Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

RGDN peptide interaction with endothelial alpha5beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles.

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.

Transforming growth factor beta3 promotes fascial wound healing in a new animal model.

HYPOTHESIS: Transforming growth factor beta(3) (TGF-beta(3)) promotes fascial wound healing in a new animal model, as measured by wound breaking strength, collagen deposition, and cellular proliferation. DESIGN/INTERVENTION: Bilateral, longitudinal incisions were made in the anterior rectus sheaths of 24 male New Zealand white rabbits. One incision was treated with 1 microg of TGF-beta(3); the contralateral incision served as a control. The wounds were harvested at 1, 2, 3, 4, 6, and 8 weeks after creation ("wounding"). MAIN OUTCOME MEASURES: Wound tissue was tested for breaking strength using a tensiometer and processed for histological examination of collagen deposition and cellular proliferation at all time points after wounding. Collagen deposition and cellular proliferation were measured in histological cross sections of wounds with Masson trichrome staining and proliferating cell nuclear antigen immunohistochemistry, respectively. RESULTS: At all time points after wounding, treatment with TGF-beta(3) significantly increased the wound breaking strength (up to 138%) and collagen deposition (up to 150%) over the control group. Cellular proliferation was increased during the first 3 weeks after wounding (up to 147%), but returned to baseline levels by the fourth week. CONCLUSIONS: Transforming growth factor beta(3) promotes fascial wound healing. In this new animal model of fascial wound healing, TGF-beta(3) increased fascia breaking strength, collagen deposition, and cellular proliferation. These results are similar to findings in cutaneous wound models and demonstrate, for the first time, a pharmacologic agent to accelerate fascial healing.

Control of smooth muscle cell proliferation and phenotype by integrin signaling through focal adhesion kinase.

Extracellular matrix proteins such as fibronectin (FN) and laminin (LM) are known to help control the growth and phenotype of vascular smooth muscle cells (VSMCs). Here we have analyzed the relationship between growth factor and integrin signaling pathways in VSMCs. Culturing porcine coronary artery smooth muscle cells (PCASMCs) on FN and LM leads to distinct effects on cell proliferation and contractile protein expression. PCASMCs cultured on FN proliferate at a higher rate than cells cultured on LM, regardless of the growth factor used to support proliferation. Moreover, cells cultured on LM show higher levels of expression of smooth muscle myosin heavy chain (a marker of smooth muscle cell differentiation) than cells cultured on FN. In contrast to the effects on proliferation and contractile protein expression, both FN and LM supported cell migration in response to PDGF. Also, both FN and LM supported activation of ERK1 and ERK2 in response to PDGF and bFGF. However, FN and LM did show a difference in their ability to support signaling through the focal adhesion kinase (FAK). PCASMCs cultured on FN show robust activation of FAK in response to either PDGF or bFGF, however, cells cultured on LM show little-to-no activation of FAK in response to the growth factors. The results show that integrin signaling pathways have a profound effect on VSMC proliferation and phenotype, and that FAK is an important intermediate in these signaling pathways. The implications of our findings on the mechanisms controlling VSMC proliferation and phenotype in pathological states such as atherosclerosis and restenosis are discussed.

Vascular smooth muscle alpha v beta 3 integrin mediates arteriolar vasodilation in response to RGD peptides.

Arteriolar vasodilation and the resultant increase in blood flow are characteristic vascular responses to tissue injury. The dilatory mediators signaling these responses are incompletely understood. We show that integrin-binding peptides containing the Arg-Gly-Asp (RGD) tripeptide sequence cause immediate and, in some instances, sustained vasodilation when applied to isolated rat cremaster arterioles. The vasodilation is dependent on interaction of the soluble RGD sequence with the alpha v beta 3 integrin expressed by smooth muscle cells in the arteriolar wall. Possible in vivo sources of soluble RGD sequences are fragments of extracellular matrix proteins that are generated after tissue injury. Indeed, protease-generated fragments of denatured collagen type I (a major source of RGD sequences) also cause cremaster arteriolar vasodilation through the alpha v beta 3 integrin. Thus, extracellular matrix protein fragments containing the RGD sequence may act as vascular wound recognition signals to regulate blood flow to injured tissue.

Role of K+ channels in arteriolar vasodilation mediated by integrin interaction with RGD-containing peptide.

Integrins are transmembrane adhesion receptors found on most cells, including vascular smooth muscle cells. Several integrins bind to the conserved amino acid sequence Arg-Gly-Asp (RGD), and synthetic RGD-containing peptides can cause endothelium-independent arteriolar vasodilation by interacting with the alphavbeta3-integrin expressed by vascular smooth muscle. We hypothesized that RGD peptide-induced vasodilation involves K+ channels. Rat cremaster arterioles were treated with cRGD (GPenGRGDSPCA) in the presence or absence of the nonselective K+ channel inhibitor tetraethylammonium (TEA, 20 mM). TEA caused arterioles to constrict by 19 +/- 5% and inhibited cRGD-induced vasodilation (n = 7, P < 0.05). Vessels preconstricted with phenylephrine (5 x 10(-7) M) showed no significant inhibition of the dilatory response to cRGD, indicating that inhibition by TEA was not related to increased vasomotor tone. Further evidence for the involvement of K+ channels was obtained by addition of 100 mM KCl (n = 5), which inhibited vasodilation caused by cRGD. Inhibition of large and small conductance, Ca2+-activated K+ channels with iberiotoxin (100 nM) or apamin (25 nM), respectively, had no effect on cRGD-induced vasodilation. Partial inhibition of vasodilation was observed with inhibitors of voltage-gated (4-aminopyridine, 1 mM), ATP-sensitive (glibenclamide, 1 microM), and inward rectifying (barium, 50 microM) K+ channels. These data support the hypothesis that integrin-signaling pathways leading to arteriolar vasodilation may involve modulation of K+ channel function.

