Optitrain: a randomised controlled exercise trial for women with breast cancer undergoing chemotherapy.

BACKGROUND: Women with breast cancer undergoing chemotherapy suffer from a range of detrimental disease and treatment related side-effects. Exercise has shown to be able to counter some of these side-effects and improve physical function as well as quality of life. The primary aim of the study is to investigate and compare the effects of two different exercise regimens on the primary outcome cancer-related fatigue and the secondary outcomes muscle strength, function and structure, cardiovascular fitness, systemic inflammation, skeletal muscle gene activity, health related quality of life, pain, disease and treatment-related symptoms in women with breast cancer receiving chemotherapy. The second aim is to examine if any effects are sustained 1, 2, and 5 years following the completion of the intervention and to monitor return to work, recurrence and survival. The third aim of the study is to examine the effect of attendance and adherence rates on the effects of the exercise programme. METHODS: This study is a randomised controlled trial including 240 women with breast cancer receiving chemotherapy in Stockholm, Sweden. The participants are randomly allocated to either: group 1: Aerobic training, group 2: Combined resistance and aerobic training, or group 3: usual care (control group). During the 5-year follow-up period, participants in the exercise groups will receive a physical activity prescription. Measurements for endpoints will take place at baseline, after 16 weeks (end of intervention) as well as after 1, 2 and 5 years. DISCUSSION: This randomised controlled trial will generate substantial information regarding the effects of different types of exercise on the health of patients with breast cancer undergoing chemotherapy. We expect that dissemination of the knowledge gained from this study will contribute to developing effective long term strategies to improve the physical and psychosocial health of breast cancer survivors. TRIAL REGISTRATION: OptiTrain - Optimal Training Women with Breast Cancer (OptiTrain), NCT02522260 ; Registration: June 9, 2015, Last updated version Feb 29, 2016. Retrospectively registered.

Negative regulation of HIF in skeletal muscle of elite endurance athletes: a tentative mechanism promoting oxidative metabolism.

The transcription factor hypoxia-inducible factor (HIF) has been suggested as a candidate for mediating training adaptation in skeletal muscle. However, recent evidence rather associates HIF attenuation with a trained phenotype. For example, a muscle-specific HIF deletion increases endurance performance, partly through decreased levels of pyruvate dehydrogenase kinase 1 (PDK-1). HIF activity is regulated on multiple levels: modulation of protein stability, transactivation capacity, and target gene availability. Prolyl hydroxylases (PHD1-3) induces HIF degradation, whereas factor-inhibiting HIF (FIH) and the histone deacetylase sirtuin-6 (SIRT6) repress its transcriptional activity. Together, these negative regulators introduce a mechanism for moderating HIF activity in vivo. We hypothesized that long-term training induces their expression. Negative regulators of HIF were explored by comparing skeletal muscle tissue from moderately active individuals (MA) with elite athletes (EA). In elite athletes, expression of the negative regulators PHD2 (MA 73.54 ± 9.54, EA 98.03 ± 6.58), FIH (MA 4.31 ± 0.25, EA 30.96 ± 7.99) and SIRT6 (MA 0.24 ± 0.07, EA 11.42 ± 2.22) were all significantly higher, whereas the response gene, PDK-1 was lower (MA 0.12 ± 0.03, EA 0.04 ± 0.01). Similar results were observed in a separate 6-wk training study. In vitro, activation of HIF in human primary muscle cell culture by PHD inactivation strongly induced PDK-1 (0.84 ± 0.12 vs 4.70 ± 0.63), providing evidence of a regulatory link between PHD activity and PDK-1 levels in a relevant model system. Citrate synthase activity, closely associated with aerobic exercise adaptation, increased upon PDK-1 silencing. We suggest that training-induced negative regulation of HIF mediates the attenuation of PDK-1 and contributes to skeletal muscle adaptation to exercise.

