Function of the vascular endothelial growth factor receptors Flt-1 and Flk-1/KDR in the alloimmune response in vivo

BACKGROUND: We have recently reported that vascular endothelial growth factor (VEGF) functions as a proinflammatory cytokine to regulate the trafficking of leukocytes into allografts in the early posttransplant period. VEGF binds two major VEGF receptors: VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Here, we wished to investigate the expression and function of VEGF receptors in the process of acute allograft rejection in vivo. METHODS: We performed fully MHC-mismatched C57BL/6 (H-2b) into BALB/c (H-2d) vascularized heterotopic murine cardiac transplants and we examined the expression of VEGF and VEGF receptors by immunohistochemistry during acute allograft rejection. Next, we treated mice with specific neutralizing monoclonal antibodies against murine VEGFR-1 and VEGFR-2 and examined their effect on the development of acute allograft rejection by histology and by analysis of graft survival. The intragraft expression of cytokines and chemokines were also evaluated by quantitative real-time PCR analysis. RESULTS: The expression of VEGF, VEGFR-1 and VEGFR-2 were significantly up-regulated during allograft rejection as compared to isografts. Administration of either anti-VEGFR-1 or anti-VEGFR-2 alone failed to inhibit allograft rejection. However, coadministration of both antibodies together inhibited leukocyte infiltration of allografts and prolonged allograft survival. Furthermore, the effect of VEGFR blockade was associated with the downregulation of intragraft cytokine and chemokine expression. CONCLUSIONS: Our data suggest that VEGF-VEGFR interactions function in the alloimmune response in vivo. Targeting VEGFRs may represent a novel therapy to protect allografts following clinical transplantation.     

Combined vascular endothelial growth factor and platelet-derived growth factor inhibition in rat cardiac allografts: beneficial effects on inflammation and smooth muscle cell proliferation

BACKGROUND: Perivascular inflammation and subsequent smooth muscle cell (SMC) proliferation are central in the development of cardiac allograft arteriosclerosis. We examined the effect of combined inhibition of proinflammatory vascular endothelial growth factor (VEGF) and SMC mitogen platelet-derived growth factor (PDGF) in rat cardiac allografts. METHODS: Heterotopic cardiac transplantations were performed between fully major histocompatibility mismatched rat strains receiving cyclosporine A immunosuppression. In situ hybridization and immunohistochemistry were performed to examine VEGF and PDGF ligand and receptor (R) expression. Protein tyrosine kinase inhibitors PTK787 and imatinib were used to inhibit VEGFR and PDGFR activity, respectively. Rat coronary artery SMC migration and proliferation assays were used to examine the effect of VEGF and PDGF and tyrosine kinase inhibitors in vitro. RESULTS: Both ligand and receptor expression of VEGF and PDGF were detected in chronically rejecting allografts. In vitro, PDGF-BB mediated rat coronary artery SMC migration and proliferation was completely inhibited with imatinib and partially with PTK787. In vivo, combined treatment with PTK787 and imatinib significantly reduced the formation of neointimal lesions in arteries of cardiac allografts at 8 weeks, producing a greater effect than either drug alone. PTK787, in contrast with imatinib, reduced the number of ED1 macrophages and PDGF-B immunoreactivity in the allografts at 4 weeks. CONCLUSIONS: Blocking VEGF and PDGF receptor signaling in cardiac allografts has distinctive effects on inflammation and SMC proliferation, suggesting that targeting both inflammation and pathologic vascular remodeling may be needed to inhibit cardiac allograft arteriosclerosis.     

Vascular endothelial growth factor expression in human chronic renal allograft rejection

BACKGROUND: Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy. METHODS: Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease. RESULTS: In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P<0.0001). In serial sections, VEGF colocalized with the expression of CD 68-positive macrophages. Significantly more macrophages were in the tubulointerstitium in tissue with chronic rejection than in those with temporary allograft dysfunction (P<0.005). Additionally, VEGF protein expression in the glomeruli and the vascular compartment of patients with chronic rejection was increased. CONCLUSION: The up-regulation of VEGF in chronic renal allograft rejection may be important in inflammation and development of fibrosis.     

