Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.

Effect of heat treatment on changes in texture, structure and properties of Thai indigenous chicken muscle

Changes in texture, microstructure, colour and protein solubility of Thai indigenous and broiler chicken Pectoralis muscle stripes cooked at different temperatures were evaluated. The change in shear value of both chicken muscles was a significant increase from 50 to 80 degrees C but no change from 80 to 100 degrees C. A significant decrease in fibre diameter was obtained in samples heated to an internal temperature of 60 degrees C and the greatest shrinkage of sarcomeres was observed with internal temperatures of 70-100 and 80-100 C for broiler and indigenous chicken muscles, respectively (P < 0.05). Cooking losses of indigenous chicken muscles increased markedly in the temperature range 80-100 C and were significantly higher than those of the broiler (P < 0.001). With increasing temperature, from 50 to 70 degrees C, cooked chicken muscle became lighter and yellower. Relationships between changes in sarcomere length, fibre diameter, shear value, cooking loss and solubility of muscle proteins were evaluated. It was found that the solubility of muscle protein was very highly correlated with the texture of cooked broiler muscle while sarcomere length changes and collagen solubility were important factors influencing the cooking loss and texture of cooked indigenous chicken muscle. (c) 2004 Elsevier Ltd. All rights reserved.

High temperature reduction of metmyoglobin in aqueous muscle extracts

Oxymyoglobin in aqueous extracts of fresh beef longissimus dorsi muscles was initially oxidised to metmyoglobin during heat treatments at temperatures in the range 50-70 degreesC. The metmyoglobin then underwent reduction to a red pigment that was shown spectrally to be identical to oxymyoglobin. The formation of oxymyoglobin involved a heat induced precipitate that when removed from the solution, allowed oxidation to metmyoglobin to occur. However, on re-addition of the precipitate further reduction to oxymyoglobin took place. Dialysis of the muscle extract prior to heating markedly inhibited the reduction but addition of NADH to the dialysate permitted further reduction. The precipitate plus NADH caused oxymyoglobin formation in the presence of metmyoglobin but neither the precipitate nor NADH alone induced this formation. It is concluded that the initial conversion of oxymyoglobin to metmyoglobin on heating fresh beef muscle extracts was reversible and that the reverse reaction depended on the presence of both NADH and a muscle protein.

Effects of high pressure/thermal treatment on lipid oxidation in beef and chicken muscle

Lipid oxidation was studied in beef and chicken muscle after high pressure treatment (0.1-800 MPa) at different temperatures (20-70 degrees C for 20 min, prior to storage at 4 degrees C for 7 days. Pressure treatment of beef samples at room temperature led to increases in TBARS values after 7 days storage at 4 degrees C; however, the increases were more marked after treatment at pressures >= 400 MPa (at least fivefold) than after treatment at lower pressures (less than threefold). Similar results were found in those samples treated at 40 degrees C, but at 60 degrees C and 70 degrees C pressure had little additional effect on the oxidative stability of the muscle. Pressure treatments of 600 MPa and 800 MPa, at all temperatures. induced increased rates of lipid oxidation in chicken muscle, but, in general, chicken muscle was more stable than beef to pressure. and the catalytic effect of pressure was still seen at the higher temperatures of 50 degrees C, 60 degrees C and 70 degrees C. The addition of 1%, Na(2)EDTA decreased TBARS values of the beef muscle during storage and inhibited the increased rates of lipid oxidation induced by pressure. The inhibition by vitamin E (0.05% w/w) and BHT (0.02% w/w), either alone or in combination, were less marked than seen with Na(2)EDTA, suggesting that transition metal ions released from insoluble complexes are of major importance in catalysing lipid oxidation in pressure-treated muscle foods. (c) 2007 Elsevier Ltd. All rights reserved.

