Haemodynamics Regulate Fibronectin Assembly via PECAM

Abstract

Fibronectin (FN) assembly and fibrillogenesis are critically important in both development and the adult organism, but their importance in vascular functions is not fully understood. Here we identify a novel pathway by which haemodynamic forces regulate FN assembly and fibrillogenesis during vascular remodelling. Induction of disturbed shear stress in vivo and in vitro resulted in complex FN fibril assembly that was dependent on the mechanosensor PECAM. Loss of PECAM also inhibited the cell-intrinsic ability to remodel FN. Gain- and loss-of-function experiments revealed that PECAM-dependent RhoA activation is required for FN assembly. Furthermore, PECAM^-/- mice exhibited reduced levels of active β1 integrin that were responsible for reduced RhoA activation and downstream FN assembly. These data identify a new pathway by which endothelial mechanotransduction regulates FN assembly and flow-mediated vascular remodelling.

Figure 1. PECAM is required for FN assembly in response to CAL.

(A) 3 weeks after CAL, sham and ligated carotid arteries were harvested, mounted en face and stained for FN. (B) FN fibril area was quantified. Scale Bar = 20 μm; n = 16 WT and 10 PECAM^−/− mice. (C) WT FN fibril angle was analyzed in sham and 3-week ligated LCAs n = 3 sham and n = 5 CAL. (D) KO FN fibril angle was analyzed in sham and 3-week ligated LCAs n = 2 sham and n = 3 CAL.

Figure 2. Disturbed shear stress induces PECAM-dependent FN assembly.

RC and KO cells were subjected to flow or kept as static controls. (A) After flow, RC and KO cells were fixed and stained for FN, followed by (B) quantification of FN fibril area. (C) Representative western blots showing DOC-insoluble FN in RC and KO ECs before or after oscillatory shear stress. Quantification of DOC-insoluble FN relative to static RC lysates is shown, and was performed after normalization to vimentin. Scale bar in (A) is 20 um. (A,B) n = 5 independent experiments; (C) n = 4 independent experiments.

Figure 3. PECAM is required for the intrinsic ability of ECs to remodel FN.

(A) FITC-conjugated FN was provided to static RC and KO cells as a tissue culture glass coat, media supplement, or both. Representative images are shown (FN was pseudocolored in red for clarity). Representative images are shown (FN was pseudocolored in red for clarity). Scale Bar = 40 μm (B) Percent fibril area was also quantified. (A,B) n = 3 independent experiments.

Figure 4. PECAM-dependent RhoA activity is required for FN assembly.

(A) RhoA-GTP levels in RC and KO cells were determined by RBD pulldown. FN assembly was assayed by DOC-insolubility. (B) Quantification of RhoA-GTP and DOC-insoluble FN is below. (C) KO cells were stimulated with LPA, a RhoA agonist. RhoA-GTP was determined by RBD pulldown and FN assembly was assayed by DOC-insolubility. (D) Quantification of RhoA-GTP and DOC-insoluble FN is below. (E) ECs were transfected with control or RhoA siRNA and Rho-GTP levels and DOC-insuble FN were assayed. (F) Quantification of RhoA-GTP and DOC-insoluble FN is below. Experiments were performed in static cells. (A,B) n = 4 independent experiments; (C,D) n = 6 independent experiments; (E,F) n = 4 independent experiments.

Figure 5. Role of β1 integrin activation in FN assembly.

(A) Active β1-integrin staining in cross sections of WT and PECAM^−/− carotid arteries harvested 5 days post-CAL. Scale Bar = 20 μm; n = 3 independent experiments. (B) ECs were transfected with control or β1 integrin siRNA and levels of GTP-RhoA and DOC-insoluble FN were assayed and quantified. (C) ECs were incubated with control IgG or the β1 integrin function blocking antibody AIIB2 before determination of FN assembly by DOC-insolubility. (B) n = at least 3 independent experiments. (C) n = 4 independent experiments.

Figure 6. Distrubed flow regulates PECAM-dependent FN assembly through a β1-integrin-RhoA pathway.

Distrubed flow regulates FN fibrillogenesis; mechanistically, this is dependent on PECAM-dependent activation of β1 integrins and RhoA.