Human and murine lymphocytes express dopamine (DA) D2-like receptors including DRD2, DRD3, and DRD4. However, their roles in rheumatoid arthritis (RA) are less clear. Here we showed that lymphocyte DRD2 activation alleviates both imbalance of T-helper (Th)17/T-regulatory (Treg) cells and inflamed symptoms in a mouse arthritis model of RA. Collagen-induced arthritis (CIA) was prepared by intradermal injection of chicken collagen type II (CII) in tail base of DBA/1 mice or Drd2 (-/-) C57BL/6 mice. D2-like receptor agonist quinpirole downregulated expression of proinflammatory Th17-related cytokines interleukin- (IL-) 17 and IL-22 but further upregulated expression of anti-inflammatory Treg-related cytokines transforming growth factor- (TGF-) β and IL-10 in lymphocytes in vitro and in ankle joints in vivo in CIA mice. Quinpirole intraperitoneal administration reduced both clinical arthritis score and serum anti-CII IgG level in CIA mice. However, Drd2 (-/-) CIA mice manifested more severe limb inflammation and higher serum anti-CII IgG level and further upregulated IL-17 and IL-22 expression and downregulated TGF-β and IL-10 expression than wild-type CIA mice. In contrast, Drd1 (-/-) CIA mice did not alter limb inflammation or anti-CII IgG level compared with wild-type CIA mice. These results suggest that DRD2 activation is involved in alleviation of CIA symptoms by amelioration of Th17/Treg imbalance.

This study aims to determine the effect of age-related cartilage turnover on the serum C-telopeptide of type II collagen (CTX-II) and osteocalcin (OC) levels in growing rabbits with and without surgically induced osteoarthritis. Twenty-four New Zealand male 3-month-old rabbits were randomized into three operated groups (n=6 per group, with surgically induced osteroarthritis in the right knee; after blood sampling, the knees were harvested following euthanization at 2, 3, and 6 months after surgery) and a control group (n=6, blood samples were obtained monthly between 3 and 15 months). Histomorphologically, the medial femoral condyles, particularly the central parts, harbored the most severe osteoarthritic changes among the operated rabbits. The serum levels of CTX-II and OC decreased in the controls from 3 to 11 months and then remained stable. No significant differences in the serum CTX-II and OC levels between the osteoarthritic rabbits and controls were observed. The osteoarthritic-to-normal ratios (ONRs, the ratios of serum CTX-II or OC levels in osteoarthritic rabbits to those of the controls at same ages) enabled an overall assessment of osteoarthritis and age-related cartilage turnover. Elevated CTX-II ONRs were observed in rabbits with mild to advanced osteoarthritis. However, the OC ONRs were unhelpful in assessing osteoarthritic growing rabbits.

Adult articular cartilage synthesises very little type II collagen in comparison to young cartilage. The age-related difference in collagen II synthesis is poorly understood. This is the first systematic investigation of age-related differences in extracellular matrix synthesis in fresh articular cartilage and following isolation of chondrocytes. A histological comparison of 3-year-old skeletally mature and 6-month-old juvenile porcine cartilage was made. Differences in collagen II, aggrecan, and Sox5, 6, and 9 mRNA and protein expression and mRNA stability were measured. Adult cartilage was found to be thinner than juvenile cartilage but with similar chondrocyte density. Procollagen α1(II) and Sox9 mRNA levels were 10-fold and 3-fold reduced in adult cartilage. Sox9 protein was halved and collagen II protein synthesis was almost undetectable and calculated to be at least 30-fold reduced. Aggrecan expression did not differ. Isolation of chondrocytes caused a drop in procollagen α1(II) and Sox9 mRNA in both adult and juvenile cells along with a marked reduction in Sox9 mRNA stability. Interestingly, juvenile chondrocytes continued to synthesise collagen II protein with mRNA levels similar to those seen in adult articular cartilage. Age-related differences in collagen II protein synthesis are due to both transcriptional and posttranscription regulation. A better understanding of these regulatory mechanisms would be an important step in improving current cartilage regeneration techniques.

