Primary Investigators: Dr. Kiran Khush, MD
Institution: Stanford University School of Medicine
Funding began in 2019.
Cardiac allograft vasculopathy (CAV) has often been referred to as the “Achilles heel” of long-term survival after HTation. By 10 years after transplant, CAV is diagnosed in 50% of adult recipients and in almost 30% of pediatric recipients. As perioperative and early survival have improved, the need to understand and manage late complications more effectively has become a major focus for research and clinical care. Indeed, CAV is the second leading cause of mortality beyond years after pediatric heart transplantation (after graft failure, which is often due to CAV) and accounts for approximately 25% of late deaths.
In the growing pediatric and adult heart transplant (HT) recipient population, CAV represents a major threat to long-term survival. Innovative studies that identify key signaling pathways in CAV development are needed to (1) deepen our knowledge of the pathogenesis of CAV, (2) identify targets for drugs that can effectively preventor treat CAV, and (3) develop noninvasive biomarkers for CAV screening and detection.
In the growing pediatric and adult heart transplant (HT) recipient population, CAV represents a major threat to long-term survival. Innovative studies that identify key signaling pathways in CAV development are needed to (1) deepen our knowledge of the pathogenesis of CAV, (2) identify targets for drugs that can effectively prevent or treat CAV, and (3) develop noninvasive biomarkers for CAV screening and detection. The long-term goal of this proposal is to translate findings from preliminary animal and human CAV models into clinically useful biomarkers and novel targets for therapeutics to prevent and treat CAV.
Our hypothesis is that leukotriene B4 (LTB4) mediates the development of CAV, that LTB4 level can be used as a biomarker for early CAV detection, and that the LTB4 signaling pathway is an important target for novel drugs that prevent and/or treat CAV.
Specific Aim 1: Define the involvement of Leukotriene B4 (LTB4) in an experimental model for CAV. Rationale: LTB4 is produced by macrophages through the conversion of LTA4 to LTB4 (Figure 2) via the enzyme leukotriene A4 hydrolase (LTA4H) and induces vascular endothelial cell apoptosis and smooth muscle cell proliferation. To assess the role of LTB4 during CAV development, we will inhibit LTA4H with the drug Bestatin (also known as ubenimex), and assess lesion formation and local inflammation, as a surrogate for CAV formation, in a well-established rat aortic transplant model. Specific Aim 2: Determine plasma LTB4 levels in HT recipients with CAV and correlate with clinical activity to determine whether plasma LTB4 levels can be used as a non-invasive CAV biomarker. Hypothesis 2a: Plasma LTB4 levels are elevated in pediatric and adult HT patients with CAV, compared to controls (healthy non-transplant controls and transplant patients without CAV) Figure 1. (A) Progression of intimal thickness of the coronary artery in a transplanted heart, representing development and progression of cardiac allograft vasculopathy (CAV); (B) Progression of allograft from normal systolic and diastolic function to restrictive physiology with severe diastolic function due to reduced distal coronary perfusion Hypothesis 2b: Plasma LTB4 levels correlate with New York Heart Association (NYHA) Class, echocardiographic measurements, and NT-proBNP levels in patients with CAV Preliminary Data: Our hypothesis is based on the recognition that the pathogenesis of CAV may share key features with that of pulmonary arterial hypertension (PAH)—another fibroproliferative disease of small arteries/arterioles. In both human and rat models of severe PAH, there is high expression of leukotriene A4 hydrolase (LTA4H), a biosynthetic enzyme of LTB4 that is produced by macrophages that accumulate in and around the pulmonary arterioles. LTB4 has been shown to directly induce endothelial cell apoptosis and smooth muscle cell proliferation, resulting in hypertrophy of the endothelium. 8,9 This excessive cell division results in arterial occlusion and pulmonary hypertension (Figure 2). Given the histologic overlap between CAV and PAH, we have previously (unpublished data) evaluated the role of macrophagederived LTB4 in the pathogenesis of CAV. We found a significant increase in macrophage accumulation and LTB4 production in coronary arterioles derived from autopsy samples from HT recipients with CAV (CAV+), compared to controls without CAV (CAV-) (Figure 3). We further detected higher LTB4 levels in the plasma of a small cohort of CAV+ HT recipients, compared to CAV- controls (Figure 4). Subsequently, in collaboration with Sonja Shrepfer, MD, PhD (Co-Investigator, Laboratory of Transplant Surgery and Immunology, UCSF), we performed allogeneic orthotopic aortic transplants in a rat model Figure 2: Leukotrienes (LT) are lipid mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism. LTB4 may function as a transcriptional regulator in the nucleus, or is transported out from the source cell and binds to its cognate receptors (BLT1 and BLT2) to initiate