Ellen C. Breen

VEGF in biological control

Vascular endothelial growth factor A (VEGF‐A) belongs to a family of heparin binding growth factors that include VEGF‐B, VEGF‐C, VEGF‐D, and placental‐like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well‐known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non‐endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non‐angiogenic processes that take place in adult organs. J. Cell. Biochem. 102: 1358–1367, 2007.

Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice

Vascular endothelial growth factor (VEGF) is required for vasculogenesis and angiogenesis during embryonic and early postnatal life. However the organ‐specific functional role of VEGF in adult life, particularly in skeletal muscle, is less clear. To explore this issue, we engineered skeletal muscle‐targeted VEGF deficient mice (mVEGF−/−) by crossbreeding mice that selectively express Cre recombinase in skeletal muscle under the control of the muscle creatine kinase promoter (MCKcre mice) with mice having a floxed VEGF gene (VEGFLoxP mice). We hypothesized that VEGF is necessary for regulating both cardiac and skeletal muscle capillarity, and that a reduced number of VEGF‐dependent muscle capillaries would limit aerobic exercise capacity. In adult mVEGF−/− mice, VEGF protein levels were reduced by 90 and 80% in skeletal muscle (gastrocnemius) and cardiac muscle, respectively, compared to control mice (P < 0.01). This was accompanied by a 48% (P < 0.05) and 39% (P < 0.05) decreases in the capillary‐to‐fibre ratio and capillary density, respectively, in the gastrocnemius and a 61% decrease in cardiac muscle capillary density (P < 0.05). Hindlimb muscle oxidative (citrate synthase, 21%; β‐HAD, 32%) and glycolytic (PFK, 18%) regulatory enzymes were also increased in mVEGF−/− mice. However, this limited adaptation to reduced muscle VEGF was insufficient to maintain aerobic exercise capacity, and maximal running speed and endurance running capacity were reduced by 34% and 81%, respectively, in mVEGF−/− mice compared to control mice (P < 0.05). Moreover, basal and dobutamine‐stimulated cardiac function, measured by transthoracic echocardiography and left ventricular micromanomtery, showed only a minimal reduction of contractility (peak +dP/dt) and relaxation (peak –dP/dt, τE). Collectively these data suggests adequate locomotor muscle capillary number is important for achieving full exercise capacity. Furthermore, VEGF is essential in regulating postnatal muscle capillarity, and that adult mice, deficient in cardiac and skeletal muscle VEGF, exhibit a major intolerance to aerobic exercise.

TNF‐α‐mediated reduction in PGC‐1α may impair skeletal muscle function after cigarette smoke exposure

Skeletal muscle dysfunction contributes to exercise limitation in COPD. In this study cigarette smoke exposure was hypothesized to increase expression of the inflammatory cytokine, TNF‐α, thereby suppressing PGC‐1α, and hence affecting down stream molecules that regulate oxygen transport and muscle function. Furthermore, we hypothesized that highly vascularized oxidative skeletal muscle would be more susceptible to cigarette smoke than less well‐vascularized glycolytic muscle. To test these hypotheses, mice were exposed to cigarette smoke daily for 8 or 16 weeks, resulting in 157% (8 weeks) and 174% (16 weeks) increases in serum TNF‐α. Separately, TNF‐α administered to C2C12 myoblasts was found to dose‐dependently reduce PGC‐1α mRNA. In the smoke‐exposed mice, PGC‐1α mRNA was decreased, by 48% in soleus and 23% in EDL. The vascular PGC‐1α target molecule, VEGF, was also down‐regulated, but only in the soleus, which exhibited capillary regression and an oxidative to glycolytic fiber type transition. The apoptosis PGC‐1α target genes, atrogin‐1 and MuRF1, were up‐regulated, and to a greater extent in the soleus than EDL. Citrate synthase (soleus—19%, EDL—17%) and β‐hydroxyacyl CoA dehydrogenase (β‐HAD) (soleus—22%, EDL—19%) decreased similarly in both muscle types. There was loss of body and gastrocnemius complex mass, with rapid soleus but not EDL fatigue and diminished exercise endurance. These data suggest that in response to smoke exposure, TNF‐α‐mediated down‐regulation of PGC‐1α may be a key step leading to vascular and myocyte dysfunction, effects that are more evident in oxidative than glycolytic skeletal muscles. J. Cell. Physiol. 222: 320–327, 2010.

