Young Seomun

Overexpression of matrix metalloproteinase-2 mediates phenotypic transformation of lens epithelial cells

Transforming growth factor-beta (TGF-beta) is known to be a causative factor in pathological fibrosis and the metastasis of cancer cells, through effects on molecules of the extracellular matrix (ECM). We evaluated the influence of TGF-beta(1) on the gene expression of matrix metalloproteinase-2 (MMP-2) in lens epithelial cells (LECs). The results showed that TGF-beta(1) induced the expression of mRNA for MMP-2 in LECs. Subsequently, in order to examine the role of MMP-2, we overexpressed MMP-2 in LECs by stable transfection. The MMP-2-overexpressing LECs showed typical indicators of a myofibroblast-like cell phenotype, such as multiple layers of cells, elongated morphology, and expression of alpha-smooth muscle actin. We also showed that an MMP inhibitor blocked the TGF-beta(1)-induced morphological change in LECs. These results demonstrate that MMP-2 plays a role in the transformation of LECs, which has implications for the pathological fibrosis of these cells.

MMP‐14 mediated MMP‐9 expression is involved in TGF‐beta1‐induced keratinocyte migration

The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP‐14 in TGF‐β1‐induced keratinocyte migration. TGF‐β1 stimulated cell migration and the expression of MMP‐2, ‐9 in HaCaT human keratinocyte cells. When we lowered MMP‐14 mRNA with siRNA, cell migration, and MMP‐9 expression decreased. Furthermore, the MMP‐14 siRNA also reduced activation of JNK in response to TGF‐β1, and a JNK‐specific inhibitor decreased both cell migration and MMP‐9 expression. Taken together, these results suggest that TGF‐β1‐induced HaCaT cell migration is mediated by MMP‐14, which regulates MMP‐9 expression via JNK signaling. J. Cell. Biochem. 104: 934–941, 2008.

Matrix metalloproteinase (MMP) and TGF-β1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGF-β) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGF-β-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins, and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.

Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling.

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins alpha2 and beta1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin beta1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

The effective control of a bleeding injury using a medical adhesive containing batroxobin

Many types of hemostatic agents have been studied for the effective control of bleeding. In this study, a powdery medical adhesive composed of aldehyded dextran and ε-poly (L-lysine) was used with the recombinant batroxobin. Batroxobin is a venomous component from the snake Bothrops atrox moojeni and catalyzes fibrinogen conversion to form soluble fibrin clots. This research aims to examine the performance of the batroxobin-containing adhesive for hemostasis, and evaluate its potential as a novel hemostatic adhesive. The fibrinogen conversion ability of batroxobin was evaluated by a fibrinogen clotting assay and a whole blood clotting assay. Both experiments demonstrated the effectiveness of the batroxobin-containing adhesive for blood clot formation. Animal experiments were also conducted. After a pricking wound was made in an ICR (imprinting control region) mouse liver, the adhesive and various concentrations of batroxobin were applied. The total amount of blood loss was reduced with increasing concentrations of batroxobin. For excessive bleeding conditions, the femoral artery wound model of SD (Sprague-Dawley) rats was adopted. With higher concentrations of batroxobin, hemostasis was more rapidly achieved. Histological analysis of the liver model also supports the hemostatic effects through fibrin clot formation. In conclusion, batroxobin and medical adhesive effectively facilitate blood coagulation, and could be developed for clinical use.

Functional improvement of hemostatic dressing by addition of recombinant batroxobin

