[doi: 10.5505/2017ichc.PP-255]

Minoxidil Effects Angiogenesis In-vivo and In-vitro Settings

¹Department of Medical Histology and Embryology, University of Health Sciences, Gulhane Faculty of Medicine, Ankara, Turkey
²Stem Cell Laboratory, Gulhane Research Institute, University of Health Sciences, Ankara, Turkey

Introduction&OBJECTIVES: Angiogenesis a process initiated in preexisting vessels. Many researches regarding cancer, diabetic retinopathy, psoriasis and some inflammatory diseases interrelated with angiogenesis have been performed to date. Angiogenesis is regulated by expression of VEGF which is a dynamic process and effected by many molecules such as; IL-6, IL-1β, TGF-β and PDGF-β. However, this mechanism is not well-characterized and it is likely that other molecules may also stimulate the expression of VEGF.
Minoxidil is a well-known hypertrichotic agent used for the treatment of androgenic alopecia. Minoxidil is reported to be involved in the development of dermal papilla vascularization via stimulation of VEGF expression. The purpose of this study was to evaluate the effect of minoxidil on angiogenesis using in vitro and in vivo model systems.
Materials&METHODS: Lohmann LSL classical type fertilized eggs were used for in vivo CAM assay and they were kept at 37 °C and 65–75% relative humidity in an incubator. The CAMs were treated with different concentrations of Minoxidil (10µM, 50µM, 100µM, 200µM). At the 24th hour of the administration the CAMs were visualized by a digital camera.
For tube formation assay; Matrigel matrix was plated in 96-well (ibidi Cat.No:89646) μ-plates. After incubation for 30 min, 40 μL HUVEC (10,000 cells/well) and 40 μL of Minoxidil (50µM - 100µM - 200µM) in a conditioned medium were placed on Matrigel and incubated at 37°C for 24 h. Following incubation, the plated wells were photographed, and the results were evaluated with “Angiosys and Wimasis” software. The proliferation inducing activity of Minoxidil was also determined through MTT assay on HUVECs. Control and vehicle groups were prepared by following the same procedure as mentioned previously except Minoxidil solution for all assays.
RESULTS: In CAM assay, minoxidil had no significant angiogenic effect compared to vehicle groups (Figure 1). Minoxidil had a statistically significant (p<0.05) proliferative effect on HUVECs. Results of tube formation assay are presented in Figure 2.
CONCLUSIONS: Although minoxidil had a slight angiogenic effect of on CAM vessels, it significantly induces angiogenesis on in-vitro assays.

---

Figure 1

Stereomicroscopic observation of CAM assay. (A-B) Control (C-D) Vehicle (E-F)10 μM (G-H) 50 μM (I-J) 100 μM (K-L) 200 μM. The blue arrows indicate drug treated sites. The black squares point out application areas after 24h. There was no difference in vascular branching when compared to vehicle and control groups. Minoxidil has a dosage-independent and statistically insignificant stimulation effect on vascular bridging.

Figure 2

Representive images of tube formation assay. The first line is the original phase-contrast images at 24th h, the second line shows the images used for analysis, the third line shows the resulting images of Wimasis-WimTube software, the fourth line shows the resulting images of Angiosys software. (A) Control (B) 50 µM (C) 100 µM (D) 200 µM (E) Vehicle (40 X). Comparing the number of tubules and junctions, areas of loops, branch sites and mean tubule length, there was a statistically significant dose-dependent increase in the minoxidil groups compared to the control and vehicle groups (p<0.05).