Oral Platelet-Derived Growth Factor and Vascular Endothelial Growth Factor Inhibitor Sunitinib Prevents Chronic Allograft Injury in Experimental Kidney Transplantation Model.

BACKGROUND: Expression of both platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) is increased during the development of chronic rejection which remains the major reason for late allograft loss in clinical kidney transplantation. Sunitinib is a tyrosine kinase inhibitor which inhibits both VEGF and PDGF receptors. Here we investigated its effect on the development of chronic rejection. METHODS: Rat aortic denudation model was used to define sunitinib dose. In vitro studies were done to investigate the effect of sunitinib on smooth muscle cell proliferation and migration. Kidney transplantations were performed from dark agouti rat strain (DA) to Wistar furth rat strain rats and syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed either with cyclosporine or with cyclosporine and sunitinib. Grafts were harvested at 5 and 90 days for histology and immunohistochemistry. Serum creatinine levels were measured weekly to monitor graft function. RESULTS: Sunitinib decreased neointimal formation and smooth muscle cell proliferation and migration in a dose-dependent manner. Sunitinib was well tolerated and almost completely prevented chronic rejection changes and preserved significantly better renal graft function after transplantation. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expressions. CONCLUSIONS: These results demonstrate that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation which indicates that sunitinib could be a potential intervention also in clinical kidney transplantation.

Epidermal growth factor receptor inhibition with erlotinib ameliorates anti-Thy 1.1-induced experimental glomerulonephritis.

BACKGROUND: Mesangial proliferative glomerulonephritis is a common glomerular disorder that may lead to end-stage renal disease. Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation and in the pathology of various renal diseases. Erlotinib is a novel, oral, highly selective tyrosine kinase inhibitor of the EGF receptor. It is clinically used to treat non-small cell lung and pancreatic cancers. Here, we investigated the effect of erlotinib on the progression of mesangioproliferative glomerulonephritis in an experimental model. METHODS: Mesangial glomerulonephritis was induced with anti-rat Thy-1.1 antibody in male Wistar rats weighing 150-160 g. Rats were treated with erlotinib (10 mg/kg/day p.o.) or vehicle only (polyethylene glycol). Native Wistar rat kidneys were used as histological controls. Serum creatinine levels were measured at day 7. Kidneys were harvested 7 days after antibody administration for histology. RESULTS: Native controls showed no histological signs of glomerular pathology. In the vehicle group, intense glomerular inflammation developed after 7 days and prominent mesangial cell proliferation and glomerular matrix accumulation was seen. Erlotinib was well tolerated and there were no adverse effects during the follow-up period. Erlotinib significantly prevented progression of the glomerular inflammatory response and glomerular mesangial cell proliferation as well as matrix accumulation when compared with the vehicle group. Erlotinib also preserved renal function. CONCLUSION: These results indicate that erlotinib prevents the early events of experimental mesangial proliferative glomerulonephritis. Therefore, inhibition of the EGF receptor with erlotinib could prevent the progression of glomerulonephritis also in clinical nephrology.

Intensive perioperative simvastatin treatment protects from chronic kidney allograft injury.

BACKGROUND/AIMS: Ischemia-reperfusion injury (IRI) and innate immune response augment adaptive immunity and may also trigger repair processes that lead to uncontrolled fibrosis and atherosclerosis as seen in chronic allograft injury. Simvastatin has been shown to protect from renal IRI in several experimental studies. The aim of this study was to examine the effect of donor simvastatin pretreatment and early initiation of recipient simvastatin treatment on chronic kidney allograft injury. METHODS: A rat renal transplantation model was used. Simvastatin was administered perorally for donor (5 mg/kg) and/or for recipient (2 mg/kg) 2 hours before transplantation and/or as daily treatment starting on the first postoperative day (2 mg/kg/day). The study included 5 groups: (1) no simvastatin, (2) donor pretreatment, (3) daily recipient treatment, (4) donor pretreatment + daily recipient treatment and (5) donor pretreatment + recipient pretreatment + daily recipient treatment. The grafts were recovered at day 90 for histopathological and immunohistochemical analysis. Kidney function was followed weekly with serum creatinine, and 24-hour urine protein was measured 60 and 90 days after transplantation. RESULTS: We found that donor and recipient simvastatin pretreatment combined with daily recipient treatment reduced graft inflammation and chronic allograft injury. Treatment using only statins started after transplantation reduced inflammation to some extent, but did not affect chronic kidney allograft injury. Pretreatment using only donor statins impaired graft function and increased proteinuria. CONCLUSION: Our data suggest that perioperative recipient statin treatment reduces inflammation and may protect the graft in the long term.

