In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

Background Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. New Method We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Results Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. Comparison with Existing Methods In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Conclusions Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease

Novel methods for spatial prediction of soil functions within landscapes (SP0531)

Previous studies showed that soil patterns could be predicted in agriculturally managed landscapes by modelling and extrapolating from extensive existing but related integrated datasets. Based on these results we proposed to develop and apply predictive models of the relationships between environmental data and known soil patterns to predict capacity for key soil functions within diverse landscapes for which there is little detailed underpinning soil information available. Objectives were: To develop a high-level framework in which the non-specialist user-community could explore questions. To generate digital soil maps for three selected catchments at a target resolution of 1:50000 to provide the base information for soil function prediction. To use a modelling approach to predict the performance of key soil functions in catchments undergoing change but where only sparse or low resolution soil survey data are available. To use a modelling approach to assess the impact of different management scenarios and/or environmental conditions on the delivery of multiple soil functions within a catchment. To create a detailed outline of the requirements for ground-truthing to test the predicted model outputs at a catchment scale. To contribute to the development of a high-level framework for decision makers

Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-β treatment would phenocopy these metabolic changes. We used 13C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify 13C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid β-oxidation. TGF-β treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-β-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-β treatment

Regulation of vascular smooth muscle cell expression and function of matrix metalloproteinases is mediated by estrogen and progesterone exposure

ObjectivePostmenopausal women receiving hormone replacement therapy (HRT) have been reported to have more adverse outcomes after vascular reconstructions, including increased intimal hyperplasia development and bypass graft failure. HRT may be affecting the pathway contributing to intimal hyperplasia. An important component of this pathway involves matrix metalloproteinases (MMPs), implicated in vascular remodeling due to their ability to degrade components of the extracellular matrix. We hypothesize that estrogen (Est) and progesterone (Prog) upregulate the MMP pathway in vascular smooth muscle cells (VSMCs) thereby increasing MMP activity and function.Methods and ResultsVSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est + Prog combination (Est/Prog), and/or doxycycline (40 μg/mL; Doxy). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis we have previously shown membrane type 1-MMP (MT1-MMP) messenger ribonucleic acid (mRNA) levels are significantly increased by Est. Here, Western blot analyses indicated MT1-MMP and MMP-2 protein levels, not tissue inhibitor of MMP-2 (TIMP-2), were increased in response to Est and Est/Prog (P < .05 vs control). In-gel zymography revealed that Est and Est/Prog resulted in increased MMP-2 activity (hormone groups, P < .05 vs control) with no significant difference among the hormone groups. VSMC migration was increased by 45 ± 14% in response to Est (P < .05 vs control), as measured using a modified Boyden chamber assay. Doxycycline significantly inhibited basal and Est/Prog-stimulated increases in MMP-2 activity (P < .05 vs control; P < .05 vs hormone groups), and partially blocked basal and hormonally stimulated migration (P < .05 vs control and Est).ConclusionEstrogen and progesterone affects the MMP pathway by increasing MMP-2 enzymatic activity, possibly via the upregulation of MT1-MMP expression without a corresponding increase in TIMP expression. This increased collagenase activity increases VSMC motility and their ability to migrate through a collagen type IV lattice. Est/Prog upregulation of MT1-MMP may contribute to the adverse effect of HRT on vascular interventions.Clinical RelevancePostmenopausal women receiving HRT have more adverse outcomes after vascular reconstructions, including intimal hyperplasia, restenosis, and decreased graft patency. MMPs play a major role in vascular remodeling due to their degradation of components of the basement membrane separating vascular cell layers. Specifically, MMP-2 has a strong affinity for collagen type IV degradation, and MT1-MMP is a transmembrane protein known to activate MMP-2 by proteolytic cleavage. Here we provide strong evidence for MT1-MMP's role in increased MMP-2 activity and increased cellular migration in VSMCs exposed to estrogen and progesterone. Manipulations of the MMP pathway specifically targeting MT1-MMP expression at the time of vascular interventions may improve outcomes in females receiving HRT

Trading in your spindles for blebs: the amoeboid tumor cell phenotype in prostate cancer

