The signs of differentiation of chondrocytes in the formation of early cartilage lesions in the elderly

Objective. To study the relationship between the activity of collagen type II cleavage and the pattern of expression of the genes responsible for chondrocyte differentiation in the areas of intact cartilaginous tissue and in those of early focal cartilage lesions similar to those observed in elderly people with osteoarthrosis (OA). Material and methods. The distal femoral articular surface of the knee joint that articulated with the patella and had focal lesions was examined in the elderly. Collagen type II cleavage was estimated by enzyme immunoassay. Gene expression was determined with reverse-transcription polymerase chain reaction. Results. The activity of collagen type II cleavage was shown to be increased in the area of age-related OA-like cartilage lesions. This was accompanied by the high expression of collagenases of metalloproteinases (MMP) 1, 14 (MT1-MMP), aggrecanases - desintegrin and MMP with thrombospondin type 1 motif (ADAMTS) 5, the cytokines of interleukins (IL) 1α/β and tumor necrosis factor-α (TNF-α), as well as the genes associated with chondrocyte hypertrophy of type X collagen (C0L10A1), MMP 13 and 9, Indian hedgehog (Ihh) and cas-pase 3 in the immediate vicinity of a lesion area. At the same time, there was a high expression of growth factors associated with the proliferation phase of chondrocytes, namely: parathyroid hormone-related peptide (PTHrP), fibroblast growth factor-2 (FGF-2), transforming growth factor β1/2 (TGF-β1/2), as well as macromolecules of matrix of type II collagen (C0L2A1) and aggrecan in both the areas adjacent to the lesions and at a considerable distance from their center. However, these areas showed no higher collagen cleavage activity. Nether higher collagen cleavage, nor excess expression of the genes examined were observed in the absolutely intact cartilage areas. Conclusion. Our studies have indicated that the area of very early age-related OA-like focal cartilage lesions exhibits enhanced type II collagen cleavage that is attended by the expression of the genes associated with chondrocyte differentiation in the embryonic growth plate

Type II collagen peptide is able to accelerate embryonic chondrocyte differentiation: an association with articular cartilage matrix resorption in osteoarthrosis

Objective: To study the effect of type II collagen peptide (CP) on the decomposition of type II collagen and the differentiation of embryonic chondrocytes of various morphotypes, isolated from the bovine growth plate. Material and methods. Bovine growth plate chondrocytes were separated into morphotypes on a Percoll gradient in accordance with different differentiation stages and then cultured in the presence or absence of 10 ßM of CP. Cartilage explants from patients with osteoarthrosis (OA) were also cultured in the presence of 10 μM of CP. Type II collagen decomposition activity was evaluated by ELISA. Gene expressions were determined by reverse transcription polymerase chain reaction (RT-PCR). Results. The cells having the highest buoyant density on a Percoll gradient (subpopulation A) were represented as early proliferative zone chondrocytes in the growth plate. In their presence of CP, there was increased expression of the genes of transforming growth factor (TGF) β2, parathyroid hormone-related peptide (PTHrP), fibroblast growth factor (FGF) 2, and cyclin B2, which were expressed in the proliferative zone of the growth plate, as well as metalloproteinase matrix (MMP) 13 and collagen decomposition activity. The large cells with the lowest buoyant density on a Percoll gradient (subpopulation B) were represented as hypertrophic chondrocytes. Cultivation of these cells in the presence of CP increased the expression of the genes associated with the terminal chondrocyte differentiation of type X collagen (COL10A1), Indian hedgehog (Ihh), core-binding factor (CBFA) 1 and TGF β1; and the expression of MMP 13 was suppressed. At the same time, collagen decomposition activity in cultured chondrocytes remained unchanged in the presence of CP. Although type II collagen was able to induce the collagen decomposition in the healthy articular cartilage explants, which was attended by the increased expression of the genes associated with the hypertrophic zone of an embryonic growth plate, it did not affect collagen decomposition rate in the cartilage of patients with OA. Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology

THE ACTIVATION OF MATRIX METALLOPROTEINASES AND CHONDROCYTE DIFFERENTIATION, WHICH ACCOMPANIES THE INDUCTION OF COLLAGEN DECOMPOSITION UNDER THE ACTION OF COLLAGEN PEPTIDE IN THE CARTILAGE OFHEALTHY INDIVIDUALS

