Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition

Abstract

Lysyl oxidase-like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial-to-mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor-β (TGF-β)-mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.

Keywords: angiogenesis; endothelial-to-mesenchymal transition; focal adhesion kinase; lysyl oxidase-like 2; protein kinase B.

Figure 1

LOXL2 regulates migration in EC. (a) LOXL2 mRNA expression is decreased in shLOXL2 expressing EC compared to shCtrl expressing EC (n = 4 + SD, Student’s t test). (b) Doxycycline‐induced overexpression of LOXL2 mRNA is confirmed by stimulating EC expressing pInducer20‐LOXL2 (Ind LOXL2) with PBS (Vehicle) or doxycycline (Dox) for 24 hr. (c) Western blot analysis demonstrating reduced LOXL2 protein expression in shLOXL2 expressing EC, and increased LOXL2 protein expression when stimulating pInducer20‐LOXL2 infected EC (Ind LOXL2), but not wildtype EC (WT), with doxycycline, using GAPDH as a loading control. (d) Representative pictures of a migration scratch assay using shCtrl and shLOXL2 expressing EC at t = 0 and t = 6 hr. (e) Quantification of shCtrl and shLOXL2 expressing endothelial cell migration over 6 hr (n = 5 + SD, Student’s t test). (f) Representative pictures of a migration scratch assay when overexpressing LOXL2 in EC at t = 0 and t = 6 hr. (g) Quantification of LOXL2 overexpressing endothelial cell migration over 6 hr (n = 3 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐buffered saline; shCtrl: shRNA control; shLOXL2: short hairpin‐mediated LOXL2 knockdown

Figure 2

LOXL2 regulates angiogenic sprouting in EC. (a) Representative pictures of an angiogenic sprouting assay using shCtrl and shLOXL2 expressing EC at t = 72 hr. (b) Quantification of sprout length in a 72 hr angiogenic sprouting assay using shCtrl and shLOXL2 expressing EC (n = 4 + SD, Student’s t test). (c) Representative pictures of an angiogenic sprouting assay in pInducer20‐LOXL2 (Ind LOXL2) expressing EC stimulated with PBS (Vehicle) or doxycycline (Dox) at t = 72 hr. (d) LOXL2 overexpression increases sprouting length in an angiogenic sprouting assay (n = 4 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐buffered saline; shCtrl: shRNA control; shLOXL2: short hairpin‐mediated LOXL2 knockdown

Figure 3

LOXL2‐induced increase in migration and sprouting is independent of enzymatic activity. (a) A schematic representation of LOXL2 (upper panel), and the catalytically inactive H26Q‐H628Q‐LOXL2 mutant (LOXL2 H626/628Q). (b) Western blot analysis demonstrating the inducible overexpression of an H26Q‐H628Q‐LOXL2 mutant in EC by stimulation with doxycycline (Dox) for 24 hr compared to PBS (Vehicle). (c) Representative pictures of a migration scratch assay when overexpressing wildtype LOXL2 (Ind LOXL2) and the H26Q‐H628Q‐LOXL2 mutant (Ind dLOXL2) in EC at t = 0 and t = 6 hr. (d) Overexpression of either wildtype LOXL2 or LOXL2 H626/628Q in EC results in comparable increased migration in scratch migration assays (n = 4 + SD, Student’s t test). (e) Representative pictures of an angiogenic sprouting assay in inducible wildtype LOXL2 or LOXL2 H626/628Q expressing EC stimulated with PBS (Vehicle) or doxycycline (Dox) at t = 72 hr. (f) Overexpression of either wildtype LOXL2 or LOXL2 H626/628Q in EC results in a comparable increase in sprouting length in angiogenic sprouting assays (n = 4 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; PBS: phosphate‐buffered saline [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

LOXL2 knockdown in EC reduces EndMT, but LOXL2 overexpression does not induce EndMT. (a) LOXL2 knockdown in EC reduces mRNA expression of mesenchymal markers α‐SMA, calponin 1, and fibronectin, and increases mRNA expression of endothelial markers PECAM‐1 and VE‐cadherin (n = 3, + SD, Student’s t test). (b) LOXL2 knockdown in EC increases protein expression of endothelial markers PECAM‐1 and VE‐cadherin, and reduces protein expression of mesenchymal markers α‐SMA and fibronectin. (c) The LOXL2 expression is increased in EC after stimulation with TGF‐β for 24 hr. (d) LOXL2 knockdown results in delayed TGF‐β‐mediated upregulation of α‐SMA in EC. (e) LOXL2 overexpression does not affect mRNA expression of mesenchymal markers α‐SMA, calponin 1, and fibronectin, or expression of endothelial markers PECAM‐1 and VE‐cadherin (n = 3, + SD, Student’s t test). (f) LOXL2 overexpression does not affect protein expression of endothelial markers PECAM‐1 and VE‐cadherin, or expression of mesenchymal markers α‐SMA and fibronectin; *p < 0.05, **p < 0.01, and ***p < 0.001. EC: endothelial cells; EndMT: endothelial‐to‐mesenchymal transition; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐buffered saline; PECAM‐1: platelet endothelial cell adhesion molecule 1; shCtrl: shRNA control; α‐SMA: α‐smooth muscle actin; shLOXL2: short hairpin‐mediated LOXL2 knockdown; TGF‐β: transforming growth factor‐β; VE‐cadherin: vascular endothelial cadherin

Figure 5

LOXL2 regulates EMT–EndMT‐associated signaling pathways PKB/Akt and FAK. (a) Knockdown of LOXL2 in EC reduces FAK Y397 phosphorylation, and reduces PKB/Akt expression and phosphorylation. (b) Overexpression of LOXL2 in EC increases FAK Y397 phosphorylation and PKB/Akt S473. (c) Overexpression of the catalytically inactive H26Q‐H628Q‐LOXL2 mutant increases FAK Y397 phosphorylation and PKB/Akt S473. EC: endothelial cells; EndMT: endothelial‐to‐mesenchymal transition; EMT: epithelial‐to‐mesenchymal transition; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; FAK: focal adhesion kinase; LOXL2: lysyl oxidase‐like 2; PKB: protein kinase B