Alan G. Contreras

Overexpression of Vascular Endothelial Growth Factor and the Development of Post-Transplantation Cancer

Cancer is an increasing and major problem after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calcineurin inhibitors on the expression of vascular endothelial growth factor (VEGF) leads to an angiogenic milieu that favors tumor growth. Here, we used 786-0 human renal cancer cells to investigate the effect of cyclosporine (CsA) on VEGF expression. Using a full-length VEGF promoter-luciferase construct, we found that CsA markedly induced VEGF transcriptional activation through the protein kinase C (PKC) signaling pathway, specifically involving PKC zeta and PKC delta isoforms. Moreover, CsA promoted the association of PKC zeta and PKC delta with the transcription factor Sp1 as observed by immunoprecipitation assays. Using promoter deletion constructs, we found that CsA-mediated VEGF transcription was primarily Sp1 dependent. Furthermore, CsA-induced and PKC-Sp1-mediated VEGF transcriptional activation was partially inhibited by von Hippel-Lindau protein. CsA also promoted the progression of human renal tumors in vivo, wherein VEGF is overexpressed. Finally, to evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we injected CT26 murine carcinoma cells (known to form angiogenic tumors) into mice with fully MHC mismatched cardiac transplants. We observed that therapeutic doses of CsA increased tumor size and VEGF mRNA expression and also enhanced tumor angiogenesis. However, coadministration of a blocking anti-VEGF antibody inhibited this CsA-mediated tumor growth. Collectively, these findings define PKC-mediated VEGF transcriptional activation as a key component in the progression of CsA-induced post-transplantation cancer.

Calcineurin Inhibitors Activate the Proto-Oncogene Ras and Promote Protumorigenic Signals in Renal Cancer Cells

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.

CD40-Induced Signaling in Human Endothelial Cells Results in mTORC2-and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo

We have examined CD40-dependent signals in endothelial cells (EC) mediating the expression of vascular endothelial growth factor (VEGF) and VEGF-induced angiogenesis. We treated confluent cultures of EC with soluble CD40L (sCD40L), and by Western blot found a marked increase in the phosphorylation of Akt, 4EBP-1, and S6K1, compared with untreated cells. EC were transfected with a full-length VEGF promoter-luciferase construct and cultured in the absence or presence of rapamycin and sCD40L. We found that rapamycin, which blocks mTORC1 and mTORC2 signaling, inhibited sCD40L-mediated transactivation of VEGF. In addition, by Western blot, we found that the transfection of EC with small interfering RNA (siRNA) to rictor (to inhibit mTORC2), and not raptor (to inhibit mTORC1), inhibited sCD40L-dependent protein expression of VEGF. In additions, we found that basal levels of phosphorylated Akt as well as VEGF were increased in EC transfected with the raptor siRNA. Also, rapamycin failed to inhibit VEGF promoter activation, as well as VEGF protein expression in EC transfected with a constitutively active construct of Akt, further demonstrating that mTORC1 is not necessary for CD40- and Akt-induced expression of VEGF. Finally, we injected human CD40L-transfected fibroblasts or mock transfectants into human skin on SCID mice. We found that the injection of CD40L transfectants, but not mock cells, resulted in VEGF expression and mediated a marked angiogenesis reaction, and this response was reduced in mice treated with rapamycin. Together, these observations indicate that mTORC2 and Akt facilitate CD40-inducible expression of VEGF in EC, which is of clinical importance in tumor growth and the progression of chronic inflammatory diseases.

Key Features of the Intragraft Microenvironment that Determine Long-Term Survival Following Transplantation

In this review, we discuss how changes in the intragraft microenvironment serve to promote or sustain the development of chronic allograft rejection. We propose two key elements within the microenvironment that contribute to the rejection process. The first is endothelial cell proliferation and angiogenesis that serve to create abnormal microvascular blood flow patterns as well as local tissue hypoxia, and precedes endothelial-to-mesenchymal transition. The second is the overexpression of local cytokines and growth factors that serve to sustain inflammation and, in turn, function to promote a leukocyte-induced angiogenesis reaction. Central to both events is overexpression of vascular endothelial growth factor (VEGF), which is both pro-inflammatory and pro-angiogenic, and thus drives progression of the chronic rejection microenvironment. In our discussion, we focus on how inflammation results in angiogenesis and how leukocyte-induced angiogenesis is pathological. We also discuss how VEGF is a master control factor that fosters the development of the chronic rejection microenvironment. Overall, this review provides insight into the intragraft microenvironment as an important paradigm for future direction in the field.

Calcineurin Inhibitors Modulate CXCR3 Splice Variant Expression and Mediate Renal Cancer Progression

Calcineurin inhibitors (CNI) are used to prevent inflammatory diseases and allograft rejection. However, little is known about the mechanism(s) underlying their ability to promote the development and recurrence of cancer. Recent studies suggested that the chemokine receptor CXCR3 may play important roles in tumorigenesis. CXCR3 has two splice variants with opposite functions: CXCR3-A promotes cell proliferation, and CXCR3-B inhibits cell growth. Here, we explored the effects of CNI on the expression and function of CXCR3 splice variants. Compared with normal renal tissues and renal epithelial cells, human renal cancer tissues and renal cancer cell lines demonstrated higher expression of CXCR3-A and markedly lower expression of CXCR3-B. In human renal cancer cells (786-0 and Caki-1) and renal epithelial cells, CNI markedly downregulated the expression of CXCR3-B, whereas expression of CXCR3-A was unchanged. This CNI-mediated downregulation of CXCR3-B resulted in increased proliferation and migration of renal cancer cells; CNI-mediated cell proliferation involved signaling through G(i) proteins, perhaps via CXCR3-A. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the expression of CXCR3-B was significantly decreased in these tumors. In summary, these observations suggest that CNI may mediate the progression of human renal cancer by downregulating CXCR3-B and by promoting proliferative signals, likely through CXCR3-A. Targeting CXCR3 splice variants or the signaling pathways downstream of CXCR3 receptors may provide a therapeutic strategy for the prevention of CNI-mediated renal cancer progression.

