Muhammad Farooq Rai

Molecular Analysis of Age and Sex-related Gene Expression in Meniscal Tears with and without a Concomitant Anterior Cruciate Ligament Tear

BACKGROUND The meniscus plays critical roles in the knee, contributing to load transmission, shock absorption, and joint stability. Little is known about gene expression in meniscal tears, particularly in relation to injury pattern and patient age and sex. The purpose of this study was to test the hypothesis that gene expression in meniscal tears varies depending on patient age and sex and whether the anterior cruciate ligament (ACL) is also torn. METHODS Meniscal tissue from twenty-eight patients with an isolated meniscal tear or a meniscal tear with a concomitant ACL tear was collected at the time of clinically indicated partial meniscectomy. Messenger RNA (mRNA) expression was examined by quantitative real-time polymerase chain reaction for molecular markers of osteoarthritis including proinflammatory cytokines (interleukin [IL]-1α, IL-1β, IL-6, and tumor necrosis factor-alpha [TNFα]), chemokines (IL-8, CCL3, CCL3L1, CXCL1, CXCL3, CXCL6, and CCL20), aggrecanases (ADAMTS-4 [a disintegrin and metalloproteinase with thrombospondin type-4 motifs] and ADAMTS-5), matrix metalloproteinases (MMP-1, MMP-3, MMP-9, and MMP-13), transcription factors (NFκB2 [nuclear factor kappa B2], NFκBIA [NF-kappa B inhibitor alpha], and IκBA [inhibitor of kappa B alpha]), and matrix components (bone morphogenetic protein [BMP]-2, type-I collagen alpha 1 [Col1a1], Col2a1, and aggrecan). RESULTS Expression of IL-1β (p = 0.02), ADAMTS-5 (p = 0.001), MMP-1 (p = 0.007), MMP-9 (p = 0.002), MMP-13 (p = 0.01), and NFκB2 (p = 0.01) was significantly higher in patients with a meniscal tear who were under the age of forty years than it was in those over the age of forty years. Similarly, the expression of ADAMTS-4 (p = 0.002), ADAMTS-5 (p = 0.02), MMP-1 (p = 0.02), and MMP-13 (p = 0.0002) was higher in patients with a meniscal tear and an ACL tear who were under the age of forty years than it was in those over forty years. In patients with a meniscal tear and an ACL tear, the expression of IL-1β (p = 0.01), TNFα (p = 0.02), MMP-13 (p = 0.004), CCL3 (p = 0.03), and CCL3L1 (p = 0.03) was significantly higher, while that of aggrecan (p = 0.03) was lower, than that in patients with a meniscal tear alone. The only sex-based difference in gene expression was higher levels of CCL3L1 in female patients (p < 0.05) of all ages with combined injuries. CONCLUSIONS AND CLINICAL RELEVANCE These findings suggest clinically relevant differences in the response of the knee to meniscal tears on the basis of patient age and sex. Elevated expression levels of arthritis-related markers indicate an increased catabolic response in patients under forty years old. Higher expression of catabolic markers in patients with meniscal and ACL tears suggests this combined injury pattern is more likely to lead to the development of osteoarthritis. Catabolic activity in meniscal tissue may predict patients who are at risk for progression of osteoarthritis following partial meniscectomy.

Heritability of articular cartilage regeneration and its association with ear wound healing in mice.

OBJECTIVE Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains. METHODS An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n=265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n=229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes. RESULTS Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2=26%; P=0.006) and ear wound closure (H2=53%; P<0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r=0.92) and RI mouse lines (r=0.86) were found to be extremely high. CONCLUSION Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration.

Transcriptome Analysis of Human Injured Meniscus Reveals a Distinct Phenotype of Meniscus Degeneration with Aging

OBJECTIVE Meniscus tears are associated with a heightened risk of osteoarthritis. This study aimed to advance our understanding of the metabolic state of injured human meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to the patients age and degree of cartilage chondrosis. METHODS The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (n = 12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. Variations in the relative changes in gene expression with age and chondrosis were analyzed, and integrated biologic processes were investigated computationally. RESULTS We identified a set of genes in torn menisci that were differentially expressed with age and chondrosis. There were 866 genes that were differentially regulated (≥1.5-fold difference and P < 0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed, while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed, and those involved in T cell differentiation and apoptosis were elevated. CONCLUSION Differences in age-related gene expression suggest that in older adults, meniscal cells might dedifferentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but they maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis.

