Expression Of Matrix Molecules Research Articles

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

Cellular behavior and extracellular matrix turnover in bovine annulus fibrosus cells under hydrostatic pressure and deviatoric strain.

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.

The application of mechanical load onto mouse tendons by magnetic restraining represses Mmp-3 expression

ObjectivesMechanical loading is crucial for tendon matrix homeostasis. Under-stimulation of tendon tissue promotes matrix degradation and ultimately tendon failure. In this study, we examined the expression of tendon matrix molecules and matrix-degrading enzymes (matrix metalloproteinases) in stress-deprived tail tendons and compared to tendons that were mechanically loaded by a simple restraining method.Data descriptionIsolated mouse tail fascicles were either floated or restrained by magnets in cell culture media for 24 h. The gene expression of tendon matrix molecules and matrix metalloproteinases in the tendon fascicles of mouse tails were examined by real-time RT-PCR. Stress deprivation of tail tendons increase Mmp3 mRNA levels. Restraining tendons represses these increases in Mmp3. The gene expression response to restraining was specific to Mmp3 at 24 h as we did not observe mRNA level changes in other matrix related genes that we examined (Col1, Col3, Tnc, Acan, and Mmp13). To elucidate, the mechanisms that may regulate load transmission in tendon tissue, we examined filamentous (F-)actin staining and nuclear morphology. As compared to stress deprived tendons, restrained tendons had greater staining for F-actin. The nuclei of restrained tendons are smaller and more elongated. These results indicate that mechanical loading regulates specific gene expression potentially through F-actin regulation of nuclear morphology. A further understanding on the mechanisms involved in regulating Mmp3 gene expression may lead to new strategies to prevent tendon degeneration.

Regenerative Capability of Human Nucleus Pulposus Cells in Degenerated Disc Under Hydrostatic Pressure Mimicking Physiologically Relevant Intradiscal Pressure In Vitro.

Isolated human nucleus pulposus (hNP) cells from the degenerated intervertebral disc (IVD) were incubated under hydrostatic pressure (HP) and evaluated for regenerative potential. To characterize metabolic turnover in hNP cells isolated from degenerated IVDs classified by Pfirrmann grade under physiologically relevant HP at high osmolality in vitro. We demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic HP followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells. hNP cells isolated from discarded tissue classified as Pfirrmann grade 2 to 3 (n = 13: age, 46.7 ± 14.0) and grade 4 (n = 13: age, 53.0 ± 11.5) were incubated under cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules and catabolic and anticatabolic proteins were evaluated. Aggrecan and collagen type II expression were significantly more upregulated under HP in grades 2 to 3 than in grade 4 tissues (both, P < 0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in grades 2 to 3, whereas a negative correlation was found in grade 4 ( P < 0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP. Resident cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading. This study explored the potential of degenerated remnant nucleus pulposus cells under a physiological environment, possibly leading to establishing strategies for IVD regeneration.

Hyper-osmolarity environment-induced oxidative stress injury promotes nucleus pulposus cell senescence in vitro.

Nucleus pulposus (NP) cell senescence is involved in disc degeneration. The in situ osmolarity within the NP region is an important regulator of disc cell’s biology. However, its effects on NP cell senescence remain unclear. The present study was aimed to investigate the effects and mechanism of hyper-osmolarity on NP cell senescence. Rat NP cells were cultured in the in situ-osmolarity medium and hyper-osmolarity medium. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was added along with the medium to investigate the role of oxidative injury. Cell cycle, cell proliferation, senescence associated β-galactosidase (SA-β-Gal) activity, telomerase activity, expression of senescence markers (p16 and p53) and matrix molecules (aggrecan and collagen II) were tested to assess NP cell senescence. Compared with the in situ-osmolarity culture, hyper-osmolarity culture significantly decreased cell proliferation and telomerase activity, increased SA-β-Gal activity and cell fraction in the G0/G1 phase, up-regulated expression of senescence markers (p16 and p53) and down-regulated expression of matrix molecules (aggrecan and collagen II), and increased intracellular ROS accumulation. However, addition of NAC partly reversed these effects of hyper-osmolarity culture on cellular senescence and decreased ROS content in NP cells. In conclusion, a hyper-osmolarity culture promotes NP cell senescence through inducing oxidative stress injury. The present study provides new knowledge on NP cell senescence and helps us to better understand the mechanism of disc degeneration.

Effect of photobiomodulation and exercise on early remodeling of the Achilles tendon in streptozotocin-induced diabetic rats.

