Marlies E.J. Reinders

Proinflammatory functions of vascular endothelial growth factor in alloimmunity

Vascular endothelial growth factor (VEGF), an established angiogenesis factor, is expressed in allografts undergoing rejection, but its function in the rejection process has not been defined. Here, we initially determined that VEGF is functional in the trafficking of human T cells into skin allografts in vivo in the humanized SCID mouse. In vitro, we found that VEGF enhanced endothelial cell expression of the chemokines monocyte chemoattractant protein 1 and IL-8, and in combination with IFN-gamma synergistically induced endothelial cell production of the potent T cell chemoattractant IFN-inducible protein-10 (IP-10). Treatment of BALB/c (H-2d) recipients of fully MHC-mismatched C57BL/6 (H-2b) donor hearts with anti-VEGF markedly inhibited T cell infiltration of allografts and acute rejection. Anti-VEGF failed to inhibit T cell activation responses in vivo, but inhibited intragraft expression of several endothelial cell adhesion molecules and chemokines, including IP-10. In addition, whereas VEGF expression was increased, neovascularization was not associated with acute rejection, and treatment of allograft recipients with the angiogenesis inhibitor endostatin failed to inhibit leukocyte infiltration of the grafts. Thus, VEGF appears to be functional in acute allograft rejection via its effects on leukocyte trafficking. Together, these observations provide mechanistic insight into the proinflammatory function of VEGF in immunity.

Angiogenesis and Endothelial Cell Repair in Renal Disease and Allograft Rejection

This review discusses the concept that the turnover and replacement of endothelial cells is a major mechanism in the maintenance of vascular integrity within the kidney. CD133+CD34+KDR+ endothelial cell progenitor cells emigrate from the bone marrow and differentiate into CD34+KDR+ expressing cells, which are present in high numbers within the circulation. These progenitor cells are available for recruitment into normal or inflamed tissues to facilitate endothelial cell repair. In several forms of renal disease, proinflammatory insults mediate oxidative stress, senescence, and sloughing of endothelial cells. A lack of growth factors or an inefficient recruitment of endothelial cell progenitors results in hypoxic tissue injury and accelerates the process of chronic renal failure. Augmentation of vascular repair by the provision of growth factors such as vascular endothelial growth factor or by the transfer of progenitor cells directly into the kidney can be protective and prevent ongoing interstitial damage. In allografts, persistent injury results in excessive turnover of graft vascular endothelial cells. Moreover, chronic damage elicits a response that is associated with the recruitment of both leukocytes and endothelial cell progenitors, facilitating an overlapping process of inflammation and angiogenesis. Because the angiogenesis reaction itself is proinflammatory, this process becomes self-sustaining. Collectively, these data indicate that angiogenesis and endothelial cell turnover are important in renal inflammatory processes and allograft rejection. Manipulation of the response may have therapeutic implications to protect against injury and chronic disease processes.

The role of the graft endothelium in transplant rejection: Evidence that endothelial activation may serve as a clinical marker for the development of chronic rejection

Abstract: In this review, we discuss the role of the allograft endothelium in the recruitment and activation of leukocytes during acute and chronic rejection. We discuss associations among endothelial activation responses, the expression of adhesion molecules, chemokines and chemokine receptors, and rejection; and we propose that endothelial vascular cellular adhesion molecule‐1 (VCAM‐1) may be used as a surrogate marker of acute rejection and allograft vasculopathy. In addition, we describe potential mechanistic interpretations of persistent endothelial cell (EC) expression of major histocompatibility complex (MHC) class II molecules in allorecognition. The graft endothelium may provide an antigen‐specific signal to transmigrating, previously activated, T cells and may induce B7 expression on locally transmigrating leukocytes to promote costimulation. Taken together, these functions of the EC provide it with a potent regulatory role in rejection and in the maintenance of T‐cell activation via the direct and/or the indirect pathways of allorecognition.

Proangiogenic Function of CD40 Ligand-CD40 Interactions

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.

Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection.

Background. Vascular endothelial growth factor (VEGF), a major angiogenesis factor, has been found to have proinflammatory properties in vivo in several chronic inflammatory diseases. However, little is known of the expression or function of VEGF in acute and chronic allograft rejection. Methods. In a cross‐sectional analysis, we evaluated the expression of VEGF by immunohistochemistry in human endomyocardial biopsies (n=101) from 10 cardiac transplant patients. We correlated expression (scores from 0‐4) with CD3+ T cell, CD68+ monocyte and macrophage infiltrates, or rejection (International Society of Heart and Lung Transplantation grades 0‐4). In addition, we evaluated the temporal patterns of VEGF expression in consecutive biopsies from seven patients (total of 74 biopsies) who were assessed for the development of graft vascular disease (GVD) by intravascular ultrasonography at 1 year posttransplantation. Results. VEGF is expressed in normal human endomyocardial biopsies at low levels and is induced (scores >1) in association with CD3+ T cells (odds ratio [OR], 19.90; P<0.001), CD68+ monocyte and macrophage infiltrates (OR, 8.49; P<0.001), and all grades of acute rejection (OR, 5.4; P<0.001). Increases in VEGF expression were persistent during the first posttransplant year in biopsies from four patients who demonstrated evidence of GVD (mean annual score of 2.3). In contrast, limited expression of VEGF was found in three patients without GVD (mean annual score 1.2). Conclusions. These findings define VEGF as an important proinflammatory cytokine after transplantation and indicate that its expression pattern might identify patients at risk for the development of GVD.

