Faculty Bibliography

Venous thromboembolism (VTE), comprised of deep vein thrombosis and pulmonary embolism, is a common health problem both in the United States and worldwide, with significant associated morbidity and mortality. Despite multiple known genetic and situational risk factors, an estimated 30% of all events remain classified as idiopathic, demonstrating a significant knowledge gap in the pathophysiology VTE. While platelets are well established as an essential contributor to thrombus formation and there has been recent interest in the role of neutrophil extracellular traps, specific cell types and pathways involved in the pathogenesis of VTE remain uncertain. In this study, our primary aims were to define a unique transcriptional signature for VTE and to identify the types of cells and specific pathways involved in development of VTE. Whole blood was collected in PAX gene tubes and RNA sequencing for coding mRNA was performed in an unbiased manner in 201 patients with prevalent VTE as well as 43 healthy controls. We used a bioinformatics approach to develop a unique signature for VTE by identifying differentially expressed genes, developing cell-type modules, and ascertaining pathways driving differentially expressed transcripts. We performed additional analyses on subgroups of patients with idiopathic VTE, patients with incident VTE, and VTE patients matched to healthy controls by age and sex. We went on to use machine learning methods to learn models that best differentiate VTE patients from healthy controls and validated it on a left out test set within our VTE population. Genes specific to neutrophils, erythrocytes, and platelets, in that order, were most significantly upregulated in patients with VTE compared to healthy controls. Genes related to T-cells were downregulated. Pathway analysis revealed upregulated neutrophil activation and degranulation, erythrocyte differentiation and homeostasis, and platelet degranulation. A gene signature of 217 transcripts was outstanding at differentiating patients with VTE versus healthy controls (AUC 0.94). Following adjustment for age, sex, and race/ethnicity our genetic signature remained significantly robust at differentiating patients with VTE versus controls (AUC 0.83). Our expression signature remained stable across patients with idiopathic VTE (AUC 0.93), and in patients who went on to develop future VTE events (AUC 0.95). In summary, we have demonstrated a whole blood transcriptional signature for prevalent and incident VTE. Genes related to neutrophils, erythrocytes, and platelets are upregulated in patients with VTE and genes related to T-cells were downregulated. These findings suggest an active role of cell types once thought to be passively entrapped within thrombus and provide new areas of study to establish the pathophysiology of VTE

Background/Purpose: Psoriatic arthritis (PsA) and Psoriasis (PsO) are chronic inflammatory diseases associated with vascular inflammation and increased CVD risk. Few studies have examined vascular inflammatory differences between PsO and PsA and how these differences may impart a different CVD risk profile. We directly investigated the vascular endothelium of patients with PsA, PsO and compared to controls to better understand the inflammatory mechanism(s) that predispose psoriatic patients to CVD risk.
Method(s): Twenty patients with psoriatic disease (PD) (mean age 45 years, 55% male, 11.2 +/- 19% body surface area (BSA) involvement) were first compared to 10 matched controls. Next, comparisons were made between PsO (n = 14, average age 50 years, 57% male, 11 +/- 22% BSA) and active PsA (n = 6, average age 36 years, 50% male, 11 +/- 10% BSA, average 2 - 3 tender/swollen joints per individual). To measure vascular endothelial health, venous endothelial cells were collected from the brachial vein using guidewires inserted through an angiocatheter and isolated with CD146-conjugated magnetic beads. Following collection, endothelial mRNA was isolated, converted to cDNA and inflammatory gene profiling performed by RT-qPCR with Taqman probes and primers. Transcripts were chosen based on in vitro gene arrays of human aortic endothelial cells co-stimulated with IL-17 and TNF-alpha.
Result(s): PD patients compared to controls showed a trend towards higher levels of hs-CRP (2.4 +/- 4 mg/dl vs. 0.8 +/- 2 mg/ dl, p = 0.08) with no overall difference noted between PsA and PsO patients (2.8 +/- 2 mg/dl vs. 2.7 +/- 4 mg/dl, p = 0.24). Transcriptomic profiling of venous endothelial cells comparing PD (PsO and PsA) to controls revealed upregulation of inflammatory cytokine- and chemokine- associated transcripts (lymphotoxin beta [4 - fold], CCL3 [11 - fold], CXCL10 [16 - fold], IL-8 [10 - fold] and IL-1beta [4 - fold], P < 0.05 for all) and transcripts related to intracellular adhesion (ICAM1 [2.4 - fold] and inflammation COX-2 [3 - fold], P < 0.05). Increased expression of the chemokine fractalkine (CX3CL1 [2.8 - fold], p < 0.05) and lymphotoxin beta [2 - fold, p = 0.10] were found in patients with active PsA compared to PsO. No differences in CCL3, CXCL10, IL-8, IL-1B, ICAM1 and COX-2 where seen between PsA and PsO patients.
Conclusion(s): Endothelial cell pro-inflammatory transcripts are upregulated in patients with active PD compared with controls. Levels of lymphotoxin beta and fractalkine, a chemokine present in inflamed arthritic synovial tissue, are greater in endothelial cells of patients with PsA than PsO. These findings may underlie increased CVD risk in patients with PD and highlight the inflammatory vascular differences between PsO and PsA

Diabetes is the greatest risk factor for the development of cardiovascular disease, which, in turn, is the most prevalent cause of mortality and morbidity in diabetics. These patients have elevations in inflammatory monocytes, a factor consistently reported to drive the development of atherosclerosis. In preclinical models of both type 1 and type 2 diabetes, studies have demonstrated that the increased production and activation of monocytes is driven by enhanced myelopoiesis, promoted by factors, including hyperglycemia, impaired cholesterol efflux, and inflammasome activation, that affect the proliferation of bone marrow precursor cells. This suggests that continued mechanistic investigations of the enhanced myelopoiesis and the generation of inflammatory monocytes are timely, from the dual perspectives of understanding more deeply the underlying bases of diabetes pathophysiology and identifying therapeutic targets to reduce cardiovascular risk in these patients.

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.

OBJECTIVE: Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. APPROACH AND RESULTS: Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1+/-12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1+/-3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and ICAM-1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1beta-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1beta-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. CONCLUSIONS: Platelet activity measurements and subsequent interleukin-1beta-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE.