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Chronic heart failure (CHF) results in central and peripheral derangements that ultimately reduce skeletal muscle O2 delivery and impair exercise tolerance. Dietary nitrate (NO3−) supplementation improves skeletal muscle vascular function and tolerance to exercise. We tested the hypothesis that NO3− supplementation would elevate exercising skeletal muscle blood flow (BF) and vascular conductance (VC) in CHF rats. Myocardial infarction (MI) was induced (coronary artery ligation) in young adult male rats. After 21 days of recovery, rats randomly received 5 days of NO3−-rich beetroot juice (CHF + BR, n = 10) or a placebo (CHF, n = 10). Mean arterial pressure (carotid artery catheter) and skeletal muscle BF (radiolabeled microspheres) were measured during treadmill exercise (20 m/min, 5% grade). CHF-induced dysfunction, as determined by myocardial infarction size (29 ± 3% and 33 ± 4% in CHF and CHF + BR, respectively) and left ventricular end-diastolic pressure (18 ± 2 and 18 ± 2 mmHg in CHF and CHF + BR, respectively), and exercising mean arterial pressure (131 ± 3 and 128 ± 4 mmHg in CHF and CHF + BR, respectively) were not different ( P > 0.05) between groups. Total exercising hindlimb skeletal muscle BF (95 ± 5 and 116 ± 9 ml·min−1·100 g−1 in CHF and CHF + BR, respectively) and VC (0.75 ± 0.05 and 0.90 ± 0.05 ml·min−1·100 g−1·mmHg−1 in CHF and CHF + BR, respectively) were 22% and 20% greater in BR-supplemented rats, respectively ( P < 0.05). During exercise, BF in 9 and VC in 10 hindlimb muscles and muscle portions were significantly greater in the CHF + BR group. These results provide strong evidence that dietary NO3− supplementation improves skeletal muscle vascular function during exercise in rats with CHF and, thus, support the use of BR as a novel therapeutic modality for the treatment of CHF.

Left ventricular diastolic dysfunction (LVDD) is one of the earliest signs for abnormal cardiac function in patients with type 2 diabetes (T2DM). It is important to explore the risk factors that will assist in identifying the severity of the LVDD in this population. We examined the influences of fitness and fatness on the level of left ventricular (LV) impairment in patients with T2DM. Twenty-five patients (age: 64.0 ± 2.5 years, body mass index [BMI] = 36.0 ± 1.5 kg/m2, mean ± standard error of measurement) with T2DM and preserved systolic function, but impaired diastolic function, mitral valve (MV) E/e′, participated in the study. LV function was assessed using a stress echocardiograph, aerobic power was assessed with a sign- and symptom-limited graded exercise test, and the fatness level was assessed using Dual-energy X-ray absorptiometry and BMI. Patients in the higher 50% of BMI had higher lateral and septal MV E/e′ (~34% and ~25%, respectively, both P < 0.001), compared to those in the lower 50% of BMI, with no difference in LV ejection fraction (LVEF) ( P > 0.05). In addition, a higher BMI correlated with a higher lateral ( r = 0.62, P < 0.001) and septal ( r = 0.56, P < 0.01) E/é. There was no such relationship for VO2peak. BMI and VO2peakwere not correlated with LV systolic function (ejection fraction). In individuals with T2DM and diastolic dysfunction, a higher BMI was associated with worsening diastolic function independent of their aerobic capacity. The data provide a simple and practical approach for clinicians to assist in the early identification and diagnostics of functional changes in the heart diastolic function in this population.

Peripheral arterial disease (PAD) results in a failure to adequately supply blood and oxygen (O2) to working tissues and presents as claudication pain during walking. Nitric oxide (NO) bioavailability is essential for vascular health and function. Plasma nitrite (NO2−) is a marker of vascular NO production but may also be a protected circulating “source” that can be converted to NO during hypoxic conditions, possibly aiding perfusion. We hypothesized that dietary supplementation of inorganic nitrate in the form of beetroot (BR) juice would increase plasma NO2−concentration, increase exercise tolerance, and decrease gastrocnemius fractional O2extraction, compared with placebo (PL). This was a randomized, open-label, crossover study. At each visit, subjects ( n = 8) underwent resting blood draws, followed by consumption of 500 ml BR or PL and subsequent blood draws prior to, during, and following a maximal cardiopulmonary exercise (CPX) test. Gastrocnemius oxygenation during the CPX was measured by near-infrared spectroscopy. There were no changes from rest for [NO2−] (152 ± 72 nM) following PL. BR increased plasma [NO2−] after 3 h (943 ± 826 nM; P ≤ 0.01). Subjects walked 18% longer before the onset of claudication pain (183 ± 84 s vs. 215 ± 99 s; P ≤ 0.01) and had a 17% longer peak walking time (467 ± 223 s vs. 533 ± 233 s; P ≤ 0.05) following BR vs. PL. Gastrocnemius tissue fractional O2extraction was lower during exercise following BR (7.3 ± 6.2 vs. 10.4 ± 6.1 arbitrary units; P ≤ 0.01). Diastolic blood pressure was lower in the BR group at rest and during CPX testing ( P ≤ 0.05). These findings support the hypothesis that NO2−-related NO signaling increases peripheral tissue oxygenation in areas of hypoxia and increases exercise tolerance in PAD.

Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm2. Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne / cm2 for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.

Plasma nitrite is a major oxidation product of nitric oxide. It has also recently been suggested to perform an endocrine-like function as a nitric oxide donor in hypoxic tissues, allowing vasodilation. Exercise performance is limited in peripheral arterial disease because of an inadequate blood supply to working tissues. We hypothesized that exercise training in peripheral arterial disease subjects will improve "plasma nitrite flux" and endothelial function, to accompany increased exercise performance. Peripheral arterial disease subjects were tested at baseline and after 3 months supervised or home exercise training. Venous blood (arm) was drawn at rest and 10 min after a maximal graded treadmill test. Samples were added to heparin and centrifuged and plasma was snap-frozen for analysis by reductive chemiluminescence. Brachial artery endothelial function was measured in response to a hyperemic stimulus (flow-mediated dilation). At 3 months the peripheral arterial disease-supervised exercise group showed increases in claudication onset pain time (+138 s, por =0.05), peak walking time (+260 s, por =0.01), VO(2peak) (1.3 ml/kg/min, por =0.05), brachial artery flow-mediated dilation (+2%, por =0.05), and plasma nitrite flux (+33% por =0.05). There were no changes in the peripheral arterial disease-home exercise group. The change in plasma nitrite flux predicted the change in claudication onset pain (r(2)=0.59, por =0.01). These findings suggest that changes in plasma nitrite are related to endothelial function and predict exercise performance in peripheral arterial disease.

