Neo-Vasculogenesis In Vivo Is Facilitated by Oxygen Sensing Mesenchymal Stem and Pogenitor Cells
Authors: Hofmann Nicole A , Ortner Anna , Jacamo Rodrigo O , Reinisch Andreas , Schallmoser Katharina , Fruewirth Margareta , Diwoky Clemens , Rohban Rokhsareh , Beham-Schmid Christine , Andreeff Michael , Strunk Dirk 
Published: October 2019

Abstract
Abstract 699

Rationale:
Vascular repair after hypoxic tissue damage requires a stringent interaction between somatic endothelial colony-forming progenitor cells (ECFCs) and mesenchymal stem and progenitor cells (MSPCs). Stem cell therapy to re-vascularize ischemic tissue has been a promising tool for various therapeutic targets including stroke, myocardial infarction and peripheral artery disease. Despite promising experimental data, therapeutic approaches employing endothelial progenitor cells have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. Hypoxia in ischemic tissue is an extensively studied key factor that influences pro- and anti-angiogenic treatment by driving the revascularization machinery. We and others have shown that despite hypoxic stimulation, ECFCs in vivo only form patent vessels in the presence of MSPCs. Here we show that MSPCs but not ECFCs are the oxygen sensors enabling vasculogenesis in vivo.

Methods:
Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. In in vitro studies progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen conditions (5% O2) and standard culture conditions (20% O2). ECFC and MSPC interaction in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelete lysate). To investigate the respective roles of MSPCs and ECFCs during vasculogenesis under hypoxia in vivo chemical and genetic inhibitors against protein synthesis (cycloheximide) and HIF-1α (YC-1, shRNA) were employed. Immune histochemistry, immune fluorescence and TUNEL assays were performed on plugs in the time course after transplantation.

Results:
In vitro studies showed that compared to 20% O2, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced at 5% O2, and even more at 1% O2. Standard culture conditions resulted in a shift in the progenitor hierarchy with an augmented number of high proliferative potential (HPP)-ECFC colonies (60±18% of total colonies) as compared to venous oxygen conditions (9±6%) and a complete loss of HPP-ECFC colonies under severe hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair function in scratch assays and the ability to form vascular-like networks in matrigel assays in vitro were diminished with declining oxygen supply. The re-oxygenation to 20% O2 of ECFCs which where precultured at 1% or 5% O2 led to enhanced proliferation, colony size and function. Single cell analysis revealed that ECFCs stabilized hypoxia-inducing factor-1α (HIF-1α) only at 1% O2 while MSPCs stabilize HIF-1α at 1% O2 as well as 5% O2 conditions. In a mouse model, subcutaneously injected ECFCs underwent apoptosis after 24h and attracted mouse leucocytes. In contrast, ECFCs co-implanted in vivo with MSPCs were rescued from apoptotic death and formed perfused human vessels 7 days after transplantation independent of matrix. Perivascular cells, but not ECFCs, were positive for HIF-1α in vivo. Inhibition of MSPCs but not ECFCs protein synthesis and HIF-1α prior to co-implantation blocked vessel formation.

Conclusion:
These data demonstrates that hypoxic ECFCs alone show reduced functuionality in vitro and form patent vessels in vivo. In contrast, MSPCs react to the low oxygen environment more sensitively than ECFCs and promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis. Surprisingly, this study shows that therapeutic vasculogenesis can occur independent of endothelial HIF stabilization and protein synthesis. This data indicate that in addition to their established role regulating hematopoiesis, MSPCs oxygen sensing is crucial during vascular regeneration. This suggests a shift of focus from endothelial cells to perivascular cells as a therapeutic target in regenerative medicine and anti-angiogenic therapy.

Disclosures:
No relevant conflicts of interest to declare.