Health-related quality of life after kidney transplantation: who benefits the most?

The influence of dialysis modalities on HRQoL before and after kidney transplantation (KT) and the role of adherence to medication on HRQoL have not been fully studied. Sixty four dialysis patients who answered the 15D HRQoL survey during dialysis were surveyed again after KT. Adherence and employment were also investigated. The mean 15D score was highest among home hemodialysis patients (HHD) and lowest among in-center hemodialysis patients (icHD). After KT, the mean 15D score improved significantly in 78.6% of peritoneal dialysis patients (PD), 47.6% of HHD, and 53.8% of icHD. Then, mean 15D score remained unchanged in 28.6% of HHD and in 23.1% of icHD patients. A deterioration in the 15D score occurred in 14.3% of PD, 23.1% of icHD, and 23.8% of HHD patients, and this was influenced by the number of pills (P = 0.04). Adherence to medication was the lowest in PD, timing being the most challenging task showing a connection to higher creatinine concentration (never forgot 1.41 mg/dl vs. forgot 2.08 mg/dl P = 0.05). Employed patients had a higher mean 15D score. The icHD and PD patients benefited the most from KT and HHD the least. Low pill burden and employment were linked to a better HRQoL.

Epidermal growth factor inhibition, a novel pathway to prevent chronic allograft injury.

BACKGROUND: Chronic allograft injury remains a major problem in clinical kidney transplantation and different growth factors participate in its development. Epidermal growth factor (EGF) affects cell proliferation and mitogenesis through its tyrosine kinase receptor. Erlotinib is an orally administered tyrosine kinase inhibitor used in clinical oncology to inhibit EGF signaling. We investigated its effect on the development of chronic allograft injury in an experimental kidney transplantation model. METHODS: Kidney transplantations were performed between Dark Agouti and Wistar Furth rats. Recipients were immunosuppressed either with cyclosporine A (CsA, 1.5 mg/kg/day subcutaneously) or with CsA and erlotinib (10 mg/kg/day orally). Kidney grafts were harvested after 5 and 90 days for histology and immunohistochemistry. Aorta denudation model was used for the erlotinib dose response study to define the optimal dose for the transplantation study. RESULTS: Epidermal growth factor expression was increased in CsA-treated allografts which developed intense chronic changes on day 90. Erlotinib ameliorated neointimal formation in the dose response study. In addition, erlotinib decreased chronic rejection changes and maintained better graft function in kidney transplantation model. Late posttransplant EGF and EGF receptor levels were reduced with erlotinib. CONCLUSION: Based on these findings, EGF mediates in part the development of chronic allograft injury. Its inhibition with erlotinib prevents chronic rejection and maintains better allograft function. Therefore, EGF blocking by erlotinib provides a novel pathway to prevent chronic allograft injury.

TGF-ß1 expression in kidney allograft protocol biopsies during cyclosporine A therapy.

BACKGROUND: TGF-ß1 expression has been described to increase along with time from transplantation and has also been linked to allograft dysfunction and toxic effects of cyclosporine. Our aim was to correlate intragraft TGF-ß1 expression with cyclosporine exposure after kidney transplantation.
 METHODS: Altogether 53 kidney allograft protocol biopsies from 42 patients on a low-dose cyclosporine-based regimen obtained at 3, 6, and 12 months were classified according to Banff and the chronic allograft damage index (CADI). TGF-ß1 expression in tubules, glomeruli, vessels, and inflammatory cells was semi-quantitatively scored and correlated with cyclosporine concentrations (C0 and C2), CADI, and graft function. 
 RESULTS: TGF-ß1 expression was mildly increased along time from transplantation, but the results were not statistically significant. TGF-ß1 expression was neither related to CADI nor to the use of ACE inhibitors/ARB. TGF-ß1 expression in the kidney was not correlated with C0 or C2 levels or kidney graft function during follow-up. 
 CONCLUSION: In protocol biopsies from patients on low-dose cyclosporine regimen, expression of TGB-ß1 was not significantly increased along time since transplantation, and did not correlate with cyclosporine exposure. Our findings suggest that the toxic effects of low-dose cyclosporine on TGF-ß expression may be milder than previously thought.