Calcyclin (S100A6) regulates pulmonary fibroblast proliferation, morphology, and cytoskeletal organization in vitro.

Calcyclin (S100A6) is a member of the S100A family of calcium binding proteins. While the precise function of calcyclin is unknown, calcyclin expression is associated with cell proliferation and calcyclin is expressed in several types of cancer phenotypes. In the present study, the functional role of calcyclin was further elucidated in pulmonary fibroblasts. Antisense S100A6 RNA expression inhibited serum and mechanical strain‐induced fibroblast proliferation. This attenuated proliferative response was accompanied by a flattened, spread cell morphology, and disruption of tropomyosin labeled microfilaments. Changes in cytoskeletal organization did not correspond with a decrease in tropomyosin levels. These observations suggest a role for calcyclin in modulating calcium dependent signaling events that regulate progression through the cell cycle. J. Cell. Biochem. 88: 848–854, 2003.

Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis

We have previously shown, using a Cre-LoxP strategy, that vascular endothelial growth factor (VEGF) is required for the development and maintenance of skeletal muscle capillarity in sedentary adult mice. To determine whether VEGF expression is required for skeletal muscle capillary adaptation to exercise training, gastrocnemius muscle capillarity was measured in myocyte-specific VEGF gene-deleted (mVEGF(-/-)) and wild-type (WT) littermate mice following 6 wk of treadmill running (1 h/day, 5 days/wk) at the same running speed. The effect of training on metabolic enzyme activity levels and whole body running performance was also evaluated in mVEGF(-/-) and WT mice. Posttraining capillary density was significantly increased by 59% (P < 0.05) in the deep muscle region of the gastrocnemius in WT mice but did not change in mVEGF(-/-) mice. Maximal running speed and time to exhaustion during submaximal running increased by 20 and 13% (P < 0.05), respectively, in WT mice after training but were unchanged in mVEGF(-/-) mice. Training led to increases in skeletal muscle citrate synthase (CS) and phosphofructokinase (PFK) activities in both WT and mVEGF(-/-) mice (P < 0.05), whereas β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity was increased only in WT mice. These data demonstrate that skeletal muscle capillary adaptation to physical training does not occur in the absence of myocyte-expressed VEGF. However, skeletal muscle metabolic adaptation to exercise training takes place independent of myocyte VEGF expression.

Exercise-induced VEGF transcriptional activation in brain, lung and skeletal muscle.

Muscle VEGF expression is upregulated by exercise. Whether this VEGF response is regulated by transcription and/or post-transcriptional mechanisms is unknown. Hypoxia may be responsible: myocyte P(O2) falls greatly during exercise and VEGF is a hypoxia-responsive gene. Whether exercise induces VEGF expression in other organs important to acute physical activity is also unknown. To address these questions, we created a VEGF-Luciferase reporter mouse and measured VEGF transcription, mRNA and protein responses to (a) acute exercise and (b) short-term hypoxia (FI(O2) = 0.06) in brain (brainstem, cerebellum, cortex, hippocampus and striatum), muscle, lung, heart and liver. Exercise increased VEGF transcription, mRNA and protein in brain (hippocampus only), lungs and skeletal muscles, but not liver or heart. Hypoxia increased VEGF expression only in brain (cortex, hippocampus and striatum). New transcription appears to be a major exercise-induced regulatory step for increasing VEGF expression in muscle, lung and brain. Hippocampal VEGF expression was the only component of the exercise response recapitulated by hypoxia equivalent to the Everest summit.