Alternative splice variant PGC-1α-b is strongly induced by exercise in human skeletal muscle.

The present study investigated whether exercise induces the expression of PGC-1α splice variants in human skeletal muscle and the possible influence of metabolic perturbation on this response. The subjects exercised one leg for 45 min with restricted blood flow (R-leg), followed by 45 min of exercise using the other leg at the same absolute workload but with normal blood flow (NR-leg). This ischemic model (R-leg) has been shown previously to induce a greater metabolic perturbation and enhance the expression of PGC-1α beyond that observed in the NR-leg. Cultured human myotubes were used to test suggested exercise-induced regulatory stimuli of PGC-1α. We showed, for the first time, that transcripts from both the canonical promoter (PGC-1α-a) and the proposed upstream-located promoter (PGC-1α-b) are present in human skeletal muscle. Both transcripts were upregulated after exercise in the R-leg, but the fold change increase of PGC-1α-b was much greater than that of PGC-1α-a. No differences were observed between the two conditions regarding the marker for calcineurin activation, MCIP1, or p38 phosphorylation. AMPK phosphorylation increased to a greater extent in the R-leg, and AICAR stimulation of cultured human myotubes induced the expression of PGC-1α-a and PGC-1α-b. AICAR combined with norepinephrine yielded an additive effect on the PGC-1α-b expression only. Our results indicate clearly that exercise can activate an upstream promoter in humans and support AMPK as a major regulator of transcripts from the canonical PGC-1α promoter and the involvement of ß-adrenergic stimulation in combination with AMPK in the regulation of PGC-1α-b.

Stimulation of beta1 integrins on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF beta-receptors.

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta-receptors in human diploid foreskin AG 1518 fibroblasts. A transient tyrosine phosphorylation of PDGF beta-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not on poly-L-lysine. In contrast EGF or PDGF alpha-receptors were not phosphorylated on tyrosine residues under these conditions. Tyrosine phosphorylation of PDGF beta-receptors induced by plating on collagen type I was inhibited by cytochalasin D and herbimycin A, unaffected by cycloheximide and enhanced by orthovanadate. Furthermore, a transient phosphorylation of PDGF beta-receptors occurred when AG 518 fibroblasts were cultured in three-dimensional collagen lattices or exposed to external strain exerted through centrifugation. The latter effect was evident already after two minutes. Clustering of cell surface beta1 integrins led to PDGF beta-receptor phosphorylation both in suspended and firmly attached AG 1518 fibroblasts. Plating of cells on collagen type I, fibronectin, and anti-beta1-integrin IgG resulted in the formation of PDGF beta-receptor aggregates as detected by immunofluorescence. Suramin or anti-PDGF-BB IgG had no effect on the plating-induced tyrosine phosphorylation of PDGF beta-receptors. PDGF-B chain mRNA, or protein, were not detected in AG 1518 fibroblasts. Our data suggest that a ligand-independent PDGF beta-receptor activation during cell adhesion and early phases of cell spreading is involved in integrin-mediated signaling in fibroblasts, and constitutes parts of a mechanism for cells to respond during the dynamic phases of externally applied tension as well as fibroblast-mediated tension during cell adhesion and collagen gel contraction.

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste.

AIMS: To monitor emissions of NH(3) and N(2)O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB). METHODS AND RESULTS: A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH(3) emissions peaked when pH started to increase. Small amounts of N(2)O and CH(4) were also produced. In total, 16% and less than 1% of the initial N was lost as NH(3)-N and N(2)O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase. CONCLUSIONS: Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH(3) emissions. Small but significant amounts of N(2)O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH(3) emissions.

Bench-scale composting of source-separated human faeces for sanitation.

