Jeffrey S. Schechner

Interferon-gamma elicits arteriosclerosis in the absence of leukocytes.

Atherosclerosis and post-transplant graft arteriosclerosis are both characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the proliferation of vascular smooth muscle cells (VSMCs) and the accumulation of extracellular matrix. They are also associated with the presence of the immunomodulatory cytokine interferon-γ (IFN-γ). Moreover, in mouse models of atheroma formation or allogeneic transplantation, the serological neutralization or genetic absence of IFN-γ markedly reduces the extent of intimal expansion. However, other studies have found that exogenous IFN-γ inhibits cultured VSMC proliferation and matrix synthesis, and reduces intimal expansion in response to mechanical injury. This discrepancy is generally explained by the idea that IFN-γ either directly activates macrophages, or, by increasing antigen presentation, indirectly activates T cells within the lesions of atherosclerosis and graft arteriosclerosis. These activated leukocytes are thought to express the VSMC-activating cytokines and cell-surface molecules that cause the observed arteriosclerotic responses. Here we have inserted pig and human arteries into the aorta of immunodeficient mice, and we show that IFN-γ can induce arteriosclerotic changes in the absence of detectable immunocytes by acting on VSMCs to potentiate growth-factor-induced mitogenesis.

Control of Apoptosis during Angiogenesis by Survivin Expression in Endothelial Cells

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Induction of human tumor-loaded dendritic cells

A preferred anti‐cancer vaccine would be tumor‐specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumors distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte‐to‐dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor‐loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

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 nontrans‐duced 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.—Shepherd, B. R., Enis, D. R., Wang, F., Suarez, Y., Pober, J. S., Schechner, J. S. Vascularization and engraftment of a human skin substitute using circulating progenitor cell‐derived endo‐thelial cells. FASEB J. 20, E1124–E1132 (2006)

Human Endothelial Cell Presentation of Antigen and the Homing of Memory/Effector T Cells to Skin

Abstract: Dermal microvascular endothelial cells (ECs) form a continuous lining that normally bars blood‐borne T lymphocytes from entering the skin, but as part of the response to foreign antigen, dermal ECs undergo alterations in their surface proteins so as to provide signals to circulating T cells that lead to their activation and recruitment. Several observations suggest that human dermal microvascular ECs may help initiate cutaneous immune reactions by presentation of cognate antigens to circulating T memory cells: (1) antigen‐specific inflammatory responses in the skin, as in other organs, involve accumulation of memory and effector T cell populations that are enriched in cells specific for the eliciting antigen; (2) recall responses to intradermal protein antigens in the skin start very rapidly within two hours of challenge; (3) dermal microvascular ECs in humans and other large mammals basally display high levels of class I and class II MHC molecules, the only known purpose of which is to present antigenic peptides to lymphocytes; (4) the lumen of dermal capillaries are narrower than the diameter of circulating T cells, ensuring surface contact; and (5) cultured human ECs effectively present antigens to resting memory T cells isolated from the circulation. Upon contact with activated T cells or their secreted products (cytokines), dermal ECs themselves become activated, increasing their capacity to recruit memory and effector T cell populations in an antigen‐independent manner. Specifically, activated ECs express inducible leukocyte adhesion molecules such as E‐selectin, ICAM‐1, and VCAM‐1; and several lines of evidence, including neutralizing antibody experiments and gene knockouts, have supported a role of these molecules in T cell recruitment. Dermal ECs have unique expression patterns of adhesion molecules that can determine the subsets of memory T cells that are recruited into the skin. For example, slow internalization of E‐selectin allows more persistent expression of this protein on the surface of dermal ECs, favoring interactions with CLA‐1+ T cells. VCAM‐1 expression, normally confined to venular EC may extend to capillaries within the dermal papillae and contribute to epidermal inflammation, recruiting α4β7 integrin‐expressing T cells that also express the cadherin‐binding integrin αEβ7. New models involving transplantation of normal and genetically modified human dermal ECs into immunodeficient mice may be used to further explore these properties.

