Impact of pulmonary exposure to gold core silver nanoparticles of different size and capping agents on cardiovascular injury.

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.

Cardioprotection via preserved mitochondrial structure and function in the mPer2-mutant mouse myocardium.

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O2 consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O2 consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.

Perfluorooctanoic acid induced-developmental cardiotoxicity: are peroxisome proliferator activated receptor α (PPARα) and bone morphorgenic protein 2 (BMP2) pathways involved?

Perfluorooctanoic acid (PFOA) is an environmental contaminant known to induce developmental toxicity in animal models through activation of the peroxisome proliferator-activated receptor α (PPARα). Previously, it was demonstrated that in ovo exposure to PFOA induced cardiotoxicity in chicken embryos and hatchlings. To investigate potential PPARα-mediated mechanisms, fertile chicken eggs were injected prior to incubation with WY 14,643, a PPARα agonist. Cardiac morphology and function were evaluated in late-stage embryos and hatchlings. Histologically, unlike PFOA, WY 14,643 did not induce thinning of the right ventricular wall. Via echocardiography, however, WY 14,643 induced effects similar to those of PFOA, including increased left ventricular wall thickness and mass, elevated heart rate, ejection fraction, fractional shortening, and decreased stroke volume. Additionally, to investigate mechanisms associated with early heart development, a separate group of fertile chicken eggs was injected prior to incubation with PFOA or WY 14,643 and in early-stage embryos, gene expression and protein concentration associated with the bone morphogenic protein (BMP2) pathway were determined. Although changes were not statistically consistent among doses, expression of BMP2, Nkx2.5, and GATA4 mRNA in early embryos was altered by PFOA exposure; however, protein concentrations of these targets were not markedly altered by either PFOA or WY 14,643. Protein levels of pSMAD1/5, a transcriptional regulator stimulated by BMPs, were altered by both PFOA and WY 14,643, but in different directions; PFOA reduced cytoplasmic pSMAD1/5, whereas WY 14,643 decreased nuclear pSMAD1/5. Taken together, these data suggest that developmental cardiotoxicity induced by PFOA likely involves both PPARα and BMP2 pathways.

Mitochondrial permeability transition in the diabetic heart: contributions of thiol redox state and mitochondrial calcium to augmented reperfusion injury.

Mitochondria from diabetic hearts are sensitized to mitochondrial permeability transition pore (PTP) opening, which may be responsible for the increased propensity for cardiac injury in diabetic hearts. The purpose of this study was to determine if redox-dependent PTP opening contributes to augmented injury in diabetic hearts, and if compounds targeted at mitochondrial PTP, ROS, and calcium influx protected diabetic hearts from injury. Hearts from control or streptozotocin-induced diabetic rats were excised for either whole-heart or isolated mitochondria experiments. Myocardial glutathione content was oxidized in diabetic hearts when compared to control, and this translated to increased oxidation of the adenine nucleotide translocase in diabetic hearts. Diabetic mitochondria displayed significantly greater sensitivity to PTP opening than non-diabetic counterparts, which was reversed with the thiol-reducing agent dithiothreitol. The thiol-oxidant diamide increased calcium sensitivity in control, but not diabetic mitochondria. Diabetic animals treated with the mitochondria-targeted ROS suppressing peptide MTP-131 also showed improved resistance to PTP opening. In separate experiments hearts underwent ex vivo ischemia/reperfusion (IR). Diabetic hearts were more susceptible to IR injury, with infarct sizes of 60 ± 4% of the area-at-risk (vs. 46 ± 2% in non-diabetics; P<0.05). Administration of the PTP blocker NIM811 (5 µM), MTP-131 (1 nM) or the mitochondrial calcium uniporter blocker minocycline (1 µM) at the onset of reperfusion reduced infarct sizes in both control and diabetic hearts. These findings suggest that augmented susceptibility to injury in the diabetic heart is mediated by redox-dependent shifts in PTP opening, and that three novel mitochondria-targeted agents administered at reperfusion may be suitable adjuvant reperfusion therapies to attenuate injury in diabetic patients.

Expansion of cardiac ischemia/reperfusion injury after instillation of three forms of multi-walled carbon nanotubes.