Immunolocalization of fibroblast growth factor-1 (FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1 (FSP-1) in inflammatory renal disease

BACKGROUND: The fibroblast growth factor (FGF) family has functions in development, cell proliferation, migration, and differentiation. While FGF-2 induces fibrosis, the role of FGF-1 in inflammation and fibrosis is less defined. We examined the expression of FGF-1 and FGF receptor (FGFR-1) to determine if renal diseases with varying etiologies of inflammation, including lupus nephritis (LN), acute interstitial nephritis (AIN) and acute rejection superimposed on chronic allograft nephropathy (CAN), showed varying patterns of expression. We also examined the expression of fibroblast-specific protein-1 (FSP-1), which has been linked to epithelial-mesenchymal transition (EMT) and fibrosis, to determine whether it was linked to potential profibrotic and inflammatory FGF-1 mechanisms. METHODS: Proliferative LN (PLN) (N= 12), nonproliferative lupus nephritis (NPLN) (N= 5), AIN (N= 6), CAN (N= 4), and normal kidneys (N= 3) were studied. FGF, FGFR-1, and FSP-1 were localized by immunohistochemistry, and intensity scored on a 0 to 3+ scale. Double staining with CD68 and separate immunohistochemical staining for CD4 and CD8 with serial sections analysis were done to identify if T lymphocytes or macrophages showed staining for FGF-1 and FGFR-1 or FSP-1. RESULTS: In normal kidneys, FGF-1 was expressed in mesangial cells (0.67 +/- 0.58), glomerular endothelial (0.67 +/- 0.58), visceral, and parietal epithelial cells (1.67 +/- 0.58). FGFR-1 showed a similar pattern of staining but also was expressed in tubular epithelium, and arterial endothelium and smooth muscle. Expression of FGF-1 was increased over normal in glomerular parenchymal cells only in CAN in podocytes (2.30 +/- 0.58 vs. 3.00 +/- 0.00) (P < 0.05) and parietal epithelial cells (1.67 +/- 0.58 vs. 2.25 +/- 0.50) (P < 0.05). Infiltrating glomerular and interstitial inflammatory cells in diseased glomeruli also expressed FGF-1 and FGFR-1. Tubular cells expressed slightly increased FGFR-1 in renal diseases vs. normal, whereas tubules remained negative for FGF-1 in diseased kidneys. FSP-1 expression was prominent in the interstitium in all kidneys with interstitial inflammation, and most prominent in CAN. Interstitial FSP-1+ cells were consistent with a myofibroblast-type morphology, and did not stain with CD-68. FSP-1 expression was closely associated with inflammatory cells expressing FGF-1 and FGFR-1. FSP-1 also showed positivity within crescents and occasional podocytes in PLN. CONCLUSION: The expression of FGF-1 and FGFR-1 in infiltrating lymphocytes and macrophages, and of FGFR-1 in tubules, is supportive, but does not prove causality, of the possibility that FGF-1 might have both autocrine and paracrine functions in renal inflammation. However, the initial stimulus for renal inflammation, whether immune complex, hypersensitivity or rejection, did not alter expression patterns of FGF-1 or its receptor. The colocalization of inflammatory infiltrates with interstitial fibrosis supports the possibility of a contribution of FGF-1 for chemotaxis and associated fibrosis, further supported by interstitial FSP-1 expression closely associated with these inflammatory cells expressing FGF-1 and FGFR-1.     

Vascular Endothelial Growth Factor Expression and Cyclosporine Toxicity in Renal Allograft Rejection

The aim of this study was to evaluate the influence of vascular endothelial growth factor (VEGF) on renal function and on development of interstitial fibrosis (IF) in renal allografts. Tubular and interstitial expressions of VEGF and TNF-α, and density of macrophages in the interstitium were examined in 92 patients with nonrejected kidneys, acute rejection (AR), chronic allograft nephropathy (CAN), borderline changes (BC) and acute cyclosporin A (CsA) toxicity. Follow-up biopsy specimens from patients with AR and BC were evaluated for development of IF. A significant difference in tubular and interstitial VEGF expressions was found between patients with AR, BC, CAN and CsA toxicity (p < 0.001). Macrophage infiltration was positively correlated with VEGF and TNF-α expressions (p < 0.001). VEGF expression increased with increasing expression of TNF-α (p < 0.001). Renal function in first 6 months after initial biopsy was better in patients with marked tubular VEGF expression (p < 0.01); however, in follow-up, development of IF and graft loss was found earlier in these patients (p < 0.01 and p < 0.05, respectively). Increased renal VEGF expression has protective properties immediately following renal allograft but allows for increased risk of early IF, and therefore poor graft outcome in the long term.     

