Vascular Endothelial Growth Factor Blockade Induces Dermal Endothelial Cell Apoptosis in a Clinically Relevant Skin Organ Culture Model

Abstract

Background/Aims: Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, plays a key role in physiological processes and is a major contributor to several diseases including cancer and psoriasis. Anti-VEGF therapies are widely used as cancer and ophthalmological treatments. There is some evidence that VEGF blockade may have utility in the management of psoriasis, although their potential has been largely unexplored. We hypothesized that a human skin organ culture could provide a stable ex vivo model in which the cutaneous microvascular network could be studied and experimentally manipulated. Methods: Punch biopsies (3 mm) of skin, donated by healthy individuals (39-72 years old, n = 5), were incubated with monoclonal antibody (mAb) to human VEGF (bevacizumab) at doses based on data from animal and clinical studies. After 3-day culture, cell death and proliferation as well as vascular endothelial cell changes were assessed using quantitative immunohistomorphometry. Results: Anti-VEGF mAb at 0.8 mg/mL induced a significant increase in cleaved caspase-3 expression in CD31+ cells (p < 0.05). None of the doses tested increased TUNEL or decreased Ki-67 expression in the basal layer of the epidermis, confirming the model's viability. In addition, the lactate dehydrogenase (LDH) assay showed no increase in LDH activity in treated samples compared to untreated control. The highest anti-VEGF mAb dose (0.8 mg/mL) induced an increase in TUNEL expression in the upper epidermis, which did not correlate with caspase-3 immunoreactivity. Further investigation revealed that anti-VEGF mAb did not change the expression of markers of terminal differentiation such as keratin 10, filaggrin, and involucrin, suggesting that VEGF depletion does not affect keratinocyte terminal differentiation. In contrast to the control group, levels of VEGF protein were undetectable in the culture supernatant of samples treated with 0.8 mg/mL of anti-VEGF mAb, suggesting sufficient dose. Conclusion: Our pilot study provides the first evidence that anti-VEGF therapy promotes endothelial cell apoptosis in human skin ex vivo. Our pragmatic human skin organ culture assay offers a valuable tool for future preclinical endothelial cell and translational microvascular network/anti-Angiogenesis research in human skin.