Sirolimus inhibits lymphangiogenesis in rat renal allografts, a novel mechanism to prevent chronic kidney allograft injury.

Lymphangiogenesis occurs in renal allografts and it may be involved in the maintenance of the alloreactive immune response and thus participate in the development of chronic kidney allograft injury. Sirolimus (SRL) has been shown to inhibit lymphangiogenesis. The aim of this study was to describe lymphangiogenesis and its regulation during the development of chronic kidney allograft injury and to investigate the effect of SRL on allograft lymphangiogenesis and chronic kidney allograft injury. A rat renal transplantation model was used. Allografts treated with cyclosporine A or with SRL were analyzed in various time points. Syngenic transplantations were used as controls. Kidney function was followed with serum creatinine. Histology was analyzed by Chronic Allograft Damage Index (CADI). Immunohistochemistry was used to detect lymphatic vessels, VEGF-C and VEGFR-3. In cyclosporine-treated allografts VEGF-C/VEGFR-3 pathway was strongly upregulated leading to extensive lymphangiogenesis 60 days after transplantation. Lymphangiogenesis correlated positively with the CADI score. Sirolimus efficiently inhibited lymphangiogenesis, improved graft function and attenuated the development of chronic kidney allograft injury when compared with cyclosporine. In conclusion, lymphangiogenesis is associated with chronic kidney allograft injury and SRL is a potent inhibitor of lymphangiogenesis in renal allografts. Inhibition of lymphatic proliferation could mediate the nephroprotective properties of SRL.

Early short-term imatinib treatment is sufficient to prevent the development of chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN), now defined as interstitial fibrosis and tubular atrophy not otherwise specified, is a near universal finding in kidney grafts by the end of the first decade posttransplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. Here, we investigated whether early short-term PDGF inhibition with imatinib could prevent CAN. METHODS: Kidney transplantations were performed from Dark-Agouti (DA) to Wistar-Furth (WF) rats and syngenic controls were done between DA rats. Allografts were immunosuppressed with cyclosporine. One group was also treated with imatinib for the first 30 days after transplantation. Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation. RESULTS: In control allografts, moderate to intense chronic changes were seen, whereas in syngenic grafts, no changes were seen. The early imatinib treatment prevented the development of CAN significantly compared to control allografts. Only few histological changes were seen. Fibrogenic growth factor ligand and receptor induction as well as inflammatory cell response was significantly inhibited by imatinib. Creatinine values of imatinib-treated allografts were also significantly lower compared to controls. CONCLUSIONS: We show that short-term imatinib treatment is sufficient to prevent CAN significantly, indicating that early PDGF induction has an important role in the pathogenesis of CAN. Here, we provide preclinical work that will need to be confirmed in patients with CAN.

Targeting lymphatic vessel activation and CCL21 production by vascular endothelial growth factor receptor-3 inhibition has novel immunomodulatory and antiarteriosclerotic effects in cardiac allografts.

BACKGROUND: Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. METHODS AND RESULTS: Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C(+) inflammatory cell and hyaluronan receptor-1 (LYVE-1)(+) lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3(+), contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein-positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8(+) effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62(+) dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4(+) T cells in chronically rejecting mouse cardiac allografts. CONCLUSIONS: These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel-targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

PDGF-A, -C, and -D but not PDGF-B increase TGF-beta1 and chronic rejection in rat cardiac allografts.

OBJECTIVE: Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A-D)--potent mesenchymal cell mitogens--in rat cardiac allografts. METHODS AND RESULTS: PDGFR-alpha mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac centhatn allografts. In acute rejection, PDGFR-alpha immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors--except that of PDGF-B ligand--was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-beta1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection. CONCLUSIONS: We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-beta1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

Tacrolimus treatment effectively inhibits progression of obliterative airway disease even at later stages of disease development.

