Aortic Transplant Model Research Articles

Repeated CXCR4 Blockade by Plerixafor Attenuates Transplant Vasculopathy in Murine Aortic Allografts.

Plerixafor, a hematopoietic stem cell mobilization agent, increases the peripheral blood content of effector and regulatory T cells and may have beneficial effects on cardiac allograft vasculopathy. The aim of the current study was to evaluate its effects in a murine aortic allograft model using different application procedures. Allogeneic donor aorta grafts (n = 8/group) from C57BL/6 mice(H2b) were abdominally transplanted into CBA mice (H2k). Plerixafor application was performed either continuously for 14 d using abdominally implanted osmotic pumps (1 mg/kg/d) or i.p. with a single dose (1 and 5 mg/kg) on day 0 or pulsed injections of 1 mg/kg on days 0, 7, 14, and 21. Cell distribution was monitored by FACS. Aortic grafts were evaluated for neointima development by Elastica-van-Gieson on day 30. Immunofluorescence and intragraft gene expression analysis were performed. On day 14, significantly fewer hematopoietic stem cells were found in the bone marrow of all plerixafor-treated mice. In the pulsed application group, significantly more hematopoietic stem cells were found in the peripheral blood on day 14 (0.045 ± 0.002%; p < 0.01 [pulsed]; versus 0.0068 ± 0.002% [control]) and also more regulatory T cells. PCR revealed lower inflammatory cytokines. The luminal occlusion was significantly reduced in the pulsed treated group (33.65 ± 8.84 versus 53.13 ± 12.41) going along with decreased neointimal CD4+ T cell and plasmacytoid dendritic cell infiltration, as well as less smooth muscle cell proliferation. The application of plerixafor attenuates chronic rejection in aortic allografts via immunomodulatory effects. Injection of repeated low-dose plerixafor is the most effective application form in the aortic transplant model.

In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7–9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Pirfenidone treatment reduces cardiac allograft vasculopathy in a mouse model

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ISHLT research grant Objectives Chronic rejection in the form of cardiac allograft vasculopathy after heart transplantation remains without effective treatment. Main characteristics of cardiac allograft vasculopathy are immigration of leukocytes and proliferation of smooth muscle cells - collectively resulting in development of a neointima narrowing the vessel lumen and leading to graft ischemia. Pirfenidone is an anti-fibrotic drug with anti-inflammatory properties that is already approved for treatment of idiopathic pulmonary fibrosis. The aim of this study was to evaluate whether application of pirfenidone leads to reduced luminal occlusion corresponding with decreased chronic rejection in a mouse transplant model. Methods Murine thoracic aortas were transplanted into the abdominal aorta of CBA mice (H2k). For allogeneic transplantation, C57BL/6 mice (H2b) were used as donors (n = 8 / group). After transplantation, mice were fed a 0.5% pirfenidone diet versus conventional diet. Preliminary tests were performed determining the blood pirfenidone level by mass spectrometry throughout the study period on days 1, 3, 7, 14, 21 and 28, respectively, as well as after obtaining the aortic grafts on day 30 for histological analysis. Results Preliminary tests confirmed the successful uptake of pirfenidone by achieving constant plasma levels of pirfenidone and its metabolite 5-carboxy-pirfenidone over the whole examination period. Pirfenidone treated murine aortic grafts showed significantly reduced neointima formation compared to control allografts (luminal occlusion: 32.92 % ± 13.02 % vs. 42.44 % ± 19.81 %, p=0.046). As expected, syngeneic aortic grafts with and without pirfenidone treatment showed no luminal occlusion. Conclusion The results indicate that pirfenidone application after aortic transplantation reduces neointima formation and thus chronic rejection in a mouse aortic transplant model. Further analyses of underlying mechanisms are currently underway.