BACKGROUND: The exceptional physical-chemical properties of carbon nanotubes have lead to their use in diverse commercial and biomedical applications. However, their utilization has raised concerns about human exposure that may predispose individuals to adverse health risks. The present study investigated the susceptibility to cardiac ischemic injury following a single exposure to various forms of multi-walled carbon nanotubes (MWCNTs). It was hypothesized that oropharyngeal aspiration of MWCNTs exacerbates myocardial ischemia and reperfusion injury (I/R injury). METHODS: Oropharyngeal aspiration was performed on male C57BL/6J mice with a single amount of MWCNT (0.01 - 100 µg) suspended in 100 µL of a surfactant saline (SS) solution. Three forms of MWCNTs were used in this study: unmodified, commercial grade (C-grade), and functionalized forms that were modified either by acid treatment (carboxylated, COOH) or nitrogenation (N-doped) and a SS vehicle. The pulmonary inflammation, serum cytokine profile and cardiac ischemic/reperfusion (I/R) injury were assessed at 1, 7 and 28 days post-aspiration. RESULTS: Pulmonary response to MWCNT oropharyngeal aspiration assessed by bronchoalveolar lavage fluid (BALF) revealed modest increases in protein and inflammatory cell recruitment. Lung histology showed modest tissue inflammation as compared to the SS group. Serum levels of eotaxin were significantly elevated in the carboxylated MWCNT aspirated mice 1 day post exposure. Oropharyngeal aspiration of all three forms of MWCNTs resulted in a time and/or dose-dependent exacerbation of myocardial infarction. The severity of myocardial injury varied with the form of MWCNTs used. The N-doped MWCNT produced the greatest expansion of the infarct at any time point and required a log concentration lower to establish a no effect level. The expansion of the I/R injury remained significantly elevated at 28 days following aspiration of the COOH and N-doped forms, but not the C-grade as compared to SS. CONCLUSION: Our results suggest that oropharyngeal aspiration of MWCNT promotes increased susceptibility of cardiac tissue to ischemia/reperfusion injury without a significant pulmonary inflammatory response. The cardiac injury effects were observed at low concentrations of MWCNTs and presence of MWCNTs may pose a significant risk to the cardiovascular system.

Influence of Intrinsic Aerobic Exercise Capacity and Sex on Cardiac Injury Following Acute Myocardial Ischemia and Reperfusion.

Purpose: Previous reports have suggested that active exercise aside, intrinsic aerobic running capacity (Low = LCR, high = HCR) in otherwise sedentary animals may influence several cardiovascular health-related indicators. Relative to the HCR phenotype, the LCR phenotype is characterized by decreased endothelial reactivity, increased susceptibility to reperfusion-induced arrhythmias following short, non-infarction ischemia, and increased diet-induced insulin resistance. More broadly, the LCR phenotype has come to be characterized as a "disease prone" model, with the HCRs as "disease resistant." Whether these effects extend to injury outcomes in an overt infarction or whether the effects are gender specific is not known. This study was designed to determine whether HCR/LCR phenotypic differences would be evident in injury responses to acute myocardial ischemia-reperfusion injury (AIR), measured as infarct size and to determine whether sex differences in infarction size were preserved with phenotypic selection. Methods: Regional myocardial AIR was induced in vivo by either 15 or 30 min ligation of the left anterior descending coronary artery, followed by 2 h of reperfusion. Global ischemia was induced in isolated hearts ex vivo using a Langendorff perfusion system and cessation of perfusion for either 15 or 30 min followed by 2 h of reperfusion. Infarct size was determined using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and normalized to area at risk in the regional model, or whole heart in the global model. Portions of the tissue were paraffin embedded for H&E staining and histology analysis. Results: Phenotype dependent differences in infarct size were seen with 15 min occlusion/2 h reperfusion (LCR > HCR, p < 0.05) in both regional and global models. In both models, longer occlusion times (30 min/2 h) produced significantly larger infarctions in both phenotypes, but phenotypic differences were no longer present (LCR vs. HCR, p = n.s.). Sex differences in infarct size were present in each phenotype (LCR male > LCR female, p < 0.05; HCR male > HCR female, p < 0.05 regardless of length of occlusion, or ischemia model. Conclusions: There is cardioprotection afforded by high intrinsic aerobic capacity, but it is not infinite/continuous, and may be overcome with sufficient injury burden. Phenotypic selection based on endurance running capacity preserved sex differences in response to both short and longer term coronary occlusive challenges. Outcomes could not be associated with differences in system characteristics such as circulating inflammatory mediators or autonomic nervous system influences, as similar phenotypic injury patterns were seen in vivo, and in isolated crystalloid perfused heart ex vivo.

Low Intrinsic Aerobic Capacity Limits Recovery Response to Hindlimb Ischemia.