BACKGROUND: Obliterative bronchiolitis (OB) is the most prominent cause of morbidity and mortality among lung transplant patients. No effective treatment for OB exists, but preliminary clinical studies have suggested that a calcineurin inhibitor, tacrolimus, may delay the development of OB when compared with standard cyclosporine-based immunosuppression. METHODS: Using a murine tracheal transplantation model, we examined the effects of tacrolimus prophylaxis and treatment on the development of obliterative airway disease (OAD). Tracheal allografts were transplanted heterotopically from BALB/c to C57 black mice into a subcutaneous pouch. The mice received different doses of tacrolimus monotherapy, ranging from 0 to 3 mg/kg/day, subcutaneously initiated at 0 (prophylaxis), 7 (early treatment) or 14 (late treatment) days. We harvested the grafts 30 days after transplantation for histologic and immunohistochemical analyses. RESULTS: We found that tacrolimus prophylaxis dose-dependently inhibited OAD, and that early treatment halts OAD progression and that late treatment delays progression. Syngeneic grafts showed no obliterative changes. Tacrolimus prophylaxis was associated with inhibition of recruitment of CD4+, CD8+ and interleukin-2R+ inflammatory cells into the allografts, suggesting a central role for interleukin-2 in the development of OAD. In addition, a dose-dependent correlation between epithelial necrosis and tracheal occlusion was observed, suggesting that epithelial injury is required for the development of OAD. When tacrolimus treatment was initiated at the time the obliterative lesion had already started to develop, it inhibited the progression of OAD significantly. CONCLUSIONS: The findings from this study suggest that tacrolimus therapy is effective during the early stages of clinical OB.

Vascular endothelial growth factor in chronic rat allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is a complex process of alloimmune responses and chronic inflammation leading to fibrosis and vasculopathy. We examined the biological role of proinflammatory vascular endothelial growth factor (VEGF) in a rat renal transplantation model of CAN. METHODS: Syngraft and allograft recipients were treated with a suboptimal dose of cyclosporine A which allows acute rejection and CAN to develop. Intragraft VEGF, VEGFR-1 and VEGFR-2 expressions were determined at 5, 14, 30 and 60 days. Protein tyrosine kinase inhibitor PTK787 was used to inhibit VEGFR activity. RESULTS: In nontransplanted kidneys and syngrafts, mild VEGF expression was observed in the glomeruli and tubuli. VEGFR-1 was detected in vascular structures and VEGFR-2 in glomeruli as well. In allografts, total intragraft VEGF expression and interstitial inflammatory cell VEGF expression were induced and correlated with the chronic allograft damage index (CADI) score. Total intragraft and interstitial inflammatory cell VEGFR-1 expression was induced and interstitial cell VEGFR-1 expression correlated with the CADI score. Blocking VEGF receptor signaling with PTK787 significantly reduced fibrosis and the CADI score, but did not affect early inflammation or VEGF, VEGFR-1, VEGFR-2 expressions compared to vehicle treated group. CONCLUSIONS: Interstitial inflammatory cell VEGF and VEGFR-1 expressions are induced during the development of CAN. Increased VEGF activity may enhance the alloimmune induced inflammatory responses leading to fibrosis and CAN.

VEGFR-1 and -2 regulate inflammation, myocardial angiogenesis, and arteriosclerosis in chronically rejecting cardiac allografts.

OBJECTIVE: Interplay between inflammation and angiogenesis is important in pathological reparative processes such as arteriosclerosis. We investigated how the two vascular endothelial growth factor receptors VEGFR-1 and -2 regulate these events in chronically rejecting cardiac allografts. METHODS AND RESULTS: Chronic rejection in mouse cardiac allografts induced primitive myocardial, adventitial, and intimal angiogenesis with endothelial expression of CD31, stem cell marker c-kit, and VEGFR-2. Experiments using marker gene mice or rats as cardiac allograft recipients revealed that replacement of cardiac allograft endothelial cells with recipient bone marrow- or non-bone marrow-derived cells was rare and restricted only to sites with severe injury. Targeting VEGFR-1 with neutralizing antibodies in mice reduced allograft CD11b+ myelomonocyte infiltration and allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardial c-kit+ and CD31+ angiogenesis in the allograft, and decreased allograft inflammation and arteriosclerosis. CONCLUSIONS: These results suggest interplay of inflammation, primitive donor-derived myocardial angiogenesis, and arteriosclerosis in transplanted hearts, and that targeting VEGFR-1 and -2 differentially regulate these pathological reparative processes.

Role of platelet-derived growth factor and vascular endothelial growth factor in obliterative airway disease.

