Evaluation of the modulating effect of epidermal growth factor receptor inhibitor cetuximab in carbon-tetrachloride induce hepatic fibrosis in rats.

Liver fibrosis, developed in almost all chronic liver injuries. Epidermal growth factor receptors (EGFR) have been thought to contribute to cirrhosis and liver fibrosis. Therefore, using a rat model of carbon tetrachloride (CCl4)-induced liver fibrogenesis, we investigated the preventive effects of cetuximab, an inhibitor of the EGF receptor (EGFR). Ameliorative effects of cetuximab were examined in rats, brought on by biweekly doses of 50 mg/kg of carbon tetrachloride (CCl4). There were a total of 24 male Long Evans rats split up into four distinct groups such as control, CCl4, control+cetuximab and CCl4+cetuximab. After two weeks of treatment with cetuximab (100 µg/kg), samples of tissue and blood were taken after all the rats had been sacrificed. Plasma samples were examined for the biochemical indicators of inflammation and oxidative stress. Histological staining on liver sections was performed for morphologic pathologies, and related genes expressions analysis were done with RT-PCR in liver tissue. The findings showed that cetuximab could raise the levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and considerably lower the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), and nitric oxide (NO). Sirius red staining and hematoxylin-eosin (H&E) displayed that cetuximab therapy reduced the inflammatory cells infiltration and enhanced fibrotic lesions. In the meantime, cetuximab therapy also dramatically reduces the expression of genes linked to inflammation in the liver tissue, including NF-кB, iNOS, IL-6, TNF-α, and TGF-ß. To sum up, the anti-inflammatory, antifibrotic, and antioxidant properties of cetuximab confer curative efficacy against liver fibrosis.

Protective effects of l-carnitine on isoprenaline -induced heart and kidney dysfunctions: Modulation of inflammation and oxidative stress-related gene expression in rats.

The aim of this study was to evaluate the effect of l-carnitine (L-CAR) treatment on isoprenaline (ISO) administered kidney and heart impairment in male Long Evans rats. Four groups of rats were engaged in this study such as control, ISO, control + L-CAR, and ISO + L-CAR, where n = 6 in each group. The rats were also provided with chow food and water ad libitum. At the end of the study, all rats were sacrificed, and blood and tissue samples were collected for bio-chemical analysis. Oxidative stress parameters and antioxidant enzyme activities were determined in plasma and tissues. Antioxidant and inflammatory genes expression were analyzed in the kidney cortex, and histopathological studies of kidney tissues were performed. This study showed that creatinine and uric acid in plasma were significantly increased in ISO-administered rats. l-carnitine treatment lowered the uric acid and creatinine level. ISO-administered rats showed increased lipid peroxidation and declined levels of antioxidant enzymes activities in kidneys and heart. l-carnitine treatment restored antioxidant enzymes activities and protect against oxidative stress in kidney and heart. This effect is correlated with the restoration of Nrf-2-HO-1 genes expression followed by increased SOD and catalase genes expression in the kidney. l-carnitine treatment also prevented the TNF-α, IL-6, and NF-кB expression in kidneys of ISO administered rats. Histopathology staining showed that l-carnitine treatment prevented kidney damage and collagen deposition in ISO administered rats. The result of this study exhibited that l-carnitine treatment reduced oxidative stress and increased antioxidant enzyme activities by enhancing antioxidant genes expression in ISO administered rats.

Crataeva nurvala Bark (Capparidaceae) Extract Modulates Oxidative Stress-Related Gene Expression, Restores Antioxidant Enzymes, and Prevents Oxidative Stress in the Kidney and Heart of 2K1C Rats.

Objective: Crataeva nurvala is a medicinal plant, which contains a wide range of polyphenolic and bioactive compounds. The aim of the study was to evaluate the renal-protective activity of Crataeva nurvala in two-kidney, one-clip (2K1C) rats. Methods: In this study, the ethanol extract of Crataeva nurvala bark at a dose of 100 mg/kg was orally used to treat 2K1C rats for four weeks. At the end of the experiment, all rats were sacrificed and tissue samples were collected for further biochemical and histological assessments. Results: This investigation showed that Crataeva nurvala treatment prevented the kidney dysfunction in 2K1C rats. Uric acid and creatinine concentration and CK-MB activities increased in 2K1C rats which were normalized by Crataeva nurvala. 2K1C rats also showed increased oxidative stress, depicted by the elevated level of MDA, NO, and APOP in plasma and tissues. Oxidative stress parameters declined in 2K1C rats by the treatment of Crataeva nurvala. These results could be attributed to the restoration of antioxidant enzyme activities such as catalase and SOD. Crataeva nurvala extracts also upregulated antioxidant gene expression in the kidneys of 2K1C rats. Moreover, several anti-inflammatory genes were suppressed by Crataeva nurvala treatment in 2K1C rats. Furthermore, fibrosis and collagen deposition in the kidneys were also lowered by the treatment of the Crataeva nurvala extract. Conclusion: The experimental data suggest that the Crataeva nurvala extract protected renal damage and oxidative stress, probably by restoring antioxidant enzymes activities in 2K1C rats.