Nitric oxide (NO) bioavailability is crucial for normal vascular endothelial function and health. Recent studies have demonstrated an endocrine role for NO equivalents that may be transported in the blood to peripheral tissue beds, where under hypoxic conditions they can liberate NO and cause vasodilation. Exercise training improves endothelial function but its effect on NO bioavailability in peripheral tissues during acute exercise stress in CVD is unclear. This paper will present evidence and discuss possible mechanisms by which NO delivery to peripheral tissues may be dysfunctional in diabetic subjects.

The promise of immunotherapy for cancer is underscored by the recent efficacy of checkpoint inhibitors, which hinder the ability of tumors to escape attack by the immune system. Patients most likely to benefit from such therapy include those whose tumors are inflamed and who express the PD-L1 checkpoint protein. It is imperative that alternative therapies are developed for patients whose tumors do not exhibit these characteristics. Using our proprietary OGAP® system, we identified a novel membrane cancer target, OX001L. OX001L expression in certain cancers is associated with poor prognosis and reduced survival. IHC studies showed substantial prevalence of OX001L across multiple tumor types, with a majority of the OX001L positive samples scoring negative for PD-L1. In non-small cell lung cancer, OX001L expression was significantly increased in tumors lacking abundant intratumoral PD-1 positive T-cell infiltrate or PD-L1 expression compared to inflamed or PD-L1 positive tumors (p Citation Format: Angelo Kaplan, Nickolas Attanasio, To Uyen T Do, Sudha Swaminathan, Arnima Bisht, San Lin Lou, Jason Allen, Robert Boyd, James E. Ackroyd, Gleb Feldman, Christian Rohlff, Rachel L. Dusek. Preclinical development of a novel antibody-drug conjugate targeting “cold” tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2772.

NO is crucial for endothelial function and vascular health. Plasma nitrite (NO(2)(-)) is the main oxidation product of NO and has been shown to reflect changes in eNOS activity. We hypothesized that plasma NO(2)(-) response to physical exercise stress along with physiological endothelial function would be reduced with increasing severity of vascular disease. Subject groups were: (a) risk factors but no vascular disease (RF); (b) Type 2 diabetes with no vascular disease (DM); (c) diagnosed peripheral arterial disease (PAD); and (d) DM+PAD. Venous blood was drawn at rest and 10min following maximal exercise. Plasma samples were analyzed by reductive chemiluminescence. Brachial diameters were imaged prior to, during and following 5min of forearm occlusion (BAFMD). There were no differences in resting plasma NO(2)(-) or BA diameters between groups. The PAD groups had lower age adjusted BAFMD responses (p0.05). Within group analysis revealed an increase in NO(2)(-) in the RF group (+39.3%), no change in the DM (-15.51%), and a decrease in the PAD (-44.20%) and PAD+DM (-39.95%). This was maintained after adjusting for age and VO(2peak) (p0.05). DeltaNO(2)(-) and BAFMD were the strongest independent predictors of VO(2peak) in multivariate linear regression. These findings suggest DeltaNO(2)(-) discriminates severity of cardiovascular disease risk, is related to endothelial function and predicts exercise capacity.

We studied group formation and anemone use in captively reared anemonefish Amphiprion frenatus. Four small fish (23–32 mm total length) were simultaneously released into a central area of the aquaria (ca. 160 l) that was divided in half with an anemone placed on each half, with the opportunity to settle on either side. Four-day trials were conducted for analyzing settlement patterns. Because of the monogamous mating system, we predicted that small fish should settle in pairs. However, all four small fish settled on the same side in 7 of 14 replicates, which was a higher than expected value under random settlement.

Aim: Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). Materials & Methods: Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. Results: DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. Conclusion: Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

It is now recognised that administration of oral nitrate (NO3(-)), in its various forms, increases the level of nitric oxide (NO) metabolites in the circulation of humans. Its application to modulate physiology and alleviate cardiovascular dysfunction in some patients is now recorded and shows particular promise in hypertension, in modifying platelet activation/aggregation, and in conditions where tissue ischaemia prevails. The potential of oral NO3(-) to modify exercise/performance via elevation of plasma nitrite concentration ([NO2(-)]) has been applied across a range of human test systems. Herein we discuss how the choice of NO3(-) source, route of administration and resulting pharmacokinetics might influence the outcome of physiological measures and potentially contribute to discrepancies in performance trials. There are but a few examples of detailed pharmacokinetic data on which the majority of researchers base their test protocols in different cohorts/settings. We compare and contrast the results of key publications with the aim of highlighting a consensus of our current understanding and critical considerations for those entering the field.

Nitric oxide (NO) bioavailability is important in vascular health, but unsuitable as a clinical measure due to biological oxidation. Total nitrogen oxides (NO(x)) are stable but background nitrate levels make it difficult to detect disease-based variation. We investigated the clinical discriminatory value of NO(x) as it relates to exercise capability (VO(2peak)) and brachial artery reactivity (BAR, an NO-dependent measure of endothelial health), in healthy (H), increased risk (RF), and known cardiovascular disease (CVD) subjects. BAR was measured using forearm occlusion/hyperemia stimulus. Subjects performed a maximal graded exercise test (GXT). Blood at rest, exercise termination, and 10 min into recovery was mixed equally with 0.1 M NaOH at 4 degrees C, filtered, and stored at -70 degrees C. NO(x) was measured by chemiluminescence. Seven of the RF group then exercise-trained for 6 months prior to retesting. The H group (n = 12) was younger, had higher VO(2peak), HDL levels, and baseline NO(x) values than the RF (n = 15) and CVD (n = 10) groups. NO(x) increased from baseline to recovery in the H group only (75.85 +/- 19.04 microM vs 97.76 +/- 31.93 microM; Por= 0.01). BAR was greater in the H versus CVD group (7.24 +/- 3.78% vs 2.59 +/- 3.53%; Por= 0.01). The relation between VO(2peak) and NO(x) recovery was significant across groups (r = 0.71, Por= 0.01). Following training the RF subjects (n = 7) increased VO(2peak) (29.98 +/- 6.74 to 33.08 +/- 5.57 ml kg(-1) min(-1); Por= 0.05), BAR (3.19 +/- 3.96 to 6.86 +/- 4.81%; Por= 0.05), and recovery NO(x) (18.29 +/- 6.46 to 66.48 + 21.11 microM; Por= 0.01). These findings suggest that plasma NO(x) following GXT discriminate cardiovascular disease status, are related to regional endothelial function, and respond favorably to exercise training.

