Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes

Background Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. Methods BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. Results In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. Conclusions Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Mechanical stimulation induced osteogenic differentiation of BMSCs through TWIST/E2A/p21 axis

The relationship between mechanical force and alveolar bone remodeling is an important issue in orthodontics because tooth movement is dependent on the response of bone tissue to the mechanical force induced by the appliances used. Mechanical cyclical stretch plays an essential role in the cell osteogenic differentiation involved in bone remodeling. However, the underlying mechanisms are unclear, particularly the molecular pathways regulated by mechanical stimulation. In the present study, we reported a dynamic change of p21 level in response to mechanical cyclical stretch, and shRNA-p21 in bone marrow mesenchymal stem cells (BMSCs) induced osteogenic differentiation. The mechanism was mediated through TWIST/E2A/p21 axis. These results supported the mechanical stimulation-induced osteogenic differentiation is negatively regulated by p21.

Bone remodeling induced by mechanical forces is regulated by miRNAs

The relationship between mechanical force and alveolar bone remodeling is an important issue in orthodontics because tooth movement is dependent on the response of bone tissue to the mechanical force induced by the appliances used. Mechanical cyclical stretch (MCS), fluid shear stress (FSS), compression, and microgravity play different roles in the cell differentiation and proliferation involved in bone remodeling. However, the underlying mechanisms are unclear, particularly the molecular pathways regulated by non-coding RNAs (ncRNAs) that play essential roles in bone remodeling. Amongst the various ncRNAs, miRNAs act as post-transcriptional regulators that inhibit the expression of their target genes. miRNAs are considered key regulators of many biologic processes including bone remodeling. Here, we review the role of miRNAs in mechanical force-induced bone metabolism.

Abstract 054: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation Into Immunogenic Dendritic Cells

We have shown that dendritic cells (DCs) from hypertensive mice and monocytes in humans accumulate highly reactive isolevuloglandins (isoLGs) or isoketals that adduct to protein lysines and promote T cell activation, specifically. Monocytes that traverse the endothelium have three different fates: reemerge into circulation as an activated monocyte; differentiate into macrophages or into monocyte-derived DCs. We hypothesized that human endothelial cells exposed to hypertensive mechanical stretch will promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive uniaxial cyclical stretch (10%) for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and the p22 phox subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also accumulated isoLG-modified proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. We also co-cultured these monocytes with T cells from the same patient and examined proliferation of the latter using CFSE. Monocytes co-cultured with 10% stretched HAECs induced a 1,500-fold increase in CD4 + T cell proliferation and a 1,300-fold increase in CD8 + T cells proliferation. In addition, monocytes co-cultured with 10% stretched HAECs had a 2-fold increase in phosphorylated STAT3 expression compared to 5% stretch cultures. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, reduced their differentiation into DCs and prevented both CD4 + and CD8 + T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into activated DCs potentially via the STAT3 pathway.

Abstract 354: Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation into Immunogenic Dendritic Cells

Fatty acid oxidation leads to formation of highly reactive isoketals that adduct to protein lysines. We have shown that dendritic cells (DCs) from hypertensive mice accumulate isoketal-adducted proteins and promote T cell activation. In hypertension, the endothelium is activated to produce reactive oxygen species and to express adhesion molecules and chemokines that attract inflammatory cells. Human monocytes that traverse the endothelium differentiate into monocyte-derived DCs upon exposure to a pro-inflammatory state. We hypothesized that human endothelial cells exposed to mechanical stretch promote conversion of human monocytes into immunogenic DCs. We co-cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive cyclical stretch (10%) using the Flexcell ® Tension System for 48 hours. We found that co-culture of monocytes with HAECs exposed to 10% mechanical stretch markedly increased monocyte mRNA expression of the Th17 polarizing cytokines IL-6, I-23A and IL-1β and monocyte chemokine CCL4 and the p22 phox subunit of the NADPH oxidase compared to 5% stretch. HAECs exposed to 10% stretch promoted monocyte in culture to differentiate into DCs, as evidenced by the surface expression of DC-SIGN, CD83 and the co-stimulatory marker CD86. These monocytes also had an accumulation of isoketal-adducted proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. Monocytes co-cultured with 10%-stretched HAECs induced a 1,500-fold increase in CD4 + T cell proliferation and a 1,300-fold increase in CD8 + T cells proliferation as monitored by CFSE compared to 5% stretch controls. Conversely, monocytes-HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor, stattic, prevented both CD4 + and CD8 + T cell proliferation. These data show that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote their differentiation into immunogenic DCs potentially via the JAK/STAT3 pathway. These findings give insight into a new mechanism of lymphocyte activation in the vascular endothelium during hypertension.

Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β2-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF.