Effects And Mechanism Of Maintaining Hypoxia Environment Of Subchondral Bone In Retarding Progression Of Post Traumatic Osteoarthritis

Objection:Abnormal bone remodeling of subchondral bone caused by over-activated osteoclasts（OC）in the early stages of post-traumatic osteoarthritis（PTOA）leads to degeneration of articular cartilage and progression of PTOA,but the mechanism is still unclear.In this study,we investigated whether inhibition of abnormal activation of subchondral bone osteoclasts could delay the progression of PTOA by knocking out the lymphocyte plasma protein 1（Lcp1）.To observe changes of oxygen partial pressure in early PTOA subchondral bone.To investigate the role of hypoxia-inducible factor-1alpha（HIF-1α）in OA cartilage degeneration;To verify that inhibiting L-Plastin（LPL）,a protein encoded by Lcp1 gene,can reduce the abnormal activation of subchondral osteoclasts and slow down the progression of osteoarthritis.Method:Anterior cruciate ligament transection（ACLT）was used to construct PTOA model in Lcp1 knockout and wild type（WT）mice.The mice were sacrificed at 2,4 and 8 weeks after ACLT.The bone volume/total volume（BV/TV）and subchondral bone plate thickness（SBP.th）of the knee subchondral bone were compared between WT and Lcp1^-/-mice via Micro-CT scan.Subchondral bone vascular volume was analyzed by angiographic Micro-CT scan.Knee tissue was fixed,decalcified and prepared in 5μm or 30μm-thick sagittal paraffin or frozen sections.The following histological staining methods were used to observe alteration of cartilage and subchondral bone:1.Hematoxylin eosin staining（HE）to calculate the ratio of articular hyaline cartilage to calcified cartilage;2.Osteoarthritis Research Society International（OARSI）grade was used to quantify cartilage degeneration via Safranin O and Fast Green staining（SOFG）;3.Tartrate Resistant Acid Phosphatase（TRAP）staining was used to count the number of osteoclasts per unit area in subchondral bone;The following cartilage anabolic and catabolic markers were used to compare the cartilage degeneration via Immunohistochemistry（IHC）staining:1.Type II collagen（COLⅡ）and aggrecan（ACAN）staining were used to observe the cartilage matrix synthesis.2.Matrix metalloproteinases 13（MMP13）and Disintegrin And Metalloproteinase with Thrombospondinc Motifs 5（ADAMTS5）staining were used to evaluate cartilage matrix degrading enzymes level;3.TypeⅩcollagen（COLⅩ）staining to observe cartilage matrix calcification.The following immunofluorescence（IF）staining were used to compare the invasion of bone vessels and nerves in cartilage and subchondral bone:1.Platelet-endothelial cell adhesion molecule（CD31）and Endomucin（Emcn）;2.The hypoxia probe Pimonidazole;3.Axonal growth inducing factor 1（NETRIN-1）;4.Calcitonin gene related peptide（CGRP）.The oxygen distribution in the knee joint of living mice was observed by Micro-PETCT scan using ¹⁸F-fluoromisonidazole(¹⁸F-FMISO)radiographic hypoxia probe.The ratio of the maximum standard uptake value（SUV）on the healthy side and affected side was calculated.The van Frey test was used to observe knee pain in mice.Soft tissue samples of human knee joints were taken from two patients with total knee replacement,and paraffin sections were prepared.IHC staining of HIF-1αwas used to compare the difference of HIF-1αexpression between normal cartilage and OA cartilage.The data of human knee cartilage chondrocytes single-cell sequencing in the GES104782 database were reanalyzed by bioinformatics analysis.Differential gene enrichment,Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis,Gene Ontology（GO）enrichment.Analysis and Gene Set Variation Analysis（GSVA）analysis were used to screen chondrocytes regulated by HIF-1αand involved in OA progression.By administration of Hif1a knockdown adeno-associated virus（AAV）in Lcp1knockout OA model mice and HIF-1αprotein stabilizer dimethyloxallyl glycine（DMOG）in wild-type OA model