Abstract 639: Deletion of Alternatively Spliced Extra Domain A of Cellular Fibronectin Stabilizes Advanced Rupture-Prone Plaques in Hyperlipidemic Mice

Background: The most important clinical manifestation of atherosclerosis is rupture of advanced plaques. The fibronectin containing alternatively spliced extra domain A (Fn-EDA) is abundant in extracellular matrix around macrophages and endothelial cells in advanced human plaques. In vitro studies suggest that Fn-EDA is a ligand for TLR4 and upregulates MMP9, which has been shown to enhance elastin degradation and promote plaque rupture in advanced plaques of hyperlipidemic apolipoprotein E-deficient (Apoe -/- ) mice. The mechanistic role of Fn-EDA in advanced plaques remains unknown. Hypothesis: Fn-EDA/TLR4 signaling in activated macrophages contributes to plaque vulnerability by upregulating MMP9 during advanced atherosclerosis. Methods and Results: We compared atherosclerotic plaques in the brachiocephalic artery (a model artery for study of plaque vulnerability) of female Fn- EDA -/- Apoe -/- and Apoe -/- mice at 50 weeks of age (44 weeks on high-fat Western diet). Fn- EDA -/- Apoe -/- mice exhibited decreased plaque size and characteristics of stable plaques, with decreased necrosis, reduced influx of activated macrophages, increased SMC staining and greater collagen content compared to Apoe -/- mice (P&lt;0.05, n=11-13 mice/group), although cholesterol and triglyceride levels, and circulating leukocyte counts were similar. Purified cellular FN, which contains EDA, potentiated the NFκB-mediated inflammatory pathway (increased phospho-NFκB p65/ NFκB p65, TNFα and IL1β) and MMP9 protein expression (gelatin zymography) in a dose-dependent manner in purified macrophages from Fn-EDA -/- Apoe -/- mice but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Using immunohistochemistry, we demonstrated colocalization of macrophage TLR4 and Fn-EDA within advanced atherosclerotic plaques in murine brachiocephalic arteries and human coronary arteries. Genetic deletion of TLR4 in Apoe -/- mice stabilized advanced plaques, and parameters of plaque stability were comparable between TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice. Conclusions: These findings suggest that Fn-EDA/TLR4 signaling in macrophages is a key mechanism that upregulates MMP9, and thereby promotes plaque vulnerability in advanced atherosclerosis. </jats:p

Abstract 332: Fibronectin Containing Extra Domain a in the Plasma, but not Tissue, Exacerbates Stroke Outcome in the Comorbid Condition of Hyperlipidemia

Background: Fibronectin-splicing variant containing extra domain A (Fn-EDA) is expressed in the endothelium of atherosclerotic arteries and elevated in circulation in at risk of patients with diabetes, hypertension, and atherosclerosis. Recent studies suggest that Fn-EDA contributes to ischemic stroke. The relative contribution of plasma versus (vs.) tissue Fn-EDA in stroke outcome remains unclear. We determined the contribution of plasma vs. tissue Fn- EDA in stroke exacerbation in the comorbid condition of hyperlipidemia. Methods: We generated Fn-EDA fl/fl AlbCre + , which express Fn-EDA in all tissues but not in plasma, on hyperlipidemic apolipoprotein E deficient ( Apoe -/- ) background. Controls were Fn- EDA fl/fl Apoe -/- mice, which express FN-EDA in all tissues and plasma. Susceptibility to stroke outcome was evaluated in male and female mice (8-10 weeks) by transient 1-hour ischemia followed by 1, 3, and 7 days of reperfusion. Quantitative assessment of stroke outcome was evaluated by measuring infarct volume (MRI), cerebral blood flow (laser speckle imaging), neurological outcome, and postischemic thrombo-inflammation (fibrin deposition, neutrophil, and inflammatory cytokines within the infarcted and surrounding region by Western blotting and immunohistochemistry). Susceptibility to thrombosis was evaluated in a FeCl 3 injury- induced carotid thrombosis model. Results: Irrespective of gender, Fn-EDA fl/fl AlbCre + Apoe -/- exhibited smaller infarcts and improved neurological outcome at days 1, 3 and 7 concomitant with improved survival, and decreased post ischemic thrombo-inflammation ( P &lt;0.05 vs. Fn-EDA fl/fl Apoe -/- ). Laser speckle imaging revealed improved regional cerebral blood flow in Fn- DA fl/fl AlbCre + Apoe -/- mice ( P &lt;0.05 vs. Fn-EDA fl/fl Apoe -/- ), suggesting that Fn-EDA present in plasma may promote intracerebral thrombosis and thereby exacerbate stroke. Using i ntravital microscopy, we found that Fn-EDA fl/fl AlbCre + Apoe -/- mice were less susceptible to experimental thrombosis ( P &lt;0.05 vs. Fn-EDA fl/fl Apoe -/- ). Conclusion: Following STAIR guidelines, we provide evidence that Fn-EDA present in the plasma, but not tissue, contribute to stroke exacerbation by promoting thrombo-inflammation. </jats:p