Introduction: In this study, we determined the influence of intrinsic exercise capacity on the vascular adaptive responses to hind limb ischemia. High Capacity Running, HCR; Low Capacity Running, LCR, rats were used to assess intrinsic aerobic capacity effects on adaptive responses to ischemia. Methods: Muscle samples from both ischemic and non-ischemic limb in both strains were compared, histologically for the muscle-capillary relationship, and functionally using microspheres to track blood flow and muscle stimulation to test fatigability. PCR was used to identify the differences in gene expression between the phenotypes following occlusive ischemia. Results: Prior to ligation, there were not significant differences between the phenotypes in the exhaustion time with high frequency pacing. Following ligation, LCR decreased significantly in the exhaustion time compare with HCRs (437 ± 47 vs. 824 ± 56, p < 0.001). The immediate decrease in flow was significantly more severe in LCRs than HCRs (52.5 vs. 37.8%, p < 0.001). VEGF, eNOS, and ANG2 (but not ANG1) gene expression were decreased in LCRs vs. HCRs before occlusion, and increased significantly in LCRs 14D after occlusion, but not in HCRs. LCR capillary density (CD) was significantly lower at all time points after occlusion (LCR 7D = 564.76 ± 40.5, LCR 14D = 507.48 ± 54.2, both p < 0.05 vs. HCR for respective time point). NCAF increased significantly in HCR and LCR in response to ischemia. Summary: These results suggest that LCR confers increased risk for ischemic injury and is subject to delayed and less effective adaptive response to ischemic stress.

Augmented Cardiac Mitochondrial Capacity in High Capacity Aerobic Running "Disease-Resistant" Phenotype at Rest Is Lost Following Ischemia Reperfusion.

Rationale: Regular active exercise is considered therapeutic for cardiovascular disease, in part by increasing mitochondrial respiratory capacity, but a significant amount of exercise capacity is determined genetically. Animal models, demonstrating either high capacity aerobic running (HCR) or low capacity aerobic running (LCR) phenotypes, have been developed to study the intrinsic contribution, with HCR rats subsequently characterized as "disease resistant" and the LCRs as "disease prone." Enhanced cardioprotection in HCRs has been variable and mutifactoral, but likely includes a metabolic component. These studies were conducted to determine the influence of intrinsic aerobic phenotype on cardiac mitochondrial function before and after ischemia and reperfusion. Methods: A total of 34 HCR and LCR rats were obtained from the parent colony at the University of Toledo, housed under sedentary conditions, and fed normal chow. LCR and HCR animals were randomly assigned to either control or ischemia-reperfusion (IR). On each study day, one HCR/LCR pair was anesthetized, and hearts were rapidly excised. In IR animals, the hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to global hypothermic ischemic arrest (80 min). Following the arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff perfusion system. Following reperfusion, the heart was weighed and the left ventricle (LV) was isolated. A midventricular ring was obtained to estimate infarction size [triphenyltetrazolium chloride (TTC)] and part of the remaining tissue (~150 mg) was transferred to a homogenation buffer on ice. Isolated mitochondria (MITO) samples were prepared and used to determine respiratory capacity under different metabolic conditions. In control animals, MITO were obtained and prepared similarly immediately following anesthesia and heart removal, but without IR. Results: In the control rats, both resting and maximally stimulated respiratory rates were higher (32 and 40%, respectively; p < 0.05) in HCR mitochondria compared to LCR. After IR, resting MITO respiratory rates were decreased to about 10% of control in both strains, and the augmented capacity in HCRs was absent. Maximally stimulated rates also were decreased more than 50% from control and were no longer different between phenotypes. Ca++ retention capacity and infarct size were not significantly different between HCR and LCR (49.2 ± 5.6 vs. 53.7 ± 4.9%), nor was average coronary flow during reperfusion or arrhythmogenesis. There was a significant loss of mitochondria following IR, which was coupled with decreased function in the remaining mitochondria in both strains. Conclusion: Cardiac mitochondrial capacity from HCR was significantly higher than LCR in the controls under each condition. After IR insult, the cardiac mitochondrial respiratory rates were similar between phenotypes, as was Ca++ retention capacity, infarct size, and arrhythmogenicity, despite the increased mitochondrial capacity in the HCRs before ischemia. Relatively, the loss of respiratory capacity was actually greater in HCR than LCR. These data could suggest limits in the extent to which the HCR phenotype might be "protective" against acute tissue stressors. The extent to which any of these deficits could be "rescued" by adding an active exercise component to the intrinsic phenotype is unknown.

Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro.

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control.

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine diminution was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete beta-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.

