Noninvasive Assessment of Pulse-Wave Velocity and Flow-Mediated Vasodilation in Anesthetized G?ttingen Minipigs

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine–midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV—but not FMD—showed acceptable interexamination variation for its potential application in porcine models.Abbreviations: FMD, flow-mediated vasodilation; FVI, integrated flow velocity; GTN, glyceryl trinitrate; PWV, pulse-wave velocity; T, transit timeCardiovascular disease has become a global challenge in public health,⁴² and the development and characterization of comparative animal models are of increasing importance. Several animal models of atherosclerosis, including porcine, have been described.^11,12,34 Due to similarities to humans in the anatomy of the cardiovascular system and metabolic physiology, pigs represent a generally useful model in regard to preclinical evaluation and pharmacology.³⁶ Assessment of changes related to atherosclerosis in vivo would be valuable in for example longitudinal assessment of drug effect, but few noninvasive methods for evaluating structural and functional changes in the arteries of pigs are available. In humans, increased arterial stiffness, which occurs with advanced age, also is caused by the pathophysiologic changes associated with atherosclerosis,²⁶ and noninvasive methods for assessing arterial stiffness have been established. The evaluation of pulse-wave velocity (PWV) by using pressure transducers, such as applanation tonometry, is a method recognized as an independent predictor for cardiovascular events in epidemiologic studies.²¹ The method evaluates the velocity with which the pulse wave is propagated through the arterial tree, with arterial stiffness causing increased velocity.^{1,22,29,37,39} Flow-mediated vasodilation (FMD), assessed by vascular ultrasonography, represents a noninvasive evaluation of endothelial-dependent vasodilation. A decrease in vasodilation as a response to increased shear stress has been recognized as a marker of endothelial dysfunction, which precedes the development of atherosclerosis.^5,7 Recent studies have shown that the FMD method is applicable in large animals (that is, dogs and horses) and that a decreased FMD response occurs in dogs with valvular heart disease.^10,15,23,28The aim of this study was to evaluate the reproducibility of methods for assessing arterial stiffness (PWV) and endothelial function (FMD response) in anesthetized Göttingen minipigs, including the influences of arterial blood pressure, heart rate, and room and body temperatures on these methods.     

Peak Oxygen Uptake after Cardiac Rehabilitation: A Randomized Controlled Trial of a 12-Month Maintenance Program versus Usual Care

Background

Exercise capacity is a strong predictor of survival in patients with coronary artery disease (CAD). Exercise capacity improves after cardiac rehabilitation exercise training, but previous studies have demonstrated a decline in peak oxygen uptake after ending a formal rehabilitation program. There is a lack of knowledge on how long-term exercise adherence can be achieved in CAD patients. We therefore assessed if a 12-month maintenance program following cardiac rehabilitation would lead to increased adherence to exercise and increased exercise capacity compared to usual care.

Materials and Methods

Two-centre, open, parallel randomized controlled trial with 12 months follow-up comparing usual care to a maintenance program. The maintenance program consisted of one monthly supervised high intensity interval training session, a written exercise program and exercise diary, and a maximum exercise test every third month during follow-up. Forty-nine patients (15 women) on optimal medical treatment were included following discharge from cardiac rehabilitation. The primary endpoint was change in peak oxygen uptake at follow-up; secondary endpoints were physical activity level, quality of life and blood markers of cardiovascular risk.

Results

There was no change in peak oxygen uptake from baseline to follow-up in either group (intervention group 27.9 (±4.7) to 28.8 (±5.6) mL·kg (-1) min (−1), control group 32.0 (±6.2) to 32.8 (±5.8) mL·kg (−1) min (−1), with no between-group difference, p = 0.22). Quality of life and blood biomarkers remained essentially unchanged, and both self-reported and measured physical activity levels were similar between groups after 12 months.

Conclusions

A maintenance exercise program for 12 months did not improve adherence to exercise or peak oxygen uptake in CAD patients after discharge from cardiac rehabilitation compared to usual care. This suggests that infrequent supervised high intensity interval training sessions are inadequate to improve peak oxygen uptake in this patient group.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01246570","term_id":"NCT01246570"}}NCT01246570     

Expression of the EGF Family in Gastric Cancer: Downregulation of HER4 and Its Activating Ligand NRG4

Gastric cancer is a major cause of cancer-related deaths in both men and women. The epidermal growth factor receptors are EGFR, HER2, HER3 and HER4. Of the four epidermal growth factor receptors, EGFR and HER2 are well-known oncogenes involved in gastric cancer. Little, however, is known about the role played by HER3 and HER4 in this disease. We obtained paired samples from the tumor and the adjacent normal tissue from the same patient undergoing surgery for gastric cancer. Using RT-qPCR, we quantified the mRNA expression of the four receptors including the HER4 splicing isoforms and all the ligands activating these receptors. Using immunohistochemistry, the protein expression of HER4 was also quantified. We found that HER2 mRNA expression was upregulated in the tumor tissue compared to the matched normal tissue (p = 0.0520). All ligands with affinity for EGFR were upregulated, whereas the expression of EGFR was unchanged. Interestingly, we found the mRNA expression of HER4 (p = 0.0002) and its ligand NRG4 (p = 0.0009) to be downregulated in the tumor tissue compared to the matched normal tissue. HER4 downregulation was demonstrated for all the alternatively spliced isoforms of this receptor. These results support the involvement of EGFR and HER2 in gastric cancer and suggest an interesting association of reduced HER4 expression with development of gastric cancer.     

