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Background Individuals with cancer experience loss of function and disability due to disease and cancer-related treatments. Physical fitness and frailty influence treatment plans and may predict cancer outcomes. Outcome measures currently used may not provide sufficiently comprehensive assessment of physical performance. Objective The objectives of this study are to: (1) describe the development of a functional measure, the Bellarmine Norton Assessment Tool (BNAT), for individuals with cancer; and (2) assess the relationship between the BNAT and the Eastern Cooperative Oncology Group (ECOG) Performance Status, a commonly used classification system by oncologists. Design This was a prospective cohort correlation study. Methods The BNAT encompasses 1 self-reported physical activity question and 4 objective tests: 2-Minute Step Test, 30-Second Sit to Stand, Timed Arm Curl, and Timed Up and Go. The BNAT score and its components were compared with ECOG Performance Status scores assigned by oncologists and analyzed for correlation and agreement. Results A total of 103 male and female individuals (ages 33–87 years) with various cancer diagnoses participated. The mean (SD) ECOG Performance Status score was 0.95 (0.87), range 0 to 3, and the mean BNAT score was 14.9 (4.3), range 5 to 24. Spearman agreement association of BNAT and ECOG Performance Status scores revealed a significant moderate negative relationship (r = −0.568). Limitations The BNAT was compared with the ECOG Performance Status, a commonly used but subjective measure. Additionally, a common data set was used for both deriving and evaluating the BNAT performance scale. Conclusions There was a moderate negative linear relationship of BNAT to ECOG Performance Status scores across all participants. Utilization of the BNAT may reflect overall physical performance and provide comprehensive and meaningful detail to influence therapeutic decisions.

BACKGROUND: There is a paucity of literature about the adverse events associated with Therapeutic Dry Needling (TDN). Much of the literature surrounding adverse events associated with TDN has been extrapolated from the acupuncture literature. Given that acupuncture and TDN are distinctly different in their application and proposed mechanisms, adverse events associated with TDN should be examined specifically. PURPOSE: To determine and report the type of adverse events associated with the utilization of TDN. STUDY DESIGN: Prospective Questionnaire METHODS: Four hundred and twenty physical therapists participated in this study. Information related to minor and major adverse events that occurred during 20,464 TDN treatment sessions was collected. Each physical therapist respondent was asked to fill out two weekly self-reported electronic surveys over a six-week period. One survey was related to “minor adverse events” (i.e. pain, bleeding, bruising), while the other was related to “major adverse events” (i.e. pneumothorax, excessive bleeding, prolonged aggravation). Following the six-week period, descriptive statistics were used to describe the adverse events (AE) associated with TDN and calculate the frequencies of those events. RESULTS: A total of 7,531 minor AE's were reported, indicating that 36.7% of the reported TDN treatments resulted in a minor AE. The top three minor AE's were bleeding (16%), bruising (7.7%), and pain during dry needling (5.9 %). The average ratio of minor AE's for all respondents across all weeks was 0.53 or approximately one event for every two patients. Twenty major AE's were reported out of the 20,494 treatments for a rate of

Pulmonary granuloma formation is a complex and poorly understood response to inhaled pathogens and particulate matter. To explore the mechanisms of pulmonary granuloma formation and maintenance, our laboratory has developed a multiwall carbon nanotube (MWCNT)-induced murine model of chronic granulomatous inflammation. We have demonstrated that the MWCNT model closely mimics pulmonary sarcoidosis pathophysiology, including the deficiency of alveolar macrophage ATP-binding cassette (ABC) lipid transporters ABCA1 and ABCG1. We hypothesized that deficiency of alveolar macrophage ABCA1 and ABCG1 would promote pulmonary granuloma formation and inflammation. To test this hypothesis, the effects of MWCNT instillation were evaluated in ABCA1, ABCG1, and ABCA1/ABCG1 myeloid-specific knockout (KO) mice. Histological examination revealed significantly larger pulmonary granulomas in ABCG1-KO and ABCA1/ABCG1 double-KO animals when compared with wild-type animals. Evaluation of BAL cells indicated increased expression of CCL2 and osteopontin, genes shown to be involved in the formation and maintenance of pulmonary granulomas. Single deficiency of alveolar macrophage ABCA1 did not affect MWCNT-induced granuloma formation or proinflammatory gene expression. These observations indicate that the deficiency of alveolar macrophage ABCG1 promotes pulmonary granulomatous inflammation and that this is augmented by additional deletion of ABCA1.

IntroductionExposure to airborne particulate matter (PM) is associated with cardiovascular disease. These outcomes are believed to originate from pulmonary oxidative stress and the systemic delivery of oxidised biomolecules (eg, aldehydes) generated in the lungs. Carnosine is an endogenous di-peptide (β-alanine-L-histidine) which promotes physiological homeostasis in part by conjugating to and neutralising toxic aldehydes. We hypothesise that an increase of endogenous carnosine by dietary supplementation would mitigate the adverse cardiovascular outcomes associated with PM exposure in humans.Methods and analysisTo test this, we designed the Nucleophilic Defense Against PM Toxicity trial. This trial will enroll 240 participants over 2 years and determine if carnosine supplementation mitigates the adverse effects of PM inhalation. The participants will have low levels of endogenous carnosine to facilitate identification of supplementation-specific outcomes. At enrollment, we will measure several indices of inflammation, preclinical cardiovascular disease and physical function. Participants will be randomly allocated to carnosine or placebo groups and instructed to take their oral supplement for 12 weeks with two return clinical visits and repeated assessments during times of peak PM exposure (June–September) in Louisville, Kentucky, USA. Statistical modelling approaches will be used to assess the efficacy of carnosine supplementation in mitigating adverse outcomes.Ethics and disseminationThis study protocol has been approved by the Institutional Review Board at the University of Louisville. Results from this study will be disseminated at scientific conferences and in peer-reviewed publications.Trial registration:NCT03314987; Pre-results

The broad list of commercial applications for multi-walled carbon nanotubes (MWCNT) can be further expanded with the addition of various surface chemistry modifications. For example, standard commercial grade MWCNT (C-grade) can be carboxylated (COOH) or nitrogen-doped (N-doped) to suite specific utilities. We previously reported dose-dependent expansions of cardiac ischemia/reperfusion (I/R) injury, 24 hours after intratracheal instillation of C-grade, COOH, or N-doped MWCNT in mice. Here we have tested the hypothesis that airway exposure to MWCNT perturbs cardiovascular adenosinergic signaling, which could contribute to exacerbation of cardiac I/R injury. One hundred microliters of Vehicle or identical suspension volumes containing 100 μg of C-grade, COOH, or N-doped MWCNT were instilled into the trachea of CD-1 ICR mice. One day later we measured cyclic adenosine monophosphate (cAMP) concentrations in cardiac tissue and evaluated arterial adenosinergic smooth muscle signaling mechanisms related to nitric oxide synthase (NOS) and cyclooxygenase (COX) in isolated aortic tissue. We also verified cardiac I/R injury expansion and examined both lung histology and bronchoalveolar lavage fluid cellularity in MWCNT exposed mice. Myocardial cAMP concentrations were reduced (p < 0.05) in the C-grade group by 17.4% and N-doped group by 13.7% compared to the Vehicle group. Curve fits to aortic ring 2-Cl-Adenosine concentration responses were significantly greater in the MWCNT groups vs. the Vehicle group. Aortic constrictor responses were more pronounced with NOS inhibition and were abolished with COX inhibition. These findings indicate that addition of functional chemical moieties on the surface of MWCNT may alter the biological responses to exposure by influencing cardiovascular adenosinergic signaling and promoting cardiac injury.

