The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several pathological conditions including atherosclerosis. It remains unclear in humans whether these elevated levels of EDA+-FN are actively contributing to disease pathogenesis, or rather simply serving as a marker associated with vascular stress and/or injury. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses We hypothesize that presence of EDA+-FN in plasma promotes inflammation and accelerates atherosclerotic plaque formation.
We generated EDA+/+/ApoE−/− mice, which contain optimized spliced sites at both splicing junctions of the EDA exon and constitutively express only EDA+-FN, and EDA−/−/ApoE−/− mice, which contain an EDA-null allele of the EDA exon and express only FN lacking EDA. ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the VerHoeffs/Van Gieson stain.
We report that atherosclerotic plaque (% of total aorta) formation in the aorta of EDA+/+/ApoE−/− mice was increased by two-fold compared to control ApoE−/− mice (P<0.0001). Deletion of the alternatively spliced EDA domain in the ApoE−/− mice (EDA−/−/ApoE−/−) significantly reduced atherosclerotic plaque formation in the aorta (P<0.05) compared to ApoE−/− mice. Total cholesterol and triglycerides levels were similar in ApoE−/−, EDA+/+/ApoE−/− and EDA−/−/ApoE−/− mice. Similarly, atherosclerotic plaque formation was significantly increased in the aortic sinus of EDA+/+/ApoE−/− mice, intermediate in control ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice (P<0.05). Additionally, we found that macrophage content, as analyzed by immunohistochemistry, was significantly elevated in the aortic root lesions of EDA+/+/ApoE−/− mice and reduced in EDA−/−/ApoE−/− mice compared to ApoE−/− mice (P<0.05). Moreover, EDA+-FN did not affect the sex-dependent regulation of atherosclerosis in ApoE−/− mice. Future experiments using EDA+/+/ApoE−/−/TLR4−/− are under progress to determine whether EDA+-FN exacerbate atherosclerosis via upregulating TLR4 signaling.
Our findings reveal that EDA+-FN is pro-inflammatory and promotes atherosclerotic lesion formation and that monitoring plasma EDA+-FN might have prognostic value in patients at high risk for atherosclerosis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.