HIF and VEGF relationships in response to hypoxia and sciatic nerve stimulation in rat gastrocnemius

To determine if hypoxia-inducible factor-1 (HIF-1) may regulate skeletal muscle vascular endothelial growth factor (VEGF) expression in response to exercise or hypoxia, rats underwent 1h sciatic nerve electrical stimulation (ES), hypoxic exposure (H) or combined stimuli. HIF-1alpha protein levels increased six-fold with maximal (8V) ES with or without H. Similar HIF-1alpha increases occurred with sub-maximal (6V and 4V) ES plus H, but not in sub-maximal ES or H alone. VEGF mRNA and protein levels increased three-fold in sub-maximal ES or H alone, six-fold in sub-maximal ES plus H, 6.3-fold with maximal ES, and 6.5-fold after maximal ES plus H. These data suggest: (1) intracellular hypoxia during normoxic exercise may exceed that during 8% oxygen breathing at rest and is more effective in stimulating HIF-1alpha; (2) HIF-1 may be an important regulator of exercise-induced VEGF transcription; and (3) breathing 8% O(2) does not alter HIF-1alpha expression in skeletal muscle, implying that exercise-generated signals contribute to the regulation of HIF-1alpha and/or VEGF.

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

Background Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. Objectives We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Methods We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. Results Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. Conclusions The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Skeletal muscle capillarity during hypoxia: VEGF and its activation.

Long-term exposure of humans and many mammals to hypoxia leads to the activation of several cellular mechanisms within skeletal muscles that compensate for a limited availability of cellular oxygen. One of these cellular mechanisms is to increase the expression of a subset of hypoxia-inducible genes, including the expression of vascular endothelial growth factor (VEGF). The VEGF promoter contains a hypoxic response element (HRE) that can bind the transcription factor, hypoxia-inducible factor-1alpha; (HIF-1alpha), and initiate transcriptional activation of the VEGF gene. VEGF gene expression is critically important for skeletal muscle angiogenesis and VEGF gene deletion in the mouse has been shown to greatly reduce skeletal muscle capillarity. However, HIF-1alpha-dependent transcriptional activation of the VEGF gene may not be the only signaling pathway that leads to increased or maintained VEGF levels under conditions of acute or long-term hypoxia. Additional mechanisms, induced during hypoxic exposure that could signal skeletal muscle VEGF activation include inflammation, possibly linked to reactive O(2) species generation, or a change in cellular energy status as reflected by AMP kinase activity. These pathways may provide quite different mechanisms for VEGF upregulation in the context of muscular activity during long-term exposure to a hypoxic environment such as occurs at high altitude. This review will accordingly discuss the potential cellular signals or stimuli resulting from hypoxic exposure that could increase myocyte VEGF expression. These cellular signals include 1) a decrease in intracellular P(O(2)), 2) skeletal muscle inflammation, associated cytokines and oxidative stress, and 3) an increase in AMP kinase activity and adenosine accompanying a reduction in cellular energy potential.

Regional differences in expression of VEGF mRNA in rat gastrocnemius following 1 hr exercise or electrical stimulation

BackgroundVascular endothelial growth factor (VEGF) mRNA levels increase in rat skeletal muscle after a single bout of acute exercise. We assessed regional differences in VEGF165 mRNA levels in rat gastrocnemius muscle using in situ hybridization after inducing upregulation of VEGF by treadmill running (1 hr) or electrical stimulation (1 hr). Muscle functional regions were defined as oxidative (primarily oxidative fibers, I and IIa), or glycolytic (entirely IIb or IId/x fibers). Functional regions were visualized on muscle cross sections that were matched in series to slides processed through in situ hybridization with a VEGF165 probe. A greater upregulation in oxidative regions was hypothesized.ResultsTotal muscle VEGF mRNA (via Northern blot) was upregulated 3.5-fold with both exercise and with electrical stimulation (P = 0.015). Quantitative densitometry of the VEGF mRNA signal via in situ hybridization reveals significant regional differences (P ≤ 0.01) and protocol differences (treadmill, electrical stimulation, and control, P ≤ 0.05). Mean VEGF mRNA signal was higher in the oxidative region in both treadmill run (~7%, N = 4 muscles, P ≤ 0.05) and electrically stimulated muscles (~60%, N = 4, P ≤ 0.05). These regional differences were not significantly different from control muscle (non-exercised, non-stimulated, N = 2 muscles), although nearly so for electrically stimulated muscle (P = 0.056).ConclusionsModerately higher VEGF mRNA signal in oxidative muscle regions is consistent with regional differences in capillary density. However, it is not possible to determine if the VEGF mRNA signal difference is important in either the maintenance of regional capillarity differences or exercise induced angiogenesis.