Lower skeletal muscle capillarization and VEGF expression in aged vs. young men.

Recently, we observed that muscle capillarization, vascular endothelial growth factor (VEGF) protein, and the VEGF mRNA response to acute exercise were lower in aged compared with young women (Croley AN, Zwetsloot KA, Westerkamp LM, Ryan NA, Pendergast aged men, Hickner RC, Pofahl WE, and Gavin TP. J Appl Physiol 99: 1875-1882, 2005). We hypothesized that similar age-related differences in muscle capillarization and VEGF expression would exist between young and aged men. Skeletal muscle biopsies were obtained from the vastus lateralis before and at 4 h after a submaximal exercise bout for the measurement of morphometry, capillarization, VEGF, KDR, and Flt-1 in seven aged (mean age 65 yr) and eight young (mean age 21 yr) sedentary men. In aged compared with young men, muscle capillary contacts and capillary-to-fiber perimeter exchange index were lower regardless of fiber type. Muscle VEGF mRNA and protein were lower in aged men both at rest and 4 h postexercise. Exercise increased muscle VEGF mRNA and protein and KDR mRNA independent of age group. There were no effects of exercise or age on muscle Flt-1 mRNA or protein or KDR protein. These results confirm that skeletal muscle capillarization and VEGF expression are lower in aged compared with young men.     

Expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle

Angiogenesis is fundamental for human endometrial development and differentiation necessary for implantation. These vascular changes are thought to be mediated by the vascular endothelial growth factor (VEGF), whose specific receptors have not been examined in detail thus far. We conducted the present study to determine, by immunocytochemistry and computerized image analysis of the functionalis, the expression and modulation of the receptors Flk-1/KDR and Flt-1, which mediate VEGF effects on endothelial mitogenicity, chemotaxis, and capillary permeability. VEGF receptors are expressed mainly in endometrial endothelial cells, with variations of intensity and number of stained capillaries related to the phase of the cycle. The number of capillaries immunostained for Flk-1/KDR was maximal in the proliferative phase (ratio Flk-1/CD34: 1), twice as high as the number of Flt-1-expressing capillaries (ratio Flt-1/CD34: 0.47). The staining intensity for Flk-1 decreased during the late proliferative and early secretory phases, to increase again in the midsecretory period. The number of Flt-1-labeled capillaries was about 2-fold higher in the secretory than in the proliferative phase; however, the proportion of Flt-1-positive cells did not change, owing to the associated increase in vascular density that characterizes progression of the functionalis from the proliferative to the secretory stage. The staining intensity for Flt-1 was higher during the late proliferative and secretory phases (especially in the midsecretory phase) and the premenstrual period. In contrast, the proportion of capillaries expressing Flk-1/KDR decreased in the secretory phase (ratio Flk-1/Von Willebrand factor: 0.55). Enhanced expression of Flk-1/KDR, and of Flt-1, on narrow capillary strands at the beginning of and during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration following menstrual shedding. The high coexpression of Flk-1/KDR and Flt-1 observed on capillaries during the midsecretory period correlates with an increase of endometrial microvascular density and of permeability characteristic of this phase of the cycle, which is a prerequisite for implantation. Finally, strong expression of Flt-1, but not Flk-1/KDR, was observed on dilated capillaries during the premenstrual period and the late proliferative phase, suggesting preferential association of Flt-1 with nonproliferating capillaries at those times; activation of this receptor by VEGF could be involved in premenstrual vascular hyperpermeability, edema, and extravasation of leukocytes. In addition to the endothelial localization, we found that epithelial cells expressed Flt-1 and Flk-1/KDR. We conclude that Flt-1 and Flk-1/KDR in the functionalis are modulated in parallel or independently according to the phase of the cycle, and that these changes are responsible for VEGF actions on endometrial vascular growth and permeability. The molecular mechanisms concerning these regulations will require further investigation.     

Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.     

Characterization of the VEGF binding site on the Flt-1 receptor.

The angiogenic growth factor VEGF binds to the receptor tyrosine kinases Flt-1 and KDR/Flk-1. Immunoglobulin (Ig)-like loop-2 of Flt-1 is involved in binding VEGF, but the contribution of other Flt-1 Ig-loops to VEGF binding remains unclear. We tested the ability of membrane-bound chimeras between the extracellular domain of Flt-1 and the cell adhesion molecule embigin to bind VEGF. VEGF bound as well to receptors containing Flt-1 loops 1-2 or 2-3 as it did to the entire Flt-1 extracellular domain. Chimeras containing only loop-2 of Flt-1 bound VEGF with 22-fold lower affinity. We conclude that high-affinity VEGF binding requires Ig-like loop-2 plus either loop-1 or loop-3. In addition, Flt-1 amino acid residues Arg-224 and Asp-231 were not essential for high-affinity binding of VEGF to membrane-bound Flt-1.     

