Abstract
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.
Highlights
De-novo vessel formation is an essential step in organ regeneration as well as pathological manifestations such as ischemia and tumorigenesis which is mainly realized by sprouting angiogenesis in adult life [1].The formation of patent neo-vascular structures by stem/progenitor cells is termed neo-vasculogenesis and requires migration of transplanted or circulating endothelial lineage cells assembling the integral lining of newly formed vessels and mesenchymal cell-derived pericytes establishing and maintaining neo-vessel stability [1,2,3]
Subcutaneous implantation of endothelial colony-forming progenitor cells (ECFCs) or mesenchymal stem/progenitor cells (MSPCs) or co-transplantation of both cell types in an established 80:20 ratio [9] was performed to study the mechanism by which these two Umbilical cord (UC)-derived mesodermal cell types contribute to neo-vasculogenesis in vivo (Figure 1)
The plugs containing a combination of MSPC+ECFC in vivo became vascularized after 2 weeks, indicating a supportive role for MSPCs acting as pericytes to stabilize the established micro-vessels as shown previously [9]
Summary
De-novo vessel formation is an essential step in organ regeneration as well as pathological manifestations such as ischemia and tumorigenesis which is mainly realized by sprouting angiogenesis in adult life [1].The formation of patent neo-vascular structures by stem/progenitor cells is termed neo-vasculogenesis and requires migration of transplanted or circulating endothelial lineage cells assembling the integral lining of newly formed vessels and mesenchymal cell-derived pericytes establishing and maintaining neo-vessel stability [1,2,3]. We used an established neo-vasculogenesis model [7,8,9] to test our hypothesis of whether proteomic profiling can provide us with information on the in vivo signaling signature early during neo-vasculogenesis. The model was selected based on our hypothesis that human cell transplantation may allow for the recovery of a signaling signature derived from the transplanted cells. Anti-human antibodies used for array profiling were used to restrict the signature information to events related to transplanted cell-derived vasculogenesis. We show that caspase-4 inhibition can block vasculogenesis in vivo, suggesting a role for caspase-4 activation in endothelial cell arrangement during vascular-like network formation in vitro as well as during experimental therapeutic neo-vasculogenesis in vivo
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