The aim of this study was to compare the treatment effects of laser photobiomodulation (LPBM) therapy and aerobic exercise on the biomechanical properties, tissue morphology and the expression of tendon matrix molecules during early remodeling of Achilles tendon (AT) injury in diabetic rats. Animals were randomly assigned to five groups: injured non diabetic (I, n = 15), injured diabetic (ID, n = 15), injured diabetic plus LPBM (IDL, n = 16), injured diabetic plus aerobic exercise (IDE, n = 16) and injured diabetic plus aerobic exercise and LPBM (IDEAL, n = 17). Type 1 diabetes was induced via a single intravenous injection of Streptozotocin at a dose of 40 mg/kg. A partial tenotomy was performed in the right AT. LPBM was performed with an indium-gallium-aluminum-phosphide 660 nm 10 mW laser device (spot size 0.04 cm2, power density 250 mW/cm2, irradiation duration 16 s, energy 0.16 J, energy density 4 J/cm2) on alternate days for a total of 9 sessions over 3 weeks (total energy 1.44 J), using a stationary contact technique to a single point over the dorsal aspect of the AT. Moderate aerobic exercise was performed on a motorized treadmill (velocity 9 m/min for 60 minutes). At 3 weeks post-injury, biomechanical analyzes as well as assessment of fibroblast number and orientation were performed. Collagen 1 (Col1) and 3 (Col3) and matrix metalloproteinases (MMPs) -3 and 13 protein distributions were studied by immunohistochemistry; while Col1 and Col3 and MMP-2 and 9 gene expression were assessed by quantitative RT-PCR (qRT-PCR). IDEAL exhibited significant increases in several biomechanical parameters in comparison to the other groups. Moreover, IDEAL presented stronger Col1 immunoreactivity when compared to ID, and weaker Col3 immunoreactivity than IDE. Both IDL and IDEAL demonstrated weaker expression of MMP-3 in comparison to I, while IDL presented no expression of MMP-13 when compared to ID. ID, IDL and IDE showed an increased number of fibroblasts in comparison to I, while IDEAL decreased the number of these cells in comparison to ID and IDE. IDL and IDEAL groups exhibited decreased angular dispersion among the fibroblasts when compared to I. The gene expression results showed that IDE demonstrated a downregulation in Col1 mRNA expression in comparison to I and ID. IDEAL demonstrated upregulation of Col1 mRNA expression when compared to IDL or IDE alone and increased MMP-2 expression when compared to IDL and IDE. MMP-9 expression was upregulated in IDEAL when compared to I, IDL and IDE. Our results suggest a beneficial interaction of combining both treatment strategies i.e., aerobic exercise and LPBM, on the biomechanical properties, tissue morphology and the expression of matrix molecules in diabetic tendons.

The Atypical Chemokine Receptor 2 Limits Progressive Fibrosis after Acute Ischemic Kidney Injury

Following renal ischemia-reperfusion injury (IRI), resolution of inflammation allows tubular regeneration, whereas ongoing inflammatory injury mediated by infiltrating leukocytes leads to nephron loss and renal fibrosis, typical hallmarks of chronic kidney disease. Atypical chemokine receptor 2 (ACKR2) is a chemokine decoy receptor that binds and scavenges inflammatory CC chemokines and reduces local leukocyte accumulation. We hypothesized that ACKR2 limits leukocyte infiltration, inflammation, and fibrotic tissue remodeling after renal IRI, thus preventing progression to chronic kidney disease. Compared with wild type, Ackr2 deficiency increases CC chemokine ligand 2 levels in tumor necrosis factor-stimulated tubulointerstitial tissue invitro. In Ackr2-deficient mice with early IRI 1 or 5 days after transient renal pedicle clamping, tubular injury was similar to wild type, although accumulation of mononuclear phagocytes increased in postischemic Ackr2-/- kidneys. Regarding long-term outcomes, Ackr2-/- kidneys displayed more tubular injury 5 weeks after IRI, which was associated with persistently increased renal infiltrates of mononuclear phagocytes, T cells, Ly6Chigh inflammatory macrophages, and inflammation. Moreover, Ackr2 deficiency caused substantially aggravated renal fibrosis in Ackr2-/- kidneys 5 weeks after IRI, shown by increased expression of matrix molecules, renal accumulation of α-smooth muscle actin-positive myofibroblasts, and bone marrow-derived fibrocytes. ACKR2 is important in limiting persistent inflammation, tubular loss, and renal fibrosis after ischemic acute kidney injury and, thus, can prevent progression to chronic renal disease.

