Alterations in hemodynamic shear stress and oxidant stress at atherosclerosis-prone sites promote a low level, chronic endothelial activation that predisposes these sites to plaque development. Work from my laboratory has identified the Nck family of signaling adaptors, including both Nck1 and Nck2, as novel regulators of endothelial activation. Nck1/2 knockdown or addition of a peptide inhibitor blunts shear and oxidant stress- induced p21 activated kinase (PAK) signaling, thereby reducing endothelial permeability, cytoskeletal remodeling, and NF-?B-driven proinflammatory gene expression. Additionally, we provide compelling evidence that PECAM-1 phosphorylation in response to shear and oxidant stress recruits Nck to endothelial cell-cell junctions, promotes Nck-dependent PAK activation, and stimulates Nck-dependent induction of proinflammatory signaling (NF-?B) and gene expression (ICAM-1, VCAM-1). These data suggest a model whereby Nck recruitment to PECAM-1 alters the local signaling milieu thereby switching PECAM-1 signaling from anti-inflammatory to pro-inflammatory. Endothelial deletion of both Nck1 and Nck2, but not individual isoforms, impairs vascular development. However, several studies have found distinct functions for Nck1 and Nck2, and disturbed flow patterns differentially affect Nck1 and Nck2 expression both in vitro and in vivo. While no studies to date have examined endothelial Nck1 or Nck2 function during pathological conditions in adults, treatment with a Nck- blocking peptide significantly blunts leukocyte recruitment and vascular permeability in multiple model systems in vivo. However, these studies cannot determine whether this peptide acts through Nck or affects endothelial function directly. Our preliminary data utilizing endothelial-specific Nck1/2 deletion in atherosclerosis-prone ApoE knockout mice show reduced proinflammatory gene expression in response to disturbed flow and reduced early plaque development. Therefore, we hypothesize that PECAM-1 phosphorylation in response to shear and oxidant stress facilitates the formation of distinct Nck-based signaling complexes that drive permeability and proinflammatory gene expression under atherogenic conditions.
In Aim 1, we will characterize PECAM-1/Nck complex formation during endothelial activation by mapping the Nck-binding sites on PECAM-1, verifying the interaction in vivo, and determining the crosstalk with local oxidant stress in this response.
In Aim 2, we will characterize the composition and function of Nck-based signaling complexes during endothelial cell activation by assessing the mechanisms of altered Nck1/Nck2 expression, testing their relative roles in endothelial cell activation, and utilizing domain mapping and spatial proteomics to characterize the Nck1/2 interactome during endothelial activation.
In Aim 3, we will determine whether endothelial Nck signaling affects endothelial activation in vivo utilizing mice containing conditional deletion of the Nck genes in endothelial cells to assess their relative roles in endothelial activation under atherogenic conditions.

Public Health Relevance

Functional alterations in the endothelial cells lining the vessel wall represent an important step in atherosclerotic initiation, progression, and clinical complications. Our research identifies members of the Nck family of signaling adaptor proteins as novel players in this process, regulating vessel permeability and proinflammatory gene expression in response to changes in the local microenvironment. Understanding the molecular mechanisms controlling endothelial cell function by the microenvironment may provide novel therapeutic targets to limit chronic inflammatory diseases, such as atherosclerosis and arthritis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL133497-02
Application #
9282802
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Olive, Michelle
Project Start
2016-06-01
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Louisiana State University Hsc Shreveport
Department
Pathology
Type
Schools of Medicine
DUNS #
095439774
City
Shreveport
State
LA
Country
United States
Zip Code
71103
Yuan, Shuai; Yurdagul Jr, Arif; Peretik, Jonette M et al. (2018) Cystathionine ?-Lyase Modulates Flow-Dependent Vascular Remodeling. Arterioscler Thromb Vasc Biol 38:2126-2136
Janjanam, Jagadeesh; Zhang, Baolin; Mani, Arul M et al. (2018) LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis. J Biol Chem 293:3088-3103
Vozenilek, Aimee E; Navratil, Aaron R; Green, Jonette M et al. (2018) Macrophage-Associated Lipin-1 Enzymatic Activity Contributes to Modified Low-Density Lipoprotein-Induced Proinflammatory Signaling and Atherosclerosis. Arterioscler Thromb Vasc Biol 38:324-334
Funk, Steven Daniel; Finney, Alexandra C; Yurdagul Jr, Arif et al. (2018) EphA2 stimulates VCAM-1 expression through calcium-dependent NFAT1 activity. Cell Signal 49:30-38
Shrestha, Bandana; Prasai, Priya K; Kaskas, Amir M et al. (2018) Differential arterial and venous endothelial redox responses to oxidative stress. Microcirculation 25:e12486
Prasai, Priya K; Shrestha, Bandana; Orr, A Wayne et al. (2018) Decreases in GSH:GSSG activate vascular endothelial growth factor receptor 2 (VEGFR2) in human aortic endothelial cells. Redox Biol 19:22-27
Al-Yafeai, Zaki; Yurdagul Jr, Arif; Peretik, Jonette M et al. (2018) Endothelial FN (Fibronectin) Deposition by ?5?1 Integrins Drives Atherogenic Inflammation. Arterioscler Thromb Vasc Biol 38:2601-2614
Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa et al. (2018) Aberrant Mitochondrial Fission Is Maladaptive in Desmin Mutation-Induced Cardiac Proteotoxicity. J Am Heart Assoc 7:
Chandra, Mini; Escalante-Alcalde, Diana; Bhuiyan, Md Shenuarin et al. (2018) Cardiac-specific inactivation of LPP3 in mice leads to myocardial dysfunction and heart failure. Redox Biol 14:261-271
Finney, Alexandra C; Funk, Steven D; Green, Jonette M et al. (2017) EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis. Circulation 136:566-582

Showing the most recent 10 out of 13 publications