We established a murine model of multiwall carbon nanotube (MWCNT)-elicited chronic granulomatous disease that bears similarities to human sarcoidosis pathology, including alveolar macrophage deficiency of peroxisome proliferator–activated receptor γ (PPARγ). Because lymphocyte reactivity to mycobacterial antigens has been reported in sarcoidosis, we hypothesized that addition of mycobacterial ESAT-6 (early secreted antigenic target protein 6) to MWCNT might exacerbate pulmonary granulomatous pathology. MWCNTs with or without ESAT-6 peptide 14 were instilled by the oropharyngeal route into macrophage-specific PPARγ-knockout (KO) or wild-type mice. Control animals received PBS or ESAT-6. Lung tissues, BAL cells, and BAL fluid were evaluated 60 days after instillation. PPARγ-KO mice receiving MWCNT + ESAT-6 had increased granulomas and significantly elevated fibrosis (trichrome staining) compared with wild-type mice or PPARγ-KO mice that received only MWCNT. Immunostaining of lung tissues revealed elevated fibronectin and Siglec F expression on CD11c(+) infiltrating alveolar macrophages in the presence of MWCNT + ESAT-6 compared with MWCNT alone. Analyses of BAL fluid proteins indicated increased levels of transforming growth factor (TGF)-β and the TGF-β pathway mediator IL-13 in PPARγ-KO mice that received MWCNT + ESAT-6 compared with wild-type or PPARγ-KO mice that received MWCNT. Similarly, mRNA levels of matrix metalloproteinase 9, another requisite factor for TGF-β production, was elevated in PPARγ-KO mice by MWCNT + ESAT-6. Analysis of ESAT-6 in lung tissues by mass spectrometry revealed ESAT-6 retention in lung tissues of PPARγ-KO but not wild-type mice. These data indicate that PPARγ deficiency promotes pulmonary ESAT-6 retention, exacerbates macrophage responses to MWCNT + ESAT-6, and intensifies pulmonary fibrosis. The present findings suggest that the model may facilitate understanding of the effects of environmental factors on sarcoidosis-associated pulmonary fibrosis.

Few investigations have been conducted on the disposition and fate of silver nanoparticles (AgNP) in pregnancy. The distribution of a single dose of polyvinylpyrrolidone (PVP)-stabilized AgNP was investigated in pregnant rats. Two sizes of AgNP, 20 and 110 nm, and silver acetate (AgAc) were used to investigate the role of AgNP diameter and particle dissolution in tissue distribution, internal dose and persistence. Dams were administered AgNP or AgAc intravenously (i.v.) (1 mg kg-1 ) or by gavage (p.o.) (10 mg kg-1 ), or vehicle alone, on gestation day 18 and euthanized at 24 or 48 h post-exposure. The silver concentration in tissues was measured using inductively-coupled plasma mass spectrometry. The distribution of silver in dams was influenced by route of administration and AgNP size. The highest concentration of silver (μg Ag g-1 tissue) at 48 h was found in the spleen for i.v. administered AgNP, and in the lungs for AgAc. At 48 h after p.o. administration of AgNP, the highest concentration was measured in the cecum and large intestine, and for AgAc in the placenta. Silver was detected in placenta and fetuses for all groups. Markers of cardiovascular injury, oxidative stress marker, cytokines and chemokines were not significantly elevated in exposed dams compared to vehicle-dosed control. NMR metabolomics analysis of urine indicated that AgNP and AgAc exposure impact the carbohydrate, and amino acid metabolism. This study demonstrates that silver crosses the placenta and is transferred to the fetus regardless of the form of silver. Copyright © 2016 John Wiley & Sons, Ltd.

Introduction The biological importance of testosterone is generally accepted by the medical community; however, controversy focuses on its relevance to sexual function and the sexual response, and our understanding of the extent of its role in this area is evolving. Aim To provide scientific evidence examining the role of testosterone at the cellular and molecular levels as it pertains to normal erectile physiology and the development of erectile dysfunction and to assist in guiding successful therapeutic interventions for androgen-dependent sexual dysfunction. Methods In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current basic science literature examining the role of testosterone in sexual function and dysfunction. Results Testosterone plays an important role in sexual function through multiple processes: physiologic (stimulates activity of nitric oxide synthase), developmental (establishes and maintains the structural and functional integrity of the penis), neural (development, maintenance, function, and plasticity of the cavernous nerve and pelvic ganglia), therapeutically for dysfunctional regulation (beneficial effect on aging, diabetes, and prostatectomy), and phosphodiesterase type 5 inhibition (testosterone supplement to counteract phosphodiesterase type 5 inhibitor resistance). Conclusion Despite controversies concerning testosterone with regard to sexual function, basic science studies provide incontrovertible evidence for a significant role of testosterone in sexual function and suggest that properly administered testosterone therapy is potentially advantageous for treating male sexual dysfunction.

e19067 Background: Individuals with cancer experience fatigue, pain and decline in function. The Bellarmine Norton Assessment Tool (BNAT) was developed to give an objective measure identifying a person’s overall functional ability through a variety of physical assessments focused on mobility and strength. The purpose of this study was to develop a referral tool to provide health care professionals an easy determinant for physical therapy referrals. The referral tool was designed as a Red, Yellow, Green light for easy interpretation and use. Methods: The BNAT is composed of 1 self-reported physical activity question and 4 objective tests: 2-Minute Step Test (2MST); 30-Second Sit-to-Stand; Timed Arm Curl; and Timed Up and Go. A previously collected data set of BNAT scores was utilized to develop the referral tool with the poorest performance corresponding to Red and the best corresponding to Green. Three variations of normative scaling examined optimal distribution for classification. The first model averaged normative data of 70-74 age group of healthy individuals. No participants in our study achieved 50% of population norms. Therefore, the Green light represented 35% of the normative values, the Yellow light ranged from 15-34% and the Red light identified < 15%. A second model used frequency histograms for each BNAT elements. Groupings were made based on the natural break of the data to depict the Red, Yellow or Green light. A third approach combined normative and frequency distribution for each element as compared to the respective BNAT scores. The outcome assigned BNAT score of 1 and 2 to Red light, 3 to Yellow light and 4 and 5 to Green light. Except for the 2MST, these models were similar for the cutoff between color categories. The third model best fit the study population with respect to the individual’s total BNAT score. Results: Of the 161 subjects, 47 subjects (29%) fell into the Red category, 81 (50%) into Yellow and 33 (21%) into Green. Using this scaling system for the total BNAT score, 13 score combinations result in Red light, 6 scores for Yellow light and 6 scores for Green light. Regardless of the model, most individuals were assigned a Yellow light. Conclusions: The literature is void of functional cut off scores and normative distributive data for the oncology population. We defined a referral tool with scaling based off normative scores and functional assessments that reflect the distribution of oncology patients. The third model may be used as a simple referral tool among multiple health care professionals aiding in a referral for physical therapy.

Interleukin-6 (IL-6) is a pleiotropic cytokine historically associated with development and/or maintenance of cardiovascular disease (CVD). Despite this long-standing association, the discrete roles of IL-6 on the pathogenesis or severity of CVD and notably acute myocardial infarction (AMI) remain unclear. Dogma contends that IL-6 mediates effects largely through direct interaction with a membrane-bound IL-6 receptor (IL-6R) on leukocytes and hepatocytes. Intriguingly, identification of a soluble receptor for IL-6 (sIL-6R) has invigorated study into a regulatory role of IL-6 trans-signaling (IL-6trans) in CVD pathogenesis. IL-6trans provokes signal transduction in cells lacking the membrane-bound receptor, including notably cardiomyocytes, and ultimately activates STAT3 gene transcription and stabilizes mitochondrial function, thereby promoting cardioprotection. In this study we hypothesized that IL-6trans plays a crucial role in the injury response to AMI by increasing circulating IL-6 and sIL-6R and inducing cardioprotection via STAT3. To test this hypothesis, male BALB/c mice were subjected to acute ischemia (30 min) of the LAD coronary artery (to mimic AMI) with/without reperfusion and with/without inclusion of a pre- or post-ischemic conditioning event in order to determine regulatory involvement of IL-6trans in acute cardioprotection. Following surgery, ischemic regions of left ventricle (LV) and sera were analyzed for IL-6, sIL-6R, and activated (phosphorylated) STAT3. Results show that AMI led to increases in serum IL-6 (p=0.0053) and sIL-6R (p=0.0023) as well as in LV phosphorylated (Tyr705) STAT3 (p=0.0059) compared to naïve LV tissues. Preliminary data from ischemic pre- and post-conditioning regimens were independently associated with increases in IL-6trans demonstrated by trending increases in LV STAT3 phosphorylation (Ser727, Tyr705) compared to naïve controls. These inclusive data on IL-6trans suggest it serves a biologically significant intrinsic role in mediating acute protection following AMI. We anticipate that continued investigation will firmly establish IL-6trans as a unique protective pathway that could be targeted in order to improve morbidity and mortality of AMI patients.