High pressure/thermal treatment effects on the texture of beef muscle

The effects of high pressure (to 800 MPa) applied at different temperatures (20-70 degreesC) for 20 min on beef post-rigor longissimus dorsi texture were studied. Texture profile analysis showed that when heated at ambient pressure there was the expected increase in hardness with increasing temperature and when pressure was applied at room temperature there was again the expected increase in hardness with increasing pressure. Similar results to those found at ambient temperature were found when pressure was applied at 40 degreesC. However, at higher temperatures, 60 and 70 degreesC it was found that pressures of 200 MPa caused large and significant decreases in hardness. The results found for hardness were mirrored by those for gumminess and chewiness. To further understand the changes in texture observed, intact beef longissimus dorsi samples and extracted myofibrils were both subjected to differential scanning calorimetry after being subjected to the same pressure/temperature regimes. As expected collagen was reasonably inert to pressure and only at temperatures of 60-70 degreesC was it denatured/unfolded. However, myosin was relatively easily unfolded by both pressure and temperature and when pressure denatured a new and modified structure was formed of low thermal stability. Although this new structure had low thermal stability at ambient pressure it still formed in both the meat and myofibrils when pressure was applied at 60 degreesC. It seems unlikely that structurally induced changes can be a major cause of the significant loss of hardness observed when beef is treated at high temperature (60-70 degreesC) and 200 MPa and it is suggested that accelerated proteolysis under these conditions is the major cause. (C) 2004 Elsevier Ltd. All rights reserved.

A comparison of the aroma volatiles and fatty acid compositions of grilled beef muscle from Aberdeen Angus and Holstein-Friesian steers fed diets based on silage or concentrates

This paper compares the volatile compound and fatty acid compositions of grilled beef from Aberdeen Angus and Holstein-Friesian steers slaughtered at 14 months, each breed fed from 6 months on either cereal-based concentrates or grass silage. Linoleic acid levels were higher in the muscle of concentrates-fed animals, which in the cooked meat resulted in increased levels of several compounds formed from linoleic acid decomposition. Levels of alpha-linolenic acid, and hence some volatile compounds derived from this fatty acid, were higher in the meat from the silage-fed steers. 1-Octen-3-ol, hexanal, 2-pentylfuran, trimethylamine, cis- and trans-2-octene and 4,5-dimethyl-2-pentyl-3-oxazoline were over 3 times higher in the steaks from the concentrates-fed steers, while grass-derived 1-phytene was present at much higher levels in the beef from the silage-fed steers. Only slight effects of breed were observed. (C) 2004 Elsevier Ltd. All rights reserved.

Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy

The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting andin vitrokinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21CIP1- and p27KIP1- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.

Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy

We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.

Differential protein expression and subcellular distribution of TGFβ1, β2 and β3 in cardiomyocytes during pressure overload-induced hypertrophy

The transforming growth factorβ(TGFβ) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFβ1,β2andβ3in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFβ1,β2andβ3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFβ1levels in LV tissue of AC rats increased significantly from d1–d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFβ3levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14–d42,P<0.03). No significant difference in TGFβ2levels were observed from SH and AC rats after operation. Antibodies to TGFβ1stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFβ1to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFβ3stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFβ3expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFβ3immunofluorescence in myocytes. Antibodies to TGFβ2stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFβ1,β2andβ3in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.

Effects of dietary fat modification on skeletal muscle fatty acid handling in the metabolic syndrome

