Dexmedetomidine represses TGF-ß1-induced extracellular matrix production and proliferation of airway smooth muscle cells by inhibiting MAPK signaling pathway.

BACKGROUND: Airway remodeling is implicated in the pathogenesis of asthma, and abnormal proliferation of airway smooth muscle cells (ASMCs) contribute to airway remodeling. Inflammatory mediator, transforming growth factor-ß1 (TGF-ß1), stimulates the proliferation of ASMCs, and is associated with airway remodeling in asthma. Dexmedetomidine (DEX) has been widely used in the adjuvant therapy of acute asthma. OBJECTIVE: The potential effects of DEX on extracellular matrix (ECM) production and proliferation of ASMCs were investigated in this study. MATERIAL AND METHODS: Human ASMCs were incubated with TGF-ß1 for 48 hours, and then treated with different concentrations of DEX for another 24 hours. Cell proliferation was detected by MTT and BrdU (5'-bromo-2'-deoxyuridine) staining. Flow cytometry was used to assess cell apoptosis, and western blot was applied to identify the underlying mechanism. RESULTS: TGF-ß1 induced increase in cell viability and bromodeoxyuridine (BrdU) positive cells in ASMCs while repressed cell apoptosis. Second, TGF-ß1-induced ASMCs were then treated with different concentrations of DEX. Cell viability of TGF-ß1-induced ASMCs was decreased by incubation of DEX. The number of BrdU positive cells in TGF-ß1-induced ASMCs was reduced by incubation of DEX. Moreover, incubation of DEX promoted cell apoptosis of TGF-ß1-induced ASMCs. Third, incubation of DEX attenuated TGF-ß1-induced increase in fibronectin, collagen I, MMP9, and versican in ASMCs. Lastly, the up-regulation of phosphorylated extracellular receptor kinase (p-ERK), phosphorylated Jun N-terminal Kinase (p-JNK), and p-p38 in TGF-ß1-induced ASMCs was reversed by incubation of DEX. CONCLUSION: DEX suppressed TGF-ß1-induced ECM production and proliferation of ASMCs through inactivation of p38 mitogen-activated protein kinase (MAPK) pathway, providing a potential strategy for prevention of asthma.

Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) culture models of chondrogenic ATDC5 cells, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Both culture models were treated with ITS+, 10mM ß-glycerophosphate (ßGP), or ITS+/10mM ßGP, which resulted in three levels of Pi abundance in cultures: basal (Pi/DNA <10ng/µg), moderate (Pi/DNA=25.3 - 32.3ng/µg), and high abundance (Pi/DNA >60ng/µg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM ßGP resulted in moderate abundance in 2D and high abundance in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters reduced phosphate-induced expression of chondrogenic markers. When 3D ITS+/ßGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

PTEN Regulates Renal Extracellular Matrix Deposit via Increased CTGF in Diabetes Mellitus.

Extracellular matrix accumulation and fibrosis are the features of diabetic nephropathy. PI3K (phosphatidylinositol 3-kinase)/Akt (protein kinase B) signal pathway and its inhibitor PTEN (phosphatase and tensin homolog deleted on chromosome 10) are revealed to modulate renal fibrosis. However, the exact mechanism is still not well known. In the present study we found that compared with normal mice, diabetic mice showed decreased PTEN, increased phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF (connective tissue growth factor), α-SMA (α-smooth muscle actin), and matricellular protein in kidney. Knocking down of PTEN caused an increase in phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in HKC cells (human renal tubular epithelial cells). Again, in vitro experiment revealed 1.89, 2.18, 1.92, 3.06, 2.06-fold increases of phospho-Akt (Ser 473), phospho-Akt (Thr 308), CTGF, secreted fibronectin, and secreted Col 3 in high glucose-stimulated HKC cells in comparison with normal control cells. Furthermore, knocking down of CTGF reversed increased secreted fibronectin and Col 3 in high glucose-treated HKC cells. Moreover, transfection of PTEN expression vector prevented high glucose-caused these changes in HKC cells. Especially, CTGF expression, secretion of fibronectin and Col 3 were, respectively, decreased by 38.81, 53.85, and 39.12%. The treatment of LY294002 inhibited phospho-Akt (Ser 473) and phospho-Akt (Thr 308) expression followed by decreased CTGF, secretory fibronectin and secretory Col 3 in high glucose-treated HKC cells. In the end our study suggests that PTEN regulates renal extracellular matrix production via activated Akt and increased CTGF in diabetes mellitus.

