Topically Applied Etamsylate: A New Orphan Drug for HHT-Derived Epistaxis (Antiangiogenesis through FGF Pathway Inhibition)

Virginia Albiñana, Guillermo Giménez-Gallego, Angela García-Mato, Patricia Palacios, Lucia Recio-Poveda, Angel-M Cuesta, José-Luis Patier, Luisa-María Botella, Virginia Albiñana, Guillermo Giménez-Gallego, Angela García-Mato, Patricia Palacios, Lucia Recio-Poveda, Angel-M Cuesta, José-Luis Patier, Luisa-María Botella

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by recurrent and spontaneous epistaxis (nose bleeds), telangiectases on skin and mucosa, internal organ arteriovenous malformations, and dominant autosomal inheritance. Mutations in Endoglin and ACVRL1 / ALK1 , genes mainly expressed in endothelium, are responsible in 90% of the cases for the pathology. These genes are involved in the transforming growth factor-β(TGF-β) signaling pathway. Epistaxis remains as one of the most common symptoms impairing the quality of life of patients, becoming life-threatening in some cases. Different strategies have been used to decrease nose bleeds, among them is antiangiogenesis. The two main angiogenic pathways in endothelial cells depend on vascular endothelial growth factor and fibroblast growth factor (FGF). The present work has used etamsylate, the diethylamine salt of the 2,5-dihydroxybenzene sulfonate anion, also known as dobesilate, as a FGF signaling inhibitor. In endothelial cells, in vitro experiments show that etamsylate acts as an antiangiogenic factor, inhibiting wound healing and matrigel tubulogenesis. Moreover, etamsylate decreases phosphorylation of Akt and ERK1/2. A pilot clinical trial (EudraCT: 2016-003982-24) was performed with 12 HHT patients using a topical spray of etamsylate twice a day for 4 weeks. The epistaxis severity score (HHT-ESS) and other pertinent parameters were registered in the clinical trial. The significant reduction in the ESS scale, together with the lack of significant side effects, allowed the designation of topical etamsylate as a new orphan drug for epistaxis in HHT (EMA/OD/135/18).

Keywords: Alk1; Endoglin; FGF pathway; HHT; TGF-β signaling pathway; antiangiogenesis; epistaxis; etamsylate.

Conflict of interest statement

Conflict of Interest None declared.

Figures

Fig. 1
Fig. 1
Effect of etamsylate on angiogenesis in HMEC-1 cells. (A) Wound healing assay. In the upper part, representative images of the different experiments and replicas are shown. In the lower part, the percentage of migration in time is shown at different doses. (B) Tubulogenesis assay. In the upper part, representative images are shown. In the lower part, the tube densities per field at different doses compared with the untreated cells are shown. Mean with the standard deviation is represented normalized versus control. Statistical significance was studied using Student'st-test. No significant differences were found in this case (ns).
Fig. 2
Fig. 2
Effect of etamsylate on angiogenesis in HUVECs. (A) Wound-healing assay. In the representative images shown, the cell migration after etamsylate administration is observed. In the lower part, the percentage of wound healing (migration) in time is represented. The mean with SD is shown. Values are normalized with control. Statistical significance was studied using Student'st-test. *p < 0.05. (B) Tubulogenesis assay. Representative images are shown, demonstrating a lower amount of tubes in the treated cells compared with untreated cells. The different tube densities are shown in a representative experiment. The mean with SD is shown. Values are normalized with control. Statistical significance was studied using Student'st-test. *p < 0.5. HUVECs, human umbilical vein endothelial cells.
Fig. 3
Fig. 3
(A) Effect of etamsylate on tubulogenesis assay in BOECs. Representative images demonstrate a lower amount of tubes in the treated cells. The different tube densities are shown in the graph. The number of closed tubules was counted in five different fields and the mean was calculated. The mean with SD is shown. Statistical significance was studied using Student'st-test (healthy:p < 0.002; HHT:p < 0.023). (B) Tubulogenesis assay FGF vs. FGF/etamsylate. HHT BOECs were treated with or without FGF and etamsylate. The number of closed tubules was counted in five different fields and the mean was calculated. The images and graph show a significant decrease in the number of closed tubes when cells are without FGF (***p < 2.6 × 10 −6 ), and when etamsylate is added in the presence of FGF (FGF/etamsylate; ***p < 1.1 × 10 −6 ) in comparison with FGF-treated cells (FGF). BOECs, blood outgrowth endothelial cells; FGF, fibroblast growth factor; HHT, hereditary hemorrhagic telangiectasia.
Fig. 4
Fig. 4
3D angiogenesis. HUVEC-coated beads were cultured in the presence or absence of etamsylate during 5 days. When etamsylate is present, fewer bonds can be observed between neighboring beads forming a network, as it occurs in control conditions. There was a significantly higher number of ramifications in control (untreated cells) compared with etamsylate-treated cells, as the graph shows (day 4, ***p < 6.5 × 10 −4 ; day 5, ***p = 8.34 × 10 −5 ). HUVECs, human umbilical vein endothelial cells.
Fig. 5
Fig. 5
Effect of etamsylate on viability and Endoglin and ALK1 RNA expression. (A) Viability of HUVECs and BOECs cells in the 0 to 100 μM range of etamsylate treatment is not affected. (B) RNA expression of ENG and ALK1 after treatment with different doses of etamsylate in HUVECs. RNA levels ofACVRL1/ALK1andEndoglinwere determined by RT-qPCR in HUVECs, after etamsylate treatments (range: 10–100 μM), and normalized with the untreated controls. Statistical significance was studied using Student'st-test. No significant difference was found. BOECs, blood outgrowth endothelial cells; HUVECs, human umbilical vein endothelial cells.
Fig. 6
Fig. 6
Analysis of Akt and Erk1 phosphorylation after etamsylate treatment. (A) Analysis of pAkt1, Akt1, and tubulin in cell lysates in HUVECs. A representative gel of three experiments performed is shown. Bands were quantified by Adobe Photoshop CS. Relative levels of pAkt1 normalized versus total Akt1. A decrease of the amount of phosphorylated protein is apparent compared with control. (B) Relative levels of pERK2 normalized versus total erk. pERK2 levels decrease when cells were treated with the highest doses of etamsylate (50–100 μM) compared with the control. HUVECs, human umbilical vein endothelial cells.
Fig. 7
Fig. 7
Levels of epistaxis severity score (ESS). (A) HHT-ESS of the 12 patients before and after the treatment. (B) HHT-ESS mean of pre- and posttreatment levels of the patients. A Student'st-test was made to compare pre- and posttreatment HHT-ESS values. Differences were significant atp < 0.01. (CandD) Analysis of FGF and VEGF, as biomarkers related to angiogenesis and bleeding. The graph shows pre- and posttreatment results. FGF, fibroblast growth factor; HHT, hereditary hemorrhagic telangiectasia; VEGF, vascular endothelial growth factor.
Fig. 8
Fig. 8
The antiangiogenic effect of etamsylate on the nose mucosa. Aspect of the nose mucosa in a patient before starting the treatment, and around the same area after 4 weeks of treatment with etamsylate (* shows the major affected area).

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