Xenobiotic exposures affect the maternal and/or in utero environment resulting in impairments in fetal development. During the period of rapid fetal growth, developing cardiovascular systems are especially vulnerable to their environment. Furthermore, fetal exposures can evoke changes in epigenetic signatures that result in permanent modifications in gene expression. This symposium focused on the intersection between maternal and fetal exposure and the developing cardiovascular system. The impact of maternal exposures on prenatal development is of major concern for regulatory agencies given the unique vulnerability of the embryo/fetus to environmental factors, the importance of vascular biology to maternal-fetal interactions, and the adverse consequences of vascular disruption to children’s health. Speakers provided data from diverse exposures: nanomaterials, particulate matter or air pollution (PM(2.5)), nicotine, and environmental chemicals. The current findings related to susceptible gestational windows for cardiovascular development and epigenetic, transcriptomic, toxicokinetic, and toxicodynamic changes in vascular physiology and cardiac function. In response to these concerns, new concepts in predictive modeling and risk assessment associated with in utero exposures were presented as future avenues of research within developmental toxicology. Finally, current applications using an Adverse Outcome Pathway framework for developmental toxicity were presented to integrate data from in vitro profiling of chemical libraries (e.g. ToxCast™) with computational models for in silico toxicology. In summary, this symposium addressed the significant threats to cardiovascular health that are associated with fetal/perinatal exposures, and offered new insights into the predictive, mechanistic, and risk assessment strategies in developmental toxicology.

Pulmonary instillation of multiwalled carbon nanotubes (MWCNT) has the potential to promote cardiovascular derangements, but the mechanisms responsible are currently unclear. We hypothesized that exposure to MWCNT would result in increased epithelial barrier permeability by 24 h postexposure and initiate a signaling process involving IL-6/gp130 transsignaling in peripheral vascular tissue. To test this hypothesis we assessed the impact of 1 and 10 μg/cm2MWCNT on transepithelial electrical resistance (TEER) and expression of barrier proteins and cell activation in vitro using normal human bronchial epithelial primary cells. Parallel studies using male Sprague-Dawley rats instilled with 100 μg MWCNT measured bronchoalveolar lavage (BAL) differential cell counts, BAL fluid total protein, and lung water-to-tissue weight ratios 24 h postexposure and quantified serum concentrations of IL-6, soluble IL-6r, and soluble gp130. Aortic sections were examined immunohistochemically for gp130 expression, and gp130 mRNA/protein expression was evaluated in rat lung, heart, and aortic tissue homogenates. Our in vitro findings indicate that 10 μg/cm2MWCNT decreased the development of TEER and zonula occludens-1 expression relative to the vehicle. In rats MWCNT instillation increased BAL protein, lung water, and induced pulmonary eosinophilia. Serum concentrations of soluble gp130 decreased, aortic endothelial expression of gp130 increased, and expression of gp130 in the lung was downregulated in the MWCNT-exposed group. We propose that pulmonary exposure to MWCNT can manifest as a reduced epithelial barrier and activator of vascular gp130-associated transsignaling that may promote susceptibility to cardiovascular derangements.

Introduction Although clinical evidence supports an association between cardiovascular/metabolic diseases (CVMD) and erectile dysfunction (ED), scientific evidence for this link is incompletely elucidated. Aim This study aims to provide scientific evidence for the link between CVMD and ED. Methods In this White Paper, the Basic Science Committee of the Sexual Medicine Society of North America assessed the current literature on basic scientific support for a mechanistic link between ED and CVMD, and deficiencies in this regard with a critical assessment of current preclinical models of disease. Results A link exists between ED and CVMD on several grounds: the endothelium (endothelium-derived nitric oxide and oxidative stress imbalance); smooth muscle (SM) (SM abundance and altered molecular regulation of SM contractility); autonomic innervation (autonomic neuropathy and decreased neuronal-derived nitric oxide); hormones (impaired testosterone release and actions); and metabolics (hyperlipidemia, advanced glycation end product formation). Conclusion Basic science evidence supports the link between ED and CVMD. The Committee also highlighted gaps in knowledge and provided recommendations for guiding further scientific study defining this risk relationship. This endeavor serves to develop novel strategic directions for therapeutic interventions.

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that induces developmental cardiotoxicity. It is detectable in late stage chicken embryos and hatchling chickens. To investigate mechanism(s) of cardiotoxicity, primary cultures of cardiomyocytes were prepared from 10-day-old chicken embryos that were (A) pre-exposed to vehicle or 2 mg of PFOA/kg of egg weight in ovo or (B) incubated with PFOA in vitro at concentrations ranging from 0 to 100 µg/mL in medium for 1 or 36 h. When viability was assessed, survival of cardiomyocytes prepared from pre-exposed embryos did not differ from vehicle controls, even under conditions of serum starvation designed to challenge the cells. However, 1 h of exposure to 100 µg/mL of PFOA in vitro and 36 h of exposure to 75 and 100 µg/mL PFOA in vitro decreased viability. When contractility was evaluated, cardiomyocytes cultured from pre-exposed embryos exhibited decreases in time to maximum departure velocity and cell length at peak contraction, whereas cardiomyocytes exposed in vitro exhibited a reduction in the 50% relaxation time at a concentration of 1 µg/mL relative to vehicle controls. Morphological assessment revealed decreased cardiomyocytes axial length following in ovo PFOA exposure and 24 h in vitro PFOA 50 µg/mL exposure. Reactive oxygen species (ROS) generation, which was evaluated only in cardiomyocytes exposed to PFOA in vitro, was significantly elevated following incubation with 50 µg/mL of PFOA for 1 h. These data indicate that while in vitro exposure to relatively high concentrations of PFOA can induce cytotoxicity and ROS, developmental cardiotoxicity observed in ovo is not likely mediated via PFOA-induced overt cytotoxicity, but likely by altering early cardiac morphologic and function processes. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1580-1590, 2016.

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([14C(U)]C60). Rats were administered [14C(U)]C60 (~0.2 mg [14C(U)]C60 kg–1 body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [14C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [14C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [14C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [14C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk. Copyright © 2015 John Wiley & Sons, Ltd.

