Interactive effects of cerium oxide and diesel exhaust nanoparticles on inducing pulmonary fibrosis

Cerium compounds have been used as a fuel-borne catalyst to lower the generation of diesel exhaust particles (DEPs), but are emitted as cerium oxide nanoparticles (CeO₂) along with DEP in the diesel exhaust. The present study investigates the effects of the combined exposure to DEP and CeO₂ on the pulmonary system in a rat model. Specific pathogen-free male Sprague–Dawley rats were exposed to CeO₂ and/or DEP via a single intratracheal instillation and were sacrificed at various time points post-exposure. This investigation demonstrated that CeO₂ induces a sustained inflammatory response, whereas DEP elicits a switch of the pulmonary immune response from Th1 to Th2. Both CeO₂ and DEP activated AM and lymphocyte secretion of the proinflammatory cytokines IL-12 and IFN-γ, respectively. However, only DEP enhanced the anti-inflammatory cytokine IL-10 production in response to ex vivo LPS or Concanavalin A challenge that was not affected by the presence of CeO₂, suggesting that DEP suppresses host defense capability by inducing the Th2 immunity. The micrographs of lymph nodes show that the particle clumps in DEP + CeO₂ were significantly larger than CeO₂ or DEP, exhibiting dense clumps continuous throughout the lymph nodes. Morphometric analysis demonstrates that the localization of collagen in the lung tissue after DEP + CeO₂ reflects the combination of DEP-exposure plus CeO₂-exposure. At 4 weeks post-exposure, the histological features demonstrated that CeO₂ induced lung phospholipidosis and fibrosis. DEP induced lung granulomas that were not significantly affected by the presence of CeO₂ in the combined exposure. Using CeO₂ as diesel fuel catalyst may cause health concerns.     

Neovibsanin B increases extracellular matrix proteins in optic nerve head cells via activation of Smad signalling pathway

The present study demonstrates the effect of neovibsanin B on the synthesis and deposition of ECM proteins and the signalling pathways used in optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells. For investigation of the signalling pathway used by neovibsanin B, ONH cells were treated with neovibsanin B. Western blot and immunostaining analyses were used to examine the phosphorylation of proteins involved in Smad and non-Smad signalling pathway. The results revealed that ONH cells on treatment with neovibsanin B showed enhanced synthesis of extracellular matrix (ECM) proteins. Neovibsanin B induced phosphorylation of canonical signalling proteins, Smad2/3. However phosphorylation of non-canonical signalling proteins, extracellular signal-regulated kinases, p38, and c-Jun N-terminal kinases (JNK) 1/2 remained unaffected. There was also increase in co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells indicating activation of the canonical Smad signalling pathway. Treatment of ONH cells with SIS3, inhibitor of Smad3 phosphorylation reversed the neovibsanin B stimulated ECM expression as well as activation of canonical pathway signalling molecules. In addition, inhibition of Smad2 or Smad3 using small interfering RNA (siRNA) also suppressed neovibsanin B stimulated ECM protein synthesis in ONH astrocytes and LC cells. Thus neovibsanin B utilizes the canonical Smad signalling pathway to stimulate ECM synthesis in human ONH cells. The neovibsanin B induced ECM synthesis and activation of the canonical Smad signalling pathway may be due to its effect on transforming growth factor-β2 (TGF-β2). However, further studies are under process to understand the mechanism.     

Transfer stamping of human mesenchymal stem cell patches using thermally expandable hydrogels with tunable cell-adhesive properties

Development of stem cell delivery system with ability of control over mutilineage differentiation and improved engraft efficiency is imperative in regenerative medicine. We herein report transfer stamping of human mesenchymal stem cells (hMSCs) patches using thermally expandable hydrogels with tunable cell-adhesive properties. The hydrogels were prepared from functionalized four arm copolymer of Tetronic^®, and the cell adhesion on the hydrogel was modulated by incorporation of fibronectin (FN) or cell-adhesive peptide (RGD). The resulting hydrogels showed spontaneous expansion in size within 10 min in response to the temperature reduction from 37 to 4°C. The adhesion and proliferation of hMSCs on FN-hydrogels were positively tunable in proportion to the amount of FN within hydrogels with complete monolayer of hMSCs (hMSC patch) being successfully achieved. The hMSC patch on the hydrogel was faced to the target substrate, which was then easily detached and re-attached to the target when the temperature was reduced from 37°C up to 4°C. We found that the transfer stamping of cell patch was facilitated at lower temperature of 4°C relative to 25°C, with the use of thinner hydrogels (0.5 mm in thickness relatively to 1.0 or 1.5 mm) and longer transfer time (>15 min). Notably, the hMSC patch was simply transferred from the hydrogel to the subcutaneous mouse skin tissue within 15 min with cold saline solution being dropped to the hydrogel. The hMSC patch following osteogenic or adipogenic commitment was also achieved with long-term culture of hMSCs on the hydrogel, which was successfully detached to the target surface. These results suggest that the hydrogels with thermally expandable and tunable cell-adhesive properties may serve as a universal substrate to harvest hMSC patch in a reliable and effective manner, which could potentially be utilized in many cell-sheet based therapeutic applications.     