Neural tube defects (NTDs) are the second most common birth defects (1 in 1000 live births) in the world. Periconceptional maternal folate supplementation reduces NTD risk by 50–70%; however, studies of folate related and other developmental genes in humans have failed to definitively identify a major causal gene for NTD. The aetiology of NTDs remains unknown and both genetic and environmental factors are implicated. We present findings from a microsatellite based screen of 44 multiplex pedigrees ascertained through the NTD Collaborative Group. For the linkage analysis, we defined our phenotype narrowly by considering individuals with a lumbosacral level myelomeningocele as affected, then we expanded the phenotype to include all types of NTDs. Two point parametric analyses were performed using VITESSE and HOMOG. Multipoint parametric and nonparametric analyses were performed using ALLEGRO. Initial results identified chromosomes 7 and 10, both with maximum parametric multipoint lod scores (Mlod) >2.0. Chromosome 7 produced the highest score in the 24 cM interval between D7S3056 and D7S3051 (parametric Mlod 2.45; nonparametric Mlod 1.89). Further investigation demonstrated that results on chromosome 7 were being primarily driven by a single large pedigree (parametric Mlod 2.40). When this family was removed from analysis, chromosome 10 was the most interesting region, with a peak Mlod of 2.25 at D10S1731. Based on mouse human synteny, two candidate genes (Meox2, Twist1) were identified on chromosome 7. A review of public databases revealed three biologically plausible candidates (FGFR2, GFRA1, Pax2) on chromosome 10. The results from this screen provide valuable positional data for prioritisation of candidate gene assessment in future studies of NTDs.

The Framingham Heart Study, initiated over 50 years ago, introduced the concept of risk factors for coronary heart disease (CHD) and has served as the standard for risk assessment over the years.1-4 Major risk factors identified by the Framingham HeartStudy, in- cluding age, sex, total cholesterol, high-density li- poprotein (HDL) cholesterol,smoking, and systolic blood pressure, have been incorporated into a scoring system thatidentifies subjects at high (>20%), interme- diate (10%–20%), and low ( 80% of the excess population risk for CHD.6-8 Recent clinical trials in high-risk subjectsdemonstrate dramatic reductions in risk (approximately 33%–50% in 5 y) with riskreduction therapies.9 This provides strong support for the concept that CHD and its sequela can be prevented by aggressive medical therapy and therapeutic lifestyle changes.Recent American Heart Association (AHA) guidelines (2002)4 for primary prevention ofcardiovascular disease and stroke recom- mend that risk-factor screening in adults shouldbegin at age 20 and should be repeated at least every 5 years in the absence of risk factors and every 2 years if risk factors are present. This panel recommends that global risk should be estimated in all adults >40 years of age. In this issue of the Journal, Cohn et al10 have proposed a method for risk assessment that focuses on measurements of early vasculardysfunction and disease markers rather than standard risk factors. Studies are ongoing intheir outpatient cardiovascular disease prevention clinic to validate the model by relating risk assessments to disease outcomes over time

Purpose: The purpose was to determine the influence of a unilateral localized short-term handgrip training protocol on brachial artery flow-mediated dilation (BAFMD) and to examine the time course of such changes. Methods: Fourteen healthy males (age: 26 ± 5.7 yr) underwent high-resolution ultrasonographic brachial artery assessments before (V1), during (V2–V7), and at the end of 4 wk (V8) of 60% maximal voluntary contraction handgrip training (20 min·d-1, 5 d·wk-1) of the nondominant arm. Results: Before training resting diameters were similar between the trained (nondominant) and untrained (dominant) arms. A 2 (trained and untrained arms) × 2 (V1 and V8) repeated measure ANOVA revealed a significant main (P = 0.02 and P = 0.03) and interaction effect (P = 0.05 and P = 0.01) for the percent and absolute change in BAFMD. BAFMD improved 62% and 70%, respectively from V1 to V8, for the percent and absolute change. Subsequent linear orthogonal polynomial contrasts indicate both the percent and absolute change in BAFMD were statistically different at V2 (end of week 1 and 4 training days) from V1. These unilateral changes were not accompanied by changes in resting artery diameter, hemodynamic measures, hematological markers, and indices of heart rate variability suggesting the change may be locally mediated. Conclusions: This study shows a localized short-term exercise-training program resulted in significant improvements in BAFMD in the trained arm compared with the untrained arm and suggests this occurred after only 4 d of training.

In cancer, T cells are exposed to persistent antigen stimulation and/or inflammatory signals. Such prolonged stimulation is often associated with deterioration of T cell effector functions resulting in a functional state termed ‘exhaustion’. Exhausted T cells are characterized by expression of multiple inhibitory receptors, loss of cytolytic function and decline in cytokine production capability. T cell exhaustion is likely associated with inefficient control of tumor progression. Identification of cell surface proteins involved in induction and maintenance of the exhausted state could inform novel immunomodulatory cancer therapies. To comprehensively investigate cell surface signature of the exhausted state and identify potential immuno-oncology (I-O) targets, we conducted in-depth expression profiling of membrane proteins from intact tumors (Colorectal, Pancreatic, Lung, Breast and Prostate cancer), different in vitro models of T cell exhaustion and primary tumor-derived lymphocytes (Colorectal, Breast and Lung cancer) by targeted proteomics for known cell type and activation markers. Using proteomic expression analysis and statistical clustering analysis methods, novel I-O targets were identified in both the in vitro models of T cell exhaustion and primary tumor-derived lymphocytes (TILs). The cancer specify of novel I-O targets was then interrogated using the expression profiling of cell membrane proteins from intact tumors. Using this approach we identified most known T cell inhibitory receptors (PD-1, CTLA-4, BTLA, TIM-3, KIRs, LAG-3 and TMIGD2) and their described ligands (B7-H1 to B7-H7). To generate more precise expression data to validate I-O targets emerging from the above discovery approaches and their known interacting partners we analyzed single tumors using an integrated evaluation methodology. Tumors were subject to multiple parallel analysis steps: (i) proteomic analysis of the tumor and TILs; (ii) TILs were subject to flow cytometry analysis for specific proteins; (iii) tumor tissue sections were subject to immuno-histochemical analysis for specific proteins. The multiple analysis platforms were integrated by key cell type and functional markers allowing the different types of information provided by each technique to be placed in an overall 3-D tumor context. We present one example of a potential I-O target identified using our integrated proteomic discovery approach which is further validated at the expression level using multiple whole tumor and TIL analyses. Citation Format: James Ackroyd, Joanne Berry, Yaoyao Fu, Jason Allen, Martin Barnes, Alexander Patterson, Angelo Kaplan, Christian Rohlff, Haining Huang, Livija Deban, Robert Boyd. Analysis of tumor infiltrating lymphocytes and in vitro exhausted T-cell models to identify unique Immuno-Oncology targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3715. doi:10.1158/1538-7445.AM2017-3715

