Vascular Biology Section

在血管对缺血应激的反应中，Vav1对HIF-1的激活是必不可少的。Vav1是小分子RhoGTP酶的酪氨酸磷酸化依赖的鸟氨酸核苷酸交换因子。它被认为仅限于正常发育的造血细胞。然而，我们首次发现Vav1在血管内皮细胞中的表达，并在应激状态下调节血管的动态平衡。血管对低氧的反应是器官功能的主要决定因素，这对心脏等重要器官尤为关键。最近，我们发现Vav1是缺氧的关键血管调节因子，对HIF-1的激活是必不可少的。它通过p38/Siah2/PHD3途径调节HIF-1a的稳定。因此，Vav1基因缺陷的小鼠在心脏缺血时容易猝死，冠状动脉内皮细胞凋亡增加。此外，Vav1与VEGFR1结合，VEGFR1携带Vav1到溶酶体，在常氧条件下进行降解。低氧通过抑制蛋白质降解上调Vav1。这些发现表明，低氧对Vav1的调节类似于HIF-1a的调节。这两种蛋白质都是结构性产生的，允许在应激发生时做出快速反应，并在常压下不断降解。缺氧可以稳定Vav1，这是HIF-1a积累所必需的。它们共同调节血管对缺氧的反应，并维持组织的动态平衡。这部作品已提交出版。Vav1在血管内皮细胞中积聚，并参与衰老相关的血管功能障碍。血管功能障碍是生物衰老的结果，是心血管疾病的主要危险因素。最近，我们观察到小鼠Vav1缺乏导致eNOS依赖的血管扩张，并对主动脉中的乙酰胆碱有更大的反应。分子分析显示，Vav1在内皮细胞中的敲除增加了Sirt1，从而使eNOS去乙酰化，进而激活eNOS，诱导NO的产生。相反，Vav1的异位表达抑制了Sirt1的表达和eNOS的激活。这些数据表明，Vav1通过抑制Sirt1对eNOS的激活具有负性作用。有趣的是，在衰老过程中，Vav1在培养的内皮细胞和小鼠主动脉中积聚，这与Sirt1减少、eNOS乙酰化/失活增加和NO产生减少有关。通过eNOS依赖的血管松弛、高血压和动脉僵硬的测量，Vav1的缺失可以保护小鼠免受衰老相关的血管功能障碍的影响。这部作品已提交出版。Vav1通过Sirt1调节间充质干细胞(MSC)在脂肪细胞和软骨细胞之间的分化。使用我们建立的Vav1报告小鼠，我们研究了Vav1在体内的表达。检测Vav1在MSCs中的表达。有趣的是，骨髓间充质干细胞中Vav1的缺失导致自发的成脂，但损害了软骨分化，相应地，Vav1缺失的小鼠表现出脂肪含量的增加和软骨的减少。在机制上，Vav1的缺失降低了Sirt1的水平，这是导致乙酰化PPARg增加的原因。由于乙酰化激活了PPARg，C/EBPA表达增加，促进了脂肪的生成。另一方面，Vav1的缺失导致Sirt1靶标乙酰化Sox9的增加。由于乙酰化抑制了Sox9的活性，导致2A1胶原蛋白的急剧减少，2A1胶原蛋白是软骨细胞分化的关键调节因子。综上所述，这项研究揭示了Vav1通过Sirt1调控MSC细胞分化命运的新功能。SIRT1去乙酰化PPARg和Sox9，这是控制脂肪细胞和软骨细胞分化的两个关键介质。PPARg和Sox9的乙酰化状态对其活性有相反的影响，从而控制细胞的命运决定。这项研究发表在2016年的《干细胞》杂志上。几年前，我们发现了一种名为NK4的新基因，它可以放大血管炎症并促进败血症的发展。然而，什么是NK4以及它如何调节细胞功能尚不清楚。小的GTP酶和RAS同源的Rap1在囊泡运输、胞吐、信号传递和细胞黏附中起着中心作用。最近，我们发现NK4，也被称为白介素32，是Rap1的晚期内体/多囊泡小体(MVB)相关的全环基金。NK4与RAP1结合，促进GDP与GTP的交换，激活RAP1，诱导肌动蛋白萌发。通过活细胞和超分辨率成像的结合，我们证明了NK4/RAP1复合体在胞吐泡中的存在。NK4/Rap1的结合需要一个在Rap GEF之间保守的小螺旋结构域，两侧是与ApoE同源的区域，这表明了一种新的全球环境基金结构。在功能上，NK4的激活导致病原体通过胞吐作用从上皮细胞和内皮细胞中排出。这些作品将被总结出来出版。

Vav1 is essential for HIF-1 activation in vascular response to ischemic stress. Vav1 is a tyrosine phosphorylation-dependent guanine nucleotide exchange factor (GEF) for small RhoGTPase. It has been thought to be restricted to hematopoietic cells in normal development. However, we for the first time found Vav1 expression in vascular endothelium and regulates vascular homeostasis under stress. Vascular response to hypoxia is a major determinant of organ function, which is particularly critical for vital organs such as the heart. Recently, we identified Vav1 as a key vascular regulator of hypoxia and essential for HIF-1 activation. It regulates HIF-1a stabilization through the p38/Siah2/PHD3 pathway. Consequently, Vav1 deficient mice are predisposed to sudden death under cardiac ischemia with increased coronary endothelial apoptosis. Moreover, Vav1 binds to VEGFR1 that carries Vav1 to lysosomes for degradation in normoxia. Hypoxia upregulates Vav1 through inhibition of protein degradation. These findings reveal that regulation of Vav1 by hypoxia is analogous to HIF-1a regulation. Both proteins are constitutively produced allowing for rapid responses when stress occurs, and constantly degraded in normoxia. Hypoxia stabilizes Vav1, which is required for HIF-1a