In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse

Abstract

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Figure 1

The behavior of HUVEC in 3D-gel culture and in synthetic vascular beds in vivo. (a) Phase-contrast microscopy of HUVEC in 3D-gel culture 24 h after suspension in a collagen fibronectin gel (×400). (b) Electron microscopy (EM) of these constructs showing the HUVEC form aggregates with a lumen-like (L, lumen) configuration cleared of matrix proteins (×10,000). (c) EM of another field of the same construct containing a cell undergoing apoptosis as demonstrated by condensation of the nucleus (×10,000). (d) HUVEC construct harvested 31 days after implantation into a SCID/beige mouse [hematoxylin/eosin (H + E) stain, ×400]. (e) UEA-1 reactivity is seen on the cells lining the vascular spaces (×200). (f) Mock construct in which HUVEC were not included in the gel (H + E stain, ×200). (g) Anti-murine CD31 reactivity (red) limited to the edges of the specimen shown in f (arrows, ×400). All magnifications are reported as original magnification before photographic enlargement.

Figure 2

The behavior of retrovirally transduced HUVEC in vitro. H + E staining of (a) EGFP- and (b) Bcl-2-transduced HUVEC, 24 h after suspension in 3D-gel culture (×200). (c) Intrinsic fluorescence of an EGFP-transduced [and (Inset) absence of signal from Bcl-2-transduced] HUVEC construct (×100). (d) Anti-Bcl-2 antibody staining of the Bcl-2-transduced [brown and (Inset) EGFP-transduced] construct at this same time point (×200). (e and f) Phase-contrast microscopy of the EGFP- (e) and Bcl-2- (f) transduced HUVEC maintained in 3D-gel culture for 36 h (×400). After 7 days in 3D-gel culture, there are no detectable viable EGFP-transduced cells (g), whereas those transduced with Bcl-2 are still organized into cords (h) (×400).

Figure 3

The behavior of retrovirally transduced HUVEC in vivo. Histology of (a) Bcl-2- and (b) EGFP-transduced HUVEC constructs harvested 31 days after implantation into a SCID/beige mouse (×1,000). UEA-1 staining (brown) of the constructs in a (c) and b (d) (×200). (e) Anti-Bcl-2 staining (brown) of a Bcl-2-transduced construct 31 days after implantation into a SCID/beige mouse (×1,000). (f) Fluorescence of the EGFP-transduced HUVEC constructs in vivo (×400). (g) Anti-murine CD31 staining (red) of a Bcl-2-transduced HUVEC construct in vivo, showing the restriction of mouse microvessels to the edge of the construct (arrow, ×400).

Figure 4

Analysis of complex vascular structures. Double immunostaining of frozen sections for UEA-1 (red) and smooth-muscle α-actin (blue) in (a) BCL-2- and (b) EGFP-transduced constructs harvested from mice 31 days after implantation (×200). (c) UEA-1 staining (Inset, anti-human CD31, both shown in brown) of a larger vessel from a Bcl-2-transduced construct 31 days after implantation (×400). (d) Smooth-muscle α-actin staining (brown) of this same construct (×400). (e) Histology of a Bcl-2-transduced construct harvested 60 days after implantation. (f) UEA-1 staining (brown) of this same construct (▸, arteriole-like structure; ◃, venule-like structure; *, capillary-like structure; ×400).

Figure 5

Electron microscopy of constructs 31 days after implantation into SCID/beige mice. (a) Untransduced HUVEC have formed perfused vessel-like structures that have inosculated with the mouse circulation, as demonstrated by the presence of erythrocytes within the lumen. The vessel has a single endothelial layer surrounded by matrix. (b) The Bcl-2-transduced HUVEC have formed more complex vessels that are now comprised of the EC layer surrounded by a second layer representing a pericyte/smooth-muscle cell. (c) This vessel, formed from Bcl-2-transduced HUVEC, shows an even more complex structure with an endothelial layer surrounded by several layers of investing cells, mimicking the anatomy of a postcapillary venule. (RBC, erythrocytes; *, investing cell; EC, endothelial cell; ×10,000).