Objective: To study the effect of type II collagen peptide (CB12-2) that is able to induce the collagen decomposition in the knee articular cartilage explants, which activates matrix metalloproteinase (MMP) and articular chondrocyte differentiation. Material and methods. Human cartilage explants were cultured in the presence of CB12-2 (amino acid residues 195-218) at a concentration of 10 μM. Type II collagen decomposition was evaluated by enzyme immunoassay. Immunohistochemical assay of type X collagen (C0L10A1) used frozen cartilage sections. Apoptotic activity was measured by terminal deoxynucleotidyl transferase-mediated dUPTnick end-labeling method (TUNEL). Gene expressions were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results. CB12-2 at high, but naturally occurring concentrations (10 μM) induced type II collagen decomposition by collagenase in the cartilage explants from a healthy individual. The peptide caused an increase in the expression of MMP 13, 1, 9, MT1-MMP, and the genes associated with the differentiation of embryo chondrocytes in the growth plate - transforming growth factor (TGF) β1/2, sex-determining region Y-box 9 (Sox9), Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTHrP), fibroblast growth factor (FGF-2); a marker for proliferation of cyclin B2 and cytokines of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1Я). At the same time the increased expression of the chondrocyte hypertrophy marker C0L10A1 was accompanied by enhanced staining for type X collagen in the frozen cartilage sections whereas the number of TUNEL-positive cells increased in the cartilage surface area as the expression of the apoptotic marker caspase 3 became higher. Conclusion. This study has shown that the induction of collagenase activity by CB12-2 in the human articular cartilage chondrocytes is attended by terminal differentiation/hypertrophy of these cells. The terminal differentiation of chondrocytes may be one of the mechanisms of chondrolysis in osteoarthrosis since it naturally occurs not only in endochondrial ossification, but also in the development of pathology

ANALYSIS OF GENE EXPRESSION IN BLOOD AS AN ADDITIONAL TOOL TO MONITOR METHOTREXATE THERAPY IN PATIENTS WITH RHEUMATOID ARTHRITIS

Objective. To assess the changes in clinical, immunological, X-ray indicators and expression of the mTOR (mammalian target of rapamycin) genes, the key regulator of cell growth and proliferation; ULK1 (autophagy marker); р21 (cyclindependent kinase inhibitor); caspase 3 (indicator of apoptotic activity); MMP9 (matrix metalloproteinase 9) and cathepsin K, which participate in joint destruction, and proinflammatory cytokine TNFα (tumor necrosis factor α) in blood of patients with rheumatoid arthritis (RA) receiving methotrexate (MT) therapy.Materials and Methods. Thirty-three RA patients (21 with positive and 12 with negative rheumatoid factor (RF), respectively; median age, 47.1 years) and 28 healthy volunteers (median age, 45.1 years) were examined. All patients have been receiving MT for 2 years. The clinical response was assessed according to the DAS28 score. ESR and the serum levels of anti-cyclic citrullinated peptide antibodies (ACPA), C-reactive protein (CRP), and RF were also determined. Degenerative changes in the joints were evaluated by X-ray examination. Gene expression was measured in peripheral blood cells using reverse transcriptase reaction and real-time polymerase chain reaction.Results. MT therapy considerably reduced the disease severity according to DAS28 score, as well as the number of swollen and painful joints both in seropositive (RF+) and seronegative (RF-) RA patients. Ten patients reached remission by the end of the study. In (RF-) RA patients, the absence of progression of joint destruction was accompanied by the absence of any significant changes in expression of MMP9 and cathepsin K, as well as a stronger suppression of TGFα (its expression became comparable to that in the control group). Patients who achieved remission showed a significant decrease in the expression level of the cathepsin K gene as compared to that at the start of the study. In (RF+) RA patients, MT therapy significantly reduced the clinical and immunological indicators; however, the increased number of erosion sites and further joint space narrowing was observed. It was accompanied by a considerable increase in the expression levels of the MMP9, cathepsin K, and TGFα genes as compared to those in healthy individuals.Conclusions. The changes in expression of the genes responsible for destruction of the hyaline cartilage and bone matrix (MMP9 and cathepsin K) and the TGFα level in blood of RA patients due to MT therapy correlate with the changes in clinical, immunological, and X-ray parameters used to evaluate patient's condition in clinical practice

IMPAIRED REGULATORY MECHANISMS OF THE mTOR SIGNALING PATHWAY IN OSTEOARTHROSIS

Objective: to study the pattern of impaired regulatory mechanisms of the mammalian target of rapamycin (TOR) signaling pathway, by monitoring gene expression in the blood of patients with osteoarthrosis (OA) at different stages of the disease. Subjects and methods. The study covered 33 outpatients with OA, 14 patients with this condition prior to knee joint endoprosthesis, and 27 healthy individuals (controls) (mean age 58.0+7.4, 56.5+8.9, and 55.0+8.3 years, respectively). Total RNA was isolated from their blood and used to determine the level of gene expression by a real-time polymerase chain reaction for AMP-activated protein kinase (AMPK), hypoxia-inducible factor-1α (HIF1α), the rate-limiting proteins of the hexosamine signaling pathway — glutamine-fructose-6-phosphate amidotransferase and acetylglucosaminyltransferase, as well as the glucose transporter GLUT1 and steps 6 and 7 glycolytic pathway components — glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-1, respectively; the lipogenesis-related genes — fatty acid synthase (FAS) and the activity of the pentose phosphate pathway — glucose-6-phosphate dehydrogenase in the blood of patients with OA at different stages of the disease. Results. Analysis of gene expressions showed that in the OA patients with a low expression of the mTOR gene (a LOW subgroup), the expression of AGT and GLUT1 genes proved to be significantly lower and that of the AMPK gene was higher than in the healthy individuals. In the OA patients with a high expression of the mTOR gene (a HIGH subgroup), the expression of all the genes under study was much higher, except for the FAS gene; moreover, the greatest expression excess as compared to the controls was observed for the AMPK and HIFlα genes. In the patients with endstage disease (an ES subgroup), the expression of all the study genes, including the FAS gene, turned out to be higher than in the healthy individuals. Conclusion. The development of OA is accompanied by a considerable decrease in the efficiency of energy metabolism. At the same time, in the patients with a low mTOR gene expression, energy deficiency may be due to decreased cellular metabolite transport. It may be caused by the deficiency of the end electron acceptor oxygen in the patients with a high mTOR gene expression and the pathological redistribution of energy substrate in favor of lipogenesis cannot be ruled out in those with end-stage disease