accumulation. Together they mediate the vascular response to hypoxia and maintain tissue homeostasis. This work has been submitted for publication. Vav1 accumulates in endothelium and contributes to aging associated vascular dysfunction. Vascular dysfunction occurs as a consequence of biological aging and is a major risk factor for cardiovascular diseases. Recently, we observed that Vav1 deficiency in mice led to eNOS dependent vascular dilation and a greater response to acetylcholine in aorta. Molecular analysis revealed that knockdown of Vav1 in endothelial cells increased Sirt1, which deacetylated eNOS, and subsequently activated eNOS and induced NO production. Conversely, ectopic expression of Vav1 inhibited Sirt1 expression and eNOS activation. These data suggest a negative role of Vav1 on eNOS activation through inhibition of Sirt1. Interestingly, Vav1 accumulates in cultured endothelial cells and mouse aorta during aging, which correlates with a reduction of Sirt1 and increased eNOS acetylation/inactivation and reduced NO production. Loss of Vav1 protected mice from aging associated vascular dysfunction as measured by eNOS dependent vascular relaxation, hypertension and arterial stiffness. This work has been submitted for publication. Vav1 regulates mesenchymal stem cell (MSC) differentiation decision between adipocyte and chondrocyte via Sirt1. Using a Vav1 reporter mice we generated, we characterized Vav1 expression in vivo. We detected expression of Vav1 in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARg. As acetylation activates PPARg, it increased C/EBPa expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2a1, a key regulator in chondrocyte differentiation. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARg and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARg and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. This work was published in Stem Cells in 2016. Several years ago, we identified a novel gene, NK4, that amplifies vascular inflammation and potentiates sepsis development. However, what is NK4 and how it regulates cellular function is not known. The small GTPase and Ras homolog Rap1 plays a central role in vesicle trafficking, exocytosis, signaling and cell adhesion. Recently, we found that NK4, also known as interleukin 32, is a late endosomal/multivesicular body (MVB) associated GEF for Rap1. NK4 bound Rap1 and promoted the exchange of GDP for GTP, activating Rap1 and eliciting actin sprouting. Through a combination of live cell and super-resolution imaging, we demonstrate the presence of the NK4/Rap1 complex at exocytotic blebs. NK4/Rap1 association required a small helical domain conserved among Rap GEFs, flanked by regions homologous to ApoE, indicating a novel GEF architecture. Functionally, activation of NK4 results in expulsion of pathogens from epithelial and endothelial cells via exocytosis. These works will be summarized for publication.