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2020 Research Focus: Prevent, Early Diagnose and Treat CAV

Enduring Hearts has funded eight (8) different research teams across the country to investigate novel strategies to prevent, diagnose early and prevent the onset of CAV in children who have received new hearts.

The projected outcomes of these research initiatives by Enduring Hearts are as follows:

  1. Targeted Ex vivo Nanotherapy for use in Cardiac Transplantation.
    1. This unique “drug”, nanotherapy, infused into the coronary arteries of the donor heart, could reduce the pre-transplant-related allograft coronary artery injury and, in turn, reduce CAV.
  2. Functional consequences of antigen specificity in CMV-responsive T cells.
    1. CM Virus infections can occur due to immune suppression following heart transplantation, causing severe coronary artery damage to a transplanted heart. This study will try to reduce CMV-mediated coronary artery injury and, in turn, CAV.
  3. The Causal Role of Axl in the Acceleration of Cardiac Allograft Vasculopathy.
    1. This experimental study will determine if a molecular signaling pathway contributes to CAV.
  4. MicroRNA Biomarkers of Allograft Rejection in Cardiac Transplantation.
    1. Damage to the coronary arteries releases signaling molecules into the blood. If unique to CAV-related injury, these biomarkers could contribute to the development of a diagnostic test for early detection of CAV.
  5. Predictive Modeling.
    1. A unique approach for the detection of early (<6 months post-transplant) microvascular injury is the goal of this project and could lead to early diagnosis of the onset of CAV.
  6. Developing a Comprehensive Biomedical Imaging Informatics Tool for Precision Care of Pediatric Heart Transplant Patients.
    1. This pilot project will develop a clinical decision support system to assist in quantifying the analyses of the heart biopsy. Such a system will improve the detection and treatment of rejection, resulting in injury which is a likely contributor to CAV.
  7. Integrating Multi-parametric Echocardiography with Computer-Assisted Analyses in Detection for Early Allograft Rejection in Pediatric Heart Transplant Recipients: A Pilot Prospective Multi-center Trial.
    1. Optimizing and standardization of echocardiogram to provide more accurate screening/detection of early rejection and reduce injury-related CAV, in pediatric heart recipients
  8. Leukotriene B4: A Potential Mediator and Biomarker for Cardiac Allograft Vasculopathy.
    1. One of the heart’s responses to injury is inflammation that can lead to CAV. CAV causes the release of a newly found signaling substance that can become a biomarker for early CAV detection. That signaling pathway will be studied as an important target for novel drugs that could prevent and/or treat CAV

In-Depth Look at the Enduring Hearts CAV Studies

1) Patel Kunal (AHA EH fully fund) Targeted Ex vivo Nanotherapy for use in Cardiac Transplantation.

Background. Almost 6 million adults in the U.S. have heart failure, and with realistically no current medical treatment options, 50% of those patients will die within 5 years of diagnosis. Consequently, cardiac transplantation represents their only viable option for survival, however this is also fraught with complications including long term immunosuppression, which is very difficult to tolerate, and ultimately a median survival of only 11 years. Additionally, due to a shortage of quality organs, only a small percentage of heart failure patients will ever receive their life-saving transplant. Our research aims to make more organs available to end-stage heart failure patients, and then make living with their new organs more tolerable.

Method. In transplantation, organs spend time stored on ice outside the body, where they incur injuries from lack of nutrients and oxygen. This damage results in priming the immune system to attack post-transplantation. We have designed a novel targeted immunosuppressant nano-molecule which has demonstrated an ability to protect donor organs prior to transplantation without the need for further administration of immune system depleting drugs in the post-transplantation period. The goal of our studies is to characterize what our nanotherapy is doing at a cellular level, and more definitively demonstrate how our treated organs are better able to function and survive while minimizing the toxicity of whole-body drug administration.