Transcriptome Analysis of Injured Human Meniscus Reveals a Distinct Phenotype of Meniscus Degeneration With Aging

OBJECTIVE Meniscus tears are associated with a heightened risk of osteoarthritis. This study aimed to advance our understanding of the metabolic state of injured human meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to the patients age and degree of cartilage chondrosis. METHODS The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (n = 12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. Variations in the relative changes in gene expression with age and chondrosis were analyzed, and integrated biologic processes were investigated computationally. RESULTS We identified a set of genes in torn menisci that were differentially expressed with age and chondrosis. There were 866 genes that were differentially regulated (≥1.5-fold difference and P < 0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed, while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed, and those involved in T cell differentiation and apoptosis were elevated. CONCLUSION Differences in age-related gene expression suggest that in older adults, meniscal cells might dedifferentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but they maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis.

Cartilage and bone changes during development of post-traumatic osteoarthritis in selected LGXSM recombinant inbred mice.

INTRODUCTION Little evidence is available on the natural course of osteoarthritis (OA) development and the genes that protect and predispose individuals to it. This study was designed to compare strain-dependent development of OA and its association with tissue regeneration in mice. Two recombinant inbred lines LGXSM-6 and LGXSM-33 generated from LG/J and SM/J intercross were used. Previous studies indicated that LGXSM-6 can regenerate both articular cartilage and ear hole punch while LGXSM-33 cannot. METHODS Transection of the medial meniscotibial ligament was performed on 10-week-old male mice to induce OA. Cartilage damage was analyzed by histology and bone morphology was evaluated using micro-computed tomography (CT). Ear punches were performed and evaluated by measurement of residual hole diameter. RESULTS Cartilage analysis showed that LGXSM-33 developed a significantly higher grade of OA than LGXSM-6. Bone analysis showed that LGXSM-33 had substantial subchondral bone and trabecular bone thickening 8 weeks post-surgery, while LGXSM-6 showed bone loss over time. We also confirmed that LGXSM-6 can heal ear tissues significantly better than LGXSM-33. CONCLUSIONS OA was found to be negatively correlated with the degree of tissue regeneration. LGXSM-33, a poor healer of ear tissues (and articular cartilage), developed more OA compared to LGXSM-6, which had better regenerative ability for ear tissues and articular cartilage. The phenotypic differences observed here are due to genetic differences further suggesting that similar sets of physiological processes and gene variants may mediate variation in OA development and tissue regeneration.

Quantification of cytokines and inflammatory mediators in a three-dimensional model of inflammatory arthritis.

Human recombinant IL-1beta and TNFalpha have been previously used to induce a cytokine response in canine chondrocytes. In order to establish this functional relation in a homologous system in vitro, we have developed both 2D and 3D models of inflammatory arthritis using canine recombinant cytokines in canine articular chondrocytes. IL-1beta and TNFalpha were cloned and subsequently expressed in Escherichia coli. The purified recombinant canine cytokines were used to simulate inflammation in vitro and the expression of typical inflammation markers such as proinflammatory cytokines (IL-1beta, IL-6, IL-8, GM-CSF and TNFalpha), enzyme mediators (MMP-3 MMP-13, iNOS, COX-2) and their catabolites (NO, PGE(2)) was measured. High expression of proinflammatory cytokines, enzyme mediators and their catabolites was only observed in IL-1beta/TNFalpha stimulated cells. We conclude that the canine IL-1beta and TNFalpha generated in this study are biologically active and equally effective in the canine cell culture systems. Inducing an inflammatory pathway by canine exogenous cytokines in canine chondrocytes provides a useful tool for the study of canine inflammatory arthritis.