In urine-diverting toilets, urine and faeces are collected separately so that nutrient content can be recycled unmixed. Faeces should be sanitized before use in agriculture fields due to the presence of possible enteric pathogens. Composting of human faeces with food waste was evaluated as a possible method for this treatment. Temperatures were monitored in three 78-L wooden compost reactors fed with faeces-to-food waste substrates (F:FW) in wet weight ratios of 1:0, 3:1 and 1:1, which were observed for approximately 20 days. To achieve temperatures higher than 15 degrees C above ambient, insulation was required for the reactors. Use of 25-mm thick styrofoam insulation around the entire exterior of the compost reactors and turning of the compost twice a week resulted in sanitizing temperatures (>or=50 degrees C) to be maintained for 8 days in the F:FW=1:1 compost and for 4 days in the F:FW=3:1 compost. In these composts, a reduction of >3 log(10) for E. coli and >4 log(10) for Enterococcus spp. was achieved. The F:FW=1:0 compost, which did not maintain >or=50 degrees C for a sufficiently long period, was not sanitized, as the counts of E. coli and Enterococcus spp. increased between days 11 and 15. This research provides useful information on the design and operation of family-size compost units for the treatment of source-separated faeces and starchy food residues, most likely available amongst the less affluent rural/urban society in Uganda.

Substrate composition and moisture in composting source-separated human faeces and food waste.

The composting of a faeces/ash mixture and food waste in relative proportions of 1:0, 1:1 and 1:3 was studied in three successive experiments conducted in Kampala, Uganda in 216 L reactors insulated with 75 mm styrofoam or not insulated. The faeces/ash mixture alone exceeded 50 degrees C for < or = 12 days in insulated reactors, but did not reach or maintain 50 degrees C in non-insulated reactors. Inclusion of food waste kept temperatures above 50 degrees C for over two weeks in insulated reactors except when the substrate was too wet. Escherichia coli and total coliform concentrations decreased below detection in material that exceeded 50 degrees C for at least six days. Enterococcus spp. decreased below detection in material that exceeded 50 degrees C for at least two weeks, but remained detectable after 1.5 months in material that exceeded 50 degrees C for less than two weeks, suggesting that a period of at least two weeks above 50 degrees C, combined with mixing, is needed to achieve sanitation. Initially substrates that were too wet proved a challenge to composting and ways of decreasing substrate moisture should be investigated. The results obtained are applicable to the management of small- to medium-scale composting of faeces/ash and food waste at household and institution levels, e.g. schools and restaurants.

Lack of sex differences in the IGF-IGFBP response to ultra endurance exercise.

The insulin-like growth factor (IGF)-IGF binding proteins (BP) and the pituitary-gonadal axes were investigated during ultra endurance exercise in 16 endurance-trained athletes (seven women). Median duration of the race was 6.3 days. Although food and drink were ad libitum, energy balance was negative. Blood samples were drawn before (PRE), at the end of (END) and 24 h after (POST24h) the race. Serum concentrations of total IGF-I (t-IGF-I) and free IGF-I (f-IGF-I) decreased by 33 (SD 38)% and 54 (19)%, respectively. The decrease in t-IGF-I appeared to be associated to the total energy deficit during the race. At END, the IGFBP-3 fragmentation and IGFBP-1 were increased but these changes did not predict changes in f-IGF-I. An increase in POST24h IGFBP-2 levels in women was the only sex difference. Testosterone was decreased by 67 (12)% in the men and estradiol became undetectable in the women without any detectable increase in LH and/or FSH. In conclusion ultra endurance exercise results in similar IGF-IGFBP responses in men and women reflecting a catabolic state. IGFBP-2 was the only exception, with increased levels in women after exercise. A concomitant decrease in gonadal hormones was not related to endocrine changes in the IGF-IGFBP axis but may be related to local changes in IGF-I expression.

Higher pH and faster decomposition in biowaste composting by increased aeration.

