Elsevier

Microvascular Research

Volume 72, Issues 1–2, July–September 2006, Pages 3-11
Microvascular Research

Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: Role of receptor transactivation

https://doi.org/10.1016/j.mvr.2006.04.004Get rights and content

Abstract

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, agents that inhibit angiogenesis have important therapeutic implications in numerous malignancies. We examined the effects of methylnaltrexone (MNTX), a peripheral mu opioid receptor antagonist, on agonist-induced human EC proliferation and migration, two key components in angiogenesis. Using human dermal microvascular EC, we observed that morphine sulfate (MS), the active metabolite, morphine-6-glucuronide (M6G), DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]enkaphalin) and VEGF induced migration which were inhibited by pretreatment with MNTX at therapeutically relevant concentration (0.1 μM). The biologically inactive metabolite morphine-3-glucuronide (M3G) did not affect EC migration. We next examined the mechanism(s) by which MNTX inhibits opioid and VEGF-induced angiogenesis using human pulmonary microvascular EC. MS and DAMGO induced Src activation which was required for VEGF receptor transactivation and opioid-induced EC proliferation and migration. MNTX inhibited MS, DAMGO and VEGF induced tyrosine phosphorylation (transactivation) of VEGF receptors 1 and 2. Furthermore, MS, DAMGO and VEGF induced RhoA activation which was inhibited by MNTX or VEGF receptor tyrosine kinase inhibition. Finally, MNTX or silencing RhoA expression (siRNA) blocked MS, DAMGO and VEGF-induced EC proliferation and migration. Taken together, these results indicate that MNTX inhibits opioid-induced EC proliferation and migration via inhibition of VEGF receptor phosphorylation/transactivation with subsequent inhibition of RhoA activation. These results suggest that MNTX inhibition of angiogenesis can be a useful therapeutic intervention for cancer treatment.

Introduction

Angiogenesis is an essential phenotype in a number of physiologic and pathologic processes including growth and development (Risau, 1997), wound healing (Arnold and West, 1991) and reproduction (Welsh and Enders, 1991, Rogers et al., 1992, Torry and Rongish, 1992). Inadequate angiogenesis contributes to ulcer formation (Folkman et al., 1991), while excessive angiogenesis contributes to the pathology of arthritis (Wilson, 2004), psoriasis (Creamer et al., 2002, Leong et al., 2005) and neoplasia (Isayeva et al., 2004, Dhanabal et al., 2005, Gaya and Rustin, 2005). In a series of now classical experiments, Folkman and colleagues demonstrated that solid tumors cannot grow larger than 2–3 mm in diameter unless they induce their own blood supply (Folkman et al., 1991, Folkman, 1995).

Although morphine and other opioids are widely used to relieve cancer pain, surprisingly, few studies have considered the effect of opioids on angiogenesis or endothelial cell biology. The role of mu receptors, known to exist on endothelial cells (Cadet et al., 2004), remains unknown. The lack of a specific peripheral opioid antagonist has largely precluded clinical trials in this area because conventional tertiary opioid antagonists reverse analgesia with the reversal of peripheral effects. The development of methylnaltrexone (MNTX), a peripheral opioid antagonist now in phase III trials, offers an opportunity to examine the effect of opioids on endothelial cell biology (Yuan, 2004).

While the mechanism of the mu opioid effect on endothelial cell migration is uncertain, it likely occurs at the membrane level. MNTX, unlike naloxone, is a charged molecule at physiological pH. Morphine acts via G-protein-coupled receptors (Law and Loh, 1999, Bailey and Connor, 2005), while other important angiogenic signals including VEGF act by receptor tyrosine kinases (Cross et al., 2003, Ferrara et al., 2003). There is growing evidence of tyrosine kinase receptor transactivation as a mechanism of G-protein-coupled receptor (GPCR) signaling (Endo et al., 2002, Fujita et al., 2005). Pertussis-toxin-dependent G-protein-coupled receptors can transactivate VEGF receptor 2/FlK1 (Zeng et al., 2003). Similarly, morphine could transactivate these receptors to promote an environment in which endothelial cells could proliferate and a tumor could grow. A recent study of mu opioid receptor knockout mice (MOR-KO) injected with T241 fibrosarcoma cells demonstrated significant differences in the incidence of tumor growth and a 10-fold increase in Flk-1 expression in MS-treated mice versus controls, versus no increase in MS-treated MOR-KO mice (Stephenson et al., 2005). This result supports the hypothesis that MS stimulates endothelial cell proliferation and promotes tumor growth, probably by transactivating FLK1 (VEGF receptor 2) phosphorylation, and it suggests that MNTX may be administered in conjunction with current therapies targeting VEGF.