17β-Estradiol/extrogen receptor β alleviates apoptosis and enhances matrix biosynthesis of nucleus pulposus cells through regulating oxidative damage under a high glucose condition

BackgroundHyperglycemia in the Diabetes mellitus (DM) patients is a potential etiology of disc degeneration. 17β-estradiol (17β-E2) supplementation plays an anti-diabetic role in DM patients. ObjectiveThis study was aimed to investigate the role and mechanism of 17β-E2 in regulating nucleus pulposus (NP) cell apoptosis and NP matrix production under a high glucose condition. MethodsRat NP cells were cultured in medium with a high glucose concentration (0.2 M). 17β-E2 was added into the culture medium to investigate its protective effects. The ERβ inhibitor PHTPP and ERβ activator ERB041 were used to investigate the effects of ERβ. NP cell apoptosis was analyzed by flow cytometry and expression of apoptosis-related molecules. NP matrix production was evaluated by expression of matrix macromolecules. Additionally, intracellular reactive oxygen species (ROS) content was also detected. ResultsCompared with the control NP cells, 17β-E2 decreased NP cell apoptosis ratio, down-regulated gene expression of Bax and caspase-3, up-regulated gene expression of Bcl-2, increased protein expression of cleaved PARP and cleaved caspase-3, and increased expression of matrix molecules (aggrecan and collagen II). Moreover, 17β-E2 decreased ROS content. Further analysis showed that ERβ inhibition partly reversed these effects of 17β-E2 whereas ERβ activation further promoted its effects. Conclusion17β-E2/ERβ interaction attenuates apoptosis and promotes matrix biosynthesis of NP cells through alleviating oxidative damage under a high glucose condition. This study provides new knowledge on strategies for retarding disc degeneration.

Upregulation of lipocalin-2 (LCN2) in osteoarthritic cartilage is not necessary for cartilage destruction in mice

Purpose: Lipocalin-2 (LCN2) has recently emerged as a novel adipokines involved in variety of physiological and pathophysiological processes. Although recent in vitro cell-based studies suggest that increased LCN2 level in OA may be detrimental, the precise in vivo role of LCN2 in OA progression has been challenging to define. Here, we performed a set of experiments aimed to elucidate in vivo role of LCN2 in the pathogenesis of OA. Methods: The expression of LCN2 were determined at the mRNA and protein levels in primary cultured mouse chondrocytes and in human and mouse OA cartilage. Experimental OA was induced in wild-type or Lcn2 knockout (KO) mice by destabilization of the medial meniscus (DMM) surgery or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8). The effect of LCN2 overexpression on the cartilage of wild-type mice was examined by IA injection of Ad-Lcn2. Primary culture mouse chondrocytes were treated with Lcn2-siRNA or were infected with Ad-Lcn2, and gene expression patterns analyzed by using reverse transcription (RT)-PCR. Results: LCN2 mRNA levels in chondrocytes was markedly increased by the pro-inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and by previously identified catabolic regulators of OA, such as hypoxia-inducible factor (HIF)-2α and components of the zinc-ZIP8-MTF1 axis. LCN2 protein levels were also markedly increased in human OA cartilage and cartilage from various experimental mouse models of OA. However, overexpression of LCN2 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Furthermore, LCN2 overexpression in mouse cartilage via IA injection of Ad-Lcn2 did not cause OA pathogenesis, and Lcn2 KO mice showed no alteration in DMM-induced OA cartilage destruction. Conclusions: Our observation collectively suggest that upregulation of LCN2 in OA cartilage is not sufficient or necessary for OA cartilage destruction in mice.

A Substance Exchanger-Based Bioreactor Culture of Pig Discs for Studying the Immature Nucleus Pulposus.

Various research models have been developed to study the biology of disc cells. Recently, the adult disc nucleus pulposus (NP) has been well studied. However, the immature NP is underinvestigated due to a lack of a suitable model. This study aimed to establish an organ culture of immature porcine disc by optimizing culture conditions and using a self-developed substance exchanger-based bioreactor. Immature porcine discs were first cultured in the bioreactor for 7 days at various levels of glucose (low, medium, high), osmolarity (hypo-, iso-, hyper-) and serum (5, 10, 20%) to determine the respective optimal level. The porcine discs were then cultured under the optimized conditions in the novel bioreactor, and were compared with fresh discs at day 14. For high-glucose, iso-osmolarity, or 10% serum, cell viability, the gene expression profile (for anabolic genes and catabolic genes), and glycosaminoglycan (GAG) and hydroxyproline (HYP) contents were more favorable than for other levels of glucose, osmolarity, and serum. When the immature discs were cultured under the optimized conditions using the novel bioreactor for 14 days, the viability of the immature NP was maintained based on histology, cell viability, GAG and HYP contents, and matrix molecule expression. In conclusion, the viability of the immature NP in organ culture could be maintained under the optimized culture conditions (high-glucose, iso-osmolarity, and 10% serum) in the substance exchanger-based bioreactor.

