Mechanism of Lysyl Oxidase‐Like 2 Regulation in Vasculature
Authors: Wang Huilei , Steppan Jochen , Jandu Sandeep , Melucci Sean , Santhanam Lakshmi 
Published: November 2023

Background

Vascular stiffening increases the risk of major adverse cardiovascular events such as strokes and heart attacks. Clinical management has been difficult because the mechanism of the development and progression of vascular stiffening is unknown. Matrix crosslinking proteins play a key role in vascular remodeling. Through a proteomic screen, Lysyl Oxidase‐Like 2 was identified as a potential therapeutic target. LOXL2 is an amine oxidase in the LOX family. It has the prototypic LOX domain which catalyzes the first step of crosslinking for extracellular matrix proteins such as collagen and elastin, as well as four Scavenger Receptor Cysteine‐Rich (SRCR) domains which are typically found in scaffolding and protein‐protein, cell‐protein interactions. In order to understand the role of LOXL2 in vascular stiffening, we studied the mechanisms by which LOXL2 is regulated in vasculature.

Methods and Results

Heterozygous LOXL2 mice were obtained from Taconic Inc. and aortic smooth muscle cells (SMCs) and endothelial cells (ECs) were isolated from wild type and LOXL2+/− litter mates. qPCR and western blotting results verified LOXL2 expression and showed that while EC from LOXL2+/− mice show significant decrease in LOXL2 expression, the SMC overexpress LOXL2. Cell adhesion was then studied with two complementary methods: Electrical Cell‐Substrate Impedance Sensing (ECIS) by the use of PicoGreen DNA quantification to determine fraction of cells adhered 1 h after seeding. Results from both methods showed that LOXL2+/− ECs adhered slower than WTs, while the LOXL2+/− SMCs adhered much more rapidly than those from WTs. Cell proliferation was also studied with ECIS and with EdU incorporation. LOXL2+/− ECs that lack LOXL2 proliferated slower than WT ECs; LOXL2+/− SMCs that overexpress LOXL2 proliferated more rapidly than WTs.

Next we studied whether LOXL2 secretion to the extracellular space is dependent on nitric oxide (NO). Treatment of human aortic ECs with the eNOS inhibitor L‐NAME, resulted in a dose dependent increase of LOXL2 secretion. In human aortic SMCs and rat thoracic SMCs (A7r5 cells), NO donor NOC18 suppressed LOXL2 secretion and ROS donor SOTs‐1 increased LOXL2 secretion.

In silico digestion of LOXL2 (ExPASY protein cutter) revealed a putative cleavage of LOXL2 by Factor Xa. In cell culture as well as in conditioned cell culture media containing overexpressed LOXL2, riveroxiban, a factor Xa specific inhibitor, dose dependently prevented cleavage of LOXL2. Conversely, the delivery of activated Factor Xa in media increased the cleavage of full length LOXL2 significantly both in cell culture and in conditioned cell culture media with overexpressed LOXL2.

Conclusion

LOXL2+/− mice are mosaic in that there is overexpression of LOXL2 in SMCs and decreased expression in ECs. The adhesion and proliferation of ECs and SMCs are positively correlated with LOXL2 expression. The secretion of LOXL2 is eNOS/NO dependent. Secreted LOXL2 is processed by Factor Xa. The consequence of these mechanisms on vascular mechanics and functions ex vivo and in vivo is the focus of ongoing work.

Support or Funding Information

Funded by StAAR Award from Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine.