Jeffrey Berger, Ph.D. - US grants

digital imaging; image processing; eye disorder diagnosis; angiography; retina disorder; eye fundus photography; mathematical model; model design /development; pathologic process; clinical research; human data;

The major long-term objective of the Reading Center for the Complications of Age-Related Macular Degeneration Prevention Trial (CAPT) is to provide reliable, systemic fundus grading and analysis of fundus features derived from photographic images of CAPT patients. This information will expand our current understanding of risk factors for vision loss associated with AMD as well as evaluate the potential efficacy of the CAPT in reducing the incidence of late complications of AMD. A variety of grading systems have been used to classify the presence and character of drusen and other clinical features of AMD. The majority of these systems utilize trained grading of stereoscopic color macular fundus photographs and fluorescein angiograms. The CAPT Reading Center System described in this proposal is a modification of the common system proposed by the International Classification and Grading System for AMD and the Wisconsin Age-related Maculopathy Grading System. 1,2 The Wisconsin System is currently used by the National Eye Institute sponsored Age-Related Eye Disease Study. Specific Aims: 1) To assess final CAPT eligibility and compliance with the CAPT treatment and retreatment protocols. 2) To monitor the immediate post-treatment and follow-up complications from stereoscopic color fundus photographs and fluorescein angiograms. 3) To grade, log and record specific baseline clinical features of AMD (for example, predominant drusen morphology drusen confluence, area of drusen involvement, focal hyperpigmentation, retinal pigment epithelial depigmentation, geographic atrophy. from stereoscopic fundus photographs grades using the CAPT Grading System. 4) To assess changes in drusen as a parameter for retreatment in the CAPT. In addition, to assess changes in other clinical features of AMD and to relate these changes and their baseline characteristics to the risk for late AMD complications, drusen regression and potentially protection from late AMD complications in treated eyes.

DESCRIPTION (provided by applicant): Peripheral artery disease is a highly prevalent condition in the United States, and a significant cause of cardiovascular morbidity and all-cause mortality. Although vascular surgical procedures are commonly performed to improve patient survival and quality of life, patients are at heightened risk of adverse perioperative cardiovascular events. Following vascular surgery, patients remain at increased risk of long-term cardiovascular events. Traditional risk factors are limited in their ability to discriminate cardiovascular risk following vascular surgery and are mostly non-modifiable. Pathological and clinical studies consistently demonstrate that platelets are a major culprit in the pathogenesis of atherothrombosis. We propose a novel, reliable, and reproducible measurement of increased platelet activity, thus allowing investigation of this clinically relevant and potentially modifiabe cardiovascular risk factor in this very high-risk population. The study hypothesis is that platelet activity measurements before vascular surgery is independently and significantly associated with 30-day cardiovascular events. We further hypothesize that platelet activity measured postoperatively is independently and significantly associated with long-term cardiovascular events. We will also correlate platelet activity with the genome expression profile to determine molecular mechanisms controlling platelet activity. The long-term goal is to identify a clinically useful assessment of platelet activity for risk stratification that may be used as a diagnostic too and a target for therapeutic intervention. The proposed study will be a prospective observational cohort study enrolling 350 subjects before non- emergent open vascular surgery of the lower extremities. Platelet activity will be measured before and after vascular surgery and will be correlated with 30-day and long-term cardiovascular events. The 30-day primary endpoint will be death, myocardial infarction, stroke and troponin I >0.1ng/ml. The long-term primary endpoint will be death, myocardial infarction, or stroke. Secondary endpoints are major bleeding, vascular thrombosis, each individual endpoint, and cardiovascular death. The mechanism of increased platelet activity in patients with peripheral artery disease will be explored; isolated platelet mRNA and microRNA expression profile in 12 patients with increased platelet activity will be compared with 12 patients with normal platelet activity. This study will provide novel data to address existing gaps in knowledge regarding the association between platelet activity measurements and incident cardiovascular events, and will ascertain whether a unique platelet RNA expression profile exists in peripheral artery disease subjects with increased platelet activity. Data obtained from this study will identify high-risk subjects before vascular surgery using a potentially modifiable risk factor. These data will provide insight into the molecular mechanisms regulating platelet activity and yield novel diagnostic tests for risk stratification an novel therapeutic targets to improve clinical outcomes in patients with peripheral artery disease undergoing vascular surgery.

