Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice

• Published in: PloS one 2018-01, Vol.13 (1), p.e0190981-e0190981
• Main Authors: Merentie, Mari, Rissanen, Riina, Lottonen-Raikaslehto, Line, Huusko, Jenni, Gurzeler, Erika, Turunen, Mikko P, Holappa, Lari, Mäkinen, Petri, Ylä-Herttuala, Seppo
• Format: Article
• Language: English
• Subjects: Angiogenesis; Antibiotics; Biology and Life Sciences; Blood vessels; Cardiomyocytes; Cloning; Complications and side effects; Dosage and administration; Doxycycline; Endothelial cells; Flow cytometry; Gene expression; Growth factors; Health sciences; Homeostasis; Ischemia; Levels; Medicine and Health Sciences; MicroRNAs; Permeability; Physiology; Proteins; Research and Analysis Methods; Ribonucleic acid; RNA; Transgenic animals; Transgenic mice; Vascular endothelial growth factor; Well being
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0190981

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.