Prediction of chronic allograft damage index of renal allografts using serum level of plasminogen activator inhibitor‐1

Abstract: Background: Plasminogen activator inhibitor‐1 (PAI‐1) plays an important role in renal fibrosis. We conducted this study to examine whether serum PAI‐1 has a role in predicting chronic allograft nephropathy (CAN). Methods: Fifty kidney transplant recipients receiving graft biopsies were enrolled. The pathologic diagnoses were acute tubular necrosis (ATN; n = 12), borderline rejection (BR; n = 7), acute rejection (ACR; n = 12), CAN (n = 11), polyomavirus nephropathy (PVN; n = 3) and others (n = 5; glomerulopathy, n = 4; calcineurin inhibitor nephropathy, n = 1). The serum level of PAI‐1 and chronic allograft damage index (CADI) score of each patient were determined. Results: The CADI score of all patients was 4 (0–10) (median and range) and in each group was ATN 0.5 (0–4), BR 2.0 (1–4), ACR 4.0 (2–8), CAN 5.0 (4–10), PVN 7 (4–8) and others 2 (0–8). The serum PAI‐1 level of each group was ATN 9.38 (2.35–14.52) ng/mL, BR 10.09 (0.61–18.06) ng/mL, ACR 11.08 (2.32–20.68) ng/mL, CAN 14.51 (3.66–21.12), PVN 14.79 (13.94–21.94) and others 16.35 (4.05–21.01) ng/mL. CADI score is associated with serum PAI‐1 activity (r = 0.405, p = 0.003) and is inversely associated with serum creatinine (r = ?0.348, p = 0.011), but not with estimated glomerular filtration rate (p = 0.124). Conclusion: Serum PAI‐1 level has the potential to be a marker to predict CADI score, the quantitative score of CAN.     

Expression of fractalkine, CX3CR1, and vascular endothelial growth factor in human chronic renal allograft rejection

AIM: Fractalkine/CX3CR1 system may contribute to the pathogenesis of renal allograft chronic rejection (CR). Vascular endothelial growth factor (VEGF) is an endothelial mitogen, which shows increased expression in inflammation and vasculopathy. This study sought describe the expression and distribution of Fractalkine/CX3CR1 and VEGF, and their relationship to human renal allograft CR. METHODS: Renal tissue from 10 patients with CR was examined for Fractalkine/CX3CR1 and VEGF protein by immunohistochemistry for comparison with patients displaying hyperacute rejection (n = 10), acute rejection (n = 10), and normal kidneys (n = 10). All patients were selected based upon histologically proven diagnoses between 1992 and 2003. RESULTS: Immunohistochemistry revealed that Fractalkine/CX3CR1 were mostly expressed in the tubulointerstitium and tubular epithelial cell basolateral membrane. Some vessels showed positive staining for Fractalkine/CX3CR1 as well as occasionally glomerular parietal wall cells. Among the CR group, VEGF was mostly expressed in tubular epithelium and the tubulointerstitium. A proportion of glomeruli and vessels had positive staining for VEGF, which was up-regulated most strikingly in the interstitial compartment in CR. There was markedly increased expression of Fractalkine/CX3CR1 and VEGF protein in the interstitium of the CR compared with other groups (P < .05). VEGF colocalized with the expression of Fractalkine/CX3CR1. CONCLUSION: Fractalkine/CX3CR1 and VEGF may play an important role in the development of interstitial fibrosis via mononuclear cell-induced cytokine production and myofibroblast stimulation in CR. Further studies are necessary to identify the role in the pathogenesis of CR.     