Abstract 374: Lack of Fibronectin Containing Extra Domain a Attenuates Acute Myocardial Ischemia/Reperfusion Injury in Hyperlipidemic Mice by Limiting Postischemic Thrombo-inflammation

Background: Reperfusion therapy is standard care for patients following acute myocardial infarction. Fibronectin-splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for toll-like-receptor 4 (TLR4), is upregulated during vascular hypertension, myocardial injury, and atherosclerosis. An earlier study done in rabbit showed that Fn-EDA accumulates more rapidly (within 24 hrs) in the reperfused hearts. Very little is known about the mechanistic role of Fn-EDA in the pathophysiology of myocardial infarction (MI) in the comorbid condition of hyperlipidemia. Methods: Susceptibility to MI outcome was evaluated in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice (male and female; 8-10 weeks old) by transient 1-hour ischemia/23 hours of reperfusion (I/R). Myocardial I/R injury outcome was evaluated by measuring infarct area, cardiac Troponin I (cTnI) levels in plasma, postischemic thrombo-inflammation (thrombi and neutrophil influx) and myocyte apoptosis. Results: Irrespective of gender, Fn-EDA -/- Apoe -/- mice (Apoe -/- mice expressing Fn deficient in EDA) exhibited smaller infarcts and decreased cTnI levels concomitant with reduced postischemic thrombosis, inflammation and myocyte apoptosis ( P &lt;0.05 vs. Apoe -/ - mice). Genetic ablation of TLR4 attenuated myocardial I/R injury outcome in Apoe -/- mice ( P &lt;0.05 vs. Apoe -/- mice), but did not further reduce in Fn-EDA -/- Apoe -/- mice, suggesting Fn-EDA modulates TLR4-dependent MI exacerbation. Bone marrow transplantation experiments revealed that nonhematopoietic cells-derived Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the hematopoietic cell. Infusion of a specific inhibitor of Fn-EDA into Apoe -/- mice significantly reduced myocardial I/R injury. Conclusions: Hyperlipidemic mice deficient in Fn-EDA exhibit TLR4-dependent reduced myocardial I/R injury that was associated with decreased thrombo-inflammatory response. These findings suggest that targeting Fn-EDA combined with thrombolytic agents could be an effective therapeutic strategy to inhibit myocardial I/R injury in patients with hyperlipidemia. </jats:p

Differential Roles of Endothelial Cell-Derived and Smooth Muscle Cell-Derived Fibronectin Containing Extra Domain A in Early and Late Atherosclerosis

