Many in vitro studies have used cell cultures to focus on the relationships between cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) isoforms. We have investigated the time-course of regulation and the role of COX-2 and iNOS in a model of experimental inflammation in mice, the air pouch injected with zymosan. This study demonstrates that there is an early acute phase (4 h) mediated mainly by eicosanoids, with high levels of prostaglandin E2 (PGE2) produced by cyclo-oxygenase-1. In addition, in the later phase (from 12 h) there is a participation of nitric oxide (NO) and PGE2 accompanied by co-induction of both iNOS and COX-2. These enzymes were detected in migrating leukocytes as well as in macrophages lining the air pouch. Administration of NS398 or indomethacin inhibited PGE2 levels and COX activity, but also nitrite levels and iNOS activity, which was accompanied by a reduction in iNOS expression. Aminoguanidine inhibited nitrite levels and iNOS activity in addition to exerting inhibitory effects on the COX pathway. Treatment of animals with dexamethasone reduced nitrite and PGE2 concentrations in air pouch exudates, as well as iNOS and COX-2 expression in migrating cells. Our results indicate that PGE2 and NO may play in vivo mutual modulatory roles in the inflammatory response caused by zymosan injection into the mouse air pouch, a suitable model to study drugs acting on those pathways.
Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50-75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P < 0.05) and impaired vasodilation (P < 0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P< 0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P < 0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1beta (interleukin-1beta) expression in intact epidermis, TNF-alpha (tumour necrosis factor-alpha) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-beta (transforming growth factor-beta) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.
Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).
The use of replication-defective adenoviruses to deliver effectively to use lactose as an energy source; (3) does transgenes into hepatocytes seems to be a promising not affect urea synthesis, plasma protein synthesis and approach to human liver gene therapy. However, the xenobiotic biotransformation activities (1A2, 2A1, 2B6, effects that the adenovirus-mediated expression of a 3A3/5). Glycolysis was moderately increased (approxforeign gene could have on the expression of other hepatic imately 20%), while gluconeogenesis decreased characteristic genes have not yet been properly examined.(approximately 20%) in transduced hepatocyte; moreover, We have investigated this problem by using human hepa-(4) the expression of inducible genes (acute-phase plasma tocytes infected with a recombinant E-1 defective adenproteins, CYPs) was not impaired in transduced human ovirus that carried a modified lacZ gene. The analysis of hepatocytes upon stimulation with IL-6 or methylcholanthe biochemical functionality of transduced cells showed trene. The results of this research support the idea that that the use of adenovirus: (1) was a very efficient way efficient expression of transgenes can be achieved in to introduce a foreign gene into human hepatocytes (80% human hepatocytes by means of adenoviral transduction, transduced cells after 1 h contact, at an MOI of 15; approxiwithout altering these characteristic hepatic biochemical mately 100% transduced cells at an MOI of 20); (2) allowed functions. the expression of the transgene to levels that enabled cells
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