UnlabelledErectile dysfunction (ED) is a major adverse effect of radical prostatectomy (RP). We conducted a randomized controlled trial to examine the efficacy of aerobic training (AT) compared with usual care (UC) on ED prevalence in 50 men (n=25 per group) after RP. AT consisted of five walking sessions per week at 55-100% of peak oxygen uptake (VO2peak) for 30-60 min per session following a nonlinear prescription. The primary outcome was change in the prevalence of ED, as measured by the International Index of Erectile Function (IIEF), from baseline to 6 mo. Secondary outcomes were brachial artery flow-mediated dilation (FMD), VO2peak, cardiovascular (CV) risk profile (eg, lipid profile, body composition), and patient-reported outcomes (PROs). The prevalence of ED (IIEF score ≤ 21) decreased by 20% in the AT group and by 24% in the UC group (difference: p=0.406). There were no significant between-group differences in any erectile function subscale (p>0.05). Significant between-group differences were observed for changes in FMD and VO2peak, favoring AT. There were no group differences in other markers of CV risk profile or PROs. In summary, nonlinear AT does not improve ED in men with localized prostate cancer in the acute period following RP.Trial registrationClinicaltrials.gov identifier NCT00620932.

Intradialytic hypertension affects ∼15% of hemodialysis patients and is associated with increased morbidity and mortality. While intradialytic hypertension is associated with increases in endothelin 1 relative to nitric oxide (NO), the cause of these imbalances is unknown. In vitro evidence suggests that altering plasma sodium levels could affect endothelial-derived vasoregulators and blood pressure (BP). Thus, we hypothesized that compared to high dialysate sodium, low dialysate sodium concentration would lower endothelin 1 levels, increase NO release, and reduce BP.3-week, 2-arm, randomized, crossover study.16 patients with intradialytic hypertension.Low (5 mEq/L below serum sodium) versus high (5 mEq/L above serum sodium) dialysate sodium concentration.Endothelin 1, nitrite (NO2(-)), and BP.Mixed linear regression was used to compare the effect of dialysate sodium (low vs high) and randomization arm (low-then-high vs high-then-low) on intradialytic changes in endothelin 1, NO2(-), and BP values.The average systolic BP throughout all hemodialysis treatments in a given week was lower with low dialysate sodium concentrations compared with treatments with high dialysate sodium concentrations (parameter estimate, -9.9 [95% CI, -13.3 to -6.4] mm Hg; P 0.001). The average change in systolic BP during hemodialysis also was significantly lower with low vs high dialysate sodium concentrations (parameter estimate, -6.1 [95% CI, -9.0 to -3.2] mm Hg; P 0.001). There were no significant differences in intradialytic levels of endothelin 1 or NO2(-) with low vs high dialysate sodium concentrations.Carryover effects limited the power to detect significant changes in endothelial-derived vasoregulators, and future studies will require parallel trial designs.Low dialysate sodium concentrations significantly decreased systolic BP and ameliorated intradialytic hypertension. Longer studies are needed to determine the long-term effects of low dialysate sodium concentrations on BP and clinical outcomes.

Chronic administration of a nitric oxide-donating drug fully corrects abnormal blood flow regulation in the mdx mouse model of dystrophinopathy. As a first step to clinical translation, we treated 6 men with Becker muscular dystrophy with a single oral dose of sodium nitrate- a simple, exogenous inorganic nitric oxide donor. We measured reflex vasoconstriction (ΔHb02, near infrared spectroscopy; evoked by lower body negative pressure) at rest and during rhythmic handgrip exercise, before and 3 hours after ~8.4 mmol of oral sodium nitrate. Before treatment, blood flow regulation was impaired in BMD, because handgrip exercise caused no attenuation of reflex vasoconstriction (ΔHbO2:-17±2 vs. -16±2 %, rest vs. HG). Remarkably, sodium nitrate supplementation more than doubled plasma nitrate and nitrite levels, and restored exercise-induced attenuation of reflex vasoconstriction (ΔHbO2: -18±1 vs. -11±2; P

High dose nitrate (NO3(-)) supplementation via beetroot juice (BR, 1 mmol/kg/day) lowers mean arterial blood pressure (MAP) and improves skeletal muscle blood flow and O2 delivery/utilization matching thereby raising microvascular O2 pressure (PO2mv). We tested the hypothesis that a low dose of NO3(-) supplementation, consistent with a diet containing NO3(-) rich vegetables (BRLD, 0.3 mmol/kg/day), would be sufficient to cause these effects. Male Sprague-Dawley rats were administered a low dose of NO3(-) (0.3 mmol/kg/day; n=12), a high dose (1 mmol/kg/day; BRHD, n=6) or tap water (control, n=10) for 5 days. MAP, heart rate (HR), blood flow (radiolabeled microspheres) and vascular conductance (VC) were measured during submaximal treadmill exercise (20 m/min, 5% grade, equivalent to ~60% of maximal O2 uptake). Subsequently, PO2mv (phosphorescence quenching) was measured at rest and during 180 s of electrically-induced twitch contractions (1 Hz, ~6 V) of the surgically-exposed spinotrapezius muscle. BRLD and BRHD lowered resting (control: 139 ± 4, BRLD: 124 ± 5, BRHD: 128 ± 9 mmHg, P0.05, BRLD vs. control) and exercising (control: 138 ± 3, BRLD: 126 ± 4, BRHD: 125 ± 5 mmHg, P0.05) MAP to a similar extent. For BRLD this effect occurred in the absence of altered exercising hindlimb muscle(s) blood flow or spinotrapezius PO2mv (rest and across the transient response at the onset of contractions, all P0.05), each of which increased significantly for the BRHD condition (all P0.05). Whereas BRHD slowed the PO2mv kinetics significantly (i.e.,mean response time, MRT; control: 16.6 ± 2.1, BRHD: 23.3 ± 4.7s) following the onset of contractions compared to control, in the BRLD group this effect did not reach statistical significance (BRLD: 20.9 ± 1.9s, P=0.14). These data demonstrate that while low dose NO3(-) supplementation lowers MAP during exercise it does so in the absence of augmented muscle blood flow, VC and PO2mv; all of which are elevated at a higher dose. Thus, in healthy animals, a high dose of NO3(-) supplementation seems necessary to elicit significant changes in exercising skeletal muscle O2 delivery/utilization.

To determine the short term effects (21 days) of 200 mg (7% standardized) Panax ginseng supplementation vs. placebo on peak aerobic exercise performance in healthy young adults, with unrestricted diets.Twenty men and eight women (age = 23.2 +/- 3.2 years, height = 175.8 +/- 8.6 cm; weight = 75.2 +/- 15.3 kg) were randomly assigned to either a Panax ginseng or placebo group for a period of 3 weeks in a double blind design. Prior to and following treatment the subjects performed a symptom limited graded exercise test on a Schwinn Airdyne ergometer. The data were analyzed using an analysis of variance.No significant treatment effect was observed for the dependent variables of VO2, exercise time, workload, plasma lactate and hematocrit at peak levels, or for heart rate and rate of perceived exertion at 150 watts, 200 watts and peak.The results of this study do not support an ergogenic effect on peak aerobic exercise performance following a 3-week supplementation period of 200 mg 7% Panax ginseng in healthy young adults with moderate exercise capacities and unrestricted diets.