Composting of source separated municipal biowaste has at several plants in Scandinavia been hampered by low pH. In this study the hypothesis that increased aeration would improve the process was tested in full-scale experiments at two large composting plants. The O2 concentrations were high (>15%) even at the low aeration rates, so the prevailing low pH was not due to an anaerobic process environment. In spite of this, increased aeration rates at the start of the process resulted in higher microbial activity, increased pH and a more stable compost product. At one plant the decomposition rate varied in proportion to the aeration rate, to the extent that the temperatures and O2 concentrations were similar during the early processes even though aeration rates varied between 10 and 50 m3/(h, m3 compost). However, increased aeration caused severe drying of the compost, but at one plant the addition of water was adequate to prevent drying. In conclusion, by increasing the aeration rates and adding water to compensate for drying, it was possible to shorten the time needed to produce a stable compost product and thus to increase the efficiency of the composting plants.

Tissue localization of beta receptors for platelet-derived growth factor and platelet-derived growth factor B chain during wound repair in humans.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.

Substrate availability limits human skeletal muscle oxidative ATP regeneration at the onset of ischemic exercise.

We have demonstrated previously that dichloroacetate can attenuate skeletal muscle fatigue by up to 35% in a canine model of peripheral ischemia (Timmons, J.A., S.M. Poucher, D. Constantin-Teodosiu, V. Worrall, I.A. Macdonald, and P.L. Greenhaff. 1996. J. Clin. Invest. 97:879-883). This was thought to be a consequence of dichloroacetate increasing acetyl group availability early during contraction. In this study we characterized the metabolic effects of dichloroacetate in a human model of peripheral muscle ischemia. On two separate occasions (control-saline or dichloroacetate infusion), nine subjects performed 8 min of single-leg knee extension exercise at an intensity aimed at achieving volitional exhaustion in approximately 8 min. During exercise each subject's lower limbs were exposed to 50 mmHg of positive pressure, which reduces blood flow by approximately 20%. Dichloroacetate increased resting muscle pyruvate dehydrogenase complex activation status by threefold and elevated acetylcarnitine concentration by fivefold. After 3 min of exercise, phosphocreatine degradation and lactate accumulation were both reduced by approximately 50% after dichloroacetate pretreatment, when compared with control conditions. However, after 8 min of exercise no differences existed between treatments. Therefore, it would appear that dichloroacetate can delay the accumulation of metabolites which lead to the development of skeletal muscle fatigue during ischemia but does not alter the metabolic profile when a maximal effort is approached.

The influence of physical training on the angiopoietin and VEGF-A systems in human skeletal muscle.

Eleven subjects performed one-legged exercise four times per week for 5 wk. The subjects exercised one leg for 45 min with restricted blood flow (R leg), followed by exercise with the other leg at the same absolute workload with unrestricted blood flow (UR leg). mRNA and protein expression were measured in biopsies from the vastus lateralis muscle obtained at rest before the training period, after 10 days, and after 5 wk of training, as well as 120 min after the first and last exercise bouts. Basal Ang-2 and Tie-1 mRNA levels increased in both legs with training. The Ang-2-to-Ang-1 ratio increased to a greater extent in the R leg. The changes in Ang-2 mRNA were followed by similar changes at the protein level. In the R leg, VEGF-A mRNA expression responded transiently after acute exercise both before and after the 5-wk training program. Over the course of the exercise program, there was a concurrent increase in basal VEGF-A protein and VEGFR-2 mRNA in the R leg. Ki-67 mRNA showed a greater increase in the R leg and the protein was localized to the endothelial cells. In summary, the increased translation of VEGF-A is suggested to be caused by the short mRNA burst induced by each exercise bout. The concurrent increase in the Ang-2-to-Ang-1 ratio and the VEGF-expression combined with the higher level of Ki-67 mRNA in the R leg indicate that changes in these systems are of importance also in nonpathological angiogenic condition such as voluntary exercise in humans. It further establish that hypoxia/ischemia-related metabolic perturbation is likely to be involved as stimuli in this process in human skeletal muscle.