A comprehensive distribution study was conducted in female rats and mice exposed to a suspension of uniformly carbon-14-labeled C60 ([14C(U)]C60). Rodents were administered [14C(U)]C60 (~0.9 mg kg−1 body weight) or 5% polyvinylpyrrolidone-saline vehicle alone via a single tail vein injection. Tissues were collected at 1 h and 1, 7, 14 and 30 days after administration. A separate group of rodents received five daily injections of suspensions of either [14C(U)]C60 or vehicle with tissue collection 14 days post exposure. Radioactivity was detected in over 20 tissues at all time points. The highest concentration of radioactivity in rodents at each time point was in liver, lungs and spleen. Elimination of [14C(U)]C60 was < 2% in urine and feces at any 24 h time points. [14C(U)]C60 and [14C(U)]C60-retinol were detected in liver of rats and together accounted for ~99% and ~56% of the total recovered at 1 and 30 days postexposure, respectively. The blood radioactivity at 1 h after [14C(U)]C60 exposure was fourfold higher in rats than in mice; blood radioactivity was still in circulation at 30 days post [14C(U)]C60 exposure in both species (

The potential uses of engineered C₆₀ fullerene (C₆₀) have expanded in recent decades to include industrial and biomedical applications. Based on clinical findings associated with particulate matter exposure and our data with multi-walled carbon nanotubes, we hypothesized that ischemia/reperfusion (I/R) injury and pharmacological responses in isolated coronary arteries would depend upon the route of exposure and gender in rats instilled with C₆₀. Male and female Sprague Dawley rats were used to test this hypothesis by surgical induction of cardiac I/R injury in situ 24 h after intratracheal (IT) or intravenous (IV) instillation of 28 μg of C₆₀ formulated in polyvinylpyrrolidone (PVP) or PVP vehicle. Serum was collected for quantification of various cytokines. Coronary artery segments were isolated for assessment of vasoactive pharmacology via wire myography. Both IV and IT exposure to C₆₀ resulted in expansion of myocardial infarction in male and female rats following I/R injury. Serum-collected post-I/R showed elevated concentrations of interleukin-6 and monocyte chemotactic protein-1 in male rats exposed to IV C₆₀. Coronary arteries isolated from male rats exposed to IT C₆₀ demonstrated augmented vasocontraction in response to endothelin-1 that was attenuated with Indomethacin. IV C₆₀ exposure resulted in impaired acetylcholine relaxation in male rats and IT C₆₀ exposure resulted in depressed vasorelaxation in response to sodium nitroprusside in female rats. Based on these data, we conclude that IT and IV exposure to C₆₀ results in unique cardiovascular consequences that may favor heightened coronary resistance and myocardial susceptibility to I/R injury.

The aim of this study was to investigate aerobic exercise training as a means to prevent erectile dysfunction (ED) and coronary artery disease (CAD) development associated with inactivity and diet-induced obesity. Male Sprague-Dawley rats were fed a Western diet (WD) or a control diet (CD) for 12 wk. Subgroups within each diet remained sedentary (Sed) or participated in aerobic interval treadmill running throughout the dietary intervention. Erectile function was evaluated under anesthesia by measuring the mean arterial pressure and intracavernosal pressure in response to electrical field stimulation of the cavernosal nerve, in the absence or presence of either apocynin, an NADPH oxidase inhibitor, or sepiapterin, a tetrahydrobiopterin precursor. Coronary artery endothelial function (CAEF) was evaluated ex vivo with cumulative doses of ACh applied to preconstricted segments of the left anterior descending coronary artery. CAEF was assessed in the absence or presence of apocynin or sepiapterin. Erectile function ( P < 0.0001) and CAEF ( P < 0.001) were attenuated in WD-Sed. Exercise preserved erectile function ( P < 0.0001) and CAEF ( P < 0.05) within the WD. Erectile function ( P < 0.01) and CAEF ( P < 0.05) were augmented by apocynin only in WD-Sed, while sepiapterin ( P < 0.05) only augmented erectile function in WD-Sed. These data demonstrate that a chronic WD induces impairment in erectile function and CAEF that are commonly partially reversible by apocynin, whereas sepiapterin treatment exerted differential functional effects between the two vascular beds. Furthermore, exercise training may be a practical means of preventing diet-induced ED and CAD development.

Increased fatty acid availability and oxidative stress are physiological consequences of exercise (Ex) and a high-fat, high-sugar (HFHS) diet. Despite these similarities, the global effects of Ex are beneficial, whereas HFHS diets are largely deleterious to the cardiovascular system. The reasons for this disparity are multifactorial and incompletely understood. We hypothesized that differences in redox adaptations following HFHS diet in comparison to exercise may underlie this disparity, particularly in mitochondria. Our objective in this study was to determine mechanisms by which heart and skeletal muscle (red gastrocnemius, RG) mitochondria experience differential redox adaptations to 12 weeks of HFHS diet and/or exercise training (Ex) in rats. Surprisingly, both HFHS feeding and Ex led to contrasting effects in heart and RG, in that mitochondrial H2O2 decreased in heart but increased in RG following both HFHS diet and Ex, in comparison to sedentary animals fed a control diet. These differences were determined to be due largely to increased antioxidant/anti-inflammatory enzymes in the heart following the HFHS diet, which did not occur in RG. Specifically, upregulation of mitochondrial thioredoxin reductase-2 occurred with both HFHS and Ex in the heart, but only with Ex in RG, and systematic evaluation of this enzyme revealed that it is critical for suppressing mitochondrial H2O2 during fatty acid oxidation. These findings are novel and important in that they illustrate the unique ability of the heart to adapt to oxidative stress imposed by HFHS diet, in part through upregulation of thioredoxin reductase-2. Furthermore, upregulation of thioredoxin reductase-2 plays a critical role in preserving the mitochondrial redox status in the heart and skeletal muscle with exercise.

Introduction. It is suggested that erectile dysfunction (ED) may be an early risk factor for cardiovascular disease. Aim. The goal of this study was to determine whether development of ED precedes the onset of coronary artery endothelial dysfunction in response to a Western diet (WD), thereby establishing whether the WD differentially impacts the endothelium in a time-dependent manner. Additionally, a goal was to determine if diet-induced ED is reversible with intracavernosal sepiapterin treatment. Methods. Male Sprague-Dawley rats were fed a WD for 4, 8, or 12 weeks, or a control diet for 8 weeks. Erectile function was evaluated by measuring the mean arterial pressure (MAP) and intracavernosal pressure (ICP) in response to electrical field stimulation of the cavernosal nerve near the major pelvic ganglion, in the absence and presence of sepiapterin. Coronary artery endothelial function was evaluated ex vivo with cumulative doses of acetylcholine (ACh) applied to segments of the left anterior descending coronary artery preconstricted with serotonin. Main Outcome Measures. Erectile function was assessed as the ICP response to electrical field stimulation (EFS), normalized to MAP. Coronary artery endothelial function was assessed as the effective concentration producing 50% of a maximal response (EC50 ) of the ACh response. Results. The ICP/MAP response to EFS was significantly attenuated following both 8 and 12 weeks of the WD compared with the control diet (P 0.05). Sepiapterin treatment augmented the ICP/MAP response in all WD groups (P 0.05). The coronary artery EC50 of the ACh response was not different from control following 4 or 8 weeks but was significantly elevated following 12 weeks of the WD (P 0.01). Conclusions. These data suggest that erectile function is reduced prior to coronary artery endothelial function in response to the WD. Improvement of erectile function with sepiapterin in WD rats indicates that nitric oxide synthase uncoupling is a key mechanism in diet-induced ED. La Favor JD, Anderson EJ, Hickner RC, and Wingard CJ. Erectile dysfunction precedes coronary artery endothelial dysfunction in rats fed a high-fat, high-sucrose, Western pattern diet. J Sex Med 2013;10:694-703.

We established a murine model of multiwall carbon nanotube (MWCNT)-induced chronic granulomatous disease, which resembles human sarcoidosis pathology. At 60 days after oropharyngeal MWCNT instillation, bronchoalveolar lavage (BAL) cells from wild-type mice exhibit an M1 phenotype with elevated proinflammatory cytokines and reduced peroxisome proliferator-activated receptor γ (PPARγ)-characteristics also present in human sarcoidosis. Based upon MWCNT-associated PPARγ deficiency, we hypothesized that the PPARγ target gene, ATP-binding cassette (ABC) G1, a lipid transporter with antiinflammatory properties, might also be repressed. Results after MWCNT instillation indicated significantly repressed ABCG1, but, surprisingly, lipid transporter ABCA1 was also repressed, suggesting a possible second pathway. Exploration of potential regulators revealed that microRNA (miR)-33, a lipid transporter regulator, was strikingly elevated (13.9 fold) in BAL cells from MWCNT-instilled mice but not sham control mice. Elevated miR-33 was also detected in murine granulomatous lung tissue. In vitro studies confirmed that lentivirus-miR-33 overexpression repressed both ABCA1 and ABCG1 (but not PPARγ) in cultured murine alveolar macrophages. BAL cells of patients with sarcoidosis also displayed elevated miR-33 together with reduced ABCA1 and ABCG1 messenger RNA and protein compared with healthy control subjects. Moreover, miR-33 was elevated within sarcoidosis granulomatous tissue. The findings suggest that alveolar macrophage miR-33 is up-regulated by proinflammatory cytokines and may perpetuate chronic inflammatory granulomatous disease by repressing antiinflammatory functions of ABCA1 and ABCG1 lipid transporters. The results also suggest two possible pathways for transporter dysregulation in granulomatous disease-one associated with intrinsic PPARγ status and the other with miR-33 up-regulation triggered by environmental challenges, such as MWCNT.

