M. Ramsperger-Gleixner

Differential Expression of Sucrose Transporter and Polyol Transporter Genes during Maturation of Common Plantain Companion Cells

The cDNAs of two sorbitol transporters, common plantain (Plantago major) polyol transporter (PLT) 1 and 2 (PmPLT1 and PmPLT2), were isolated from a vascular bundle-specific cDNA library from common plantain, a dicot plant transporting Suc plus sorbitol in its phloem. Here, we describe the kinetic characterization of these sorbitol transporters by functional expression in Brewers yeast (Saccharomyces cerevisiae) and in Xenopus sp. oocytes and for the first time the localization of plant PLTs in specific cell types of the vascular tissue. In the yeast system, both proteins were shown to be uncoupler sensitive and could be characterized as low-affinity and low-specificity polyol symporters. The Km value for the physiological substrate sorbitol is 12 mm for PmPLT1 and even higher for PmPLT2, which showed an almost linear increase in sorbitol transport rates up to 20 mm. These data were confirmed in the Xenopus sp. system, where PmPLT1 was analyzed in detail and characterized as a H+ symporter. Using peptide-specific polyclonal antisera against PmPLT1 or PmPLT2 and simultaneous labeling with the monoclonal antiserum 1A2 raised against the companion cell-specific PmSUC2 Suc transporter, both PLTs were localized to companion cells of the phloem in common plantain source leaves. These analyses revealed two different types of companion cells in the common plantain phloem: younger cells expressing PmSUC2 at higher levels and older cells expressing lower levels of PmSUC2 plus both PLT genes. The putative role of these low-affinity transporters in phloem loading is discussed.

Attenuation of transplant arteriosclerosis with clopidogrel is associated with a reduction of infiltrating dendritic cells and macrophages in murine aortic allografts.

Background. Monotherapy with clopidogrel reduced the formation of transplant arteriosclerosis in a murine aortic allograft model. However, the underlying immunologic mechanisms are still unknown. Methods. Fully major histocompatibility complex-mismatched C57BL/6 (H2b) donor aortas were transplanted into CBA.J (H2k) recipients and mice received different doses (1, 10, and 20 mg/kg) of clopidogrel, an antagonist of the P2Y12 ADP receptor on platelets, or control saline for 30 days. Blood was analyzed for changes in adhesion molecule and sCD40L concentrations by ELISA. Grafts were analyzed by histology, morphometry, and immunofluorescence on day 30 after transplantation. Intragraft cytokine mRNA production was analyzed by reverse-transcriptase polymerase chain reaction on day 14 after transplantation. Results. Treatment with clopidogrel resulted in significantly decreased blood concentrations of sCD40L and P-selectin after transplantation. Cellular analysis of the aortic transplant revealed fewer numbers of infiltrating dendritic cells (CD205+) and macrophages (F4/80+) after application of clopidogrel, whereas T-cells within the graft were unaltered. In addition cellular P-/E-selectin, ICAM-1, and platelet-derived-growth-factor (PDGF)-&bgr; surface expression were significantly reduced as compared with untreated controls. Intragraft mRNA expression confirmed these results and showed significant lower production of P-/E-selectin, ICAM-1, and PDGF-&bgr; after treatment with clopidogrel. Antiglycoprotein-Ib and antiglycoprotein VI had no beneficial effect on the development of transplant arteriosclerosis. Conclusion. This report shows that application of clopidogrel after transplantation results in a reduction in adhesion molecule expression within the blood and transplant tissue and is associated with reduced transendothelial migration of dendritic cells and macrophages within the vascular wall.

Combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis in murine aortic allografts

Our group has shown that platelet inhibition with clopidogrel, an antagonist of the P2Y12 adenosine diphosphate receptor on platelets, reduced the formation of transplant arteriosclerosis. The aim of this study was to investigate whether a combination of cyclosporin or everolimus with clopidogrel has a beneficial effect on the development of transplant arteriosclerosis. Fully MHC mismatched C57Bl/6 (H2b) donor aortas were transplanted into CBA.J (H2k) recipients and mice received either clopidogrel alone (1 mg/kg/day) or in combination with cyclosporin (2 mg/kg/day) or everolimus (0.05 mg/kg/day). Grafts were analysed by histology and morphometry on day 30 after transplantation. In mice treated with clopidogrel alone, transplant arteriosclerosis was significantly reduced [intima proliferation 56 ± 11% vs. 81 ± 7% (control)/n = 7]. Daily application of everolimus reduced the development of transplant arteriosclerosis compared with untreated controls [intima proliferation of 29 ± 9% vs. 81 ± 7% (control)/n = 7]. Strikingly, combination of clopidogrel and everolimus almost abolished the formation of transplant arteriosclerosis [intima proliferation: 11 ± 8% vs. 81 ± 7% (control)/n = 7]. By contrast, combination of cyclosporin and clopidogrel compared with clopidogrel alone showed no additive effect. These results demonstrate that combination of platelet‐ and mammalian target of Rapamycin‐inhibition can dramatically reduce the development of transplant arteriosclerosis.