Rheumatoid arthritis (RA) is a serious autoimmune disease caused by chronic inflammation of connective tissues. The basic principle of RA treatment is aimed to reduce joint inflammation. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in different mouse inflammatory diseases. In this study, we investigated the prophylactic effect of PG on the progression of RA using an experimental mouse model, collagen-induced arthritis (CIA). Clinical and histological severity of CIA was attenuated by daily oral administration of PG. In the joints of PG-administered mice, infiltration of macrophages and neutrophils and also osteoclast accumulation were limited. In comparison to nonadministered mice, anti-collagen antibodies in the sera of PG-administered mice did not alter. On the other hand, local expression of interleukin-17A (IL-17A), IL-6, IL-1 β, interferon- γ (IFN- γ), C-C chemokine ligand 2 (CCL2), C-X-C chemokine ligand 1 (CXCL1), and CXCL2 in the joints of PG-administered mice decreased. Moreover, in the response of type II collagen- (CII-) restimulation ex vivo, IL-17A and IFN- γ production by splenocytes from PG-administered mice was less than that of control mice. These data suggested that daily ingested PG attenuated CIA pathogenesis by modulating immune response of splenocytes to CII stimulation and local production inflammatory cytokines and chemokines in the joints.

Treatment of tracheal stenosis is occasionally performed in combination with wound healing modulators to manipulate new extracellular matrix (ECM) formation and prevent fibrosis. Hyaluronic acid (HA) and collagen-polyvinylpyrrolidone (collagen-PVP) decrease fibrosis in experimental tracheal healing. However, they have not been used clinically as their effect on ECM components, which modify tracheal scarring, has not been described. Objective. To evaluate the effect of the application of HA, collagen-PVP, a mixture of HA and collagen-PVP (HA+collagen-PVP), and mitomycin C on the expression of decorin, matrix metalloproteinase 1 (MMP1), and MMP9, as well as the type of collagen and deposits formed in the scar after resection and end-to-end anastomosis (REEA) of the cervical trachea using an experimental model. Materials and Methods. Thirty dogs underwent REEA of the cervical trachea and were treated with different wound healing modulators: group I (n = 6), control; group II (n = 6), HA; group III (n = 6), collagen-PVP; group IV (n = 6), HA+collagen-PVP; and group V (n = 6), mitomycin C. The dogs were evaluated clinically and endoscopically for 4 weeks. Subsequently, macroscopic and microscopic changes, expression of ECM proteins, and collagen deposition in tracheal scars were analysed. Results. Groups II, III, and IV showed reduced endoscopic, macroscopic, and microscopic inflammation, improved neovascularization, high decorin expression (p < 0.01, analysis of variance (ANOVA)), and moderate expression of MMP1 (p < 0.003, ANOVA) and type I and III collagen (p < 0.05, Kruskal-Wallis). Groups IV and V developed fewer collagen deposits (p < 0.001, ANOVA). Conclusion. Treatment with HA and collagen-PVP improved post-REEA healing by increasing neovascularization, stimulating the expression of decorin, and regulating the expression of MMP1, as well as type I and III collagen and their deposition.

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Diagnosis of intervertebral disc degeneration (IVDD) is challenging at the early stage. The cartilage oligomeric matrix protein (COMP) and extracellular matrix degradation products of C-telopeptide of type II collagen (CTX-II) serve as markers for the serological diagnosis of IVDD. Oxidative stress might cause IVDD and matrix degeneration.

