Maria Luisa Carnazza

Characterization of apoptosis in articular cartilage derived from the knee joints of patients with osteoarthritis.

PurposeThe present study was conduced in order to analyse the molecular changes during the apoptotic cascade in knee articular cartilage of patients with OA.MethodArticular cartilage specimens were assessed by histology (Haematoxylin and Eosin), histochemistry (Masson’s Trichromic and Alcian Blue), immunohistochemistry through TRAIL, DR5 and Caspase-3, TUNEL and Hoechst staining in fresh isolated chondrocytes.ResultsHistology results demonstrated the structural alterations in the articular knee cartilage with OA, and histochemistry results demonstrated the presence of matrix calcification and a proteoglycans reduction. Immunohistochemistry staining showed that structural alterations, matrix calcification and a proteoglycans reduction coincided with an increase in apoptotic cells when compared to normal cartilage; however, this cellular mechanism of death was demonstrated by TUNEL and Hoechst 33258 staining in fresh isolated chondrocytes.ConclusionIn this study, we demonstrated an apoptosis activation by the extrinsic pathway in OA cartilage. The apoptosis-positive cells might be due to a protection mechanism after sublethal injury, in particular, represented by an increased survival of chondrocytes that are able to participate in the repair process.

The effects of physical activity on apoptosis and lubricin expression in articular cartilage in rats with glucocorticoid-induced osteoporosis.

Glucocorticoids are considered the most powerful anti-inflammatory and immunomodulating drugs. However, a number of side-effects are well documented in different diseases, including articular cartilage, where increases or decreases in the synthesis of hormone-dependent extracellular matrix components are seen. The objective of this study has been to test the effects of procedures or drugs affecting bone metabolism on articular cartilage in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity with treadmill and vibration platform training on articular cartilage. The animals were divided into 5 groups, and bone and cartilage evaluations were performed using whole-body scans and histomorphometric analysis. Lubricin and caspase-3 expression were evaluated by immunohistochemistry, Western blot analysis and biochemical analysis. These results confirm the beneficial effect of physical activity on the articular cartilage. The effects of drug therapy with glucocorticoids decrease the expression of lubricin and increase the expression of caspase-3 in the rats, while after physical activity the values return to normal compared to the control group. Our findings suggest that it might be possible that mechanical stimulation in the articular cartilage could induce the expression of lubricin, which is capable of inhibiting caspase-3 activity, preventing chondrocyte death. We can assume that the physiologic balance between lubricin and caspase-3 could maintain the integrity of cartilage. Therefore, in certain diseases such as osteoporosis, mechanical stimulation could be a possible therapeutic treatment. With our results we can propose the hypothesis that physical activity could also be used as a therapeutic treatment for cartilage disease such as osteoarthritis.

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.

PACAP and VIP prevent apoptosis in schwannoma cells

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.

Expression of bone morphogenetic protein-6 and transforming growth factor-β1 in the rat brain after a mild and reversible ischemic damage

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.

Acute injury affects lubricin expression in knee menisci: An immunohistochemical study

The aim of this study was to investigate for the first time lubricin expression in intact menisci and in menisci from patients with recent knee joint injury using histology, immunohistochemistry, Western blotting and gene expression analysis, to provide insights into pathological processes affecting meniscal tissue. Lubricin expression was studied in vivo in 20 patients (14 males and 6 females) with recent joint injury subjected to arthroscopic partial meniscectomy and in vitro in fibroblast-like cells from meniscus tissue to establish whether it is down-regulated following acute traumatic knee injury. The control group consisted of cadaver donors with normal menisci. Histology demonstrated a normal tissue without structural changes in control samples and structural alterations and clefts in injured menisci. Very strong lubricin immunohistochemical staining was observed in intact menisci; in contrast weak staining was seen in injured menisci. Western blot and mRNA expression analysis also demonstrated strong lubricin expression in control cells and a negligible amount of lubricin in injured fibroblast-like cells. Our data provide information concerning the immediate in vivo response to injury of human knee menisci by documenting early changes in the boundary-lubricating ability of synovial fluid and articular cartilage integrity. These findings may provide the biological basis for developing novel medical therapies to be applied before surgical treatment to preserve tissue function and prevent cartilage damage.

Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: An in vivo and in vitro study

Osteoarthritis (OA) of the knee is a major problem in our society. The development of new treatment options for OA is limited, because the pathophysiological mechanisms are not clearly understood, especially on the molecular level. Aquaporin 1 (AQP1) is a specific protein channels for water transport; it is expressed in articular chondrocytes, human synovitis, in chondrocytes of patients with rheumatoid arthritis or OA and in chondrocyte-like cells of human intervertebral disc. The aim of this study was to investigate the expression of AQP1, through immunohistochemistry, immunocytochemistry and Western blot, in experimentally induced OA knee menisci. AQP1 was studied in vivo in knee OA menisci from 36 rats that underwent medial or lateral meniscectomy, and in vitro on fibrochondrocytes derived from knee OA menisci rats. OA in rats was experimentally induced and tested by histomorphometric analysis. Histological results demonstrated structural alterations in OA menisci accompanied by a very strong AQP1 immunohistochemical and immunocytochemical staining. The Western blot analysis confirmed a strong expression of AQP1 in OA fibrochondrocytes cells. The results of the present research suggest that an activation of AQP1, induced by the OA process, may represent an endogenous mechanism, which can be used to control the tissue degeneration within OA articular joints.

β-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate β-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of β-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong β-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak β-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of β-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using β-defensin-4 as a relevant marker.

Ameliorative effect of PACAP and VIP against increased permeability in a model of outer blood retinal barrier dysfunction.

Breakdown of outer blood retinal barrier (BRB) due to the disruption of tight junctions (TJs) is one of the main factors accounting for diabetic macular edema (DME), a major complication of diabetic retinopathy. Previously it has been shown that PACAP and VIP are protective against several types of retinal injuries. However, their involvement in the maintenance of outer BRB function during DME remains uncovered. Here, using an in vitro model of DME, we explored the effects of both PACAP and VIP. Human retinal pigment epithelial cells (ARPE19) were cultured for 26 days either in normal glucose (5.5 mM, NG) or in high glucose (25 mM, HG). In addition, to mimic the inflammatory aspect of the diabetic milieu, cells were also treated with IL-1β (NG+IL-1β and HG+IL-1β). Effects of PACAP or VIP on cells permeability were evaluated by measuring both apical-to-basolateral movements of fluorescein isothyocyanate (FITC) dextran and transepithelial electrical resistance (TEER). Expression of TJ-related proteins was evaluated by immunoblot. Results demonstrated that NG+IL-1β and, to a greater extent, HG+IL-1β significantly increased FITC-dextran diffusion, paralleled by decreased TEER. PACAP or VIP reversed both of these effects. Furthermore, HG+IL-1β-induced reduction of claudin-1 and ZO-1 expression was reversed by PACAP and VIP. Occludin expression was not affected in any of the conditions tested. Altogether, these finding show that both peptides counteract HG+IL-1β-induced damage in ARPE19 cells, suggesting that they might be relevant to the maintenance of outer BRB function in DME.

Expression of β-defensin-4 in “an in vivo and ex vivo model” of human osteoarthritic knee meniscus

PurposeTo investigate, for the first time, the expression of β-defensins-4, by immunohistochemistry and western blotting, in OA meniscus versus control meniscus, thus providing new insights into the physiological processes of meniscus repairing.Methodβ-defensins-4 was studied in vivo, in knee osteoarthritic menisci obtained from 30 patients (20 men and 10 women) who underwent isolated arthroscopic partial medial or lateral meniscectomy, and in vitro on fibrochondrocyte cells from human OA knee menisci. The study was conducted using morphological, immunohistochemical, and Western blot analysis.ResultsThe histological results demonstrated structural alterations and cracks of OA menisci accompanied by a very strong β-defensin-4 immunohistochemical staining. The Western blot analysis confirmed also a strong expression of β-defensin-4 in OA fibrochondrocyte cells.ConclusionThe present study suggests an activation of β-defensin-4 induction, in human knee meniscus induced by the OA inflammatory process. It may represent an endogenous antibiotic defense mechanism accompanied by an intrinsic effort of tissue remodeling in OA articular joints. In conclusion, the present paper suggests the clinical relevance of β-defensin-4 in the prospective of future alternative medical treatment for OA.