Abstract
Angiogenesis is a critical process involved in normal physiology. Pathological angiogenesis is observed in vascular diseases and neoplasia. The propeptide domain of LOX (LOX-PP) has been shown to inhibit tumorigenesis in various cancers. In this study, we explored the role of both overexpressed and recombinant LOX-PP in naïve human umbilical vein endothelial cell with the addition of vascular endothelial growth factor (VEGF). Primarily, we observed a significant reduction in the angiogenesis signaling pathways upon LOX-PP overexpression by proteomic analysis. Further functional analysis showed that the VEGF induced cell proliferation, migration, adhesion and tube formation was inhibited by LOX-PP. Moreover, LOX-PP arrested cells at S-phase, reduced F-actin levels and decreased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK). The anti-angiogenic effect of LOX-PP was further confirmed by the reduction in the vascular network formation in chick chorioallantoic membrane (CAM). These results indicate that inhibition of angiogenesis events is not only achieved by overexpressing LOX-PP but also by addition of rLOX-PP. Taken together our findings discovered the anti-angiogenic role of LOX-PP in endothelial cells which suggests that harnessing this potential can be a promising strategy to inhibit angiogenesis.
Highlights
Angiogenesis is a dynamic process that involves cell proliferation, migration, adhesion and tube formation in endothelial cells orchestrated by proangiogenic mediators and anti angiogenic factors[1]
The His - tag cleaved Lysyl oxidase (LOX)-PP was confirmed by mass spectrometry (Fig. 1d) and the purified protein was used for antibody production
The tumor suppressor activity of LOX was mapped to the propeptide region of the protein and lysyl oxidase propeptide (LOX-PP) was considered as an attractive anti-angiogenesis candidate molecule[14]
Summary
HUVECs which were treated with VEGF showed increased cell proliferation which was significantly (p = 0.001) inhibited by LOX-PP overexpression. HUVECs treated with VEGF showed significant increase in tube length, when compared to CTRL This was significantly inhibited in LOX-PP (42.45%; p = 0.0005) overexpressed cells (Fig. 8a,b). No significant change was observed in EV transfected cells This was further confirmed by adding rLOX-PP to HUVECs (Fig. 10a) which significantly decreased pFAK (p = 0.05; Fig. 10b) and pERK (p = 0.01; Fig. 10c). Cells treated with rLOX-PP (Fig. 10d) significantly decreased pFAK (p = 0.05; Fig. 10e) and pERK (p = 0.01; Fig. 10f) levels even in the absence of VEGF stimulation suggesting its direct role in downregulating angiogenesis signaling. This data demonstrated the uptake of rLOX-PP by HUVECs which was localized in the cytoplasm
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