Pseudoachondroplasia (PSACH) is an autosomal dominant osteochondrodysplasia caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Accurate clinical diagnosis of PSACH is sometimes difficult. Here, we identified a novel COMP mutation (c.1675G>A, p.Glu559Lys) in a Chinese PSACH family. We detected the plasma levels of COMP and type II collagen (CTX-II) in the four affected individuals. The results showed the levels of plasma COMP significantly decreased and plasma CTX-II significantly increased in the three PSACH patients with COMP mutation. However, both plasma levels of COMP and CTX-II were not to have found significant difference between the presymptomatic carrier and the age-matched subjects. In vitro analysis and immunofluorescence displayed wild type COMP homogenously expressed in cytoplasm, but mutant proteins were irregularly accumulated inside the HEK-293 cells. Western blot revealed that the quantity of the mutant COMP was more compared to wild type COMP in cells after transfection for 12 hours and 24 hours. Subsequently, 3D structural analysis showed three changes have taken place in secondary structure of the mutant COMP. In conclusion, the novel mutation of COMP may result in intracellular accumulation of the mutant protein. Decreased plasma COMP and increased plasma CTX-II may potentially serve as diagnostic markers of PSACH but may not be applicable in the presymptomatic carrier.

We investigated the effects of Wenxin Keli (WXKL) on the Calcium/Calmodulin dependent kinase II (CaMK II) signal transduction pathway with transverse aortic constriction (TAC) rats. Echocardiographic measurements were obtained 3 and 9 weeks after the surgery. Meanwhile, the action potentials (APDs) were recorded using the whole-cell patch clamp technique, and western blotting was used to assess components of the CaMK II signal transduction pathway. At both 3 and 9 weeks after treatment, the fractional shortening (FS%) increased in the WXKL group compared with the TAC group. The APD90 of the TAC group was longer than that of the Sham group and was markedly shortened by WXKL treatment. Western blotting results showed that the protein expressions of CaMK II, phospholamban (PLB), and ryanodine receptor 2 (RYR2) were not statistically significant among the different groups at both treatment time points. However, WXKL treatment decreased the protein level and phosphorylation of CaMK II (Thr-286) and increased the protein level and phosphorylation of PLB (Thr-17) and the phosphorylation of RYR2 (Ser-2814). WXKL also decreased the accumulation of type III collagen fibers. In conclusion, WXKL may improve cardiac function and inhibit the arrhythmia by regulating the CaMK II signal transduction pathway.

The knee osteoarthritis is a common joint disease that causes pain and inconvenience. Clinically, patients with knee osteoarthritis often have response points on the gastrocnemius. Gastrocnemius plays an essential role in stabilizing joints and changing gait and pace, which also has a close relationship with the knee joint. The objective of this study is to determine changes in the tibiofemoral joint after medial and lateral gastrocnemius injury. Rabbits were divided into a medial gastrocnemius injury group, a lateral gastrocnemius injury group, and a control group with two intervals: 6 and 8 weeks after modeling of the semisevered gastrocnemius. The gastrocnemius was weighed and sectioned for histology. The joint space and subchondral bone were observed using X-ray and microcomputed tomography. The cartilage was observed histologically using Safranin O fast green and Masson and immunohistochemically using antibodies to collagen type II, matrix metalloproteinase 13, and integrin beta1. Results showed muscle fiber atrophy, and fibrotic changes occurred after gastrocnemius semidissociation. After gastrocnemius injury, the femoral condyle of the tibiofemoral joint produced abnormal sclerosis and bone degeneration. The pathological changes of cartilage included disordered or reduced cell alignment, cartilage matrix loss, and collagen loss due to decreased collagen type II and increased matrix metalloproteinase 13 activity. The increase of integrin beta1 in the injured group may be related to mechanical conduction process. The results suggest that gastrocnemius injury is an essential factor in tibiofemoral arthritis.

Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA- (miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype.

