124 PATHOPHYSIOLOGIC CHANGES IN LOXL-1 KNOCKOUT MOUSE WITH PELVIC FLOOR DYSFUNCTION INDUCE A COMPENSATORY AND ABERRANT ELASTIN REGENERATIVE RESPONSE BY VAGINAL SMOOTH MUSCLE CELLS

Abstract

INTRODUCTION AND OBJECTIVES: Impaired elastic matrix remodeling during pregnancy and after parturition may be linked to pelvic organ prolapse (POP). In lysyl oxidase like-1 (LOXL1) knockout (KO) mice, POP develops after vaginal delivery and correlates with disorganized elastin clusters. The goal of this project was to investigate the alterations in new elastic matrix assembly by vaginal smooth muscle cells (SMC) in age-matched nulliparious (N), multiparous nonprolapsed (MNP) and multiparous prolapsed (MP) LOXL1 KO mice. METHODS: Primary vaginal SMC cultures from all LOXL1 KO mice expressed the SMC markers -actin, SM22, caldesmon, smoothelin and tropomyosin. Passage 1 SMC from each group were cultured for 21 days in DMEM/F12 medium with 10% fetal bovine serum, and then assayed for increase in DNA content, collagen matrix, and alkalisoluble, and insoluble matrix elastin. RT-PCR was used to assess expression of collagen, tropoelastin, fibrillin-1, and lysyl oxidase (LOX). Elastic matrix ultrastructure and fiber integrity was assessed using TEM. RESULTS: SMC from MP mice were smaller, less fusiform, and proliferated more rapidly than SMC from N mice. SMC from MNP mice were larger, more spread, and less proliferative than SMC from MP mice. Elastin, LOX, and collagen mRNA expression were reduced in SMC from MNP mice, and were upregulated in SMC from MP mice compared to N mice. Fibrillin mRNA levels were significantly higher in SMC from MNP mice, while LOX mRNA levels were lower in SMC from multiparous mice. SMC from MNP mice synthesized less elastic matrix per well & per cell than did SMC from MP or N mice. Elastin deposits were sporadic in cultures of SMC from MNP mice. A few aberrant fibers were seen in cultures of SMC from N mice. Such fibers were more numerous in cultures of SMC from MP mice. CONCLUSIONS: New elastic matrix synthesis by LOXL1 KO vaginal SMC diminishes with pregnancy and multi-parity. SMC from MP mice exhibit a more activated, proliferative, and remodeling phenotype than SMC from MNP or N mice. Elastic fiber assembly by MNP LOXL1 KO mice is not compromised by reduced fibrillin deposition, but by poor crosslinking from reduced LOX mRNA and the lack of LOXL1. Reduced elastic matrix regenerative repair due to suppression of elastin mRNA by SMC in MNP mice may lead to POP. Pathophysiologic changes in LOXL1 KO mice induce a compensatory and aberrant elastin regenerative response by vaginal SMC. Therapies to regenerate healthy matrix may have application to POP.