(173) - Blocking Signaling Pathways Like Notch and Hedgehog Reduces Chronic Rejection in a Murine Aortic Transplant Model

(173) - Blocking Signaling Pathways Like Notch and Hedgehog Reduces Chronic Rejection in a Murine Aortic Transplant Model

Allograft Model of Aortic Arch Segment Grafting to Abdominal Aorta Through End-to-Side Anastomosis in Mice

The mouse aortic transplantation model is a valuable tool for investigating the mechanisms of atherosclerosis regression, but few laboratories can generate it due to the operation difficulty, especially for the style of end-to-side anastomosis, which facilitates syngeneic heterotopic transplanting a plaque-rich aortic arch into the abdominal aorta. Here we provide a modified protocol for generating this allograft model, which is capable of overcoming several critical surgical challenges such as separating a longer abdominal aorta segment, reducing bleeding and thrombosis, optimizing aortotomy, and improving end-to-side anastomosis to guarantee a potent graft. By transplanting plaque-rich aortic arches into the abdominal aorta of wildtype mice, a high operation success rate (over 90%) was noted with aortic clamping time under 60 min, the graft potency was satisfactory evidenced by examinations of micro-CT, ultrasound, and lower limb blood flow measurement, while a significant atherosclerosis regression was observed in the grafts at 1 week after transplantation.Graphical

EphB4 monomer inhibits chronic graft vasculopathy in an aortic transplant model

T cells and macrophages play an important role in the formation of allograft vasculopathy, which is the predominant form of chronic rejection in cardiac transplants. Arteries express Ephrin-B2 as a marker of arterial identity, whereas circulating monocytes express the cognate receptor EphB4, which facilitates monocyte adhesion to the endothelial surface. Adherent monocytes transmigrate and differentiate into macrophages that activate T cells and are a main source of tissue damage during rejection. We hypothesized that inhibition of Ephrin-B2-EphB4 binding would decrease immune cell accumulation within a transplanted graft and prevent allograft vasculopathy. We used EphB4 monomer to inhibit Ephrin-B2-EphB4 binding in a rat infrarenal aortic transplant model. Rats treated with EphB4 monomer had fewer macrophages and T cells in the aortic allografts at 28 days, as well as significantly less neointima formation. These data show that the Ephin-B2-EphB4 axis may be an important target for prevention or treatment of allograft vasculopathy.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 enables prevention of transplant arteriosclerosis.

The loss of vascular wall cells in allotransplanted arteries is the initial event leading to transplant arteriosclerosis (TA) and ensuing loss of allograft function. Pharmacological agents able to prevent TA are currently lacking. We previously showed that selective inhibition of the immunoproteasome prevented the chronic rejection of renal allografts. However, the role and mechanisms of selective inhibition of a single immunoproteasome subunit to prevent immune-mediated vascular allograft rejection and TA is not clear. The effect and potential mechanism of combined or individual inhibition of peptidolytically active immunoproteasome LMP7 (β5i) and LMP2 (β1i) subunits on immune rejection-mediated TA was investigated using the epoxyketone inhibitor ONX 0914, and the recently developed LMP7-selective inhibitor KZR-329 and LMP2-selective inhibitor KZR-504 in a rat aorta transplantation model. We find that co-inhibition of LMP7 and LMP2 in allogeneic recipients significantly suppressed T-cell activation and function by expressing inhibitory surface markers and then activating inhibitory signals. Moreover, co-inhibition of LMP7 and LMP2 substantially reduced the number of immunoglobulin G-secreting cells and plasma cells and production of alloantibodies through activating the unfolded protein response and incapacitating the survival niche of plasma cells in the bone marrow. Consequentially, the accumulation of inflammatory cytokines, complement, and antibodies is reduced and the apoptosis of vascular wall cells decreased in aortic allografts via LMP7 and LMP2 co-inhibition with ONX 0914 treatment or combined KZR-329 and KZR-504 treatment. However, neither individual inhibition of LMP7 by KZR-329 nor individual inhibition of LMP2 by KZR-504 showed suppression of immune rejection and TA. We define a critical role of LMP7 and LMP2 in TA and strongly propose co-inhibition of both immunoproteasome subunits as promising therapeutic approach to suppress TA and allograft rejection.