The role of renin–angiotensin system in the chronic allograft nephropathy: an immunohistochemical study

Abstract

Background: Evidences suggest a role of renin–angiotensin system (RAS) in the development of chronic allograft injury. Methods: We correlated intrarenal angiotensin-converting enzyme, angiotensin II (Angio II) and transforming growth factor β₁ (TGFβ₁) expression in 58 biopsies-proven chronic allograft nephropathy (CAN) with tissue injury and allograft survival. Results: The biopsies with CAN were graded according to Banff classification as I (22 cases), II (17) and III (19); 27 biopsies also showed a mononuclear inflammatory infiltrate in scarred areas. There were increased expression of angiotensin converting-enzyme (ACE), Angio II and TGFβ₁ mainly in tubulointerstitial compartment in the group with CAN; there was no association of Angio II and TGFβ₁ expression with interstitial fibrosis. There were no significant differences of ACE, Angio II and TGFβ₁ expression between the patients treated and untreated with RAS blockade, and with the graft outcome. Interstitial inflammatory infiltrate had positive correlation with interstitial fibrosis and significant impact on graft survival at 8 years. Conclusions: Our study showed in a group of cases with CAN a high percentage of inflammatory infiltrate that correlated with interstitial fibrosis and graft outcome. The chronic inflammatory changes in these cases did not show significant association with local RAS expression.     

Growth inhibition of orthotopic anaplastic thyroid carcinoma xenografts in nude mice by PTK787/ZK222584 and CPT-11

BACKGROUND: A preclinical evaluation of CPT-1 (Camptosar, irinotecan) and PTK787/ZK222584, a vascular endothelial growth factor receptor (VEGFR-2) tyrosine kinase inhibitor, as therapeutic agents against anaplastic thyroid carcinoma (ATC) was performed in vitro and in an orthotopic model of ATC in nude mice. METHODS: The cytotoxic and cytostatic effects of CPT-11 on ATC cell lines were evaluated. The antitumor effects of CPT-11 in combination with PTK787/ZK222584 on orthotopic ATC xenografts in nude mice were also studied. RESULTS: CPT-11 demonstrated significant antiproliferative effects on ATC cell lines. In vivo, PTK787/ZK222584, CPT-11, and the two agents together produced 61%, 82%, and 89% decrease in tumor growth, respectively. The differences in tumor volume between CPT-11 and CPT-11 + PTK787/ZK222584 groups were not statistically significant. PTK787/ZK222584 inhibited the phosphorylation of VEGFR-2 on tumor endothelium and decrease the tumor microvessel density. CONCLUSIONS: The camptothecin class of chemotherapeutic agents and antiangiogenic agents such as PTK787/ZK222584 warrant further study as novel therapeutic agents against ATC.     

Protective effects of atorvastatin on chronic allograft nephropathy in rats

OBJECTIVE: Chronic allograft nephropathy (CAN) is the leading cause of late kidney allograft loss. Recent studies have suggested that atorvastatin (ATO) may interact with the acute inflammatory process in the renal interstitium and suppress the proliferation of mesangial cells. We hypothesized that ATO could also inhibit the chronic inflammatory process and prevent the progression of CAN. MATERIALS AND METHODS: Fisher (F344) kidneys were orthotopically transplanted into Lewis rat recipients. Lewis-to-Lewis rat kidney transplantation was served as the syngeneic control (Syn group). Allograft recipients were randomized and treated with cyclosporine A alone (Allo group) or in combination with ATO (15 or 30 mg/kg/d intrgastric, respectively, the low dose treatment group/high dose treatment group [LT/HT] groups). Renal function and the urine protein excretion were analyzed. Animals were sacrificed 20 weeks posttransplantation for histological and immunohistochemical studies, as well as analysis of mRNA levels of cytokines and chemokines. RESULTS: Renal function progressively deteriorated and substantial proteinuria developed in the Allo group compared with the Syn group. ATO-treated rats had significantly higher creatinine clearance rate and less amount of proteinuria. Histological examination revealed obvious features of CAN in the Allo group, whereas LT/HT groups demonstrated minimal glomerulosclerosis, interstitial fibrosis, intimal thickening, and tubular atrophy. The numbers of infiltrating mononuclear cells (ED1+, CD8+, and CD68+) decreased markedly, and the intragraft expression of transforming growth factor beta1 (TGF-beta1) and collagen III were also significantly attenuated in the LT/HT groups, as compared with the Allo group. The mRNA levels of proinflammatory cytokines (interleukin-2, interferon-gamma, interleukin-10), chemokines (RANTES, MCP-1), and profibrotic genes (TGF-beta1, collagen III) were significantly down-regulated in ATO-treated rats. CONCLUSION: Atorvastatin showed excellent favorable effects on blocking renal inflammation and fibrosis, and thus, efficiently inhibited the development and progression of CAN, which might improve the long-term survival rate of renal allografts.     

