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Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4 + T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs., (© 2021 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

Cytokine activation of cultured human vascular endothelial cells renders them hyperadhesive for blood leukocytes. Co-incubation of freshly isolated, unstimulated human blood neutrophils with confluent cytokine-activated human endothelial monolayers for 90 minutes results in extensive endothelial detachment and destruction of monolayer integrity. In contrast, unactivated endothelial monolayers remain intact. Using this in vitro model, we have explored the neutrophil-effector mechanisms involved in this injury. Coincubation in the presence of a serine protease inhibitor (phenylmethylsulfonyl fluoride) or specific elastase inhibitors (Ala-Ala-Pro-Val-chloromethyl ketone or alpha-1-protease inhibitor) markedly diminished injury. In contrast, scavengers or inhibitors of oxygen-derived free radicals (superoxide dismutase, catalase, mannitol, or sodium azide) were not protective. Purified human neutrophil elastase mimicked the effect of the neutrophils suggesting a key role for elastase in the neutrophil-mediated injury in this model. Interfering with direct neutrophil-endothelial cell contact by interposing a microporous barrier insert prevented endothelial cell detachment. Furthermore, this neutrophil-mediated detachment could be inhibited with interleukin-8, an action correlated with a decrease in neutrophil adhesion to activated endothelial monolayers. By defining the role of endothelial activation in neutrophil-mediated injury, this in vitro model may provide useful insights into potential therapeutic interventions designed to prevent disruption of the endothelial barrier function.

The ability of angiotensin II and arachidonic acid to release immunoreactive prostaglandins into venous and ureteral effluents of rabbit isolated perfused kidneys was examined 7 days after suprarenal aortic coarctation (SRAC) or sham operation (SHAM). Renal vascular responses to angiotensin II were significantly enhanced in SRAC and accompanied by an enhanced venous efflux of bioassayable prostaglandins. Angiotension II-induced release of immunoreactive PGE2, PGF2 alpha, 6-keto PGF1 alpha and TxB2 into the venous effluent was exaggerated in SRAC. As angiotensin II did not stimulate TxB2 efflux in the SHAM group the induction of TxB2 release by SRAC is particularly noteworthy. These changes in eicosanoid release in response to angiotensin II were not mimicked by arachidonic acid administration. These results suggest that in renovascular hypertension angiotensin II-induced prostaglandin release is primarily augmented in the vascular compartment and is consistent with the sensitivity of renal function to cyclooxygenase inhibitors in renovascular hypertension.