Jingming Zhou

Activation of Indian hedgehog promotes chondrocyte hypertrophy and upregulation of MMP-13 in human osteoarthritic cartilage

OBJECTIVE The objectives of this study were to (1) determine the correlation between osteoarthritis (OA) and Indian hedgehog (Ihh) expression, and (2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and matrix metalloprotease (MMP)-13 in human OA cartilage. DESIGN OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by Western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples was analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. RESULTS Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r(2) = 0.80) and Ihh intensity (r(2) = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor cyclopamine had the opposite effect. CONCLUSIONS Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression.

Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice

IntroductionPrevious observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency.MethodsTo evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreERT2; Ihhfl/fl). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates.ResultsOnly mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh.ConclusionsThese findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.

Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis.

To determine if supplemental intraarticular α2‐macroglobulin (α2M) has a chondroprotective effect in a rat model of osteoarthritis (OA).

Indian hedgehog in synovial fluid is a novel marker for early cartilage lesions in human knee joint.

To determine whether there is a correlation between the concentration of Indian hedgehog (Ihh) in synovial fluid (SF) and the severity of cartilage damage in the human knee joints, the knee cartilages from patients were classified using the Outer-bridge scoring system and graded using the Modified Mankin score. Expression of Ihh in cartilage and SF samples were analyzed with immunohistochemistry (IHC), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, we detected and compared Ihh protein levels in rat and mice cartilages between normal control and surgery-induced osteoarthritis (OA) group by IHC and fluorescence molecular tomography in vivo respectively. Ihh expression was increased 5.2-fold in OA cartilage, 3.1-fold in relative normal OA cartilage, and 1.71-fold in OA SF compared to normal control samples. The concentrations of Ihh in cartilage and SF samples was significantly increased in early-stage OA samples when compared to normal samples (r = 0.556; p < 0.001); however, there were no significant differences between normal samples and late-stage OA samples. Up-regulation of Ihh protein was also an early event in the surgery-induced OA models. Increased Ihh is associated with the severity of OA cartilage damage. Elevated Ihh content in human knee joint synovial fluid correlates with early cartilage lesions.

Indian Hedgehog, a critical modulator in osteoarthritis, could be a potential therapeutic target for attenuating cartilage degeneration disease

Abstract The Hedgehog (Hh) family of proteins consists of Indian hedgehog (Ihh), sonic hedgehog (Shh), and desert hedgehog (Dhh). These proteins serve as essential regulators in a variety of developmental events. Ihh is mainly produced and secreted by prehypertrophic chondrocytes and regulates chondrocyte hypertrophy and endochondral bone formation during growth plate development. Tissue-specific deletion of the Ihh gene (targeted by Col2a1-Cre) causes early lethality in mice. Transgenic mice with induced Ihh expression exhibit increased chondrocyte hypertrophy and cartilage damage resembling human osteoarthritis (OA). During OA development, chondrocytes recapitulate the differentiation process that happens during the fetal status and which does not occur to an appreciable degree in adult articular cartilage. Ihh expression is up-regulated in human OA cartilage, and this upregulation correlates with OA progression and changes in chondrocyte morphology. A genetic study in mice further showed that conditional deletion of Ihh in chondrocytes attenuates OA progression, suggesting the possibility that blocking Ihh signaling can be used as a therapeutic approach to prevent or delay cartilage degeneration. However, Ihh gene deletion is currently not a therapeutic option as it is lethal in animals. RNA interference (RNAi) provides a means to knockdown Ihh without the severe side effects caused by chemical inhibitors. The currently available delivery methods for RNAi are nanoparticles and liposomes. Both have problems that need to be addressed. In the future, it will be necessary to develop a safe and effective RNAi delivery system to target Ihh signaling for preventing and treating OA.