downstream signaling. Leukotriene B4 (LTB4) specifically contributes to vascular remodeling resulting in endothelial cell (EC) apoptosis and smooth muscle cell (SMC) proliferation Figure 3: Representative immunofluorescence images from coronary arteriole sections stained with p5-LO (left, LTB4 marker, green), and CD68 (middle, macrophage marker, red), and merged images (right). DAPI (blue) identifies nuclei. Differential interference contrast (DIC) highlights vascular structures. Top panel: HT recipient with CAV, bottom panel: HT recipient without CAV. Note increase in arteriolar macrophage accumulation and LTB4 production in CAV+ patients Figure 4: Plasma LTB4 levels in 5 HT recipients with CAV (left) and 5 patients without CAV (right) measured at 4-6 weeks post-transplant (green) and at one-year post-transplant (blue). Comparison of LTB4 levels at 1 year post-transplant shows a significantly higher mean LTB4 level in patients who have developed early CAV (p=0.03). of CAV (Figure 5) and showed significant increase in production of 5-LO (enzyme involved in the LTB4 synthetic pathway) and CD68 staining (macrophage marker) in rats that developed CAV (data not shown). Finally, treatment of rats after allogeneic aortic transplantation with ubenimex (Bestatin), a well-tolerated oral drug that inhibits LTA4H production and blocks LTB4 formation, prevented CAV development (Figure 6). Additional studies measuring LTB4 levels in HT recipients with and without CAV are required to determine whether LTB4 is indeed an accurate non-invasive CAV biomarker. These experiments comprise an important first step for further elucidation of the role of LTB4 in the pathogenesis of CAV. Such studies may ultimately lead to strategies for CAV prevention, early CAV detection, and therapeutic targets to improve the prognosis of HT recipients with this deadly, life-threatening complication. Methods: Specific Aim 1: Define the involvement of Leukotriene B4 (LTB4) in an experimental model for CAV. Rationale: To assess the role of LTB4 in CAV development, we will inhibit LTA4H (see Figure 2) with Bestatin and assess lesion formation and local inflammation, and will characterize the overall systemic immune status. Experimental design: Our lab has developed and established a well-recognized rat aortic transplant model to study CAV.10 Compared to the heterotopic HT model, the aortic transplant model has the advantage that histopathological analysis is drastically facilitated since transplant vasculopathy may be examined in one single vessel with a defined diameter instead of the exploration of numerous small cardiac vessels of varying sizes. The aorta from Fischer 344 rats will be transplanted into the orthotopic position of a Lewis rat. CAV lesions develop in the transplanted graft over time, leading to gradual narrowing of the lumen. Syngeneic (Lewis to Lewis) transplants will serve as negative controls. Implanted grafts will be harvested after 28 days for subsequent histopathological, immunohistochemical, and molecular biological analyses. ELISPOT, donor specific antibody assays and CyTOF will be used to characterize and assess the systemic immune status. Study Groups: Male Lewis and Fischer 344 rats will be used as presented in Table 1: Figure 5. (Left) Orthotopic aortic transplant from Fischer-to-Lewis rat model (allogenic) and Lewis-to-Lewis (syngeneic) over a 30-day period. (Right) Histopathology results comparing the allogeneic (n=11) and syngeneic (n=6) transplants with respect to aortic myointimal area, showing a significant increase in thickness in the intima after allogenic transplantation, representing CAV in this animal model (p 0.01) Figure 6. (Left) Orthotopic aortic transplant from Fischer-to-Lewis rat model (allogeneic, left), Lewis-toLewis (syngeneic, right), and allogeneic rats treated with ubenimex (bestatin) over a 30-day period. (Right) Histopathology results comparing the allogeneic (n=11), syngeneic (n=5), and ubenimextreated alogeneic transplants (n=6) showing significant reduction in myointimal area in rats treated with ubenimex (p90% power to detect a significant difference in LTB4 levels between the groups, with α=5% using a two sided two-sample t-test: a difference in LTB4 of 153 pg/ml between Null Hypothesis (Ho) μCAV+ = μCAV- = 142 pg/ml and Alternative Hypothesis (HA) μCAV+ =294 pg/ml with estimated SD=158 pg/ml. Basic demographic characteristics, echo measures, and NT-proBNP level will be compared between the groups using Student’s t-test with a Tukey correction for multiple hypothesis testing. The ANOVA method will be used to compare the means of more than 2 populations (CAV+, CAV-, and healthy non-transplant controls). Significance and Future Directions: We will use the data and results obtained from these preliminary studies to apply for larger-scale funding mechanisms that will enable our group to further characterize the role of LTB4 in development of CAV. Ultimately, our aim is to identify reliable non-invasive biomarkers for CAV monitoring, and to test to role of novel agents such as Bestatin for CAV prevention in pediatric and adult HT recipients.
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