Recent studies suggest additive effects of environmental pollutants and microbial antigens on respiratory disease. We established a granuloma model in which instilled multiwall carbon nanotubes (MWCNT) elicit granulomatous pathology. We hypothesized that mycobacterial antigen ESAT-6, a T cell activator associated with tuberculosis and sarcoidosis, might alter pathology. Wild-type C57Bl/6 mice received MWCNT with or without ESAT-6 peptide. Controls received vehicle (surfactant-PBS) or ESAT-6 alone. Mice were evaluated 60 days later for granulomas, fibrosis, and bronchoalveolar lavage (BAL) cell expression of inflammatory mediators (CCL2, MMP-12, and Osteopontin). Results indicated increased granulomas, fibrosis, and inflammatory mediators in mice receiving the combination of MWCNT+ESAT-6 compared to MWCNT or vehicle alone. ESAT-6 alone showed no significant effect on these pathological endpoints. However, CD3 (+) lymphocyte infiltration of lung tissue increased with MWCNT+ESAT-6 versus MWCNT alone. Findings suggest that concurrent exposure to microbial antigen and MWCNT exacerbates chronic pulmonary disease.

Background: Silver nanoparticles (AgNP) have garnered much interest due to their antimicrobial properties, becoming one of the most utilized nano-scale materials. However, any potential evocable cardiovascular injury associated with exposure has not been reported to date. We have previously demonstrated expansion of myocardial infarction after intratracheal (IT) instillation of carbon-based nanomaterials. We hypothesized pulmonary exposure to Ag core AgNP induces a measureable increase in circulating cytokines, expansion of cardiac ischemia-reperfusion (I/R) injury and is associated with depressed coronary constrictor and relaxation responses. Secondarily, we addressed the potential contribution of silver ion release on AgNP toxicity. Methods: Male Sprague-Dawley rats were exposed to 200 μl of 1 mg/ml of 20 nm citrate-capped Ag core AgNP, 0.01, 0.1, 1 mg/ml Silver Acetate (AgAc), or a citrate vehicle by intratracheal (IT) instillation. One and 7 days following IT instillation the lungs were evaluated for inflammation and the presence of silver; serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and coronary artery reactivity were assessed. Results: AgNP instillation resulted in modest pulmonary inflammation with detection of silver in lung tissue and alveolar macrophages, elevation of serum cytokines: G-CSF, MIP-1α, IL-1β, IL-2, IL-6, IL-13, IL-10, IL-18, IL-17α, TNFα, and RANTES, expansion of I/R injury and depression of the coronary vessel reactivity at 1 day post IT compared to vehicle treated rats. Silver within lung tissue was persistent at 7 days post IT instillation and was associated with an elevation in cytokines: IL-2, IL-13, and TNFα and expansion of I/R injury. AgAc resulted in a concentration dependent infarct expansion and depressed vascular reactivity without marked pulmonary inflammation or serum cytokine response. Conclusions: Based on these data, IT instillation of AgNP increases circulating levels of several key cytokines, which may contribute to persistent expansion of I/R injury possibly through an impaired vascular responsiveness.

The growing use of multi-walled carbon nanotubes (MWCNTs) across industry has increased human exposures. We tested the hypothesis that pulmonary instillation of MWCNTs would exacerbate cardiac ischaemia/reperfusion (I/R) injury. One day following intratracheal instillation of 1, 10 or 100 μg MWCNT in Sprague-Dawley rats, we used a Langendorff isolated heart model to examine cardiac I/R injury. In the 100 μg MWCNT group we report increased premature ventricular contractions at baseline and increased myocardial infarction. This was associated with increased endothelin-1 (ET-1) release and depression of coronary flow during early reperfusion. We also tested if isolated coronary vascular responses were affected by MWCNT instillation and found trends for enhanced coronary tone, which were dependent on ET-1, cyclooxygenase, thromboxane and Rho-kinase. We concluded that instillation of MWCNTs promoted cardiac injury and depressed coronary flow by invoking vasoconstrictive mechanisms involving ET-1, cyclooxygenase, thromboxane and Rho-kinase.

Nanoparticles (NP) are highly applicable in a variety of technological and biomedical fields because of their unique physicochemical properties. The increased development and utilization of NP has amplified human exposure and raised concerns regarding their potential to generate toxicity. The biological impacts of NP exposures have been shown to be dependent on aerodynamic size, chemical composition, and the route of exposure (oral, dermal, intravenous, and inhalation), while recent research has demonstrated the cardiovascular (CV) system as an important site of toxicity. Proposed mechanisms responsible for these effects include inflammation, oxidative stress, autonomic dysregulation, and direct interactions of NP with CV cells. Specifically, NP have been shown to impact vascular endothelial cell (EC) integrity, which may disrupt the dynamic endothelial regulation of vascular tone, possibly altering systemic vascular resistance and impairing the appropriate distribution of blood flow throughout the circulation. Cardiac consequences of NP-induced toxicity include disruption of heart rate and electrical activity via catecholamine release, increased susceptibility to ischemia/reperfusion injury, and modified baroreceptor control of cardiac function. These and other CV outcomes likely contribute to adverse health effects promoting myocardial infarction, hypertension, cardiac arrhythmias, and thrombosis. This review will assess the current knowledge regarding the principle sites of CV toxicity following NP exposure. Furthermore, we will propose mechanisms contributing to altered CV function and hypothesize possible outcomes resulting in decrements in human health. WIREs Nanomed Nanobiotechnol 2012, 4:691–702. doi: 10.1002/wnan.1194 For further resources related to this article, please visit the WIREs website.

C oncern about the use of nanomaterials has increased signifi cantly in recent years due to potentially hazardous impacts on human health. Mast cells are critical for innate and adaptive immune responses, often modulating allergic and pathogenic conditions. Mast cells are well known to act in response to danger signals through a variety of receptors and pathways including IL-33 and the IL-1-like receptor ST2. Here, the involvement of mast cells and the IL-33/ST2 axis in pulmonary and cardiovascular responses to multi-walled carbon nanotube (MWCNT) exposure are examined. Toxicological effects of MWCNTs are observed only in mice with a suffi cient population of mast cells and are not observed when mast cells are absent or incapable of responding to IL-33. Our fi ndings establish for the fi rst time that mast cells and the IL-33/ST2 axis orchestrates adverse pulmonary and cardiovascular responses to an engineered nanomaterial, giving insight into a previously unknown mechanism of toxicity. This novel mechanism of toxicity could be used for assessing the safety of engineered nanomaterials and provides a realistic therapeutic target for potential nanoparticle induced toxicities.

