Abstract
Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.
Highlights
Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis. It is effectively up-regulated in hypoxia and it binds to VEGF-receptor 1 and VEGF-receptor 2, the latter of which is the most important receptor mediating angiogenesis
The sh1 construct with a normal TRE promoter (T-1) proved to be the most efficient of the tested constructs, since in response to dox treatment it depleted the secreted VEGF-A protein level to 26% when compared to control cells transduced with a control vector and treated with dox (Fig 2A)
VEGF-A is the most important growth factor affecting the vasculature and its growth [1,2,3]. It is the primary target for several pro-angiogenic therapies for treating ischemia [4] and for anti-angiogenic therapies for treating cancer and age-related macular degeneration [2,12,34]
Summary
Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis. In addition to promoting growth it acts as a survival factor for vascular endothelial cells and is a strong enhancer of vascular permeability [1,2,3,4]. VEGF-A is expressed in all vascularized tissues suggesting that low physiological levels of VEGF-A are needed for the maintenance of general vascular homeostasis [7,8]. Physiological levels of VEGF-A maintain vascular homeostasis and protection while higher levels induce physiological vascular growth. Very high levels of VEGF-A promote aberrant vascular growth with significant tissue edema [4,8]
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