A. Wayne Orr, Ph.D.

Associate Professor
Ph.D., 2002, University of Alabama at Birmingham
Department of Pathology
LSU Health Sciences Center
BRI Rm 6-21
1501 Kings Hwy
Shreveport, LA 71103
Office phone: 318.675.5462
Lab phone: 318.675.5463
Fax: 318.675.8144
Email: aorr@lsuhsc.edu

Major Research Interests: Despite advances in treatments over the past several decades, cardiovascular disease remains the leading cause of death in developed countries worldwide. Atherosclerosis, a chronic inflammatory disease of the vessel wall, is responsible for more than 80% of all cardiovascular disease related deaths. Current models suggest that the local accumulation of low density lipoproteins (LDL, “bad cholesterol”) in the vessel wall promotes an inflammatory response characterized by endothelial cell activation and leukocyte recruitment. Local inflammation may also promote plaque rupture by degradingand weakening the protective fibrotic cap overlying the plaque. Transition from a quiescent endothelial monolayer to a site of local endothelial cell activation requires specific stimulation from soluble factors, local hemodynamics, and adhesive interactions with the local microenvironment. While current cardiovascular disease therapies target the systemic soluble risk factors, the role of hemodynamics and adhesive interactions are less well characterized and have not yet been utilized for therapeutic value. Our lab studies how cell-cell and cell-matrix adhesions affect endothelial cell activation and atherosclerosis. Through a better understanding of the molecular mechanisms regulating endothelial cell activation, we hope to identify novel therapeutic targets to limit cardiovascular disease.

Cell-Matrix Interactions – Our research has shown an important role for matrix composition in modulating the cellular response to multiple atherogenic stimuli. Early during atherogenesis in vivo, wound-associated transitional matrix proteins (ex. fibronectin, fibrinogen) become deposited into the endothelial cell basement membrane. In vitro, these transitional matrix proteins enhance the ability of atherogenic stimuli, such as disturbed flow patterns and oxidized LDL, induce endothelial cell dysfunction, while components of the basement membrane (ex. collagen, laminin) limit endothelial cell dysfunction. We hypothesize that signals from cell-matrix interactions serve as a form of tissue memory, helping cells place atherogenic stimuli into a temporal context by indicating whether the tissue is actively remodeling. Our current research seeks to understand the factors regulating matrix remodeling during atherogenesis (ex. hyperglycemia) and to identify the molecular mechanisms different matrix proteins utilize to affect endothelial cell dysfunction. This research is currently funded by an NIH R01 award.

Diabetic matrix remodeling – Cardiovascular complications, especially the formation of atherosclerotic plaques, account for the majority of diabetes-associated mortality. Elevated blood glucose (hyperglycemia) in diabetics stimulates atherosclerosis by disrupting the protective endothelial cell layer that lines the blood vessels. While hyperglycemia is systemic and should affect the vasculature uniformly, atherosclerotic plaques preferentially form in certain regions of the vasculature exposed to disturbed blood flow, suggesting flow patterns regulate hyperglycemia-induced endothelial cell dysfunction. Interestingly, hyperglycemia stimulates the production of transitional matrix proteins in cells in culture. We hypothesize that hyperglycemia promotes transitional matrix deposition specifically at sites of disturbed flow and that these matrix proteins enhance hyperglycemia-induced endothelial cell dysfunction. This research is currently funded by an American Diabetes Association Junior Faculty Award.

Cell-Cell Interactions – The ephrin and Eph family of cell-cell adhesion molecules are well characterized regulators of vascular and neuronal development and are implicated in numerous tissue remodeling responses in adult organisms. However, a role for Eph and ephrin signaling has never been described for atherosclerosis. We have recently demonstrated that the ephrins and their Eph receptors show altered expression and activation patterns in response to atherogenic agents in vitro, and show enhanced expression in both mouse models of atherosclerosis and human atherosclerotic plaques. Current data suggests that signaling through these Eph receptors may facilitate the local inflammatory response. Our current research seeks to identify the ephrins and Eph receptors involved in atherosclerosis and characterize their role in atherosclerotic progression.

Ongoing cardiovascular-related projects:

1. Matrix-specific signaling in endothelial cell dysfunction.
Brief description: Cells adapt to extracellular signals, such as the composition of the extracellular matrix and local biomechanical stimuli, to match cellular function to environmental context. During the early stages of atherosclerosis, endothelial activation by circulating atherogenic risk factors is critically regulated by shear stress, the frictional force generated from blood flow. Work from my laboratory has found that changes in the subendothelial matrix composition similarly alter the endothelial response to both shear stress and soluble stimuli (e.g. oxidized LDL). The goal of this project is to characterize the interplay between signaling responses activated in endothelial cells on normal basement membrane proteins (e.g. protein kinase A, eNOS) and those activated on pathogenic transitional matrix proteins such as fibronectin (e.g. p21 activated kinase, NF-κB).   In addition, this project seeks to identify how matrix composition regulates oxLDL-induced endothelial cell dysfunction and determine whether inhibiting specific cell-matrix receptors affect atherosclerotic plaque formation in mouse model systems.
Names of collaborators / collaborating institutions: Christopher Kevil (Pathology), James Traylor Jr. (Pathology), Richard Hynes (MIT), and Andrew Mazar (Northwestern University).
Funding source: NIH R01 grant (8/15/10- 5/31/15)

