The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis

Abstract

Objectives-Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics. Methods and Results-The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo. Key Words: Rho GTPase Ⅲ ADMA Ⅲ DDAH I Ⅲ VEGF Ⅲ angiogenesis H igh plasma levels of ADMA are associated with cardiovascular disorders including atherosclerosis, hypertension, homocysteinemia, diabetes mellitus, insulin resistance, and hypercholesterolemia. 1,2 ADMA and the related methylarginine analogue monomethylarginine (L-NMMA) inhibit NO production by competing with L-arginine for binding to NOS. 2 Levels of methylarginines are regulated by the enzymes DDAH I and II, which metabolize these factors. 2 DDAH I heterozygote (HT) knockout mice have raised ADMA levels and a hypertensive phenotype as a consequence of reduced NO signaling. 3 Further, dysregulation of the ADMA/DDAH pathway is associated with impaired angiogenesis, which contributes to disease pathology. In a mouse model of hypercholesterolemia, DDAH I activity is inhibited by lipid-induced oxidative stress, leading to elevated ADMA levels, reduced NO production, and impaired angiogenesis. 4 ADMA metabolism through DDAH overexpression promotes angiogenesis in vitro, 5,6 promotes sprouting from aortic rings, 7,8 and enhances angiogenesis in tumors and after ischemia, 6,9 but the mechanisms are not fully understood. Conclusion-The

Endothelial Dimethylarginine Dimethylaminohydrolase 1 Is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis

“…[33][34][35] We wanted to confirm that this local effect of endothelial DDAH1 deletion still occurred in our new mouse strain. Aortic rings were taken from DDAH1 Figure IIIA and IIIB in the online-only Data Supplement).…”

“…34,39 These inhibitory effects of endogenous or exogenous ADMA on endothelial cell function can be attenuated by overexpression of DDAH1 and recapitulated by global deletion of DDAH1. To determine the contribution of endothelial DDAH1 expression to these effects, we examined the impact of endothelium-specific DDAH1 deletion in several models of angiogenesis.…”

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo

“…For example, Ddah1 encodes an enzyme involved in the metabolism of methylarginines, molecules that inhibit nitric oxide synthase 35 and regulate VEGF-mediated angiogenesis. 36 Other examples include the fragilis gene family members Ifitm2 (fragilis3) and Ifim3 (fragilis1), which regulate the migration of primordial germ cells 37 ; Sox5, which encodes an HMG domain transcription factor related to Sry 38 ; and Muscleblind-1, a regulator of alternative splicing in muscle development. 39 Future studies should help to clarify the functions of these genes in the EryP lineage.…”

Role of Asymmetric Methylarginine and Connexin 43 in the Regulation of Pulmonary Endothelial Function

“…2,5 ADMA has been shown to induce pulmonary endothelial leakage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro, with these effects being prevented by overexpression of DDAHI. 4,6 Exogenous or lysophosphatidylcholineinduced ADMA was shown to decrease connexin (Cx) 43 expression and gap junctional communication 7 in human umbilical vein endothelial cells, suggesting a link between elevated ADMA level and progression of atherosclerosis. However, the role of other vascular Cxs or functional implications of ADMA-induced dysregulation of gap junctional communication has not been elucidated.…”