Department of Pathology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS One. 2010 Oct 25;5(10):e13612. doi: 10.1371/journal.pone.0013612.
Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.
METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.
CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.
新血管生成需要发芽的内皮细胞（EC）整合形成新的血管网络。然而，血管内皮生长因子-A（VEGF）诱导的病理性血管生成中，血管结构缺陷，有盲端的血管整合不良，这限制了 VEGF 在治疗性血管生成中的应用，并加重了缺血相关的病变。在这里，我们研究了过度活跃的钙蛋白酶活性是否导致了异常的 VEGF 新生血管结构和整合。钙蛋白酶是一种细胞内钙依赖性、非溶酶体半胱氨酸蛋白酶家族，通过对许多底物的蛋白水解来调节细胞功能。
方法/主要发现：在 VEGF 驱动的血管生成的小鼠皮肤模型中，用显性负性（DN）钙蛋白酶-I 逆转录病毒转导促进了新生血管的整合和管腔形成，减少了盲端，并改善了血管灌注。中等剂量的钙蛋白酶抑制剂-I 对 VEGF 驱动的血管生成的改善作用类似于 DN 钙蛋白酶-I。相反，用野生型（WT）钙蛋白酶-I 逆转录病毒转导则消除了新生血管的整合和管腔形成。在体外，用 DN 钙蛋白酶-I 或钙蛋白酶抑制剂-I 适度抑制钙蛋白酶活性可增加内皮细胞（EC）中的微管稳定蛋白 tau，增加微管的平均长度，增加肌动蛋白索的长度，并增加血管索的连通性。相反，WT 钙蛋白酶-I 则减少了 tau，使微管崩溃，破坏了肌动蛋白索，并抑制了索网络的整合。与微管对于血管网络整合的重要性一致，微管稳定剂紫杉醇支持血管索的整合，而用诺考达唑溶解微管则使索网络崩溃。
结论/意义：这些发现表明，VEGF 诱导的钙蛋白酶活性和细胞骨架动力学的损伤，导致了 VEGF 诱导的新生血管无法正常形成和整合。因此，钙蛋白酶是纠正与病理性血管生成相关的关键血管缺陷和改善 VEGF 治疗性血管生成的重要靶点。