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

ObjectivesTo assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone.MethodsOsteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats.ResultsAfter mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups.ConclusionsThese results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis

In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the invivo functions of Atrogin-1 in mouse models of OA. The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1β, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice.

Upregulation of lipocalin-2 (LCN2) in osteoarthritic cartilage is not necessary for cartilage destruction in mice

Lipocalin-2 (LCN2) is a recently characterized adipokine that is upregulated in chondrocytes treated with pro-inflammatory mediators and in the synovial fluid of osteoarthritis (OA) patients. Here, we explored the invivo functions of LCN2 in OA cartilage destruction in mice. The expression levels of LCN2 were determined at the mRNA and protein levels in primary cultured mouse chondrocytes and in human and mouse OA cartilage. Experimental OA was induced in wild-type (WT) or Lcn2-knockout (KO) mice by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing hypoxia-inducible factor (HIF)-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or LCN2 (Ad-Lcn2). The effect of LCN2 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Lcn2. LCN2 mRNA levels in chondrocytes were markedly increased by the pro-inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and by previously identified catabolic regulators of OA, such as HIF-2α and components of the zinc-ZIP8-MTF1 axis. LCN2 protein levels were also markedly increased in human OA cartilage and cartilage from various experimental mouse models of OA. However, overexpression of LCN2 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Furthermore, LCN2 overexpression in mouse cartilage via IA injection of Ad-Lcn2 did not cause OA pathogenesis, and Lcn2 KO mice showed no alteration in DMM-induced OA cartilage destruction. Our observations collectively suggest that upregulation of LCN2 in OA cartilage is not sufficient or necessary for OA cartilage destruction in mice.

Estrogen receptor beta modulates breast cancer cells functional properties, signaling and expression of matrix molecules

Estrogen receptors have pivotal roles in breast cancer growth and progression. ERα has been clearly shown to play key role in hormone-dependent breast cancer properties, but little is known for the isoform ERβ. To evaluate the role of ERβ, we established stably transfected ERβ-suppressed MDA-MB-231 breast cancer cells by knocking down the human ERβ gene, using specific shRNA lentiviral particles. As observed by scanning electron microscopy, the ERβ suppression induces significant phenotypic changes in these cells, as compared to the control cells. Notably, the down-regulation of ERβ decreases the expression of the mesenchymal markers fibronectin and vimentin, whereas it increases the expression levels of the epithelial marker E-cadherin and cell junctions. These alterations are followed by reduced levels of the functional cell properties that promote the aggressiveness of these cells, such as proliferation, migration, spreading capacity, invasion and adhesion on collagen I. Notably, the down-regulation of ERβ reduces the migration of breast cancer cells through the tyrosine kinase receptors EGFR/IGF-IR and the JAK/STAT signaling pathways. Moreover, ERβ has a crucial role on the gene expression of several matrix mediators, including the proteoglycans syndecans-2/-4 and serglycin, several matrix metalloproteinases, plasminogen activation system components and receptor tyrosine kinases. These data clearly show that ERβ plays a crucial role in the cell behavior and ECM composition of the highly aggressive MDA-MB-231 cells and opens a new area of research to further understand its role and to improve pharmaceutical targeting of the non-hormone dependent breast cancer.

Biochemical and toxicological evaluation of nano-heparins in cell functional properties, proteasome activation and expression of key matrix molecules.

