Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis.

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Changes in midbrain pain receptor expression, gait and behavioral sensitivity in a rat model of radiculopathy.

Intervertebral disc herniation may contribute to inflammatory processes that associate with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait and the expression of key pain receptors in the midbrain in a rodent model of radiculopathy. Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in rats (NP-treated, n=12). Tail NP was discarded in sham-operated animals (n=12). Mechanical allodynia, weight-bearing, and gait were evaluated in all animals over time. At 1 and 4 weeks after surgery, astrocyte and microglial activation was tested in DRG sections. Midbrain sections were similarly evaluated for immunoreactivity to serotonin (5HT(2B)), mu-opioid (µ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the duration of the study. Astroctye activation was observed at DRGs only at 4 weeks after surgery. Findings for pain receptors in the midbrain of NP-treated rats included an increased expression of 5HT(2B) at 1, but not 4 weeks; increased expression of µ-OR and mGluR5 at 1 and 4 weeks (periaqueductal gray region only); and no changes in expression of mGluR4 at any point in this study. These observations provide support for the hypothesis that the midbrain responds to DRG injury with a transient change in receptors regulating pain responses.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Evaluating intra-articular drug delivery for the treatment of osteoarthritis in a rat model.

Osteoarthritis (OA) is a degenerative joint disease that can result in joint pain, loss of joint function, and deleterious effects on activity levels and lifestyle habits. Current therapies for OA are largely aimed at symptomatic relief and may have limited effects on the underlying cascade of joint degradation. Local drug delivery strategies may provide for the development of more successful OA treatment outcomes that have potential to reduce local joint inflammation, reduce joint destruction, offer pain relief, and restore patient activity levels and joint function. As increasing interest turns toward intra-articular drug delivery routes, parallel interest has emerged in evaluating drug biodistribution, safety, and efficacy in preclinical models. Rodent models provide major advantages for the development of drug delivery strategies, chiefly because of lower cost, successful replication of human OA-like characteristics, rapid disease development, and small joint volumes that enable use of lower total drug amounts during protocol development. These models, however, also offer the potential to investigate the therapeutic effects of local drug therapy on animal behavior, including pain sensitivity thresholds and locomotion characteristics. Herein, we describe a translational paradigm for the evaluation of an intra-articular drug delivery strategy in a rat OA model. This model, a rat interleukin-1beta overexpression model, offers the ability to evaluate anti-interleukin-1 therapeutics for drug biodistribution, activity, and safety as well as the therapeutic relief of disease symptoms. Once the action against interleukin-1 is confirmed in vivo, the newly developed anti-inflammatory drug can be evaluated for evidence of disease-modifying effects in more complex preclinical models.

Autonomic cardiovascular dysregulation as a potential mechanism underlying depression and coronary artery bypass grafting surgery outcomes.

BACKGROUND: Coronary artery bypass grafting (CABG) is often used to treat patients with significant coronary heart disease (CHD). To date, multiple longitudinal and cross-sectional studies have examined the association between depression and CABG outcomes. Although this relationship is well established, the mechanism underlying this relationship remains unclear. The purpose of this study was twofold. First, we compared three markers of autonomic nervous system (ANS) function in four groups of patients: 1) Patients with coronary heart disease and depression (CHD/Dep), 2) Patients without CHD but with depression (NonCHD/Dep), 3) Patients with CHD but without depression (CHD/NonDep), and 4) Patients without CHD and depression (NonCHD/NonDep). Second, we investigated the impact of depression and autonomic nervous system activity on CABG outcomes. METHODS: Patients were screened to determine whether they met some of the study's inclusion or exclusion criteria. ANS function (i.e., heart rate, heart rate variability, and plasma norepinephrine levels) were measured. Chi-square and one-way analysis of variance were performed to evaluate group differences across demographic, medical variables, and indicators of ANS function. Logistic regression and multiple regression analyses were used to assess impact of depression and autonomic nervous system activity on CABG outcomes. RESULTS: The results of the study provide some support to suggest that depressed patients with CHD have greater ANS dysregulation compared to those with only CHD or depression. Furthermore, independent predictors of in-hospital length of stay and non-routine discharge included having a diagnosis of depression and CHD, elevated heart rate, and low heart rate variability. CONCLUSIONS: The current study presents evidence to support the hypothesis that ANS dysregulation might be one of the underlying mechanisms that links depression to cardiovascular CABG surgery outcomes. Thus, future studies should focus on developing and testing interventions that targets modifying ANS dysregulation, which may lead to improved patient outcomes.

