Emilie Roudier

HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage

3‐Hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase inhibitors, statins, are widely used cholesterol‐lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53‐specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody‐specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3‐kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5‐fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA‐damaged cells, half‐lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin‐treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR‐dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.

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.

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.

Inhibition of Proliferation, Migration and Proteolysis Contribute to Corticosterone-Mediated Inhibition of Angiogenesis

The angiostatic nature of pharmacological doses of glucocorticoid steroids is well known. However, the consequences of pathophysiological elevation of endogenous glucocorticoids are not well established. In the current study, we hypothesized that the angiostatic effect of corticosterone, an endogenous glucocorticoid in rodents, occurs through multi-faceted alterations in skeletal muscle microvascular endothelial cell proliferation, migration, and proteolysis. Chronic corticosterone treatment significantly reduced the capillary to fiber ratio in the tibialis anterior muscle compared to that of placebo-treated rats. Corticosterone inhibited endothelial cell sprouting from capillary segments ex vivo. Similarly, 3-dimensional endothelial cell spheroids treated with corticosterone for 48 hours showed evidence of sprout regression and reduced sprout length. Endothelial cell proliferation was reduced in corticosterone treated cells, coinciding with elevated FoxO1 and reduced VEGF production. Corticosterone treated endothelial cells exhibited reduced migration, which correlated with a reduction in RhoA activity. Furthermore, corticosterone treated endothelial cells in both 3-dimensional and monolayer cultures had decreased MMP-2 production and activation resulting in decreased proteolysis by endothelial cells, limiting their angiogenic potential. Promoter assays revealed that corticosterone treatment transcriptionally repressed MMP-2, which may map to a predicted GRE between −1510 and −1386 bp of the MMP-2 promoter. Additionally, Sp1, a known transcriptional activator of MMP-2 was decreased following corticosterone treatment. This study provides new insights into the mechanisms by which pathophysiological levels of endogenous glucocorticoids may exert angiostatic effects.

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.

Considering the role of pyruvate in tumor cells during hypoxia.

Impairment of oxygen supply occurs in many pathological situations. In the case of cancer, both chronic and acute hypoxic areas are found in the tumor. Tumor hypoxia is associated with poor clinical prognoses and is correlated with tumor growth and metastasis development. Pyruvate is a common metabolite, as it is an end-product of glycolysis and an energy substrate for the mitochondrial Krebs cycle. It is also well known for its protective properties against stressful conditions, particularly hypoxia. Its presence determines cellular fate when there is a lack of oxygen. Interestingly, pyruvate metabolism is altered during cancer development. For years, this was assumed to be a consequence of malignant transformation. However, it now is becoming clear that pyruvate could contribute to cancer progression. The role of pyruvate during hypoxia has been widely studied in non-tumor tissues and cells; it is less documented whether or not the protective effect of pyruvate could also take place in cancer cells. If so, pyruvate might be deleterious for cancer patients. The present paper reviews data that highlight the role of pyruvate in cancer cells and tumors during hypoxic stress.

Murine double minute-2 expression is required for capillary maintenance and exercise-induced angiogenesis in skeletal muscle

Exercise‐induced angiogenesis is a key determinant of skeletal muscle function. Here, we investigated whether the E3 ubiquitin ligase murine double minute‐2 (Mdm2) exerts a proangiogenic function in exercised skeletal muscle. Mdm2 hypomorphic (Mdm2Puro/Δ7‐9) mice have a 60% reduction in Mdm2 expression compared with that in wild‐type animals. Capillary staining on muscle sections from Mdm2Puro/Δ7‐9 sedentary mice with a wild‐type or knockout background for p53 revealed that deficiency in Mdm2 resulted in 20% capillary regression independently of p53 status. In response to one bout of exercise, protein expression of the proangiogenic vascular endothelial growth factor‐A (VEGF‐A) was increased by 64% in muscle from wild‐type animals, and endothelial cell outgrowth from exercised muscle biopsy samples cultured in a 3‐dimensional collagen gel was enhanced by 37%. These proangiogenic responses to exercise were impaired in exercised Mdm2Puro/Δ7‐9 mice. Prolonged exercise training resulted in increased Mdm2 protein expression (+49%) and capillarization (+24%) in wild‐type muscles. However, exercise training‐induced angiogenesis was abolished in Mdm2Puro/Δ7‐9 mice. Finally, exercise training restored Mdm2, VEGF‐A, and capillarization levels in skeletal muscles from obese Zucker diabetic fatty rats compared with those in healthy animals. Our results define Mdm2 as a crucial regulator of capillary maintenance and exercise‐induced angiogenesis in skeletal muscle.—Roudier, E., Forn, P., Perry, M. E., Birot, O. Murine double minute‐2 expression is required for capillary maintenance and exercise‐induced angiogenesis in skeletal muscle. FASEB J. 26, 4530–4539 (2012). www.fasebj.org

Forkhead BoxO transcription factors restrain exercise‐induced angiogenesis

The growth of new capillaries, angiogenesis, within skeletal muscle occurs only after weeks of repeated aerobic exercise. Paradoxically, large increases in pro‐angiogenic factors such as vascular endothelial growth factor occur with a single exercise bout. The mechanisms underlying the substantial lag in the angiogenic response remain to be elucidated. We detected concomitant increases in the angiostatic Forkhead Box ‘O’ transcription factors FoxO1 and FoxO3a and the matrix protein thrombospondin‐1 following a single bout of exercise, but these responses were repressed after 10 days of repeated exercise. This observation led us to hypothesize that FoxO proteins delay the initiation of exercise‐induced angiogenesis. Endothelial cell‐directed deletion of FoxO proteins abolished the increase in thrombospondin‐1 following a single exercise bout, and resulted in a substantially accelerated angiogenic response. This study identifies an intrinsic endothelial‐specific FoxO signalling pathway that opposes the onset of physiological angiogenesis within healthy exercising skeletal muscle and demonstrates that endothelial cell FoxO proteins are critical determinants of the angiogenic capacity within skeletal muscle.