The glycosaminoglycan heparin and its derivatives act strongly on blood coagulation, controlling the activity of serine protease inhibitors in plasma. Nonetheless, there is accumulating evidence highlighting different anticancer activities of these molecules in numerous types of cancer. Nano-heparins may have great biological significance since they can inhibit cell proliferation and invasion as well as inhibiting proteasome activation. Moreover, they can cause alterations in the expression of major modulators of the tumor microenvironment, regulating cancer cell behavior. In the present study, we evaluated the effects of two nano-heparin formulations: one isolated from porcine intestine and the other from the sea squirt Styela plicata, on a breast cancer cell model. We determined whether these nano-heparins are able to affect cell proliferation, apoptosis and invasion, as well as proteasome activity and the expression of extracellular matrix molecules. Specifically, we observed that nano-Styela compared to nano-Mammalian analogue has higher inhibitory role on cell proliferation, invasion and proteasome activity. Moreover, nano-Styela regulates cell apoptosis, expression of inflammatory molecules, such as IL-6 and IL-8 and reduces the expression levels of extracellular matrix macromolecules, such as the proteolytic enzymes MT1-MMP, uPA and the cell surface proteoglycans syndecan-1 and -2, but not on syndecan-4. The observations reported in the present article indicate that nano-heparins and especially ascidian heparin are effective agents for heparin-induced effects in critical cancer cell functions, providing an important possibility in pharmacological targeting.

Alcohol Differentially Alters Extracellular Matrix and Adhesion Molecule Expression in Skeletal Muscle and Heart

The production of fibrosis in response to chronic alcohol abuse is well recognized in liver but has not been fully characterized in striated muscle and may contribute to functional impairment. Therefore, the purpose of this study was to use an unbiased discovery-based approach to determine the effect of chronic alcohol consumption on the expression profile of genes important for cell-cell and cell-extracellular matrix (ECM) interactions in both skeletal and cardiac muscle. Adult male rats were pair-fed an alcohol-containing liquid diet or control diet for 24weeks, and skeletal muscle (gastrocnemius) and heart were collected in the freely fed state. A pathway-focused gene expression polymerase chain reaction array was performed on these tissues to assess mRNA content for 84 ECM proteins, and selected proteins were confirmed by Western blot analysis. In gastrocnemius, alcohol feeding up-regulated the expression of 11 genes and down-regulated the expression of 1 gene. Alcohol increased fibrosis as indicated by increased mRNA and/or protein for collagens α1(I), α2(I), α1(III), and α2(IV) as well as hydroxyproline. Alcohol also increased α-smooth muscle actin protein, an index of myofibroblast activation, but no concomitant change in transforming growth factor-β was detected. The mRNA and protein content for other ECM components, such as integrin-α5, L-selectin, PECAM, SPARC, and ADAMTS2, were also increased by alcohol. Only laminin-α3 mRNA was decreased in gastrocnemius from alcohol-fed rats, while 66 ECM- or cell adhesion-related mRNAs were unchanged by alcohol. For heart, expression of 16 genes was up-regulated, expression of 3 genes was down-regulated, and 65 mRNAs were unchanged by alcohol; there were no common alcohol-induced gene expression changes between heart and skeletal muscle. Finally, alcohol increased tumor necrosis factor-α and interleukin (IL)-12 mRNA in both skeletal and cardiac muscle, but IL-6 mRNA was increased and IL-10 mRNA decreased only in skeletal muscle. These data demonstrate a fibrotic response in striated muscle from chronic alcohol-fed rats which is tissue specific in nature, suggesting different regulatory mechanisms.

Novel nano-composite biomimetic biomaterial allows chondrogenic and osteogenic differentiation of bone marrow concentrate derived cells

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of osteochondral lesions. For this purpose, the ideal scaffold should possess the right properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, mechanical strength, and flexibility. In addition, it should be easy to handle and serve as a template for chondrocyte and bone cells guiding both cartilage and bone formation. The aim of the present study was to estimate the chondrogenic and osteogenic capability of bone marrow concentrated derived cells seeded onto a novel nano-composite biomimetic material. These properties have been evaluated by means of histological, immunohistochemical and electron microscopy analyses. The data obtained demonstrated that freshly harvested cells obtained from bone marrow were able, once seeded onto the biomaterial, to differentiate either down the chondrogenic and osteogenic pathways as evaluated by the expression and production of specific matrix molecules. These findings support the use, for the repair of osteochondral lesions, of this new nano-composite biomimetic material together with bone marrow derived cells in a "one step" transplantation procedure.

Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells.

BackgroundThree-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells.MethodsHuman AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13.ResultsAn initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan.ConclusionsWe failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.