Inhibitory effect of TGF-ß peptide antagonist on the fibrotic phenotype of human hypertrophic scar fibroblasts.

CONTEXT: TGF-ß plays a central role in hypertrophic scar (HS) formation and development. OBJECTIVE: This study investigated the role of a TGF-ß antagonist peptide in inhibiting fibrotic behavior of human HS-derived fibroblasts (HSFs). MATERIALS AND METHODS: HSFs were seeded at a density of 3.1 × 10(4)/cm(2) and were subjected to treatment of peptide antagonist (30 µM) or TGF-ß receptor inhibitor LY2109761 (10 µM) or without treatment followed by the analyses of quantitative PCR, Elisa, in vitro wounding and fibroblast-populated collagen lattice (FPCL) assays. RESULTS: qPCR and Elisa analyses showed that the peptide could, respectively, reduce the gene (at 48 h) and protein (at 72 h) expression levels of collagen I (86 ± 4.8%; 56.6 ± 7.3%), collagen III (73 ± 10.7%; 43.7 ± 7.2%), fibronectin (90 ± 8.9%; 21.1 ± 2.8%), and TGF-ß1 (85 ± 9.3%; 25.0 ± 9.4%) as opposed to the non-treated group (p < 0.05), as the LY2109761 group similarly did. Cell proliferation was also significantly inhibited at day 5 (CCK-8 assay) by both peptide and LY2109761 treatments compared with the non-treated group (p < 0.05). The peptide also significantly inhibited cell migration as opposed to blank control at 24 h (43 ± 6.7% versus 60 ± 2.1%, p < 0.05) and at 48 h (63.9 ± 3.1% versus 95 ± 4.1%, p < 0.05). Similar to LY2109761, the peptide antagonist significantly reduced HS FPCL contraction compared with the non-treated group with significant differences in surface area at 48 h (0.71 ± 0.06 cm(2) versus 0.51 ± 0.06 cm(2), p < 0.05) and at 72 h (0.65 ± 0.02 cm(2) versus 0.42 ± 0.01 cm(2), p < 0.05). CONCLUSION: The TGF-ß antagonist peptide may serve as an important drug for HS prevention and reduction given the obvious benefits of good biosafety, low cost, and easy manufacture and delivery.

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/ß-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a ß-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/ß-catenin signaling.

Developing a tissue engineered repair material for treatment of stress urinary incontinence and pelvic organ prolapse-which cell source?

AIMS: Synthetic non-absorbable meshes are widely used to augment surgical repair of stress urinary incontinence (SUI) and pelvic organ prolapse (POP); however, there is growing concern such meshes are associated with serious complications. This study compares the potential of two autologous cell sources for attachment and extra-cellular matrix (ECM) production on a biodegradable scaffold to develop tissue engineered repair material (TERM). METHODS: Human oral fibroblasts (OF) and human adipose-derived stem cells (ADSC) were isolated and cultured on thermo-annealed poly-L-lactic acid (PLA) scaffolds for two weeks under either unrestrained conditions or restrained (either with or without intermittent stress) conditions. Samples were tested for cell metabolic activity (AlamarBlue® assay), contraction (serial photographs analyzed with image J software), total collagen production (Sirius red assay), and production of ECM components (immunostaining for collagen I, III, and elastin; and scanning electron microscopy) and biomechanical properties (BOSE tensiometer). Differences were statistically tested using two sample t-test. RESULTS: Both cells showed good attachment and proliferation on scaffolds. Unrestrained scaffolds with ADSC produced more total collagen and a denser homogenous ECM than OF under same conditions. Restrained conditions (both with and without intermittent stress) gave similar total collagen production, but improved elastin production for both cells, particularly OF. The addition of any cell onto scaffolds led to an increase in biomechanical properties of scaffolds compared to unseeded scaffolds. CONCLUSIONS: OF and ADSC both appear to be suitable cell types to combine with biodegradable scaffolds, in the development of a TERM for the treatment of SUI and POP. Neurourol. Urodynam. 33:531-537, 2014. © 2013 Wiley Periodicals, Inc.

Radial matrix constraint influences tissue contraction and promotes maturation of bi-layered skin equivalents.