Objective: The extracellular matrix of atherosclerotic arteries contains abundant deposits of cellular Fn-EDA (fibronectin containing extra domain A), suggesting a functional role in the pathophysiology of atherosclerosis. Fn-EDA is synthesized by several cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs), which are known to contribute to different stages of atherosclerosis. Although previous studies using global Fn-EDA-deficient mice have demonstrated that Fn-EDA is proatherogenic, the cell-specific role of EC versus SMC-derived-Fn-EDA in atherosclerosis has not been investigated yet. Approach and Results: To determine the relative contribution of different pools of Fn-EDA in atherosclerosis, we generated mutant strains lacking Fn-EDA in the ECs (Fn-EDA EC-KO ) or smooth muscle cells (Fn-EDA SMC-KO ) on apolipoprotein E-deficient ( Apoe −/− ) background. The extent of atherosclerotic lesion progression was evaluated in whole aortae, and cross-sections of the aortic sinus in male and female mice fed a high-fat Western diet for either 4 weeks (early atherosclerosis) or 14 weeks (late atherosclerosis). Irrespective of sex, Fn-EDA EC-KO , but not Fn-EDA SMC-KO mice, exhibited significantly reduced early atherogenesis concomitant with decrease in inflammatory cells (neutrophil and macrophage) and VCAM-1 (vascular cell adhesion molecule-1) expression levels within the plaques. In late atherosclerosis model, irrespective of sex, Fn-EDA SMC-KO mice exhibited significantly reduced atherogenesis, but not Fn-EDA EC-KO mice, that was concomitant with decreased macrophage content within plaques. Lesional SMCs, collagen content, and plasma inflammatory cytokines (TNF-α [tumor necrosis factor-α] and IL-1β [interleukin-1β]), total cholesterol, and triglyceride levels were comparable among groups. Conclusions: EC-derived Fn-EDA contributes to early atherosclerosis, whereas SMC-derived Fn-EDA contributes to late atherosclerosis. </jats:sec

Targeting Myeloid-Cell Specific Integrin α9β1 Inhibits Arterial Thrombosis in Mice

Background: Coordinated interactions between neutrophils, platelets and endothelial cells contribute towards the development of arterial thrombosis. Neutrophils along with platelets are the first immune cells that are recruited at the site of endothelial activation/injury or infection. Recent studies have suggested that neutrophils modulate thrombosis via several mechanisms, including NETosis (formation of neutrophil extracellular traps). The integrin α9 is highly expressed on neutrophils while platelets do not express it. The integrin α9 up-regulated upon neutrophil activation and is implicated in stable adhesion and transmigration. The mechanisms underlying the role of integrin α9 towards the progression of arterial thrombosis has not been explored yet. Objective: To elucidate the mechanistic insights into the role of myeloid-cell specific integrin α9 in neutrophil adhesion and arterial thrombosis. Methods: We generated novel myeloid-specific α9-/- mice (α9fl/fl LysMcre+l-) by crossing α9fl/fl with LysMcr+/+mice. Littermates α9fl/flLysMcre-l-mice were used as controls. Standardized in vitro assays were used to evaluate the role of integrin α9 in neutrophil mediated platelet aggregation, NETosis and Cathepsin-G release. Susceptibility to arterial thrombosis and hemostasis was evaluated in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy and tail bleeding assay respectively. Results: α9fl/flLysMCre+/-mice developed smaller thrombi (~40% occlusion), when compared with α9fl/flmice (~80% occlusion, 10 minutes post-FeCl3 induced injury). The mean time to complete occlusion was significantly prolonged in α9fl/flLysMCre+/-mice (P&amp;lt;0.05 vs α9fl/fl mice). Consistent with this, α9fl/flLysMCre+/-mice displayed significantly decreased platelet mean fluorescence intensity (MFI) and reduced rate of thrombus growth in laser injury-induced thrombosis model (P&amp;lt;0.05 vs. α9fl/fl mice). Together, these results suggest that myeloid cell-specific integrin α9 contributes to the experimental thrombosis at arterial shear rates. Monocytes depletion experiments demonstrated a minimal role for monocyte in progression of arterial thrombosis. In vitro mechanistic studies demonstrated a reduction in neutrophil-mediated platelet aggregation and cathepsin-G secretion in myeloid cell-specific integrin α9-/- mice, when compared with litter-mates control wild-type mice. Notably, the percentage of cells releasing NETs was markedly reduced in myeloid cell-specific integrin α9-/- mice that was concomitant with reduced MPO levels in carotid thrombus of α9fl/flLysMCre+/-mice. Together, these results suggest most likely integrin α9 expressed on neutrophils, but not monocytes, promotes arterial thrombosis. Comparable tail bleeding time between α9fl/flLysMcreand littermate α9fl/fl mice suggested that myeloid-cell specific deficiency of integrin α9 does not alter hemostasis. Conclusion: These findings reveal a novel role for integrin α9 in modulation of arterial thrombosis. While the clinical implications of these findings remains to be explored, we suggest that targeting integrin α9 may reduce post reperfusion thrombo-inflammatory injury, following acute myocardial infarction or stroke. Disclosures No relevant conflicts of interest to declare. </jats:sec