Triglyceride-rich lipoprotein lipolysis products increase blood-brain barrier transfer coefficient and induce astrocyte lipid droplets and cell stress.

Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer's disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increase the BBB transfer coefficient (Ki = permeability × surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found upregulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced endoplasmic reticulum stress protein C/EBP homologous protein (CHOP), and the NF-κB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient (Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

Diagnosis and Control of a LPAI H5N8 Outbreak in a Japanese Quail (Coturnix coturnix japonica) Commercial Flock in the Central Valley of California.

In April 2014 an outbreak of low pathogenic avian influenza H5N8 North American genetic lineage was diagnosed in a commercial quail operation in Stanislaus County, California. Sudden increase in mortality prompted the submission of 20 Japanese quail hens (Coturnix c. japonica) to the California Animal Health and Food Safety Laboratory, Turlock Branch. Oropharyngeal and cloacal swabs tested positive for influenza A virus H5N8 by real-time reverse transcription-polymerase chain reaction. The virus was subsequently isolated. In vivo assay and sequencing of the hemagglutinin protein cleavage site classified the virus as a North American genetic lineage of low pathogenicity for chickens. Following the diagnosis, a rapid and coordinated response took place to contain the outbreak. The affected premise was depopulated, cleaned, and disinfected. Three areas from the affected premises-a 3 kilometer (km) radius (High Risk Zone), a 3-10 km area (Buffer Zone), and a 10-20 km (Surveillance Zone)-were established for avian influenza testing of commercial and noncommercial poultry operations. Surveillance testing and rapid control measures were successful in the control and eradication of the outbreak and revealed no area of spread of the virus from the index flock. This report describes the history, diagnosis, surveillance, and control measures applied to manage this outbreak.

Induction of ATF3 gene network by triglyceride-rich lipoprotein lipolysis products increases vascular apoptosis and inflammation.

OBJECTIVE: Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. APPROACH AND RESULTS: We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. CONCLUSIONS: This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Single-molecule quantification of lipotoxic expression of activating transcription factor 3.

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500 h(-1). After three hours we detected 33,090 ± 3491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ∼3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates.

Inflammatory dilated cardiomyopathy in Abcg5-deficient mice.

Dilated cardiomyopathy (DCM) in A/J mice homozygous for the spontaneous thrombocytopenia and cardiomyopathy (trac) mutation results from a single base pair change in the Abcg5 gene. A similar mutation in humans causes sitosterolemia with high plant sterol levels, hypercholesterolemia, and early onset atherosclerosis. Analyses of CD3+ and Mac-3+ cells and stainable collagen in hearts showed inflammation and myocyte degeneration in A/J-trac/trac mice beginning postweaning and progressed to marked dilative and fibrosing cardiomyopathy by 140 days. Transmission electron microscopy (TEM) demonstrated myocyte vacuoles consistent with swollen endoplasmic reticulum (ER). Myocytes with cytoplasmic glycogen and irregular actinomyosin filament bundles formed mature intercalated disks with normal myocytes suggesting myocyte repair. A/J-trac/trac mice fed lifelong phytosterol-free diets did not develop cardiomyopathy. BALB/cByJ-trac/trac mice had lesser inflammatory infiltrates and later onset DCM. BALB/cByJ-trac/trac mice changed from normal to phytosterol-free diets had lesser T cell infiltrates but persistent monocyte infiltrates and equivalent fibrosis to mice on normal diets. B- and T-cell-deficient BALB/cBy-Rag1(null) trac/trac mice fed normal diets did not develop inflammatory infiltrates or DCM. We conclude that the trac/trac mouse has many features of inflammatory DCM and that the reversibility of myocardial T cell infiltration provides a novel model for investigating the progression of myocardial fibrosis.

Evolutionary design of explainable algorithms for biomedical image segmentation.

An unresolved issue in contemporary biomedicine is the overwhelming number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. However, these frameworks require large human-annotated datasets for training and the resulting "black box" models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming-based computational strategy that generates fully transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets. This Few-Shot Learning method confers tremendous flexibility, speed, and functionality to this approach. We then deploy Kartezio to solve a series of semantic and instance segmentation problems, and demonstrate its utility across diverse images ranging from multiplexed tissue histopathology images to high resolution microscopy images. While the flexibility, robustness and practical utility of Kartezio make this fully explicable evolutionary designer a potential game-changer in the field of biomedical image processing, Kartezio remains complementary and potentially auxiliary to mainstream Deep Learning approaches.