Lung granulomas are associated with numerous conditions, including inflammatory disorders, exposure to environmental pollutants, and infection. Osteopontin is a chemotactic cytokine produced by macrophages, and is implicated in extracellular matrix remodeling. Furthermore, osteopontin is up-regulated in granulomatous disease, and osteopontin null mice exhibit reduced granuloma formation. Animal models currently used to investigate chronic lung granulomatous inflammation bear a pathological resemblance, but lack the chronic nature of human granulomatous disease. Carbon nanoparticles are generated as byproducts of combustion. Interestingly, experimental exposures to carbon nanoparticles induce pulmonary granuloma-like lesions. However, the recruited cellular populations and extracellular matrix gene expression profiles within these lesions have not been explored. Because of the rapid resolution of granulomas in current animal models, the mechanisms responsible for persistence have been elusive. To overcome the limitations of previous models, we investigated whether a model using multiwall carbon nanoparticles would resemble chronic human lung granulomatous inflammation. We hypothesized that pulmonary exposure to multiwall carbon nanoparticles would induce granulomas, elicit a macrophage and T-cell response, and mimic other granulomatous disorders with an up-regulation of osteopontin. This model demonstrates: (1) granulomatous inflammation, with macrophage and T-cell infiltration; (2) resemblance to the chronicity of human granulomas, with persistence up to 90 days; and (3) a marked elevation of osteopontin, metalloproteinases, and cell adhesion molecules in granulomatous foci isolated by laser-capture microdissection and in alveolar macrophages from bronchoalveolar lavage. The establishment of such a model provides an important platform for mechanistic studies on the persistence of granuloma.

Cerium oxide (CeO₂) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO₂ exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-Kit(W-sh) mast cell deficient mice following CeO₂ nanoparticle instillation. C57BL/6 mice instilled with CeO₂ exhibited mild pulmonary inflammation. However, B6.Cg-Kit(W-sh) mice did not display a similar degree of inflammation following CeO₂ instillation. Moreover, C57BL/6 mice instilled with CeO₂ exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-Kit(W-sh) mice. In vitro CeO₂ exposure resulted in increased production of PGD₂, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO₂ nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

135 Background: Individuals with cancer may experience loss of function, disability and fatigue. There is interest in team-based functional and physical assessment as part of a comprehensive oncology care plan. The Bellarmine Norton Assessment Tool (BNAT) was developed to provide an objective physical assessment tool for interprofessional use. The purpose of this study was to evaluate the BNAT and correlate its outcomes with ECOG scores across groups diagnosed with pancreatic (P), breast (B) and lung (L) cancers. Methods: The BNAT was developed by the Bellarmine University Physical Therapy Program utilizing validated physical assessment tests. It consists of 1 subjective question about physical activity frequency followed by 4 objective tests: 2-Minute Step Test (2MST), 30 Sec Sit-to-Stand, Timed Arm Curl (TAC) and Timed Up and Go. The ECOG was administered by attending oncologists during the same visit as BNAT testing. 18 participants were identified 6 in each of the diagnosis groups from a recruitment cohort of 57. The avg. age for P was 68.8±9 yrs 4♀,2♂; B 71.5±15.8 yrs 6♀; L 65.0±12.4 yrs 3♀,3♂. Results: The combined mean ± SD ECOG score was 1.2±1.0 (range 0-3) while the combined BNAT score was 15.4±6.5 (range 7-29). Mean BNAT score by diagnosis: P 16.6±7.4; B 13.0±5.5; & L 18.3±5.7. Spearman correlation analysis of BNAT and ECOG scores revealed a significant (p = 0.0001) negative correlation of agreement (r = -0.79). Both B and L cancers had significant (p < 0.05) correlation of agreement (B r = -0.71 & L r = -0.88). TAC correlation to ECOG was strongest in L group (r = -0.86), but 2MST was strongest for P group (r = -0.62). There was a reciprocal relationship of slopes between TAC and 2MST based on diagnosis. L steepest slope in TAC (-6.2 with deviation from 0, p = 0.014) while P had steepest slope with 2MST (-13.1). Slope for B group consistently fell between the 2 other diagnoses. Conclusions: There was a strong linear relationship of BNAT, TAC & 2MST to ECOG scores across the 3 cohorts of diagnoses. The BNAT subcomponents have varying degrees of contribution to the total that can be attributed to diagnosis-related functional deficits, thus giving providers information to determine appropriate care.

Introduction The combination of independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are collectively manifested in a condition known as metabolic syndrome X (MSX). However, the regulatory mechanisms responsible for the erectile dysfunction (ED) are not fully understood. Clinical studies suggest that a pleiotropic effect of statin's ability to enhance vascular relaxation might be through an impact on nitric oxide signaling or through a regulation of RhoA activation. Aim We hypothesized that regulatory aspects of short‐term statin therapy involve the alteration of the RhoA/Rho‐kinase signaling cascade and will reverse the ED seen in a rat model of MSX. Main Outcome Measures The magnitude and sensitivity of the voltage‐dependent maintenance of intracavernosal blood pressure and mean arterial blood pressure. These responses were correlated with tissue protein and mRNA expression levels of RhoA and Rho kinases. Methods Erectile function was evaluated by assessing voltage‐dependent stimulation of the cavernosal nerve in 16–20 weeks old lean and obese‐diabetic Zucker rats treated with 5 mg/kg/day of rosuvastatin intraperitoneally for 3 days. Cavernosal tissue RhoA and Rho‐kinases expression levels were evaluated by real‐time reverse transcriptase‐polymerase chain reaction, Western blot. Results The voltage‐dependent erectile responses were suppressed by >30% in the obese‐diabetic Zucker rat. The 3‐day treatment with rosuvastatin partially restored the erectile response. The Rho‐kinase inhibitor, H‐1152, dose dependently increased the erectile responses and shifted the voltage sensitivity with statin treatment. Analysis of protein expression levels suggested elevation of RhoA and Rho kinases in obese‐diabetics and statin treatment lowering Rho‐kinase II. The RhoA and Rho‐kinase II mRNA levels were significantly reduced in the rosuvastatin‐treated obese‐diabetic animals. Conclusions. These results support a hypothesis that short‐term statin therapy may lower RhoA/Rho‐kinase expression levels and improve cavernosal blood pressure response to Rho‐kinase inhibition and voltage‐stimulation, and reversing an augmented vasoconstricted state associated with diabetes and/or hypertension in MSX. Wingard CJ, Moukdar F, Prasad RY, Cathey BL, and Wilkinson L. Reversal of voltage‐dependent erectile responses in the Zucker obese‐diabetic rat by rosuvastatin‐altered RhoA/Rho‐kinase signaling.

The vascular activity of scutellarin (SCU), a flavonoid isolated from a Chinese traditional medicinal plant, was investigated in isolated thoracic aortic rings of mice. SCU-induced dose-dependent relaxation of phenylephrine (1 microM) stimulated contractions. This relaxation was reduced by endothelium removal, significantly reduced by both the nitric oxide synthase inhibitor (Nomega-nitro-L-arginine methylester, 300 microM) and slightly limited by the soluble guanylyl cyclase inhibitor (1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one, 100 microM). The catalase inhibitor (3-amino-1,2,4-triazole, 50 mM) augmented the constriction and blocked the lowest SCU concentration relaxation, whereas catalase addition was without effect. Preincubation with 300 and 1000 microM SCU significantly suppressed the contractile dose-response to phenylephrine, causing both a significant rise in half maximal effective concentration and a decrease in the maximal developed force. Western blot analysis showed that SCU inhibition of contraction was independent of reductions in myosin light chain phosphorylation. These results suggested that SCU relaxation was predominantly endothelium dependent and likely involved the catalase-sensitive nitric oxide synthase signaling pathway, without loss of myosin phosphorylation. The potential clinical use of SCU may prove to be effective in increasing vasoreactivity, independently of smooth muscle contractile activity that is mediated by the 20-kDa myosin light chain phosphorylation.

