Mutations in VEGFA are associated with congenital left ventricular outflow tract obstruction

Left ventricular outflow tract obstruction (LVOTO) comprises a spectrum of stenotic lesions. Previous studies have shown that the vascular endothelial growth factor (VEGF) signaling system plays a critical role in cardiac cushion formation, vasculogenesis, and angiogenesis. We hypothesize that VEGFA may be a potential candidate gene associated with the spectrum of LVOTO lesions. However, it remains unclear whether the VEGFA gene is responsible for the development of LVOTO malformations. In this study, we identified three exon mutations in the VEGFA gene in three of 192 nonsyndromic LVOTO patients, and the overall mutation frequency was 1.6% (3/192). The c.454C>T (p.Arg152X) nonsense mutation and c.19_22dupGACA (p.Thr8ArgfsX78) internal tandem duplication mutation each introduced a premature stop codon and are predicted to produce a truncated VEGFA protein. The c.998G>A missense mutation changes a highly conserved arginine to a glutamine at residue 333 (p.Arg333Gln). These mutations were carried by some family members, and average penetrance was 33.3%. The present study suggests, for the first time to our knowledge, that VEGFA mutations may be associated with congenital LVOTO malformations. We provide evidence that LVOTO is likely oligogenic.     

Higher mortality in heterozygous neuropilin-1 mice after cardiac pressure overload

We previously identified that neuropilin-1 (NP-1) was a co-receptor of vascular endothelial growth factor receptor 2 (VEGFR2) and confirmed that NP-1 knockout mice were embryonic lethal due to impairment of vascular development, while VEGF was reported to be involved in the progression of heart failure. However, it is unknown whether NP-1 has any influence on cardiac function, and it also remains poor understood concerning cardiac expression of NP-1 and its interaction with other VEGF receptors in the heart. Here, we first showed that NP-1 heterozygous mice had significantly higher mortality due to either acute or chronic heart failure in response to left ventricular pressure overload. We also observed that NP-1 mRNA and protein were expressed in both neonatal rat cardiomyocytes and adult murine heart. Furthermore, we found that NP-1 formed complexes with VEGFR1 and VEGFR2, respectively, in cardiomyocytes. These findings suggest that NP-1 should play beneficial role in heart failure.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.     

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

Expression of angiostatin and its related factors in the plasma of newborn pigs with hypoxia and reoxygenation

Little is known about angiostatin and its related factors in the hypoxia-reoxygenation of neonates. In this study we compared the effect of 21% and 100% reoxygenation on temporal changes in the plasma level of these factors in newborn piglets subjected to hypoxia. Newborn piglets were subjected to 2 h hypoxia followed by 1 h of reoxygenation with either 21% or 100% oxygen and observed for 4 days. On day 4 of recovery in 100% hypoxic-reoxygenated group, there were increases in total angiostatin, plasminogen/plasmin and MMP-2 levels, and decreases in VEGF levels (vs. respective baseline levels, all P <0.001), whereas no significant temporal changes were found in the 21% hypoxic-reoxygenated and sham-operated groups. Angiostatin levels correlated positively with the levels of MMP-2 and HIF-1alpha and negatively with VEGF levels in 100% hypoxic-reoxygenated group (all P <0.05). In comparison to 21% oxygen, neonatal resuscitation with 100% oxygen was found to increase the levels anti-angiogenic factors.     

N-cadherin is required for neural crest remodeling of the cardiac outflow tract

Cardiac neural crest cells undergo extensive cell rearrangements during the formation of the aorticopulmonary septum in the outflow tract. However, the morphogenetic mechanisms involved in this fundamental process remain poorly understood. To determine the function of the Ca2+-dependent cell adhesion molecule, N-cadherin, in murine neural crest, we applied the Cre/loxP system and created mouse embryos genetically mosaic for N-cadherin. Specifically, deletion of N-cadherin in neural crest cells led to embryonic lethality with distinct cardiovascular defects. Neural crest cell migration and homing to the cardiac outflow tract niche were unaffected by loss of N-cadherin. However, N-cadherin-deficient neural crest cells were unable to undergo the normal morphogenetic changes associated with outflow tract remodeling, resulting in persistent truncus arteriosus in the majority of mutant embryos. Other mutant embryos initiated aorticopulmonary septum formation; however, the neural crest cells were unable to elongate and align properly along the midline and remained rounded with limited contact with their neighbors. Interestingly, rotation of the outflow tract was incomplete in these mutants suggesting that alignment of the channels is dependent on N-cadherin-generated cytoskeletal forces. A second cardiac phenotype was observed where loss of N-cadherin in the epicardium led to disruption of heterotypic cell interactions between the epicardium and myocardium resulting in a thinned ventricular myocardium. Thus, we conclude that in addition to its role in myocardial cell adhesion, N-cadherin is required for neural crest cell rearrangements critical for patterning of the cardiac outflow tract and in the maintenance of epicardial-myocardial cell interactions.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

