Benjamin R. Shepherd

Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence

Prohibitin 1 (PHB1) is a highly conserved protein that is mainly localized to the inner mitochondrial membrane and has been implicated in regulating mitochondrial function in yeast. Because mitochondria are emerging as an important regulator of vascular homeostasis, we examined PHB1 function in endothelial cells. PHB1 is highly expressed in the vascular system and knockdown of PHB1 in endothelial cells increases mitochondrial production of reactive oxygen species via inhibition of complex I, which results in cellular senescence. As a direct consequence, both Akt and Rac1 are hyperactivated, leading to cytoskeletal rearrangements and decreased endothelial cell motility, e.g., migration and tube formation. This is also reflected in an in vivo angiogenesis assay, where silencing of PHB1 blocks the formation of functional blood vessels. Collectively, our results provide evidence that PHB1 is important for mitochondrial function and prevents reactive oxygen species–induced senescence and thereby maintains the angiogenic capacity of endothelial cells.

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 Effector Memory CD4 + T Cells Directly Recognize Allogeneic Endothelial Cells In Vitro and In Vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (TEM) and central memory (TCM) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ TEM (CD45RO+CCR7−CD62L−) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ TCM (CD45RO+CCR7+CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ TEM secrete more IFN-γ within 48 h in response to allogeneic ECs than do TCM. In contrast, TEM and TCM secrete comparable amounts of IFN-γ in response to allogeneic monocytes (Mo). In the same cultures, both TEM and TCM produce IL-2 and proliferate in response to IFN-γ-treated allogeneic human EC or Mo, but TCM respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-γ secretion by CD4+ TEM cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-γ production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic TEM production of IL-2 but not IFN-γ. We conclude that human CD4+ TEM directly recognize and respond to allogeneic EC in vitro by secreting IFN-γ and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ TEM can mediate allogeneic EC injury in vivo.

Dual delivery of VEGF and MCP-1 to support endothelial cell transplantation for therapeutic vascularization

Transplantation of endothelial cells (EC) for therapeutic vascularization is a promising approach in tissue engineering but has yet to be proven effective in clinical trials. This cell-based therapy is hindered by significant apoptosis of EC upon transplantation as well as poor recruitment of host mural cells to stabilize nascent vessels. Here, we address these deficiencies by augmenting endothelial cell transplantation with dual delivery of vascular endothelial growth factor (VEGF) - to improve survival of transplanted EC - and monocyte chemotactic protein-1 (MCP-1) - to induce mural cell recruitment. We produced alginate microparticles that deliver VEGF and MCP-1 with distinct release kinetics and that can be integrated into a collagen/fibronectin (protein) gel construct for delivery of EC. Combined delivery of VEGF and MCP-1 increased functional vessel formation from transplanted EC and also led to a higher number of smooth muscle cell-invested vessels than did EC therapy alone. Despite the well-known role of MCP-1 in inflammation, these beneficial effects were accomplished without a long-term increase in monocyte/macrophage recruitment or a shift to a pro-inflammatory (M1) macrophage phenotype. Overall, these data suggest a potential benefit of combined delivery of MCP-1 and VEGF from EC-containing hydrogels as a strategy for therapeutic vascularization.

Explant outgrowth, propagation and characterization of human pericytes.

Microcirculation (2010) 17, 367–380. doi: 10.1111/j.1549‐8719.2010.00038.x

Engineering of multifunctional gels integrating highly efficient growth factor delivery with endothelial cell transplantation

Transplantation of Bcl‐2‐transduced human umbilical vein endothelial cells (ECs) in protein gels into the gastrocnemius muscle improves local reperfusion in immunodeficient mouse hosts with induced hind limb ischemia. We tested the hypothesis that incorporation of local, sustained growth factor delivery could enhance and accelerate this effect. Tissue engineering scaffolds often use synthetic polymers to enable controlled release of proteins, but most synthetic delivery systems have major limitations, most notably hydrophobicity and inefficient protein loading. Here, we report the development of a novel alginate‐based delivery system for vascular endothelial growth factor‐A165 (VEGF) that exhibits superior loading efficiency and physical properties to previous systems in vitro. In vivo, VEGF released from alginate microparticles within protein gels was biologically active and, when combined with EC transplantation, led to increased survival of transplanted cells at 28 days. The composite graft described also improved early (14 days) tissue perfusion and late (28 days) muscle myoglobin expression, a sign of recovery from ischemia, compared with EC transplantation and VEGF delivery separately. We conclude that our improved approach to sustained VEGF delivery in tissue engineering is useful in vivo and that the integration of high efficiency protein delivery enhances the therapeutic effect of protein gel‐based EC transplantation.—Jay, S. M., Shepherd, B. R., Bertram, J. P., Pober, J. S., Saltzman, W. M. Engineering of multifunctional gels integrating highly efficient growth factor delivery with endothelial cell transplantation. FASEB J. 22, 2949–2956 (2008)

Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells.

Collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) implanted in the abdominal walls of immunodeficient mice form mature microvessels invested by host-derived smooth muscle cells (SMC) by 8 weeks. We tested the hypothesis that coengraftment of human aortic SMC (HASMC) could accelerate vessel maturation. To prevent SMC-mediated gel contraction, we polymerized the gel within a nonwoven poly(glycolic acid) (PGA) scaffold. Implanted grafts were evaluated at 15, 30, and 60 days. Acellular PGA-supported protein gels elicited a macrophage-rich foreign body reaction and transient host angiogenic response. When transplanted alone, HASMC tightly associated with the fibers of the scaffold and incorporated into the walls of angiogenic mouse microvessels, preventing their regression. When transplanted alone in PGA-supported gels, Bcl-2-HUVEC retained the ability to form microvessels invested by mouse SMC. Interestingly, grafts containing both Bcl-2-HUVEC and HASMC displayed greater numbers of smooth muscle alpha-actin-expressing cells associated with human EC-lined arteriole-like microvessels at all times examined and showed a significant increase in the number of larger caliber microvessels at 60 days. We conclude that SMC coengraftment can accelerate vessel development by EC and promote arteriolization. This strategy of EC-SMC coengraftment in PGA-supported protein gels may have broader application for perfusing bioengineered tissues.

Alloimmunity to Human Endothelial Cells Derived from Cord Blood Progenitors

There is considerable interest in exploiting circulating endothelial progenitor cells (EPCs) for therapeutic organ repair. Such cells may be differentiated into endothelial cells (ECs) in vitro and then expanded for use in tissue engineering. Vessel-derived ECs are variably immunogenic, depending upon tissue source, and it is unknown whether ECs derived from cord blood EPCs are able to initiate an allogeneic response. In this study, we compare the phenotype and alloantigenicity of human cord blood progenitor cell-derived ECs with HUVECs isolated from the same donors. Human cord blood progenitor cell-derived ECs are very similar to HUVECs in the expression of proteins relevant for alloimmunity, including MHC molecules, costimulators, adhesion molecules, cytokines, chemokines, and IDO, and in their ability to initiate allogeneic CD4+ and CD8+ memory T cell responses in vitro and in vivo. These findings have significant implications for the use of cord blood EPCs in regenerative medicine or tissue engineering.

Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the Akt pathway.

Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial‐denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm2) for up to 24 h and compared to control SMC (0 dynes/cm2). SMC density was assessed by cell counting, DNA synthesis by 3[H]‐thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl‐2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in 3[H]‐thymidine incorporation (n = 3, P = 0.003), and 15‐fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl‐2 phosphorylation was increased by 1.8‐fold (n = 3, P = 0.01). Caspase‐3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD‐fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively‐active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury. J. Cell. Physiol. 216: 389–395, 2008.

Development of a humanized mouse model to study the role of macrophages in allograft injury.

Background. Nearly half of all infiltrating leukocytes in rejecting human allografts are macrophages, yet, in comparison with T cells, much less is known about the contribution of this cell type to rejection. Our laboratory has previously described models of rejection of human skin or artery grafts in immunodeficient mouse hosts mediated by adoptively transferred allogeneic T cells. However, mature human monocyte/macrophages have consistently failed to engraft in these animals. Here, we describe the introduction of human CD68+ macrophages into irradiated immunodeficient mice by transplantation of enriched CD34+ hematopoietic stem-cells isolated from peripheral blood of G-colony-stimulating factor pretreated adults. Methods. We investigated strains of immunodeficient mice bearing human tissue grafts (skin and artery) inoculated with 1×106 human CD34+ adult hematopoietic stem cells, peripheral blood monuclear cells autologous to the CD34 donor, or both for human cell engraftment. Results. In the absence of T cells, CD68+ CD14+ macrophages infiltrate allogeneic human skin but produce little injury or thrombosis. Both responses are enhanced when combined with adoptive transfer of T cells autologous to the hematopoietic stem cells as exemplified by the induction of the macrophage activation marker CD163. CD68+ macrophages also infiltrate allogeneic arterial interposition grafts, producing intimal expansion and calcification in the absence of T cells. Conclusions. These new models may be used to study the role of human macrophages in transplant rejection and other pathologies in vivo.