(775c) Directing Vascular Regeneration in-Situ

Recently our group demonstrated
that immobilized VEGF can capture circulating endothelial cells from the blood in-vitro.
Furthermore, we have demonstrated proof of concept by implanting a-cellular
tissue engineered vessels (A-TEVs) comprised of SIS immobilized with heparin
and vascular endothelial growth factor (VEGF) into the arterial system of sheep
which remained patent (92%, n=12) for 3mo (Fig.1). Upon analysis, the lumen of
these grafts was comprised of a fully functional endothelium as early as 1mo post
implantation. This study sought to identify the type of cells that are captured
by VEGF on the lumen of A-TEVs in-vivo and understand how these cells
turn into an endothelial (EC) monolayer that is capable of maintaining patency in-vivo.
A-TEV implantations were performed as previously published. In-vivo
Analysis: Fixed explants of 1wk, 1mo, 3mo, and 6mo VEGF functionalized
A-TEVs are assessed via IHC for MC and EC markers. In-vitro Capture under
Flow: Capture of VEGFR expressing cells from blood under flow will be assessed
in a microfluidic device with a flat surface comprised of chitosan, heparin,
and VEGF (CHV) as previously published. In-vitro differentiation: Blood
borne mononuclear cells that are captured on surface immobilized VEGF are
coaxed to differentiate into EC with a combination of soluble and biophysical
signals. A-TEVs were implanted as interpositional grafts into the arterial
circulation of an ovine animal model. As early as 1mo post-implantation, the
graft lumen was fully endothelialized as shown by IHC for EC markers, CD144 and
eNOS. At the same time, luminal cells co-expressed leukocyte markers CD14 and
CD163 (Fig. 3). To understand these results, we performed cell capture
experiments under flow using microfluidic devices. Interestingly, blood
mononuclear cells expressing high levels of VEGF receptors were captured on CHV
surfaces with high specificity under a range of shear stresses. Initially,
these cells expressed high levels of CD14 and CD16. Under the right conditions
they were coaxed to differentiate into an EC phenotype as shown by expression
of CD144 and eNOS (Fig.2), additional IC analysis, WB, qRT-PCR, and flow
cytometry. We will also discuss the role of soluble signals and biophysical
forces in transdifferentiation of blood cells into EC that maintain graft
patency. We demonstrate the ability of VEGF functionalized surfaces to capture cells
directly from the blood in-vitro and in vivo. In the presence of
the right biochemical and biophysical signals these cells differentiate into EC
like cells that maintain graft patency and vascular function. Our results shed
light into the process of vascular tissue regeneration in situ using the
body’s regenerative capacity.