Engraftment of a vascularized human skin equivalent

Clinical performance of currently avail able 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 equiva lents in vivo. Enhancement of vascularization by Bcl‐2 overexpression was demonstrated by seeding human acellular dermis grafts with human umbilical vein en dothelial cells (HUVEC) transduced with the survival gene Bcl‐2 or an EGFP control transgene, and subcuta neous 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 api cal 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, acceler ated the rate of graft vascularization. Successful trans plantation of such vascularized human skin equivalents should enhance clinical utility, especially in recipients with impaired angiogenesis.—Schechner, J. S., Crane, S. K., Wang, F., Szeglin, A. M., Tellides, G., Lorber, M. I., Bothwell, A. L. M., Pober, J. S. Engraftment of a vascularized human skin equivalent. FASEB J. 17, 2250‐2256 (2003)

Cytoprotection of Human Umbilical Vein Endothelial Cells Against Apoptosis and CTL-Mediated Lysis Provided by Caspase-Resistant Bcl-2 Without Alterations in Growth or Activation Responses

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-κB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Interferon-γ 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)‐γ 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‐γ‐producing cytokine profile. Neutralization of IFN‐γ inhibits the arterial and intimal expansion, whereas administration of IFN‐γ enhances these effects. The nonredundant role of IFN‐γ in T‐cell‐dependent remodeling of human coronary arteries demonstrated here presents a new therapeutic target for preservation of vessel lumen in arteriosclerosis.

HUMAN ALLOGENEIC VASCULAR REJECTION AFTER ARTERIAL TRANSPLANTATION AND PERIPHERAL LYMPHOID RECONSTITUTION IN SEVERE COMBINED IMMUNODEFICIENT MICE1

BACKGROUND Interspecies differences create important shortcomings in existing animal models used to describe in vivo events responsible for allograft rejection. Alloimmune destruction of human dermal microvessels, histologically consistent with rejection, has been demonstrated in human skin-grafted severe combined immunodeficient (SCID) mice receiving allogeneic human peripheral blood mononuclear cells (PBMC). We have now documented human alloimmune injury in a vascularized, SCID-human arterial transplantation model. METHODS Fresh human artery was used to replace the CB.17 SCID/beige mouse infrarenal aorta. Seven days later, 3x10(8) human PBMC were administered intraperitoneally, and lymphocyte engraftment was considered successful when circulating human CD3+ cells were later identified in peripheral blood. RESULTS Forty-six of 49 (94%) mice undergoing transplantation survived, including 14 controls with arterial grafts receiving no PBMC. Twenty-eight of 32 mice demonstrated circulating human CD3+ cells, 14 days after PBMC administration. Animals were killed at 14, 21, or 28 days after receiving allogeneic PBMC, and arteries were recovered for histology and immunohistology. All 14 control mice had patent transplanted grafts with normal vascular histology and no lymphoid infiltration. Damage to transplanted arteries among lymphocyte-engrafted mice was apparent by 14 and 21 days in some animals, whereas 16 of 22 exhibited moderate to severe intimal, medial, and/or adventitial lymphocytic infiltration with intimal expansion by day 28. The infiltrate consisted of HLA-A, -B, -C+, and -DR+, human CD3+ cells, approximately equally distributed as CD4+ and CD8+ subsets. Some infiltrating lymphocytes were cytolytic cells as demonstrated by perforin staining. The endothelium of transplanted human arteries exhibited endothelialitis, and the endothelial cells stained intensely with anti-HLA-A, -B, -C and anti-HLA-DR antibodies. The expanded intima was predominantly smooth muscle cells, staining positively for smooth muscle alpha-actin, HLA-A, -B, -C and HLA-DR. Medial necrosis was not observed. CONCLUSION The results provide evidence of alloimmune-mediated vascular rejection in this human arterial transplantation model.

Dermal Microvascular Injury in the Human Peripheral Blood Lymphocyte Reconstituted-Severe Combined Immunodeficient (HuPBL-SCID) Mouse/Skin Allograft Model Is T Cell Mediated and Inhibited by a Combination of Cyclosporine and Rapamycin

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.