In vivo perfusion of human skin substitutes with microvessels formed by adult circulating endothelial progenitor cells.

BACKGROUND: At present, tissue-engineered human skin substitutes (HSSs) mainly function as temporary bioactive dressings due to inadequate perfusion. Failure to form functional vascular networks within the initial posttransplantation period compromises cell survival of the graft and its long-term viability in the wound bed. OBJECTIVES: Our goal was to demonstrate that adult circulating endothelial progenitor cells (EPCs) seeded onto HSS can form functional microvessels capable of graft neovascularization and perfusion. MATERIALS AND METHODS: Adult peripheral blood mononuclear cells (PBMCs) underwent CD34 selection and endothelial cell (EC) culture conditions. After in vitro expansion, flow cytometry verified EC phenotype before their incorporation into HSS. After 2 weeks in vivo, immunohistochemical analysis, immunofluorescent microscopy, and microfil polymer perfusion were performed. RESULTS: CD34+ PBMCs differentiated into EPC demonstrating characteristic EC morphology and expression of CD31, Tie-2, and E-selectin after TNFalpha-induction. Numerous human CD31 and Ulex europaeus agglutinin-1 (UEA-1) microvessels within the engineered grafts (HSS/EPCs) inosculated with recipient murine circulation. Limitation of murine CD31 immunoreactivity to HSS margins showed angiogenesis was attributable to human EPC at 2 weeks posttransplantation. Delivery of intravenous rhodamine-conjugated UEA-1 and microfil polymer to HSS/EPCs demonstrated enhanced perfusion by functional microvessels compared to HSS control without EPCs. CONCLUSION: We successfully engineered functional microvessels in HSS by incorporating adult circulating EPCs. This autologous EC source can form vascular conduits enabling perfusion and survival of human bioengineered tissues.

Vascularization and engraftment of a human skin substitute using circulating progenitor cell-derived endothelial cells.

We seeded tissue engineered human skin substitutes with endothelial cells (EC) differentiated in vitro from progenitors from umbilical cord blood (CB-EC) or adult peripheral blood (AB-EC), comparing the results to previous work using cultured human umbilical vein EC (HUVEC) with or without Bcl-2 transduction. Vascularized skin substitutes were prepared by seeding Bcl-2-transduced or nontransduced HUVEC, CB-EC, or AB-EC on the deep surface of decellularized human dermis following keratinocyte coverage of the epidermal surface. These skin substitutes were transplanted onto C.B-17 SCID/beige mice receiving systemic rapamycin or vehicle control and were analyzed 21 d later. CB-EC and Bcl-2-HUVEC formed more human EC-lined vessels than AB-EC or control HUVEC; CB-EC, Bcl-2-HUVEC, and AB-EC but not control HUVEC promoted ingrowth of mouse EC-lined vessels. Bcl-2 transduction increased the number of human and mouse EC-lined vessels in grafts seeded with HUVEC but not with CB-EC or AB-EC. Both CB-EC and AB-EC-induced microvessels became invested by smooth muscle cell-specific alpha-actin-positive mural cells, indicative of maturation. Rapamycin inhibited ingrowth of mouse EC-lined vessels but did not inhibit formation of human EC-lined vessels. We conclude that EC differentiated from circulating progenitors can be utilized to vascularize human skin substitutes even in the setting of compromised host angiogenesis/vasculogenesis.

Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.

Implants of collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVECs) induce the formation of human endothelial cell (EC)/murine vascular smooth muscle cell (VSMC) chimeric vessels in immunodeficient mice. Microfil casting of the vasculature 60 d after implantation reveals highly branched microvascular networks within the implants that connect with and induce remodeling of conduit vessels arising from the abdominal wall circulation. Approximately 85% of vessels within the implants are lined by Bcl-2-positive human ECs expressing VEGFR1, VEGFR2, and Tie-2, but not integrin alpha(v)beta(3). The human ECs are seated on a well formed human laminin/collagen IV-positive basement membrane, and are surrounded by mouse VSMCs expressing SM-alpha actin, SM myosin, SM22alpha, and calponin, all markers of contractile function. Transmission electron microscopy identified well formed EC-EC junctions, chimeric arterioles with concentric layers of contractile VSMC, chimeric capillaries surrounded by pericytes, and chimeric venules. Bcl-2-HUVEC-lined vessels retain 70-kDa FITC-dextran, but not 3-kDa dextran; local histamine rapidly induces leak of 70-kDa FITC-dextran or India ink. As in skin, TNF induces E-selectin and vascular cell adhesion molecule 1 only on venular ECs, whereas intercellular adhesion molecule-1 is up-regulated on all human ECs. Bcl-2-HUVEC implants are able to engraft within and increase perfusion of ischemic mouse gastrocnemius muscle after femoral artery ligation. These studies show that cultured Bcl-2-HUVECs can differentiate into arterial, venular, and capillary-like ECs when implanted in vivo, and induce arteriogenic remodeling of the local mouse vessels. Our results support the utility of differentiated EC transplantation to treat tissue ischemia.

Bcl-2 transduction protects human endothelial cell synthetic microvessel grafts from allogeneic T cells in vivo.