The pathogenesis of posttraumatic osteoarthritis (PTOA) remains unrevealed. We speculate that cartilage crack caused by joint trauma will induce local abnormal tensile stress, leading to change in extracellular matrix (ECM) expression of chondrocytes, cartilage degeneration, and initiation of osteoarthritis. Finite element model was used to examine whether the local tensile stress could be produced around the crack. Cell experiments were conducted to test the effect of tensile strain on chondrocyte ECM expression. Animal tests in rabbits were carried out to examine the change around the cartilage crack. The results indicated that the local tensile stress was generated around the crack and varied with the crack angles. The maximum principal tensile stress was 0.59 MPa around the 45° crack, and no tensile stress was found at 90°. 10% tensile strain could significantly promote type I collagen mRNA expression and inhibit type II collagen and aggrecan (the proteoglycan core protein) mRNA expression. Type I collagen was detected around the 45° crack region in the cartilage with no change in type II collagen and proteoglycan. We conclude that the local tensile stress produced around the cartilage crack can cause the change in cartilage matrix expression which might lead to cartilage degeneration and initiation of osteoarthritis. This study provides biomechanical-based insight into the pathogenesis of PTOA and potentially new intervention in prevention and treatment of PTOA.

The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0) and Passage 1 (P1) cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

Alport syndrome (AS) is a monogenic disease of the basement membrane (BM), resulting in progressive renal failure due to glomerulonephropathy, variable sensorineural hearing loss, and ocular anomalies. It is caused by mutations in the collagen type IV alpha-3 gene (COL4A3), the collagen type IV alpha-4 gene (COL4A4), and the collagen type IV alpha-5 gene (COL4A5), which encodes type IV collagen α3, α4, and α5 chains, respectively. To explore the disease-related gene in a four-generation Chinese Han pedigree of AS, exome sequencing was conducted on the proband, and a novel deletion mutation c.499delC (p.Pro167Glnfs*36) in the COL4A5 gene was identified. This mutation, absent in 1,000 genomes project, HapMap, dbSNP132, YH1 databases, and 100 normal controls, cosegregated with patients in the family. Neither sensorineural hearing loss nor typical COL4A5-related ocular abnormalities (dot-and-fleck retinopathy, anterior lenticonus, and the rare posterior polymorphous corneal dystrophy) were present in patients of this family. The phenotypes of patients in this AS family were characterized by early onset-age and rapidly developing into end-stage renal disease (ESRD). Our discovery broadens the mutation spectrum in the COL4A5 gene associated with AS, which may also shed new light on genetic counseling for AS.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

The influences of angiotensinase C on ethanol-induced left ventricular (LV) systolic function were assessed in spontaneously hypertensive rats (SHRs). SHRs were fed by a liquid diet with or without ethanol for 49 days. The normotensive Wistar Kyoto rats (WKY) were fed by the liquid diet without ethanol and used as control. We evaluated LV systolic function, angiotensinase C mRNA and protein expressions, activation of the renin-angiotensin system (RAS), and the gene expressions of LV collagen (Col) III a1 and matrix metalloproteinases- (MMP-) 9. Compared to the WKY, LV systolic dysfunction (expressed by decreased fractional shortening and ejection fraction) was observed in the SHRs before ethanol treatment and further deteriorated by ethanol treatment. In the ethanol-treated SHRs, the following were observed: downregulations of angiotensinase C mRNA and protein, increased RAS activity with low collagen production as evidenced by angiotensin II and angiotensin type 1 receptor (AT1R) protein upregulation, AT1aR mRNA downregulation, and an MMP-9 mRNA expression upregulation trend with the downregulation of Col III a1 mRNA expression in LV. We conclude that chronic ethanol regimen is sufficient to promote the enhanced RAS activity-induced decrease in the production of cardiac collagen via downregulated angiotensinase C, leading to the further deterioration of LV systolic dysfunction in SHRs.

