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,,1,
,,2,,3
* Department of Pathology,
Department of Dermatology,
Department of Immunobiology, and
Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, Connecticut, USA
2Correspondence: Yale University School of Medicine, 295 Congress Ave., Boyer Center for Molecular Medicine Rm. 454, New Haven, CT 06510, USA. E-mail: jordan.pober{at}yale.edu
SPECIFIC AIMS
The success of wound healing following engraftment of a commercial skin substitute (Apligraf) is currently limited by graft ischemic loss due to inadequate vascularization of the implanted tissue, a problem compounded by the impaired angiogenesis and vasculogenesis that are commonly seen in patients with nonhealing skin ulcers. Here we examine (a) the ability of human endothelial cells (EC), differentiated and expanded from circulating endothelial progenitor cells in human cord blood (CB) or in healthy volunteer donor adult blood (AB), to promote the vascularization of skin substitutes following implantation onto immunodeficient mice; (b) the effects of transducing CB-EC or AB-EC with Bcl-2 on the process of vascularization, a manipulation that improves vessel development induced by human umbilical vein EC (HUVEC); and (c) the effects of rapamycin treatment of the recipient animal on skin substitute vascularization, an agent that impairs angiogenesis and vasculogenesis.
PRINCIPAL FINDINGS
1. Both CB-derived and AB-derived EPC can routinely give rise to well differentiated EC
For CB, this simply involves plating of mononuclear cells under culture conditions [EGM-2 medium supplemented with vascular endothelial growth factor (VEGF)] that favor differentiation and outgrowth of EC. For healthy adult volunteer donors, blood mononuclear cells must be concentrated by leukapheresis and progenitors enriched by immunoselection for CD34 prior to culture. CB-EC colonies are detectable within 5–7 d, and AB-EC colonies appear between 21 and 35 d. Once differentiated, both cell types grow to form confluent monolayers of cobblestone-shaped cells expressing VE-cadherin at the cell junctions and vWF within cytoplasmic granules. Both EC types express CD34, VEGFR2, CD31, binding sites for Ulex Europeus agglutinin I, and TNF-inducible E-selectin (CD62E) on their surface but lack CD133, CD45, CD18, and CD14. Both cell types appear capable of multiple rounds of cell division and expansion. These markers and behaviors are consistent with well-differentiated EC, derived from true EPC, and are not consistent with monocytes that have acquired EC markers in response to culture with VEGF.
2. Seeding of tissue engineered human skin substitutes with CB-EC or AB-EC leads to development of human and mouse EC-lined vessels within engrafted constructs
Human CB-EC or AB-EC were used to seed the deep surface of decellularized human dermis that had been re-epithelialized with a human keratinocyte multilayer derived from neonatal foreskin, as this laboratory has previously described using HUVEC. Human skin substitutes were transplanted onto the backs of C.B-17-SCID/Bg mice for 3 wk, at which time the mice were euthanized and the grafts were analyzed by routine histology and immunostaining. Skin substitutes constructed with keratinocytes, but without EC-seeding, developed a very limited microvascular network, which was limited to host-derived vessels found largely at the graft periphery (Fig 1
A). By contrast to keratinocyte-only controls, human skin substitutes seeded with either CB-EC or AB-EC contained both mouse EC-lined and human EC-lined microvessels (Fig. 1D, E
). CB-EC formed more vessels than did AB-EC at the three week time point (Fig. 2
). Although HUVEC also formed human EC-lined microvessels at levels comparable to that seen with AB-EC, HUVEC-seeded skin substitutes showed many fewer mouse EC-lined vessels than did constructs seeded with CB-EC or AB-EC. The presence of smooth muscle cell-specific alpha actin-positive cells was observed to coat the EC-lined microvessels throughout human skin substitutes containing CB-EC and AB-EC, a sign of microvascular maturation (Fig. 1f
).
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3. Overexpression of Bcl-2 had no effect on the number of human EC-lined vessels found in human skin substitutes seeded with CB-EC or AB-EC
In the experiments described here, and as previously reported by our laboratory, seeding of human skin substitutes with HUVEC that had been transduced with a retrovirus expressing a caspase-resistant Bcl-2 enhanced the number of human EC-lined vessels found in the skin substitutes, compared with nontransduced controls (Fig. 2A
). Further, increased numbers of mouse EC-lined microvessels were observed in skin substitutes seeded with Bcl-2-transduced HUVEC, compared with nontransduced HUVEC controls (Fig. 2d
). Bcl-2 transduction had no effect, however, on the number of human or mouse EC-lined vessels found in human skin substitutes seeded with CB-EC or AB-EC (Fig. 2B, C, E, F
). Overall, the number of human EC-lined vessels found in skin substitutes seeded with CB-EC was approximately equal to, if not greater than, the number of human EC-lined vessels found in skin substitutes seeded with Bcl-2-transduced HUVEC. Bcl-2 transduction also did not increase the extent of microvessel coverage by smooth muscle cell-specific alpha-actin-expressing cells.
4. Rapamycin inhibits host-dependent vascularization but does not inhibit the formation of human EC-lined vessels
Five days after transplantation of the tissue-engineered skin substitutes, animals were either treated with the antiproliferative, immunosuppressive agent rapamycin, or vehicle control. Rapamycin, which inhibits cell division, impairs wound healing and angiogenesis. Systemic administration of rapamycin had no effect on the number of human EC-lined microvessels found within the engrafted human skin substitutes seeded with CB-EC, AB-EC, or HUVEC, with or without transduced Bcl-2 (Fig. 2A-C
). The number of mouse EC-lined microvessels present within the human skin substitutes was significantly reduced, however, when compared to vehicle-treated controls (Fig. 2D-F
). In the case of synthetic skin substitutes formed without EC seeding, rapamycin treatment essentially prevented revascularization and led, in many animals to graft necrosis and sloughing. Graft injury was not increased in constructs containing human EC.
CONCLUSIONS AND SIGNIFICANCE
EC, differentiated and expanded from circulating endothelial progenitor cells, can be used to promote vascularization of tissue engineered human skin substitutes. Our strategy and the outcomes reported here are schematized in Fig. 3
. Within the transplanted skin substitute there is development of microvessels from both the implanted human EC and from angiogenesis/vasculogenesis of the recipient host tissue. Unlike the case of HUVEC, Bcl-2 transduction does not increase the potential of CB-EC or AB-EC to form human EC-lined vessels or to promote the invasion of the graft by mouse EC-lined vessels. Most significantly, the development of vessels in tissue engineered skin substitutes containing human EC is not prevented in a setting of impaired host angiogenesis/vasculogenesis, suggesting clinical utility of our approach for patients with nonhealing skin ulcers.
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FOOTNOTES
1 Current address: Southern Illinois University School of Medicine, Department of Internal Medicine, Springfield, Illinois, USA. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5682fje
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