Angiogenic growth factor response to acute systemic exercise in human skeletal muscle.

We investigated whether acute systemic exercise increases vascular endothelial growth factor (VEGF), VEGF receptor (KDR and Flt-1) mRNA, and VEGF protein in sedentary humans. Twelve sedentary subjects were recruited and performed 1 h of acute, cycle ergometer exercise at 50% of maximal oxygen consumption. Muscle biopsies were obtained from the vastus lateralis before exercise and at 0, 2, and 4 h postexercise. Acute exercise significantly increased VEGF mRNA at 2 and 4 h and increased KDR and Flt-1 mRNA at 4 h postexercise. The sustained increase in VEGF mRNA through 4 h and the increases in KDR and Flt-1 at 4 h are different from their respective time course responses in rats. In contrast to the increase in VEGF mRNA postexercise, VEGF protein levels were decreased at 0 h postexercise. These results provide evidence in humans that 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different from those previously reported in rats (Gavin TP and Wagner PD. Acta Physiol Scand 175: 201-209, 2002); and 3) VEGF protein is decreased immediately after exercise.     

Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.     

Effect of captopril on skeletal muscle angiogenic growth factor responses to exercise.

Acute exercise increases vascular endothelial growth factor (VEGF), transforming growth factor-beta(1) (TGF-beta(1)), and basic fibroblast growth factor (bFGF) mRNA levels in skeletal muscle, with the greatest increase in VEGF mRNA. VEGF functions via binding to the VEGF receptors Flk-1 and Flt-1. Captopril, an angiotensin-converting enzyme inhibitor, has been suggested to reduce the microvasculature in resting and exercising skeletal muscle. However, the molecular mechanisms responsible for this reduction have not been investigated. We hypothesized that this might occur via reduced VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 gene expression at rest and after exercise. To investigate this, 10-wk-old female Wistar rats were placed into four groups (n = 6 each): 1) saline + rest; 2) saline + exercise; 3) 100 mg/kg ip captopril + rest; and 4) 100 mg/kg ip captopril + exercise. Exercise consisted of 1 h of running at 20 m/min on a 10 degrees incline. VEGF, TGF-beta(1), bFGF, Flk-1, and Flt-1 mRNA were analyzed from the left gastrocnemius by quantitative Northern blot. Exercise increased VEGF mRNA 4.8-fold, TGF-beta(1) mRNA 1.6-fold, and Flt-1 mRNA 1.7-fold but did not alter bFGF or Flk-1 mRNA measured 1 h after exercise. Captopril did not affect the rest or exercise levels of VEGF, TGF-beta(1), bFGF, and Flt-1 mRNA. Captopril did reduce Flk-1 mRNA 30-40%, independently of exercise. This is partially consistent with the suggestion that captopril may inhibit capillary growth.     

The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.     

Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA.

Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.     

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.

The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.     

Identification of vascular endothelial growth factor receptor-binding protein in the venom of eastern cottonmouth. A new role of snake venom myotoxic Lys49-phospholipase A2

Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.     

Effects of Long Term Supplementation of Anabolic Androgen Steroids on Human Skeletal Muscle

The effects of long-term (over several years) anabolic androgen steroids (AAS) administration on human skeletal muscle are still unclear. In this study, seventeen strength training athletes were recruited and individually interviewed regarding self-administration of banned substances. Ten subjects admitted having taken AAS or AAS derivatives for the past 5 to 15 years (Doped) and the dosage and type of banned substances were recorded. The remaining seven subjects testified to having never used any banned substances (Clean). For all subjects, maximal muscle strength and body composition were tested, and biopsies from the vastus lateralis muscle were obtained. Using histochemistry and immunohistochemistry (IHC), muscle biopsies were evaluated for morphology including fiber type composition, fiber size, capillary variables and myonuclei. Compared with the Clean athletes, the Doped athletes had significantly higher lean leg mass, capillary per fibre and myonuclei per fiber. In contrast, the Doped athletes had significantly lower absolute value in maximal squat force and relative values in maximal squat force (relative to lean body mass, to lean leg mass and to muscle fiber area). Using multivariate statistics, an orthogonal projection of latent structure discriminant analysis (OPLS-DA) model was established, in which the maximal squat force relative to muscle mass and the maximal squat force relative to fiber area, together with capillary density and nuclei density were the most important variables for separating Doped from the Clean athletes (regression  =  0.93 and prediction  =  0.92, p<0.0001). In Doped athletes, AAS dose-dependent increases were observed in lean body mass, muscle fiber area, capillary density and myonuclei density. In conclusion, long term AAS supplementation led to increases in lean leg mass, muscle fiber size and a parallel improvement in muscle strength, and all were dose-dependent. Administration of AAS may induce sustained morphological changes in human skeletal muscle, leading to physical performance enhancement.     