Mitogen-activated protein kinase p38 induces HDAC4 degradation in hypertrophic chondrocytes

Histone deacetylase 4 (HDAC4) is a critical negative regulator for chondrocyte hypertrophy by binding to and inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. It is unclear how HDAC4 expression and stability are regulated during growth plate development. We report here that inhibition of mitogen-activated protein kinase (MAPK) p38 by dominant negative p38 or p38 inhibitor prevents HDAC4 degradation. Mutation of a potential caspase-2 and 3 cleavage site Asp289 stabilizes HDAC4 in chondrocytes. In contrast, constitutively active MAPK kinase 6 (constitutive activator of p38) transgenic mice exhibit decreased HDAC4 content in vivo. We also observed that p38 stimulates caspase-3 activity in chondrocytes. Inhibition of p38 or caspases reduced HDAC4 degradation. HDAC4 inhibited Runx2 promoter activity in a dose-dependent manner and caspase inhibitors further enhanced this inhibition by preventing HDAC4 degradation. Overall, these results demonstrate that p38 promotes HDAC4 degradation by increasing caspase-mediated cleavage, which releases Runx2 from a repressive influence of HDAC4 and promotes the chondrocyte hypertrophy and bone formation.

Identification of α1-Antitrypsin as a Potential Candidate for Internal Control for Human Synovial Fluid in Western Blot.

Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.

A novel therapeutic strategy for cartilage diseases based on lipid nanoparticle-RNAi delivery system

Background Cartilage degeneration affects millions of people but preventing its degeneration is a big challenge. Although RNA interference (RNAi) has been used in human trials via silencing specific genes, the cartilage RNAi has not been possible to date because the cartilage is an avascular and very dense tissue with very low permeability. Purpose The objective of this study was to develop and validate a novel lipid nanoparticle (LNP)-siRNA delivery system that can prevent cartilage degeneration by knocking down specific genes. Methods LNP transfection efficiency was evaluated in vitro and ex vivo. Indian Hedgehog (Ihh) has been correlated with cartilage degeneration. The in vivo effects of LNP-Ihh siRNA complexes on cartilage degeneration were evaluated in a rat model of surgery-induced osteoarthritis (OA). Results In vitro, 100% of chondrocytes were transfected with siRNA in the LNP-siRNA group. In accordance with the cell culture results, red positive signals could be detected even in the deep layer of cartilage tissue cultures treated by LNP-beacon. In vivo data showed that LNP is specific for cartilage, since positive signals were detected by fluorescence molecular tomography and confocal microscopy in joint cartilage injected with LNP-beacon, but not on the surface of the synovium. In the rat model of OA, intraarticular injection of LNP-Ihh siRNA attenuated OA progression, and PCR results showed LNP-Ihh siRNA exerted a positive impact on anabolic metabolism and negative impact on catabolic metabolism. Conclusion This study demonstrates that our LNP-RNAi delivery system has a significantly chondroprotective effect that attenuates cartilage degeneration and holds great promise as a powerful tool for treatment of cartilage diseases by knocking down specific genes.

Blockade of hypoxia-induced CXCR4 with AMD3100 inhibits production of OA-associated catabolic mediators IL-1β and MMP-13

Binding of the chemokine stromal cell-derived factor-1 (SDF-1) to its receptor C-X-C chemokine receptor type 4 (CXCR4) results in receptor activation and the subsequent release of matrix metalloproteinases (MMPs) that contribute to osteoarthritis (OA) cartilage degradation. As hypoxia is a defining feature of the chondrocyte microenvironment, the present study investigated the possible mechanism through which SDF-1 induces cartilage degradation under hypoxic conditions. To do this, OA chondrocyte cultures and patient tissue explants pretreated with the CXCR4 inhibitor, AMD3100 were incubated with SDF-1. It was identified that hypoxic conditions significantly elevated the expression of CXCR4 in osteoarthritic chondrocytes relative to normoxic conditions. Furthermore, SDF-1 elevated MMP-13 mRNA levels and proteinase activity. It also elevated the mRNA and protein levels of runt-related transcription factor 2, and induced the release of glycosaminoglycans and the inflammatory cytokine, interleukin-1β. By contrast, such changes did not occur to an appreciable degree in cells that were pretreated with AMD3100. The results of the present study demonstrate that even under hypoxic conditions, where CXCR4 expression is significantly elevated in chondrocytes, AMD3100 effectively blocks this receptor and protects chondrocytes from OA-induced catabolism, suggesting that the successful inhibition of CXCR4 may be an effective approach for OA treatment.

Identification of Alpha 2 Macroglobulin (A2M) as a master inhibitor of cartilage degrading factors that attenuates post-traumatic osteoarthritis progression

To determine if supplemental intraarticular α2‐macroglobulin (α2M) has a chondroprotective effect in a rat model of osteoarthritis (OA).