Every allograft needs a silver lining

The development of chronic allograft rejection is based on the hypothesis that cumulative, time-dependent tissue injury eventually leads to a fibrotic response. In this issue of the JCI, Babu and colleagues found that alloimmune-mediated microvascular loss precedes tissue damage in murine orthotopic tracheal allografts (see the related article beginning on page 3774). The concept that injury to the endothelium may precede airway fibrosis suggests that interventions to maintain vascular integrity may be important, especially in the case of lung transplantation. Further, for all solid organ allografts, it is possible that the key to long-term allograft survival is physiological vascular repair at early times following transplantation.

Organ-specific differences in the function of MCP-1 and CXCR3 during cardiac and skin allograft rejection.

Background. Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known. Methods. Fully major histocompatibility complex–mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1−/− (H-2b) and CXCR3−/− (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days. Results. Cardiac allografts in MCP-1−/− recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1−/− grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3−/− recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3−/− donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3−/− donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3−/− skin grafts were used as donors in the SCID model of rejection. Conclusion. Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.

Liver transplantation in Latin America: The state-of-the-art and future trends

We reviewed the current status of liver transplantation in Latin America. We used data from the Latin American and Caribbean Transplant Society and national organizations and societies, as well as information obtained from local transplant leaders. Latin America has a population of 589 million (8.5% of world population) and more than 2,500 liver transplantations are performed yearly (17% of world activity), resulting in 4.4 liver transplants per million people (pmp) per year. The number of liver transplantations grows at 6% per year in the region, particularly in Brazil. The top liver transplant rates were found in Argentina (10.4 pmp), Brazil (8.4 pmp), and Uruguay (5.5 pmp). The state of liver transplantation in some countries rivals those in developed countries. Model for End-Stage Liver Disease-based allocation, split, domino, and living-donor adult and pediatric transplantations are now routinely performed with outcomes comparable to those in advanced economies. In contrast, liver transplantation is not performed in 35% of Latin American countries and lags adequate resources in many others. The lack of adequate financial coverage, education, and organization is still the main limiting factor in the development of liver transplantation in Latin America. The liver transplant community in the region should push health care leaders and authorities to comply with the Madrid and Istambul resolutions on organ donation and transplantation. It must pursue fiercely the development of registries to advance the science and quality control of liver transplant activities in Latin America.

Probability of deceased donor kidney transplantation based on % PRA

UNLABELLED Sensitization to HLA antigens creates an obstacle for the accessibility and success of kidney transplantation (KT). Highly sensitized patients have longer waiting times and some may never receive a KT. AIM To determine the probability of patients on the deceased donor (DD) waiting list to receive a KT based on the panel reactive antibody percentage (% PRA) in our center. METHODS The DD waiting list from our institution was analyzed from 01/05 to 08/12 documenting the clinical variables from donor and potential recipients (ABO blood group), lymphocyte cross-match [CxM (CDC-AHG)] results, highest % PRA determination, and time on the waiting list. The patients were classified into 4 groups based on the % PRA: 0%, 1-19%, 20-79% and 80-100%. The data was analyzed using odds ratio and logistic regression (significant p<0.05). RESULTS 58 DD (F:M 34:24, ABO group O=35, A=13, B=10) and 179 potential recipients were analyzed (F:M 98:81, ABO group O=127, A=33, B=19, participating 4.2 ± 3.8 times with different donors to receive KT). The mean PRA for the whole group was 22 ± 32%, median [md] 0 (0-98). A total of 100 patients received KT (mean waiting time 2.2 ± 1.7 years, 12 days-7 years) and their mean % PRA was 11.6 ± 24, md 0 (0-94) vs. 31.4 ± 37 md 8.5 (0-98) in those who have not received a KT. An association between the % PRA group and KT (p<0.003) was observed. The probability of receiving KT with a 0% PRA vs. >0% was higher (OR 2.12, 1.17-3.84). There was no difference between the 0% vs. 1-19% group (OR 1); differences were observed between 0% vs. 20-79% (OR 2.5, 1.18-5.3) and 0% vs. 80-100% (OR 5, 1.67-14.9). For every percent increase in the PRA above 20%, the risk of not receiving a KT increased by 5% (1-9, p<0.01). CONCLUSIONS The probability of receiving a DD kidney transplant is inversely related to the % PRA although a higher risk for not receiving a KT becomes evident with a PRA >20%.

mTOR—Understanding the Clinical Effects

The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. The TOR kinase, which was originally discovered in yeast, is also expressed in human cells as mammalian TOR (mTOR). In this review, we focus on how mTOR-inducible signals function in cell protection and cell survival of effector and regulatory T cells as well as its role in endothelial cell biology. We evaluate how signaling is important for vascular endothelial cell growth, survival, and proliferation; and we consider how the function of mTOR in endothelial cells may be clinically important in the rejection process. Understanding the biology of mTOR allows clinicians to use mTOR inhibitors optimally as therapeutics following solid organ transplantation.