Application of inflammation-responsive promoter for an in vitro arthritis model

OBJECTIVE The application of inflammation-regulated therapeutic gene expression in arthritis conditions increases the efficiency of gene therapy by self-limiting the transgene. Incidentally, constitutive overexpression of transgenes typically leads to detrimental effects in disease conditions; therefore, regulation of expression is warranted. We undertook this study to validate a new gene therapy approach using a cell culture-based inflammation model and a novel self-limiting, inflammation-responsive promoter construct. METHODS We designed a self-limiting promoter construct that expresses an antiinflammatory gene (interleukin-4 [IL-4]) only in the presence of inflammation. Our construct featured a truncated promoter sequence of cyclooxygenase 2 (COX-2) upstream of the IL-4 gene. We triggered inflammation in vitro in articular chondrocytes by applying the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) together exogenously, and we studied the extent of IL-4 expression and its effect on the inflammatory cascade. RESULTS Using articular chondrocytes, we showed that our COX-2 promoter construct expressed IL-4 only in the presence of IL-1beta and TNFalpha. IL-4 expressed in the presence of IL-1beta and TNFalpha down-regulated a series of inflammation mediators, prostaglandins, and matrix metalloproteinases. CONCLUSION The use of this construct for the expression of antiinflammatory genes allows production of a therapeutic gene product that is controlled by the severity of the disease. The effectiveness of this promoter construct for combating inflammation makes it a suitable candidate for the development of a new local gene therapy strategy for the treatment of osteoarthritis, in which IL-1beta and TNFalpha trigger a signal cascade that elevates COX-2 levels.

The chemokine receptor CCR5 plays a role in post-traumatic cartilage loss in mice, but does not affect synovium and bone

OBJECTIVE C-C chemokine receptor type 5 (CCR5) has been implicated in rheumatoid arthritis and several inflammatory diseases, where its blockade resulted in reduced joint destruction. However, its role in modulating cartilage and bone changes in post-traumatic osteoarthritis (OA) has not yet been investigated. In this study, we investigated changes in articular cartilage, synovium and bone in a post-traumatic OA model using CCR5-deficient (CCR5(-/-)) mice. METHOD Destabilization of the medial meniscus (DMM) was performed on the right knee of 10-week old CCR5(-/-) and C57BL/6J wild-type (WT) mice to induce post-traumatic OA. The contralateral left knee served as sham-operated control. Knee joints were analyzed at 4-, 8- and 12-weeks after surgery to evaluate cartilage degeneration and synovitis by histology, and bone changes via micro-CT. RESULTS Our findings showed that CCR5(-/-) mice exhibited significantly less cartilage degeneration than WT mice at 8- and 12-weeks post-surgery. CCR5(-/-) mice showed some altered bone parameters 18- and 22-weeks of age, but body size and weight were not affected. The effect of CCR5-ablation was insignificant at all time points post-surgery for synovitis and for bone parameters such as bone volume/total volume, connectivity density index (CDI), structure model index (SMI), subchondral bone plate thickness, and trabecular bone number, thickness and spacing. CONCLUSION These findings suggest that CCR5(-/-) mice developed less cartilage degeneration, which may indicate a potential protective role of CCR5-ablation in cartilage homeostasis. There were no differences in bone or synovial response to surgery suggesting that CCR5 functions primarily in cartilage during the development of post-traumatic OA.

Molecular Characterization of Articular Cartilage from Young Adults with Femoroacetabular Impingement