A single bout of exercise activates matrix metalloproteinase in human skeletal muscle.

The aims of this study were 1) to characterize changes in matrix metalloproteinase (MMP), endostatin, and vascular endothelial growth factor (VEGF)-A expression in skeletal muscle in response to a single bout of exercise in humans; and 2) to determine if any exchange of endostatin and VEGF-A between circulation and the exercising leg is associated with a change in the tissue expression or plasma concentration of these factors. Ten healthy males performed 65 min of cycle exercise, and muscle biopsies were obtained from the vastus lateralis muscle at rest and immediately and 120 min after exercise. In the muscle biopsies, measurements of mRNA expression levels of MMP-2, MMP-9, MMP-14, and tissue inhibitor of metalloproteinase; VEGF and endostatin protein levels; and MMP activities were performed. Femoral arterial and venous concentrations of VEGF-A and endostatin were determined before, during, and 120 min after exercise. A single bout of exercise increased MMP-9 mRNA and activated MMP-9 protein in skeletal muscle. No measurable increase of endostatin was observed in the skeletal muscle or in plasma following exercise. A concurrent increase in skeletal muscle VEGF-A mRNA and protein levels was induced by exercise, with no signs of peripheral uptake from the circulation. However, a decrease in plasma VEGF-A concentration occurred following exercise. Thus 1) a single bout of exercise activated the MMP system without any resulting change in tissue endostatin protein levels, and 2) the increased VEGF-A protein levels are due to changes in the skeletal muscle tissue itself. Other mechanisms are responsible for the observed exercise-induced decrease in VEGF-A in plasma.

Potential nitrification and denitrification and the corresponding composition of the bacterial communities in a compact constructed wetland treating landfill leachates.

Constructed wetlands can be used to decrease the high ammonium concentrations in landfill leachates. We investigated nitrification/denitrification activity and the corresponding bacterial communities in landfill leachate that was treated in a compact constructed wetland, Tveta Recycling Facility, Sweden. Samples were collected at three depths in a filter bed and the sediment from a connected open pond in July, September and November 2004. Potential ammonia oxidation was measured by short-term incubation method and potential denitrification by the acetylene inhibition technique. The ammonia-oxidising and the denitrifying bacterial communities were investigated using group-specific PCR primers targeting 16S rRNA genes and the functional gene nosZ, respectively. PCR products were analysed by denaturing gradient gel electrophoresis and nucleotide sequencing. The same degree of nitrification activity was observed in the pond sediment and at all levels in the filter bed, whereas the denitrification activity decreased with filter bed depth. Denitrification rates were higher in the open pond, even though the denitrifying bacterial community was more diverse in the filter bed. The ammonia-oxidising community was also more varied in the filter bed. In the filter bed and the open pond, there was no obvious relationship between the nitrification/denitrification activities and the composition of the corresponding bacterial communities.

Local changes in the insulin-like growth factor system in human skeletal muscle assessed by microdialysis and arterio-venous differences technique.

IGF-I plays a direct role in whole body glucose homeostasis primarily by stimulating skeletal muscle glucose uptake. IGF-I is also involved in exercise induced muscle hypertrophy. Knowledge regarding local changes in muscle IGF-I bioavailability and its regulation by IGFBPs at rest and during exercise is limited. We have therefore explored changes in total IGF-I levels as well as circulating IGFBP levels and their post-translational modifications over an exercising leg. For the first time we have determined IGF-I levels in exercising skeletal muscle microdialysate in an attempt to assess local IGF-I bioavailability. Eighteen healthy young men performed one legged knee-extension exercise during 45min. Blood samples were taken from the femoral artery and vein of the exercising leg. No significant differences between arterial and venous concentrations of total IGF-I or IGFBP-1 were detected over the leg at any time. IGF-I concentrations increased significantly during exercise in the artery but not in the vein. Total IGFBP-1 increased after exercise in both artery and vein. The increase in non-plus less phosphorylated forms of IGFBP-1 was less pronounced and did not reach statistical significance. The proportion of fragmented IGFBP-3 (IGFBP-3 proteolysis) assessed by Western immunoblotting did not change significantly during or after exercise. Although optimization and validation of IGF-I determinations in muscle microdialysate (md) will be required, our first results using this technique demonstrate a significant 2-fold increase in mdIGF-I collected during and after exercise. We conclude that determination of A-V-differences appears to be of limited value in the assessments of local muscle change in the IGF-system. A substantial release of IGF-I during short time is required to detect significant change in the large circulating store of IGF-I. We suggest that an optimized and validated microdialysis technique for determination of local IGF-I may be advantageous in future studies.

