modification on interleukin‐10 promoter

During the pathogenesis of early pulmonary arterial hypertension (PAH), pulmonary arterial adventitial fibroblast act as an initiator and mediator of inflammatory processes that predispose vessel walls to excessive vasoconstriction and pathogenic vascular remodeling. Emerging studies report that Yin Yang‐1 (YY‐1) plays important roles in inflammatory response and vascular injury. Our recent study finds that activation of CD40 ligand (CD40L)–CD40 signaling promotes pro‐inflammatory phenotype of pulmonary adventitial fibroblasts. However, whether YY‐1 is involved in CD40L–CD40 signaling‐triggered inflammatory response in pulmonary adventitial fibroblasts and its underlying mechanism is still unclear. Here, we show that soluble CD40L (sCD40L) stimulation promotes YY‐1 protein expression and suppresses anti‐inflammatory cytokine, interleukin 10 (IL‐10) expression in pulmonary adventitial fibroblasts, while YY‐1 knockdown prevents sCD40L‐mediated reduction of IL‐10 expression via enhancing IL‐10 gene transactivation. Further, we find that sCD40L stimulation significantly increases histone H3 tri‐methylation at lysine 27 (H3K27me3) modification on IL‐10 promoter in pulmonary adventitial fibroblasts, and YY‐1 knockdown prevents the effect of sCD40L on IL‐10 promoter by reducing the interaction with enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, binding to IL‐10 promoter. Moreover, we find that sCD40L stimulation promotes YY‐1 protein, but not messenger RNA (mRNA) expression, via decreasing N6‐methyladenosine methylation on YY‐1 mRNA to suppress YTHDF2‐medicated mRNA decay. Overall, this in‐depth study shows that the activation of CD40L‐CD40 signaling upregulates YY‐1 protein expression in pulmonary adventitial fibroblasts, which results in increasing YY‐1 and EZH2 binding to the IL‐10 promoter region to enhance H3K27me3 modification, eventually leading to suppression of IL‐10 transactivation. This study first uncovers the roles of YY‐1 on CD40L‐CD40 signaling‐triggered inflammatory response in pulmonary adventitial fibroblasts.     

低氧时肺动脉内皮细胞单层通透性变化

目的和方法：研究肺动态内皮细胞（ＰＡＥＣ）在低氧性肺水肿（ＨＰＥ）发生中的作用机制。采用ＯＰＡＥＣ体外培养的方法，观察了ＰＡＥＣ生长状态和特征性蛋白因子ⅧＲ：Ａｇ的变化，并利用ＰＡＥＣ融合单层通透性模型研究了低氧对ＰＡＣＥ融合单层的通透性的影响。结果：ＰＡＥＣ生长数量无明显变化，但细胞的生长质量，ⅧＲ：Ａｇ阳性细胞数明显下降，ＰＡＣＥ通透性明显增加，ＣａＭ阻断剂ＴＦＰ只能部分抑制０这种通透性增加。     

内源性过氧亚硝基阴离子介导脂多糖导致肺动脉内皮细胞损伤

在培养的牛肺动脉内皮细胞(bovine pulmonary artery endothelial cells,BPAECs)水平上,观察脂多糖(lipopolysaccharide,LPS)对BPAECs诱生过氧亚硝基阴离子(peroxynitrite,ONOO~-)能力及内皮源性ONOO~-在LPS致BPAECs损伤中的作用。结果显示:(1)LPS剂量依赖性地引起BPAECs诱生ONOO~-生成标志物硝基酪氨酸(nitrotyrosine,NT)的荧光强度(即ONOO~-)明显增多,NT阳性细胞数和百分率也明显增多或增高(P<0.05);iNOS选择性抑制剂氨基胍(AG)明显抑制LPS诱生ONOO~-增多(P<0.05),而NT阳性细胞数和百分率分别减少或降低,但无明显差异。(2)在LPS作用下BPAECs培养上清中的MDA含量和LDH活性明显增多和增高,呈现剂量依赖性效应。加AG后MDA含量明显降低(P<0.001),LDH活性呈降低趋势。(3)LPS可诱导BPAECs凋亡明显增多,用EB荧光染色后可见细胞染色质浓集、核变小等凋亡征象。AG可导致LPS引起的BPAECs凋亡明显减少,但仍明显高于溶剂组。LPS可导致BPAECs线粒体呼吸抑制及膜电位下降。上述结果表明,LPS可引起BPAECs生成ONOO~-增多,ONOO~-参与介导LPS所致BPAECs过氧化损伤与细胞凋亡。     

Protease‐activated receptor (PAR)‐2 is required for PAR‐1 signalling in pulmonary fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease‐activated receptor (PAR)‐1 and PAR‐2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin‐induced lung fibrosis is diminished in both PAR‐1 and PAR‐2 deficient mice. We thus have been suggested that combined inactivation of PAR‐1 and PAR‐2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR‐1 and PAR‐2 agonists in the absence or presence of specific PAR‐1 or PAR‐2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild‐type and PAR‐2 deficient mice with or without a specific PAR‐1 antagonist (P1pal‐12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR‐1 and/or PAR‐2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR‐1 and PAR‐2 did not show any additive effects on these pro‐fibrotic responses. Strikingly, PAR‐2 deficiency as well as pharmacological PAR‐1 inhibition reduced bleomycin‐induced pulmonary fibrosis to a similar extent. PAR‐1 inhibition in PAR‐2 deficient mice did not further diminish bleomycin‐induced pulmonary fibrosis. Finally, we show that the PAR‐1‐dependent pro‐fibrotic responses are inhibited by the PAR‐2 specific antagonist. Targeting PAR‐1 and PAR‐2 simultaneously is not superior to targeting either receptor alone in bleomycin‐induced pulmonary fibrosis. We postulate that the pro‐fibrotic effects of PAR‐1 require the presence of PAR‐2.     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.