Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Use of a Hemoglobin-Trapping Approach in the Determination of Nitric Oxide in In Vitro and In Vivo Systems

We describe methods for measuring the release of nitric oxide (NO) derived from organic nitrates in vitro, using triple wavelength and difference spectrophotometry in the presence and absence of concentric microdialysis probes. These methods are based on the ability of NO to oxidize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in aqueous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organic nitrate, increased MetHb concentration in 45 min from 2.47 ± 0.47 to 4.15 ± 0.12 M (p < 0.05) and decreased OxyHb concentration from 2.13 ± 0.35 to 0.33 ± 0.26 M (p < 0.05) at 37°C. At 27°C, the OxyHb concentration was not significantly altered (2.04 ± 0.23 to 1.60 ± 0.04 M) by ISDN, nor was the MetHb concentration (from 2.68 ± 0.50 to 2.59 ± 0.25 M). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentrations in 30 min from 4.21 ± 0.26 to 6.00 ± 0.56 M (p < 0.05) at 37°C, and from 4.23 ± 0.39 to 5.90 ± 0.43 M (p < 0.01) at 27°C. SNP also decreased OxyHb concentrations in 30 min from 1.99 ± 0.32 to 0.13 ± 0.12 M (p < 0.01) at 37°C, and from 2.25 ± 0.31 to 0.13 ± 0.09 M (p < 0.01) at 27°C. Difference spectrophometry indicated that 0.25-5 mM SNP significantly increased NO production in a dose-dependent fashion. This hemoglobin-trapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric microdialysis probes. The NO concentration following exposure to SNP was 530 ± 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in vivo microdialysis, we implanted concentric microdialysis probes into hippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats were 11 ± 2 nM and 23 ± 9 nM, respectively, while in the cerebellum NO concentrations were 28 ± 9 nM and 41 ± 20 nM, respectively. These results demonstrate that microdialysis using a novel hemoglobin-trapping technique possesses adequate sensitivity to measure the NO levels produced from organic nitrates in aqueous solutions, and further document the applicability of this approach to in vivo systems.     

Medical Students’ Opinions About the Commercialization of Healthcare: A Cross-Sectional Survey

There are serious concerns about the commercialization of healthcare and adoption of the business approach in medicine. As market dynamics endanger established professional values, healthcare workers face more complicated ethical dilemmas in their daily practice. The aim of this study was to investigate the willingness of medical students to accept the assertions of commercialized healthcare and the factors affecting their level of agreement, factors which could influence their moral stance when market demands conflict with professional values. A cross-sectional study was conducted in three medical schools in Turkey. The study population consisted of first-, third-, and sixth-year students, and 1,781 students participated in total. Students were asked to state if they agreed with the assertions of commercialized healthcare. Of all students, 87.2 per cent agreed with at least one of the assertions, and one-fifth (20.8 per cent) of them agreed with more than half of the assertions. First-year students significantly agreed more with some assertions than third- and sixth-year students. Being female, having mid-level family income, choosing medicine due to idealistic reasons, and being in the third or sixth years of medical study increased the probability of disagreement. Also, studying in a medical school that included integrated lectures on health policies, rights related to health, and health inequities, along with early field visits, increased the probability of disagreement. This study suggests that agreement with the assertions of commercialized healthcare might be prevalent among students at a considerable level. We argue that this level of agreement is not compatible with best practice in professional ethics and indicates the need for an educational intervention in order to have physicians who give priority to patients’ best interests in the face of market demands.     

Iron and iron/manganese ratio in forage from Icelandic sheep farms: relation to scrapie

This study was undertaken in order to examine whether any connection existed between the amounts of iron in forage and the sporadic occurrence of scrapie observed in certain parts of Iceland. As iron and manganese are considered antagonistic in plants, calculation of the Fe/Mn ratios was also included by using results from Mn determination earlier performed in the same samples. Forage samples (n = 170) from the summer harvests of 2001–2003, were collected from 47 farms for iron and manganese analysis. The farms were divided into four categories: 1. Scrapie-free farms in scrapie-free areas (n = 9); 2. Scrapie-free farms in scrapie-afflicted areas (n = 17); 3. Scrapie-prone farms (earlier scrapie-afflicted, restocked farms) (n = 12); 4. Scrapie-afflicted farms (n = 9). Farms in categories 1 and 2 are collectively referred to as scrapie-free farms. The mean iron concentration in forage samples from scrapie-afflicted farms was significantly higher than in forage samples from farms in the other scrapie categories (P = 0.001). The mean Fe/Mn ratio in forage from scrapie-afflicted farms was significantly higher than in forage from scrapie-free and scrapie-prone farms (P < 0.001). The results indicated relative dominance of iron over manganese in forage from scrapie-afflicted farms as compared to farms in the other categories. Thus thorough knowledge of iron, along with manganese, in soil and vegetation on sheep farms could be a pivot in studies on sporadic scrapie.     

Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ

Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well differentiated receptor neurons. The mean resting potential was -58+/-14 mV (n=39). The contribution of the sodium pump current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 microM dihydro-ouabain. The input resistance was in the range of 1-6 GOmega and depolarizing current pulses of a few pA were sufficient to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content. Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor cells were depolarized by diluted rat male urine.      

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF₁₆₅ molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration-dependent manner, with micromolar affinity, and with a 2:1 peptide: VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide-VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.