Significance. If successful, our drug nanotherapy has the potential to decrease the toxicity associated with current transplant-related immunosuppressive medications, while simultaneously improving long term heart transplant outcomes. That is, heart transplant patients would live longer, healthier lives while requiring less intensive maintenance regimens to remain free from transplant-specific cardiovascular disease. Lastly, the prospect also exists that this pre-treatment of hearts prior to transplant could demonstrate an ability to recover marginal or sub-optimal organs that would otherwise go untransplanted, thereby increasing the donor pool and ultimately allowing more heart failure patients to receive the lifeline they so desperately need.

2) Lauren Higdon (AHA EH fully fund) Functional consequences of antigen specificity in CMV-responsive T cells

 Background. Cytomegalovirus (CMV) causes organ rejection and vascular disease in transplant recipients, potentially leading to death. CMV infects over half of the general population by adulthood and, like chicken pox, persists for life. It does not cause disease in healthy people, due in part to the action of white blood cells called T cells. These cells are blocked in transplant patients by the immunosuppression used to prevent organ rejection. Antiviral medication protects transplant patients from CMV, but is not completely effective and often causes side effects. Some patients have T cells that could protect against CMV if their immunosuppression were reduced. Our goal is to identify these patients by predicting the efficacy of T cell responses to CMV in heart, kidney, and lung transplant patients.

Method. The long-term research question is to understand how to use T cells to protect transplant recipients from CMV without also attacking the donor organ. This proposal focuses on the question: how do transplant and immunosuppression change T cell responses to CMV? We will use innovative research techniques to study T cells shortly after transplantation and six months later. First, we will examine changes in T cell activity through analysis of small molecules that T cells use to function. We will also analyze the nature of T cell recognition of CMV by analyzing expression of genes that are key to T cell activity. Through these approaches we will gain understanding of the changes that occur in T cell immunity after transplantation, which is the first crucial step towards our long-term goal.

Significance. The long-term impact of this research is to enable personalized treatment for transplant patients. CMV can cause severe disease in transplant patients, including vascular damage to a transplanted heart. For about 20% of patients, disease occurs even with preventive antiviral medication. Our studies could help develop diagnostic tools and fine-tune post-transplant therapies. For example, we may be able to predict if a transplant patient would benefit from reduced immunosuppression, without risking transplant rejection. In addition, understanding the role of a patient’s immune system in protection against CMV may contribute to treatment of other opportunistic infections. This study therefore offers great potential to improve therapeutic outcomes and quality of life after transplantation.

3) Palak Shah (AHA EH fully fund) MicroRNA Biomarkers of Allograft Rejection in Cardiac Transplantation

Background. In cardiac transplantation, there are no reliable, blood-based markers to accurately detect heart transplant rejection or coronary disease. After transplant, patients are subjected to repetitive invasive procedures (~10-17) to screen for rejection. These procedures are associated with patient discomfort, significant healthcare cost, and carry a risk of complications. Also, the tissue obtained at the time of the biopsy is often interpreted incorrectly. Coronary disease of the transplanted heart is often silent clinically until it is too advanced to be treated effectively; this accounts for 30% of the mortality after transplant. The angiogram is not sensitive enough to detect disease until it is too advanced. A non-invasive marker of earlier coronary disease is urgently needed.

Methods. We propose developing a blood-based microRNA panel to replace two common invasive procedures employed after heart transplant to detect rejection and coronary disease: the endomyocardial biopsy and coronary angiogram. We will use next-generation sequencing technologies to identify specific microRNAs that can be detected in the blood and accurately diagnose rejection and coronary disease. We will accomplish this by evaluating blood-based microRNAs in 500 heart transplant patients from an existing study. Using statistical tools, we will identify key microRNAs that can be used in a blood-based panel to screen patients after heart transplant for rejection and coronary disease. This panel of microRNAs could replace the biopsy and angiogram to manage patients after heart transplant.

Significance.  Our work will lead to the development of a diagnostic test that can screen the blood of patients after heart transplant for rejection and coronary disease. This will significantly reduce patient morbidity and healthcare costs, as it will eliminate the need for an invasive endomyocardial biopsy or heart catheterization. Additionally, because microRNAs are not only markers of disease but can mediate disease as well, this proposal will identify microRNAs that might cause heart transplant rejection and coronary disease. These microRNAs can then be targeted in the future to develop new drugs to prevent or treat rejection and coronary disease. Additionally, these microRNAs may be implicated in rejection of other transplanted organs (e.g. kidneys, lung).