The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates the agonist-occupied beta-adrenergic receptor to promote rapid receptor uncoupling from Gs, thereby attenuating adenylyl cyclase activity. Beta ARK-mediated receptor desensitization may reflect a general molecular mechanism operative on many G-protein-coupled receptor systems and, particularly, synaptic neurotransmitter receptors. Two distinct cDNAs encoding beta ARK isozymes were isolated from rat brain and sequenced. The regional and cellular distributions of these two gene products, termed beta ARK1 and beta ARK2, were determined in brain by in situ hybridization and by immunohistochemistry at the light and electron microscopic levels. The beta ARK isozymes were found to be expressed primarily in neurons distributed throughout the CNS. Ultrastructurally, beta ARK1 and beta ARK2 immunoreactivities were present both in association with postsynaptic densities and, presynaptically, with axon terminals. The beta ARK isozymes have a regional and subcellular distribution consistent with a general role in the desensitization of synaptic receptors.

Association of a peripheral blood metabolic profile with coronary artery disease and risk of subsequent cardiovascular events.

BACKGROUND: Molecular tools may provide insight into cardiovascular risk. We assessed whether metabolites discriminate coronary artery disease (CAD) and predict risk of cardiovascular events. METHODS AND RESULTS: We performed mass-spectrometry-based profiling of 69 metabolites in subjects from the CATHGEN biorepository. To evaluate discriminative capabilities of metabolites for CAD, 2 groups were profiled: 174 CAD cases and 174 sex/race-matched controls ("initial"), and 140 CAD cases and 140 controls ("replication"). To evaluate the capability of metabolites to predict cardiovascular events, cases were combined ("event" group); of these, 74 experienced death/myocardial infarction during follow-up. A third independent group was profiled ("event-replication" group; n=63 cases with cardiovascular events, 66 controls). Analysis included principal-components analysis, linear regression, and Cox proportional hazards. Two principal components analysis-derived factors were associated with CAD: 1 comprising branched-chain amino acid metabolites (factor 4, initial P=0.002, replication P=0.01), and 1 comprising urea cycle metabolites (factor 9, initial P=0.0004, replication P=0.01). In multivariable regression, these factors were independently associated with CAD in initial (factor 4, odds ratio [OR], 1.36; 95% CI, 1.06 to 1.74; P=0.02; factor 9, OR, 0.67; 95% CI, 0.52 to 0.87; P=0.003) and replication (factor 4, OR, 1.43; 95% CI, 1.07 to 1.91; P=0.02; factor 9, OR, 0.66; 95% CI, 0.48 to 0.91; P=0.01) groups. A factor composed of dicarboxylacylcarnitines predicted death/myocardial infarction (event group hazard ratio 2.17; 95% CI, 1.23 to 3.84; P=0.007) and was associated with cardiovascular events in the event-replication group (OR, 1.52; 95% CI, 1.08 to 2.14; P=0.01). CONCLUSIONS: Metabolite profiles are associated with CAD and subsequent cardiovascular events.

Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.

Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation.

Localization of Metal Electrodes in the Intact Rat Brain Using Registration of 3D Microcomputed Tomography Images to a Magnetic Resonance Histology Atlas.

