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.

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.

Dexmedetomidine represses TGF-beta1-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-β1 (TGF-β1), 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-β1 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-β1 induced increase in cell viability and bromodeoxyuridine (BrdU) positive cells in ASMCs while repressed cell apoptosis. Second, TGF-β1-induced ASMCs were then treated with different concentrations of DEX. Cell viability of TGF-β1-induced ASMCs was decreased by incubation of DEX. The number of BrdU positive cells in TGF-β1-induced ASMCs was reduced by incubation of DEX. Moreover, incubation of DEX promoted cell apoptosis of TGF-β1-induced ASMCs. Third, incubation of DEX attenuated TGF-β1-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-β1-induced ASMCs was reversed by incubation of DEX.Conclusion DEX suppressed TGF-β1-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 (AU)

MiR-143-3p controls TGF-ß1-induced cell proliferation and extracellular matrix production in airway smooth muscle via negative regulation of the nuclear factor of activated T cells 1.

MicroRNAs (miRNAs) are small noncoding RNAs that function in diverse biological processes. However, little is known about the precise role of microRNAs in the functioning of airway smooth muscle cells (ASMCs). Here, we investigated the potential role and mechanisms of the miR-143 -3p on proliferation and the extracellular matrix (ECM) protein production of ASMCs. We demonstrated that miR-143-3p was aberrantly lower in ASMCs isolated from individuals with asthma than in individuals without asthma. Meanwhile, TGF-ß1 caused a marked decrease in a time-dependent manner in miR-143-3p expression in ASMCs from asthmatics. Additionally, the overexpression of miR- 143-3p robustly reduced TGF-ß1-induced ASMCs proliferation and downregulated CDK and cyclin expression, whereas the inhibition of miR-143-3p significantly enhanced ASMCs proliferation and upregulated the level of CDKs and cyclins. Re-expression of miR-143-3p attenuated ECM protein deposition reflected as a marked decrease in the expression of type I collagen and fibronectin, whereas miR-143-3p downregulation caused an opposite effect on the expression of type I collagen and fibronectin. Moreover, qRT-PCR and western blot analysis indicated that miR-143-3p negatively regulated the expression of nuclear factor of activated T cells 1 (NFATc1). Subsequent analyses demonstrated that NFATc1 was a direct and functional target of miR-143-3p, which was validated by the dual luciferase reporter assay. Most importantly, the overexpression of NFATc1 effectively reversed the inhibition of miR-143-3p on TGF-ß1-induced proliferation, and strikingly abrogated the effect of miR-143-3p on the expression of CDK4 and Cyclin D1. Together, miR-143-3p may function as an inhibitor of asthma airway remodeling by suppressing proliferation and ECM protein deposition in TGF-ß1-mediated ASMCs via the negative regulation of NFATc1 signaling, suggesting miR-143-3p as a potential therapeutic target for asthma.

Meroterpenoid enantiomers from Ganoderma sinensis.

Zizhines A-F (1-6), six pairs of new meroterpenoid enantiomers and a known meroterpenoid (7) were isolated from the fruiting bodies of Ganoderma sinensis. The structures and absolute configurations of the new substances were assigned by spectroscopic and computational methods. All the compounds apart from 7 were evaluated for their inhibition on extracellular matrix component (fibronectin) generation by using TGF-ß1-induced rat kidney tubular epithelial cells. Although none of them was found to be active in these cells, the present findings add new facets for the chemistry of Ganoderma.

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.

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.