Enhanced regulatory gene expressions in the blood and articular cartilage of patients with rheumatoid arthritis

Objective: to study the expression ratio of the non-tissue specific regulatory genes mTOR, р21, ATG1, caspase 3, tumor necrosis factor-а (TNF-а), and interleukin-6 (IL-6), as well as matrix metalloproteinase 13 (MMP-13) and X type collagen (COL10A1), cartilage resorption-associated MMP13 and COL10A1 in the blood and knee articular cartilage in patients with rheumatoid arthritis (RA). Subjects and methods. Twenty-five specimens of the distal femoral articular cartilage condyles were studied in 15 RA patients (mean age 52.4+9.1 years) after endoprosthetic knee joint replacement and in 10 healthy individuals (mean age 36.0+9.1 years) included into the control group. Twenty-eight blood samples taken from 28 RA patients (aged 52+7.6 years) prior to endoprosthetic knee joint replacement and 27 blood samples from healthy individuals (mean age 53.6+8.3 years; a control group) were also analyzed. Real-time quantitative polymerase chain reaction was applied to estimate the expression of the mTOR, p21, ATG1, caspase 3, TNF-а, IL- 6, COL0A1, and MMP-13 genes. The levels of a protein equivalent in the p70-S6K(activated by mTOR), p21, and caspase 3 genes concerned was measured in the isolated lymphocyte lysates, by applying the commercially available ELISA kits. Total protein in the cell extracts was determined using the Bradford assay procedure. Results. The cartilage samples from patients with end-stage RA exhibited a significantly higher mTOR, ATG1, p21, TNFа, MMP-13, and COL10A1 gene expressions than did those from the healthy individuals. At the same time, IL6 gene expression was much lower than that in the control group. The expressions of the mTOR, ATG1, p21, TNFа, and IL 6 genes in the blood of RA patients were much greater than those in the donors. Caspase 3 expression did not differ essentially in the bloods of the patients with RA and healthy individuals. The bloods failed to show MMP-13 and COL10A1 expressions. High mTOR and p21 gene expressions were accompanied by the elevated concentrations of the corresponding proteins in the cell lysates of the patients with RA compared to the controls. Conclusion. The findings suggest for the first time that regulatory mTOR, ATG1, p21, and TNFа gene expressions are enhanced in the blood and articular cartilage of RA patients. These changes are accompanied by the increased expression of MMP 13 gene that is responsible for articular cartilage resorption. Therefore, the higher expression of the examined regulatory genes in the blood of RA patients may be indicative of articular cartilage degradation

Association of transforming growth factor (TGF) p1 T(861-20)C polymorphism with bone mineral density and TGFip gene expression in postmenopausal osteoporosis

Objective: to study the mechanism for the involvement of ТGFβ1 T(861-20)C in bone resorption in postmenopausal osteoporosis (OP). Material and methods. DNA from 158 postmenopausal women and patients with OP and from 89 healthy age-matched women was examined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis. Bone mineral density (BMD) was estimated by dual-energy X-ray absorptiometry. Standard biochemical protocols were used to detect alkaline phosphatase activity and calcium and phosphorus levels in serum. Total RNA was isolated from the peripheral blood of 32 patients with OP and 39 healthy donors and used for real-time PCR study. Results. No significant differences were found in the frequency of individual alleles and genotypes between the OP group and control donors. The minor T allele frequency was 0.27. There was a significant correlation of ТGFβ1 T(861-20)C polymorphism with low lumbar spine BMD (r=0.18; p=0.025) in Russian patients with OP. Age-adjusted (Z-score) BMD in CC genotype carriers turned to be significantly lower than that in CT and TT genotype carriers. This was accompanied by lower ТGFβ1 gene expression in the peripheral blood of CC genotype carriers (n=10) as compared to the combined group of carriers of two other genotypes (n=22) in the OP group (p=0.03). No changes in ТGFβ1 gene expression were seen in healthy women who were CC genotype carriers (n=18) as compared to the combined representatives of two other genotypes (n=21). Overall, the OP group exhibited significantly lower ТGFβ1 gene expression than the healthy controls (p=0.04). Conclusion. The association of ТGFβ1 (861-20)CC genotype with lower lumbar spine BMD in patients with OP is attended by decreased ТGFβ1 gene expression. Therefore, ТGFβ1 T(861-20)C polymorphism may be a predictor for the development of OP and the more severe form of the disease may be expected in (861-20)CC genotype carriers