血清胆红素、NO、ET-1、VEGF水平联合检测对冠心病诊断价值

摘要 目的：探讨血清胆红素、一氧化氮（NO）、内皮素-1（ET-1）、血管内皮生长因子（VEGF）水平联合检测对冠心病（CHD）的诊断价值。方法：选取2019年12月至2020年12月在我院治疗的冠心病患者65例作为观察组;另选取同期参与健康体检的60例健康人群作为对照组。对所有患者总胆红素水平（TBIL）、直接胆红素水平（DBIL）、NO、ET-1、VEGF表达水平进行检测,计算间接胆红素水平（IBIL）,采用受试者工作特征曲线（ROC）下面积（AUC）比较血清胆红素、VEGF、ET-1和NO单独和联合检测对冠心病的诊断价值,采用单因素和多因素logistic回归分析法分析冠心病的影响因素。结果：与对照组相比,观察组患者血清IBIL、DBIL、TBIL、VEGF和NO检测结果均显著降低,ET-1检测结果显著升高（P＜0.05）。血清胆红素、VEGF、ET-1和NO对冠心病联合检测的AUC值显著高于单独检测值（P＜0.05）。观察组患者高血压、糖尿病、血脂异常、吸烟、肥胖的发生率明显高于对照组（P＜0.05）。多因素非条件logistic回归分析结果显示,高血压、糖尿病、血脂异常、吸烟、肥胖、血清胆红素、VEGF、ET-1和NO均可作为评估冠心病的危险因素,其中血清检测指标的关联强度依次为ET-1、VEGF、NO、胆红素（P＜0.05）。结论：冠心病患者血清胆红素代谢异常,血管内皮功能指标紊乱,是导致患者动脉粥样硬化的重要原因。血清胆红素、NO、ET-1、VEGF水平联合检测对冠心病诊断效能明显优于单个指标,且可作为冠心病发病风险的有效指标。     

肾癌患者血清VEGF的水平及其与临床病理分期的关系

目的:探讨肾癌患者血清血管内皮生长因子(VEGF)的水平及其与临床病理分期的关系。方法:选择2013年1月至2015年1月在我院行手术治疗的肾癌患者56例为观察组,选择同期在我院进行健康体检的正常成人50例作为对照,所有患者的诊断均经病理切片证实,对所有研究对象,采集其清晨空腹静脉血,用酶联免疫吸附法(ELISA)检测血清中的VEGF和VEGF受体-1(VEGFR-1)。结果:观察组血清中VEGF和VEGFR-1的浓度分别为(132.75±68.31)ng/mL和(33.76±15.39)ng/mL,均显著高于对照组,差异有统计学差异(均P0.05);不同分期患者血清中VEGF和VEGFR-1浓度差异有统计学意义(均P0.05),病理分期增加,VEGF与VEGFR-1的浓度增加,VEGF与VEGFR-1呈正相关(r=0.625,P0.05)。结论:血清VEGF水平可用于诊断RCC,且对于预判RCC的病例分期具有重要价值。     

Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue

Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 degrees C for 15 min. The control (n=23) and heated (n=55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart.     

Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone

Pigment epithelial derived factor (PEDF) is one of the most effective inhibitors of angiogenesis described so far, especially in controlling the growth of blood vessels in the eye. We now describe the localization of PEDF in regions of active bone formation in the mid-gestation mouse embryo and its specific and high levels of secretion by osteoblasts. PEDF is detected to a lesser extent in osteoclasts as well. The proangiogenic factors, VEGF and its receptors VEGF-R1 and VEGF-R2, are also expressed by both osteoblasts and osteoclasts. These findings suggest that bone angiogenesis and matrix remodeling may be mediated both by PEDF and by VEGF.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