ABSTRACT Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by heterogeneity of presentation, an undulating course, and a remarkably elevated risk for premature cardiovascular disease. Platelets have been understudied as a relevant contributor to premature atherosclerosis in SLE. Yet these cells, which contain transcripts and the necessary molecular machinery to conduct translation, are intercellular regulators of inflammation and immune activation and play a key role in atherothrombosis. Platelets express low affinity type 2 receptor (Fc?RIIA) whose ligand is the Fc portion of IgG. A single amino acid substitution, H131R, in the extracellular ligand binding domain increases the affinity for IgG and may account for individual variation in platelet activation, specifically gain of function. Leveraging a well-characterized SLE cohort, we identified a significant enrichment of carotid plaque in patients carrying at least one copy of the variant compared to those homozygotic for the ancestral gene. In a second SLE cohort, a significant increase in monocyte-platelet aggregates (MPA) in patients carrying the variant versus ancestral gene was observed. Although not yet assessed for genotype, SLE platelets exhibited a hyperreactive phenotype relative to healthy donors. Accordingly, it is hypothesized that platelet activity measurements and platelet-derived coding and non-coding RNA are significantly influenced by Fc?RIIA genotype. Two aims provide complementary studies to evaluate the underlying mechanism of increased platelet activity in SLE. In Specific Aim 1 the relationship between SLE platelet phenotype and transcriptome in the context of Fc?RIIA genotype will be investigated. To test the influence of Fc?RIIA genotype on platelets, a multidimensional panel of platelet activity markers representing different pathophysiological mechanisms will be measured, including: light transmission aggregometry (LTA); leukocyte- and monocyte-platelet aggregates; reticulated platelets; platelet receptor expression of PAC-1, CD40 ligand, P-selectin; platelet size; and the platelet transcriptome. Readouts will be assessed and compared in three groups of SLE subjects: H/H homozygotes, R/H heterozygotes, and R/R homozygotes. In Specific Aim 2 the goal is to molecularly assess platelet reactivity dependent on Fc?RIIA ligation and the impact of genotype on the phenotype of the vascular targets, endothelial cells (ECs) and macrophages. In contrast to Specific Aim 1, the approach utilizes platelets from healthy controls (H/H, R/H, and RR) since platelets from SLE patients may be intrinsically coated with immune complexes (ICs) accounting for baseline reactivity. Co-treatment of platelets with anti-CD9 will serve as a proxy of ICs and Fc?RIIA dependency approached using blocking antibodies. ECs and macrophages co-cultured with anti-CD9 platelets will be assessed for both pro-inflammatory and protective signaling cascades. Identification of a novel biomarker to gauge response to therapy, and/or to be used to identify patients at increased risk for premature cardiovascular disease and likely to benefit from anti-platelet agents, would be an advance.