4) 2018. K Clinton (AHA-CoFunding). The causal role of Axl in the acceleration of cardiac allograft vasculopathy

Background. Cardiac Allograft Vasculopathy (CAV) is the most significant contributor to long term transplant rejection, despite advances in immunotherapy. A mechanistic understanding of the cellular processes underlying CAV is therefore necessary for the targeted development of better therapies to promote long term tolerance. The process of allorecognition involves transplant antigen presentation by either donor or recipient antigen presenting cells (APCs) to resident T-cells. The TAM family of receptors tyrosine kinases (Tyro, Axl and MerTK) have be widely studied in the promotion of engulfment of apoptotic cells and downstream transduction of intracellular signaling that influence cell proliferation and anti-inflammatory immune response. Dysfunction of TAMs is implicated in a variety of disease states including cancer, viral infection and autoimmune diseases; however, their influence on transplant tolerance and subsequent CAV development is not well understood.

Methods: Hypothesis 1. I hypothesize that TAMs are important regulators of graft related immune response and repair and that Axl in particular promotes progression of CAV. Preliminary data by our group support the notion of contrasting roles of Axl and MerTK in cardiac transplant survival. While MerTK ameliorates inflammatory responses and promotes tolerance, Axl contributes to vasculopathy, observed through increased intimal thickening and vascular smooth muscle cell proliferation.

Methods: Hypothesis 2. Due to their importance in antigen presentation and T-cell activation, I further hypothesize that Axl-dependent signaling in dendritic cells promotes the differentiation and proliferation of vascular smooth muscle cells and fibroblasts, which are contribute to vasculopathy.

Methods. To address this, I will utilize a well-established single MHC-II mismatch vascularized heart allograft model of CAV, making use of mice with targeted depletion of Axl. Flow cytometric and histological analyses will be carried out to assess vasculopathy through immune cell infiltration, intimal thickness and extracellular matrix staining. Furthermore, inflammatory cytokine levels will also be assessed by standard biochemical assays (ELISA, Western blotting). Long term grafts survival will be evaluated for rejection by routine palpitations. The efficacy of pharmacological inhibition of Axl will also be evaluated.

Significance. Such studies will provide invaluable insight into the general of allograft tolerance that will enable more precise therapeutic targeting in the future. I expect to gain significant knowledge about hybrid blood pump technology and its clinical relevance to treat pediatric patients, in alignment with the mission of the American Heart Association.

5) M Sleiman (AHA Co-Fund) Strategies to Improve Early Injury and Promote Long Term Tolerance post Cardiac Transplants.

Background. Cardiac transplant (Tx) patients need immunosuppressants (IMS) to maintain graft survival. These IMS can be highly toxic and have failed to control long term tolerance. Therefore, we need to better understand mechanisms of graft injury and design better and less toxic therapies. Brain death (BD) injury (BDI) and ischemia reperfusion (IR) injury (IRI) are two unavoidable factors that exacerbate graft injury and there is currently no intervention to control them. Our lab has previously shown that the complement system (C) plays an important role in both types of injury making C inhibition a promising therapeutic strategy. Given the several homeostatic functions of C, it is important to limit its inhibition to graft tissue. We have characterized several C inhibitors (inhs) that target heart grafts by means of single chain antibodies (scFv) that recognize neoepitopes expressed on grafts after BD and IR. Another approach is the use of regulatory T-cells (Treg) where injecting mice with graft specific Tregs can establish long term survival. An emerging method to confer specificity is the use of chimeric antigen receptor (CAR): a membrane receptor (usually a graft alloantigen-specific scFv) linked to T-cell intracellular signaling domains.