RATIONALE: Platelet-derived growth factor (PDGF) is an important smooth muscle cell mitogen, and vascular endothelial growth factor (VEGF) is a known angiogenic and proinflammatory growth factor. We hypothesized that specific therapy aimed at these growth factors might inhibit the development of experimental obliterative airway disease (OAD). METHODS: In fully mismatched rat tracheal allografts, we used imatinib and PTK/ZK, either alone or in combination, to block PDGF and VEGF receptor protein tyrosine kinase (RTK) action, respectively. Prophylaxis was initiated at the time of transplantation. Early treatment was commenced on Day 7 during the inflammatory phase and late treatment on Day 14 during the fibroproliferative phase of OAD. No immunosuppression was administered. MEASUREMENTS AND MAIN RESULTS: Prophylaxis with either PTK/ZK or imatinib alone significantly reduced OAD, and combined prophylaxis completely prevented its development. Early treatment with PTK/ZK and imatinib also effectively reduced the development of OAD. Late treatment failed to show significant efficacy. Blocking VEGF RTK action with PTK/ZK reduced the activation of allograft blood vessels and the number of lymph vessels in the allograft airway wall, and significantly diminished allograft inflammation, whereas PDGF blockade with imatinib inhibited the growth of smooth muscle cells in the proliferating lesion. CONCLUSIONS: Combined prophylactic PDGF and VEGF RTK blockade completely prevents the development of OAD. Also, when early treatment with PTK/ZK and imatinib is commenced during the inflammatory phase of OAD development, it significantly attenuates the development of tracheal occlusion, suggesting that these drugs could potentially be used to treat bronchiolitis obliterans syndrome in its early phase.

Angiogenic growth factors in cardiac allograft rejection.

Normal adult vasculature is in a quiescent state. In transplanted hearts, peri- and postoperative ischemic and alloimmune stimuli may be interpreted as inadequate tissue perfusion leading to activation of angiogenic signaling. Although this may have protective functions, improper activation of cardiac allograft endothelial cells and smooth muscle cells may actually result in impaired survival of cardiac allografts. In this paper, we review the current knowledge on angiogenic growth factors, vascular endothelial growth factor, angiopoietins, and platelet-derived growth factor in cardiac allografts. We also discuss the potential for therapies aimed at angiogenic growth factors in preventing and treating cardiac allograft rejection and transplant coronary artery disease.

Inhibition of tumor necrosis factor-alpha attenuates myocardial remodeling in rat cardiac allografts.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) elicits a wide range of pro-inflammatory activities on target cells and mediates diverse cardiovascular processes ranging from heart failure to atherosclerosis. Recently, we demonstrated that TNF-alpha regulates the platelelet-derived growth factor (PDGF)-A/PDGF-Ralpha activation pathway in rat cardiac allograft arteriosclerosis. The aim of this study was to determine the kinetics and biologic role of TNF-alpha and its receptors, TNF-R1 and TNF-R2, in rat cardiac allografts. METHODS: Heterotopic heart transplantations were performed from Dark Agouti to Wistar-Furth rats. In the acute rejection model, recipients were given no immunosuppression and grafts were removed 5 days after transplantation. In the chronic rejection model, cyclosporine (CsA) was administered and grafts were removed at 60 days. To investigate the functional role of TNF-alpha in chronic rejection, recipients received recombinant human soluble TNF receptor p80/IgG1 Fc fusion protein (rhu TNF-R2:Fc). RESULTS: During acute and chronic rejection, an increase in intragraft TNF-alpha and TNF-R2 mRNA expression was recorded, but not TNF-R1 mRNA expression. Prominent induction of TNF-alpha and TNF-R2 immunoreactivity was localized to medial cells of coronary arteries and interstitial inflammatory cells, whereas cardiomyocytes showed moderate immunoreactivity to TNF-alpha and its receptors. Inhibition of the TNF-alpha-mediated pathway by TNF-R2:Fc did not affect the incidence or intensity of arteriosclerotic lesions in rat cardiac allografts; however, it significantly inhibited myocardial remodeling with a concomitant decrease in myocardial TNF-alpha expression but not intragraft PDGF immunoreactivity. CONCLUSIONS: We conclude that inhibition of TNF-alpha attenuates myocardial remodeling but is not rate-limiting for arteriosclerotic lesion formation.

Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy.

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.