The 104-123 amino acid sequence of the beta-domain of von Hippel-Lindau gene product is sufficient to inhibit renal tumor growth and invasion.

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in patients with VHL disease and in the majority of patients with sporadic renal cell carcinomas (RCCs). RCCs are dependent on insulin-like growth factor-1 receptor-mediated signaling for tumor growth and invasion in vivo. Reintroduction of the VHL gene product (pVHL) can inhibit on insulin-like growth factor-I receptor-mediated signaling in RCC cells in vitro through interaction with protein kinase C delta and is mediated by a specific amino acid sequence (104-123) in the beta-domain of the pVHL. In the present study, the amino acid sequence (104-123) of the pVHL was conjugated to the protein transduction domain of HIV-TAT protein (TATFLAGVHL-peptide) to facilitate entry into cells, and we demonstrate that this amino acid region of VHL is sufficient to block proliferation and invasion of 786-O renal cancer cells in vitro. Furthermore, daily i.p. injections with the TATFLAGVHL peptide retarded and, in some cases, caused partial regression of renal tumors that were implanted in the dorsal flank of nude mice. Treatment with this peptide also inhibits the invasiveness of renal tumors. A 56% decrease in the proliferative index in tumors treated with the TATFLAGVHL-peptide versus control-peptide-treated mice was observed. Taken together, these results show the novel importance of a 20-amino acid sequence of the beta-domain of the VHL gene product capable of inhibiting tumor growth and invasion. These results lay the foundation for a unique approach toward treating RCCs using this small-molecular-weight peptide fused to the TAT-sequence, which may, in the future, be used alone or in conjunction with other therapies.

Effects of trace element addition on process stability during anaerobic co-digestion of OFMSW and slaughterhouse waste.

This study used semi-continuous laboratory scale biogas reactors to simulate the effects of trace-element addition in different combinations, while degrading the organic fraction of municipal solid waste and slaughterhouse waste. The results show that the combined addition of Fe, Co and Ni was superior to the addition of only Fe, Fe and Co or Fe and Ni. However, the addition of only Fe resulted in a more stable process than the combined addition of Fe and Co, perhaps indicating a too efficient acidogenesis and/or homoacetogenesis in relation to a Ni-deprived methanogenic population. The results were observed in terms of higher biogas production (+9%), biogas production rates (+35%) and reduced VFA concentration for combined addition compared to only Fe and Ni. The higher stability was supported by observations of differences in viscosity, intraday VFA- and biogas kinetics as well as by the 16S rRNA gene and 16S rRNA of the methanogens.

Disordered cellular migration and angiogenesis in cd39-null mice.