Several downstream signaling mechanisms have been implicated in agonist-induced angiogenesis including Src (Alper and Bowden, 2005, Tanimoto et al., 2002). Src (pp60src, c-Src tyrosine kinase) is a non-receptor tyrosine kinase that contains an amino-terminal myristolyation site, Src Homology (SH) sites (i.e. SH2 and SH3), a tyrosine kinase catalytic domain and regulatory tyrosine phosphorylation sites (Alper and Bowden, 2005). Certain GPCR, including the S1P receptor, can transactivate the VEGF receptor through activation of Src (Tanimoto et al., 2002).

One important signaling molecule involved in angiogenesis is the small G-protein RhoA (Aepfelbacher et al., 1997, Cascone et al., 2003, Hoang et al., 2004, Liu and Senger, 2004). With certain angiogenic signals, RhoA is converted from its inactive (GDP-bound) to active (GTP-bound) form via catalysis from certain Rho Guanine Nucleotide Exchange Factors (RhoGEFs) (Sah et al., 2000, Fukuhara et al., 2001, Hakoshima et al., 2003). The active form of RhoA can bind to and activate other important signaling molecules involved in angiogenesis including the serine/threonine kinase, ROCK (Singleton and Bourguignon, 2002, Croft et al., 2004, Hyvelin et al., 2005). Furthermore, activation of RhoA has been shown to regulate VEGF-induced angiogenesis (van Nieuw Amerongen et al., 2003).

In this study, we have demonstrated that opioids induce Src-dependent VEGF receptor transactivation (tyrosine phosphorylation) and RhoA activation resulting in increased microvascular endothelial cell (EC) proliferation and migration. Furthermore, methylnaltrexone (MNTX) inhibits both opioid- and VEGF-mediated VEGF receptor tyrosine phosphorylation, RhoA activation and angiogenesis. These results suggest that MNTX may be useful as a potential anti-angiogenic agent.

Section snippets

Cell culture and reagents

Human dermal microvascular endothelial cells (Cell Systems, Kirkland, WA) and human pulmonary microvascular endothelial cells from Cambrex (Walkersville, MD) were cultured as previously described in EBM-2 complete medium from Cambrex at 37°C in a humidified atmosphere of 5% CO2, 95% air, with passages 6–10 used for experimentation (Garcia et al., 2001, Lingen, 2002). Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO). Reagents for SDS-PAGE electrophoresis were

Effects of opioids and methylnaltrexone (MNTX) on human dermal microvascular endothelial cell migration

Using the endothelial cell migration assay, we found that MS caused a concentration-dependent increase in endothelial migration. Naloxone and MNTX alone had no effect on endothelial cell migration over a wide range of concentrations (Fig. 1A). At clinically relevant concentrations of morphine, the magnitude of the effect was approximately 70% of that achieved by VEGF. Endothelial cell migration was induced by morphine in concentrations as low as 10 7 M (Fig. 1B). Morphine-based endothelial cell

Discussion

Taken as a whole, our findings suggest a model in which the peripheral mu opioid receptor antagonist MNTX attenuates opioid and VEGF-induced VEGF receptor and RhoA activation. This attenuation is important for the inhibitory role of MNTX on opioid and VEGF-mediated angiogenesis (Fig. 7). The exact mechanism(s) by which MNTX exerts these effects are currently being investigated in our laboratory.

In a previous study, MOR agonists had a significant effect on neovascularization and proliferation of

Acknowledgments

This work was supported in part by the NIH grants DE12322 (MWL), DE00470 (MWL) and DE015830 (MWL).

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