Attenuation of myocardial injury in mice with functional deletion of the circadian rhythm gene mPer2.

Variations in circadian rhythms are evident in the incidence of cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. The suprachiasmatic nucleus is the central circadian pacemaker that regulates the daily rhythm of peripheral organs. Diurnal rhythms have more recently been shown to exist in myocardial tissue and are involved in metabolism and contractile function. Thus we sought to determine whether the functional deletion of the circadian rhythm mouse periodic gene 2 (mPer2) would protect the heart against ischemic injury. Nonreperfused myocardial infarction was induced in anesthetized, ventilated C57 (n = 17) and mPer2 mutant (mPer2-M; n = 15) mice via permanent ligation of the left anterior descending coronary artery. At 4 days post-myocardial infarction, we observed a 43% reduction of infarct area in mPer2-M mice compared with wild-type mice. This is coincident with 25% less macrophage infiltration, 43% higher capillary density, 17% increase in hypertrophy, and 15% less cardiomyocyte apoptosis in the infarct zone. Also, matrix metalloproteinase-9 was expressed in inflammatory cells in both groups, but total protein was 40% higher in wild-type mice, whereas it was not elevated in mPer2-M mice in response to injury. The functional deletion of the mPer2 gene reduces the severity of myocardial infarct injury by limiting the inflammatory response, reducing apoptosis, and inducing cardiomyocyte hypertrophy, thus preserving cardiac function. These findings collectively imply that the disruption of the circadian clock gene mPer2 is protective. Understanding the interactions between circadian rhythm genes and cardiovascular disease may provide insights into potential preventative and therapeutic strategies for susceptible populations.

Nitric oxide and catalase-sensitive relaxation by scutellarin in the mouse thoracic aorta.

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.

What is the role of peptide fragments of collagen I and IV in health and disease?

Collagen is the most abundant protein in mammalian systems; it can be found in organs such as bones, the liver, kidney, heart, teeth, and skin. Collagen provides the necessary structural framework for tissues in which it is found. However, if there are any alterations in the delicate balance of collagen types in the extracellular matrix (ECM), then problems arise. For example, increasing collagen I:III ratio would provide additional rigidity to tissue structure, whereas decreasing this ratio would provide elasticity and flexibility to the tissue. The proper function of tissues is reliant on this scale not tipping too far in either direction. Major players in the process of ECM remodeling, both normal and adverse, are the fibroblast cells via the secretion of collagen precursors and matrix metalloproteinases, with the latter responsible for ECM degradation. The collagen peptides created by the proteolytic cleavage of these collagen fibrils, while once thought to have an absence of function, have been shown over recent years to potentiate and regulate a variety of cellular processes acting through integrin receptors. Many collagen peptides have been identified from many different collagen types and have been shown to regulate processes such as cell proliferation, migration, apoptosis, and reduce angiogenesis. The collagen peptides of interest are those generated from the primary collagen type of tissue interstitial matrix, collagen type I, and the basement membrane, collagen type IV. Thus, this review looks to highlight some examples of unorthodox functional roles of collagen and its peptides in regulating physiological health and disease.

Airway Exposure to Modified Multi-walled Carbon Nanotubes Perturbs Cardiovascular Adenosinergic Signaling in Mice.

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 h 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. 100 µL 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. 1 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.

PPAR-gamma expression in animals subjected to volume overload and chronic Urotensin II administration.

Activation of PPAR-gamma through the administration of glitazones has shown promise in preserving function following cardiac injury, although recent evidence has suggested their use may be contraindicated in the case of severe heart failure. This study tested the hypothesis that PPAR-gamma expression increases in a time dependent manner in response to chronic volume overload (VO) induced heart failure. Additionally, we attempted to determine what effect 4 week administration of Urotensin II (UTII) may have on PPAR-gamma expression. VO induced heart failure was produced in Sprague-Dawley rats (n=32) by aorta-caval fistula. Animals were sacrificed at 1, 4, and 14 weeks following shunt creation. In a separate set of experiments, animals were administered 300 pmol/kg/h of UTII for 4 weeks, subjected to 4 weeks of volume overload, or given UTII+VO. Densitometric analysis of left ventricular (LV) protein demonstrated PPAR-gamma expression was significantly ((*)p<0.05) upregulated at 4 and 14 weeks (31.5% and 37%, respectively) post-fistula formation compared to control values. PPAR-gamma activation was decreased in the 4 and 14 week (39.16% and 42.4%, respectively), but not in the 1-week animals, and these changes did not correlate with NF-kappaB activity. Animals given UTII either with or without VO demonstrated increased expression of PPAR-gamma as did animals subjected to 4 week VO alone. Animals given UTII either with or without VO had decreased activity vs. control. These data suggest PPAR-gamma may play a role in the progression of heart failure, however, the exact nature has yet to be determined.