A prospective study comparing cytomegalovirus antigenemia, DNAemia and RNAemia tests in guiding pre-emptive therapy in thoracic organ transplant recipients.

We evaluated the usefulness of DNAemia and mRNAemia tests in guiding the pre-emptive therapy against cytomegalovirus (CMV) infections in thoracic organ transplant recipients using antigenemia test as the reference. Seven lung (LTR) and 14 heart (HTR) transplant recipients were prospectively monitored for CMV by antigenemia, DNAemia (Cobas Amplicor PCR Monitor) and pp67-mRNAemia (NASBA) tests. However, only the antigenemia test guided pre-emptive therapy with cut-off levels of >or=2 and >or=5-10 pp65-positive leukocytes/50 000 leukocytes in the LTRs and HTRs, respectively. CMV DNAemia was detected in 26/28 (93%) and RNAemia in 17/28 (61%) of the CMV antigenemias requiring antiviral therapy (P = 0.01). Optimal DNAemia levels (sensitivity/specificity) estimated from receiver-operating characteristic curve to achieve maximal sum of sensitivity and specificity were 400 (75.9/92.7%), 850 (91.3/91.3%) and 1250 (100/91.5%) copies/ml for the antigenemia of 2, 5 and 10 pp65-positive leukocytes, respectively. The sensitivities of nucleic acid sequence-based amplification (NASBA) were 25.9%, 43.5% and 56.3% in detecting the same cut-off levels of antigenemia. In thoracic organ transplant recipients, the Cobas PCR assay is comparable with the antigenemia test in guiding pre-emptive therapy against CMV infections when threshold levels of over 5 pp65-antigen-positive leukocytes are used as the reference. In contrast, the low sensitivity of NASBA limits its usefulness in the guidance of pre-emptive therapy.

Bronchoscopy in the diagnosis and surveillance of respiratory infections in lung and heart-lung transplant recipients.

Fiberoptic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) and transbronchial biopsies (TBB) is a widely used method to detect respiratory infections and to differentiate them from other postoperative complications in lung transplant (LTX) recipients, but the usefulness of surveillance FOBs is not yet established. The aim of this study was to evaluate the usefulness of FOB in the diagnosis and surveillance of infections in LTX recipients. We reviewed all the consecutive 609 FOBs performed on 40 lung or heart-LTX recipients between February 1994 and November 2002. The overall diagnostic yield was 115/190 (61%) and 43/282 (15%) for clinically indicated and surveillance FOBs respectively (P < 0.001). Infection was established by bronchoscopic samples in 96/190 (50.5.%) of the clinically indicated FOBs and 34/282 (12.1%) of the surveillance FOBs (P < 0.001). The diagnostic yield of the clinically indicated FOBs was highest (72%) from 1 to 6 months post-transplant (P = 0.04). Pneumocystis carinii was detected in 23 (4.9%) of the bronchoscopic specimens and 15 (65%) of the P. carinii infections were detected during adequate chemoprophylaxis. To conclude, in LTX recipients clinically indicated FOB has a good diagnostic yield in detecting infections and other postoperative complications, whereas the information received from surveillance FOB has remained less significant. With current prophylaxis and screening strategies FOB is still required to detect P. carinii infections.

Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. We investigated the role of vascular endothelial growth factor (VEGF) in the development of OB in rat tracheal allografts. In nonimmunosuppressed allografts, VEGF mRNA and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells with progressive loss of epithelium and airway occlusion compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF(164) gene increased early epithelial cell proliferation and regeneration but increased microvascular remodeling and lymphangiogenesis and luminal occlusion by more than 50% compared with AdlacZ-treated allografts. Although VEGF receptor inhibition decreased early epithelial regeneration in noninfected allografts, it reduced microvascular remodeling, lymphangiogenesis, intragraft traffic of CD4(+) and CD8(+) T cells, and the degree of luminal occlusion. Simultaneous VEGF gene transfer and platelet-derived growth factor receptor inhibition with imatinib preserved respiratory epithelium and totally prevented luminal occlusion. In conclusion, our findings indicate that VEGF has a dual role in transplant OB. Our results suggest that VEGF may protect epithelial integrity. On the other hand, VEGF may enhance luminal occlusion by increasing the recruitment of mononuclear inflammatory cells with platelet-derived growth factor acting as a final effector molecule in this process.