Background The uses of engineered nanomaterials have expanded in biomedical technology and consumer manufacturing. Furthermore, pulmonary exposure to various engineered nanomaterials has, likewise, demonstrated the ability to exacerbate cardiac ischemia reperfusion (I/R) injury. However, the influence of particle size or capping agent remains unclear. In an effort to address these influences we explored response to 2 different size gold core nanosilver particles (AgNP) with two different capping agents at 2 different time points. We hypothesized that a pulmonary exposure to AgNP induces cardiovascular toxicity influenced by inflammation and vascular dysfunction resulting in expansion of cardiac I/R Injury that is sensitive to particle size and the capping agent. Methods Male Sprague–Dawley rats were exposed to 200 μg of 20 or 110 nm polyvinylprryolidone (PVP) or citrate capped AgNP. One and 7 days following intratracheal instillation serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and isolated coronary artery and aorta segment were assessed for constrictor responses and endothelial dependent relaxation and endothelial independent nitric oxide dependent relaxation. Results AgNP instillation resulted in modest increase in selected serum cytokines with elevations in IL-2, IL-18, and IL-6. Instillation resulted in a derangement of vascular responses to constrictors serotonin or phenylephrine, as well as endothelial dependent relaxations with acetylcholine or endothelial independent relaxations by sodium nitroprusside in a capping and size dependent manner. Exposure to both 20 and 110 nm AgNP resulted in exacerbation cardiac I/R injury 1 day following IT instillation independent of capping agent with 20 nm AgNP inducing marginally greater injury. Seven days following IT instillation the expansion of I/R injury persisted but the greatest injury was associated with exposure to 110 nm PVP capped AgNP resulted in nearly a two-fold larger infarct size compared to naïve. Conclusions Exposure to AgNP may result in vascular dysfunction, a potentially maladaptive sensitization of the immune system to respond to a secondary insult (e.g., cardiac I/R) which may drive expansion of I/R injury at 1 and 7 days following IT instillation where the extent of injury could be correlated with capping agents and AgNP size. Electronic supplementary material The online version of this article (doi:10.1186/s12989-016-0159-z) contains supplementary material, which is available to authorized users.

Epidemiological studies have linked ambient particulate matter (PM) levels to an increased incidence of adverse cardiovascular events. Yet little is definitively known about the mechanisms accounting for the cardiovascular events associated with PM exposure. The goal of this study was to determine the effects of ultrafine (

Myostatin (GDF8) and dystrophin are critical molecules for muscle organisation. Myostatin is involved in regulating muscle growth and development, whereas dystrophin is part of the dystrophin-glycoprotein complex (DGC), which anchors the cytoskeleton to the sarcolemma. We examined temporalis muscle morphology and function in myostatin deficient and dystrophin deficient (Mdx) mice in order to determine how myostatin and dystrophin affect bite force and muscle fibre composition. Bite forces from 4-month-old myostatin-/-, dystrophin deficient (Mdx) and normal control mice were measured by load cell and field stimulation of the temporalis muscle. Tissue sections were stained with haemotoxylin and eosin (H&E) and the periodic acid-Schiff reaction (PAS) to assess morphology and fibre type differences. A positive relationship between bite force and muscle mass for both genetic models was observed. Both Mstn-/- and Mdx mice showed significant elevation in bite force and muscle mass. Histological examination revealed greater muscle fibre cross-sectional area variability in Mdx mice (ANOVA, F=5.6, P

Constriction of the penile vasculature prevents erection and is largely mediated by physiological agonists. We hypothesized that protein kinase C (PKC) may act as a regulator of penile vascular tone. Studies were designed to identify PKC isoforms present and to investigate their roles in phenylephrine-induced muscle contraction in the isolated rat corpora cavernosa. We demonstrated the presence of PKCalpha, beta, gamma, epsilon, delta, eta, and iota in rat corpora cavernosa and a subcellular distribution, which favored a membrane association for PKCalpha, beta, delta, and iota. Phenylephrine (3 microM) generated an active stress of 9.6 +/- 1.5 mN/mm2 and was associated with a significant increase of PKCalpha and PKCiota immunoreactivity in the particulate fraction. The amount of PKCalpha and PKCiota in the particulate fraction rose by 36 +/- 4.4 and 51 +/- 2.2% with phenylephrine stimulation. Furthermore, the phenylephrine concentration-response curve was potentiated in the presence of phorbol 12-myristate13-acetate (PMA) (0.1 microM), a PKC activator (EC50: phenylephrine 1.0 +/- 0.8 microM vs phenylephrine + PMA 0.3 +/- 0.1 microM) and inhibited in the presence of chelerythrine chloride (30 microM), a PKC inhibitor (EC50: phenylephrine 1.0 +/- 0.8 microM vs phenylephrine + chelerythrine chloride 5.7 +/- 2.4 microM). Based on these results, we suggest a potential role for PKCalpha and PKCiota in phenylephrine-induced smooth muscle tone of the rat cavernosum.

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 ± 0.040; castrate, 0.492 ± 0.022; testosterone, 0.681 ± 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 ± 0.091; castrate, 0.782 ± 0.081; testosterone treated, 0.894 ± 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([(14) C(U)]C60 ). Rats were administered [(14) C(U)]C60 (~0.2 mg [(14) C(U)]C60 kg(-1) body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [(14) C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [(14) C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [(14) C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [(14) C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk.

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that induces developmental cardiotoxicity. It is detectable in late stage chicken embryos and hatchling chickens. To investigate mechanism(s) of cardiotoxicity, primary cultures of cardiomyocytes were prepared from 10-day-old chicken embryos that were (A) pre-exposed to vehicle or 2 mg of PFOA/kg of egg weight in ovo or (B) incubated with PFOA in vitro at concentrations ranging from 0 to 100 µg/mL in medium for 1 or 36 h. When viability was assessed, survival of cardiomyocytes prepared from pre-exposed embryos did not differ from vehicle controls, even under conditions of serum starvation designed to challenge the cells. However, 1 h of exposure to 100 µg/mL of PFOA in vitro and 36 h of exposure to 75 and 100 µg/mL PFOA in vitro decreased viability. When contractility was evaluated, cardiomyocytes cultured from pre-exposed embryos exhibited decreases in time to maximum departure velocity and cell length at peak contraction, whereas cardiomyocytes exposed in vitro exhibited a reduction in the 50% relaxation time at a concentration of 1 µg/mL relative to vehicle controls. Morphological assessment revealed decreased cardiomyocytes axial length following in ovo PFOA exposure and 24 h in vitro PFOA 50 µg/mL exposure. Reactive oxygen species (ROS) generation, which was evaluated only in cardiomyocytes exposed to PFOA in vitro, was significantly elevated following incubation with 50 µg/mL of PFOA for 1 h. These data indicate that while in vitro exposure to relatively high concentrations of PFOA can induce cytotoxicity and ROS, developmental cardiotoxicity observed in ovo is not likely mediated via PFOA-induced overt cytotoxicity, but likely by altering early cardiac morphologic and function processes. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1580-1590, 2016.

Ablation of the premigratory cardiac neural crest (CNC) from the chick embryo results in a malformed outflow tract vasculature termed persistent truncus arteriosus (PTA). In addition, loss of the CNC disrupts myocardial excitation–contraction (EC) coupling, decreases intracellular Ca2+ transients, and depresses force generation. We examined if similar defects occurred in the neural crest-derived smooth muscle of the aortic arch in a test of the hypothesis that loss of elements from the CNC disrupts EC coupling and force production in the smooth muscle of the tunica media of the aortic arch. Aortic arch segments from chicks (embryonic day 15) displaying PTA generated ~43% of stress generated by the aortic arch from sham-operated control embryos during potassium depolarization. The depressed force response was associated with a twofold lower Fura-2 transient. In contrast, force and steady-state Fura-2 signals during endothelin-1 stimulation were unchanged. The differences seen in stress generation with potassium depolarization between sham and PTA displaying embryos were not seen in the descending aorta, a tissue not derived from the neural crest. Protein content and immunostaining revealed no differences in the content of actin, myosin, or dihydropyridine receptor from sham or PTA aortic arch. Our results suggest that the CNC is required for normal aortic arch smooth muscle function and support the hypothesis that the loss of CNC impacts the force generating ability, in part by disruption of the EC-coupling processes and altering Ca2+-handling.

