Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Systems Nutrigenomics Reveals Brain Gene Networks Linking Metabolic and Brain Disorders.

Nutrition plays a significant role in the increasing prevalence of metabolic and brain disorders. Here we employ systems nutrigenomics to scrutinize the genomic bases of nutrient-host interaction underlying disease predisposition or therapeutic potential. We conducted transcriptome and epigenome sequencing of hypothalamus (metabolic control) and hippocampus (cognitive processing) from a rodent model of fructose consumption, and identified significant reprogramming of DNA methylation, transcript abundance, alternative splicing, and gene networks governing cell metabolism, cell communication, inflammation, and neuronal signaling. These signals converged with genetic causal risks of metabolic, neurological, and psychiatric disorders revealed in humans. Gene network modeling uncovered the extracellular matrix genes Bgn and Fmod as main orchestrators of the effects of fructose, as validated using two knockout mouse models. We further demonstrate that an omega-3 fatty acid, DHA, reverses the genomic and network perturbations elicited by fructose, providing molecular support for nutritional interventions to counteract diet-induced metabolic and brain disorders. Our integrative approach complementing rodent and human studies supports the applicability of nutrigenomics principles to predict disease susceptibility and to guide personalized medicine.

Effect of interleukin-1beta and dehydroepiandrosterone on the expression of lumican and fibromodulin in fibroblast-like synovial cells of the human temporomandibular joint.

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 µM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in synovial tissue in OA and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (µ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint.

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7 kb long cDNA contains an open reading frame of 1131 nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.