Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells

Background and Purpose Cardiovascular disease associated with antiretroviral therapy (ART) has become a major clinical challenge for HIV-positive patients. However, the role of ART in blood vessel growth is largely unknown. Here, we examined an integral component of ART, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), and investigated their effects on key microvascular functions, including angiogenesis and lymphangiogenesis. Experimental Approach The angiogenesis/lymphangiogenesis capability of endothelial cells (ECs) was evaluated using migration, proliferation and tube formation assays in vitro, and mouse ear and Matrigel plug assays in vivo. Expression of signalling molecules and mitochondrial antioxidant catalases were determined using western blotting. Receptor tyrosine kinase (RTK) internalization and endocytosis were examined using flow cytometry and confocal immunofluorescence microscopy, respectively. Mitochondrial DNA copy number and ROS were determined using quantitative real-time PCR and MitoSOX staining, respectively. Key Results Pharmaceutical doses of NRTIs (AZT, TDF and 3TC) inhibited angiogenesis and lymphangiogenesis both in vivo and in vitro by affecting the proliferation and migration of ECs. Correspondingly, NRTIs treatment selectively attenuated the endothelial RTK signal activation and transduction, VEGFR2 and FGFR1 pathways in vascular ECs and VEGFR3 pathway in lymphatic ECs. Mechanically, TDF and 3TC restrained RTKs endocytosis into early endosome but not internalization, while AZT blocked the protein maturation of RTKs. Upon NRTIs treatment, excessive ROS derived from dysfunctional mitochondria was detected, and the MnSOD mimic MnTMPyP rescued the angiogenic/lymphangiogenic defects induced by NRTIs. Conclusions and Implications NRTIs negatively regulate angiogenesis and lymphangiogenesis by inducing mitochondrial oxidative stress and subsequently impairing RTK signalling in ECs.