Murine cytomegalovirus infection leads to increased levels of transplant arteriosclerosis in a murine aortic allograft model.

Introduction. Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model. Methods. Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR. Results. After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%±9.6% [MCMV+] vs. 43.9%±5.1% [MCMV−]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%±7.3% [MCMV+] vs. 20.2%±1.7% [MCMV−]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4+, CD8+, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant. Conclusion. These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

Delayed therapy with clopidogrel and everolimus prevents progression of transplant arteriosclerosis and impairs humoral alloimmunity in murine aortic allografts

OBJECTIVES It was previously shown that the combination of clopidogrel and everolimus reduced the development of transplant arteriosclerosis. The aim of this study was to investigate whether delayed onset of treatment, similar to the clinical situation after heart transplantation, inhibits progression of transplant arteriosclerosis. METHODS Fully allogeneic C57BL/6 (H2-b) donor aortas were transplanted into CBA.J (H2-k) recipients treated with clopidogrel and everolimus alone or in combination starting on Days 1, 7 or 14. Grafts were analysed by histology and alloantibodies were detected by fluorescence activated cell sorting after transplantation. RESULTS Delayed platelet inhibition with clopidogrel reduced the development of transplant arteriosclerosis [neointima formation (Day 14): 50±4 vs 84±9% (control)]. The combination of clopidogrel and everolimus almost abolished formation of transplant arteriosclerosis when therapy was started on Day 1 [neointima formation (Day 1): 14±5 vs 84±9% (control)] and also showed a remarkable reduction in both delayed treatment groups [neointima formation (Day 7): 24±7 vs 84±9% (control); neointima formation (Day 14): 28±11 vs 84±9% (control)]. Platelet inhibition alone and in combination with everolimus resulted in reduced alloantibody production. CONCLUSIONS These results demonstrate that delayed treatment with clopidogrel and everolimus-representative of a clinical setting-prevents the progression of transplant arteriosclerosis and impairs humoral immunity in this experimental model.

Clopidogrel reduces post-transplant obliterative bronchiolitis

Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction (CLAD). The aim of this study was to investigate if platelet inhibition by clopidogrel has an influence on the formation of obliterative bronchiolitis, the histopathological correlate to bronchiolitis obliterans syndrome, present in the majority of patients suffering from CLAD. C57Bl/6(H2b) donor tracheas were orthotopically transplanted into CBA.J(H2k). Mice received different doses of clopidogrel alone or in combination with tacrolimus or everolimus. Grafts were analyzed by histology and immunofluorescence method on postoperative days 15, 30 or 60. Cytokines were analyzed by real‐time polymerase chain reaction on postoperative day 21 and alloantibodies by FACS. Mice treated with 20 mg/kg/day clopidogrel for 30 days showed reduced obliteration [34.40 ± 3.76% (20 mg/kg/day clopidogrel) vs. 49.92 ± 2.11% (control), n = 5, P < 0.05]. Platelet inhibition resulted in significant lower infiltration of T cells and macrophages, and we also found significantly lower expression of IL‐12, IL‐4, IL‐6, TNF‐α, TGF‐β, PDGFβ, MCP1, P‐/E‐selectin, ICAM1 and CD40L after treatment with clopidogrel. Combination of 1 mg/kg/day clopidogrel and 0.05 mg/kg/day everolimus or 12 mg/kg/day tacrolimus revealed a synergistic effect. Humoral immunity as manifested by donor‐specific alloantibody secretion was also impaired after treatment with clopidogrel. Here, we can show that platelet inhibition by clopidogrel as a single treatment and in combination with tacrolimus or everolimus reduced the development of fibrosis and obliteration in tracheal allografts.

Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model

OBJECTIVES The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. METHODS MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). RESULTS Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P < 0.01]. In addition, experimental groups revealed a down-regulation of E-selectin (-57%) and MCP1 (-33%) expression after ICA pretreatment compared with controls, going along with decreased T-cell [1.4% CD4+ T-cell infiltration vs 8.4% (control) and 4.9% CD8+ T-cell infiltration vs 10.7% (control)], dendritic cell (0.6% dendritic cells infiltration vs 1.9% infiltration(control)] and macrophage infiltration [4.8% macrophages (ICA) vs 10.9% (control)] within vascular grafts. CONCLUSIONS These data of an animal transplant model show that the pharmaceutical activation of HIF with endogenous up-regulation of protective target genes leads to adaptation of the graft to low oxygen-saturation and hereby attenuates the development of transplant arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

Microvascular integrity plays an important role for graft survival after experimental skin transplantation

