From the Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden (M.C., B.L., K.A., A.Á.-A., A.A.T., E.D., C. Betsholtz, K.G.).
Biotech Research and Innovation Center, University of Copenhagen, Denmark (C. Brakebusch).
Circ Res. 2019 Apr 12;124(8):1240-1252. doi: 10.1161/CIRCRESAHA.118.314300.
Aberrant formation of blood vessels precedes a broad spectrum of vascular complications; however, the cellular and molecular events governing vascular malformations are not yet fully understood.
Here, we investigated the role of CDC42 (cell division cycle 42) during vascular morphogenesis and its relative importance for the development of cerebrovascular malformations.
To avoid secondary systemic effects often associated with embryonic gene deletion, we generated an endothelial-specific and inducible knockout approach to study postnatal vascularization of the mouse brain. Postnatal endothelial-specific deletion of Cdc42 elicits cerebrovascular malformations reminiscent of cerebral cavernous malformations (CCMs). At the cellular level, loss of CDC42 function in brain endothelial cells (ECs) impairs their sprouting, branching morphogenesis, axial polarity, and normal dispersion within the brain tissue. Disruption of CDC42 does not alter EC proliferation, but malformations occur where EC proliferation is the most pronounced during brain development-the postnatal cerebellum-indicating that a high, naturally occurring EC proliferation provides a permissive state for the appearance of these malformations. Mechanistically, CDC42 depletion in ECs elicited increased MEKK3 (mitogen-activated protein kinase kinase kinase 3)-MEK5 (mitogen-activated protein kinase kinase 5)-ERK5 (extracellular signal-regulated kinase 5) signaling and consequent detrimental overexpression of KLF (Kruppel-like factor) 2 and KLF4, recapitulating the hallmark mechanism for CCM pathogenesis. Through genetic approaches, we demonstrate that the coinactivation of Klf4 reduces the severity of vascular malformations in Cdc42 mutant mice. Moreover, we show that CDC42 interacts with CCMs and that CCM3 promotes CDC42 activity in ECs.
We show that endothelial-specific deletion of Cdc42 elicits CCM-like cerebrovascular malformations and that CDC42 is engaged in the CCM signaling network to restrain the MEKK3-MEK5-ERK5-KLF2/4 pathway.
血管异常形成先于广泛的血管并发症；然而，调控血管畸形的细胞和分子事件尚不完全清楚。
本研究旨在探讨 CDC42（细胞分裂周期 42）在血管形态发生中的作用及其对脑血管畸形发生的相对重要性。
为避免胚胎基因缺失常伴有的继发性全身效应，我们构建了一种内皮细胞特异性和诱导型基因敲除方法，以研究小鼠脑的血管新生。新生期内皮细胞特异性敲除 Cdc42 可引发类似脑动静脉畸形（CAVM）的脑血管畸形。在细胞水平，CDC42 功能缺失会损害脑内皮细胞（EC）的出芽、分支形态发生、轴向极性和在脑组织中的正常弥散。CDC42 的缺失并不改变 EC 的增殖，但这些畸形出现在脑发育过程中 EC 增殖最显著的部位——新生小脑，这表明高自然发生的 EC 增殖为这些畸形的出现提供了一种许可状态。在机制上，EC 中 CDC42 的耗竭会引起 MEKK3（丝裂原激活蛋白激酶激酶激酶 3）-MEK5（丝裂原激活蛋白激酶激酶 5）-ERK5（细胞外信号调节激酶 5）信号的增加，继而导致 KLF（Kruppel 样因子）2 和 KLF4 的过度表达，这再现了 CAVM 发病机制的标志性机制。通过遗传方法，我们证明 Klf4 的共缺失可降低 Cdc42 突变小鼠中血管畸形的严重程度。此外，我们表明 CDC42 与 CAVM 相互作用，并且 CCM3 在 EC 中促进 CDC42 活性。
本研究表明，内皮细胞特异性敲除 Cdc42 可引发类似 CAVM 的脑血管畸形，并且 CDC42 参与了 CCM 信号网络，以抑制 MEKK3-MEK5-ERK5-KLF2/4 通路。