VAC 技术治疗兔耳缺血性创面的实验研究

目的:观察间歇和持续负压下缺血创面不同处理与愈合的关系。方法:实验前1天,用脱毛剂(Nair,美国)对兔耳背脱毛。动物用1%戊巴比妥钠耳缘静脉注射麻醉(30 mg/kg体重),固定于手术台。75%乙醇消毒双侧耳背皮肤。距耳根3-3.5cm处分离、结扎兔耳中央神经血管束。在耳背中部形成直径2.5cm全层皮肤缺损创面(保留软骨膜)[1]。止血后置动物于特制木盒内。42只大白兔共84个创面,随机分为-50mmHg-75mmHg和-100mmHg 3大组,分别施以间歇负压(运行2分钟,停1分钟)和持续负压组。实验分别运用-50mmHg,-75mmHg,-100mmHg三个不同负压值进行连续、间歇治疗兔耳缺血性创面,观察伤后1,3,7,10,14,20d创面愈合情况,取伤后7d组织标本进行Western blot、HE染色,观察VEGF(vascular endothelial growth factor)的表达及创面上皮的再生和肉芽组织生长情况[1]。以及各时间点细胞凋亡的检测。结果:-50mmHg(纱布+海绵)间歇负压引流技术治疗兔耳缺血性创面的愈合最快,-75mmHg治疗组次之,-100mmHg治疗组创面愈合最慢。在同一时间点上,-50mmHg治疗组与-75mmHg,-100mmHg治疗组和空白对照组之间相比,能够更快地促进创面VEGF的表达和肉芽组织的再生,毛细血管增多。封闭负压治疗能够降低创面组织细胞的凋亡的发生。结论:(1)封闭负压治疗能够促进缺血创面的肉芽组织再生及VEGF的表达,减少创面组织细胞的凋亡的发生;(2)-50mmHg间歇封闭负压治疗效果最好。     

Macrophages of hemangioblastic lineage invade the lens vesicle-ectoderm interspace during closure and detachment of the avian embryonic lens

Summary An area of cell death is apparent in the lens vesicle margin and the lens stalk during closure and detachment of the lens anlage from the cephalic ectoderm. Free phagocytic cells closely associated with this area of cell death have been interpreted as cells migrating from the lens epithelium. Scanning and transmission electron microscopy, light-microscopic histochemical staining for acid phosphatase and immunostaining using MB1 (a monoclonal antibody specific for quail endothelial and hemopoietic cells) of chimeras of chick embryo and quail yolk sac were used to analyze these lens vesicle-associated free phagocytic cells. The cells have morphological features identical to those of macrophages in other embryonic tissues. In contrast to epithelial cells phagocytosing cell debris, they exhibit strong acid phosphatase activity, a feature typical of macrophages. In addition, free phagocytic cells are MB1 positive in chick embryo-quail yolk sac chimeras, hence they proceed from cells of hemangioblastic lineage originating in the yolk sac. These results indicate that the lens vesicle-associated free phagocytic cells are macrophages. Observations of MB1 positive amoeboid cells in the juxta-retinal mesenchyme and on the borders of the optic cup suggest that these macrophages migrate through the mesenchyme surrounding the eye primordium. Macrophages are seen in both the interspace between lens vesicle and ectoderm and in the lumen of the lens as well as within both the ectoderm and the lens epithelium. In these locations they remove cell debris, and thereby contribute to the complete disappearance of the area of cell death. Macrophages remain in the lens vesicle-ectoderm interspace until developmental stages at which it is invaded by corneal endothelial cells.     

Roles of erbB4, rhombomere-specific, and rhombomere-independent cues in maintaining neural crest-free zones in the embryonic head

Within the developing vertebrate head, the migration of neural tube-derived neural crest cells (NCCs) through the cranial mesenchyme is patterned into three streams, with mesenchyme adjacent to rhombomeres (r)3 and r5 maintained NCC-free. The receptor tyrosine kinase erbB4 is expressed within r3 and r5 and is required to maintain the r3-adjacent NCC-free zone in mouse embryos. In this study, we demonstrate that the extent of r3 involvement in patterning mouse NCC migration is restricted to the same dorsolateral region regulated by erbB4. In chick embryos, we show that erbB4 signaling similarly maintains the r3-adjacent NCC-free zone. However, although r5 expresses erbB4, this is insufficient to maintain the r3-adjacent NCC-free zone in grafting experiments where r5 replaced r3, indicating that erbB4 requires additional factors at the A-P level of r3 to pattern NCC migration. Furthermore, we show that the r5-adjacent NCC-free zone is maintained independently of r5, but requires surface ectoderm. Finally, we demonstrate that avian cranial surface ectoderm is patterned molecularly, with dorsolateral surface ectoderm at the levels of r2/3 and r7 expressing the sulfatase QSulf1 in quail, or the orthologue CSulf1 in chick. Aberrant NCC migration into r3-adjacent mesenchyme correlated with more focused QSulf1 expression in r2/3 surface ectoderm.