Expression of transforming growth factor-beta1 and hypoxia-inducible factor-1alpha in renal transplantation

Chronic allograft nephropathy (CAN) includes pathologic changes of interstitial fibrosis, tubular atrophy, and fibrous intimal thickening. Transforming growth factor (TGF)-beta1 is a fibrogenic cytokine involved in renal allograft fibrosis. Hypoxia-inducible factor (HIF)-1alpha is induced as an adaptive response to hypoxia triggering the production of fibrogenic cytokines such as TGF-beta1. Between January 1995 and February 2005, we performed 71 renal allograft biopsies in 61 recipients. Immunohistochemical studies were performed with an immunoperoxidase technique using as the primary antibody either a rabbit anti-human TGF-beta1 polyclonal or a mouse anti-human HIF-1alpha monoclonal reagent. The glomerular TGF-beta1 expression in recipients diagnosed with glomerulonephritis was significantly greater than other pathologic groups (P < .05), and the glomerular TGF-beta1 expression in the heavy proteinuria group (>/=2.5 g/d) was significantly greater than the low proteinuria group (<1.0 g/d; P < .05). The tubular and interstitial TGF-beta1 and HIF-1alpha expressions in CAN were greater than in other groups (P < .05). The tubular TGF-beta1 expression among the graft loss group was significantly greater than the graft function group (P < .05).     

Vascular endothelial growth factor (VEGF) ligand and receptor induction in rat renal allograft rejection

BACKGROUND: Acute rejection is the single most important risk factor for the subsequent development of chronic allograft nephropathy (CAN), which is still the primary reason for late allograft loss in kidney transplantation. Vascular endothelial growth factor (VEGF) is a proangiogenic factor that has an important role in the development and maintenance of physiological endothelium. While its role has been characterized in the pathology of diabetic nephropathy and preeclampsia, its role in the development of acute and chronic allograft rejection remains unclear. METHODS: Kidney transplantations were performed from DA to WF rats and syngeneic control transplantations were performed between DA rats. Normal kidneys were used as controls to evaluate physiological VEGF and VEGFR-1 expression. Allografted rats were immunosuppressed with cyclosporine (CsA) (1.5 mg/kg/d subcutaneously); and no immunosuppression was given to syngeneic grafts. Grafts were harvested at 5 and 90 days after transplantation for histology and immunohistochemistry (VEGF, VEGFR-1). RESULTS: In normal kidneys VEGF ligand and receptor expression was almost nonexistent. Only mild glomerular, arterial, and tubular VEGF expression was seen. In syngeneic grafts, no histological signs of acute or chronic rejection were seen, whereas characteristics of both acute and chronic rejection were seen in CsA-treated allografts. Altough VEGF expression was increased in syngenic grafts when compared to controls it still remained mild in both the early and the late posttransplant period. In CsA-treated allografts moderate VEGF expression was seen already 5 days after transplantation; the expression increased at 90 days after transplantation. The same pattern was also discovered for VEGFR-1 expression although the difference was not as remarkable after 5 days. CONCLUSIONS: Our results demonstrated that VEGF ligand and receptor expression was increased in both acute and chronic rejection. Our data suggested that VEGF may have an important role in the pathology of chronic rejection. Based on our findings VEGF inhibition could be a potential intervention to prevent CAN in clinical kidney transplantation.     

PTK787/ZK222584 Combined with Interferon Alpha and 5-Fluorouracil Synergistically Inhibits VEGF Signaling Pathway in Hepatocellular Carcinoma

Background

The prognosis of patients with hepatocellular carcinoma (HCC) and portal vein tumor thrombus remains poor. We previously reported the beneficial effects of interferon alpha (IFN) and 5-fluorouracil (5-FU) combination therapy for these patients. We showed that the mechanism of therapy was regulation of vascular endothelial growth factor (VEGF). Here, we combined IFN/5-FU therapy with the VEGF receptor–selective inhibitor PTK787/ZK222584 (PTK/ZK) and examined the antitumor effects and the mechanism of action.

