Up‐regulation of the chemo‐attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments

Abstract

To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemoattractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of proinflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture.

Platelet-rich plasma protects rat chondrocytes from interleukin-1β-induced apoptosis

“…IL-1β can be secreted by chondrocytes, mononuclear cells, osteoblasts and synovial cells. Previous studies have reported that IL-1β may stimulate apoptosis of chondrocytes and tendon cells (22,23) by upregulating the expression of Bax and caspase-3 (24), and suppressing the expression of Bcl-2 and PARP (25). Several studies (21,26,27) have demonstrated that IL-1β promotes catabolic activity of chondrocytes through two major mechanisms: IL-1β suppresses the synthesis of major extracellular matrix proteins, such as aggrecan and COL2; and IL-1β stimulates the release of several proteolytic enzymes, including MMPs such as MMP1, MMP3 and MMP13.…”

The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors

“…Immunofluorescence analysis was carried out to assess the expression of MAP LC3 in bone biopsies and LC3 and Apaf‐1 in the cell cultures. MLO‐Y4 cells, cultured in different conditions in multiwell chamber slides, and bone biopsy samples were treated and observed as previously described . Samples were incubated with the following antibodies: rabbit anti‐MAP LC3 (Code n. sc‐134226), mouse anti‐Apaf‐1 (Code n. sc‐135836, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); goat anti‐rabbit Cy3 (Code n. C2306) and sheep anti‐mouse FITC (Code n. F2883, Sigma‐Aldrich).…”

“…The confocal imaging was performed on a Leica TCS SP2 AOBS confocal laser scanning microscope (Leica S.p.A.). To quantify MAP LC3‐positive cells, 5 to 6 slides were examined at 40× magnification for each condition, in order to count almost 1000 cells as previously described . All results are expressed as the mean % ± SD.…”

“…MLO-Y4 cells, cultured in different conditions in multiwell chamber slides, and bone biopsy samples were treated and observed as previously described. (38) Samples were incubated with the following antibodies: rabbit anti-MAP LC3 (Code n. sc-134226), mouse anti-Apaf-1 (Code n. sc-135836, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); goat anti-rabbit Cy3 (Code n. C2306) and sheep anti-mouse FITC (Code n. F2883, Sigma-Aldrich). After being washed, samples were counterstained with 1 g/mL DAPI (4',6-diamidin-2-fenilindolo) and then mounted with an anti-fade medium (0.21M DABCO (1,4- aminomethane, pH 8.0).…”

“…To quantify MAP LC3-positive cells, 5 to 6 slides were examined at 40Â magnification for each condition, in order to count almost 1000 cells as previously described. (38) All results are expressed as the mean % AESD. The mean values of the groups were compared using ANOVA, followed by Bonferroni and Student-Newman-Keuls tests.…”