Molecular and Cellular Pharmacology
Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial proliferation, arterial relaxation, vascular permeability and angiogenesis by dobesilate

https://doi.org/10.1016/j.ejphar.2011.06.015Get rights and content

Abstract

Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis and vascular permeability which is associated with many pathological processes. 2,5-hydroxybenzene sulfonate (DHBS; dobesilate) is a small molecule with anti-angiogenic activity that has been described as an inhibitor of fibroblast growth factors (FGF). The aim of the present study was to evaluate the effects of DHBS on VEGF-induced actions. The effects of DHBS were evaluated on VEGF-induced proliferation in human umbilical vein endothelial cells (HUVEC) and rat aorta relaxation, as well as on in vivo VEGF-induced skin vascular permeability and neovascularization in rats. DHBS at 50 and 100 μM concentration significantly inhibited the proliferation of HUVEC induced by VEGF (10 ng/ml), without significantly affecting HUVEC proliferation in the absence of VEGF. Rapid VEGF-induced activation of Akt in HUVEC was also prevented by DHBS (100 μM). Additionally, DHBS (2 μM) specifically inhibited the relaxation of rat aorta induced by VEGF (0.1 to 30 ng/ml), but not endothelium-dependent relaxation to acetylcholine (1 nM to 10 μM). The in vivo enhancement of vascular permeability caused by VEGF injection (50 μl at 10 ng/ml) in rat skin was also inhibited by DHBS co-administration (200 μM) (74.8 ± 3.8% inhibition of dye extravasation). Administration of DHBS (200 mg/kg/day; i.p.) also reduced VEGF-induced angiogenesis in vivo. DHBS inhibits main responses elicited in vitro and in vivo by VEGF. As a dual antagonist of VEGF and FGF activities, DHBS could be of therapeutic interest in the treatment of diseases related to VEGF/FGF overproduction and excessive angiogenesis.

Introduction

Angiogenesis is the formation of new blood vessels from the existing vasculature and is vital for reproduction, development and repair (Folkman, 1995). However, excessive angiogenesis is associated with a large number of pathological processes, including growth, invasion and metastasis in malignancies (Hanahan and Folkman, 1996). Angiogenesis is finely regulated by pro-angiogenic and anti-angiogenic stimuli. When this regulation is disbalanced, either favoring pro-angiogenic factors or limiting anti-angiogenic factors, pathological angiogenesis occurs. Vascular endothelial growth factor (VEGF) is, together with fibroblast growth factors (FGF), one of the most important promoters of angiogenesis. In addition to stimulating endothelial cell proliferation and migration (Byrne et al., 2005), VEGF is known to cause vasodilation (Liu et al., 2002) and to increase vascular permeability (Bates et al., 2002). All these actions contribute to the pathophysiological processes related to VEGF over-activity.

Increased production of VEGF has been observed in diseases characterized by excessive angiogenesis, namely age-related macular degeneration (Bhutto et al., 2006), diabetic retinopathy (Aiello et al., 1994), rheumatoid arthritis (Lee et al., 2001), psoriasis (Detmar et al., 1994), rosacea (Smith et al., 2007) and inflammatory bowel disease (Tolstanova et al., 2009). Elevated VEGF production by solid tumors is associated with increased tumor vascularization, metastasis, chemoresistance and poor prognosis in cancer processes (Foekens et al., 2001, Karayiannakis et al., 2002). Therapies directed to selectively neutralize VEGF have been proved to be effective in treating cancer (Shih and Lindley, 2006) and ocular neovascularization (Lynch and Cheng, 2007). However, the anti-angiogenic agents available nowadays, including antibodies or aptamers directed against VEGF or its receptor and tyrosine kinase inhibitors, present inconveniences for the administration, are expensive, and most importantly, are associated with serious side effects (Johnson et al., 2004).

2,5-dihydroxybenzene sulfonate (DHBS; dobesilate) is a small synthetic molecule that has been widely used in treating diabetic retinopathy and chronic venous insufficiency with a good safety profile (Allain et al., 2004, Ribeiro et al., 2006). However, the mechanism of action of this drug has not been completely elucidated. Recently, it has been demonstrated that DHBS binds to the heparin-binding domain of FGF-1 reducing its activity (Fernandez et al., 2010) and DHBS has been proved to be effective for the treatment of rosacea (Cuevas and Arrazola, 2005b) and psoriasis (Cuevas and Arrazola, 2005a), two clinical manifestations related to excessive angiogenesis and over-expression of VEGF.

The aim of the present study was to evaluate, using both in vitro and in vivo approaches, the ability of DHBS to antagonize the stimulatory actions of VEGF on endothelial cell proliferation, vasodilation, vascular permeability and angiogenesis.

Section snippets

Human endothelial cell culture

Freshly delivered human umbilical cord specimens were collected from the Hospital de Getafe in Madrid, as approved by the Hospital Ethics Committee. Human umbilical vein endothelial cells (HUVEC) were obtained by treatment with 0.1% type II collagenase and characterized by both morphology and positive staining for factor VIII, as previously described (Peiro et al., 2007). Cells were routinely cultured in M199 medium supplemented with 2% endothelial cell growth supplement (ECGS) and 20% fetal

Effects of DHBS on VEGF-induced proliferation of human endothelial cells

In the presence of 1% fetal calf serum, the treatment of cultured HUVEC for 72 h with VEGF (10 ng/ml) resulted in increased cell proliferation (Fig. 1A). The simultaneous addition of DHBS concentration-dependently antagonized the VEGF-induced proliferation of HUVEC. This effect was significant with DHBS from 50 μM concentration, while 100 μM DHBS completely abolished the proliferative stimulus driven by VEGF (Fig. 1A). In contrast, DHBS, at 50 or 100 μM, did not significantly modify the

Discussion

DHBS has been used for more than 30 years in the treatment of diabetic retinopathy, showing consistent efficacy (Ribeiro et al., 2006). Nevertheless, the mechanism responsible for this effect has been poorly elucidated. DHBS has been considered as an angioprotective agent and has been shown to decrease blood viscosity and vascular permeability in patients with diabetic retinopathy (Berthet et al., 1999). In addition, it has been reported to possess antioxidant capacity that could account for

Conclusion

The present study demonstrates that DHBS is an efficient in vitro and in vivo inhibitor of VEGF signaling pathway and VEGF-induced actions, including endothelial cell proliferation, arterial vasodilatation, vascular permeability and angiogenesis. These findings provide a rationale for explaining, at least in part, its therapeutic properties. The dual inhibitory effects of DHBS on VEGF and FGF activities makes this compound a promising candidate for treating a variety of deleterious conditions

Acknowledgments

This work was supported by grants from Action Medicines SL, Madrid, Spain, and from Ministerio de Ciencia e Innovación, Spain (SAF2008-01291; SAF2008-00942). Tania Romacho is the recipient of a fellowship from Ministerio de Educación y Ciencia.

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