title: Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.
abstract: The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. 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. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin. The fibronectin-dependent component of the integrated muscle contraction-dependent arteriolar vasodilatation is coupled through an endothelial cell-dependent signalling pathway. 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,8-10 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.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA.|Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA.
authors.forename: Ingrid H
authors.initials: IH
authors.lastname: Sarelius
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Titus
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA.
authors.forename: Nir
authors.initials: N
authors.lastname: Maimon
authors.affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA.
authors.forename: William
authors.initials: W
authors.lastname: Okech
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Wilke-Mounts
authors.affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Brennan
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY, USA.|Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA.
authors.forename: Denise C
authors.initials: DC
authors.lastname: Hocking
mesh_terms: D000818:Animals; D001160:Arterioles / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D042783:Endothelial Cells / Q000502:physiology; D004730:Endothelium, Vascular / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology*; D005353:Fibronectins / Q000502:physiology*; D006493:Heparin / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000098:blood supply / Q000502:physiology*; D010455:Peptides / Q000502:physiology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D014664:Vasodilation / Q000502:physiology
chemical_list: D005353:Fibronectins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006493:Heparin; D002118:Calcium