Proposed. We propose to develop & characterize a CAR Treg that targets all grafts (i.e. not antigen specific) by targeting long-lived C activation products that are deposited on grafts. The aims of our study are the following:

Specific Aim 1. Determine the role of C-dependent BDI & IRI on alloimmune response & cardiac graft rejection. We will investigate the role of C in early post-Tx injury & subsequent alloimmune response in a clinically relevant mouse heart allograft model using B4-Crry, a C inhibitor we have already characterized in cardiac IRI. We will expand on our previous study to incorporate BDI, immunosuppressive protocols, & the shaping of alloimmunity & rejection.

Specific Aim 2. Characterize graft targeted CAR Tregs & investigate their ability to induce tolerance. Our targeting moiety will be C receptor 2 (CR2), that targets the C degradation product C3d deposited on grafts. Mouse T-cells will be modified to express a CR2-CAR in trans with FoxP3 to force a Treg phenotype. The Tregs will be tested for their inhibitory function & characterized in a mouse cardiac allograft model.

6) S. Weber (direct) Antibody Mediated Allograft Injury Following Pediatric Heart Transplantation: Mechanistic Insights and Predictive Modeling - Direct Funding Program

Background. Long-term results of transplantation in children remain disappointing. Though early results have improved, ongoing graft loss occurs at a steady rate of 2-3% per year after the first year with minimal evidence of

improvement over the last two decades. An emerging literature suggests that donor-specific HLA antibodies (DSA), whether present pre-transplant or developing de novo after transplant, are key drivers of chronic graft loss. The CTOTC cardiac consortium has focused on the role of HLA antibodies in children, and enrollment in two clinical trials has been completed (CTOTC-04 and CTOTC-09). We will leverage the comprehensive CTOTC-09 clinical database, with its detailed cardiac phenotyping, and the substantial laboratory archive of endomyocardial biopsy (EMB) and serum samples stored in the study core laboratories, to perform further mechanistic studies as outlined in the following specific aims. In Specific Aim 1, detailed phenotyping of donor-specific HLA antibodies (DSA) in the pre- and early post-transplant period, as well as donor/recipient HLA epitope matching, will be performed. We hypothesize that DSA phenotype (complement fixing ability, titer and IgG subclass) and HLA mismatch determined by epitope load, can predict adverse allograft outcomes more effectively than the presence/absence of DSA with strength (MFI) determinations. Specific Aim 2 focuses on the development of an automated, transport-based morphometry system that can be easily and widely applied to routine EMB. We hypothesize that capillary injury from antibody-mediated rejection leads to interstitial capillary remodeling that can be reliably detected using a combination of endothelial cell (CD34) staining and automated transport-based morphometry on whole slide images.

This combined approach will enable detection of early (<6 months) capillary shape changes characteristic of graft syndromes leading to microvascular remodeling and subsequent capillary loss that contribute to late graft dysfunction.

In our final aim, Specific Aim 3, we will attempt to synthesize all the information from the first three aims, along with demographic and clinical information from donor and recipient, to develop a risk prediction model that can be applied in the early months after transplantation to predict late graft outcomes. Successful accomplishment of these goals should facilitate a transition towards a precision medicine approach to pediatric cardiac transplantation, including optimal wait list management and individualized peri- and post-operative care.

7) S Desphande (direct) Developing a Comprehensive Biomedical Imaging Informatics Tool for Precision Care of Pediatric Heart Transplant Patients - Direct Funding Program

Background. Heart transplantation is considered a therapy for end-stage heart failure. In pediatric patients,

approximately 400 heart transplants are performed every year in North America [1, 2]. Over the past two decades, there has been improvement only in early survival, i.e. the first year after heart transplantation in children. The most common cause of mortality and morbidity in the pediatric heart transplant population is rejection of the donor heart by the recipient [3]. Early and accurate identification of rejection is critical for preserving the transplanted organ, guiding therapy and ultimately, saving the life of the patient. Unfortunately, providers are unable to rely on clinical signs of rejection because patients often remain asymptomatic during early stages of disease progression until they become compromised hemodynamically. The gold standard for diagnosing heart transplant rejection, established by the International Society of Heart and Lung Transplant (ISHLT), is direct pathologic analysis of endomyocardial biopsy (EMB) samples [4] routinely performed in the cardiac catheterization laboratory under general anesthesia. Once venous access is established, a bioptome is inserted into the sheath and guided across the tricuspid valve into the right ventricle. Four EMB samples are taken from the septal wall of the right ventricle during each biopsy procedure.