Toll-Like Receptor 9-Activation during Onset of Myocardial Ischemia Does Not Influence Infarct Extension

Aim

Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequence of TLR9 activation timed to the onset of ischemia is not known.

Methods and Results

The TLR9-agonist; CpG B was injected i.p. in C57BL/6 mice immediately after induction of ischemia (30 minutes). Final infarct size, as well as area-at-risk, was measured after 24 hours of reperfusion. CpG B injection resulted in a significant increase in circulating granulocytes and monocytes both in sham and I/R mice. Paradoxically, clear evidence of reduced cardiac infiltration of both monocytes and granulocytes could be demonstrated in I/R mice treated with CpG B (immunocytochemistry, myeloperoxidase activity and mRNA expression patterns). In addition, systemic TLR9 activation elicited significant alterations of cardiac inflammatory genes. Despite these biochemical and cellular changes, there was no difference in infarct size between vehicle and CpG B treated I/R mice.

Conclusion

Systemic TLR9-stimulation upon onset of ischemia and subsequent reperfusion does not alter final infarct size despite causing clear alterations of both systemic and cardiac inflammatory parameters. Our results question the clinical usefulness of TLR9 activation during cardiac I/R.     

Home-based aerobic interval training improves peak oxygen uptake equal to residential cardiac rehabilitation: a randomized, controlled trial

Aerobic capacity, measured as the peak oxygen uptake, is a strong predictor of survival in cardiac patients. Aerobic interval training (AIT), walking/running four times four minutes at 85-95% of peak heart rate, has proven to be effective in increasing peak oxygen uptake in coronary heart disease patients. As some patients do not attend organized rehabilitation programs, home-based exercise should be an alternative. We investigated whether AIT could be performed effectively at home, and compared the effects on peak oxygen uptake with that observed after a standard care, four-week residential rehabilitation. Thirty patients undergoing coronary artery bypass surgery were randomized to residential rehabilitation or home-based AIT. At six months follow-up, peak oxygen uptake increased 4.6 (±2.7) and 3.9 (±3.6) mL·kg(-1) min(-1) (both p<0.005, non-significant between-group difference) after residential rehabilitation and AIT, respectively. Quality of life increased significantly in both groups, with no statistical significant difference between groups. We found no evidence for a different treatment effect between patients randomized to home-based AIT compared to patients attending organized rehabilitation (95% confidence interval -1.8, 3.5). AIT patients reported good adherence to exercise training. Even though these first data indicate positive effects of home-based AIT in patients undergoing coronary artery bypass surgery, more studies are needed to provide supporting evidence for the application of this rehabilitation strategy. Trial registration: ClinicalTrials.gov NCT00363922.     

Assays for urinary biomarkers of oxidatively damaged nucleic acids

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Moderate multiple parentage and low genetic variation reduces the potential for genetic incompatibility avoidance despite high risk of inbreeding

Background

Polyandry is widespread throughout the animal kingdom. In the absence of direct benefits of mating with different males, the underlying basis for polyandry is enigmatic because it can carry considerable costs such as elevated exposure to sexual diseases, physical injury or other direct fitness costs. Such costs may be balanced by indirect genetic benefits to the offspring of polyandrous females. We investigated polyandry and patterns of parentage in the spider Stegodyphus lineatus. This species experiences relatively high levels of inbreeding as a result of its spatial population structure, philopatry and limited male mating dispersal. Polyandry may provide an opportunity for post mating inbreeding avoidance that reduces the risk of genetic incompatibilities arising from incestuous matings. However, multiple mating carries direct fitness costs to females suggesting that genetic benefits must be substantial to counter direct costs.

Methodology/Principal Findings

Genetic parentage analyses in two populations from Israel and a Greek island, showed mixed-brood parentage in approximately 50% of the broods. The number of fathers ranged from 1–2 indicating low levels of multiple parentage and there was no evidence for paternity bias in mixed-broods from both populations. Microsatellite loci variation suggested limited genetic variation within populations, especially in the Greek island population. Relatedness estimates among females in the maternal generation and potentially interacting individuals were substantial indicating full-sib and half-sib relationships.

Conclusions/Significance

Three lines of evidence indicate limited potential to obtain substantial genetic benefits in the form of reduced inbreeding. The relatively low frequency of multiple parentage together with low genetic variation among potential mates and the elevated risk of mating among related individuals as corroborated by our genetic data suggest that there are limited actual outbreeding opportunities for polyandrous females. Polyandry in S. lineatus is thus unlikely to be maintained through adaptive female choice.     

Application of Measurements of Transepithelial Electrical Resistance of Intestinal Epithelial Cell Monolayers To Evaluate Probiotic Activity

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.