Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings.

Long-term survival of cardiac transplant recipients continues to be severely limited by the development of a pathological, chronic rejection process, termed allograft vasculopathy (AV). This remains to be the case despite dramatic improvements in the areas of surgical techniques, pre- and post-operative care, and immunosuppression. To model the clinical setting we used calcineurin inhibitor (CNI) immunosuppression, the cornerstone of post-transplant immunosuppression, in a murine aortic interposition transplant model for our analysis of AV development. This model mimics the presentation of AV in human cardiac transplants through the development of a progressively occlusive neointimal lesion. Our previous work in this model has demonstrated that CD8+, but not CD4+, T cells play a role in neointimal lesion formation. Further investigation also highlighted a specific requirement for either CD8+ T cell-derived IFN-γ or direct cytotoxicity in the development of lesion formation. In the current study we confirmed that CD8+ T cell-derived IFN-γ also leads to the loss of medial smooth muscle cells, an event which inversely correlates with lesion formation. The Fas/FasL direct cytotoxic pathway was also significantly involved in neointimal lesion formation and medial remodeling. This work clarified the pathways utilized by CD8+ T cells in their role as mediators of AV development. Recognizing the threat that CD8+ T cells pose to cardiac transplant recipients in the presence of CNI immunosuppression, and a growing concern with the presence of anti-donor memory T cells in transplant recipients, we next explored the development of memory CD8+ T cells in the presence of CNI immunosuppression. We first established that memory CD8+ T cells could not develop when CNI immunosuppression was initiated immediately post-challenge. Next, we hypothesized that the clinical practice of CNI delay post-transplant would permit the development of de novo memory CD8+ T cells. Immediate and early initiation was sufficient to prevent the development of de novo memory CD8+ T cells. However, later delay to within a clinically practiced timeframe did permit the development of de novo memory CD8+ T cells. Our analysis revealed that this population demonstrated equivalent functionality to de novo memory CD8+ T cells generated in the absence of CNI immunosuppression.

BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

Coronary atherosclerosis is by far the most frequent cause of ischemic heart disease. Intravascular ultrasound (IVUS) along with virtual histology (VH) is a useful tool for quantification of coronary plaque buildup and provides new insights into the diagnosis of coronary disease. Rupture of vulnerable plaque causing acute coronary syndromes, coronary remodeling maintaining lumen size and plaque phenotype revealing pathological severity are among the most important topics related to atherosclerosis. In this thesis, variations of IVUS-VH-derived thin-cap fibroatheroma (TCFA) definitions are proposed to evaluate the plaque rupture, which is further analyzed in a layered manner; statins effects on coronary remodeling are comprehensively assessed with the implementation of automated IVUS segmentation and registration of IVUS pullbacks based on baseline and 1-year followup datasets; plaque phenotypes are determined and analyzed morphologically and compositionally on segmental basis using the same serial datasets. In addition, our research involves another important coronary disease — coronary allograft vasculopathy (CAV) which is a frequent complication of heart transplantation (HTx). Another intra-coronary imaging modality — intravascular optical coherence tomography (IVOCT) for quantifying CAV is involved. We present an optimal and automated 3-D graph search approach for the simultaneous IVOCT multi-layer segmentation by transforming the 3-D segmentation problem into finding a minimum-cost closed set in a weighted graph. Furthermore, a computer-aided just-enough-interaction refinement method is proposed to help achieve fully satisfactory 3-D segmentation of IVOCT images. We believe this is the first work that provides a fast, efficient and accurate solution for IVOCT multi-layer assessment in the context of CAV. The major contributions of this thesis are: (1) Proving that IVUS-VH-derived TCFA prevalence may be overestimated, and elucidating the potential loss of plaque material during rupture, (2) providing a comprehensive understanding of remodeling in the context of both changing the remodeling direction and changing the remodeling extent, and demonstrating the statin therapy effects on remodeling across patients, based on automated segmentation of IVUS images and registration of serial data, (3) showing that the pathological intimal thickening is the most active plaque phenotype in terms of plaque composition changes and plaque vulnerability progression, and (4) developing and validating a method for multi-layer 3-D segmentation of IVOCT images within a novel interactive environment.

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