Resveratrol treatment modulates several antioxidant and anti-inflammatory genes expression and ameliorated oxidative stress mediated fibrosis in the kidneys of high-fat diet-fed rats.

Objective: Resveratrol is a polyphenolic compound that possesses strong antioxidant and anti-inflammatory activities. This study evaluated the effects of resveratrol on oxidative stress, fibrosis and multiple genes regulation in the kidneys of high fat (HF) diet-fed rats. Methods: Wistar rats were fed with HF diet for eight weeks. These rats were also treated with resveratrol for eight weeks. Finally, kidney tissue samples were isolated from all sacrificed rats. The histological changes, creatinine and uric acid levels, oxidative stress parameters such as malondialdehyde (MDA), nitric oxide, and advanced oxidation protein product (AOPP) levels were analyzed. The antioxidant enzymes such as catalase, superoxide dismutase (SOD) activities and reduced glutathione (GSH) levels; gene expression of inflammatory and fibrosis-related genes namely, inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), transforming growth factor beta1 (TGF-ß1), and collagen-1 were assessed. Moreover, gene expression of oxidative stress-related genes such as nuclear factor erythroid 2-related factor 2 (Nrf-2), SOD, catalase, and glutathione reductase, were also assessed. Results: HF diet-fed rats showed increased creatinine and uric acid levels in plasma which were lowered by resveratrol treatment. The study findings also revealed that resveratrol counterbalanced the oxidative stress and prevented the expression of the inflammatory genes; restored the catalase and SOD activities followed by the up-regulation of antioxidant genes expression in the kidneys of HF diet-fed rats. HF diet caused the Nrf-2 down-regulation followed by the decreased expression of HO-1 and HO-2 genes, which was restored by resveratrol treatment. Moreover, the histological assessment showed lipotoxicity and increased fibrosis in the kidneys of HF diet-fed rats. Resveratrol prevented the kidney fibrosis probably by limiting oxidative stress, inflammation, and down-regulating TGF-ß1 mediated signaling pathway. Conclusion: In conclusion, resveratrol treatment showed beneficial effects in preventing oxidative stress and fibrosis in the kidneys of HF diet-fed rats probably by modulating the gene expression of oxidative stress and inflammation related factors and enzymes.

Metformin treatment reverses high fat diet- induced non-alcoholic fatty liver diseases and dyslipidemia by stimulating multiple antioxidant and anti-inflammatory pathways.

PURPOSE: This current study investigated the effect of metformin treatment on hepatic oxidative stress and inflammation associated with nonalcoholic fatty liver disease (NADLD) in high fat diet (HFD) fed rats. METHOD: Wistar rats were fed with a HFD or laboratory chow diet for 8 weeks. Metformin was administered orally at a dose of 200 mg/kg. Body weight, food and water intake were recorded on daily basis. Oral glucose tolerance test (OGTT), biochemical analysis and histological examinations were conducted on plasma and tissue samples. Antioxidant and anti-inflammatory mRNA expression was analyzed using reverse transcription polymeric chain reaction (RT-PCR). RESULTS: Metformin treatment for 8 weeks prevented HFD-induced weight gain and decreased fat deposition in HFD fed rats. Biochemical analysis revealed that metformin treatment significantly attenuated nitro-oxidative stress markers malondialdehyde (MDA), advanced protein oxidation product (APOP), and excessive nitric oxide (NO) levels in the liver of HFD fed rats. Gene expression analysis demonestrated that metformin treatment was associated with an enhanced expression of antioxidant genes such as Nrf-2, HO-1, SOD and catalase in liver of HFD fed rats. Metformin treatment also found to modulate the expression of fat metabolizing and anti-inflammatory genes including PPAR--γ, C/EBP-α, SREBP1c, FAS, AMPK and GLUT-4. Consistent with the biochemical and gene expression data, the histopathological examination unveiled that metformin treatment attenuated inflammatory cells infiltration, steatosis, hepatocyte necrosis, collagen deposition, and fibrosis in the liver of HFD fed rats. CONCLUSION: In conclusion, this study suggests that metformin might be effective in the prevention and treatment of HFD-induced steatosis by reducing hepatic oxidative stress and inflammation in the liver.