Objective: In the metabolic syndrome (MetS), increased fat storage in ‘nonadipose’ tissues such as skeletal muscle may be related to insulin resistance (‘lipid overflow’ hypothesis). The objective of this study was to examine the effects of dietary fat modification on the capacity of skeletal muscle to handle dietary and endogenous fatty acids (FAs). Subjects and Methods: In total, 29 men with the MetS were randomly assigned to one of four diets for 12 weeks: a high-fat saturated fat diet (HSFA, n=6), a high-fat monounsaturated fat diet (HMUFA, n=7) and two low-fat high-complex carbohydrate diets supplemented with (LFHCCn−3, n=8) or without (LFHCC, n=8) 1.24 g per day docosahexaenoic and eicosapentaenoic acid. Fasting and postprandial skeletal muscle FA handling was examined by measuring arteriovenous concentration differences across the forearm muscle. [2H2]-palmitate was infused intravenously to label endogenous triacylglycerol (TAG) and free fatty acids in the circulation and subjects received a high-fat mixed meal (2.6 MJ, 61 energy% fat) containing [U-13C]-palmitate to label chylomicron-TAG. Results: Postprandial circulating TAG concentrations were significantly lower after dietary intervention in the LFHCCn−3 group compared to the HSFA group (ΔiAUC −139±67 vs 167±70 μmol l−1 min−1, P=0.009), together with decreased concentrations of [U-13C]-labeled TAG, representing dietary FA. Fasting TAG clearance across forearm muscle was decreased on the HSFA diet, whereas no differences were observed in postprandial forearm muscle FA handling between diets. Conclusion: Chronic manipulation of dietary fat quantity and quality did not affect forearm muscle FA handling in men with the MetS. Postprandial TAG concentrations decreased on the LFHCCn−3 diet, which could be (partly) explained by lower concentration of dietary FA in the circulation.

Influence of birth weight on gene regulators of lipid metabolism and utilization in subcutaneous adipose tissue and skeletal muscle of neonatal pigs.

Epidemiological studies suggest that low-birth weight infants show poor neonatal growth and increased susceptibility to metabolic syndrome, in particular, obesity and diabetes. Adipose tissue development is regulated by many genes, including members of the peroxisome proliferator-activated receptor (PPAR) and the fatty acid-binding protein (FABP) families. The aim of this study was to determine the influence of birth weight on key adipose and skeletal muscle tissue regulating genes. Piglets from 11 litters were ranked according to birth weight and 3 from each litter assigned to small, normal, or large-birth weight groups. Tissue samples were collected on day 7 or 14. Plasma metabolite concentrations and the expression of PPARG2, PPARA, FABP3, and FABP4 genes were determined in subcutaneous adipose tissue and skeletal muscle. Adipocyte number and area were determined histologically. Expression of FABP3 and 4 was significantly reduced in small and large, compared with normal, piglets in adipose tissue on day 7 and in skeletal muscle on day 14. On day 7, PPARA and PPARG2 were significantly reduced in adipose tissue from small and large piglets. Adipose tissue from small piglets contained more adipocytes than normal or large piglets. Birth weight had no effect on adipose tissue and skeletal muscle lipid content. Low-birth weight is associated with tissue-specific and time-dependent effects on lipid-regulating genes as well as morphological changes in adipose tissue. It remains to be seen whether these developmental changes alter an individual's susceptibility to metabolic syndrome.

Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism

Adult skeletal muscle possesses a resident stem cell population called satellite cells which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration but is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. Here the process of satellite cell migration has been investigated revealing that they undergo two distinct phases of movement; firstly under the basal lamina and then rapidly increasing their velocity when on the myofibre surface. Most significantly we show that satellite cells move using a highly dynamic blebbing based mechanism and not via lamellopodia mediated propulsion. We show that nitric oxide and non-canonical Wnt signalling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.

A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity

The major component of skeletal muscle is the myofibre. Genetic intervention inducing over-enlargement of myofibres beyond a certain threshold through acellular growth causes a reduction in the specific tension generating capacity of the muscle. However the physiological parameters of a genetic model that harbours reduced skeletal muscle mass have yet to be analysed. Genetic deletion of Meox2 in mice leads to reduced limb muscle size and causes some patterning defects. The loss of Meox2 is not embryonically lethal and a small percentage of animals survive to adulthood making it an excellent model with which to investigate how skeletal muscle responds to reductions in mass. In this study we have performed a detailed analysis of both late foetal and adult muscle development in the absence of Meox2. In the adult, we show that the loss of Meox2 results in smaller limb muscles that harbour reduced numbers of myofibres. However, these fibres are enlarged. These myofibres display a molecular and metabolic fibre type switch towards a more oxidative phenotype that is induced through abnormalities in foetal fibre formation. In spite of these changes, the muscle from Meox2 mutant mice is able to generate increased levels of specific tension compared to that of the wild type.