COL10A1 is a novel factor in the development of choroidal neovascularization.

With the dramatic rise in the aging population, researching age-related macular degeneration (AMD), especially the severe form neovascular AMD (nAMD), has become more important than ever. In this study, we found that collagen type X was increased in retina-choroid tissue of mice with laser-induced choroidal neovascularization (CNV) based on immunohistofluorescence. RNA sequencing and bioinformatic analyses were performed to compare the retina-choroid tissue complex of the CNV mouse model to normal controls. Collagen type X alpha 1 chain (Col10a1) was among the most significantly upregulated genes, and the results were validated with an animal model at the mRNA and protein levels by quantitative real-time polymerase chain reaction (qPCR) and western blotting, respectively. COL10A1 was also upregulated in human retinal microvascular endothelial cells (HRMECs), human umbilical vein endothelial cells (HUVECs), RPE19 cells and RF/6A cells under hypoxic conditions. Next, in vitro and in vivo experiments were performed to study the effect of COL10A1 on neovascularization. siRNA knockdown of COL10A1 suppressed the proliferation and tube formation ability of HRMECs under hypoxic conditions. Snail family transcriptional repressor 1 (SNAIL1) and angiopoietin-2 (ANGPT2) were downregulated in COL10A1 knockdown HRMECs under hypoxic conditions and thus were potential downstream genes. Significant decreases in CNV leakage and CNV lesion area, as assessed by fundus fluorescein angiography (FFA) and immunofluorescence of choroidal flat mounts, respectively, were observed in a mouse model intravitreally injected with anti-collagen X monoclonal antibody (mAb) compared to the controls. In conclusion, COL10A1 promotes CNV formation and may represent a new candidate target for the treatment and diagnosis of nAMD and other neovascular diseases.

Effect of miR-26a-5p on gastric cancer cell proliferation, migration and invasion by targeting COL10A1.

OBJECTIVE: This study explored the effect of miR-26a-5p on cell proliferation, migration, and invasion in gastric cancer by targeting COL10A1. MATERIALS AND METHODS: First, differentially expressed genes were identified from microarray GSE103236 data of human gastric cancer. Then, qRT-PCR was carried out to detect the expression levels of COL10A1 and miR-26a-5p in gastric cancer cells and normal cases. The CCK-8 method was used to test cell proliferation. The colony formation assay was performed for the examination of the cell colony-forming ability, and transwell was applied for the detection of cell migration and invasion. Subsequently, the targeted relationship between miR-26a-5p and COL10A1 was identified by bioinformatics methods and further verified by Dual-Luciferase assay. The rescue experiment was finally conducted to validate the miR-26a-5p-dependent mechanism on cell proliferation, migration, and invasion via targeting COL10A1. RESULTS: COL10A1 was found to be highly expressed in gastric cancer cells, while miR-26a-5p was poorly expressed. Silencing COL10A1 inhibited cell proliferation, migration, and invasion in gastric cancer. Besides, miR-26a-5p could function on gastric cancer cells by reducing COL10A1. As well, the rescue experiment suggested that the down-regulation of COL10A1 could reverse the inhibitory effect of miR-26a-5p on gastric cancer cells. CONCLUSIONS: Collectively, miR-26a-5p can potentiate proliferation, migration, and invasion of gastric cancer cells by targeting COL10A1.

Halofuginone attenuates articular cartilage degeneration by inhibition of elevated TGF­ß1 signaling in articular cartilage in a rodent osteoarthritis model.

Osteoarthritis (OA) is the most common degenerative condition of the weight­bearing joints worldwide without effective medical therapy. In order to investigate whether administration of halofuginone (HF) may attenuate OA, the present study allocated 3­month­old male mice into Sham group, vehicle­treated anterior cruciate ligament transection (ACLT) group and HF­treated ACLT group. The present study determined that HF treatment reduced the expression of matrix metallopeptidase­13 and collagen X in articular cartilage. Additionally, it lowered the Osteoarthritis Research Society International­Modified Mankin score and prevented the loss of articular cartilage from Safranin O and Fast Green staining. HF reduced the progression of osteoarthritis by downregulating abnormally elevated TGF­ß1 activity in articular cartilage. Administration of HF may be a potential preventive therapy for OA.

Phenol red inhibits chondrogenic differentiation and affects osteogenic differentiation of human mesenchymal stem cells in vitro.

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.

Beta2-adrenergic receptors expressed on murine chondrocytes stimulate cellular growth and inhibit the expression of Indian hedgehog and collagen type X.

The sympathetic nervous system has been demonstrated to have a role in regulating bone remodeling through beta-adrenergic receptors (beta-AR) expressed on osteoblasts. Studies using beta(2)-adrenergic receptor agonists in vivo have also suggested an effect on endochondral bone development; however, it was not clear if this effect was mediated through osteoblasts or chondrocytes. To more thoroughly examine the role of beta-AR in chondrocytes we characterized the expression and signal transduction systems activated by beta-AR in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Using RT-PCR and immunohistochemistry we found that the chondrocytes expressed only beta(2)-AR. The receptors were coupled to stimulation of adenylyl cyclase, phosphorylation of the cyclic AMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2). Stimulation of ERK1/2 was transient and limited by the concomitant stimulation of the mitogen-activated protein kinase phosphatase (MKP-1). Isoproterenol stimulated the growth of chondrocytes as assessed by increased incorporation of [(3)H]-thymidine into the cells. The cellular expression of two markers of chondrocyte differentiation, Indian hedgehog, expressed in pre-hypertrophic cells and collagen type X, expressed in hypertrophic chondrocytes, were both significantly inhibited after incubation with isoproterenol. Collectively, these findings demonstrate regulation of chondrocytes through beta(2)-AR expressed on the cells that stimulate their growth and inhibit their differentiation, indicating that the sympathetic nervous system may be an important regulator of embryonic cartilage development.

Selective inhibition of type X collagen expression in human mesenchymal stem cell differentiation on polymer substrates surface-modified by glow discharge plasma.

Recent evidence indicates that a major drawback of current cartilage- and disc-tissue engineering is that human mesenchymal stem cells (MSCs) rapidly express type X collagen-a marker of chondrocyte hypertrophy associated with endochondral ossification. Some studies have attempted to use growth factors to inhibit type X collagen expression, but none to date has addressed the possible effect of the substratum on chondrocyte hypertrophy. Here, we sought to examine the growth and differentiation potential of human MSCs cultured on two polymer types, polypropylene and nylon-6, both of which have been surface-modified by glow discharge plasma treatment in ammonia gas. Cultures were performed for up to 14 days in Dulbecco's modified Eagle medium + 10% fetal bovine serum. Commercial polystyrene culture dishes were used as control. Reverse transcriptase-polymerase chain reaction was used to assess the expression of types I, II, and X collagens and aggrecan using gene-specific primers. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. Types I and X collagens, as well as aggrecan, were found to be constitutively expressed by human MSCs on polystyrene culture dishes. Whereas both untreated and treated nylon-6 partially inhibited type X collagen expression, treated polypropylene almost completely inhibited its expression. These results indicate that plasma-treated polypropylene or nylon-6 may be a suitable surface for inducing MSCs to a disc-like phenotype for tissue engineering of intervertebral discs in which hypertrophy is suppressed.