Olivier Birot

Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

BackgroundCytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins.ResultsAlthough fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples.ConclusionReal-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression.

Exercise‐Induced Expression of Vascular Endothelial Growth Factor mRNA in Rat Skeletal Muscle is Dependent on Fibre Type

In this study, we quantified the expression of the vascular endothelial growth factor (VEGF) gene in individual muscle fibres at the end of a single 90 min run of 20−25 m min−1, at 10 % incline. In addition, we evaluated the co‐ordinated expression of several hypoxia‐sensitive genes, including the ORP‐150 gene. Individual fibres were taken from rat plantaris muscle, either at the end of a single bout of exercise or at rest, and classified as Type I, IIa, IIx or IIb, according to the expression of myosin heavy chain (MHC) isoforms. VEGF mRNA levels increased by 90 % in exercising whole plantaris in comparison with those in control muscle (P < 0.001), while the VEGF protein content increased by 72 % (P < 0.05). Using real‐time PCR analysis, an accurate and reproducible method for quantification of mRNA levels, a marked rise in VEGF transcript levels was observed at the end of exercise in individual myofibres (P < 0.05), providing the first direct evidence that VEGF transcripts increase in muscle cells after a single bout of exercise. This exercise‐induced increase in VEGF transcript levels was specifically observed in type IIb myofibres, which are predominantly glycolytic and more susceptible to local hypoxia than oxidative myofibres such as type I or IIa fibres (110 %, P < 0.05). Moreover, treadmill exercise increased the expression of two hypoxia‐sensitive genes. The levels of mRNA for Flt‐1, a VEGF‐specific receptor, and those for ORP‐150, a chaperone essential for the secretion of mature VEGF, increased in whole plantaris muscles (108 and 92 %, respectively, P < 0.05). Taken together, these findings are consistent with the suggestion that hypoxia could be one of the mechanisms involved in exercise‐induced capillary growth.

Importance of anti-angiogenic factors in the regulation of skeletal muscle angiogenesis.

Please cite this paper as: Olfert and Birot (2011). Importance of Anti‐angiogenic Factors in the Regulation of Skeletal Muscle Angiogenesis. Microcirculation 18(4), 316–330.

Angio‐adaptation in unloaded skeletal muscle: new insights into an early and muscle type‐specific dynamic process

With a remarkable plasticity, skeletal muscle adapts to an altered functional demand. Muscle angio‐adaptation can either involve the growth or the regression of capillaries as respectively observed in response to endurance training or muscle unloading. Whereas the molecular mechanisms that regulate exercise‐induced muscle angiogenesis have been extensively studied, understanding how muscle unloading can in contrast lead to capillary regression has received very little attention. Here we have investigated the consequences of a 9 day time course hindlimb unloading on both capillarization and expression of angio‐adaptive molecules in two different rat skeletal muscles. Both soleus and plantaris muscles were atrophied similarly. In contrast, our results have shown different angio‐adaptive patterns between these two muscles. Capillary regression occurred only in the soleus, a slow‐twitch and oxidative postural muscle. Conversely, the level of capillarization was preserved in the plantaris, a fast‐twitch and glycolytic muscle. We have also measured the time course protein expression of key pro‐ and anti‐angiogenic signals (VEGF‐A, VEGF‐B, VEGF‐R2, TSP‐1). Our results have revealed that the angio‐adaptive response to unloading was muscle‐type specific, and that an integrated balance between pro‐ and anti‐angiogenic signals plays a determinant role in regulating this process. In conclusion, we have brought new evidence that measuring the ratio between pro‐ and anti‐angiogenic signals in order to evaluate muscle angio‐adaptation was a more accurate approach than analysing the expression of molecular factors taken individually.

Fibre-type specificity of interleukin-6 gene transcription during muscle contraction in rat : association with calcineurin activity

In this study, we quantified the transcription of the interleukin‐6 (IL‐6) gene in individual fibres and the associated changes in calcineurin activity assessed at the cellular level during prolonged muscle contraction. Individual myofibres were isolated from plantaris and soleus muscles of rats at the end of an exhaustive running exercise test (n= 10), categorized according to their myosin heavy chain isoform content, and compared to those of resting rats (n= 10). Using real‐time PCR analysis in individual fibres, a marked rise in IL‐6 transcript levels occurred in type I and IIa fibres at the end of exercise (P < 0.05). Transcription of the gene encoding for the modulatory calcineurin‐interacting protein‐1 (MCIP‐1), a sensitive indicator of calcineurin activity, also mainly increased in type I and IIa fibres (P < 0.05). Moreover, a slight increase in MCIP‐1 mRNA levels was observed in type IIx (P < 0.05). Fibre types determined by immunohistochemistry were qualitatively examined for glycogen content using periodic acid–Shiff staining, and no direct relationship was found, at the cellular level, between glycogen content, fibre‐type and IL‐6 transcription. Our data clearly suggest that IL‐6 gene transcription was mainly observed in early recruited myofibres and that contraction‐induced IL‐6 transcription could be associated with enhanced calcineurin activity.