BACKGROUND Femoroacetabular impingement is a frequent cause of hip pain and may lead to secondary osteoarthritis, yet little is known about the molecular events linking mechanical hip impingement and articular cartilage degeneration. The first goal of this study was to quantify the expression of inflammatory cytokine and chemokine, matrix-degrading, and extracellular matrix genes in articular cartilage harvested from control hips and hips with femoroacetabular impingement and end-stage osteoarthritis. The second goal was to analyze the relative expression of these genes in articular cartilage harvested at various stages of osteoarthritis. METHODS Cartilage samples were obtained from thirty-two hips undergoing hip preservation surgery for femoroacetabular impingement or hip arthroplasty. Three control cartilage samples were also analyzed. Specimens were graded intraoperatively with regard to the severity of cartilage damage, the radiographic osteoarthritis grade was recorded, and quantitative RT-PCR (real-time polymerase chain reaction) was performed to determine relative gene expression. RESULTS Except for interleukin-1β (IL-1β) and CXCL2, the mRNA (messenger RNA) expression of all other chemokine (IL-8, CXCL1, CXCL3, CXCL6, CCL3, and CCL3L1), matrix-degrading (matrix metalloproteinase [MMP]-13 and ADAMTS-4), and structural matrix (COL2A1 [collagen, type II, alpha] and ACAN [aggregan]) genes was higher overall in cartilage from hips with femoroacetabular impingement compared with hips with osteoarthritis and normal controls. The differences reached significance (p ≤ 0.05) for seven of these ten quantified genes, with CXCL3, CXCL6, and COL2A1 being elevated in the femoroacetabular impingement group compared with only the control group and IL-8, CCL3L1, ADAMTS-4, and ACAN being elevated compared with both the osteoarthritis and control groups. When samples were grouped according to the stage of the degenerative cascade, mRNA expression was relatively higher in one of the two middle stages of femoroacetabular impingement (chondromalacia or cleavage/thinning), with the difference reaching significance for IL-8, CXCL2, CXCL3, CCL3L1, and ACAN. ACAN expression was diminished in hips with osteoarthritis compared with femoroacetabular impingement but elevated compared with the control articular cartilage. CONCLUSIONS Articular cartilage from the impingement zone of hips with femoroacetabular impingement (and particularly those hips in the cleavage/thinning stage) expressed higher levels of certain inflammatory, anabolic, and catabolic genes, representing a heightened metabolic state. CLINICAL RELEVANCE The articular cartilage from the impingement zone of hips with femoroacetabular impingement was metabolically hyperactive, supporting the concept that such impingement is a structural precursor to hip osteoarthritis.

Suppression of NF-κB activity via nanoparticle-based siRNA delivery alters early cartilage responses to injury.

Significance Osteoarthritis is a common debilitating joint disease that affects millions in the United States and for which there are few therapeutic options. Critical barriers to the successful development of osteoarthritis treatment include limited understanding of the pathways governing early cartilage degradation and ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Using a peptidic nanoparticle carrying siRNA that specifically suppresses NF-κB, we show that early antiinflammatory intervention reduces chondrocyte death caused by joint injury, a known predisposing factor for osteoarthritis. The peptidic nanoparticle deeply penetrates human cartilage to deliver its therapeutic cargo to the chondrocytes, demonstrating its ability to permeate the dense cartilage matrix. This approach promises to overcome the barriers to effectively treat osteoarthritis. Osteoarthritis (OA) is a major cause of disability and morbidity in the aging population. Joint injury leads to cartilage damage, a known determinant for subsequent development of posttraumatic OA, which accounts for 12% of all OA. Understanding the early molecular and cellular responses postinjury may provide targets for therapeutic interventions that limit articular degeneration. Using a murine model of controlled knee joint impact injury that allows the examination of cartilage responses to injury at specific time points, we show that intraarticular delivery of a peptidic nanoparticle complexed to NF-κB siRNA significantly reduces early chondrocyte apoptosis and reactive synovitis. Our data suggest that NF-κB siRNA nanotherapy maintains cartilage homeostasis by enhancing AMPK signaling while suppressing mTORC1 and Wnt/β-catenin activity. These findings delineate an extensive crosstalk between NF-κB and signaling pathways that govern cartilage responses postinjury and suggest that delivery of NF-κB siRNA nanotherapy to attenuate early inflammation may limit the chronic consequences of joint injury. Therapeutic benefits of siRNA nanotherapy may also apply to primary OA in which NF-κB activation mediates chondrocyte catabolic responses. Additionally, a critical barrier to the successful development of OA treatment includes ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Here, we show that the peptide–siRNA nanocomplexes are nonimmunogenic, are freely and deeply penetrant to human OA cartilage, and persist in chondrocyte lacunae for at least 2 wk. The peptide–siRNA platform thus provides a clinically relevant and promising approach to overcoming the obstacles of drug delivery to the highly inaccessible chondrocytes.