| 19482300 |
Raffetto JD, Barros YV, Wells AK, Khalil RA: MMP-2 induced vein relaxation via inhibition of [Ca2+] e-dependent mechanisms of venous smooth muscle contraction. J Surg Res. 2010 Apr;159(2):755-64. Epub 2008 Oct 24.
Role of RGD peptides.. BACKGROUND: Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of varicose veins. We have shown that MMP-2 causes relaxation of venous segments and suggested a role of venous smooth muscle (VSM) hyperpolarization; however, the downstream mechanisms are unclear. We tested whether MMP-2 induced venous relaxation involves inhibition of the Ca (2+) mobilization mechanisms of VSM contraction due to generation of Arg-Gly-Asp (RGD)-containing peptides. METHODS: Circular segments of inferior vena cava (IVC) were isolated from male Sprague-Dawley rats, suspended between two wires in a tissue bath, and isometric contraction was measured. Contraction data in mg/mg tissue were presented as means +/- SEM. RESULTS: In IVC incubated in normal Krebs (2.5 mM Ca (2+)), the alpha-adrenergic agonist phenylephrine (Phe, 10 (-5) M) caused initial peak (133.2 +/- 17.5) followed by a maintained contraction (73.4 +/- 11.6), that was inhibited by MMP-2 (1 microg/mL) to 32.4 +/- 12.8 in 30 min. The inhibitory effects of MMP-2 were reversible by washing the tissue with Krebs or in the presence of the MMP inhibitors TIMP-1 (1 microg/mL), Ro 28-2653, and BB-94 (10 (-6) M), and were not associated with changes in IVC structure, demonstrating specificity. Angiotensin II (AngII, 10 (-6) M) caused a monophasic contraction (114.2 +/- 12.2), that was also inhibited by MMP-2 (66.0 +/- 7.4), suggesting a post-receptor effect on the downstream mechanisms of VSM contraction. To test the role of Ca (2+) release from the sarcoplasmic reticulum, IVC was incubated in Ca (2+)-free 2 mM ethylene glycol-bis (2-aminoethyl ether-N,N,N',N'-tetra-acetic acid (EGTA) Krebs with or without MMP-2. In Ca (2+)-free Krebs, caffeine did not cause contraction, suggesting a limited role of the Ca (2+)-induced Ca (2+)-release mechanism, and Phe and AngII caused a small contraction (7.2 +/- 1.7 and 14.9 +/- 2.8) that was slightly increased by MMP-2 (10.4 +/- 3.0 and 33.8 +/- 10.0), suggesting little effect on IP (3)-induced Ca (2+) release. To test the role of Ca (2+) entry through membrane channels, after eliciting a transient Phe contraction in nominally 0 Ca (2+) Krebs, increasing concentrations of CaCl (2) (0.1, 0.3, 0.6, 1, 2.5 mM) were added and the extracellular Ca (2+) concentration [Ca (2+)](e)-contraction relationship was constructed. The [Ca (2+)](e)-contraction relation was reduced in MMP-2 treated IVC, suggesting inhibition of Ca (2+) entry. In IVC treated with MMP-2, the Ca (2+) channel blocker diltiazem (10 (-5) M) did not cause any further inhibition of Phe contraction, suggesting that Ca (2+) entry is already inhibited by MMP-2. To test whether MMP-2 actions involve generation of RGD and modulation of integrin receptors, experiments where repeated in IVC segments saturated with RGD (10 (-5) M), or pretreated with the alpha (v) beta (3) integrin blocker cyclo (Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (cyclo-RGD). RGD-peptide caused only small relaxation of Phe contracted IVC (6.4 +/- 3.4%), and addition of MMP-2 to RGD-treated IVC caused further relaxation (69.7 +/- 3.0%). Pretreatment of IVC with cyclo-RGD did not significantly affect MMP-2 induced relaxation (55.0 +/- 5.0%). CONCLUSIONS: In rat IVC, MMP-2 attenuates [Ca (2+)](e)-dependent VSM contraction without affecting Ca (2+) release from intracellular Ca (2+) stores. MMP-2 induced VSM relaxation may not involve RGD generation or activation of alpha (v) beta (3) integrin receptor. MMP-2 induced inhibition of the Ca (2+) entry mechanism of VSM contraction may play a role in the venous dilation associated with varicose vein formation. |
1(0,0,0,1) |