Simultaneous neural recordings taken from multiple areas of the rodent brain are garnering growing interest due to the insight they can provide about spatially distributed neural circuitry. The promise of such recordings has inspired great progress in methods for surgically implanting large numbers of metal electrodes into intact rodent brains. However, methods for localizing the precise location of these electrodes have remained severely lacking. Traditional histological techniques that require slicing and staining of physical brain tissue are cumbersome, and become increasingly impractical as the number of implanted electrodes increases. Here we solve these problems by describing a method that registers 3-D computerized tomography (CT) images of intact rat brains implanted with metal electrode bundles to a Magnetic Resonance Imaging Histology (MRH) Atlas. Our method allows accurate visualization of each electrode bundle's trajectory and location without removing the electrodes from the brain or surgically implanting external markers. In addition, unlike physical brain slices, once the 3D images of the electrode bundles and the MRH atlas are registered, it is possible to verify electrode placements from many angles by "re-slicing" the images along different planes of view. Further, our method can be fully automated and easily scaled to applications with large numbers of specimens. Our digital imaging approach to efficiently localizing metal electrodes offers a substantial addition to currently available methods, which, in turn, may help accelerate the rate at which insights are gleaned from rodent network neuroscience.

Quantitative mapping of trimethyltin injury in the rat brain using magnetic resonance histology.

The growing exposure to chemicals in our environment and the increasing concern over their impact on health have elevated the need for new methods for surveying the detrimental effects of these compounds. Today's gold standard for assessing the effects of toxicants on the brain is based on hematoxylin and eosin (H&E)-stained histology, sometimes accompanied by special stains or immunohistochemistry for neural processes and myelin. This approach is time-consuming and is usually limited to a fraction of the total brain volume. We demonstrate that magnetic resonance histology (MRH) can be used for quantitatively assessing the effects of central nervous system toxicants in rat models. We show that subtle and sparse changes to brain structure can be detected using magnetic resonance histology, and correspond to some of the locations in which lesions are found by traditional pathological examination. We report for the first time diffusion tensor image-based detection of changes in white matter regions, including fimbria and corpus callosum, in the brains of rats exposed to 8 mg/kg and 12 mg/kg trimethyltin. Besides detecting brain-wide changes, magnetic resonance histology provides a quantitative assessment of dose-dependent effects. These effects can be found in different magnetic resonance contrast mechanisms, providing multivariate biomarkers for the same spatial location. In this study, deformation-based morphometry detected areas where previous studies have detected cell loss, while voxel-wise analyses of diffusion tensor parameters revealed microstructural changes due to such things as cellular swelling, apoptosis, and inflammation. Magnetic resonance histology brings a valuable addition to pathology with the ability to generate brain-wide quantitative parametric maps for markers of toxic insults in the rodent brain.

A model of sequential heart and composite tissue allotransplant in rats.

BACKGROUND: Some of the 600,000 patients with solid organ allotransplants need reconstruction with a composite tissue allotransplant, such as the hand, abdominal wall, or face. The aim of this study was to develop a rat model for assessing the effects of a secondary composite tissue allotransplant on a primary heart allotransplant. METHODS: Hearts of Wistar Kyoto rats were harvested and transplanted heterotopically to the neck of recipient Fisher 344 rats. The anastomoses were performed between the donor brachiocephalic artery and the recipient left common carotid artery, and between the donor pulmonary artery and the recipient external jugular vein. Recipients received cyclosporine A for 10 days only. Heart rate was assessed noninvasively. The sequential composite tissue allotransplant consisted of a 3 x 3-cm abdominal musculocutaneous flap harvested from Lewis rats and transplanted to the abdomen of the heart allotransplant recipients. The abdominal flap vessels were connected to the femoral vessels. No further immunosuppression was administered following the composite tissue allotransplant. Ten days after composite tissue allotransplantation, rejection of the heart and abdominal flap was assessed histologically. RESULTS: The rat survival rate of the two-stage transplant surgery was 80 percent. The transplanted heart rate decreased from 150 +/- 22 beats per minute immediately after transplant to 83 +/- 12 beats per minute on day 20 (10 days after stopping immunosuppression). CONCLUSIONS: This sequential allotransplant model is technically demanding. It will facilitate investigation of the effects of a secondary composite tissue allotransplant following primary solid organ transplantation and could be useful in developing future immunotherapeutic strategies.

Genome-wide association study of perioperative myocardial infarction after coronary artery bypass surgery.