BACKGROUND: Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 is the major ectonucleotidase of endothelial cells and monocytes and catalyzes phosphohydrolysis of extracellular nucleoside diphosphates (NDP) and triphosphates (NTP, eg, ATP and UTP). Deletion of cd39 causes perturbations in the hydrolysis of NTP and NDP in the vasculature. Activation of P2 receptors appears to influence endothelial cell chemotactic and mitogenic responses in vitro. Therefore, aberrant regulation of nucleotide P2 receptors may influence angiogenesis in cd39-null mice. Methods and Results- In control mice, implanted Matrigel plugs containing growth factors were rapidly populated by monocyte/macrophages, endothelial cells, and pericytes, with the development of new vessels over days. In cd39-null mice, migrating cells were completely confined to the tissue-Matrigel interface in a clearly stratified manner. Absolute failure of new vessel ingrowth was consistently observed in the mutant mice. Linked to these findings, chemotaxis of cd39-null monocyte/macrophages to nucleotides was impaired in vitro. This abnormality was associated with desensitization of nucleotide receptor P2Y-mediated signaling pathways. CONCLUSIONS: Our findings demonstrate a role for NTPDase1 and phosphohydrolysis of extracellular nucleotides in the regulation of the cellular infiltration and new vessel growth in a model of angiogenesis.

Prognostic significance of the microvascular count in colorectal cancer.

PURPOSE: To investigate the potential correlations between a high microvascular count and the survival rate in colorectal cancer. MATERIALS AND METHODS: Three markers for endothelial cells--Ulex Europaeus Lectin (UEA), a polyclonal anti-von Willebrand factor (vWF) antibody, and a monoclonal anti-CD31 antibody (all from Dakopatts, Glostrup, Denmark)--were used for immunohistochemical detection of microvessels in whole-mount sections from 15 colorectal cancers. Areas with higher microvascular density were homogeneously distributed in the sections, regardless of the marker used. The anti-vWF antibody was subsequently used for quantification of microvessels in full-cross tumor biopsies collected from 212 consecutive surgical specimens. The correlations between the mean number of microvessels in areas with the highest microvascular density and tumor differentiation, tumor stage according to Dukes', and survival time were investigated. RESULTS: A significantly longer survival time was shown for patients who had tumors with a mean of more than 10 anti-vWF-positive microvessels, as compared with those who had < or = five. Tumors with a microvascular count between six and 10 microvessels behaved in-between. There was no correlation between the number of microvessels and tumor differentiation or Dukes' stage. CONCLUSION: The number of microvessels measurable in tumor biopsies seems to be a prognostic predictor independent of Dukes' stage in colorectal cancer. However, our results are opposite to the findings in other tumor types investigated so far; we found that a high microvascular count predicted a longer survival time, rather than a shorter one. Determination of the microvascular count can be of importance in therapy selection even before, or immediately after, surgery, ie, before Dukes' stage is known.

VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise.

VEGF-A contributes to muscle tissue angiogenesis following aerobic exercise training. The temporal response of the VEGF-A isoforms and their target receptors has not been comprehensively profiled in human skeletal muscle. We combined submaximal exercise with and without reduced leg blood flow to establish whether ischemia-induced metabolic stress was an important physiological stimuli responsible for regulating the VEGF-A system in humans. Nine healthy men performed two 45-min bouts of one-leg knee-extension exercise, with and without blood flow restriction. Muscle biopsies were obtained at rest and 2 and 6 h after exercise. Expression (mRNA) of the VEGF-A splice variants and related receptors [VEGF receptor (VEGFR)-1, VEGFR-2, and neuropilin-1] was determined by using qPCR. VEGF-A(total) expression increased more robustly after exercise with reduced blood flow, and initially this principally reflected an increase in VEGF-A(165). Six hours after exercise, there was a relatively greater increase in VEGF-A(189), and this response was not influenced by blood flow conditions. VEGFR-1 mRNA expression increased 2 h after exercise, and neuropilin-1 expression was transiently reduced, while all three receptors increased by 6 h. There was no evidence for the expression of the inhibitory VEGF-A(165B) variant in human skeletal muscle. Our study, reflecting both VEGF-A ligand and receptors, implicates metabolic perturbation as a regulator of human muscle angiogenesis and demonstrates that VEGF-A splice variants are distinctly regulated. Our findings also indicate that all three receptor genes exhibit different pretranslational regulation, in response to exercise in humans.