Objective. Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor-inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA. Methods. Expression of Cyr61 in synovial tissue (ST) and in FLS was

Chitosan has been shown to be a promising scaffold for various applications in tissue engineering. In this study, a chitosan-gelatin complex was fabricated as a scaffold by a freezing and lyophilizing technique. Chitosan's structure and characteristics are similar to those of glycosaminoglycan (GAG) and its analogs, and possesses various biological activities, whereas gelatin can serve as a substrate for cell adhesion, differentiation, and proliferation. With the use of autologous chondrocytes isolated from pig's auricular cartilage and seeded onto the chitosan-gelatin scaffold, elastic cartilages have been successfully engineered at the porcine abdomen subcutaneous tissue. After 16 weeks of implantation, the engineered elastic cartilages have acquired not only normal histological and biochemical, but also mechanical properties. The tissue sections of the engineered elastic cartilages showed that the chondrocytes were enclosed in the lacuna, similar to that of native cartilage. The presence of elastic fibers in the engineered cartilages was also demonstrated by Vehoeff's staining, and immunohistochemical staining confirmed the presence of type II collagen in the engineered cartilages. Quantitatively, the GAG in the engineered cartilages reached 90% of the concentration in native auricular cartilage. Furthermore, biomechanical analysis demonstrated that the extrinsic stiffness of the engineered cartilages reached 85% of the level in native auricular cartilage when it was harvested at 16 weeks. Thus, this study demonstrated that the chitosan-gelatin complex may serve as a suitable scaffold for cartilage tissue engineering.

Context Uterine decidualization is critical to embryonic implantation and sustained pregnancy. Objective To evaluate the role of gap junction intercellular communications and connexin (Cx) proteins in the morphological and biochemical differentiation of decidualized human endometrial stromal cell (ESC) cultures. Design Translational cell biological study. Setting Academic medical center. Patients Endometrial tissue was provided by five healthy reproductive age women on no hormonal medication, undergoing laparoscopy in the early proliferative phase of the menstrual cycle. Interventions Endometrial biopsy under general anesthesia, establishment and decidualization of ESC with 10 nM 17β-estradiol, 100 nM progesterone and 0.5 mM dibutyryl cAMP (E/P/c), and manipulation of gap junctions in vitro via a combination of pharmacological or transgenic approaches. Main Outcome Measures Decidualized ESC evaluated morphologically for epithelioid transformation, gap junctions by dye diffusion and Cx43, prolactin, VEGF and IL-6 expression by RT-PCR, Western and ELISA methods. Results Cx43 accumulation and functional gap junctions between decidualized ESC increase concomitantly with morphological differentiation following E/P/c treatment. Disruption of gap junctions using pharmacological inhibitors or Cx43 shRNA prevents morphological differentiation and inhibits prolactin and VEGF secretion. By contrast, IL-6 secretion from decidualized ESC is augmented by both approaches. Conclusions The findings suggest that decidualized ESC function as a coordinated secretory organ to regulate embryonic implantation via intercellular cooperation mediated by gap junctions. When adjacent cells can communicate through these junctions, decidual prolactin and VEGF secretion appears to be optimized for vascular development of the placental bed. Conversely, when intercellular communications are disrupted, angiogenesis is impaired and an inflammatory state is induced.

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Previous studies revealed that gap junction intercellular communication (GJIC) among uterine stromal cells plays critical roles in modulating decidualization, neovasularization, and embryo implantation. Connexin (Cx) proteins are the major component of gap junctions and Cx43 is the most widely expressed connexin in endometrium. Phosphorylation of Cx43 was found to impair gap junction communication in this tissue. Using primary human endometrial stromal cells (ESCs) and a stable high telomerase-expressing ESC transfectant (T-HESC), we found that retinoic acid (RA) altered the phosphorylation status of Cx43 protein such that there was a decrease in the phosphorylated (P1 and P2) species accompanied by an increase in the non-phosphorylated (P0) form. This process is dependent on protein phosphatase 2A (PP2A) activity since selective PP2A inhibitors prevented the ability of RA to dephosphorylate Cx43. Although RA had no effect on total PP2A expression or activity, it significantly increased the intracellular association of Cx43 and PP2A. Inhibition of transcription and protein synthesis by actinomycin D and cycloheximide, respectively, had no effect on the RA-induced changes in the Cx43 phosphorylation pattern. Furthermore, BMS493, a potent antagonist of the classical RA-mediated transcriptional pathway, did not inhibit RA-induced Cx43 dephosphorylation. Our data indicate that RA stimulates physical association of PP2A with Cx43, resulting in the dephosphorylation of Cx43 and, as a consequence, up-regulation of GJIC in ESCs. This process is independent of new mRNA and protein synthesis and suggests a novel mechanism by which aberrant retinoid metabolism can explain certain reproductive disorders manifested by dysfunctional endometrial cell GJIC.

Vascular endothelial growth factor (VEGF) and endometrial angiogenesis play a critical role in successful embryonic implantation. Despite many studies of the effects of estrogen and progesterone on VEGF expression, its focal regulation at the site of implantation is unknown. Retinoic acid (RA) has been reported to regulate VEGF in a variety of cell types. Because localized RA synthesis occurs within the periimplantation endometrium, we tested the possibility that RA regulates VEGF production in endometrial stromal cells. Using primary and telomerase-immortalized human endometrial stromal cells, we determined that RA alone did not alter constitutive levels of VEGF production, but markedly amplified secretion when the cells were cotreated with activators of VEGF gene transcription (12-O-tetradecanoyl phorbol-13-acetate, TPA; TGF-beta; and IL-1beta). Whereas TPA or TGF-beta alone stimulated VEGF promoter activity and up-regulated mRNA levels, significant protein secretion was detected only after RA was added to the culture systems. Analysis of retinoids in secretory phase endometrial biopsies indicated that endogenous RA accumulated at concentrations sufficient to induce VEGF secretion. Polyribosome profile analysis showed that the addition of RA to transcriptional activators of VEGF shifted the translational suppressed VEGF mRNA transcripts into larger polyribosome complexes engaged in active translation. Although the precise mechanism(s) of the RA effect remains to be defined, it appears to be mediated by reactive oxygen species; the antioxidant N-acetylcysteine inhibited RA+TPA-stimulated secretion of VEGF by more than 80%. Together, our results demonstrate that in human endometrial stromal cells, RA can combine with transcriptional activators of VEGF to augment VEGF secretion through a translational mechanism of action mediated by reactive oxygen species. These findings suggest a link between the spatiotemporal changes of retinoid synthesis in the periimplantation stroma and the capacity to quickly up-regulate focal VEGF secretion needed to induce early angiogenic events of pregnancy.

Prognosis for patients with early stage kidney cancer has improved, but the treatment options for patients with locally advanced disease and metastasis remain few. Understanding the molecular mechanisms that regulate invasion and metastasis is critical for developing successful therapies to treat these patients. Proinflammatory prostaglandin E 2 plays an important role in cancer initiation and progression via activation of cognate EP receptors that belong to the superfamily of G proteincoupled receptors. Here we report that prostaglandin E 2 promotes renal cancer cell invasion through a signal transduction pathway that encompasses EP4 and small GTPase Rap. Inactivation of Rap signaling with Rap1GAP, like inhibition of EP4 signaling with ligand antagonist or knockdown with shRNA, reduces the kidney cancer cell invasion. Human kidney cells evidence increased EP4 and decreased Rap1GAP expression levels in the malignant compared with benign samples. These results support the idea that targeted inhibition of EP4 signaling and restoration of Rap1GAP expression constitute a new strategy to control kidney cancer progression.

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