Target induction of heme oxygenase-1 attenuates anti-HLA I antibody induced transplant vasculopathy in an allogeneic aortic transplantation model in mice

Abstract Introduction After solid organ transplantation every second patient develops transplant vasculopathy (TV), which still remains the major obstacle for long-term graft survival. The main risk factor for developing TV are de novo synthesized donor-specific antibodies binding molecules of the MHC complex (HLA complex in humans, respectively) on the surface of endothelial cells (EC). EC become activated, vascular smooth muscle cells proliferate, and monocytes are recruited, contributing the development of the neointima a hallmark of TV. We have demonstrated that the anti-HLA I antibody (w6/32) significantly increases transmigration of monocytes across an endothelial monolayer in vitro. These interactions were dependent on modulating the anti-inflammatory enzyme heme oxygenase (HO)-1. Purpose The aim of the project is to investigate which adhesion molecule might mediate the antibody-induced transmigration of monocytes sensitive to HO-1 modulation and whether this modulation has beneficial effects in an allogenic aortic transplantation model in mice. Methods Before w6/32 stimulation, HO-1 was modulated by pretreating Human Coronary Artery Endothelial Cells (HCAEC) either with CoPPIX as a HO-1 activator or with statins to test for a therapeutic approach. Expression of a variety of established adhesion receptors in HCAEC was screened after HO-1 modulation and w6/32 stimulation. Antibody-induced transmigration and adhesion of THP-1 were quantified in the presence of CD62E-blocking antibodies. Thoracic aorta was transplanted into the abdominal aorta between fully MHC mismatched mice and applying the corresponding anti-MHC I antibody (SF1-1.1) induced TV. Mice were treated with CoPPIX, statins or CORM to evaluate the effects of HO-1 modulation or its metabolite CO on TV development. Statistical evaluation was performed with t-test or ANOVA. Results HO-1 modulation in HCAEC diminished w6/32-induced THP-1 transmigration. CD62E-expression in HCAECs was enhanced after w6/32 stimulation on mRNA and protein level, but preincubation with CoPPIX or statins reduced these effects. Incubation of HCAEC with a CD62E-blocking antibody after w6/32 stimulation significantly diminished the rate of transmigration normalized to isotype-stimulated HCAEC (isotype 100%, w6/32 + IgG 226% vs. w6/32 + CD62E-block 138%, p&amp;lt;0.05.). Treatment of mice with CoPPIX resulted in a significant reduction of the SF1-1.1-induced number of transmigrated monocytes (SF1-1.1: 10 vs. CoPPIX: 5.4; p&amp;lt;0.05). Application of statins (SF1-1.1: 10 vs. statin 6.6; p&amp;lt;0.05) or CORM (CORM: 6.1 vs. iCORM: 11.3, p&amp;lt;0.05) significantly reduces monocyte number as well. In all treatment groups, the SF1-1.1-induced neointima formation was ameliorated. Conclusion w6/32-induced monocyte transmigration is CD62E-dependent. SF1-1.1-induced monocytic infiltrate and intimal hyperplasia in a murine aortic transplantation model is also CD62E dependent and amenable to HO-1 modulation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This sudy was funded by the German Research Foundation to Jan Larmann

315.14: The Stem Cell Mobilizator Plerixafor Reduces Transplant Vasculopathy in a Murine Aortic Allograft Model