Human skin equivalents (HSEs) serve as important tools for mechanistic studies with human skin cells, drug discovery, pre-clinical applications in the field of tissue engineering and for skin transplantation on skin defects. Besides the cellular and extracellular matrix (ECM) components used for HSEs, physical constraints applied on the scaffold during HSEs maturation influence tissue organization, functionality, and homogeneity. In this study, we introduce a 3D-printed culture insert that exposes bi-layered HSEs to a static radial constraint through matrix adhesion. We examine the effect of various diameters of the ring-shaped culture insert on the HSE's characteristics and compare them to state-of-the-art unconstrained and planar constrained HSEs. We show that radial matrix constraint of HSEs regulates tissue contraction, promotes fibroblast and matrix organization that is similar to human skin in vivo and improves keratinocyte differentiation, epidermal stratification, and basement membrane formation depending on the culture insert diameter. Together, these data demonstrate that the degree of HSE's contraction is an important design consideration in skin tissue engineering. Therefore, this study can help to mimic various in vivo skin conditions and to increase the control of relevant tissue properties.

Chsy1 deficiency reduces extracellular matrix productions and aggravates cartilage injury in osteoarthritis.

Osteoarthritis (OA) is a kind of degenerative joint disease marked by the destruction of articular cartilage due to the degeneration of chondrocytes. CHSY1, one of the glycosyltransferases, is involved in the synthesis of chondroitin sulfate. Herein, we found that the expression of Chsy1 was decreased in the knee cartilage of OA rats. In order to investigate the role of CHSY1 in chondrogenesis and OA, we established a Chsy1 stable knockdown cell line in mouse ATDC5 chondrocytes by lentivirus. It was found that Chsy1 deficiency resulted in a reduction of extracellular matrix production in chondrocytes and a promotion of endochondral osteogenesis, which was indicated by the decreased expression of early chondrocytes genes (Col2a1, Sox9), and the increased expression of cartilage hypertrophy genes (Col10a1, Runx2, Mmp13, Mmp3). The expression trend of these genes is considered to be the characteristic of osteoarthritis. In addition, knockdown of Chsy1 could upregulate BMP signaling in differentiated chondrocytes, whereas Chsy1 overexpression had opposite effects. The reduction of extracellular matrix production and the promotion of endochondral osteogenesis by Chsy1 knockdown could be rescued by BMP signaling inhibitor LDN193189. Furthermore, the abnormally enhanced BMP signaling and the high expression of OA biomarker Mmp3 in primary cells of OA rats could be rescued by either LDN193189 or Chsy1 overexpression. These results implicate a role for Chsy1 in regulating extracellular matrix production and endochondral osteogenesis through BMP signaling; and a lack of Chsy1 could aggravate the cartilage damage of osteoarthritis.

Technological robot-Machine tool collaboration for agile production.

The flexibility and efficiency in parts production can be significantly increased through the technological cooperation of industrial robots and machine tools. The paper presents an approach in which a robot, in addition to the classic handling tasks, enhance machine tools by additional manufacturing technologies and thus beneficially supports workpiece machining. This can take place in various configurations, starting with pre- and final machining by the robot outside the machine, through sequential cooperative machining of the workpiece clamped in the machine, to parallel, synchronized machining of a workpiece in the machine. The approach results in a novel type of collaborative manufacturing equipment for matrix production that will improve the versatility, efficiency and profitability in production.

Differential mineralization of human dental pulp stem cells on diverse polymers.

In tissue engineering, biomaterials are used as scaffolds for spatial distribution of specific cell types. Biomaterials can potentially influence cell proliferation and extracellular matrix formation, both in positive and negative ways. The aim of the present study was to investigate and compare mineralized matrix production of human dental pulp stem cells (DPSC), cultured on 17 different well-characterized polymers. Osteogenic differentiation of DPSC was induced for 21 days on biomaterials using dexamethasone, L-ascorbic-acid-2-phosphate, and sodium ß-glycerophosphate. Success of differentiation was analyzed by quantitative RealTime PCR, alkaline phosphatase (ALP) activity, and visualization of calcium accumulations by alizarin red staining with subsequent quantification by colorimetric method. All of the tested biomaterials of an established biomaterial bank enabled a mineralized matrix formation of the DPSC after osteoinductive stimulation. Mineralization on poly(tetrafluoro ethylene) (PTFE), poly(dimethyl siloxane) (PDMS), Texin, LT706, poly(epsilon-caprolactone) (PCL), polyesteramide type-C (PEA-C), hyaluronic acid, and fibrin was significantly enhanced (p<0.05) compared to standard tissue culture polystyrene (TCPS) as control. In particular, PEA-C, hyaluronic acid, and fibrin promoted superior mineralization values. These results were confirmed by ALP activity on the same materials. Different biomaterials differentially influence the differentiation and mineralized matrix formation of human DPSC. Based on the present results, promising biomaterial candidates for bone-related tissue engineering applications in combination with DPSC can be selected.