Lysyl Oxidase-Like 2 Protects against Progressive and Aging Related Knee Joint Osteoarthritis in Mice

Abstract

Background: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. Methods: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1β in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. Results: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1β-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. Conclusion: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.

Molecular Insights Into Lysyl Oxidases in Cartilage Regeneration and Rejuvenation

“…Further, studies have indicated the upregulation of LOXL2 in OA cartilage in response to injury, which may be considered as a naturally protective response that promotes anabolism while inhibiting specific catabolic response during OA pathophysiology (Alshenibr et al, 2017;Bais and Goldring, 2017). Likewise, a recent study further confirmed that systemic adenovirus-delivered LOXL2 expression or LOXL2 genetic overexpression both exhibited chondroprotective effects through inhibition of catabolic factors and IL-1β-induced NF-κB signaling in mice (Tashkandi et al, 2019). Also, Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed enhanced anabolic responses after LOXL2 transduction, including increased expression of sex determining region Y-box containing gene 9 (SOX9), aggrecan (ACAN), and COL2A1, whilst reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinases and inhibited chondrocyte apoptosis (Alshenibr et al, 2017).…”

“…The decreased expression of LOXs may attribute to reduction of HIF-1 activity in aging organisms (Rivard et al, 2000;Ceradini et al, 2004). Meanwhile, LOXL2 has recently been demonstrated as a potential chondroprotective factor in aging related joint osteoarthritis, mainly through inducing anabolic gene expression and attenuating catabolic genes (Bais and Goldring, 2017;Tashkandi et al, 2019). Thus, restoration of collagen and elastic fiber synthesis in juvenile ECM components though regulation of signaling pathways governing LOX expression may become a promising therapeutic approach for amelioration of aging-associated cartilage degeneration and enhanced cartilage regeneration.…”

LOXL2 promotes aggrecan and gender-specific anabolic differences to TMJ cartilage

“…As described in our earlier studies 19 , 20 , adenoviruses for LOXL2 expression co-expressing Red Fluorescent Protein (RFP) was used for verification of virus transduction and efficient of delivery Ad-CMV-RFP-CMV-hLOXL2-His; referred to as Ad-RFP-LOXL2), and the empty vector control (Ad-RFP-Empty) were custom synthesized (ADV-214438) by Vector Biolabs, PA. These adenovirus particles were amplified in 293 T cells and quantified using an adenovirus quantification kit (Cell Biolabs).…”

“…Total RNA was extracted by the Trizol protocol according to the manufacturer’s instructions (Qiagen) as described earlier 19 , 20 . RT-qPCR analysis was performed using TaqMan gene expression assays from Life Technologies, according to a standard protocol 33 .…”

“…Subsequently, we determined that LOXL2 expression is critical for chondrogenic differentiation and proteoglycan deposition in a chondrocyte culture model 18 . More recently, we identified LOXL2 as an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint in vitro and in vivo 19 , 20 . We developed a model in which human TMJ cartilage was implanted in nude mice in combination with a specialized matrix of the basement membrane.…”