Monocyte-endothelium-smooth muscle cell interaction in co-culture: proliferation and cytokine productions in response to advanced glycation end products.

BACKGROUND: During hyperglycemia, reducing sugars react with the amino groups to form Amadori products which then form advanced glycation end-products (AGEs). Studies have shown that the AGEs and the receptor binding generated reactive oxygen species, and triggered secretion of cytokines contributing to the local regulations of proliferation and inflammation in cells. Interaction of vessel wall cells and monocytes may trigger the processes leading to atherosclerosis. We evaluated the effects of AGEs on smooth muscle cell (SMC) proliferation and cytokine synthesis in co-cultures of human monocytes (THP-1), endothelial cells (HUVEC) and aortic vascular smooth muscle cell (SMC) to clarify the effects of AGEs on vascular cells and to investigate the mechanisms of arteriosclerosis. METHODS: Glycolaldehyde-induced AGEs (glycol-AGEs) was prepared. The THP-1 and HUVEC were cultured with SMC in transwell plates with 100 µg/ml of glycol-AGEs for 24 to 48 h. RESULTS: The proliferation of SMC was induced by glycol-AGEs in the co-culture system. Moreover, SMC treated with glycol-AGEs also expressed interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the level of cytokines expression was significantly elevated in the co-culture system of HUVEC and THP-1 when treated with glycol-AGEs. CONCLUSION: These results suggest that employing a co-culture system is necessary to investigate the synergistic effects of AGEs on intercellular cellular interactions and it creates a more in vivo-like environment for AGEs implicated atherosclerosis research. GENERAL SIGNIFICANCE: All three cell types are required to be investigated together to understand the effects of AGEs on intercellular interactions occurring among 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-beta1-induced keratinocyte migration. TGF-beta1 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-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.

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 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 kappaB (NF-kappaB) 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% CO(2) atmosphere at 37 degrees C. NF-kappaB translocation was confirmed by immunohistochemistry and Western blot analysis. The effects of sulfasalazine and SN50 peptide, inhibitors of NF-kappaB activation, were observed by light microscopy and scanning electron microscopy. RESULTS: NF-kappaB was found in the cytoplasm of nonproliferated lens epithelial cells. However, NF-kappaB moved to the nucleus in proliferation cells, proliferating-cell-nuclear-antigen-positive cells. This translocation was inhibited by treatment with sulfasalazine or NF-kappaB SN50 peptide. These inhibitors also blocked lens epithelial cell migration from the equatorial to the posterior capsule. CONCLUSION: NF-kappaB controls proliferation and regulates the growth of lens epithelial cells. In this study, sulfasalazine and NF-kappaB SN50 peptide inhibited cell proliferation in the capsular bag model. These results suggest that the regulation of NF-kappaB in lens epithelial cells may modulate posterior capsule opacification.

Coagulant Effect and Tolerability of Yeast-Produced Recombinant Batroxobin in Healthy Adult Subjects.

BACKGROUND AND OBJECTIVE: Batroxobin, a snake venom thrombin-like enzyme, converts fibrinogen into fibrin by cleaving fibrinopeptide A. It is used for hemostasis; however, the supply of native batroxobin is limited. Therefore, we developed a recombinant batroxobin (r-batroxobin) from Pichia pastoris and evaluated its pharmacodynamics and safety in humans. METHODS: A randomized, double-blind, placebo-controlled, single ascending-dose study was performed. Eight healthy subjects were enrolled in each r-batroxobin dose group (2.5, 5.0, or 10.0 BU/2.0 mL administered intravenously), and randomized to receive r-batroxobin (n = 6) or matching placebo (n = 2). Safety was evaluated during the study, and pharmacodynamics was assessed using prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen level. RESULTS: All subjects in each cohort completed the study. No significant changes in PT or aPTT occurred after intravenous r-batroxobin administration. Compared with the placebo group, the fibrinogen level in all r-batroxobin dose groups decreased significantly to 8.68-33.57% from the baseline within 12 h (p ≤ 0.05). The TT in the 5.0 and 10.0 BU/2.0 mL groups significantly increased to 7.53-18.48% from baseline within 12 h compared with that of the placebo group (p ≤ 0.05), whereas that of the 2.5 BU/2.0 mL group exhibited non-significant changes compared with the placebo group. No serious adverse events occurred. CONCLUSIONS: A single intravenous injection of r-batroxobin within a dose range of 2.5-10.0 BU/2.0 mL was well tolerated and resulted in a significant decrease in fibrinogen and prolongation of TT. REGISTRATION: This study is registered at the Clinical Research Information Service (CRIS, http://cris.nih.go.kr ), number KCT0002518.