PROJECTSUMMARY/ABSTRACT Systemic lupus erythematosus (SLE) is a complex autoimmune disease that poses several challenges to the clinician, including heterogeneity of presentation, undulating course, and a significantly elevated risk for vascular dysfunction and premature cardiovascular disease. Traditional risk factors are limited in their ability to discriminate cardiovascular risk in patients with SLE. Platelets, which contain transcripts and the necessary molecular machinery to conduct translation, are intercellular regulators of atherothrombosis, vascular dysfunction, inflammation, and immune activation. Activated platelets can induce endothelial cells and monocytes to produce inflammatory cytokines and chemokines resulting in vascular injury and subsequent atherogenesis. Platelets have been understudied as a relevant contributor to vascular dysfunction and premature cardiovascular disease in SLE. We propose a complementary set of studies to fully evaluate the mechanistic role of platelets in patients with SLE. The array of studies will include platelet activity measurements, coding and non-coding RNA profiles, platelets as effector cells regulating endothelial cell and leukocyte activity in vitro, and measurement of vascular health in vivo using brachial artery reactivity testing. The proposed approach will include a cross sectional study of 200 SLE patients to cover the full spectrum of organ involvement and disease activity. We will also enroll 50 age- sex- and race/ethnicity- matched disease controls. The study hypotheses are that (1) platelet activity measurements and platelet-derived coding and noncoding RNA are significantly influenced by disease activity and clinical phenotype, (2) SLE platelets will induce inflammatory, thrombogenic, and adhesive gene expression and consequent reactivity in endothelial cells, monocytes/macrophages, and vascular smooth muscle cells, and (3) SLE platelet phenotype and transcriptome will significantly associate with impaired vascular function. Longitudinal follow-up in 50 patients representative of both active and quiescent disease will allow us to ascertain whether biologic readouts track with a specific subset of patients and whether the readouts change over time. This study will provide novel data to address existing gaps in knowledge regarding the association between platelet activity measurements, the platelet transcriptome, and platelets as effector cells and vascular health across the clinical spectrum of SLE. This study will ascertain whether there is a unique platelet RNA expression profile in SLE with increased platelet activity and/or with impaired vascular health. Data obtained from this study will identify SLE patients at increased risk for vascular dysfunction and cardiovascular disease by investigating a potentially modifiable risk factor. These data should provide insight into the molecular mechanisms regulating platelet activity in SLE, novel diagnostic tests for risk stratification, and therapeutic targets to improve clinical outcomes.

PROJECT SUMMARY/ABSTRACT An estimated 15?20 million people in the United States have peripheral artery disease (PAD). Despite advances in medical therapy, PAD remains associated with considerable cardiac and limb morbidity and mortality. Currently, more invasive procedures are performed in the lower extremities than in the heart, demonstrating increasing costs to the system of advanced PAD. Although the pathogenesis of coronary artery disease (CAD) is well characterized, the pathophysiology of PAD is less understood and the mechanism(s) that regulate this complex disorder remain uncertain. While antiplatelet therapy (as a class effect) decreases the incidence and complications from PAD, we noted that the effectiveness of antiplatelet therapy differs between PAD and other vascular phenotypes. In contrast to CAD, aspirin was not particularly effective in PAD nor was there clinical benefit with more potent P2Y12 inhibition. Clearly, new directions are needed to better understand the role of platelets in PAD pathogenesis and identify new therapeutic targets. Our group demonstrated the importance in coding and noncoding RNAs in regulating platelet activity. Leveraging our established cohort of PAD patients with well-phenotyped platelet activities, we demonstrated the importance of platelet?leukocyte interactions (in contrast to platelet?platelet aggregation) in the pathogenesis of PAD. Moreover, we identified an aberrant post-transcriptional regulation of platelets in PAD and demonstrated that platelets play a central effector role in activating monocytes and fostering inflammation in PAD. Here, we propose to comprehensively investigate the relationship between (1) platelet activity, (2) the platelet transcriptome, and (3) effector cell properties in patients with PAD. We will analyze stored platelet samples from >1,000 patients with longitudinal follow-up, many of whom provided serial collections. Leveraging these valuable platelet samples, we will focus on identifying novel platelet transcripts associated with vascular phenotypes and incident cardiovascular events. For example, we will compare patients with (1) PAD vs. other vascular phenotypes (e.g., CAD, carotid artery stenosis, abdominal aortic aneurysm), (2) stable PAD vs. CLI, and (3) incident cardiac (myocardial infarction) vs. limb (major amputation) events. Mechanistic studies using both cultured megakaryocytes and animal models with platelet-specific knock-in and knock-out of candidate genes will characterize how these processes are regulated. We are also well positioned to validate our findings in well-established local, national, and international cohorts. Our data suggest these types of studies can provide conceptual advances in our understanding of the mechanisms influencing the pathogenesis and severity of PAD. These insights could be leveraged to design clinical biomarkers and therapeutic strategies to treat and prevent vascular disease and its life-threatening complications.