Effects of flavonoid derivatives on human microvascular endothelial cells

<p>Some natural compounds, including flavonoids, are active in vasculature re-growth during hair follicle disruption, but their effects have not been yet evaluated directly on microvascular endothelial cells. Skin vascularisation regulates the physiological blood supply required for hair growth and its dysregulation is the basis of several human diseases. Follicle-derived vascular endothelial growth factor (VEGF) release from follicular keratinocytes promotes perifollicular vascularisation and increases follicle and hair size, while blockade of VEGF-mediated angiogenesis leads to impaired hair growth. Here, we tested three flavonoids, namely visnadin (VSD), hesperidin (HSP) and baicalin (BC), on cultured human microvascular endothelial cells (HMEC), comparing their effects with minoxidil (MXD), a synthetic drug broadly used in the treatment of androgenetic alopecia. The response to these compounds was assayed in terms of endothelial survival, proliferation, tubulogenesis and proangiogenic signalling. We show that BC promotes HMEC proliferation, while both VSD and MXD enhance tubulogenesis. Interestingly, only HSP increases VEGFR-2 phosphorylation.</p

Experimental design.

<p>Control group (CTRL) cardiomyocytes were superfused/reperfused with Tyrode standard solution for 25 min. Ischemic/reperfused group (I/R): superfusion with Tyrode standard for 5 min, ischemic buffer (IB) for 15 min, and Tyrode standard (reperfusion) for 5 min. Ischemic + Cst group (Cst): superfusion with Tyrode standard + 5 nM Cst for 5 min, IB + 5 nM Cst for 15 min, and Tyrode standard alone for 5 min (reperfusion). Wortmannin (Cst + Wm), L-NMMA (Cst + L-NMMA) and ODQ (Cst + ODQ) groups: cardiomyocytes were treated as Cst group, in the presence of 100 nM Wm, 1 mM L-NMMA or 100 μM ODQ together with 5 nM Cst.</p

Cst maintains eNOS and PLN activation during reperfusion.

<p>A) Fluorescence confocal images of cardiomyocytes labeled with anti phospho-eNOS¹¹⁷⁹ antibody in CTRL, I/R, Cst and Cst+Wm groups. B) Statistical analysis of the simulated I/R experiments. C) Fluorescence confocal images of cardiomyocytes labeled with anti phospho-PLN^{Thr 17} (green fluorescence) antibody and with anti PLN antibody (red fluorescence) in CTRL, I/R, Cst and Cst+Wm groups. D) Statistical analysis of the simulated I/R experiments. For both eNOS and PLN, fluorescence intensity (mean ± S.E.M. from at least 25 cells; n = 5 experiments) was expressed as percentage respect I/R group for each treatment at the end of reperfusion phase.</p

Cst preserves mitochondrial membrane potential in cardiomyocytes undergoing I/R.

<p>A) Fluorescence confocal images of cardiomyocytes labeled with JC-1 fluorescent probe at t5, t20 and t25 min of each experiment. B) Statistical analysis of green/red ratio (green: monomeric form; red: aggregate) of fluorescence variation. These values were referred to the end of the reperfusion phase. Results are presented as mean ± S.E.M. from at least 25 cells; n = 5 experiments.</p