Advancing age is associated with an increase in physical impairment, functional limitations, disability, and loss of independence. Regular physical activity conveys health benefits, but the yield on physical function in the elderly, is less clear. Current exercise guidelines are focused predominantly on aerobic programs despite evidence that age-associated declines are mediated by peripheral tissue changes. The Fit for Life trial proposes a new paradigm of exercise training for the elderly that uses a low-mass high-repetition training regimen specifically focused on peripheral tissue beds or body regions (Regional Specific Training Stimulus — RSTS). RSTS is designed to deliver a localized stimulus to the peripheral vasculature, bone and muscle, without imposing a significant central cardiorespiratory strain. The purpose of this study is three-fold; 1) to derive effect sizes from the RSTS intervention by which to power a subsequent larger, confirmatory trial; 2) to assess fidelity of the RSTS intervention; and 3) to assess the interrelationship of the primary endpoints of physical impairment/fitness (VO2peak, 1 repetition maximal contraction) and function (Senior Fitness Test scores) following two versions of a 4 + 8 week protocol. Men and women over 70 years, at risk for losing independence will be randomized to either 4 weeks of RSTS or “aerobic” exercise, followed by an identical 8 weeks of progressive whole-body training (aerobic plus resistance). The guiding hypothesis is that the magnitude of adaptation after 12 weeks will be greatest in those initially randomized to RSTS. Possible mediators of the intervention effect — physical impairment/fitness and function relationship, including vascular function, muscle mass, strength, and physiology will also be assessed.

Acoustic Radiation Force Impulse (ARFI) imaging has shown improved visualization of arterial structures that may provide for a more reliable assessment of atherosclerotic risk than conventional B-mode imaging. Existing ARFI imaging methods, however, are limited due to the presence of clutter that can increase bias and jitter in estimates of tissue deformation. We have recently developed a novel pulse inversion harmonic tracking method to suppress clutter degradation in ultrasonic tissue displacement estimates. In this work, harmonic B-mode and harmonic ARFI images of in vivo carotid arteries are compared with fundamental images obtained using conventional techniques. A certified reader measured the intima-media-thickness (IMT) in B-mode images and the adventitia-intima-media-thickness (AIMT) in ARFI images. The yield and the length of the artery amenable for analysis were larger in ARFI images compared to B-mode images. Intra-reader measurements of ARFI AIMT in the distal wall were more reproducible than B-mode IMT measurements. Qualitatively, harmonic ARFI images showed improved delineation of the vessel-lumen interface compared to fundamental ARFI images. These results suggest that harmonic ARFI imaging may provide a reliable method for monitoring arterial thickening to predict the occurrence of clinical events.

Solid tumors comprises of two distinct compartments: cancer cells and the stroma that the cancer cells induce and are dispersed in. This stroma contains stromal (Fibroblasts, endothelial and pericyte cells) and infiltrating immune cells (Lymphocytes, macrophages, granulocytes and myeloid derived suppressor cells). Recent oncology research has implicated these stroma cells as promoters of tumor progression and there is thus a strong need to profile the cell membrane proteins present on these cells. For tumors which do not contain infiltrating T cells (non-immunogenic tumors) it has been recognized that it will be necessary to employ therapeutic agents to disrupt the tumor micro-environment (TME); allowing the release and processing of tumor antigens by antigen presenting cells, entry and migration T cells into intrastromal sites and potent activation of T cell responses to tumor antigens. We have conducted in-depth proteomic profiling of tumors from 14 different solid cancer indications with varying degrees of stroma involvement to characterize their immune and stromal cell composition. The data from this work was analyzed in OGAP®; a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify membrane protein expression levels and profiles. It is one of the world's largest proprietary cell-membrane focused proteomic database, with data on over 5000 cancer membrane proteins. Purified stroma cell populations were also profiled to facilitate the interpretation of whole tumor proteomic data. Cell membrane proteins present in tumors from different cancer indications were analyzed by mass spectrometry. We classified tumors as immunogenic or non-immunogenic using validated T cell markers (CD3, CD4, CD8 and CD69). We then profiled the non-immunogenic tumors for fibroblast, macrophage and B cell type markers to try and identify strong stroma cell expression patterns not detected in normal tissues. Importantly, most current antibody therapeutic stroma cell targets (CSF-1R, FAP and VEGF-A) were re-discovered. Several novel stroma cell therapeutic targets were identified which if targeted by a therapeutic antibody have the potential to enhance the T cell immune response in tumors resistant to current immunotherapy. Citation Format: James Ackroyd, Jason Allen, Jo Berry, Lisa Roesch, Martin Barnes, Arnima Bisht, Christian Rohlff, Keith Wilson, Dee Aud, Robert Boyd. The use of proteomics to analyse whole tumors and identify unique stroma cell targets for antibody-based therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 753.

Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice.

Dietary nitrate (NO(3)(-)) supplementation, via its reduction to nitrite (NO(2)(-)) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O(2) cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO(3)(-) supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO(3)(-) supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Male Sprague-Dawley rats (3-6 months) were administered either NO(3)(-) (via beetroot juice; 1 mmol kg(-1) day(-1), BR n = 8) or untreated (control, n = 11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabelled microsphere infusions) were measured during submaximal treadmill running (20 m min(-1), 5% grade). BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mm, P0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml min(-1) (100 g)(-1), P0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml min(-1) (100 g)(-1) mmHg(-1), P0.05) were greater in rats that received BR compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percentage type IIb + d/x muscle fibres (blood flow: r = 0.74, vascular conductance: r = 0.71, P0.01 for both). These data support the hypothesis that NO(3)(-) supplementation improves vascular control and elevates skeletal muscle O(2) delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO(3)(-) supplementation improves metabolic control.

Peripheral artery disease (PAD) represents a burgeoning form of cardiovascular disease associated with significant clinical morbidity and increased 5 year cardiovascular disease mortality. It is characterized by impaired blood flow to the lower extremities, claudication pain and severe exercise intolerance. Pathophysiological factors contributing to PAD include atherosclerosis, endothelial cell dysfunction, and defective nitric oxide metabolite physiology and biochemistry that collectively lead to intermittent or chronic tissue ischemia. Recent work from our laboratories is revealing that nitrite/nitrate anion and nitric oxide metabolism plays an important role in modulating functional and pathophysiological responses during this disease. In this review, we discuss experimental and clinical findings demonstrating that nitrite anion acts to ameliorate numerous pathophysiological events associated with PAD and chronic tissue ischemia. We also highlight future directions for this promising line of therapy.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12).

