Abstract
Abstract only
Background and Hypothesis:
Fibronectin (FN) containing the alternatively-spliced extra domain A (EDA
+
-FN) is absent in the arteries or circulation of healthy humans and mice, but specifically expressed in the endothelium of atherosclerotic arteries and elevated in circulation during pathological settings including stroke and atherosclerosis. We tested the hypothesis that EDA
+
-FN exacerbates atherosclerosis.
Model and Methods:
We generated EDA
hi/hi
/ApoE
-/-
mice (over express EDA
+
-FN in plasma) and EDA
-/-
/ApoE
-/-
mice (EDA-deleted allele of FN). ApoE
-/-
mice, which express EDA
+
-FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings including stroke and atherosclerosis, were used as control. Male mice (n=14/group) were fed a high-fat Western diet beginning at 6 weeks until they were sacrificed at 5 and 8 months of age. Extent of atherosclerosis was evaluated in whole aortae (Oil Red O stain) and in the cross section area of the aortic sinus (Verhoeff-Van Gieson stain). Markers of inflammation were quantified by immunohistochemistry.
Results:
We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA
hi/hi
/ApoE
-/-
mice were increased by 2-fold, concomitant with increased plaque inflammatory content (neutrophils, macrophages, and NF-κB p65 expression; P<0.01 vs. ApoE
-/-
). Conversely, EDA
-/-
/ApoE
-/-
mice had smaller plaques (P<0.01 vs. ApoE
-/-
mice), concordant with decreased inflammatory content within plaque. Because EDA
+
-FN activates toll-like receptor 4 (TLR4)
in vitro
, we investigated the role of TLR4 in the EDA
+
-FN-mediated accelerated atherosclerosis. Genetic ablation of TLR4 in EDA
hi/hi
/ApoE
-/-
mice partially reversed exacerbated atherosclerosis (P<0.05 vs. EDA
hi/hi
/ApoE
-/-
mice), whereas TLR4 deficiency in EDA
-/-
/ApoE
-/-
had no effect. Total cholesterol and triglycerides levels were similar among groups. Finally, we report co-localization of EDA
+
-FN with TLR4 on macrophages in human coronary atherosclerotic plaque samples.
Conclusions:
Endogenous EDA
+
-FN exacerbates atherosclerosis via TLR4. Targeting EDA
+
-FN/TLR4 pathway may have the potential to reduce atherosclerosis and improve outcomes in patients at high risk for coronary artery disease.