Histopathological and biomechanical evaluation of tenocyte seeded allografts on rat Achilles tendon regeneration

Tendon injuries in humans as well as in animals' veterinary medicine are problematic because tendon has poor regenerative capacity and complete regeneration of the ruptured tendon is never achieved. In the last decade there has been an increasing need of treatment methods with different approaches. The aim of the current study was to improve the regeneration process of rat Achilles tendon with tenocyte seeded decellularized tendon matrices. For this purpose, Achilles tendons were harvested, decellularized and seeded as a mixture of three consecutive passages of tenocytes at a density of 1 × 10⁶ cells/ml. Specifically, cells with different passage numbers were compared with respect to growth characteristics, cellular senescence and collagen/tenocyte marker production before seeding process. The viability of reseeded tendon constructs was followed postoperatively up to 6 months in rat Achilles tendon by histopathological and biomechanical analysis. Our results suggests that tenocyte seeded decellularized tendon matrix can significantly improve the histological and biomechanical properties of tendon repair tissue without causing adverse immune reactions. To the best of our knowledge, this is the first long-term study in the literature which was accomplished to prove the use of decellularized matrix in a clinically relevant model of rat Achilles tendon and the method suggested herein might have important implications for translation into the clinic.     

Quantum dots-based in situ molecular imaging of dynamic changes of collagen IV during cancer invasion

Cancer invasion and metastasis remains the root cause of mortality. This process involves alterations of tumor microenvironment, particularly the remodeling of extracellular matrix, characterized by collagen IV uncoiling, degradation, fragments deposition and cross-linking. Quantum dots-labeled molecular probes are promising platforms to simultaneously study several subtle changes of key biomolecules, because of their unique optical and chemical properties. Here we report on a quantum dots-based imaging technology to study key components in tumor microenvironment during cancer progression, so as to gain new insights into the role of collagen IV plays, to define the cancer “invasion unit” and to develop the “pulse-mode” of cancer invasion.     

Oxidized low-density lipoprotein-induced CD147 expression and its inhibition by high-density lipoprotein on platelets in vitro

Introduction

Matrix metalloproteinases (MMPs) are believed to progressively degrade the collagenous components of the protective fibrous cap, leading to atherosclerotic plaque rupture or destabilization. Oxidized low-density lipoprotein (ox-LDL) enhances the release of CD147, known as the extracellular MMP inducer, from coronary smooth muscle cells. However, whether ox-LDL can induce platelet CD147 expression is unknown. Therefore, we investigated the influence of ox-LDL and high-density lipoprotein (HDL) on CD147 expression on human platelets.

Materials and Methods

Washed platelets were incubated with ox-LDL (or native LDL) and HDL or anti-LOX-1 monoclonal antibody prior to incubation with ox-LDL. In parallel, buffer (PBS) was added to washed platelets as a control. The expression levels of CD147, CD62P, CD63 and Annexin V were assessed by flow cytometry, and soluble CD147 from the platelets was assessed by an enzyme-linked immunosorbent assay. Laser scanning microscopy (LSM) and transmission electron microscopy (TEM) were used to visualize the morphological changes and granule release, respectively, from the platelets.

Results

Platelets treated with ox-LDL exhibited a significant increase in the expression of CD147 (or Annexin V), followed by increases in CD62P and CD63, compared with the control group. In contrast, HDL or anti-LOX-1 monoclonal antibody decreased these effects. The expression of soluble CD147 increased as the concentration of ox-LDL used to treat the platelets increased. After exposure to ox-LDL, morphological changes and granule release in the platelets were visualized by LSM and TEM. Additionally, the TEM revealed that HDL inhibits alpha-granule release.

Conclusions

In platelets, ox-LDL stimulates the release of CD147 via binding to LOX-1, whereas HDL inhibits this effect. This finding could provide new insights concerning the influence of ox-LDL and HDL on plaque stability by the up-regulation of CD147 on platelets.     

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells

Objective

Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts.

Methods

We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically.