Supplementation of cumin seed powder prevents oxidative stress, hyperlipidemia and non-alcoholic fatty liver in high fat diet fed rats.

The present investigation was an attempt to evaluate the hypoglycemic, lipid-lowering, antioxidant and hepatoprotective effects of cumin (Cuminum cyminum family: Apiaceae) supplementation in high fat (HF) diet fed rats. Male Wistar rats were divided into four groups, such as control, control+ cumin, HF and HF+ cumin. Oral glucose tolerance test, plasma lipids, oxidative stress parameters, antioxidant enzymes activities, and liver dysfunction marker enzyme activities were evaluated. Additionally, histological staining of liver tissue was performed to evaluate the inflammatory cells infiltration, iron deposition and fibrosis. The current investigation demonstrated that 1% (w/w) supplementation of cumin powder significantly reduced HF diet-induced glucose intolerance, epididymal and mesenteric fat wet weights and lipid parameters like triglycerides, total cholesterol and low-density lipoproteins. Oxidative stress-related biomarkers including thiobarbituric acid reactive substances (TBARS), nitric oxide (NO) and advanced oxidation protein product (APOP) were also reduced by cumin supplementation. Moreover, HF-diet increased the activity of hepatic biomarker enzymes such as alanine transaminase (ALT) and alkaline phosphatase (ALP) activities which were significantly reduced by cumin powder supplementation. On the other hand, cumin powder supplementation was able to restore the reduced glutathione level with parallel augmentation of the antioxidant enzymes activities such as superoxide dismutase (SOD) and catalase in liver of HF diet-fed rats. Additionally, histological assessments confirmed that cumin powder supplementation also normalized the fat droplet deposition and inflammatory cells infiltration in the liver of HF diet-fed rats. This study suggests that cumin powder supplementation ameliorates dyslipidemia, oxidative stress and hepatic damage in HF diet-fed rats.

Flacourtia indica fruit extract modulated antioxidant gene expression, prevented oxidative stress and ameliorated kidney dysfunction in isoprenaline administered rats.

This study evaluated the effect of Flacourtia indica fruit extract against isoprenaline (ISO) induced renal damage in rats. This investigation showed that ISO administration in rats increased the level oxidative stress biomarkers such as malondialdehyde (MDA), nitric oxide (NO), advanced protein oxidation product (APOP) in kidneys followed by a decrease in antioxidant enzymes functions. Flacourtia indica fruit extract, which is rich in strong antioxidants, also reduced the MDA, NO and APOP level in kidney of ISO administered rats. Inflammation and necrosis was also visible in kidney section of ISO administered rats which was significantly prevented by atenolol and Flacourtia indica fruit extract. Moreover, atenolol and Flacourtia indica fruit extract also modulated the genes expressions related to inflammation and oxidative stress in kidneys. The beneficial effects could be attributed to the presence of a number of phenolic antioxidants. This study suggests that Flacourtia indica fruit extract may prevent kidney dysfunction in ISO administered rats, probably by preventing oxidative stress and inflammation.

Leaf powder supplementation of Senna alexandrina ameliorates oxidative stress, inflammation, and hepatic steatosis in high-fat diet-fed obese rats.

Obesity is an enduring medical issue that has raised concerns around the world. Natural plant extracts have shown therapeutic potential in preventing oxidative stress and inflammation related to obesity complications. In this study, Senna alexandrina Mill. leaves were utilized to treat high-fat diet-related metabolic disorders and non-alcoholic fatty liver diseases. Plasma biochemical assays were conducted to determine the lipid profiles and oxidative stress parameters, and the gene expression of antioxidant enzymes and inflammatory mediators was measured. Histological stained livers of high-fat diet-fed rats were observed. S. alexandrina leaf powder supplementation prevented the increase in cholesterol and triglyceride levels in high-fat diet-fed rats. Moreover, S. alexandrina leaves also reduced lipid peroxidation and nitric oxide production in these rats. Prevention of oxidative stress by S. alexandrina leaf supplementation in high-fat diet-fed rats is regulated by enhancing the antioxidant enzyme activity, followed by the restoration of corresponding gene expressions, such as NRF-2, HO-1, SOD, and CAT. Histological staining provides further evidence that S. alexandrina leaf supplementation prevents inflammatory cell infiltration, lipid droplet deposition, and fibrosis in the liver of high-fat diet-fed rats. Furthermore, this investigation revealed that S. alexandrina leaf supplementation controlled non-alcoholic fatty liver disease by modulating the expression of fat metabolizing enzymes in high-fat diet-fed rats. Therefore, S. alexandrina leaf supplementation inhibits fatty liver inflammation and fibrosis, suggesting its usefulness in treating non-alcoholic steatohepatitis. Thus, this natural leaf extract has potential in treatment of obesity related liver dysfunction.