Prostate cancer (PCa) remains a principal cause of mortality in developed countries. Because no clinical interventions overcome resistance to androgen ablation therapy, management of castration resistance and metastatic disease remains largely untreatable. Metastasis is a multistep process in which tumor cells lose cell-cell contacts, egress from the primary tumor, intravasate, survive shear stress within the vasculature and extravasate into tissues to colonize ectopic sites. Tumor cells reestablish migratory behaviors employed during nonneoplastic processes such as embryonic development, leukocyte trafficking and wound healing. While mesenchymal motility is an established paradigm of dissemination, an alternate, ‘amoeboid’ phenotype is increasingly appreciated as relevant to human cancer. Here we discuss characteristics and pathways underlying the phenotype, and highlight our findings that the cytoskeletal regulator DIAPH3 governs the mesenchymal-amoeboid transition. We also describe our identification of a new class of tumor-derived microvesicles, large oncosomes, produced by amoeboid cells and with potential clinical utility in prostate and other cancers

Contemporary outcomes of vertebral artery injury

ObjectiveVertebral artery injury (VAI) associated with cervical trauma is being increasingly recognized with more aggressive screening. Disparate results from previous literature have led to uncertainty of the significance, natural history, and optimal therapy for VAI.MethodsTo understand the natural history and treatment outcomes from our experience, we performed a retrospective, single-center review from a level I trauma center for the previous 10 years of all VAI. Injuries were identified from search of an administrative trauma database, a resident-run working database, and all radiology dictations for the same period. All VAI were classified according to segmental involvement, Denver grading scale, and laterality. Analysis of associated injuries, demographics, neurologic outcome, mortality, length of stay, treatment plan, and follow-up imaging was also performed.ResultsFifty-one patients with VAI were identified from 2001 to 2011 from a total of 36,942 trauma admissions (0.13% incidence). Associated injuries were significant with an average New Injury Severity Score of 29.6. Penetrating trauma occurred in 14%. Cervical spine fracture was present in 88% with VAI. Diagnosis was obtained with computed tomographic angiography (CTA) in 95%. Screening was prompted by injury pattern or high-risk mechanism in all cases. Injuries classified according to the Denver grading scale were grade I = 24%, grade II = 35%, grade III = 4%, grade IV = 35%, and grade V = 2%. Distribution across segments included V1 = 18%, V2 = 67%, V3 = 31%, and V4 = 6%. Only one posterior circulation stroke was attributable to VAI. Overall mortality was 8%, with each mortality being associated with significant other organ injuries. Treatment rendered for VAI was antiplatelet therapy (50%), observation (31%), warfarin (17%), and stent (2%). There were no significant differences between treatment groups on any variable with the exception of body mass index (P = .047). Follow-up was obtained for 13% (n = 6) of survivors. The CTA demonstrated injury stability in four patients and resolution in two patients. Accuracy of the administrative trauma database was 53% compared with 96% for the resident-run working database.ConclusionsNeurologic sequelae attributable to VAI were rare. Grade of VAI or vertebral artery segment did not correlate with morbidity. We did not observe any differences in short-term outcomes between systemic anticoagulation and antiplatelet therapy. Of those patients seen at follow-up, injury resolution or stability was documented by CTA. A conservative approach with either observation or antithrombotic therapy is suggested. If the natural history of VAI includes a very low stroke rate, then therapies with a lower therapeutic index, such as systemic anticoagulation, in the severely injured trauma patient are not supported. Our search strategy urges awareness of the limitations of administrative databases for retrospective vascular study

