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

Abstract

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 Ca2+ 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 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 EGTA) Krebs with or without MMP-2. In Ca2+-free Krebs, caffeine did not cause contraction, suggesting 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 [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-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%). Pretreatement of IVC with cyclo-RGD did not significantly affect MMP-2 induced relaxation (55.0±5.0%). Conclusions In rat IVC, MMP-2 attenuates [Ca2+]e-dependent VSM contraction, without affecting Ca2+ release from intracellular Ca2+ stores. MMP-2 induced VSM relax...

Matrix Metalloproteinases as Regulators of Vein Structure and Function: Implications in Chronic Venous Disease

“…MMPs do not inhibit phenylephrine-induced aortic contraction in Ca 21 -free solution, suggesting that they do not inhibit the Ca 21 release mechanism (Chew et al, 2004). On the other hand, MMP-2 and -9 cause aortic relaxation by inhibiting Ca 21 influx (Chew et al, 2004), and MMP-2 inhibits Ca 21 -dependent contraction in rat IVC (Raffetto et al, 2010a). It has been proposed that, during substrate degradation, MMPs may produce Arg-Gly-Asp (RGD)-containing peptides, which could bind to a v b 3 integrin receptors and inhibit Ca 21 entry into VSM (Waitkus-Edwards et al, 2002).…”

“…It has been proposed that, during substrate degradation, MMPs may produce Arg-Gly-Asp (RGD)-containing peptides, which could bind to a v b 3 integrin receptors and inhibit Ca 21 entry into VSM (Waitkus-Edwards et al, 2002). This is unlikely, as RGD peptides do not affect IVC contraction (Raffetto et al, 2010a). The mechanism by which MMPs inhibit Ca 21 entry could involve direct effects on Ca 21 or K 1 channels.…”

“…MMPs are expressed in the arterial system, and could have significant effects on the arterial structure and function as those discussed in the veins. For example, MMP-2 and -9 cause inhibition of Ca 21 entry-dependent mechanisms of contraction not only in rat IVC (Raffetto et al, 2010a), but also in rat aorta (Chew et al, 2004). However, veins differ from arteries in their structure and function, and the effects of MMPs on the veins should not always be generalized to the arteries.…”

Varicose Remodeling of Veins Is Suppressed by 3‐Hydroxy‐3‐Methylglutaryl Coenzyme A Reductase Inhibitors

“…Consequently, an increase in wall stress stimulates expression of MMP2 and elevates gelatinase activity in the media of affected veins and human venous SMCs (HUVSMCs) 9, 10. Likewise, MMP9 activity is increased in varicose veins of human patients11 and in rat veins on exposure to higher pressure levels 12. Blocking the activity of AP‐1 by specific decoy oligodeoxynucleotides that mimic the DNA binding site of AP‐1 in an animal model was successful in preventing pressure‐induced varicose‐like venous remodeling 9.…”

Pregnancy-associated adaptations in [Ca2+]i-dependent and Ca2+sensitization mechanisms of venous contraction: implications in pregnancy-related venous disorders

“…Although several studies have examined the pregnancy-related changes in arterial function and contraction mechanism, little is known regarding the changes in venous function during pregnancy. Also, while some studies have examined the mechanisms of VSM contraction in male and female virgin rat veins (67,68,72,93), whether these mechanisms are altered during pregnancy is unclear. The present study was designed to test the hypothesis that venous tissue reactivity is altered during pregnancy, and that the pregnancy-associated changes in venous function reflect underlying changes in [Ca 2ϩ ] i and the Ca 2ϩ -dependent and/or Ca 2ϩ -sensitization mechanisms of venous contraction.…”