Minoxidil weakens newly synthesized collagen in fibrotic synoviocytes from osteoarthritis patients

Abstract

Purpose: Synovial fibrosis (SFb) formation and turnover attributable to knee osteoarthritis (KOA) can impart painful stiffness and persist following arthroplasty. To supplement joint conditioning aimed at maximizing peri-operative function, we evaluated the antifibrotic effect of Minoxidil (MXD) on formation of pyridinoline (Pyd) cross-links catalyzed by Plod2-encoded lysyl hydroxylase (LH)2b that strengthen newly synthesized type-I collagen (COL1) in fibroblastic synovial cells (FSCs) from KOA patients. MXD was predicted to decrease Pyd without significant alterations to Col1a1 transcription by FSCs stimulated with transforming growth factor (TGF)β1.

Methods: Synovium from 10 KOA patients grouped by SFb severity was preserved for picrosirius and LH2b histology or culture. Protein and RNA were purified from fibrotic FSCs after 8 days with or without 0.5 µM MXD and/or 4 ng/mL of TGFβ1. COL1 and Pyd protein concentrations from ELISA and expression of Col1a1, Acta2, and Plod2 genes by qPCR were compared by parametric tests with α = 0.05.

Results: Histological LH2b expression corresponded to SFb severity. MXD attenuated COL1 output in KOA FSCs but only in the absence of TGFβ1 and consistently decreased Pyd under all conditions with significant downregulation of Plod2 but minimal alterations to Col1a1 and Acta2 transcripts.

Conclusions: MXD is an attractive candidate for local antifibrotic pharmacotherapy for SFb by compromising the integrity of newly formed fibrous deposits by FSCs during KOA and following arthroplasty. Targeted antifibrotic supplementation could improve physical therapy and arthroscopic lysis strategies aimed at breaking down joint scarring. However, the effect of MXD on other joint-specific TGFβ1-mediated processes or non-fibrotic components requires further investigation.

Keywords: Arthroplasty; Collagen type I; Fibrosis; Knee; Lysyl hydroxylase; Minoxidil; Osteoarthritis; Pyridinoline; Synovial fibroblasts; Synovium.

Fig. 1

(A, C) The severity of fibrous collagen deposition, or SFb, (green) measured from confocal photomicrographs of PS-stained, paraffin sections from the synovium of KOA patients is relative to (B, D) LH2b (red) immunolabeled content (E) measured as pixel area of LH2b signal over total tissue area of serial sections from those stained with PS. Scale bars = 100 µm; ns = not significant (F, G) COL1 and Pyd output measured by ELISA from homogenates of FSCs grouped by low (gray circles) or high (black circles) SFb, represented by gray or black circles, associate to histological SFb severity values

Fig. 2

A Immunocytochemical detection of intracellular COL1 production (green) and SMA-positive stress fibers (red) increase in a dose-dependent manner 48 h after inoculation of unstimulated (US) HFLS with recombinant TGFβ1. Bar = 25 μm (B) Changes in proliferation rate between samples that remained non-treated (NT) and those stimulated with TGFβ1 and treated with MXD individually or in combination was measured by immunocytochemistry of Ki67-positive (red) nuclei over total DAPI (gray)-stained nuclei. Bar = 25 μm (C) Expression of target genes measured by qPCR from RNA extracted from HFLS treated under various conditions for 8-days was analyzed by ΔΔCt method and log scaled following statistics. D, E COL1 and Pyd output were measured by sandwich and competitive ELISA, respectively, from homogenized HFLS in the presence and absence of MXD and/or TGFβ1. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, no asterisk: p > 0.05

Fig. 3

FSCs from patients grouped by high (black circles) and low (gray circles) SFb remained untreated (NT) or were treated with MXD in the presence or absence of TGFβ1 stimulation. A-A” COL1 output was measured and compared between groups and the effect of MXD treatment on unstimulated or TGFβ1-stimulated FSCs from each patient is shown. B-B” The effect on Pyd content was also measured under the same conditions. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, no asterisk: p > 0.05

Fig. 4

Comparison of ΔΔCt ratios to evaluate changes in expression of select genes fundamental to COL1 synthesis, myofibroblast transition, and LH2b expression for processing of Pyd cross-links in KOA patient FSCs under the different experimental conditions. Data represent mean ± SEM for n = 10 KOA patient primary FSCs run in duplicate. *p ≤ 0.05, **p ≤ 0.01, no asterisk: p > 0.05