mice,we observed the above-mentioned cartilage and subchondral bone change indexes to verify that subchondral bone osteoclast activation-mediated vascularization leads to the progression of osteoarthritis through degradation of HIF-1α.By administration of Oroxylin A（Oxy A）in WT arthritis model mice and observing the above-mentioned cartilage and subchondral bone change indexes,we verified that targeting LPL protein in subchondral osteoclasts could delay the progression of osteoarthritis.Result:We found that osteoclasts and LPL protein expression were rarely seen in normal cartilage and subchondral bone,but LPL protein appeared transiently in the subchondral bone in early stages of PTOA.The timing and spatial localization of its appearance was consistent with osteoclasts which peaked at 2 weeks,began to decline at 4 weeks,and returned to normal levels at 8 weeks after ACLT.Comparing with WT mice,we invested that Lcp1^-/-mice exhibited reduced cartilage degeneration:1.increased ratio of hyaline cartilage and calcified cartilage,2.decreased OARSI grade,3.increased cartilage anabolic products,4.decreased cartilage catabolic products;Lcp1^-/-mice exhibited reduced bone remodeling of subchondral bone:1.increased subchondral bone volume at 2 weeks after ACLT;Lcp1^-/-mice showed reduced subchondral bone vascular invasion.1.subchondral bone angiography showed less vascular increase than WT group,2.decreased H-type vascular increase;Lcp1^-/-mice decreased sensory nerve invasion of subchondral bone:1.decreased expression of neuroinduction factor NETRIN-1,2.decreased CGRP-positive nerve fibers,3.increased claw reduction threshold in mice von Frey test pin prick test.In vivo ¹⁸F-FMISO PETCT scans and hypoxic probe IF staining showed that the hypoxic environment of articular subchondral bone and cartilage was altered in the early stages of PTOA.Knockout Lcp1 impeded osteoclast-mediated angiogenesis,inhibited elevated oxygen partial pressure levels in subchondral bone,and maintained the hypoxic microenvironment of the joint.Oxygen-mediated ubiquitination and subsequent degradation of HIF-1αplay an important role in the progression of OA.We used single-cell sequencing analysis of human articular chondrocytes and identified a new subpopulation of hypertrophic chondrocytes highly associated with the development of OA named OA-associated hypertrophic chondrocyte（OA-HTC）.GSVA analysis revealed that this group of cells had two-fold greater activation of the OA signaling pathway than other cells but downregulation of HIF-1 signaling pathway.OA-HTC are secreting matrix degrading enzymes such as MMP13 and COLⅩin OA cartilage.Knockdown of Hif1a by AAV abrogated the cartilage protective effect of Lcp1 knockdown,but did not affect the protection of subchondral bone.Stabilization of HIF-1αby DOMG,an inhibitor of proline hydroxylase 2（PHD2）,slowed cartilage degeneration,while abnormal bone remodeling in subchondral bone was not attenuated.Finally,we applied Oxy A,an inhibitor of LPL,to inhibit the activation of subchondral bone osteoclasts and found that Oxy A attenuated subchondral bone remodeling,delayed cartilage degeneration,reduced subchondral bone nerve invasion,and alleviated pain in mice.Conclusion:1.In subchondral bone of early PTOA,Lcp1 knockdown inhibits osteoclast activation and delays osteoarthritis progression.2.In subchondral bone of early PTOA,osteoclast-mediated H-type angiogenesis elevates p O2 levels.3.Elevated p O2 levels degrade HIF-1αand destabilize chondrocyte differentiation phenotype and metabolism.4.HIF-1αis a key molecule downstream of abnormal subchondral bone remodeling in PTOA and is a potential therapeutic target.5..LPL is a therapeutic target to inhibit osteoclast activation in subchondral bone in the early stages of PTOA and Oxy A as an inhibitor of LPL protein can retard the progression of PTOA and is a potential disease-modifying drug for OA（DMOADs）.