A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration

Vascular endothelial growth factor (VEGF) has two highly homologous tyrosine kinase receptors: Flt-1 (VEGFR-1) and KDR (VEGFR-2). KDR is strongly phosphorylated on tyrosines and can transmit mitogenic and motogenic signals following VEGF binding, while Flt-1 is markedly less effective in mediating such functions. To dissect the regions that account for the differences between the two receptors, we generated a series of chimeric Flt-1-KDR molecules. We found that the juxtamembrane region of Flt-1 prevents key signaling functions. When the juxtamembrane region of Flt-1 is replaced by that of KDR, Flt-1 becomes competent to mediate endothelial cell migration and phosphatidylinositol 3'-kinase activation in response to VEGF. Further mutational analysis shows that a short divergent sequence is responsible for such repressor function. However, mutant Flt-1 receptors lacking this sequence do not transmit effective proliferative signals, suggesting that this receptor function is regulated separately. These results define a novel functional domain that serves to repress Flt-1 activity in endothelial cells.     

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.     

Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.     

Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle

Adult skeletal muscle undergoes adaptation in response to endurance exercise, including fast-to-slow fiber type transformation and enhanced angiogenesis. The purpose of this study was to determine the temporal and spatial changes in fiber type composition and capillary density in a mouse model of endurance training. Long-term voluntary running (4 wk) in C57BL/6 mice resulted in an approximately twofold increase in capillary density and capillary-to-fiber ratio in plantaris muscle as measured by indirect immunofluorescence with an antibody against the endothelial cell marker CD31 (466 ± 16 capillaries/mm² and 0.95 ± 0.04 capillaries/fiber in sedentary control mice vs. 909 ± 55 capillaries/mm² and 1.70 ± 0.04 capillaries/fiber in trained mice, respectively; P < 0.001). A significant increase in capillary-to-fiber ratio was present at day 7 with increased concentration of vascular endothelial growth factor (VEGF) in the muscle, before a significant increase in percentage of type IIa myofibers, suggesting that exercise-induced angiogenesis occurs first, followed by fiber type transformation. Further analysis with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact manifested transiently in type IIb + IId/x fibers at the time (day 7) of significant increase in total capillary density. These findings suggest that endurance training induces angiogenesis in a subpopulation of type IIb + IId/x fibers before switching to type IIa fibers. adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor     

Ovarian steroids in endometrial angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is fundamental for human endometrial development and differentiation, which are necessary for implantation. This vascular process is supposed to be mainly mediated by the vascular endothelial growth factor (VEGF), also named vascular permeability factor (VPF). We report here the expression and modulation of VEGF and its receptors, Flk-1/KDR and Flt-1, in the functionalis throughout the menstrual cycle. Using immunocytochemistry, VEGF is localized in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles. The localization of VEGF on the endothelium correlates with the presence of Flt-1 and Flk-1/KDR receptors on vascular structures, including capillary strands that have not yet formed a lumen and that have been previously described in tumors as angiogenic capillaries. The strongest immunoreactivity for both VEGF and Flk-1/KDR receptor on endothelial cells is detected in the proliferative and midsecretory phases. Enhanced expression of VEGF and its Flk-1 receptors on narrow capillary strands during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration from the residual basal layer following menstrual shedding of the functionalis. The vascular expression of Flt-1 is more important in the secretory than in the proliferative phase, associated with a high microvascular density and an increase in vascular permeability in the implantation period. Consistently with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E(2)), or E(2) + progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner. These results demonstrate that the expression of VEGF and its receptors is cyclically modulated by ovarian steroids, and that this endothelial growth factor acts on the endothelium in a paracrine fashion to control endometrial angiogenesis and permeability.