Correction of the bleeding time with lyophilized platelet infusions in dogs on cardiopulmonary bypass.

Lyophilized canine platelets were infused in a single large bolus dose into splenectomized dogs after 2 hours' perfusion on cardiopulmonary bypass to test their possible efficacy in restoring hemostasis after compromise of platelet function. The vessel bleeding time (VBT) was monitored by venipuncture of the exposed jugular vein. During cardiopulmonary bypass, platelet counts fell quickly and the VBTs became prolonged over baseline. Infusion of lyophilized platelets reconstituted in normal saline occurred just before or immediately after weaning from the cardiopulmonary bypass pump. The results showed consistent and persistent lowering of the VBTs by the infused lyophilized platelets. Controls showed continuously prolonged VBTs. The weighted average VBT in infused subjects was significantly lower than the average in controls: 3 minutes 10 seconds versus 6 minutes 59 seconds, respectively (t test, P= .01). These results in this setting indicate the possible effectiveness of similar human lyophilized platelet preparations in reducing postoperative bleeding in open heart surgery.

Are There Limitations to Exercise Benefits in Peripheral Arterial Disease?

Substantial evidence exists indicating that inactivity contributes to the progression of chronic disease, and conversely, that regular physical activity can both prevent the onset of disease as well as delay the progression of existing disease. To that end "exercise as medicine" has been advocated in the broad context as general medical care, but also in the specific context as a therapeutic, to be considered in much the same way as other drugs. As there are non-responders to many medications, there also are non-responders to exercise; individual who participate but do not demonstrate appreciable improvement/benefit. In some settings, the stress induced by exercise may aggravate an underlying condition, rather than attenuate chronic disease. As personalized medicine evolves with ready access to genetic information, so too will the incorporation of exercise in the context of those individual genetics. The focus of this brief review is to distinguish between the inherent capacity to perform, as compared to adaptive response to active exercise training in relation to cardiovascular health and peripheral arterial disease.

Hemodynamic effects of chronic urotensin II administration in animals with and without aorto-caval fistula.

Urotensin II (UTII) is a potent vasoactive peptide. Recent studies have demonstrated increased expression of both UTII and its receptor (UTR) expression in end-stage congestive heart failure (CHF), but it is unclear whether UTII and UTR are late stage markers of decompensation, or earlier adaptive responses. The purpose of this study was to measure the effects of chronic UTII administration in normal and volume overloaded animals. Chronic 4 weeks administration of UTII produced decreases in hemodynamic function in animals not subjected to volume overload while returning function to control levels in animals with overload. Expression levels of calcium regulatory proteins phospholamban (PLN), sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), and Na(+)/Ca(2+) exchanger (NCX) were measured to determine if administration of UTII resulted in aberrant Ca(2+) handling. Changes in protein expression revealed that UTII influenced Ca(2+) handling proteins in normal animals although these changes are not seen in the volume overload.

Cardiac and vascular changes in mice after exposure to ultrafine particulate matter.

Increased ambient air particulate matter (PM) concentrations are associated with risk for myocardial infarction, stroke, and arrhythmia, and ultrafine PM (UFPM) might be particularly toxic to the cardiovascular system. Recent epidemiological studies are beginning to offer mechanistic insights, yet the rodent model remains a valuable tool to explore potential mechanisms. This article reviews a series of studies from our laboratory demonstrating the promise of mouse models to link health effects to biological mechanisms. Specifically, data from 6- to 10-wk-old male ICR mice exposed to intratracheal instillation of 100 microg of UFPM collected from the Chapel Hill, NC airshed are described. Studies of ischemia/reperfusion, vascular function, and hemostasis are described. In summary, UFPM exposure doubles the size of myocardial infarction attendant to an episode of ischemia and reperfusion while increasing postischemic oxidant stress. UFPM alters endothelial-dependent and -independent regulation of systemic vascular tone; increases platelet number, plasma fibrinogen, and soluble P-selectin levels; and reduces bleeding time, implying enhanced thrombogenic potential. Taking these findings together, this model of acute UFPM exposure in the mouse indicates that UFPM induces a prothrombotic state and decreases vasomotor responsiveness, thereby offering insight into how UFPM could contribute to vascular events associated with thrombosis and ischemia and increasing the extent of infarction.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.