T cell interactions with vascular endothelial cells (EC) are of central importance for immune surveillance of microbes and for pathological processes such as atherosclerosis, allograft rejection, and vasculitis. Animal (especially rodent) models incompletely predict human immune responses, in particular with regard to the immunological functions of EC, and in vitro models may not accurately reflect in vivo findings. In this study, we describe the development of an immunodeficient SCID/bg murine model combining a transplanted human synthetic microvascular bed with adoptive transfer of human T lymphocytes allogeneic to the cells of the graft that more fully recapitulates T cell responses in natural tissues. Using this model, we demonstrate that transduced Bcl-2 protein in the engrafted EC effectively prevents injury even as it enhances T cell graft infiltration and replication.

Calciphylaxis associated with acute, reversible renal failure in the setting of alcoholic cirrhosis.

We describe a case of calciphylaxis in a 47-year-old man with alcohol-induced end-stage liver disease and acute renal failure secondary to hepatorenal syndrome. Possible contributing factors included transiently impaired renal function, protein C and S deficiencies, elevated calcium-phosphate product, hyperphosphatemia, low serum albumin, repeated albumin infusions, and elevated alkaline phosphatase level.

Tissue engineering: creation of long-lasting blood vessels.

The construction of stable blood vessels is a fundamental challenge for tissue engineering in regenerative medicine. Although certain genes can be introduced into vascular cells to enhance their survival and proliferation, these manipulations may be oncogenic. We show here that a network of long-lasting blood vessels can be formed in mice by co-implantation of vascular endothelial cells and mesenchymal precursor cells, by-passing the need for risky genetic manipulations. These networks are stable and functional for one year in vivo.

IL-11 protects human microvascular endothelium from alloinjury in vivo by induction of survivin expression.

IL-11 can reduce tissue injury in animal models of inflammation but the mechanism(s) is unknown. When C.B-17 SCID/beige mice bearing human skin grafts are injected i.p. with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) delays the time course of graft microvessel loss without reducing the extent of T cell infiltration. Protective actions of IL-11 are most pronounced on day 15. IL-11 has no effect on T cell activation marker, effector molecule, cytokine expression, or endothelial ICAM-1 expression. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and endothelial cells. Topical application of survivin antisense oligonucleotide down-regulates survivin expression in both cell types and largely abrogates the protective effect of IL-11. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than anti-inflammatory or immunomodulatory effect mediated through induction of survivin.

Interferon-gamma plays a nonredundant role in mediating T cell-dependent outward vascular remodeling of allogeneic human coronary arteries.

Vascular remodeling (change in vessel diameter) rather than intimal hyperplasia is the most important predictor of luminal loss in immune-mediated arterial injury, yet little is known about its mechanisms. Here, we show that outward vascular remodeling and intimal thickening, two manifestations of arteriosclerosis with opposing effects on luminal size, result from immune effector mechanisms that are T-cell dependent and interferon (IFN)-gamma mediated. In our in vivo model of human coronary artery injury by allogeneic peripheral blood mononuclear cells, both processes occur concurrently and are characterized by T-cell infiltrates with a predominantly IFN-gamma-producing cytokine profile. Neutralization of IFN-gamma inhibits the arterial and intimal expansion, whereas administration of IFN-gamma enhances these effects. The nonredundant role of IFN-gamma in T-cell-dependent remodeling of human coronary arteries demonstrated here presents a new therapeutic target for preservation of vessel lumen in arteriosclerosis.

Engraftment of a vascularized human skin equivalent.

Clinical performance of currently available human skin equivalents is limited by failure to develop perfusion. To address this problem we have developed a method of endothelial cell transplantation that promotes vascularization of human skin equivalents in vivo. Enhancement of vascularization by Bcl-2 overexpression was demonstrated by seeding human acellular dermis grafts with human umbilical vein endothelial cells (HUVEC) transduced with the survival gene Bcl-2 or an EGFP control transgene, and subcutaneous implantation in immunodeficient mice (n=18). After 1 month the grafts with Bcl-2-transduced cells contained a significantly greater density of perfused HUVEC-lined microvessels (55.0/mm3) than controls (25.4/mm3,P=0.026). Vascularized skin equivalents were then constructed by sequentially seeding the apical and basal surfaces of acellular dermis with cultured human keratinocytes and Bcl-2-transduced HUVEC, respectively. Two weeks after orthotopic implantation onto mice, 75% of grafts (n=16) displayed both a differentiated human epidermis and perfusion through HUVEC-lined microvessels. These vessels, which showed evidence of progressive maturation, accelerated the rate of graft vascularization. Successful transplantation of such vascularized human skin equivalents should enhance clinical utility, especially in recipients with impaired angiogenesis.

Immunopathology of human T cell responses to skin, artery and endothelial cell grafts in the human peripheral blood lymphocyte/severe combined immunodeficient mouse.

Blood vessels and their endothelial lining are major stimulators and targets of the rejection response. The immunological properties of human endothelial cells differ significantly from those of other species and new models are needed for proper study of human vessels in the transplant setting. We have employed the human peripheral blood lymphocyte/severe combined immunodeficiency (huPBL/SCID) mouse for this purpose. We describe here our results involving transplantation of human skin, human artery and cultured human endothelial cells. We also describe our more limited experience using porcine skin and artery transplantation to study human T cells responses to pig endothelium.