Potential blood pressure- (BP-) lowering mechanisms of the DASH dietary pattern were measured in 20 unmedicated hypertensive adults in a controlled feeding study. At screening, participants averaged 4 4 . 3 ± 7 . 8 years, BMI 3 3 . 9 ± 6 . 6 Kg/m2, and BP 1 4 4 . 2 ± 9 . 3 8 / 8 8 . 5 ± 6 . 0 3 mmHg. All consumed a control diet for one week, then were randomized to control or DASH for another two weeks (week one and two). With DASH, but not controls, SBP fell by 1 0 . 6 5 ± 1 2 . 8 9 ( 𝑃 = 0 . 0 2 3 ) and 9 . 6 0 ± 1 1 . 2 3 ( 𝑃 = 0 . 0 3 9 ) mmHg and DBP by 5 . 9 5 ± 8 . 0 1 ( 𝑃 = 0 . 0 6 9 ) and 8 . 6 0 ± 9 . 1 3 mmHg ( 𝑃 = 0 . 0 1 1 ) at the end of week one and two, respectively. Univariate regressions showed that changes in urinary sodium/potassium ratio ( 𝛽 = 1 . 9 9 ) and plasma renin activity ( 𝛽 = − 1 5 . 7 8 ) and percent change in plasma nitrite after hyperemia were associated with SBP changes at week one (all 𝑃 < 0 . 0 5 ). Plasma nitrite following hyperemia showed a treatment effect ( 𝑃 = 0 . 0 1 4 ) and increased at week two ( 𝑃 = 0 . 0 0 1 ). Pulse wave velocity decreased over time with DASH (trend 𝑃 = 0 . 0 1 9 ), and reached significance at week two ( 𝑃 = 0 . 0 2 6 ). This response may be mediated by an improvement in upregulation of nitric oxide bioavailability. Early natriuresis and reductions in oxidative stress cannot be ruled out. Future studies are needed to verify these findings, assess the possibility of earlier effects, and examine other potential mediators.

Objective— Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, causing claudication and exercise intolerance. The mechanism(s) by which exercise training improves functional capacity is not understood. This study tested the hypothesis that in PAD patients who undergo supervised exercise training, increases in capillary density (CD) in calf muscle take place before improvements in peak oxygen uptake (VO 2 ). Methods and Results— Thirty-five PAD patients were randomly assigned to 12 weeks of directly supervised or home-based exercise training. Peak VO 2 testing and gastrocnemius muscle biopsies were performed at baseline and after training. CD (endothelial cells/mm 2 ) was measured using immunofluorescence staining. After 3 weeks of directly supervised training, patients had an increase in CD (216±66 versus 284±77, P 2 . However, after 12 weeks, peak VO 2 increased (15.3±2.8 versus 16.8±3.8, P 2 before and after directly supervised training. Conclusion— Changes in CD in ischemic muscle with training may modulate the response to training, and those changes precede the increase in VO 2 .

The aim of this study was to determine if skeletal muscle capillary density is lower in patients with peripheral artery disease (PAD) and if capillary density relates to functional limitations. PAD patients with intermittent claudication (IC) have a decreased exercise tolerance due to exercise-induced muscle ischemia. Despite the apparent role diminished arterial flow has in this population, the degree of walking pain and functional limitation is not entirely explained by altered hemodynamics of the affected limbs. We hypothesized that skeletal muscle capillary density is lower in PAD and is related to the functional impairment observed in this population. Sixty-four patients with PAD and 56 controls underwent cardiopulmonary exercise testing and a gastrocnemius muscle biopsy. A subset of these patients (48 PAD and 47 controls) underwent peak hyperemic flow testing via plethysmography. Capillary density in PAD patients was lower compared with controls ( P < 0.001). After adjustment for several baseline demographic imbalances the model relating capillary density to peak oxygen consumption (V̇o2) remained significant ( P < 0.001). In PAD subjects, capillary density correlated with peak V̇o2, peak walking time (PWT), and claudication onset time (COT). Peak hyperemic blood flow related to peak V̇o2 in both PAD and control subjects. PAD is associated with lower capillary density, and capillary density is related to the functional impairment as defined by a reduced peak V̇o2, PWT, and COT. These findings suggest that alterations in microcirculation may contribute to functional impairment capacity in PAD.

Stroke is the third leading cause of death and long-term disability in the USA. Currently, surgical intervention decisions in asymptomatic patients are based upon the degree of carotid artery stenosis. While there is a clear benefit of endarterectomy for patients with severe (> 70%) stenosis, in those with high/moderate (50–69%) stenosis the evidence is less clear. Evidence suggests ischemic stroke is associated less with calcified and fibrous plaques than with those containing softer tissue, especially when accompanied by a thin fibrous cap. A reliable mechanism for the identification of individuals with atherosclerotic plaques which confer the highest risk for stroke is fundamental to the selection of patients for vascular interventions. Acoustic radiation force impulse (ARFI) imaging is a new ultrasonic-based imaging method that characterizes the mechanical properties of tissue by measuring displacement resulting from the application of acoustic radiation force. These displacements provide information about the local stiffness of tissue and can differentiate between soft and hard areas. Because arterial walls, soft tissue, atheromas, and calcifications have a wide range in their stiffness properties, they represent excellent candidates for ARFI imaging. We present information from early phantom experiments and excised human limb studies to in vivo carotid artery scans and provide evidence for the ability of ARFI to provide high-quality images which highlight mechanical differences in tissue stiffness not readily apparent in matched B-mode images. This allows ARFI to identify soft from hard plaques and differentiate characteristics associated with plaque vulnerability or stability.

Atherosclerosis develops in an environment of endothelial injury and inflammation. Circulating endothelial progenitor cells (EPCs) are required for vascular repair and restoration of normal endothelial function. We tested the hypothesis that the nonselective cyclooxygenase (COX) inhibitor aspirin (ASA) exerts an effect on circulating EPCs. Methods As part of a larger study evaluating the effect of aspirin dose in primary and secondary prevention, subjects (n = 32) were assigned randomly to either 81 mg or 325 mg aspirin daily for two months, and circulating mononuclear cells were enumerated at the beginning of the study and after 2 months using fluorescent antibodies against CD34 and CD133 as well as based on aldehyde dehydrogenase (ALDH) activity. Brachial artery endothelial function via flow-mediated dilation (BAFMD) and light transmittance platelet aggregometry in response to physiologic agonists was also determined. Results Subjects taking aspirin at the time of study entry had a lower numbers of CD133+/34+ cells compared to those not previously exposed (0.01% vs. 0.05% of MNCs, P < 0.03). After 2 months, subjects randomized to 81 vs. 325 mg of ASA had no significant differences in the median numbers of EPCs, although mean numbers trended lower in the high dose group. Patients on chronic ASA therapy continued to have lower numbers of EPCs. Similar effects were observed in CD34 and CD 133 single-positive cells, as well as ALDHbr cells. BAFMD did not differ nor change significantly over time between aspirin dose groups. All patients had decreased ex vivo platelet aggregation in response to arachidonic acid and ADP stimulation. Conclusions Our preliminary studies suggest that aspirin exerts a time-dependent effect on circulating EPCs. Short-term exposure to differing doses of ASA had indeterminate effects on EPCs levels, suggesting that time of ASA exposure may play a more important role than dose. Determining the responsible mechanism(s) and the overall clinical relevance of these findings will require further investigation.