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.

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.

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.

Induction of p21Cip1-mediated G2/M arrest in H2O2-treated lens epithelial cells.

PURPOSE: Oxidative damage is one of the major factors associated with the formation of age-related cataract and with senescence of various cell types. Although the effects of oxidative stress are complex, we focused on whether oxidative damage affects control of the cell cycle in lens epithelial cells. METHODS: BrdU labeling and FACS analysis were used to investigate the effect of H2O2 on the cell cycle of HLE B-3 cells. In addition, western and Northern blot analysis were performed to assess the expression of cell cycle regulatory proteins and transfection with siRNA was used to knock out expression of p21Cip1. The activation of MAPK family members by oxidative stress was assessed using antibodies to detect the activated forms. To confirm the effect of H2O2 on an ex vivo model, its effect on cultures of the lenses of 3-week-old SD rats were examined. The localization and expression of PCNA and p21Cip1 in the rat lenses were analyzed by immunohistochemistry. RESULTS: FACS analysis showed that H2O2 treatment induced G2/M phase arrest of HLE B-3 cells. p21Cip1 was strongly induced by H2O2, whereas expression of other cell cycle genes was unchanged. Attenuation of p21Cip1 expression using siRNA reduced the H2O2 induced G2/M arrest. Furthermore, JNK and ERK were activated by H2O2 and their specific inhibitors SP600125 (for JNK) and U0126 (for ERK1/2) prevented p21Cip1 expression and blocked cell cycle arrest. H2O2 treatment of a rat lens organ culture also caused an increase in p21Cip1. However, H2O2 treatment lowered the levels of p27Kip1, cdc2, and PCNA in the rat lens culture, unlike in the HLE B-3 cells. CONCLUSIONS: The accumulation of p21Cip1 in lenses exposed to oxidative stress may play a role as a defensive mediator of oxidative damage, an indicator for senescence or aging, or an inducer for the formation of cataract. This finding links oxidative stress with p21Cip1-mediated control of the cell cycle in lens epithelial cells.

Possible role of amyloid beta-(1-40)-BSA conjugates in transdifferentiation of lens epithelial cells.

We investigated whether amyloid beta(Abeta) aggregates have transforming growth factor beta- like cytokine activity and cause transdifferentiation of lens epithelial cells, leading to certain types of cataract. In order to mimic Abetaaggregates, Abeta-(1-40) was crosslinked to bovine serum albumin (BSA) with disuccinimidyl suberate according to a previously described procedure. When human lens epithelial B-3 (HLE B-3) cells were treated with the Abeta-(1-40)-BSA conjugates, we observed the translocation of Smad-3, as well as the induced mRNA levels of fibronectin (FN), collagen type I (Col I), smooth muscle actin (SMA) and matrix metalloproteinase-2 (MMP-2). In addition, we investigated the morphology of rat whole lens cultured for 5 days in the presence of Abeta-(1-40)-BSA, and the immunohistochemical localizations of Abeta-(1-40)/amyloid precursor protein (APP) in human clinical tissues beneath the anterior capsules. In rat whole lens cultures, treatment with Abeta-(1-40)-BSA produced a transformed morphology that had multiple layers of lens epithelial cells. To compare the anterior capsules in anterior subcapsular cataracts with those in nuclear cataracts, immunohistochemical studies of Abeta/APP in human clinical tissues revealed that the predominant immunostaining of Abeta occurs in the anterior epithelial plaques, which likely produces the abnormal extracellular matrix. Thus, these findings suggest that Abeta aggregates in vivo are possibly involved in the regulatory process by which lens epithelial cells may transdifferentiate into fibroblast-like cells, as well as help understand the mechanisms which lead to certain types of cataractogenesis.

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.

Matrix metalloproteinase (MMP) and TGF beta 1-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 (TGFbeta) 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 TGFbeta-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.