Although a number of natural materials have been used as hemostatic agents, many substances do not act quickly enough. Here, we created a novel dressings using collagen and chitosan with recombinant batroxobin (r-Bat) to promote faster and more effective hemostasis. We hypothesized that r-Bat would promote synergetic blood coagulation because it contains a blood coagulation active site different than those of collagen and chitosan. Our results suggest that each substances can maintain hemostatic properties while in the mixed dressings and that our novel hemostatic dressings promotes potent control of bleeding, as demonstrated by a whole blood assay and rat hemorrhage model. In a rat femoral artery model, the scaffold with a high r-Bat concentration more rapidly controlled excessive bleeding. This novel dressings has enormous possible for rapidly controlling bleeding and it improves upon the effect of collagen and chitosan used alone. Our novel r-Bat dressings is a possible candidate for improving preoperative care and displays promising properties as an absorbable agent in hemostasis. STATEMENT OF SIGNIFICANCE Despite the excellent hemostatic properties of collagen and chitosan pads, they reported to brittle behavior and lack sufficient hemostatic effect within relevant time. Therefore, we created a novel pad using collagen and chitosan with recombinant batroxobin (r-Bat). r-Bat acts as a thrombin-like enzyme in the coagulation cascade. Specifically, r-Bat, in contrast to thrombin, only splits fibrinopeptide A off and does not influence other hemostatic factors or cells, which makes it clinically useful as a stable hemostatic agent. Also the materials in the pad have synergetic effect because they have different hemostatic mechanisms in the coagulation cascade. This report propose the novel hemostatic pad isreasonable that a great potential for excessive bleeding injury and improve effects of natural substance hemostatic pad.

The Role of Nuclear Factor Kappa B in Lens Epithelial Cell Proliferation Using a Capsular Bag Model

Purpose: This study was performed to elucidate the role of nuclear factor ĸB (NF-ĸB) in lens epithelial cell proliferation in the capsular bag model for this study. Methods: Capsular bags were prepared from porcine eyes and cultured in a 5% CO2 atmosphere at 37°C. NF-ĸB translocation was confirmed by immunohistochemistry and Western blot analysis. The effects of sulfasalazine and SN50 peptide, inhibitors of NF-ĸB activation, were observed by light microscopy and scanning electron microscopy. Results: NF-ĸB was found in the cytoplasm of nonproliferated lens epithelial cells. However, NF-ĸB moved to the nucleus in proliferation cells, proliferating-cell-nuclear-antigen-positive cells. This translocation was inhibited by treatment with sulfasalazine or NF-ĸB SN50 peptide. These inhibitors also blocked lens epithelial cell migration from the equatorial to the posterior capsule. Conclusion: NF-ĸB controls proliferation and regulates the growth of lens epithelial cells. In this study, sulfasalazine and NF-ĸB SN50 peptide inhibited cell proliferation in the capsular bag model. These results suggest that the regulation of NF-ĸB in lens epithelial cells may modulate posterior capsule opacification.

Recombinant batroxobin-coated nonwoven chitosan as hemostatic dressing for initial hemorrhage control

The choice of hemostat is determined by the situation and the degree of hemorrhage. One common hemostat, the nonwoven dressing, is easy to handled and controls severe bleeding on wider wounds. In this study, chitosan-based nonwoven dressings with recombinant batroxobin (rBat) were used as efficacious hemostatic dressing agents. Hemostatic agents need to absorb blood quickly in the early stages of blood coagulation cascade to rapidly and effectively control of excessive hemorrhages. To date, most studies of hemostatic agents focused on a single material and hemostats composed of multiple materials have not been studied sufficiently. Thus, we made a chitosan dressing coated with rBat and investigated the microstructure, mechanical properties, hemostatic efficacy, and clotting properties of the coated dressing. Our results showed that the rBat had a synergetic effect on chitosan that improved blood coagulation. Furthermore, the dressing had excellent bleeding control in an Sprague-Dawley (SD) rat femoral artery hemorrhage model. In conclusion, hemostasis can be improved by combining a chitosan-based nonwoven dressing with other agents, and rBat-coated chitosan-based nonwoven dressings have enormous potential to improve blood coagulation.

Acute and repeated dose (28 days) toxicity studies in rats and dogs of recombinant batroxobin, a snake venom thrombin-like enzyme expressed from Pichia pastoris

Abstract Recombinant batroxobin is a thrombin‐like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague‐Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance‐related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin. HighlightsRecombinant batroxobin was purified from the culture broth of P. pastoris with multi‐gram quantity under GMP regulation.Single and 28 days repeated dose toxicity in rats and dogs were examined.Lethal dose, maximum tolerated dose, and no adverse effect level were determined.Clinical signs, hematological parameters, clinical chemistry, and histopathological findings were evaluated.