Methods

We studied two HCC cell lines, PLC/PRF/5 and HuH7, and a human umbilical vein endothelial cell line, HUVEC. We studied the effects of IFN/5-FU with or without PTK/ZK in growth inhibition assays, immunohistochemistry, Western blot analysis, and immunocytochemistry.

Results

In a HuH7 xenograft model, the combination of PTK/ZK and IFN/5-FU significantly inhibited proliferation, induced apoptosis, decreased microvessel density, reduced the number of tumor cells that expressed VEGF receptor 2 (VEGFR-2), and repressed the phosphorylation of Akt in vivo. In HCC cells and HUVECs in vitro, IFN/5-FU plus PTK/ZK repressed the expression of VEGFR-2 and repressed the phosphorylation of VEGFR, Akt, Erk, and p38MAPK.

Conclusions

VEGF signaling inhibition enhanced the antitumor effects of IFN/5-FU therapy on HCC cells and endothelial cells via Erk, Akt, and p38MAPK pathways.

     

The aetiology and pathogenesis of chronic allograft nephropathy

Renal transplantation is the ultimate form of renal replacement therapy, and is the treatment of choice for many patients with end-stage renal failure. The advent of calcineurin inhibitor based immunosuppression resulted in the 1-year renal allograft failure rate dropping from around 50% twenty years ago to less than 10% in more recent times. Despite a massive improvement in renal allograft survival in the first year following transplantation 10-year graft survival can be as low as 50%. Chronic allograft nephropathy (CAN) is recognised as the main cause of renal allograft failure following the first year after transplantation. The diagnosis of CAN can only be made histologically. Typically biopsy specimens in grafts with CAN demonstrate an overall fibrotic appearance effecting the vascular endothelium, renal tubules, interstitium, and glomerulus. The risk factors for CAN are divided into alloimmune and alloimmune independent. Alloimmune dependent factors include acute cellular rejection, severity of rejection, subclinical rejection and HLA mismatch. Alloimmune independent factors such as delayed graft function, donor age, Cytomegalovirus infection, donor/recipient co-morbidity and of course calcineurin inhibitor toxicity are important in the development of CAN. The pathogenesis of CAN is complex, multifactorial, and unfortunately incompletely understood. There are a number of pivotal steps in the initiation and propagation of the fibrosis seen in biopsy specimens from kidneys with CAN. Endothelial activation in response to one or more of the aforementioned risk factors stimulates leukocyte activation and recruitment. Recruited leukocytes subsequently infiltrate through the endothelium and induce key effector cells to secrete excessive and abnormal extracellular matrix (ECM). Enhanced deposition of ECM is a histological hallmark of CAN. This paper aims to present a concise yet accurate and up-to-date review of the literature concerning the aetiological factors and pathological processes which are present in the generation of CAN.     

Rat cytomegalovirus infection and chronic kidney allograft rejection

Abstract  To investigate the effect of cytomegalovirus (CMV) infection on the development of experimental chronic kidney allograft rejection, orthotropic kidney allografts from DA donors (Ag- RTl^al) to WF (Ag- RTl^u) recipients were used. The rats received cyclosporine A (CsA) for 12 weeks. A group of recipients was infected with 10⁵ plaque-forming units of rat CMV (RCMV), and another group was left non-infected and used as controls. The grafts were removed 12 weeks after transplantation. RCMV infection significantly enhanced the development of chronic kidney allograft rejection as follows: the intensity of interstitial inflammation ( P < 0.025), particularly the degree of pyroninophilic cells in the inflammatory infiltrate ( P < 0.025); the glomeruli mesangial matrix increase ( P < 0.05) and capillary basement membrane thickening ( P < 0.01); the extent of endothelial cell swelling ( P < 0.025) and intimae proliferation (P < 0.025) in the graft vasculature; and the extent of tubular epithelial atrophy ( P < 0.025). The chronic allograft damage index (CADI) was significantly increased to 4.2 ± 0.9 in RCMV-infected allograft, compared with 0.8 ± 0.4 in non-infected ( P < 0.02). At the molecular level, RCMV infection significantly increased vascular endothelial ( P < 0.05) and tubular epithelial ( P < 0.01) ICAM-1 expression. Viral antigens were detected in tubular epithelial cells and in some inflammatory cells.