Decrease In Collagen Expression Research Articles

Normal aging in human lumbar discs: An ultrastructural comparison.

The normal aging of the extracellular matrix and collagen content of the human lumbar intervertebral disc (IVD) remains relatively unknown despite vast amounts of basic science research, partly because of the use of inadequate surrogates for a truly normal, human IVD. Our objective in this study was to describe and compare the morphology and ultrastructure of lumbar IVDs in 2 groups of young (G1—<35 years) and elderly (G2—>65 years). Thirty L4-5 and L5-S1 discs per group were obtained during autopsies of presumably-asymptomatic individuals and analyzed with magnetic resonance imaging (MRI), a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. As expected, a mild to moderate degree of degeneration was present in G1 discs and significantly more advanced in G2. The extracellular matrix of G2 discs was significantly more compact with an increase of cartilaginous features such as large chondrocyte clusters. Elastic fibers were abundant in G1 specimens and their presence correlated more with age than with degeneration grade, being very rare in G2. SEM demonstrated persistence of basic structural characteristics such as denser lamellae with Sharpey-type insertions into the endplates despite advanced age or degeneration grades. Immunohistochemistry revealed type II collagen to be the most abundant type followed by collagen IV. All collagen types were detected in every disc sector except for type X collagen. Statistical analysis demonstrated a general decrease in collagen expression from G1 to G2 with an annular- and another nuclear-specific pattern. These results suggest modifications of IVD morphology do not differ between the anterior or posterior annulus but are more advanced or happen earlier in the posterior areas of the disc. This study finally describes the process of extracellular matrix modification during disc degeneration in an unselected, general population and demonstrates it is similar to the same process in the cervical spine as published previously.

Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

Anti-Fibrotic Effects of Class I HDAC Inhibitor, Mocetinostat Is Associated with IL-6/Stat3 Signaling in Ischemic Heart Failure.

Background: Recent studies have linked histone deacetylases (HDAC) to remodeling of the heart and cardiac fibrosis in heart failure. However, the molecular mechanisms linking chromatin remodeling events with observed anti-fibrotic effects are unknown. Here, we investigated the molecular players involved in anti-fibrotic effects of HDAC inhibition in congestive heart failure (CHF) myocardium and cardiac fibroblasts in vivo. Methods and Results: MI was created by coronary artery occlusion. Class I HDACs were inhibited in three-week post MI rats by intraperitoneal injection of Mocetinostat (20 mg/kg/day) for duration of three weeks. Cardiac function and heart tissue were analyzed at six week post-MI. CD90+ cardiac fibroblasts were isolated from ventricles through enzymatic digestion of heart. In vivo treatment of CHF animals with Mocetinostat reduced CHF-dependent up-regulation of HDAC1 and HDAC2 in CHF myocardium, improved cardiac function and decreased scar size and total collagen amount. Moreover, expression of pro-fibrotic markers, collagen-1, fibronectin and Connective Tissue Growth Factor (CTGF) were reduced in the left ventricle (LV) of Mocetinostat-treated CHF hearts. Cardiac fibroblasts isolated from Mocetinostat-treated CHF ventricles showed a decrease in expression of collagen I and III, fibronectin and Timp1. In addition, Mocetinostat attenuated CHF-induced elevation of IL-6 levels in CHF myocardium and cardiac fibroblasts. In parallel, levels of pSTAT3 were reduced via Mocetinostat in CHF myocardium. Conclusions: Anti-fibrotic effects of Mocetinostat in CHF are associated with the IL-6/STAT3 signaling pathway. In addition, our study demonstrates in vivo regulation of cardiac fibroblasts via HDAC inhibition.

Mir‐27b regulates expression of fibrotic genes by targeting Gremlin 1

Fibrosis is a chronic disease that causes excessive deposition of scar tissue in the afflicted organs. TGF‐β plays a central role in this process by stimulating the production of extracellular matrix proteins such as collagen. MicroRNAs (miRNAs) have been implicated in the control of both fibrosis and TGF‐β signaling. The goal of the present study is to identify and characterize new miRNAs that regulate TGF‐β‐induced fibrosis. TGF‐β‐mediated fibrotic responses in A549 lung epithelial cells were used for functional profiling of miRNAs by screening a library of miRNA inhibitors for its effects on TGF‐β‐induced type I collagen expression. The inhibition of one miRNA in particular, miR‐27b, caused a significant increase in collagen expression. Additional experiments showed that increasing miR‐27b levels resulted in a significant decrease in collagen expression. Computational analysis revealed that a predicted target of this miRNA is Gremlin 1, a BMP antagonist known to play an important role in fibrosis. A549 cells treated with TGF‐β exhibited an increase in Gremlin 1 expression and a corresponding decrease in miR‐27b expression, suggesting that TGF‐β regulates Gremlin 1 via miR‐27b. Modulating miR‐27b levels affected Gremlin 1 expression. Subsequent experiments using a luciferase reporter construct that contained either the intact or the mutated 3′UTR of Gremlin 1 confirmed that Gremlin 1 is a direct target of miR‐27b. We have also shown that reducing Gremlin 1 levels with either an siRNA or miR‐27b results in a similar reduction of TGF‐β‐induced fibrotic genes such as type I collagen, CTGF and PAI‐1. In summary, we have demonstrated that miR‐27b targets Gremlin 1, and that this regulation is likely to play a critical role in TGF‐β‐mediated fibrosis.

Increased TG2 Expression Can Result in Induction of Transforming Growth Factor β1, Causing Increased Synthesis and Deposition of Matrix Proteins, Which Can Be Regulated by Nitric Oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-κB activation, resulting in the increased expression of transforming growth factor (TGF) β₁. In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFβ₁ in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-κB and TGFβ1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor <i>S</i>-nitroso-<i>N</i>-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-κB and TGFβ₁ production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Control of human articular chondrocyte differentiation by reduced oxygen tension

Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes.

The secreted protein thrombospondin 2 is an autocrine inhibitor of marrow stromal cell proliferation.

Marrow stromal cells (MSCs) are obtained in increased number from mice in which the thrombospondin 2 (TSP2) gene is disrupted, and these cells show increased DNA synthesis in vitro. To examine more closely the role of TSP2 in the physiology and osteogenic differentiation of MSCs, an in-depth characterization of TSP2-null MSCs was conducted. Determination of TSP2 protein content by Western analysis and RNA levels by reverse-transcription polymerase chain reaction (RT-PCR) indicated that MSCs are the primary source of TSP2 in the marrow and secrete abundant TSP2 into culture medium. Morphologically, the TSP2-null and wild-type (WT) cell populations were similar and by flow cytometry contained equivalent numbers of CD44+, Mac1+, intercellular adhesion molecule-1 (ICAM-1+), and ScaI+ cells. TSP2-null cells showed delayed mineralization associated with an increased rate of proliferation. Consistent with this finding, there was a decrease in expression of collagen and osteocalcin RNA by TSP2-null MSCs on day 7 and increased osteopontin expression on day 7 and day 14. In add-back experiments, recombinant TSP2 produced a dose-dependent decrease in proliferation. This reduction was associated with an accumulation of TSP2-treated cells in the G1 phase of the cell cycle and did not result from an increase in apoptosis. When TSP2 treatment was terminated, the cell population reentered the S phase. We conclude that the increased endosteal bone formation observed in TSP2-null mice results primarily from the failure of TSP2 to regulate locally MSC cell cycle progression.

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.