Etoricoxib treatment prevented body weight gain and ameliorated oxidative stress in the liver of high-fat diet-fed rats.

The main focus of this study was to determine the role of etoricoxib in counterbalancing the oxidative stress, metabolic disturbances, and inflammation in high-fat (HF) diet-induced obese rats. To conduct this study, 28 male Wistar rats (weighing 190-210 g) were distributed randomly into four groups: control, control + etoricoxib, HF, and HF + etoricoxib. After 8 weeks of treatment with etoricoxib (200 mg/kg), all the animals were sacrificed followed by the collection of blood and tissue samples in order to perform biochemical tests along with histological staining on hepatic tissues. According to this study, etoricoxib treatment prevented the body weight gain in HF diet-fed rats. Furthermore, rats of HF + etoricoxib group exhibited better blood glucose tolerance than the rats of HF diet-fed group. In addition, etoricoxib also markedly normalized HF diet-mediated rise of hepatic enzyme activity. Etoricoxib treatment lowered the level of oxidative stress indicators significantly with a parallel augmentation of antioxidant enzyme activities. Furthermore, etoricoxib administration helped in preventing inflammatory cell invasion, collagen accumulation, and fibrotic catastrophe in HF diet-fed rats. The findings of the present work are suggestive of the helpful role of etoricoxib in deterring the metabolic syndrome as well as other deleterious pathological changes afflicting the HF diet-fed rats.

Canagliflozin ameliorates renal oxidative stress and inflammation by stimulating AMPK-Akt-eNOS pathway in the isoprenaline-induced oxidative stress model.

Diabetes is a leading cause of chronic kidney disease, and the high prevalence of sympathetic nervous system (SNS) hyperactivity in diabetic patients makes them further susceptible to SNS-mediated oxidative stress and accelerated kidney damage. Here, we investigated if canagliflozin can reverse isoprenaline (ISO)-induced renal oxidative damage in rats, a model that mimics SNS overstimulation-induced organ injuries in humans. We found that ISO administration elevates renal oxidative stress markers including malondialdehyde (MDA), advanced protein oxidation product (APOP), myeloperoxidase (MPO) and nitric oxide (NO), while depleting levels of endogenous antioxidants such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH). Strikingly, canagliflozin treatment of ISO-treated rats not only prevents elevation of oxidative stress markers but also rescues levels of depleted antioxidants. Our results also show that canagliflozin stimulates antioxidant/anti-inflammatory signaling pathways involving AMP-activated protein kinase (AMPK), Akt and eNOS, and inhibits iNOS and NADPH oxidase isoform 4 (NOX4), all of which are associated with oxidative stress and inflammation. Further, canagliflozin prevents ISO-induced apoptosis of kidney cells by inhibiting Bax protein upregulation and caspase-3 activation. Histological examination of kidney sections reveal that canagliflozin attenuates ISO-mediated increases in inflammatory cell infiltration, collagen deposition and fibrosis. Finally, consistent with these findings, canagliflozin treatment improves kidney function in ISO-treated rats, suggesting that the antioxidant effects may be clinically translatable.

Canagliflozin attenuates isoprenaline-induced cardiac oxidative stress by stimulating multiple antioxidant and anti-inflammatory signaling pathways.

The antidiabetic drug canagliflozin is reported to possess several cardioprotective effects. However, no studies have investigated protective effects of canagliflozin in isoprenaline (ISO)-induced cardiac oxidative damage-a model mimicking sympathetic nervous system (SNS) overstimulation-evoked cardiac injuries in humans. Therefore, we investigated protective effects of canagliflozin in ISO-induced cardiac oxidative stress, and their underlying molecular mechanisms in Long-Evans rat heart and in HL-1 cardiomyocyte cell line. Our data showed that ISO administration inflicts pro-oxidative changes in heart by stimulating production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In contrast, canagliflozin treatment in ISO rats not only preserves endogenous antioxidants but also reduces cardiac oxidative stress markers, fibrosis and apoptosis. Our Western blotting and messenger RNA expression data demonstrated that canagliflozin augments antioxidant and anti-inflammatory signaling involving AMP-activated protein kinase (AMPK), Akt, endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In addition, canagliflozin treatment attenuates pro-oxidative, pro-inflammatory and pro-apoptotic signaling mediated by inducible nitric oxide synthase (iNOS), transforming growth factor beta (TGF-ß), NADPH oxidase isoform 4 (Nox4), caspase-3 and Bax. Consistently, canagliflozin treatment improves heart function marker in ISO-treated rats. In summary, we demonstrated that canagliflozin produces cardioprotective actions by promoting multiple antioxidant and anti-inflammatory signaling.

