Vascular Biology Section

Vav1对HIF-1在血管缺血应激反应中的激活至关重要。Vav1是小RhoGTPase的酪氨酸磷酸化依赖的鸟嘌呤核苷酸交换因子（GEF）。它一直被认为局限于正常发育的造血细胞。然而，我们首次在血管内皮中发现了Vav1的表达，并在逆境下调节血管稳态。血管对缺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重要。最近，我们发现Vav1是缺氧的关键血管调节剂，对HIF-1的激活至关重要。它通过p38/Siah2/PHD3途径调节HIF-1a的稳定。因此，Vav1缺陷小鼠易在心脏缺血下猝死，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降解。缺氧通过抑制蛋白质降解而上调Vav1。这些发现表明缺氧对Vav1的调节类似于HIF-1a的调节。这两种蛋白质都是组成性产生的，允许在应激发生时快速反应，并在常氧条件下不断降解。缺氧稳定Vav1，这是HIF-1a积累所必需的。它们共同介导血管对缺氧的反应并维持组织稳态。这项工作已提交出版。Vav1在内皮细胞中积累，有助于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，我们观察到小鼠的Vav1缺乏导致eNOS依赖性血管扩张和对主动脉乙酰胆碱的更大反应。分子分析显示，内皮细胞中Vav1的敲低增加Sirt1，使eNOS去乙酰化，随后激活eNOS并诱导NO的产生。相反，异位表达Vav1抑制Sirt1表达和eNOS激活。这些数据表明，Vav1通过抑制Sirt1对eNOS激活起负作用。有趣的是，随着年龄的增长，Vav1在培养的内皮细胞和小鼠主动脉中积累，这与Sirt1的降低、eNOS乙酰化/失活的增加和NO产生的减少有关。通过eNOS依赖性血管松弛、高血压和动脉僵硬来测量，Vav1的缺失可以保护小鼠免受衰老相关的血管功能障碍。这项工作已提交出版。Vav1通过Sirt1调控脂肪细胞和软骨细胞间充质干细胞（MSC）的分化决定。使用我们培育的Vav1报告小鼠，我们在体内表征了Vav1的表达。我们检测了MSCs中Vav1的表达。有趣的是，MSCs中Vav1的缺失导致自发脂肪生成，但软骨分化受损，因此Vav1缺失的小鼠脂肪含量增加，软骨减少。从机制上讲，Vav1的缺失降低了Sirt1的水平，这是乙酰化ppar增加的原因。乙酰化激活PPARg，增加C/EBPa表达，促进脂肪形成。另一方面，Vav1的缺失导致Sirt1靶点乙酰化Sox9的增加。由于乙酰化抑制Sox9活性，它导致胶原蛋白2a1的急剧减少，胶原蛋白2a1是软骨细胞分化的关键调节因子。总之，这项研究揭示了Vav1通过Sirt1调节MSC细胞分化命运决定的新功能。Sirt1使PPARg和Sox9脱乙酰，这是控制脂肪细胞和软骨细胞分化的两种关键介质。PPARg和Sox9的乙酰化状态对其活性有相反的影响，从而控制细胞命运的决定。这项研究发表在2016年的《干细胞》杂志上。几年前，我们发现了一种新的基因NK4，它可以放大血管炎症并促进败血症的发展。然而，NK4是什么以及它如何调节细胞功能尚不清楚。小的GTPase和Ras同源物Rap1在囊泡运输、胞吐、信号传导和细胞粘附中起核心作用。最近，我们发现NK4，也被称为白细胞介素32，是Rap1的晚期内体/多泡体（MVB）相关的GEF。NK4结合Rap1，促进GDP与GTP交换，激活Rap1，引发肌动蛋白发芽。通过活细胞和超分辨率成像的结合，我们证明了NK4/Rap1复合物在胞外泡中的存在。NK4/Rap1关联在Rap GEF中需要一个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GEF结构。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。这些作品将整理出版。

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.