In sarcoidosis, a chronic granulomatous disease of unknown etiology, alveolar macrophages are deficient in the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Pulmonary fibrosis may accompany sarcoidosis and increase morbidity and mortality. We developed a murine model of granulomatous disease elicited by instillation of multiwall carbon nanotubes (MWCNT) and reported increased granulomas in macrophage-specific PPARγ KO mice compared to wild type mice. Because mycobacterial antigens can associate with sarcoidosis, we hypothesized that both T effector cells and pulmonary fibrosis may be exacerbated by PPARγ deficiency and exposure to Mycobacterial Early Secreted Antigenic Target Protein 6 (ESAT-6). Modified Ashcroft scoring of Trichrome staining indicated increased pulmonary fibrosis in mice receiving MWCNT + ESAT-6 compared to MWCNT alone. No changes occurred with PBS or ESAT-6. Analysis of mediastinal lymph nodes revealed increased frequency and number of Th1 cells with corresponding reductions in Th2 cells in PPARγ KO mice after MWCNT, compared to wild-type controls. Notably, the number of Th1, Th2 and Th17 cells were each increased in PPARγ KO after MWCNT + ESAT-6 compared to after administration of MWCNT alone. Our findings suggest that: (1) PPARγ deficiency exacerbates Th1 activation and pulmonary fibrogenic pathways; and (2) the murine MWCNT model may offer novel insights into fibrotic transformation and possible therapeutic options.

Pregnancy is a unique physiological state, in which C60 fullerene is reported to be distributed in both maternal and fetal tissues. Tissue distribution of C60 differs between pregnant and non-pregnant states, presumably due to functional changes in vasculature during pregnancy. We hypothesized that, polyvinylpyrorrolidone (PVP) formulated C60 (C60/PVP) increases vascular tissue contractility during pregnancy by increasing Rho-kinase activity. C60/PVP was administered intravenously to pregnant and non-pregnant female Sprague Dawley rats. Vascular responses were assessed using wire myography 24 hours post-exposure. Increased stress generation was observed in uterine artery, thoracic aorta and umbilical vein. Rho-Rho-kinase mediated force maintenance was increased in arterial segments from C60/PVP exposed pregnant rats when compared to PVP exposed rats. Our findings suggest that intravenous exposure to C60/PVP during pregnancy increases vascular tissue contractility of the uterine artery through elements of Rho-Rho-kinase signaling during late stages of pregnancy.

A covalent regulatory mechanism involving Ca2+-dependent cross-bridge phosphorylation determines both the number of cycling cross bridges and cycling kinetics in mammalian smooth muscle. Our objective was to determine whether a similar regulatory mechanism governed smooth muscle contraction from a poikilothermic amphibian in a test of the hypothesis that myosin regulatory light chain (MRLC) phosphorylation could modulate shortening velocity. We measured MRLC phosphorylation of Rana catesbiana urinary bladder strips at 25°C in tonic contractions in response to K+ depolarization, field stimulation, or carbachol stimulation. The force-length relationship was characterized by a steep ascending limb and a shallow descending limb. There was a rapid rise in unloaded shortening velocity early in a contraction, which then fell and was maintained at low rates while high force was maintained. In support of the hypothesis, we found a positive correlation of the level of myosin phosphorylation and an estimate of tissue shortening velocity. These results suggest that MRLC phosphorylation in amphibian smooth muscle modulates both the number of attached cross bridges (force) and the cross-bridge cycling kinetics (shortening velocity) as in mammalian smooth muscle.

A recent report from this laboratory (Chitaley K, Wingard C, Webb R, Branam H, Stopper V, Lewis R, and Mills T. Nature Medicine 7: 119–122, 2001) showed that inhibition of Rho-kinase increased the erectile response (intracavernosal pressure and mean arterial pressure) by a process that does not require nitric oxide or cGMP. The present study investigated whether vasoconstrictor agents, which are active in the penis, act via the Rho-kinase pathway. Western analysis revealed RhoA and Rho-kinase protein in the penis. Treatment with the selective Rho-kinase inhibitor Y-27632 significantly increased the magnitude of the erectile response. Intracavernous administration of endothelin-1 (ET-1; 50 pmol) or methoxamine (10 μg/kg) reduced the erectile response to autonomic stimulation. If Y-27632 was given before ET-1 or methoxamine, the vasoconstrictor effect was reduced, and intracavernosal pressure and mean arterial pressure remained elevated. However, when given after methoxamine, Y-27632 had a reduced vasodilatory effect, and Y-27632 had no vasodilatory effect when given after ET-1. These findings suggest that ET-1 and methoxamine increase Rho-kinase activity in the cavernous circulation and support the hypothesis that the vasoconstriction that maintains the penis in the nonerect state is mediated, in part, by the Rho-kinase pathway.

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of bovine carotid artery smooth muscle contractions and is associated with increased phosphorylation of the small heat shock-related protein (HSP20). Previous reports have shown that human umbilical artery smooth muscle is uniquely resistant to cyclic nucleotide-dependent relaxation, and HSP20 is not phosphorylated. In this investigation, we determined the phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. In carotid artery, activation of cyclic nucleotide-dependent signaling pathways inhibited contractile responses to serotonin but did not inhibit myosin light chain phosphorylation or oxygen consumption. The inhibition of contraction was associated with increases in HSP20 phosphorylation. In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways did not inhibit serotonin-induced contraction or myosin light chain phosphorylation. The lack of contractile inhibition in umbilical artery was not associated with significant increases in HSP20 phosphorylation. In conclusion, cyclic nucleotide-dependent contractile inhibition is independent of the inhibition of myosin light chain phosphorylation or oxygen consumption but does correlate with increased HSP20 phosphorylation.

Multi-walled carbon nanotubes (MWCNTs) are increasingly used in industry and in nanomedicine raising safety concerns, especially during unique life-stages such as pregnancy. We hypothesized that MWCNT exposure during pregnancy will increase vascular tissue contractile responses by increasing Rho kinase signaling. Pregnant (17–19 gestational days) and non-pregnant Sprague Dawley rats were exposed to 100 μg/kg of MWCNTs by intratracheal instillation or intravenous administration. Vasoactive responses of uterine, mesenteric, aortic and umbilical vessels were studied 24 hours post-exposure by wire myography. The contractile responses of the vessel segments were different between the pregnant and non-pregnant rats, following MWCNT exposure. Maximum stress generation in the uterine artery segments from the pregnant rats following pulmonary MWCNT exposure was increased in response to angiotensin II by 4.9 mN/mm2 (+118%), as compared to the naïve response and by 2.6 mN/mm2 (+40.7%) as compared to the vehicle exposed group. Following MWCNT exposure, serotonin induced approximately 4 mN/mm2 increase in stress generation of the mesenteric artery from both pregnant and non-pregnant rats as compared to the vehicle response. A significant contribution of the dispersion medium was identified as inducing changes in the contractile properties following both pulmonary and intravenous exposure to MWCNTs. Wire myographic studies in the presence of a Rho kinase inhibitor and RhoA and Rho kinase mRNA/protein expression of rat aortic endothelial cells were unaltered following exposure to MWCNTs, suggesting absent/minimal contribution of Rho kinase to the enhanced contractile responses following MWCNT exposure. The reactivity of the umbilical vein was not changed; however, mean fetal weight gain was reduced with dispersion media and MWCNT exposure by both routes. These results suggest a susceptibility of the vasculature during gestation to MWCNT and their dispersion media-induced vasoconstriction, predisposing reduced fetal growth during pregnancy.

The purpose of this study was to investigate NADPH oxidase as a common mechanism underlying both erectile dysfunction (ED) and coronary artery disease (CAD) in inactivity and diet-induced obesity. ...