 

However, studies in recent years have demonstrated that rejection is complicated that current staging methods may not be adequate. In most pediatric centers, the heart transplant patients undergo multiple biopsies in the first-year post-transplantation, and at least biannually thereafter for the duration of the allograft. Patients that demonstrate clinical signs of organ rejection or heart failure are subjected to more frequent biopsies. Due to their fragile hemodynamic state, they are placed at greater risk. Classically, rejection is staged based on lymphocytic infiltration of the myocardium. This acute cellular rejection (ACR) is reported based on hematoxylin and eosin (H&E) staining of EMB that have been fixed and paraffin embedded. Based on ISHLT 2004 grading system, ACR is graded 0 through 3R, with 3R reflecting the most severe form. Unfortunately, this simplistic and subjective categorization ignores another mechanism of cardiac transplant rejection Antibody-mediated rejection (AMR) that has been identified to play an important role in severe rejection. AMR is characterized by neutrophil infiltration, interstitial hemorrhage, and severe necrotizing vasculitis. Diagnosis relies on immunohistochemical (IHC) staining of the complement cascade components C4D and CD68, and the presence of donor-specific antibodies in the blood of recipients. AMR is an important entity in the pediatric transplant population because a large percentage have a history of congenital heart disease, and thus have already been exposed to multiple alloantigens through prior blood transfusions and surgeries.

 

The main purpose of the ISHLT grading system is to classify the type and degree of rejection and to provide some clinical guidance for therapy based on the severity of rejection [5]. However, this pathologic evaluation has some shortcomings. One most significant limitation is that there is subjectivity in the grading of a given biopsy specimen [6]. This is evident based on studies that have shown significant interrater variabilities for both ACR and AMR [7]. Some of the variations may stem from the fact that rejection is often patchy and may therefore be underestimated or overestimated based on routine scanning of a particular “field of view” in a given slide that may contain anywhere from 1 – 6 individual specimens of a biopsy sample. A second equally important limitation stems from the observation that the grade of rejection based on histopathology or immunostaining does not seem to correlate with either the clinical scenario or the clinical course of graft survival [8-10].

 

Advances in machine learning and biologic imaging analysis have provided additional tools in evaluating

various pathologies. Our prior study has shown that quantitative image analysis can provide objective

Georgia Tech and Children’s Healthcare of Atlanta assessment of morphology in histopathologic images [13-15]. The goal of this pilot project is to develop clinical decision support systems (CDSSs) that can assist in making rejection staging decisions by quantifying morphological properties in biopsy slides for heart transplant patients

with the following specific aims:

(1) To develop a reliable representation system for patch-level images with deep learning technique such as convolutional neural networks and multi-scale context-aware features.

(2) To construct a predictive modeling framework that predicts heart transplant rejection types and grades by combining patch-level images.

(3) To establish a new scoring system for the assessment of heart transplant rejection by integrating histopathological image analysis with clinical data and available biomarkers (cell-free DNA).

8) B Wisotzkey (direct). Integrating Multi-parametric Echocardiography with Computer Assisted Analyses in Detection for Early Allograft Rejection in Pediatric Heart Transplant Recipients: A Pilot Prospective Multicenter Trial. - Direct Funding Program