Results

Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p < 0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone.

Conclusion

3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.     

Origin of myofibroblasts in the fibrotic liver in mice

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl₄) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP⁺ cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl₄ liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.Chronic liver injury of many etiologies results in liver fibrosis. There are two general types of chronic liver diseases, hepatocellular (injury to hepatocytes, such as chronic viral hepatitis and nonalcoholic steatohepatitis) and cholestatic (obstruction to bile flow, such as primary biliary cirrhosis and primary sclerosing cholangitis) (1). Experimental rodent models of liver fibrosis mimic these two types of chronic liver injuries: Repeated carbon tetrachloride (CCl₄) administration produces hepatocelluar injury, and common bile duct ligation (BDL) produces cholestatic injury (2). In all chronic liver diseases, myofibroblasts are embedded in the fibrous scar and are the source of this excessive extracellular matrix (ECM). Myofibroblasts, which are not present in normal liver, are characterized by distinct morphology, contractility with intracellular stress fibers [α-smooth muscle actin (α-SMA), nonmuscle myosin, and vimentin], and secretion of extracellular matrix (fibronectin and fibrillar collagens) (1, 2).The cells of origin of hepatic myofibroblasts are unresolved, and perhaps the fibrosis induced by different types of liver injury results from different fibrogenic cells. Hepatic myofibroblasts may originate from bone marrow (BM)-derived mesenchymal cells and fibrocytes, but only a small contribution of BM-derived cells to the myofibroblast population has been detected in experimental liver fibrosis (3–5). Another potential source of myofibroblast is epithelial-to-mesenchymal transition (EMT), in which epithelial cells acquire a mesenchymal phenotype and may give rise to fully differentiated myofibroblasts. However, recent cell fate mapping studies have failed to detect any hepatic myofibroblasts originating from hepatocytes, cholangiocytes, or epithelial progenitor cells (3, 6–10). Thus, the major sources of myofibroblasts in liver fibrosis are the endogenous liver mesenchymal cells, which consist of portal fibroblasts and hepatic stellate cells.Quiescent hepatic stellate cells (qHSCs) are located in the space of Disse, store retinoids in lipid droplets, and express neural markers, such as glial fibrillary acidic protein (GFAP), synaptophisin, and nerve growth factor receptor p75 (1). In response to injury, qHSCs down-regulate vitamin A-containing lipid droplets and neural markers, and differentiate into α-SMA–expressing myofibroblasts (1, 2). Portal fibroblasts normally comprise a small population of the fibroblastic cells that surround the portal vein to maintain integrity of portal tract. They were first described as “mesenchymal cells not related to sinusoids,” and since then have been called “periductular fibroblasts” or portal/periportal mesenchymal cells” (11) and implicated by association in the pathogenesis of cholestatic liver injury. In response to chronic injury, portal fibroblasts may proliferate, differentiate into α-SMA–expressing myofibroblasts, and synthesize extracellular matrix (11–14).The contribution of portal fibroblasts (PFs) to liver fibrosis of different etiologies is not well understood, mainly because of difficulties in isolating PFs and myofibroblasts. The most widely used method of PF isolation from rats is based on liver perfusion with enzymatic digestion followed by size selection (15). Cell outgrowth from dissected bile segments is still used to isolate mouse PFs, and after 10–14 d in culture, PFs undergo progressive myofibroblastic activation (16). The disadvantage of this technique is that it requires multiple passaging and prolong culturing (11). A more physiological method of PF culturing in a precision-cut liver slice is designed to maintain cell–cell and cell–matrix interactions and mimic natural microenvironment of PFs, but it does not enable the study of purified PFs (17). Therefore, only a few markers of PFs are available to identify PFs in the myofibroblast population, including gremlin, Thy1, fibulin 2, interleukin 6 (IL-6), elastin, the ecto-AT-Pase nucleoside triphosphate diphosphohydrolase-2 (NTPD2), and coffilin 1. In addition, the lack of desmin, cytoglobin, α2-macroglobulin, neural proteins (GFAP, p75, synaptophysin), and lipid droplets distinguishes PFs from HSCs (1, 17–21).Our study uses transgenic reporter mice and new flow cytometry protocols to identify the origin of myofibroblasts and quantify their numbers in two murine models of chronic liver injury (BDL and CCl₄). Our study demonstrates that the origin of the myofibroblasts is determined by the type of liver injury. As previously reported using other methodologies, HSCs are the major source of myofibroblasts in CCl₄ liver injury. In contrast, most of the myofibroblasts at the onset of BDL-induced liver injury originate from activated PFs (aPFs).