Remodelling of the peripheral lung tissue and fibrotic foci are the main pathologies of idiopathic pulmonary fibrosis (IPF), a disease that is difficult to treat. TGF-β activation of peripheral lung fibroblasts is indicated as the major cause of tissue remodelling in IPF and is resulting in fibroblast hyperplasia and deposition of extracellular matrix. Soluble guanylate cyclase (sGC) stimulators combined with cyclic AMP (cAMP) activators have been reported to reduce proliferation and matrix deposition in other conditions than IPF. Therefore, this drug combination may present a novel therapeutic concept for IPF. This study investigated the effect of BAY 41-2272 and forskolin on remodelling parameters in primary human lung fibroblasts. The study determined TGF-β induced proliferation by direct cell counts after 3 days; and deposition of collagen type-I, type III, and fibronectin. BAY 41-2272 significantly reduced TGF-β induced fibroblast proliferation, but did not reduce viability. This inhibitory effect was further supported by forskolin. Both BAY 41-2272 and forskolin alone reduced TGF-β induced collagen and fibronectin de novo synthesis as well as deposition. This effect was significantly stronger when the two compounds were combined. Furthermore, the TGF-β induced expression of fibrilar α-smooth muscle actin was reduced by BAY 41-2272 and this effect was strengthened by forskolin. In addition, BAY 41-2272 and forskolin reduced TGF-β induced β-catenin. All effects of BAY 41-2272 were concentration dependent. The findings suggest that BAY 41-2272 in combination with cAMP stimulation may present a novel therapeutic strategy to reduce tissue remodelling in IPF.

Repetitive brief ischemia and reperfusion (I/R) is associated with ventricular dysfunction in pathogenesis of murine ischemic cardiomyopathy and human hibernating myocardium. We investigated the role of matricellular protein osteopontin-1 (OPN) in murine model of repetitive I/R. One 15-min LAD-occlusion followed by reperfusion was performed daily over 3, 5, and 7 consecutive days in C57/Bl6 wildtype- (WT-) and OPN(-/-)-mice (n = 8/group). After echocardiography hearts were processed for histological and mRNA-studies. Cardiac fibroblasts were isolated, cultured, and stimulated with TGF- β 1. WT-mice showed an early, strong, and cardiomyocyte-specific osteopontin-expression leading to interstitial macrophage infiltration and consecutive fibrosis after 7 days I/R in absence of myocardial infarction. In contrast, OPN(-/-)-mice showed small, nontransmural infarctions after 3 days I/R associated with significantly worse ventricular dysfunction. OPN(-/-)-mice had different expression of myocardial contractile elements and antioxidative mediators and a lower expression of chemokines during I/R. OPN(-/-)-mice showed predominant collagen deposition in macrophage-rich small infarctions. We found lower induction of tenascin-C, MMP-9, MMP-12, and TIMP-1, whereas MMP-13-expression was higher in OPN(-/-)-mice. Cultured OPN(-/-)-myofibroblasts confirmed these findings. In conclusion, osteopontin seems to modulate expression of contractile elements, antioxidative mediators, and inflammatory response and subsequently remodel in order to protect cardiomyocytes in murine ischemic cardiomyopathy.

To investigate the effect of small interfering RNA targeting mechanosensitive ion channel protein Piezo1 (Piezo1-siRNA) on abnormal chondrocyte proliferation exposed to mechanical stretch.

We evaluated the effects of mechanical stimulation on the osteogenic differentiation of human intraoral mesenchymal stem and progenitor cells (MSPCs) using the Flexcell FX5K Tension System that mediated cyclic tensile stretch on the cells. MSPCs were isolated from human mandibular retromolar bones and characterized using flow cytometry. The positive expression of CD73, CD90, and CD105 and negativity for CD14, CD19, CD34, CD45, and HLA-DR confirmed the MSPC phenotype. Mean MSPC doubling time was 30.4 ± 2.1 hrs. The percentage of lactate dehydrogenase (LDH) release showed no significant difference between the mechanically stimulated groups and the unstimulated controls. Reverse transcription quantitative real-time PCR revealed that 10% continuous cyclic strain (0.5 Hz) for 7 and 14 days induced a significant increase in the mRNA expression of the osteogenesis-specific markers type-I collagen (Col1A1), osteonectin (SPARC), bone morphogenetic protein 2 (BMP2), osteopontin (SPP1), and osteocalcin (BGLAP) in osteogenic differentiated MSPCs. Furthermore, mechanically stimulated groups produced significantly higher amounts of calcium deposited into the cultures and alkaline phosphatase (ALP). These results will contribute to a better understanding of strain-induced bone remodelling and will form the basis for the correct choice of applied force in oral and maxillofacial surgery.