OBJECTIVES: Identification of patient subpopulations susceptible to develop myocardial infarction (MI) or, conversely, those displaying either intrinsic cardioprotective phenotypes or highly responsive to protective interventions remain high-priority knowledge gaps. We sought to identify novel common genetic variants associated with perioperative MI in patients undergoing coronary artery bypass grafting using genome-wide association methodology. SETTING: 107 secondary and tertiary cardiac surgery centres across the USA. PARTICIPANTS: We conducted a stage I genome-wide association study (GWAS) in 1433 ethnically diverse patients of both genders (112 cases/1321 controls) from the Genetics of Myocardial Adverse Outcomes and Graft Failure (GeneMAGIC) study, and a stage II analysis in an expanded population of 2055 patients (225 cases/1830 controls) combined from the GeneMAGIC and Duke Perioperative Genetics and Safety Outcomes (PEGASUS) studies. Patients undergoing primary non-emergent coronary bypass grafting were included. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome variable was perioperative MI, defined as creatine kinase MB isoenzyme (CK-MB) values ≥10× upper limit of normal during the first postoperative day, and not attributable to preoperative MI. Secondary outcomes included postoperative CK-MB as a quantitative trait, or a dichotomised phenotype based on extreme quartiles of the CK-MB distribution. RESULTS: Following quality control and adjustment for clinical covariates, we identified 521 single nucleotide polymorphisms in the stage I GWAS analysis. Among these, 8 common variants in 3 genes or intergenic regions met p<10(-5) in stage II. A secondary analysis using CK-MB as a quantitative trait (minimum p=1.26×10(-3) for rs609418), or a dichotomised phenotype based on extreme CK-MB values (minimum p=7.72×10(-6) for rs4834703) supported these findings. Pathway analysis revealed that genes harbouring top-scoring variants cluster in pathways of biological relevance to extracellular matrix remodelling, endoplasmic reticulum-to-Golgi transport and inflammation. CONCLUSIONS: Using a two-stage GWAS and pathway analysis, we identified and prioritised several potential susceptibility loci for perioperative MI.

Persistent Homology Analysis of Brain Artery Trees.

New representations of tree-structured data objects, using ideas from topological data analysis, enable improved statistical analyses of a population of brain artery trees. A number of representations of each data tree arise from persistence diagrams that quantify branching and looping of vessels at multiple scales. Novel approaches to the statistical analysis, through various summaries of the persistence diagrams, lead to heightened correlations with covariates such as age and sex, relative to earlier analyses of this data set. The correlation with age continues to be significant even after controlling for correlations from earlier significant summaries.

Platelet Counts, Acute Kidney Injury, and Mortality after Coronary Artery Bypass Grafting Surgery.

BACKGROUND: Cardiac surgery requiring cardiopulmonary bypass is associated with platelet activation. Because platelets are increasingly recognized as important effectors of ischemia and end-organ inflammatory injury, the authors explored whether postoperative nadir platelet counts are associated with acute kidney injury (AKI) and mortality after coronary artery bypass grafting (CABG) surgery. METHODS: The authors evaluated 4,217 adult patients who underwent CABG surgery. Postoperative nadir platelet counts were defined as the lowest in-hospital values and were used as a continuous predictor of postoperative AKI and mortality. Nadir values in the lowest 10th percentile were also used as a categorical predictor. Multivariable logistic regression and Cox proportional hazard models examined the association between postoperative platelet counts, postoperative AKI, and mortality. RESULTS: The median postoperative nadir platelet count was 121 × 10/l. The incidence of postoperative AKI was 54%, including 9.5% (215 patients) and 3.4% (76 patients) who experienced stages II and III AKI, respectively. For every 30 × 10/l decrease in platelet counts, the risk for postoperative AKI increased by 14% (adjusted odds ratio, 1.14; 95% CI, 1.09 to 1.20; P < 0.0001). Patients with platelet counts in the lowest 10th percentile were three times more likely to progress to a higher severity of postoperative AKI (adjusted proportional odds ratio, 3.04; 95% CI, 2.26 to 4.07; P < 0.0001) and had associated increased risk for mortality immediately after surgery (adjusted hazard ratio, 5.46; 95% CI, 3.79 to 7.89; P < 0.0001). CONCLUSION: The authors found a significant association between postoperative nadir platelet counts and AKI and short-term mortality after CABG surgery.