Purpose: Effector and regulatory T cells (Tregs) are main cell populations in the context of cardiac allograft vasculopathy (CAV). The immunostimulant plerixafor mobilizes hematopoietic stem cells and may boost T cell. Therefore, the aim of this study was to evaluate if a single or repeated application as well as continuous treatment with plerixafor increases peripheral bone marrow derived stem cells including Tregs leading to tolerance induction and thus reducing chronic rejection in a mouse allograft transplant model. Methods: Fully allogeneic mouse aortas from C57BL/6 mice (H2b) were abdominally transplanted into CBA mice (H2k). The continuous plerixafor application [1mg/kg/d] was carried out for 14 days using implanted small osmotic pumps. Single dose (s.c.) injection of 1mg/kg/d or 5mg/kg/d were done at day 1 after transplantation (Tx.) and pulsed injections [1mg/kg/d] were conducted on days 1, 7, 14, and 21 after Tx. The control allograft group received vehicle loaded osmotic pumps. Stem cell mobilization was monitored by FACS. Recipients were sacrificed on day 14 for intragraft gene expression analysis or on day 30 for histological measurements. Results: Murine aortic grafts with pulsed plerixafor injections showed significantly reduced neointima proliferation compared to control allografts (33.65%±8.84% vs.53.13% ±12.41%; p<0.05). The single shot and continuous treatment groups exhibited no improvement concerning neointima formation vs. the untreated group. First FACS analysis revealed significantly less hematopoietic stem cells (HSC) in the bone marrow of plerixafor treated mice vs. the control at day 14 after Tx. Though, there were significantly more HSCs in the peripheral blood on day 30 after Tx, with the pulsed injection even doubling HSCs [0.0152%±0.008; p < 0.005 (pulsed); 0.0046%±0.002%; p < 0.01 (pump); 0.0076%±0.001%; p < 0.01 (single dose of 1mg/kg); 0.0039%±0.0017%; p < 0.1 (single dose of 5mg/kg) vs. 0.0018%±0.0016% (control)]. Intragraft gene expression results showed clearly reduced IFNγ expression and a significantly increase of IL-4, IL-10 and TGFβ. Conclusion: The data suggests that pulsed, continued and single dose application of plerixafor leads to potent stem cell mobilization and hereby the repeated treatment with plerixafor reduces neointima formation in a mouse aortic transplant model. Manfred-Roth Stiftung. Forschungsstiftung Medizin, FAU Erlangen-Nürnberg.

Stem Cell Mobilization by Plerixafor Has an Affirmatory Effect on the Development of Transplant Vasculopathy in a Murine Aortic Allograft Model

<h3>Purpose</h3> Effector and regulatory T cells (Tregs) are main cell populations in the context of cardiac allograft vasculopathy (CAV). The immunostimulant plerixafor mobilizes hematopoietic stem cells and may boost T cells. Therefore, the aim of this study was to evaluate if a single, repeated, or continous treatment with plerixafor increases peripheral bone marrow derived stem cells including Tregs leading to tolerance induction and thus reducing chronic rejection in a mouse allograft transplant model. <h3>Methods</h3> Fully allogeneic mouse aortas from C57BL/6 mice (H2^b) were abdominally transplanted into CBA mice (H2^k). The continuously plerixafor application [1mg/kg/d] was carried for 14 days using implanted small osmotic pumps. Single dose (s.c.) injection of 1mg/kg/d or 5mg/kg/d were done at day 1 after transplantation (Tx.) and pulsed injections [1mg/kg/d] were conducted on days 1, 7, 14, and 21 after Tx. The control allograft group received vehicle loaded osmotic pumps. Stem cell mobilization was monitored by FACS. Recipients were sacrificed on day 14 for intragraft gene expression analysis or on day 30 for histological measurements. <h3>Results</h3> Murine aortic grafts with pulsed plerixafor injections showed significantly reduced neointima proliferation compared to control allografts (33.65%±8.84% vs.53.13% ±12.41%; p<0.05). The single shot and continuous treatment groups exhibited no improvement concerning neointima formation. First FACS analysis revealed significantly less hematopoietic stem cells (HSC) in the bone marrow of plerixafor treated mice vs. the control at day 14 after Tx. There were significantly more HSCs in the peripheral blood on day 30 after Tx, with the pulsed injection even doubling HSCs [0.0152%±0.008; p<0.005 (pulsed); 0.0046%±0.002%; p<0.01 (pump); 0.0076%±0.001%; p<0.01 (single dose 1mg/kg); 0.0039%±0.0017%; p<0.1 (single dose 5mg/kg) vs. 0.0018%±0.0016% (control)]. Preliminary intragraft gene expression results showed clearly reduced IFNγ expression and a significantly increase of IL-4, IL-10 and TGFβ. <h3>Conclusion</h3> These data suggest that both the continued and injected application of plerixafor leads to potent stem cell mobilization and hereby the repeated treatment with plerixafor reduces neointima formation in a mouse aortic transplant model. Further analysis are under progress.

