Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow

Abstract

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Conclusions: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

Role of pericytes in the development of cerebral cavernous malformations

“…41 Fibronectin is expressed during development, especially in vascular morphogenesis, and vascular injury. 42 , 43 , 44 Pericytes are expressed ten times more fibronectin than ECs. 45 Furthermore, RNA-seq analysis results revealed that the fibronectin mRNA level was upregulated in pericytes and downregulated in Ccm1 conditional knockout ECs ( Table 1 ).…”

Splice Factor Polypyrimidine tract-binding protein 1 (Ptbp1) is Required for Immune Priming of the Endothelium in Atherogenic Disturbed Flow Conditions

“…In mouse embryonic fibroblasts, Ptbp1 was bound to mRNA encoding vinculin and alpha-actinin – bringing them to the spreading periphery, and was required for cell spreading 37 . In response to LDF patterns and in early atherogenesis, fibronectin is deposited beneath the endothelium of arteries 6,10,38 . Fibronectin that is deposited, either from the plasma or the endothelium, promotes NFκB activity and myeloid recruitment and plaque in these regions 6,39 .…”

“…Virus supernatant was added to recipient mAECs with polybrene (8ug/mL), and cells were selected with puromycin for 4 days, confirming a MOI<0.3 and complete killing of uninfected cells. In experiments examining induction of Icam and Vcam in the endothelium and protein and RNA analysis, recipient cells were TetOn-Sv40 aortic endothelial cells, prepared as previously described 9,10 . In experiments in which eGFP reported NFkB signaling activity, recipient cells had been infected with a modified version of an NFkB reporter construct, engineered to express RFP constitutively and eGFP upon activation of the 4x NFkB binding motif.…”

“…Alterations in the levels and activities of RNA-binding splice factors can allow a cell to rapidly modify the composition of signaling pathways. For example, increased inclusion of alternative exons EIIIA and EIIIB in fibronectin is linked to alterations in extracellular matrix composition, integrin binding, and increased NFκB signaling 7,10 . Thus, we hypothesize that alternative splicing mediates acute endothelial priming during the arterial response to LDF.…”

Molecular ZIP codes in targeted drug delivery

“…A ZC can arise from an ECM protein in two ways: by an increase in an ECM protein or a specific splice variant, or by increased accessibility to a probe of a protein or protein epitope in tumor ECM. Fibronectin and tenascin are among the ECM proteins with multiple splice variants ( 57 , 58 ). Some of these are predominantly expressed in embryonic and fetal tissues, and their re-expression in tumors makes them targets for drug delivery into tumors ( 58 – 61 ).…”