Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.     

What is the optimal prophylaxis for treatment of cardiac allograft vasculopathy?

Coronary artery disease in the transplanted heart, also known as cardiac allograft vasculopathy (CAV), is one of the major causes of mortality late after transplantation. It affects up to 50% of all heart transplant recipients within 5 years of surgery. The mechanisms of CAV are multifactorial and include both immune and nonimmune factors. Ischemia of the graft at the time of transplantation is one of the more important nonimmune factors, because this leads to endothelial cell injury. Immune factors involving cellular and humoral rejection can further insult the vascular endothelial cell, leading to a cascade of immunologic responses. The optimal treatment prophylaxis for CAV has not been established. The treatment approach to this major post-transplant complication includes modification of risk factors through medical therapies and strategies. The early use of diltiazem and/or pravastatin or simvastatin has been demonstrated to be effective in reducing the development of CAV, but does not completely prevent it. There are many ongoing studies involving newer immunosuppressive agents that may hold promise for the future.     

Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.     

Inhibition of natural killer cells results in acceptance of cardiac allografts in CD28-/- mice

Successful transplantation of allogeneic organs is an important objective in modern medicine. However, sophisticated immune defense mechanisms, primarily evolved to combat infections, often work against medical transplantation. To investigate the roles of natural and adaptive immune responses in transplant rejection, we functionally inactivated key effector systems of the innate (NK cells) and the adaptive immune system (CD28-mediated costimulation of T cells) in mice. Neither of these interventions alone led to acceptance of allogeneic vascularized cardiac grafts. In contrast, inhibition of NK-receptor-bearing cells combined with CD28-costimulation blockade established long-term graft acceptance. These results indicate a concerted interplay between innate and adaptive immune surveillance for graft rejection. Thus we suggest that inactivation of NK-receptor-bearing cells could be a new strategy for successful survival of solid-organ transplants.     

Effects of T cell frequency and graft size on transplant outcome in mice

The features that determine whether graft-reactive T lymphocytes develop into effector cells capable of mediating organ destruction are not well understood. To investigate potential factors involved in this process, we first confirmed that female recipient mice acutely rejected minor Ag-disparate male skin, but not heart transplants. Despite this difference in outcome, heart and skin transplantation induced antidonor T cell responses of similar magnitude, specificity, and cytokine profile. The heart-graft-primed T cells transiently infiltrated the graft and ultimately induced the development of chronic transplant vasculopathy. Increasing the frequency of donor-reactive T cells by presensitization or by using TCR (CD8+ antimale)-transgenic recipients did not mediate acute rejection but accelerated the pace and severity of the vasculopathy. Surprisingly, decreasing the tissue mass of the donor heart by 50% resulted in acute rejection of these smaller grafts without increasing the frequency of antidonor effector T cells in the recipients. In complementary studies, placement of one or two male skin grafts on a single recipient did not affect the frequency or cytokine profile of the induced antimale T cell repertoire. Nonetheless, the recipients of single grafts acutely rejected the transplanted skin while the recipients of two skin grafts did not. These results provide new insight into the pathogenesis of transplant vasculopathy and provide an explanation for the difference in outcome between murine skin and heart transplants by highlighting the novel concept that the efficiency of transplant-reactive T cell immunity is heavily influenced by the tissue burden it encounters at the effector stage.     

Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy

IL-17 is a signature cytokine of Th17 cells, a recently described subset of effector CD4 T cells implicated in the development of several pathologies. We have examined the role of IL-17 in regulating endothelial NO synthase (eNOS) expression in human vascular endothelial cells (ECs) because of the key role of eNOS in determining the pathological outcome of immune-mediated vascular diseases. In cultured ECs, IL-17 increased expression of eNOS, eNOS phosphorylation at Ser(1177), and NO production. The induction of eNOS expression by IL-17 was prevented by the pharmacological inhibition of NF-κB, MEK, and JNK, as well as by small interfering RNA-mediated gene silencing of these signaling pathways. The expression of IL-17 was then examined by immunohistochemistry in human arteries affected by transplant vasculopathy (TV), a vascular condition that is a leading reflection of chronic heart transplant rejection. IL-17 was expressed by infiltrating leukocytes in the intima of arteries with TV, and the majority of IL-17-positive cells were T cells. The number of IL-17-positive cells was not correlated with the intima/media ratio, but was negatively correlated with the amount of luminal occlusion. There was also a significant positive correlation between the number of IL-17-positive cells and the density of eNOS-expressing luminal ECs in arteries with TV. Altogether, these findings show that IL-17 induces the expression of eNOS in human ECs and that this may facilitate outward expansion of arteries afflicted with TV.     

