Alteration in angiogenic and anti-angiogenic forms of vascular endothelial growth factor-A in skeletal muscle of patients with intermittent claudication following exercise training

The aims of this study were twofold: (1) to identify whether peripheral artery disease (PAD) patients had increased muscle concentration of angiogenic VEGF-A, anti-angiogenic VEGF???b or VEGF receptor 1 (VEGF-R1) when compared with control subjects, and (2) to evaluate whether exercise training in PAD patients was associated with changes in muscle concentration of VEGF-A, VEGF???b or VEGF-R1. At baseline, 22 PAD and 30 control subjects underwent gastrocnemius muscle biopsy. Twelve PAD patients were treated with supervised exercise training (SET) and underwent muscle biopsy after 3 weeks and 12 weeks of training and had sufficient tissue to measure VEGF-A, VEGF???b and VEGF-R1 concentrations in skeletal muscle lysates by ELISA. Muscle concentrations of VEGF-A and VEGF???b were similar in PAD patients versus controls at baseline. At both time points after the start of SET, VEGF-A levels decreased and there was a trend towards increased VEGF???b concentrations. At baseline, VEGF-R1 concentrations were lower in PAD patients when compared with controls but did not change after SET. Skeletal muscle concentrations of VEGF-A are not different in PAD patients when compared with controls at baseline. SET is associated with a significant reduction in VEGF-A levels and a trend towards increased VEGF???b levels. These somewhat unexpected findings suggest that further investigation into the mechanism of vascular responses to exercise training in PAD patients is warranted.     

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes

The maintenance of sarcoplasmic reticulum Ca^2 + ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca^2 +-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.     

缺氧诱导热休克蛋白70-2表达的分子机制

目的 观察缺氧对HepG32细胞中热休克蛋白(HSP)70-2(基因名为HSPA2)表达的影响,探讨缺氧条件下缺氧诱导因子(HIF)-1在转录水平调控HSP70-2表达的具体机制. 方法 以物理缺氧法和化学缺氧诱导剂(DFO或CoC12)诱导法分别模拟肿瘤缺氧环境,Western blot检测HIF-1α和HSP70-2蛋白表达的变化,荧光素酶报告基因分析技术检测缺氧对HSPA2基因的激活作用.HIF-1α抑制剂(YC-1)或HIF-1α小干扰RNA(siRNA)处理后检测缺氧HepG2细胞中HSP70-2的表达变化.构建一系列截短和突变的HSPA2基因启动子荧光素酶报告基因质粒,将其与HIF-1α siRNA共转染HepG2细胞,荧光素酶分析技术检测HSPA2启动子活性的变化,寻找HIF-1在HSPA2启动子上的结合位点.各组间比较用方差分析,两组间比较用t检验. 结果 缺氧培养6 h后,HepG2细胞中HIF-1α和HSP70-2表达增加,其表达量随着缺氧培养时间的延长而逐渐增加(F=77.369,P<0.01;F=108.854,P<0.01).各组分别加终浓度为0、50、100 μmol/L的化学缺氧诱导剂DFO或终浓度为0、100、200 μmol/L的CoCl2,培养24 h后检测,随着DFO浓度增加,HIF-1α和HSP70-2蛋白表达逐渐增加(F=443.174,P<0.01;F=589.238,P<0.01);随着COCl2浓度增加,HIF-1 α和HSP70-2蛋白表达也逐渐增加(F=637.724,P<0.01;F=692.918,P<0.01).与常氧培养相比,缺氧培养后HSPA2启动子活性增加7.09倍(t=43.551,P<0.01).随着YC-1浓度升高,HIF-1α表达受到明显抑制(F=883.614,P<0.01),HSP70-2表达水平也相应下降(F=218.112,P<0.01).转染HIF-1α siRNA后HIF-1α表达受到明显抑制(F=577.130,P<0.01),HSP70-2表达水平也相应下降(F=465.598,P<0.01).构建系列截短的HSPA2启动子报告基因载体转染HepG2细胞后检测,转染HSPA2(-1114/+62)-LUC和HSPA2(-653/+62)-LUC细胞的相对荧光素酶活性无明显改变,但转染HSPA2(-385/+62)-LUC细胞的相对荧光素酶活性较前明显降低(t=13.717,P<0.01).分别构建突变缺氧反应元件(HRE)1和HRE2的HSPA2启动子报告基因载体,转染HepG2细胞后检测,突变HRE1后HSPA2启动子活性显著降低(t=10.954,P<0.01),但是突变HRE2后HSPA2启动子活性无明显改变.结论 缺氧显著上调HepG2细胞中HSP70-2的表达,其机制可能是HIF-1与HSPA2启动子上HRE1结合后激活该基因的转录.     