This study examined the effects of age-associated increases in fibrinogen on brachial artery flow-mediated dilation in 30 healthy nonsmokers. The findings demonstrate an inverse association between normal plasma fibrinogen levels and vascular function (r = -0.56, p = 0.001), suggesting elevated plasma fibrinogen may decrease the artery's responsiveness to certain vasodilatory signals, such as shear stress.

Acoustic radiation force impulse (ARFI) imaging characterizes the mechanical properties of tissue by measuring displacement resulting from applied ultrasonic radiation force. In this paper, we describe the current status of ARFI imaging for lower-limb vascular applications and present results from both tissue-mimicking phantoms and in vivo experiments. Initial experiments were performed on vascular phantoms constructed with polyvinyl alcohol for basic evaluation of the modality. Multilayer vessels and vessels with compliant occlusions of varying plaque load were evaluated with ARFI imaging techniques. Phantom layers and plaque are well resolved in the ARFI images, with higher contrast than B-mode, demonstrating the ability of ARFI imaging to identify regions of different mechanical properties. Healthy human subjects and those with diagnosed lower-limb peripheral arterial disease were imaged. Proximal and distal vascular walls are well visualized in ARFI images, with higher mean contrast than corresponding B-mode images. ARFI images reveal information not observed by conventional ultrasound and lend confidence to the feasibility of using ARFI imaging during lower-limb vascular workup.

Atherosclerotic disease in the carotid artery is a risk factor for stroke. The susceptibility of atherosclerotic plaque to rupture, however, is challenging to determine by any imaging method. In this study, acoustic radiation force impulse (ARFI) imaging is applied to atherosclerotic disease in the carotid artery to determine the feasibility of using ARFI to noninvasively characterize carotid plaques. ARFI imaging is a useful method for characterizing the local mechanical properties of tissue and is complementary to B-mode imaging. ARFI imaging can readily distinguish between stiff and soft regions of tissue. High-resolution images of both homogeneous and heterogeneous plaques were observed. Homogeneous plaques were indistinguishable in stiffness from vascular tissue. However, they showed thicknesses much greater than normal vascular tissue. In heterogeneous plaques, large and small soft regions were observed, with the smallest observed soft region having a diameter of 0.5 mm. A stiff cap was observed covering the large soft tissue region, with the cap thickness ranging from 0.7–1.3 mm.

The recent clinical success of the mAb therapeutics targeting immune checkpoint inhibitor proteins (PD-1/PD-L1, CTLA-4) has led to an increased appreciation of the potential of utilizing the immune system in oncology. There are two major strategies to elicit either a novel immune anti-tumor response or to reactivate a pre-existing anti-tumor response: by releasing a checkpoint inhibitory pathway via cell surface receptors (such as PD-1/PD-L1, CTLA-4) or by activation of co-stimulatory receptors (such as CD40, OX40, or GITR). Both of these strategies of immune modulation utilize cell surface receptors, and the targeting of antibody therapeutics with the appropriate functional activity to those receptors, to modify immune cell responses and allow for anti-tumor activity. The identification of novel immune-modulatory receptors with the potential to be immune-oncology therapeutic targets could be of high value to this anti-tumor approach. OGAP is a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify membrane protein expression levels and profiles. Specifically, it currently holds information on ∼16,000 human proteins sequenced, ∼7,000 membrane proteins, ∼35 tissues/organs, and ∼17 cancers. OGAP is fed by a proprietary sample preparation and processing workflow that relies on state-of-the-art high-throughput mass spectrometry and data processing to provide quantitative information on over 4,000 membrane-enriched proteins. OGAP has been used to identify novel oncology therapeutic targets for both ADC and BiTE-like approaches. Utilizing OGAP, membrane proteins present in tumors from five cancer indications (Pancreatic, Lung, Breast, Colorectal and Esophageal cancer) and multiple normal tissues or cells were analyzed. Validated immune cell markers (such as CD8, OX40, CD79B, TLR1, TLR2, TLR4, TLR7, CD56, CD204 and CD207) were profiled across different normal and tumor proteomic data sets. This analysis demonstrated we detect key immune cell markers in tumors and that different immune cell populations are found in tumors from the same or different cancer indications. The proteomic data sets were next analyzed for the presence of validated immuno-oncology targets (TIM3, PDL-1 and B7-H3). The expression patterns of these immune-oncology targets were analyzed to try and identify a unique protein signature. Using this protein expression profiling approach we identified most known immune-oncology targets, validating this as an approach to identify a pool of candidate novel immune-oncology targets. To further develop our approach we also used sequence based homology searching of uncharacterized membrane proteins to improve the quality of the pool of candidate novel immune-oncology targets. Several potential novel immune-oncology targets will be presented. Citation Format: Jim Ackroyd, Arnima Bisht, Jason Allen, Lindsey Hudson, Martin Barnes, Christian Rohlff, Keith Wilson, Robert Boyd, Dee Aud. The use of proteomics to analyze whole tumors and identify unique immuno-oncology targets for antibody-based therapeutics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1321. doi:10.1158/1538-7445.AM2015-1321

Non-invasive characterization of focal and dif- fuse atherosclerosis is difficult with existing techniques, but clinically important in guiding the selection of drug, surgical and other treatments. We describe new non-invasive tech- niques for monitoring vascular health with ARFI imaging. Custom ECG-gated beam sequencing was implemented on the Siemens SONOLINE Antares scanner to measure radiation force induced displacements for in vivo vascular tissue. We have developed layered polyvinyl alcohol cryogel (PVA- C) vessel phantoms to mimic arterial layers in the vascular wall. A custom phantom with layers of varying stiffness but homogeneous echogenicity is statically pressurized over the physiological range, and then imaged with acoustic radiation force. Layers are well visualized in the corresponding ARFI images with little contrast seen in the matched B-mode image. Displacement data is examined over the physiological range and suggests that PVA-C demonstrates some nonlinear behavior over physiologically realistic pressures. We present the preliminary results of a clinical pilot study involving twenty-four volunteers with no predisposition for peripheral vascular disease (PVD), and fifteen patients with diagnosed PVD. Both proximal and distal vascular walls are well visualized in the corresponding ARFI displacement images. Plaques that are present in the B-mode image are also well resolved in the corresponding ARFI image, with ARFI displacement images showing higher contrast between the plaque and surrounding vascular tissue than the conventional B-mode image.