Metabolic reprogramming of myeloid-derived suppressor cells in the context of organ transplantation

Myeloid-derived suppressor cells (MDSCs) are naturally occurring leukocytes that develop from immature myeloid cells under inflammatory conditions that were discovered initially in the context of tumor immunity. Because of their robust immune inhibitory activities, there has been growing interest in MDSC-based cellular therapies for transplant tolerance induction. Indeed, various pre-clinical studies have introduced in vivo expansion or adoptive transfer of MDSC as a promising therapeutic strategy leading to a profound extension of allograft survival due to suppression of alloreactive T cells. However, several limitations of cellular therapies using MDSCs remain to be addressed, including their heterogeneous nature and limited expansion capacity. Metabolic reprogramming plays a crucial role for differentiation, proliferation and effector function of immune cells. Notably, recent reports have focused on a distinct metabolic phenotype underlying the differentiation of MDSCs in an inflammatory microenvironment representing a regulatory target. A better understanding of the metabolic reprogramming of MDSCs may thus provide novel insights for MDSC-based treatment approaches in transplantation. In this review, we will summarize recent, interdisciplinary findings on MDSCs metabolic reprogramming, dissect the underlying molecular mechanisms and discuss the relevance for potential treatment approaches in solid-organ transplantation.     

Urinary cytology in renal transplant recipients with stable graft functions

The utility of routine urinary cytology in renal transplant recipients was investigated. Slides of 79 urine samples obtained from 59 renal transplant patients shortly after transplantation and of 275 urine sediments from 126 patients who had received a transplant before 1978 were screened for abnormal urothelial cells. None of the samples taken within one year of transplantation contained malignant cells. For five patients transplanted before 1978, repeated cytologic examinations showed malignant cells, but neither urologic examination nor clinical nor postmortem follow-up studies revealed a tumor. In all five cases, the abnormal cells disappeared from repeat samples within two to three months. None of the other 121 patients, with repeatedly normal urinary cytologies, exhibited a urinary tract carcinoma in the 24-month follow-up period. It would appear that the cytologic findings in the urine of renal transplant patients who have received long-term immunosuppressive medication may be transiently abnormal.     

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.     

A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation

Epstein–Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies.     

Differential Expression of Proteoglycans in Tissue Remodeling and Lymphangiogenesis after Experimental Renal Transplantation in Rats

Background

Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction.

Methods

Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence.

Findings

Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function.

Interpretation

Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.     

Modelling-informed cell-seeded nerve repair construct designs for treating peripheral nerve injuries

Millions of people worldwide are affected by peripheral nerve injuries (PNI), involving billions of dollars in healthcare costs. Common outcomes for patients include paralysis and loss of sensation, often leading to lifelong pain and disability. Engineered Neural Tissue (EngNT) is being developed as an alternative to the current treatments for large-gap PNIs that show underwhelming functional recovery in many cases. EngNT repair constructs are composed of a stabilised hydrogel cylinder, surrounded by a sheath of material, to mimic the properties of nerve tissue. The technology also enables the spatial seeding of therapeutic cells in the hydrogel to promote nerve regeneration. The identification of mechanisms leading to maximal nerve regeneration and to functional recovery is a central challenge in the design of EngNT repair constructs. Using in vivo experiments in isolation is costly and time-consuming, offering a limited insight on the mechanisms underlying the performance of a given repair construct. To bridge this gap, we derive a cell-solute model and apply it to the case of EngNT repair constructs seeded with therapeutic cells which produce vascular endothelial growth factor (VEGF) under low oxygen conditions to promote vascularisation in the construct. The model comprises a set of coupled non-linear diffusion-reaction equations describing the evolving cell population along with its interactions with oxygen and VEGF fields during the first 24h after transplant into the nerve injury site. This model allows us to evaluate a wide range of repair construct designs (e.g. cell-seeding strategy, sheath material, culture conditions), the idea being that designs performing well over a short timescale could be shortlisted for in vivo trials. In particular, our results suggest that seeding cells beyond a certain density threshold is detrimental regardless of the situation considered, opening new avenues for future nerve tissue engineering.     

Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy

The development of microsurgical techniques has facilitated the establishment of fully vascularized cardiac transplantation models in small mammals. A particularly useful model that has evolved for the study of cardiac allograft vasculopathy (CAV) is a heterotopic (abdominal) vascularized murine cardiac transplantation model. Using this model has permitted the elucidation of genetic, immune and non-immune factors contributing to the development of this inexorable pathological condition, which compromises half of all human cardiac transplants. This protocol details methods for performing the transplant, histomorphometric assessment of the graft vasculature and functional evaluation of the transplanted heart. In experienced hands, the surgical procedure requires approximately 75 min to complete, and vasculopathy results are obtained at 2 months. This model entails a fully vascularized implantation technique in which the donor ascending aorta and pulmonary artery are sutured end-to-side to the recipient abdominal aorta and inferior vena cava, respectively. As this model reliably reproduces immunological and non-immunological features of CAV, investigators can thoroughly explore contributory mechanisms, diagnostic modalities and therapeutic approaches to its mitigation.     

Serp-1, a viral anti-inflammatory serpin,regulates cellular serine proteinase and serpin responses to vascular injury

Complex DNA viruses have tapped into cellular serpin responses that act as key regulatory steps in coagulation and inflammatory cascades. Serp-1 is one such viral serpin that effectively protects virus-infected tissues from host inflammatory responses. When given as purified protein, Serp-1 markedly inhibits vascular monocyte invasion and plaque growth in animal models. We have investigated mechanisms of viral serpin inhibition of vascular inflammatory responses. In vascular injury models, Serp-1 altered early cellular plasminogen activator (tissue plasminogen activator), inhibitor (PAI-1), and receptor (urokinase-type plasminogen activator) expression (p < 0.01). Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in human endothelial cells. Treatment of endothelial cells with antibody to urokinase-type plasminogen activator and vitronectin blocked Serp-1-induced changes. Significantly, Serp-1 blocked intimal hyperplasia (p < 0.0001) after aortic allograft transplant (p < 0.0001) in PAI-1-deficient mice. Serp-1 also blocked plaque growth after aortic isograft transplant and after wire-induced injury (p < 0.05) in PAI-1-deficient mice indicating that increase in PAI-1 expression is not required for Serp-1 to block vasculopathy development. Serp-1 did not inhibit plaque growth in uPAR-deficient mice after aortic allograft transplant. We conclude that the poxviral serpin, Serp-1, attenuates vascular inflammatory responses to injury through a pathway mediated by native uPA receptors and vitronectin.     

Importance of insulin resistance to vascular repair and regeneration

Metabolic insulin resistance is apparent across a spectrum of clinical disorders, including obesity and diabetes, and is characterized by an adverse clustering of cardiovascular risk factors related to abnormal cellular responses to insulin. These disorders are becoming increasingly prevalent and represent a major global public health concern because of their association with significant increases in atherosclerosis-related mortality. Endogenous repair mechanisms are thought to retard the development of vascular disease, and a growing evidence base supports the adverse impact of the insulin-resistant phenotype upon indices of vascular repair. Beyond the impact of systemic metabolic changes, emerging data from murine studies also provide support for abnormal insulin signaling at the level of vascular cells in retarding vascular repair. Interrelated pathophysiological factors, including reduced nitric oxide bioavailability, oxidative stress, altered growth factor activity, and abnormal intracellular signaling, are likely to act in conjunction to impede vascular repair while also driving vascular damage. Understanding of these processes is shaping novel therapeutic paradigms that aim to promote vascular repair and regeneration, either by recruiting endogenous mechanisms or by the administration of cell-based therapies.     

Calcium signaling and oxidant stress in the vasculature

Recent evidence suggests that oxidant stress plays a major role in several aspects of vascular biology. Oxygen free radicals are implicated as important factors in signaling mechanisms leading to vascular pathologies such as postischemic reperfusion injury and atherosclerosis. The role of intracellular Ca(2+) in these signaling events is an emerging area of vascular research that is providing insights into the mechanisms mediating these complex physiological processes. This review explores sources of free radicals in the vasculature, as well as effects of free radicals on Ca(2+) signaling in vascular endothelial and smooth muscle cells. In the endothelium, superoxides enhance and peroxides attenuate agonist-stimulated Ca(2+) responses, suggesting differential signaling mechanisms depending on radical species. In smooth muscle cells, both superoxides and peroxides disrupt the sarcoplasmic reticulum Ca(2+)-ATPase, leading to both short- and long-term effects on smooth muscle Ca(2+) handling. Because vascular Ca(2+) signaling is altered by oxidant stress in ischemia-related disease states, understanding these pathways may lead to new strategies for preventing or treating arterial disease.     

