Endothelial k-RasV12 Expression Induces Capillary Deficiency Attributable to Marked Tube Network Expansion Coupled to Reduced Pericytes and Basement Membranes.
Endothelial k-RasV12 Expression Induces Capillary Deficiency Attributable to Marked Tube Network Expansion Coupled to Reduced Pericytes and Basement Membranes.
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DOI：
10.1161/atvbaha.121.316798
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Davis GE
中科院分区：
文献类型：
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作者：
Sun Z;Kemp SS;Lin PK;Aguera KN;Davis GE
我们试图确定激活k-Ras突变k-RasV 12的EC表达如何影响它们形成管腔和管的能力，以及在毛细血管组装过程中与周细胞相互作用的能力。使用定义的生物测定，其中人类EC经历可观察到的小管形成，发芽行为，周细胞招募到EC内衬管，周细胞诱导的EC基膜沉积，我们评估了EC k-RasV 12表达对这些关键过程的影响，这些关键过程是正确的毛细血管网络形成所必需的。这种突变，这是经常看到在人类EC脑动静脉畸形，被发现显着加重EC管腔形成机制，强烈加速细胞内空泡形成，空泡融合和管腔扩张，并减少发芽行为，导致过度加宽的管网相比，控制EC。这些异常管表现出强烈的减少周细胞招聘和周细胞诱导的EC基膜相比，对照组，与纤维连接蛋白，IV型胶原蛋白和串珠素沉积的缺陷。对这些k-RasV 12 EC管形成期间的信号传导的分析揭示了Src、Pak 2、b-Raf、Erk和Akt活化的强烈增强以及PKCε、MT 1-MMP、乙酰化微管蛋白和CDCP 1（大多数是已知的EC管腔调节剂）表达的增加。MT 1-MMP、Src、Pak、Raf、Mek激酶、Cdc 42/Rac 1和Notch的药理学阻断显著干扰这些EC的腔和管形成。总的来说，这项新的工作表明，EC表达的k-RasV 12破坏毛细血管组装，由于显着过度的管腔形成加上强烈减少周细胞募集和基底膜沉积，这是关键的致病特征，使血管系统发展动静脉畸形。
We sought to determine how EC expression of the activating k-Ras mutation, k-RasV12, affects their ability to form lumens and tubes and interact with pericytes during capillary assembly. Using defined bioassays where human ECs undergo observable tubulogenesis, sprouting behavior, pericyte recruitment to EC-lined tubes, and pericyte-induced EC basement membrane deposition, we assessed the impact of EC k-RasV12 expression on these critical processes that are necessary for proper capillary network formation. This mutation, which is frequently seen in human ECs within brain arteriovenous malformations, was found to markedly accentuate EC lumen formation mechanisms, with strongly accelerated intracellular vacuole formation, vacuole fusion and lumen expansion, and with reduced sprouting behavior, leading to excessively widened tube networks compared to control ECs. These abnormal tubes demonstrate strong reductions in pericyte recruitment and pericyte-induced EC basement membranes compared to controls, with deficiencies in fibronectin, collagen type IV and perlecan deposition. Analyses of signaling during tube formation from these k-RasV12 ECs reveals strong enhancement of Src, Pak2, b-Raf, Erk and Akt activation and increased expression of PKCε, MT1-MMP, acetylated tubulin and CDCP1 (most are known EC lumen regulators). Pharmacologic blockade of MT1-MMP, Src, Pak, Raf, Mek kinases, Cdc42/Rac1, and Notch markedly interferes with lumen and tube formation from these ECs. Overall, this novel work demonstrates that EC expression of k-RasV12 disrupts capillary assembly due to markedly excessive lumen formation coupled with strongly reduced pericyte recruitment and basement membrane deposition, which are critical pathogenic features predisposing the vasculature to develop arteriovenous malformations.