Background: Cardiac allograft rejection remains a leading cause of morbidity and mortality affecting longevity in adult and pediatric heart transplant recipients. Early detection and treatment of rejection before overt graft dysfunction develops is paramount to ensuring long-term symptom-free survival. Current practices rely on endomyocardial biopsies (EMB) which limit surveillance and are both invasive and costly. However, no single echocardiographic parameter has had sufficient predictive power for rejection detection. ECHO, a non-invasive computer-assisted analysis algorithm with standardized risk scoring for deviations of multiple m-mode and tissue Doppler imaging (TDI) parameters, has been clinically validated for EMB-based acute cellular rejection (ACR) detection in single centers. However, widespread use of ECHO has been limited primarily because of technical factors. Recently ECHO has been enhanced (ECHO-E) with migration to the MatLab language, which supports every major operating system, native DICOM support and b-splines for tracking LV wall movement.  This removes the need for a digitizer and creates program flexibility for beta testing emerging echocardiographic parameters that could also detect early antibody-mediated rejection (AMR). Specific aim 1 of this pilot study is to provide a preliminary test of the primary hypothesis that ECHO-E will be equivalent, or non-inferior, to EMB in detecting both ACR and AMR.  

Methods/Analyses: ECHO-E will be used at six high volume pediatric heart transplant centers with experience using ECHO within their rejection detection protocols. Enrolled recipients will be followed for the first 13 months post-transplant. Each institution’s pre-defined rejection surveillance protocol of clinic visits, echocardiograms, and EMBs will be continued thus minimizing confounding perturbations except for ECHO-E. Each EMB will include ECHO-E scoring blinded to the EMB results. Specific aim 1 is to determine if ECHO-E scoring predicts EMB-based rejection defined as ISHLT grade > 2R (ACR) cellular and/or ISHLT grade > pAMR1h antibody-mediated rejection (AMR). Specific aim 2 is to determine each centers’ number of treated rejection and hemodynamically-compromising treated rejection episodes during this 13-month ECHO-E study. Our hypothesis is that ECHO-E is at least equivalent to rejection detection practices over the prior 5 years.  Specific aim 3 is to determine the additive predictive value(s) to ECHO-E of each of the following: inclusion of emerging imaging techniques, specifically TDI-derived E/e’ and global longitudinal strain (GLS) and strain rate (GLSR) by 3D-speckle tracking imaging; and/or inclusion of changes in relevant laboratory values, such as B-type natriuretic peptide and de novo donor specific antibodies. We hypothesize that some/all of these emerging imaging techniques and laboratory tests will add positive predictive value to ECHO-E for both ACR and AMR.

Significance: This will be the first prospective multi-center study comparing ECHO-E to EMB with power optimized by a minimum of approximately 360 EMB + ECHO-E with 18 ACR and/or AMR episodes in an estimated 60 pediatric heart transplant recipients. This project will also be the first prospective multi-center testing of additive predictive values to ECHO-E of including emerging echocardiographic and laboratory markers of rejection. As a pilot, this study will also test the performance characteristics and capabilities of the specific multi-center study design including its implementation processes, metrics, limited standardization of immunosuppression and surveillance, and multiple equipment vendors that would germane for planning and funding a larger multi-center study.

Impact:  When properly validated, ECHO-E optimized with GLS could offer a standardized echocardiographic strategy for rapid, non-invasive patient-centric screening and detection of early allograft ACR and AMR in pediatric heart recipients.  With enhancements, ECHO-E is more user friendly permitting wider access by other pediatric transplant centers. With validation, ECHO-E guidance could result in fewer EMBs for rejection surveillance of pediatric recipients.

9) K Khush (EH Transplant Longevity Award) Leukotriene B4: A Potential Mediator and Biomarker for Cardiac Allograft Vasculopathy

Background. 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. CAV is an accelerated fibroproliferative disease process that affects vascular smooth muscle cells and the intercellular matrix and is distinct from focal traditional atherosclerotic disease. The development of CAV involves the complex interplay of immune and non-immune mechanisms. Immune mechanisms are related to “foreign” endothelium from the transplanted heart that expresses human leukocyte antigens (HLA) that activate the recipient’s immune response. Invoked immune mechanisms include proliferation of T-lymphocytes with inflammatory cytokine release, in addition to macrophage recruitment and production of growth factors that affect the coronary vessels.

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Methods. 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.