Implication of matrix metalloproteinase 7 and the noncanonical wingless-type signaling pathway in a model of kidney allograft tolerance induced by the administration of anti-donor class II antibodies

In rats, tolerance to MHC-incompatible renal allografts can be induced by the administration of anti-donor class II Abs on the day of transplantation. In this study we explored the mechanisms involved in the maintenance phase of this tolerance by analyzing intragraft gene expression profiles by microarray in long-term accepted kidneys. Comparison of the gene expression patterns of tolerated to syngeneic kidneys revealed 5,954 differentially expressed genes (p < 0.05). Further analysis of this gene set revealed a key role for the wingless-type (WNT) signaling pathway, one of the pivotal pathways involved in cell regulation that has not yet been implicated in transplantation. Several genes within this pathway were significantly up-regulated in the tolerated grafts, particularly matrix metalloproteinase 7 (MMP7; fold change > 40). Analysis of several other pathway-related molecules indicated that MMP7 overexpression was the result of the noncanonical WNT signaling pathway. MMP7 expression was restricted to vascular smooth muscle cells and was specific to anti-class II Ab-induced tolerance, as it was undetectable in other models of renal and heart transplant tolerance and chronic rejection induced across the same strain combination. These results suggest a novel role for noncanonical WNT signaling in maintaining kidney transplant tolerance in this model, with MMP7 being a key target. Determining the mechanisms whereby MMP7 contributes to transplant tolerance may help in the development of new strategies to improve long-term graft outcome.     

Adult progenitor cells in vascular remodeling during atherosclerosis

The mobilization and recruitment of bone marrow-derived, circulating or tissue resident progenitor cells giving rise to smooth muscle-like cells have been implicated in neointima hyperplasia after arterial injury and in accelerated forms of arterial lesion formation, e.g., transplant arteriopathy or graft vasculopathy. By contrast, convincing evidence has emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. In patients with coronary artery disease, the number and function of EPCs have been linked with an improved endothelial function or regeneration, but have been inversely correlated with cardiovascular risk. In animal models, however, the injection of bone marrow cells or EPCs, or the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotypes, whereas the contribution of bone marrow-derived smooth muscle progenitors to primary atherosclerosis appears to be rather confined. Here, we discuss crucial biochemical cues, namely chemokines, adhesion molecules, growth factors and pharmacological means that guide and control the context-specific mobilization, recruitment and fate of vascular progenitor cells in arterial remodeling during atherosclerosis.     

Role of IL-18 in transplant biology

Since pro-inflammatory cytokine IL-18 and its receptor (IL-18R) are closely involved in regulating both adaptive and innate immune responses, it is conceivable that they might play an important role in organ transplantation. IL-18 can stimulate lymphocytes to produce the IFN-γ and regulate macrophage activity, thereby increasing the expression of proinflammatory cytokines including IL-1β, IL-6, CCL4 (macrophage inflammatory protein-1β), CXCL2 (macrophage inflammatory protein-2), and CCL2 (monocyte chemotactic protein-1). Nevertheless, the IL-18 signaling pathway and its underlying mechanisms remain obscure in transplant biology. This review is to summarize recent advances in our knowledge about the IL-18 signaling pathway and to analyze their functions in transplant-related biology. It was found that IL-18/IL-18R signaling pathway contributed to vascular transplantation, ischemmia/reperfusion, acute kidney injury, and acute rejection of kidney/liver/heart transplantation. IL-18 was a potential CYP3A expression modulator and was capable of affecting tacrolimus pharmacokinetics. Neutralizing IL-18 by its inhibitor IL-18 binding protein could efficiently suppress the production of injury-associated cytokines such as IL-6, TNF-α, IFN-γ, CXCL10 (IFN-γ-inducible protein10), and CX₃CL1 (fractalkine) and improve allograft function. Blockade of IL-18 signaling could regulate cardiomyocyte apoptosis and inhibit Th17 cells differentiation. Alteration of IL-18 levels was suggested as a biomarker for predicting ongoing allograft outcome. All these activities could deepen our understanding of immunobiological role of IL-18 and its receptor in the field of organ transplantation. Intervention of IL-18 signaling pathway might be utilized as a therapeutic strategy in clinic.