Gender Differences in Bed Rest: Preliminary Analysis of Vascular Function

Orthostatic intolerance is a recognized consequence of spaceflight. Numerous studies have shown that women are more susceptible to orthostatic intolerance following spaceflight as well as bed rest, the most commonly used ground-based analog for spaceflight. One of the possible mechanisms proposed to account for this is a difference in vascular responsiveness between genders. We hypothesized that women and men would have differing vascular responses to 90 days of 6-degree head down tilt bed rest. Additionally, we hypothesized that vessels in the upper and lower body would respond differently, as has been shown in the animal literature. Thirteen subjects were placed in bedrest for 90 days (8 men, 5 women) at the Flight Analogs Unit, UTMB. Direct arterial and venous measurements were made with ultrasound to evaluate changes in vascular structure and function. Arterial function was assessed, in the arm and leg, during a reactive hyperemia protocol and during sublingual nitroglycerin administration to gauge the contributions of endothelial dependent and independent dilator function respectively. Venous function was assessed in dorsal hand and foot veins during the administration of pharmaceuticals to assess constrictor and dilator function. Both gender and day effects are seen in arterial dilator function to reactive hyperemia, but none are seen with nitroglycerin. There are also differences in the wall thickness in the arm vs the leg during bed rest, which return toward pre-bed rest levels by day 90. More subjects are required, especially females as there is not sufficient power to properly analyze venous function. Day 90 data are most underpowered

Acute effects of cannabinoids on addiction endophenotypes are moderated by genes encoding the CB1 receptor and FAAH enzyme

Understanding genetic factors that contribute to cannabis use disorder (CUD) is important, but to date, findings have been equivocal. Single‐nucleotide polymorphisms (SNPs) in the cannabinoid receptor 1 gene (CNR1; rs1049353 and rs806378) and fatty acid amide hydrolase (FAAH) gene (rs324420) have been implicated in CUD. Their relationship to addiction endophenotypes such as cannabis‐related state satiety, the salience of appetitive cues, and craving after acute cannabinoid administration has not been investigated. Forty‐eight cannabis users participated in a double‐blind, placebo‐controlled, four‐way crossover experiment where they were administered treatments in a randomized order via vaporization: placebo, Δ9‐tetrahydrocannabinol (THC) (8 mg), THC + cannabidiol (THC + CBD) (8 + 16 mg), and CBD (16 mg). Cannabis‐related state satiety, appetitive cue salience (cannabis and food), and cannabis craving were assessed each day. Participants were genotyped for rs1049353, rs806378, and rs324420. Results indicated that CNR1 rs1049353 GG carriers showed increased state satiety after THC/THC + CBD administration in comparison with placebo and reduced the salience of appetitive cues after THC in comparison with CBD administration; A carriers did not vary on either of these measures indicative of a vulnerability to CUD. CNR1 rs806378 CC carriers showed greater salience to appetitive cues in comparison with T carriers, but there was no evidence for changes in state satiety. FAAH rs324420 A carriers showed greater bias to appetitive cues after THC, in comparison with CC carriers. FAAH CC carriers showed reduced bias after THC in comparison with CBD. No SNPs modulated craving. These findings identify candidate neurocognitive mechanisms through which endocannabinoid system genetics may influence vulnerability to CUD

Beyond Spheroids and Discs: Classifications of CANDELS Galaxy Structure at 1.4 < z < 2 via Principal Component Analysis

Important but rare and subtle processes driving galaxy morphology and star-formation may be missed by traditional spiral, elliptical, irregular or S\'ersic bulge/disk classifications. To overcome this limitation, we use a principal component analysis of non-parametric morphological indicators (concentration, asymmetry, Gini coefficient, $M_{20}$, multi-mode, intensity and deviation) measured at rest-frame $B$-band (corresponding to HST/WFC3 F125W at 1.4 $10^{10} M_{\odot}$) galaxy morphologies. Principal component analysis (PCA) quantifies the correlations between these morphological indicators and determines the relative importance of each. The first three principal components (PCs) capture $\sim$75 per cent of the variance inherent to our sample. We interpret the first principal component (PC) as bulge strength, the second PC as dominated by concentration and the third PC as dominated by asymmetry. Both PC1 and PC2 correlate with the visual appearance of a central bulge and predict galaxy quiescence. PC1 is a better predictor of quenching than stellar mass, as as good as other structural indicators (S\'ersic-n or compactness). We divide the PCA results into groups using an agglomerative hierarchical clustering method. Unlike S\'ersic, this classification scheme separates compact galaxies from larger, smooth proto-elliptical systems, and star-forming disk-dominated clumpy galaxies from star-forming bulge-dominated asymmetric galaxies. Distinguishing between these galaxy structural types in a quantitative manner is an important step towards understanding the connections between morphology, galaxy assembly and star-formation.Comment: 31 pages, 24 figures, accepted for publication in MNRA