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

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.

Crosstalk between Stem and Progenitor Cellular Mediators with Special Emphasis on Vasculogenesis

“…In a recent study, subcutaneous co-transplantation of ECFC and MSPC in immune deficient mice resulted in establishment of perfused microvessels two weeks after transplantation. The microvessels remained stable and functional even after 6 months in vivo [48].…”

“…MSPCs can differentiate into pericytes [58,59] wrapping around blood vessels in order to support their structure and provide stability [14,43,48]. MSPCs home to the outer wall of the microvessels and arteries in many organs, including spleen, liver, kidney, lung, pancreas, and brain [40,60].…”

“…[61,62]. The potential of tissue-specific endothelial colony forming progenitor cells in contributing to vasculogenesis has been identified in a number of studies [48,63,64].…”

“…Several signaling pathways have been identified to regulate endothelial progenitor-mesenchymal stem and progenitor crosstalk during vasculogenesis such as calcium calmodulin and focal adhesion protein kinase signaling pathway [48] whereas other signaling events have been studied through which hematopoietic stem and endothelial progenitor cells communicate within the vascular microenvironment e.g. SDF-1 (CXCL12)/CXCR4 signaling [77][78][79][80], vascular endothelial growth factor signaling (VEGF) [81][82][83][84], Tie2/ Ang-1 signaling [85,86] , hedgehog [87][88][89] and Notch signaling [90,91], as well as Wingless (Wnt) signaling [92][93][94][95].…”

Transplantation of Endothelial Progenitor Cells Solely Leads to Development of Functional Neo-vesselsin vivo

“…In development phase of embryo, vessel formation occurs in alignment with re-arrangement circumstances through which the development of new vasculatures and pruning of others takes place (Risau and Flamme, 1995). Vessel formation takes place presumably upon the interaction of cellular secreted factors including various signaling components (Dasari et al, 2010, Dumont et al, 1992, Rohban et al, 2013. We used an established vessel formation model (Au et al, 2008, Mead et al, 2008 to test our hypothesis of whether neo-vasculogenesis requires the combination of ECFC and MSPC or can be performed only through ECFC transplantation.…”