Ephrin B2 Regulates Inflammatory Cell Accumulation in a Rat Aortic Transplant Model

Ephrin B2 Regulates Inflammatory Cell Accumulation in a Rat Aortic Transplant Model

Assessment of ultrasmall nanocluster for early and accurate detection of atherosclerosis using positron emission tomography/computed tomography

The development of atherosclerosis therapy is hampered by the lack of molecular imaging tools to identify the relevant biomarkers and determine the dynamic variation in vivo. Here, we show that a chemokine receptor 2 (CCR2) targeted gold nanocluster conjugated with extracellular loop 1 inverso peptide (AuNC-ECL1i) determines the initiation, progression and regression of atherosclerosis in apolipoprotein E knock-out (ApoE−/−) mouse models. The CCR2 targeted 64Cu-AuNC-ECL1i reveals sensitive detection of early atherosclerotic lesions and progression of plaques in ApoE−/− mice. CCR2 targeting specificity was confirmed by the competitive receptor blocking studies. In a mouse model of aortic arch transplantation, 64Cu-AuNC-ECL1i accurately detects the regression of plaques. Human atherosclerotic tissues show high expression of CCR2 related to the status of the disease. This study confirms CCR2 as a useful marker for atherosclerosis and points to the potential of 64Cu-AuNC-ECL1i as a targeted molecular imaging probe for future clinical translation.

Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

[Figure: see text].

Cytomegalovirus chemokine receptor M33 knockout reduces chronic allograft rejection in a murine aortic transplant model

BackgroundNumerous studies suggest that cytomegalovirus (CMV) infection may act as isolated risk factor in the development of cardiac allograft vasculopathy (CAV). Viral G protein-coupled receptors (GPCRs) are thought to contribute to the pathogenic changes associated with CMV infection. The aim of this study was to investigate the role of murine cytomegalovirus GPCR M33 in the development of CAV in a murine aortic allograft model. MethodsMHC I-mismatched aortas of C.B10 (H2b) mice were transplanted into BALB/c (H2d) recipients, which were either mock-infected, infected with wild type (WT) MCMV or MCMV with a deleted M33-receptor gene (delM33). Persistence of cytomegalovirus infection was confirmed by qPCR and by luciferase assay to ensure active viral replication. Grafts were harvested on days 21 and 37 for intragraft mRNA expression and histological analysis. ResultsActive viral replication was demonstrated and MCMV presence was confirmed by PCR within spleen, liver, salivary glands, lung and the aortic transplant. Infection with delM33 resulted in significantly less intimal proliferation compared to WT-MCMV but more pronounced proliferation than in mock-infected allografts (32.19% [delM33] vs. 41.71% [WT-MCMV] vs. 24.33% [MCMV-]). Intragraft expression of most analyzed genes was significantly increased in infected mice. VCAM-1, ICAM-1, PDGFβ, CXCR3 and Granzyme B were distinctly less expressed in grafts of delM33 infected compared to WT infected mice. Cellular infiltration revealed reduced dendritic cells and T cells in grafts infected with delM33 compared to WT MCMV. ConclusionsThese data suggest that the MCMV encoded receptor M33 plays an important role as a viral effector mechanism contributing to the development of CAV in a murine aortic transplant model.

Combined abdominal heterotopic heart and aorta transplant model in mice.