2. EphA/ephrinA interactions in atherosclerotic inflammation.
Brief description: The goal of this project is to test the hypothesis that EphA2 critically regulates endothelial and macrophage function to potentiate atherosclerotic plaque formation. We will test this hypothesis by determining whether EphA2 contributes to atherosclerotic plaque formation in ApoE knockout mice (Aim 1) and characterizing how EphA2 expression on endothelial cells and monocytes affect monocyte homing responses (Aim 2).
Names of collaborators / collaborating institutions: Christopher Kevil (Pathology), James Traylor Jr. (Pathology), Andrew Yurochko (Microbiology), Jie Chen (Vanderbilt University), and Alessandra Vitelli (Okairos).
Funding source: American Heart Association Grant-in-Aid (7/1/13- 6/31/15)

Planned cardiovascular-related projects:

1. Matrix stiffness in the endothelial response to shear stress.
Brief description: During diet-induced and especially diabetes-induced atherosclerosis, the extracellular matrix in the vessel wall stiffens. This stiffer matrix likely affects how the endothelial cells respond to changes in matrix composition as well as their response to biomechanical force such as shear stress. However, the role of matrix stiffness in the endothelial response to flow has not been explored. Future work would utilize cell culture models that accurately alter matrix stiffness and shear stress simultaneously to determine the subsequent effect on endothelial cell function.

2. EphA2 affects lipid metabolism in mouse models of atherosclerosis.
Brief description: Mice deficient for the EphA2 receptor have alterations in their circulating plasma triglyceride levels and circulating cholesterol as measured by commercial ELISA kits. Future work would utilize FPLC to gain a better understanding of the plasma lipid content in these mice with follow up studies on hepatic lipid metabolism, adipose tissue function, and endothelial lipoprotein lipase activity.

3. Oxidant stress-induced signaling through the Nck pathway.
Brief description: Shear stress-induced proinflammatory gene expression requires both reactive oxygen species and the serine/threonine kinase activated kinase (PAK). PAK activation requires its binding partner Nck, and blunting the interaction between PAK and Nck blocks the endothelial inflammatory response to shear stress, exogenous hydrogen peroxide (H2O2), and hypoxia/reoxygenation injury. Interestingly, a peptide inhibitor of the PAK/Nck interaction significantly reduces both inflammation and permeability in the cremaster muscle model of ischemia-reperfusion injury. Future work would characterize the post-translational modifications that regulate activation of the PAK/Nck pathway and characterize the role of PAK/Nck interactions in other oxidant stress-driven pathologies. In addition to the peptide inhibitor, we have developed mice that lack the Nck1/Nck2 genes specifically in the endothelium.

Methods and Available Resources:

  • Parallel plate flow chamber for stimulating cells with laminar and oscillatory shear stress
  • Endothelial isolation, transformation, transfection, and culture
  • Isolation and culture of bone marrow-derived macrophages
  • Mouse models of atherosclerosis and analysis of plaque formation at multiple vascular sites
  • Partial carotid ligation model to induce disturbed flow patterns in the common carotid artery
  • Mouse models of diabetes and macrovascular complications of diabetes
  • Alzet osmotic pump implantation
  • Bone marrow transplantation for chimeric mice generation
  • Tissue processing and immunohistochemical staining
  • mRNA isolation and analysis by qRT-PCR
  • Site-directed mutagenesis for biochemical analysis of protein function
  • Signaling pathway analysis by Western blot, immunoprecipitation, IP kinase assays, Proximity Ligation Assays, and ELISA
  • Analysis of integrin activation status and expression.
  • Bank of atherosclerotic plaques isolated from human patients.
  • Atherosclerotic plaques isolated from mouse models of hypercholesterolemia-induced and hyperglycemica-induced atherosclerosis.
  • mRNA isolated from mouse atherosclerotic plaques and various mouse organs.
  • mRNA from human peripheral blood monocytes isolated from ~10 different subjects.
  • mRNA from endothelial cells isolated from different vascular beds (aorta, coronary artery, umbilical vein, saphenous vein) taken from at least 4 different subjects.
  • Endothelial cells including conditionally transformed (temperature-sensitive) mouse aortic endothelial cells (MAECs) and bovine aortic endothelial cells (BAECs)
  • Tissue processing, embedding, and cutting equipment.
  • qRT-PCR primers for analysis of integrin and Eph/ephrin expression as well as a large variety of proinflammatory, atherogenic, and extracellular matrix proteins (both human-specific and mouse-specific primers)
  • DNA constructs (constitutively active, dominant negative, activation assay, luciferase) for analysis of a variety of signaling pathways, including multiple RhoGTPases, MAP kinases, protein kinase A, NF-κB, and NFAT.

Publications: http://www.ncbi.nlm.nih.gov/pubmed?term=orr%20aw%5BAuthor%5D