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.

Effect of vascular endothelial growth factor and its receptor KDR on the transendothelial migration and local trafficking of human T cells in vitro and in vivo

In these studies, we find that the vascular endothelial growth factor (VEGF) receptor KDR is expressed on subsets of mitogen-activated CD4(+) and CD8(+) T cells in vitro. We also found that KDR colocalizes with CD3 on mitogen-activated T cells in vitro and on infiltrates within rejecting human allografts in vivo. To evaluate whether VEGF and KDR mediate lymphocyte migration across endothelial cells (ECs), we used an in vitro live-time transmigration model and observed that both anti-VEGF and anti-KDR antibodies inhibit the transmigration of both CD4(+) and CD8(+) T cells across tumor necrosis factor α (TNFα)-activated, but not unactivated ECs. In addition, we found that interactions among CD4(+) or CD8(+) T cells and TNFα-activated ECs result in the induction of KDR on each T cell subset, and that KDR-expressing lymphocytes preferentially transmigrate across TNFα-activated ECs. Finally, using a humanized severe combined immunodeficient mouse model of lymphocyte trafficking, we found that KDR-expressing lymphocytes migrate into human skin in vivo, and that migration is reduced in mice treated with a blocking anti-VEGF antibody. These observations demonstrate that induced expression of KDR on subsets of T cells, and locally expressed VEGF, facilitate EC-dependent lymphocyte chemotaxis, and thus, the localization of T cells at sites of inflammation.

Mesenchymal Stromal Cell Therapy for Cardio Renal Disorders

Chronic kidney disease carries a very high mortality risk, in particular from cardiac diseases. Often heart failure and renal failure coincide, a phenomenon referred to as the cardio renal syndrome. In recent years, it has become clear that not only fibrotic repair but also restoration of damaged kidney and heart can occur and the use of cell therapy has been advanced as a means to activate endogenous repair mechanisms or even to re-introduce repairing tissue. In this perspective, mesenchymal stromal cells are of particular interest, since these cells have both immune modulating and reparative functions and are on the brink of entering the clinical arena. Indeed, MSCs can trigger numerous therapeutic biologic processes that contribute to both renal and cardiac repair; however exact mechanisms of actions are largely unknown. In the present review we have made a critical appraisal of the data available with respect to origin and function of MSCs, and we discuss both preclinical as well as clinical evidence on their therapeutic potential in kidney and heart disease.

The role of mesenchymal stromal cells in chronic transplant rejection after solid organ transplantation.

Purpose of reviewDespite impressive improvements in short-term survival after solid organ transplantation (SOT), the long-term outcome remains an important challenge. In recent years, it has become evident that ongoing alloreactivity, cellular and/or humoral, may play a critical role in the premature loss of kidney allografts. Clinical trials with mesenchymal stromal cells (MSCs) are currently underway for the treatment of various inflammatory disorders. Due to their immunosuppressive and reparative properties MSC immunotherapy constitutes an attractive intervention for chronic rejection, while avoiding the undesired adverse effects associated with excessive use of immunosuppressive drugs. Recent findingsThe immunoregulatory properties of MSCs in both cellular and antibody-mediated inflammatory models and diseases have highlighted their potential to treat chronic rejection after SOT. Moreover, MSCs can also induce tissue regeneration and repair due to their antifibrotic and angiogeneic properties. Studies in experimental animals after SOT support the potential for MSCs to treat or prevent ongoing alloreactivity and the first clinical trials in transplant recipients are underway. SummaryMSCs are arising as promising cellular immunotherapy in transplant recipients and carry the potential to prevent or reduce the consequences of chronic or ongoing alloimmune injury.

Mesenchymal stromal cells to prevent fibrosis in kidney transplantation.

Purpose of reviewKidney transplantation has improved the life expectancy and quality of life for patients with end-stage renal failure. However, despite the impressive improvements in short-term outcome parameters because of better and more potent immunosuppressive drugs, the long-term survival of renal allografts has changed little over the last decades. Sustained inflammation in the areas of interstitial fibrosis and tubular atrophy (IFTA) is a strong predictor of allograft failure. Mesenchymal stromal cells (MSCs) have potent anti-inflammatory and reparative properties, and could thus play a role in controlling these processes. Recent findingsLocal resident MSCs and exogenous MSCs have been implicated in the repair of the injured kidney, mostly by their paracrine functions. In the experimental models and clinical trials, first results with MSCs for the treatment of inflammation and IFTA suggest beneficial effects. SummaryEndogenously and exogenously administered MSCs might enhance the intrinsic reparative capabilities of the kidney in transplant recipients and maybe developed as a tool to control both inflammation and fibrosis.