Brachial artery flow-mediated dilation (BAFMD) has been proposed as a measurement of the degree and severity of cardiovascular disease. The purpose of this study was to (1) evaluate the associations between BAFMD and homocysteine, folate, vitamin B(12), vitamin B(6); (2) examine the influence of 5,10-methylenetetrahydrofolate reductase (MTHFR) genotypes on homocysteine levels and BAFMD; and (3) evaluate the effect of homocysteine on the baseline diameter of the vessel vs BAFMD. A total of 174 healthy research subjects were examined for BAFMD, homocysteine, folate, vitamin B(12), vitamin B(6), and MTHFR genotype, nucleotide 677 C--T. The data indicated a significant inverse correlation between homocysteine and BAFMD (r = -0.1763, P = .02). There was a significant difference in BAFMD between MTHFR genotype groups (P = .01) (T/T vs C/C, P = .042; C/C vs C/T, P = .13; T/T vs C/T, P = .003). Homocysteine was significantly associated with the baseline brachial artery diameter (r = 0.1878, P = .013). The data confirmed a significant inverse correlation between baseline diameter and BAFMD (r = -0.3321, P = .0001). Regression analysis indicated that the MTHFR genotype, homocysteine, and age were significant predictors of BAFMD (P = .0001, r(2) = 0.118). When the baseline brachial diameter was incorporated into the model, the effect of homocysteine on BAFMD disappeared. The present data indicate an association between homocysteine and BAFMD and reduced BAFMD in individuals with the MTHFR nucleotide 677 T/T genotype, despite similar blood values for folate and homocysteine. Finally, the data suggest that the effect of homocysteine on vascular reactivity is in part a consequence of its influence on baseline brachial artery diameter.

Acoustic Radiation Force Impulse (ARFI) imaging is a useful method for characterizing the local mechanical properties of tissue. ARFI imaging uses high energy, focused ultrasound pulses to generate small displacements (1-10 mum) in tissue. The response of tissue to these localized forces is observed using a series of imaging pulses, which track displacement of the tissue. ARFI imaging is complementary to B-mode imaging, and can readily distinguish between stiff and soft regions of tissue with high contrast. Currently, physicians cannot easily determine whether atherosclerotic plaques in carotid arteries are vulnerable to rupture. We are investigating the use of ARFI imaging to characterize atherosclerotic plaque in carotid arteries and to guide the treatment of these plaques. We have created custom beam sequences on a Siemens Antarestrade scanner to implement ARFI imaging. Five patients with atherosclerotic plaque and four volunteers without known atherosclerotic plaque underwent B-Mode and ARFI scanning of their carotid arteries. Measurements of the complete vessel wall (adventitial layer plus IMT) with ARFI imaging are shown to be consistent with measurements of the intima-media thickness (IMT) in normal B-mode scans. In plaque-filled vessels, the mean near-wall IMT was 0.92 mm and the mean far-wall IMT was 1.24 mm. In ARFI imaging, vessel walls have mean thicknesses of 2.17 mm and 2.17 mm for the near and far walls, respectively. For volunteers without plaques, the near and far wall IMT measurements were 0.52 and 0.54 mm, respectively and the ARFI vessel thicknesses were 1.39 and 1.26 mm. Both IMT measurements and ARFI measurements were significantly (p < 0.005) greater for patients with plaques. We demonstrate high-resolution ARFI images of carotid plaques depicting their complex mechanical structure, which often involve regions of soft and hard plaque. We discuss methods of improving the contrast and resolution of ARFI vascular images

The measurement of nitric oxide (NO) bioavailability is of great clinical interest in the assessment of vascular health. However, NO is rapidly oxidized to form nitrite and nitrate and thus its direct detection in biological systems is difficult. Venous plasma nitrite (nM concentrations) has been shown to be a marker of forearm NO production following pharmacological stimulation of the endothelium utilizing acetylcholine (Ach). In the present study, we demonstrate, within 15 apparently healthy subjects (34.1 +/- 7.3 years), that reactive hyperemia of the forearm, a physiological endothelial stimulus, results in a 52.5% increase in mean plasma nitrite concentrations (415 +/- 64.0 to 634 +/- 57.1 nM, P = 0.015). However, plasma nitrite is readily oxidized to nitrate within plasma, and thus its utility as a marker of NO production within the clinical setting may be limited. Alternatively, NOx (predominantly nitrate) is relatively stable in plasma (microM concentrations), but is produced by sources other than the vasculature and has been shown to be unsuitable as a measure of localized NO production. We reasoned that the principle source of NOx generation during exercise is NO production and thus have examined the change in NOx following treadmill exercise stress. In this study, 12 apparently healthy subjects showed an increase (from baseline) in venous NOx at peak effort and during recovery (12 +/- 9.1 and 17 +/- 15.3 microM respectively, P0.05). In contrast, 10 subjects with cardiovascular disease showed no significant increases. Additionally, a correlation between VO(2peak) and the change in circulating NOx (r(2) = 0.4585, Por = 0.01) indicated the subjects who could exercise hardest also produced the most NO.

Peripheral artery disease (PAD) is a form of cardiovascular disease (CVD) caused by atherosclerosis which leads to arterial obstruction and decreased blood flow to the lower extremities. The most frequent clinical symptomatic manifestation of PAD is intermittent claudication (IC) defined as, pain in 1 or both legs during exercise that is relieved with rest. This inability to match oxygen delivery to tissue demands during physical activity severely debilitates subjects with PAD such that approximately 1/3rd have pain ambulating in their home. These patients suffer a markedly impaired quality of life and a high perception of disability. Although measures of conduit vessel and gross limb blood flow are used to diagnose PAD, they show a poor relationship with functional capacity. It appears that the key to increasing functionality in IC patients may lie at the resistance arteries, arterioles and capillaries that serve the skeletal muscle tissue distal to the site of stenosis. These are the vessels which are responsible for much of the oxygen delivery and become hypoxic during the increased demands for perfusion accompanying physical exertion. In this talk we discuss the role of endogenous and exogenous nitrate, nitrite, and nitric oxide pathways and their impact to; (a) acutely improve oxygenation to areas of ischemia and; (b) chronically increase vessel growth to these ischemic areas. This promising therapy may allow for greater exercise tolerance, ease the burden of exercise compliance and facilitate greater improvements in function and quality of life in PAD patients.