Wound epithelialization deficits in the transforming growth factor-alpha knockout mouse.

In vitro, transforming growth factor-alpha is an important factor controlling epithelial cell proliferation and migration. However, the transforming growth factor-alpha knockout mouse has shown no wound epithelialization defect in tail amputation and full-thickness back wounds. To resolve this disparity, we combined a full-thickness head wound and a partial-thickness ear wound on the transforming growth factor-alpha knockout mouse for analysis of wound epithelialization with or without granulation tissue formation. Three-millimeter ear wounds were made on the transforming growth factor-alpha knockout and heterozygous control mice. Full-thickness head wounds were made using a 6-mm trephine on the crown of the skull. In the ear model, transforming growth factor-alpha knockout mice had significantly larger epithelial gaps versus control at post-operative day 3 and 5. Epithelial thickness at the wound edge of transforming growth factor-alpha deficient mice was also depressed at post-operative day 3 and post-operative day 5 compared to control mice. On post-operative day 8, most wounds of both groups were epithelialized. In contrast, no difference in epithelial gap or new granulation tissue was found in the head model. The data support the concept that transforming growth factor-alpha plays a significant early role in wound epithelialization in vivo but its deficit is compensated if accompanied by granulation tissue formation. The data further show the importance of appropriate wound models to address the role of vulnerary factors.

Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide.

It has previously been shown that synthetic peptides containing the sequence arginine-glycine-aspartic acid (RGD) cause vasodilation by activation of alpha(v)beta3-integrin present on vascular smooth muscle (VSM) cells. The purpose of this study was to determine whether this dilatory effect is mediated by a reduction in VSM cytosolic Ca2+ concentration ([Ca2+]i). First-order arterioles from the rat cremaster were isolated, cannulated, and pressurized. [Ca2+]i was quantitated from the ratio of emitted fluorescence intensity during alternate excitation of fura 2-loaded vessels at 340 and 380 nm. Cyclo(-Arg-Gly-Asp-D-Phe-Val) (cycloRGD; 0.21-210 microM) produced a concentration-dependent dilation of arterioles that had developed basal myogenic tone. Over the entire concentration range tested, [Ca2+]i decreased from 91 +/- 6 to 27 +/- 4 nM (69.7 +/- 5.0% reduction). In association with the decrease in [Ca2+]i, arteriolar lumen diameter increased from 89 +/- 8 to 184 +/- 8 pm (89.8 +/- 1.8% dilation). At intermediate concentrations, cycloRGD induced rhythmic spiking of Ca2+ superimposed on the concentration-dependent lowering of basal [Ca2+]i. These data directly link integrin activation with alterations in Ca2+ regulation, the net effect of which is a reduction in [Ca2+]i. These data further suggest that integrins, through their role in mediating cellular attachment to the extracellular matrix and in cellular signaling involving Ca2+, could provide a logical link to mechanotransduction and myogenic phenomena.

Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model.

BACKGROUND: Gene therapy has shown limited efficacy for treating congenital diseases, partly due to temporary gene expression and host immune responses. Such results suggest that gene therapy is ideal for chronic wound treatment where limited duration of target gene expression is required. This study tested the wound healing effects of topically applied platelet-derived growth factor (PDGF)-A or -B chain DNA plasmids embedded within a collagen lattice. MATERIALS AND METHODS: Four 6-mm dermal ulcer wounds were created in the ears of young adult New Zealand White rabbits made ischemic by division of the central and rostral arteries. Wounds were treated with lyophilized collagen containing PDGF-B DNA (1.0-3.0 mg), PDGF-A DNA (1.0 mg), irrelevant DNA (1.0 mg), or collagen alone. Wounds were dressed and harvested after 10 days for measurement of granulation tissue formation, epithelialization, and wound closure. Results were evaluated with a paired two-tailed Student t test, with P values < 0.05 considered significant. RESULTS: PDGF-B DNA increased new granulation tissue (NGT) formation up to 52% and epithelialization 34% compared with controls. Wound closure was increased up to threefold. At 1.0 mg DNA, PDGF-A and PDGF-B stimulated similar responses. No difference in NGT or epithelialization was seen between control groups. CONCLUSIONS: PDGF DNA gene therapy is effective at accelerating wound healing in ischemic dermal ulcers and provides a viable alternative to peptide growth factor therapy.