Allograft vasculopathy (AV) remains a major obstacle to long-term allograft survival. While the mouse aortic transplantation model has been proven as a useful tool for study of the pathogenesis of AV, simultaneous transplantation of the aorta alongside the transplantation of another organ may reveal more clinically relevant mechanisms that contribute to the pathogenesis of chronic allograft rejection. Therefore, we developed a combined abdominal heart and aorta transplantation model in mice which benefits from reducing animal and drug utilization, while providing an improved model to study the progressive nature of AV. The middle of the infrarenal aorta of the recipient mouse was ligatured between the renal artery and its bifurcation. Proximal and distal aortotomies were performed at this site above and below the ligature, respectively, for the subsequent anastomoses of the donor aorta and heart grafts to the recipient infrarenal aorta in an end-to-side fashion. The distal anastomotic site of the recipient infrarenal aorta was connected with the outlet of the donor aorta. Uniquely, the proximal anastomotic site on the recipient infrarenal aorta was shared to connect with both the inlet of the donor aorta and the inflow tract to the donor heart. The outflow tract from the donor heart was connected to the recipient inferior vena cava (IVC). The median times for harvesting the heart graft, aorta graft, recipient preparation and anastomosis were 11.5, 8.0, 9.0 and 40.5 min, respectively, resulting in a total median ischemic time of 70 min. The surgery survival rate was more than 96% (29/30). Both the syngeneic C57Bl/6 aorta and heart grafts survived more than 90 days in 29 C57Bl/6 recipients. Further, Balb/c to C57Bl/6 allografts treated with anti-CD40L and CTLA4.Ig survived more than 90 days with a 100% (3/3) survival rate. (3/3). This model is presented as a new tool for researchers to investigate transplant immunology and assess immunosuppressive strategies. It is possible to share a common anastomotic stoma on the recipient abdominal aorta to reconstruct both the aorta graft entrance and heart graft inflow tract. This allows for the study of allogeneic effects on both the aorta and heart from the same animal in a single survival surgery.

The Canonical Wnt Pathway in the Pathogenesis of Cardiac Allograft Vasculopathy

The canonical wnt pathway with its central signaling molecule β-catenin is important for tissue development and homeostasis and its dysregulation leads to many diseases including pathologies of the cardiovascular system. Inhibition of the wnt pathway has the potential to protect against cardiac allograft vasculopathy (CAV), because it reduces the proliferation of smooth muscle cells (SMCs), which is a main pathomechanism of CAV and causes neointima formation and vessel occlusion. For in vitro experiments human (hu) and murine (mu) aortic SMCs were grown in the presence of increasing concentrations of wnt inhibitors, such as XAV-939, ICG-001 or PKF118-310. Cell proliferation was measured as the extent of PrestoBlue Cell Viability Reagent metabolism and expression of representative wnt target genes was assessed by Taqman PCR. Additionally, the wnt inhibitors were used in the mouse model of aortic transplantation (n=8 mice/group) and the extent of neointima formation as an indicator for CAV was analyzed. We also measured the expression of β-catenin (immunofluorescence staining), wnt target genes (Taqman PCR) and several inflammatory cytokines (Taqman PCR) in the transplant. In vitro experiments revealed dose-dependent proliferation inhibition in huSMCs and muSMCs after treatment with each wnt inhibitor. PKF118-310 induced cell death in the highest concentrations tested. While XAV-939 had only minor influence on the expression of the wnt target genes cyclin D1, axin2 and p21, ICG-001 and PKF118-310 induced the expression of p21 and cyclin D1 and reduced the expression of axin2. Interestingly, in vivo experiments showed no effect of XAV-939 and ICG-001 on neointima formation, whereas neointimal amounts of β-catenin were significantly reduced. ICG-001 also reduced the expression of inflammatory cytokines (INFγ, TNFα and IL-6) and of p21, but did not influence cyclin D1 expression. XAV-939 had no relevant influence on cytokine expression. Results for PKF118-310 and wnt target gene expression in transplants after XAV-939 treatment are currently assessed. Strong anti-proliferative effects were found in vitro for all three wnt inhibitors, but this did not lead to reduced CAV development in a mouse model of vessel transplantation. Effects on SMCs themselves after ICG-001 and PKF118-310 seem indeed mediated via regulation of wnt target genes.