Voluntary running exercise prevents β-cell failure in susceptible islets of the Zucker diabetic fatty rat

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving β-cell function is uncertain. We evaluated the role of physical activity on β-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with β-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key β-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining β-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and β-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered β-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

Identification of a Mechanism Underlying Regulation of the Anti-Angiogenic Forkhead Transcription Factor FoxO1 in Cultured Endothelial Cells and Ischemic Muscle

Chronic limb ischemia, a complication commonly observed in conjunction with cardiovascular disease, is characterized by insufficient neovascularization despite the up-regulation of pro-angiogenic mediators. One hypothesis is that ischemia induces inhibitory signals that circumvent the normal capillary growth response. FoxO transcription factors exert anti-proliferative and pro-apoptotic effects on many cell types. We studied the regulation of FoxO1 protein in ischemic rat skeletal muscle following iliac artery ligation and in cultured endothelial cells. We found that FoxO1 expression was increased in capillaries within ischemic muscles compared with those from rats that underwent a sham operation. This finding correlated with increased expression of p27(Kip1) and reduced expression of Cyclin D1. Phosphorylated Akt was reduced concurrently with the increase in FoxO1 protein. In skeletal muscle endothelial cells, nutrient stress as well as lack of shear stress stabilized FoxO1 protein, whereas shear stress induced FoxO1 degradation. Endogenous FoxO1 co-precipitated with the E3 ubiquitin ligase murine double minute-2 (Mdm2) in endothelial cells, and this interaction varied in direct relation to the extent of Akt and Mdm2 phosphorylation. Moreover, ischemic muscles had a decreased level of Mdm2 phosphorylation and a reduced interaction between Mdm2 and FoxO1. Our results provide novel evidence that the Akt-Mdm2 pathway acts to regulate endothelial cell FoxO1 expression and illustrate a potential mechanism underlying the pathophysiological up-regulation of FoxO1 under ischemic conditions.

Blunted muscle angiogenic training-response in COPD patients versus sedentary controls

The impaired skeletal muscle of chronic obstructive pulmonary disease (COPD) patients reduces exercise capacity. Similar to the oxidative muscle fibres, the angio-adaptation of muscle to training may be blunted in these patients, as in other chronic conditions. We therefore compared muscle functional responses and angio-adaptations after training in COPD patients and sedentary healthy subjects (SHS). 24 COPD patients (forced expiratory volume in 1 s 45.6±17.5% predicted) and 23 SHS (<150 min·week−1 of moderate-to-vigorous exercise) completed a 6-week rehabilitation programme based on individualised moderate-intensity endurance training. Histomorphological muscle analysis and measurements of pro-angiogenic vascular endothelial growth factor (VEGF)-A and anti-angiogenic thrombospondin (TSP)-1 were conducted before and after training. COPD patients and SHS showed improved symptom-limited oxygen consumption and muscle endurance, although improvements were lower in COPD patients (+0.96±2.4 versus +2.9±2.6 mL·kg−1·min−1, p<0.05, and +65% versus +108%, p=0.06, respectively). The capillary-to-fibre (C/F) ratio increased less in COPD patients than SHS (+16±10% versus +37±20%, p<0.05) and no fibre type switch occurred in COPD patients. The VEGF-A/TSP-1 ratio increased in COPD patients and SHS (+65% versus +35%, p<0.05). Changes in C/F and symptom-limited oxygen consumption were correlated (r=0.51, p<0.05). In addition to a lack of fibre switch, COPD patients displayed a blunted angiogenic response to training.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle.

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre+, FoxO1,3,4L/L, referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

Angiomotin p80/p130 ratio: a new indicator of exercise‐induced angiogenic activity in skeletal muscles from obese and non‐obese rats?

Skeletal muscle capillarisation responds to physiological and pathological conditions with a remarkable plasticity. Angiomotin was recently identified as a new pro‐angiogenic molecule. Angiomotin is expressed as two protein isoforms, p80 and p130. Whereas p80 stimulates endothelial cell migration and angiogenesis, p130 is rather characteristic of stabilized and matured vessels. To date, how angiomotin expression is physiologically regulated in vivo remains largely unknown. We thus investigated (1) whether angiomotin was physiologically expressed in skeletal muscle; (2) whether exercise training, known to stimulate muscle angiogenesis, affected angiomotin expression; and (3) whether such regulation was altered in obesity, a pathological situation often associated with an impaired angiogenic activity and some capillary rarefaction in skeletal muscle. Two models of obesity were used: a high fat diet regime and Zucker Diabetic Fatty rats (ZDF). Our results provide evidence that angiomotin was expressed both in capillaries and myofibres. In non‐obese rats, the p80 isoform was increased in plantaris muscle in response to endurance training whereas p130 was unaffected. In obese animals, no change was observed for p80 whereas training significantly decreased p130 expression. Exercise training induced angiogenesis in plantaris from both obese and non‐obese rats, possibly through the modulation of angiomotin level and its consequences on RhoA–ROCK signalling. In conclusion, any increase in p80 or decrease in p130, as respectively observed in non‐obese and obese animals, led to an increased ratio between p80 and p130 isoforms. This increased angiomotin p80/p130 ratio might then directly reflect the enhanced angiogenic ability of skeletal muscle in response to exercise training.