Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.

体内治疗性新血管生成（therapeutic neo-vasculogenesis）可通过人类内皮集落形成祖细胞（human endothelial colony-forming progenitor cells, ECFCs）与间充质干/祖细胞（mesenchymal stem/progenitor cells, MSPCs）联合移植实现。其具体分子机制尚未完全阐明，这一短板阻碍了新型干细胞疗法的开发。本研究推测，可通过蛋白质组学分析（proteomic profiling）获取人类新血管生成早期阶段的体内信号特征。因此，研究团队将ECFCs与MSPCs单独或联合皮下移植至免疫缺陷小鼠体内。通过抗体芯片蛋白质组学分析（antibody microarray proteomic profiling），对移植后24小时内的早期细胞信号通路进行检测分析。同时通过平行移植实验，验证了血管形成与维持能力可长达24周。蛋白质组学分析显示，包括半胱天冬酶（caspases）、钙/钙调蛋白依赖性蛋白激酶、DNA依赖性蛋白激酶（DNA protein kinase）、人类ErbB2受体酪氨酸激酶（human ErbB2 receptor-tyrosine kinase）以及丝裂原活化蛋白激酶（mitogen-activated protein kinases）在内的多种调控分子发生了显著变化。研究从芯片结果中筛选出半胱天冬酶-4（Caspase-4）作为潜在治疗靶点，可在体外调控血管网络形成，同时在体内调节治疗性新血管生成。作为原理验证实验，阻断Caspase-4及广谱半胱天冬酶后，体外内皮网络形成能力显著减弱，体内治疗性新血管生成能力也明显降低。离体蛋白质组学分析由此揭示了一套信号特征，可用于筛选调控体内新血管生成的治疗靶点。