Hypoxia-Inducible Factor 1-Alpha Release After Intracoronary Versus Intramyocardial Stem Cell Therapy in Myocardial Infarction

We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1α) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8?±?1.2?×?10⁶) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22?±?4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1α was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1α expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1α increased immediately post-MI (from 278?±?127 to 631?±?375 pg/ml, p?<?0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1α significantly (p?<?0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1α expression in the infarcted area was higher in Group IM than in Group IC or S (1,963?±?586 vs. 1,307?±?392 vs. 271?±?110 activity per square millimeter, respectively, p?<?0.05), while the border zone contained similarly lower level of HIF-1α, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1α was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1α. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1α more effectively than does intracoronary delivery.     

缺氧诱导因子-2α对脂代谢的调节作用

缺氧诱导因子-2α（HIF-2α）是一种在缺氧条件下广泛存在于NSL动物和人体内的氧敏感性转录因子,通过调节缺氧诱导基因表达参与组织细胞对低氧的适应性应答,其表达是机体适应低氧的关键环节和起始步骤。近年研究发现HIF-2a可能与脂代谢过程有密切联系。本文就HIF-2α对脂肪酸代谢和胆固醇代谢的调节作用作一综述。     

HIF-1α在小鼠ACTH垂体腺瘤细胞AtT-20的抗凋亡作用

目的 观察缺氧诱导因子(HIF)-1α在小鼠垂体促肾上腺皮质激素腺瘤细胞AtT-20生长增殖中的作用,验证HIF-1α能否保护AtT-20细胞免于低氧诱导的细胞凋亡.方法 不同浓度的CoCl2诱导AtT-20细胞发生低氧,MTT方法观察CoCl2促进细胞生长增殖的作用,实时定量PCR和Western印迹检测常氧和低氧环境下HIF-1α mRNA和蛋白水平的改变;细胞转染HIF-1α-siRNA后,CoCl2低氧诱导,实时定量PCR和Western印迹验证siRNA下调HIF-1α的效率,凋亡检测系统Annexin V-FITC和TUNEL法检验细胞凋亡情况.结果 在一定浓度(≤100 μmoL/L)和时间(≤48 h)范围内,CoCl2可以促进AtT-20细胞的生长增殖,并呈浓度和时间正相关性;HIF-1α-siRNA转染后,CoCl2低氧诱导处理的细胞发生凋亡的比率大幅度上升(P＜0.05).结论 HIF-1α在AtT-20细胞生长增殖中发挥抗凋亡的作用.     

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.     

High-fat feeding induces angiogenesis in skeletal muscle and activates angiogenic pathways in capillaries

High-fat diet (HFD) increases fatty acid oxidation in skeletal muscles. We hypothesized that this leads to increased oxygen demand and thus to increased capillarization. We determined the effects of high-fat diet on capillarization and angiogenic factors in skeletal muscles of mice that were either active or sedentary. Fifty-eight C57BL/6 J mice were divided into four groups: low-fat diet sedentary (LFS), low-fat diet active (LFA), high-fat diet sedentary (HFS), and high-fat diet active (HFA). The mice in active groups were housed in cages with running wheels and the sedentary mice were housed in similar cages without running wheels. After 19 weeks HFS, LFA and HFA had higher capillary density and capillary-to-fiber-ratio in quadriceps femoris muscles than LFS. Capillarization was similar in HFS and HFA. To reveal possible mechanisms of HFD induced angiogenesis, we measured protein and mRNA levels of angiogenic factors VEGF-A, HIF-1α, PGC-1α and ERRα. VEGF-A protein levels were higher in muscles of HFS, LFA and HFA compared to LFS. However, no significant differences were observed between HFA and HFS. Protein levels of HIF-1α, PGC-1α, and ERRα were similar in all groups. However, the mRNA expression of HIF-1α and VEGF-A was up-regulated in capillaries but not in muscle fibers of HFS. The sedentary and active mice groups had similar mRNA expression levels of angiogenesis regulators studied. We conclude that high-fat feeding induces angiogenesis in skeletal muscle and up-regulates the gene expression of HIF-1α and VEGF-A in capillaries.     