Effects of Initial Boost with TGF-Beta 1 and Grade of Intervertebral Disc Degeneration on 3D Culture of Human Annulus Fibrosus Cells

Introduction Three-dimensional (3D) culture in porous biomaterials and stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated if an initial boost with transforming growth factor-β1 (TGF-β1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells. Materials and Methods Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of nonabsorbable poly vinylidene fluoride. An initial boost of TGF-β1 or TGF-β1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan, type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13. Results An initial boost with TGF-β1 or TGF-β1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from, both, slightly degenerated or severely degenerated tissue, are capable of significant upregulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower upregulations in some matrix molecules such as aggrecan. Conclusion We failed to show a supportive effect of an initial boost with TGF-β1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems. Disclosure of Interest A. A. Hegewald has no conflict of interest and C. Kaps is an employee of TransTissue Technologies GmbH, which develops products in the field of regenerative medicine.

Sprifermin (rhfgf18) has an anabolic effect on human osteoarthritic chondrocytes involving fgfr3 and erk1/2 but not p38α and JNKS

Purpose: Restoring the articular surface of degraded or largely injured cartilage necessitates cartilage re-cellularization and the stimulation of cartilage matrix molecule production. Sprifermin (currently in a phase II clinical trial for knee osteoarthritis) is a growth factor that is involved in chondrogenesis and exerts an anabolic effect on cartilage. Using a 3D cell culture system, we previously demonstrated the ability of Sprifermin to stimulate cartilage matrix molecule expression and to promote the chondrocyte phenotype in bovine and porcine articular chondrocytes. In the present work, we aimed to confirm this effect in osteoarthritic chondrocytes of human origin (hOA). In parallel, the pathways involved in Sprifermin signaling were investigated in porcine chondrocytes. Methods: Primary osteoarthritic chondrocytes were isolated from the cartilage of patients who underwent total knee replacement. Cells were cultivated for a few days in monolayer first and then for one week in scaffold-free 3D culture before starting the treatment. The latter consisted in the incubation with Sprifermin [100 ng/mL] permanently or one day/week for a total period of four weeks. Results were compared to a control culture without Sprifermin. Biochemical assays, quantitative PCR (qPCR) and histology were used to characterize the 3D constructs. FGFR expression by qPCR and activation of various kinases (with the human phospho-MAPK array kit from R&D Systems) upon Sprifermin stimulation were assessed on freshly isolated hOA chondrocytes. The effects of a panel of inhibitors (MAPK inhibitors and one pan-FGFR inhibitor) were tested in porcine cells. For this, freshly isolated chondrocytes from a pig hip were cultivated for 7 days in monolayer with 100 ng/mL of Sprifermin in presence of three concentrations of the inhibitors (0.1, 0.3 and 1 μM or 1, 3 and 10 μM depending on their potency). Sprifermin has been previously shown to strongly decrease the collagen I expression (monitored by qPCR) and to promote a round cell morphology (evaluated by actin staining) of porcine chondrocytes in monolayer. These parameters were used to evaluate the impact of the various inhibitors. Results: A 3D scaffold-free culture was used to test the effect of Sprifermin on hOA chondrocytes. In this setting Sprifermin [1day/week] has been found to have a positive effect on the cell content and to greatly increase the size and matrix content of the 3D constructs. In accordance with the literature, FGFR1 and 3 exhibited the highest expression among FGFRs in hOA chondrocytes. In addition, Sprifermin effects were suppressed by FGFR inhibition. In porcine chondrocytes cultivated in monolayer, Sprifermin strongly inhibited collagen I expression and promoted a round cell shape. Both effects were completely inhibited in presence of the pan-FGFR inhibitor PD173074 in a dose-dependent manner. Because FGF18 does not bind FGFR1, in chondrocytes Sprifermin signaling involves mainly FGFR3. Upon incubation with Sprifermin [100 ng/mL], ERK1/2 was activated as well as p38γ, but not p38α or the JNKs. The ERK inhibitor PD325901 completely inhibited the effect of Sprifermin on cell morphology at 100 nM. The JNK inhibitor (SR3306) and p38 inhibitors (BIRB-796 and SB203580) did not have any effect. The effect of these inhibitors on collagen I expression is still under investigation. Taken together these results indicate that ERK1/2 is involved in Sprifermin signaling whereas p38α and JNKs are not. Conclusions: As observed in previous studies with bovine and porcine chondrocytes, Sprifermin was found to exert anabolic effects in hOA chondrocytes. These effects seem to be mediated by FGFR3 and ERK1/2 activation. Others demonstrated that ERK1/2 is also involved in FGF2-FGFR1 signaling but in this case led to a catabolic response. This difference can be possibly explained by the fact that JNK and p38 are also activated by FGF2-FGFR1 signaling but not by FGF18-FGFR3.