BACKGROUND Every transplanted organ relies on a reliable and sound vascular system. Therefore, our study focused on the investigation if platelet inhibition alone or combined with mTOR-inhibition has a beneficial effect on the microvascular integrity in allogeneic murine skin grafts. METHODS Skin transplantation was performed from fully MHC-mismatched C57BL/6 (H-2b) donors to CBA/J (H-2k) recipient mice. Skin allograft recipients were assigned to several experimental groups and either treated with clopidogrel alone, everolimus alone or a combination of both. Graft survival was evaluated and transplants were harvested after 8 days and analyzed for CD31 and C4d by immunohistochemistry. RESULTS Untreated allografts showed a reduced amount of CD31 on postoperative day 8 as well as an increase in C4d compared to isografts. All treated animals showed a significant improvement regarding CD31 [1577.7 ± 200.4 (clopidogrel)/1702.8 ± 151.1 (clopidogrel + everolimus) vs. 479.7 ± 184.2 (control), n = 8, p b 0.05] and C4d [420.9 ± 70.9 (clopidogrel)/324.5 ± 77.3 (clopidogrel + everolimus) vs. 772.4 ± 159.7 (control), n = 8, p b 0.05]. In addition, skin grafts of animals treated with clopidogrel and everolimus survived significantly longer compared to untreated controls [19.2 ± 4.2 d vs. 12.8 ± 2.4 d, n= 10, p b 0.05]. CONCLUSION In this study we could show that clopidogrel alone and in combination with everolimus substantially improved microvascular integrity and resulted in increased survival time of skin grafts.

Reduction of Transplant Arteriosclerosis After Treatment With Mycophenolate Mofetil and Ganciclovir in a Mouse Aortic Allograft Model

OBJECTIVES Transplant arteriosclerosis is a major obstacle for long-term allograft survival in heart transplant. The aim of this study was to investigate potential synergistic effects of combined treatment with mycophenolate mofetil and ganciclovir on the development of transplant arteriosclerosis, presence of regulatory T cells, and expression of donor specific alloantibodies. MATERIALS AND METHODS Donor aortas from C57BL/6 (H2b) mice that were fully mismatched to the major histocompatibility complex were transplanted into CBA (H2k) mouse recipients. Groups of mice received mycophenolate mofetil (100 or 300 mg/kg, oral), ganciclovir (10 or 72 mg/kg, intraperitoneal), or a mycophenolate mofetil and ganciclovir combination. Grafts were analyzed by histology and morphometry on day 30 after transplant. Numbers of regulatory T cells and donor-specific alloantibodies were examined by fluorescence-activated cell sorting analysis of splenic tissue and peripheral blood. RESULTS Mycophenolate mofetil (100 mg/kg) and ganciclovir (10 mg/kg and 72 mg/kg) did not show effects on transplant arteriosclerosis formation or alloantibody production. However, groups treated with mycophenolate mofetil (300 mg/kg) or a low- or high-dose mycophenolate mofetil and ganciclovir combination had significantly reduced transplant arteriosclerosis and alloantibody levels. Expression of regulatory T cells within the spleen was similar between all experimental groups and untreated controls. CONCLUSIONS The combination of mycophenolate mofetil and ganciclovir significantly reduced the development of transplant arteriosclerosis in a mouse abdominal aortic allograft model. This effect may be a result of decreased alloantibody production.

Reduction of obliterative bronchiolitis (OB) by prolyl-hydroxylase-inhibitors activating hypoxia-inducible transcription factors in an experimental mouse model.

BACKGROUND Obliterative bronchiolitis (OB) is the major limiting factor for long-term survival after lung transplantation. As previously shown, donor treatment with a PHD-inhibitor activating hypoxia-inducible transcription factors (HIFs) prevents graft injury both in an allogenic kidney and aortic allograft transplant model. The aim of this study was to investigate the effect of HIF activation with a PHD-inhibitor on the development of OB. METHODS Fully MHC-mismatched C57BL/6 (H-2b) donor tracheas were orthotopically transplanted into CBA/J (H-2k) recipients. Donor animals received a single dose of PHD-inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40mg/kg i.p.) or vehicle control 4h before transplantation. Transplanted tracheas were harvested 14 or 30days after transplantation and were analyzed by histology, by immunofluorescence and by rtPCR for mRNA expression. RESULTS Donor pre-conditioning with ICA resulted in HIF accumulation and induction of HIF target genes: HO-1, VEGF, MIF, TGFβ, and EpoR, which persisted during different times of ischemia. Grafts of vehicle treated controls showed substantially more luminal obliteration on postoperative day 30 in contrast to groups pre-treated with ICA [luminal obliteration 29.2±5% (ICA) vs. 36.7±8% (control), p<0.01]. We found significantly lower expression of TNFα, PDGFß, MCP-1, E-selectin, and ICAM-1 14days after ICA premedication. In addition ICA pre-treated groups revealed decreased T-cell and macrophage infiltration in tracheal grafts on days 30 after transplantation (p<0.05). CONCLUSION Pre-treatment with ICA effectively reduced obliterative bronchiolitis. Our data suggest that activation of hypoxia-inducible transcription factors (HIFs) and thereby adaptation to low oxygen prevents the development of OB and allograft injury. Pharmaceutical inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves further clinical evaluation.