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation. Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

Thalidomide ameliorate graft chronic rejection in an allogenic kidney transplant model

Chronic T cell mediated rejection (TCMR), which is characterized by infiltration of the interstitium by T cells and macrophages, still remains a major barrier to the long-term survival of kidney transplantation. Our recent report indicated that thalidomide can attenuate graft arteriosclerosis in an aortic transplant model. In this study, we investigated the effect of thalidomide on chronic TCMR in a rat model of kidney transplantation.Fischer or Lewis kidney allografts were transplanted into Lewis recipient rats. After kidney transplantation, recipient rats were divided into 3 groups: the isograft (Iso) group, allograft (Allo) group, and thalidomide (Tha) group. Rats were sacrificed at 8 weeks after kidney transplantation, and blood and kidney samples were collected. Serum concentrations of creatinine (SCr),interleukin (IL)-2, IL-6, IL-17, and TNF-α in recipients were determined, and flow cytometry was used to detect the percentages of CD4+CD25+, CD4+ Foxp3+and CD4+Th17+ cell subsets in the peripheral blood. Grafts were procured for histopathological examination, and the expressions of α-SMA, transforming growth-β1 (TGF-β1), and VEGF in kidney grafts were investigated using Western blot. Thalidomide treatment significantly ameliorated chronic rejection, reduced renal allograft tissue damage, and decreased serum creatinine levels. Attenuation of chronic TCMR was due to the prohibited production of inflammatory cytokines, altered distribution of the CD4+ CD25+ FoxP3+ regulatory T (Treg) and CD4+ Th17+ cells in the peripheral blood, and decreased expression of TGF-β1, α-SMA, and VEGF in the kidney graft. These results demonstrated that thalidomide could effectively ameliorate chronic TCMR in a rat kidney transplant model.

Employing the Sirolimus-Eluting Poly (Propylene Carbonate) Mesh for the Prevention of Arteriovenous Graft Stenosis in Rats.

Poly (propylene carbonate, PPC) is a new member of the aliphatic polyester family. An outstanding feature of PPC is that it produces mainly water and carbon dioxide when degraded in vivo, causing minimal side effects. This unique property together with excellent biocompatibility and biodegradability makes PPC a promising material for drug delivery. In this study, we explored the effect of the sirolimus (an inhibitor of cell growth)-eluting PPC mesh on graft stenosis and its possible mechanisms in a rat arteriovenous grafting model. The PPC mesh was prepared by electrospinning. A jugular vein to abdominal aortic autograft transplantation model was established in rats. The graft was then treated by wrapping with the drug mesh or the drug-free mesh or left untreated. Four weeks posttransplantation, neointima was measured with hematoxylin and eosin staining, matrix metalloproteinase-2 (MMP-2), and MMP-9, and proliferating cell nuclear antigen (PCNA) in the grafts were assayed by Western blotting and immunohistochemistry, respectively. In vitro rat aortic adventitial fibroblast cell (RAAFC) migration was assessed using the Boyden chamber assay, and phospho-mammalian target of rapamycin (mTOR) levels in RAAFCs were determined by Western blotting. Animals with the drug mesh had an intimal area index of 4.87% ± 0.98%, significantly lower than that of the blank group (14.21% ± 2.56%) or the PPC group (15.03% ± 2.35%, both P < .05). The sirolimus mesh markedly suppressed MMP-2 and MMP-9 expression, decreased PCNA-positive cell numbers, inhibited RAAFC migration, and reduced phospho-mTOR levels. Our data suggest that the sirolimus-eluting PPC mesh might be potentially applied for the management of grafting stenosis.