MMP-2 Induced Vein Relaxation via Inhibition of [Ca2+]e-Dependent Mechanisms of Venous Smooth Muscle Contraction. Role of RGD Peptides

BackgroundMatrix 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 Ca2+ mobilization mechanisms of VSM contraction due to generation of Arg-Gly-Asp (RGD)-containing peptides.MethodsCircular 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.ResultsIn IVC incubated in normal Krebs (2.5 mM Ca2+), the α-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 μg/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 μg/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 Ca2+ release from the sarcoplasmic reticulum, IVC was incubated in Ca2+-free 2 mM ethylene glycol-bis(2-aminoethyl ether-N,N,N',N'-tetra-acetic acid (EGTA) Krebs with or without MMP-2. In Ca2+-free Krebs, caffeine did not cause contraction, suggesting a limited role of the Ca2+-induced Ca2+-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 IP3-induced Ca2+ release. To test the role of Ca2+ entry through membrane channels, after eliciting a transient Phe contraction in nominally 0 Ca2+ Krebs, increasing concentrations of CaCl2 (0.1, 0.3, 0.6, 1, 2.5 mM) were added and the extracellular Ca2+ concentration [Ca2+]e-contraction relationship was constructed. The [Ca2+]e-contraction relation was reduced in MMP-2 treated IVC, suggesting inhibition of Ca2+ entry. In IVC treated with MMP-2, the Ca2+ channel blocker diltiazem (10−5M) did not cause any further inhibition of Phe contraction, suggesting that Ca2+ 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 αvβ3 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%).ConclusionsIn rat IVC, MMP-2 attenuates [Ca2+]e-dependent VSM contraction without affecting Ca2+ release from intracellular Ca2+ stores. MMP-2 induced VSM relaxation may not involve RGD generation or activation of αvβ3 integrin receptor. MMP-2 induced inhibition of the Ca2+ entry mechanism of VSM contraction may play a role in the venous dilation associated with varicose vein formation.