A simple strategy for robust preparation and characterisation of hydrogels derived from chitosan and amino functional monomers for biomedical applications.

Molecular interactions of amino functional (AF) monomers with chitosan (CS) lead to the formation of external stimuli responsive hydrogels (HGs). These have the potential to produce biomaterials with novel properties by a simple blending approach. Six independent AF monomers such as diethylenetriamine (DETA), bis(3-aminopropyl)amine (BAPA), 3,3'-diamino-N-methyldipropyleamine (DAMPA), hexamethylenediamine (HMDA), N,N-dimethylethylamine (DMEA) and diethylamine (DEA) with distinct functional groups and chain lengths were designed to form stable HGs at physiological pH. Such AF monomers are able to form HGs within a short time (in the range from 10 to 19 seconds) by physically interacting with CS. This is an alternative to the covalently crosslinking reaction process, providing cost effective HG biomaterials. HG complexes were characterized by rheometry, differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. The interaction between AF monomers and the CS polymer has been discussed and the results have been confirmed by FTIR analysis. The storage modulus (G'), loss modulus (G'') and complex viscosity (η*) were evaluated for all HGs using a rheometer, and the ratios of CS and the particular AF monomer were optimized for stable HG formation. The swelling ratio was evaluated using a simple method and was found to be directly related to the structure of the AF monomer, pH and temperature. These HGs were utilised for encapsulation, and the release of active molecules (e.g., reactive red 120 (RR120) as a model compound) was measured at low pH 5.5, physiological pH 7.4 and high pH 9.5. The cell viability and cellular compatibility of the HGs were evaluated in vitro cell culture, demonstrating that all the five different types of HGs support cellular compatibility, attachment and growth. The physical mixing of AF monomers with CS is expedited for the development of new bespoke economically viable biomaterials.

Designing Smart Biomaterials for Tissue Engineering.

The engineering of human tissues to cure diseases is an interdisciplinary and a very attractive field of research both in academia and the biotechnology industrial sector. Three-dimensional (3D) biomaterial scaffolds can play a critical role in the development of new tissue morphogenesis via interacting with human cells. Although simple polymeric biomaterials can provide mechanical and physical properties required for tissue development, insufficient biomimetic property and lack of interactions with human progenitor cells remain problematic for the promotion of functional tissue formation. Therefore, the developments of advanced functional biomaterials that respond to stimulus could be the next choice to generate smart 3D biomimetic scaffolds, actively interacting with human stem cells and progenitors along with structural integrity to form functional tissue within a short period. To date, smart biomaterials are designed to interact with biological systems for a wide range of biomedical applications, from the delivery of bioactive molecules and cell adhesion mediators to cellular functioning for the engineering of functional tissues to treat diseases.

Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue-engineering strategies; translational issues.

Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l-lactic acid)-poly(ε-caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow-derived skeletal stem cells (SSCs) were implanted into mid-shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro-CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical-sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale-up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale-up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase.

Stimulation of fat storage by prostacyclin and selective agonists of prostanoid IP receptor during the maturation phase of cultured adipocytes.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

Fabrication of polymeric biomaterials: a strategy for tissue engineering and medical devices.

Polymeric biomaterials have a significant impact in today's health care technology. Polymer hydrogels were the first experimentally designed biomaterials for human use. In this article the design, synthesis and properties of hydrogels, derived from synthetic and natural polymers, and their use as biomaterials in tissue engineering are reviewed. The stimuli-responsive hydrogels with controlled degradability and examples of suitable methods for designing such biomaterials, using multidisciplinary approaches from traditional polymer chemistry, materials engineering to molecular biology, have been discussed. Examples of the fabrication of polymer-based biomaterials, utilized for various cell type manipulations for tissue re-generation are also elaborated. Since a highly porous three-dimensional scaffold is crucially important in the cellular process, for tissue engineering, recent advances in the effective methods of scaffold fabrication are described. Additionally, the incorporation of factor molecules for the enhancement of tissue formation and their controlled release is also elucidated in this article. Finally, the future challenges in the efficient fabrication of effective polymeric biomaterials for tissue regeneration and medical device applications are discussed.

Stabilizing hepatocellular phenotype using optimized synthetic surfaces.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes.

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4-6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.

Cultured preadipocytes undergoing stable transfection with cyclooxygenase-1 in the antisense direction accelerate adipogenesis during the maturation phase of adipocytes.

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.