侵袭性功能性垂体腺瘤患者低氧诱导因子1α诱导血管生成相关基因的表达及临床意义

背景研究表明,垂体肿瘤患者低氧诱导因子1α(HIF-1α)表达增加,且HIF-1α表达与肿瘤侵袭性密切相关,但具体调控机制尚需要进一步探讨。目的分析侵袭性功能性垂体腺瘤患者HIF-1α诱导血管生成相关基因的表达及临床意义。方法选取2016年1月至2020年9月唐山市人民医院神经外科门诊收入院并接受手术治疗的功能性垂体腺瘤患者58例,根据Hardy-Wilson分级和Knosp分类方法将其分为侵袭性组(n=28)和无侵袭性组(n=30)。比较两组患者临床资料,HIF-1α、血管内皮生长因子A(VEGF-A)、表皮生长因子受体(EGFR)、CD31、Ki-67表达情况及免疫组化评分。结果侵袭性组患者有视野缺损、肿瘤体积≥6.92 cm^(3)、全切及次全切者所占比例及肿瘤复发率高于无侵袭性组(P<0.05)。侵袭性组患者中HIF-1α、VEGF-A、EGFR、CD31、Ki-67高表达者所占比例高于无侵袭性组(P<0.05)。侵袭性组患者HIF-1α、EGFR、VEGF-A、CD31、Ki-67免疫组化评分高于无侵袭性组(P<0.05)。结论侵袭性功能性垂体腺瘤患者HIF-1α、VEGF-A、EGFR呈高表达,其机制可能为低氧环境下HIF-1α表达增加后上调VEGF-A、EGFR表达,从而促进血管生成,增加肿瘤侵袭性。     

葡萄糖对不同氧浓度下内皮祖细胞表达低氧诱导因子-1α的影响

目的 观察葡萄糖对低氧（1%氧浓度）和常氧（21%氧浓度）状态下内皮祖细胞表达低氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）的影响,探讨低氧在糖尿病周围血管病发生中的可能作用.方法 常规培养健康人外周血来源的内皮祖细胞,加入不同浓度葡萄糖（5、10、33 mmol/L）分为A、B、C 3组,并在常氧和低氧条件下分别进行培养.实时荧光定量逆转录-聚合酶链反应（RT-PCR）法检测内皮祖细胞表达HIF-1α及VEGF的水平,免疫印迹法检测HIF-1α蛋白表达,ELISA法检测VEGF分泌水平.结果 （1）低氧组表达HIF-1α mRNA（A、B和C组分别为1.25±0.34、1.35±0.26和0.75±0.22）高于常氧组（A、B和C组分别为1.03 ±0.25、1.21±0.28和0.61±0.17）,差异具有统计学意义（A、B和C组t值分别为1.96,2.11,1.89,均P＜0.05）;而在相同氧浓度下,B组的内皮祖细胞表达HIF-1α mRNA高于A组和C组,差异具有统计学意义（低氧组F=23.54,P＜0.01;常氧组F=29.46,P＜0.01）.（2）在相同葡萄糖浓度下,低氧组和常氧组VEGF的蛋白表达差异无统计学意义（t值分别为0.933、1.258和1.001,均P＞0.05）.在低氧浓度下,VEGF的蛋白表达A、B和C组分别为2953±237、2473±205和1768±195,差异具有统计学意义（F=137.43,P=0.000）;在常氧浓度下,A、B和C组VEGF蛋白分别为2868±247、2377±197和1844±203,F=97.96,P=0.000.结论 高糖对低氧条件下的内皮祖细胞具有毒性作用,能减弱其表达HIF-1α和VEGF,可能在糖尿病周围血管病的发生中发挥一定作用.     

Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST

Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1α (HIF-1α)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.     

Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells

OBJECTIVE: Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested (a) whether reactive oxygen species (ROS) and c-Src play roles in hypoxic induction of HIF-1alpha protein and PAI-1 gene expression in the rabbit aortic smooth muscle cell line C2/2 cells and primary cultures of rat aortic smooth muscle cells, and (b) how mitochondria act on the hypoxia-induced signaling mechanism. METHODS AND RESULTS: Hypoxic exposure of C2/2 cells increased H2O2 generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. Catalase, a scavenger of H2O2, inhibited the hypoxia-induced ROS generation and PAI-1 gene expression. Src kinase inhibitors PP1 and PP2 inhibited hypoxia-induced HIF-1alpha protein and PAI-1 gene expression. Ablation of mitochondrial respiration by rotenone abolished hypoxia-induced ROS generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. CONCLUSION: Induction of HIF-1alpha protein and PAI-1 gene expression in response to hypoxia was regulated by ROS production and c-Src activation in vascular smooth muscle cells. Mitochondria linked the hypoxic signal to c-Src, which in turn led to HIF-1alpha protein and PAI-1 gene expression. These results provide evidence that hypoxia induces the ROS-mediated and c-Src-dependent signaling cascades which are closely associated with angiogenesis and thrombosis in atherosclerotic vasculature.     

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. 2000;96:2775-2783)