The cell-dependent polymerization of intercellular fibronectin fibrils can stimulate cells to self-assemble into multicellular structures. The local physical cues that support fibronectin-mediated cellular self-assembly are largely unknown. Here, fibronectin matrix analogs were used as synthetic adhesive substrates to model cell-matrix fibronectin fibrils having different integrin-binding specificity, affinity, and/or density. We utilized this model to quantitatively assess the relationship between adhesive forces derived from cell-substrate interactions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Results indicate that the strength of initial, rather than mature, cell-substrate attachments correlates with the ability of substrates to support fibronectin-mediated cellular self-assembly. The cellular response to soluble fibronectin was bimodal and independent of the integrin-binding specificity of the substrate; increasing soluble fibronectin levels above a critical threshold increased aggregate cohesion on permissive substrates. Once aggregates formed, continuous fibronectin polymerization was necessary to maintain cohesion. During self-assembly, soluble fibronectin decreased cell-substrate adhesion strength and induced aggregate cohesion via a Rho-dependent mechanism, suggesting that the balance of contractile forces derived from fibronectin fibrils within cell-cell versus cell-substrate adhesions controls self-assembly and aggregate cohesion. Thus, initial cell-substrate attachment strength may provide a quantitative basis with which to build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates.

Key pointsr The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms.r In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. Abstract Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin-and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg −1 ) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 μm, n = 12 and 17.2 ± 2.4 μm, n = 14, respectively) whereas mutation of the active sequence (R 613 WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 μm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 μm, n = 12) that was significantly decreased (to 7.0 ± 2.7 μm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,[8][9][10] and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca 2+ (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.

During wound repair, soluble fibronectin is converted into biologically active, insoluble fibrils via a cell-mediated process. This fibrillar, extracellular matrix (ECM) form of fibronectin stimulates cell processes critical to tissue repair. Nonhealing wounds show reduced levels of ECM fibronectin fibrils. The objective of this study was to produce a small, recombinant wound supplement with the biological activity of insoluble fibronectin fibrils. A chimeric fibronectin fragment was produced by inserting the integrin-binding Arg-Gly-Asp (RGD) loop from the tenth type III repeat of fibronectin (FNIII10) into the analogous site within the heparin-binding, bioactive fragment of the first type III repeat (FNIII1H). FNIII1H was tested for its ability to support cell functions necessary for wound healing, and then evaluated for its capacity to accelerate healing of full-thickness dermal wounds in diabetic mice. , FNIII1H supported cell adhesion, proliferation, and ECM fibronectin deposition. Application of FNIII1H to dermal wounds of diabetic mice significantly enhanced wound closure compared with controls (73.9% ±4.1% vs. 58.1% ±4.7% closure on day 9, respectively), and significantly increased granulation tissue thickness (2.88 ± 0.75-fold increase over controls on day 14). Recombinant proteins designed to functionally mimic the ECM form of fibronectin provide a novel therapeutic approach to circumvent diminished fibronectin fibril formation by delivering ECM fibronectin signals in a soluble form to chronic wounds. A small, chimeric fibronectin protein was developed. FNIII1H demonstrated enhanced bioactivity and stimulated wound repair in a murine model of chronic wounds.

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