Seasonal influences on CAPs exposures: differential responses in platelet activation, serum cytokines and xenobiotic gene expression.

Increasing evidence suggests a role for a systemic pro-coagulant state in the pathogenesis of cardiac dysfunction subsequent to inhalation of airborne particulate matter (PM). We evaluated platelet activation, systemic cytokines and pulmonary gene expression in mice exposed to concentrated ambient particulate matter (CAPs) in the summer of 2008 (S08) and winter of 2009 (W09) from the San Joaquin Valley of California, a region with severe PM pollution episodes. Additionally, we characterized the PM from both exposures including organic compounds, metals, and polycyclic aromatic hydrocarbons. Mice were exposed to an average of 39.01 µg/m(3) of CAPs in the winter and 21.7 µg/m3 CAPs in the summer, in a size range less than 2.5 µm for 6 h/day for 5 days per week for 2 weeks. Platelets were analyzed by flow cytometry for relative size, shape, CD41, P-selectin and lysosomal associated membrane protein-1 (LAMP-1) expression. Platelets from W09 CAPs-exposed animals had a greater response to thrombin stimulation than platelets from S08 CAPs-exposed animals. Serum cytokines were analyzed by bead based immunologic assays. W09 CAPs-exposed mice had elevations in IL-2, MIP-1α, and TNFα. Laser capture microdissection (LCM) of pulmonary vasculature, parenchyma and airways all showed increases in CYP1a1 gene expression. Pulmonary vasculature showed increased expression of ICAM-1 and Nox-2. Our findings demonstrate that W09 CAPs exposure generated a greater systemic pro-inflammatory and pro-coagulant response to inhalation of environmentally derived fine and ultrafine PM. Changes in platelet responsiveness to agonists, seen in both exposures, strongly suggests a role for platelet activation in the cardiovascular and respiratory effects of particulate air pollution.

Comparative gene responses to collected ambient particles in vitro: endothelial responses.

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 µg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

Inhibition of miR-99a-5p prevents allergen-driven airway exacerbations without compromising type-2 memory responses in the intestine following helminth infection.

Acute exacerbations (AE) of asthma, remain one of the biggest concerns for patients living with asthma. As such, identifying the causes, the molecular mechanisms involved and new therapeutic interventions to prevent AE is a high priority. Immunity to intestinal helminths involves the reactivation of type-2 immune responses leading to smooth muscle contraction and mucus hypersecretion-physiological processes very similar to acute exacerbations in the airways following allergen exposure. In this study, we employed a murine model of intestinal helminth infection, using Heligmosomoides polygyrus, to identify miRNAs during active expulsion, as a system for the identification of miRNAs that may contribute to AE in the airways. Concomitant with type-2 immunity and expulsion of H. polygyrus, we identified miR-99a-5p, miR-148a-3p and miR-155-5p that were differentially regulated. Systemic inhibition of these miRNAs, alone or in combination, had minimal impact on expulsion of H. polygyrus, but inhibition of miR-99a-5p or miR-155-5p significantly reduced house dust mite (HDM)-driven acute inflammation, modelling human acute exacerbations. Immunological, pathological and transcriptional analysis identified that miR-155-5p or miR-99a-5p contribute significantly to HDM-driven AE and that transient inhibition of these miRNAs may provide relief from allergen-driven AE, without compromising anti-helminth immunity in the gut.

Experimental re-infected cats do not transmit SARS-CoV-2.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Viral RNA and antigen detected in the respiratory tract of the primary SARS-CoV-2 infected cats at early DPCs were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against re-infection.

Differential cellular responses to protein adducts of naphthoquinone and monocrotaline pyrrole.

Protein-xenobiotic adducts are byproducts of xenobiotic metabolism. While there is a correlation between protein adduction and target organ toxicity, a cause and effect relationship is not often clear. Naphthoquinone (NQ) and monocrotaline pyrrole (MCTP) are two pneumotoxic electrophiles that form covalent adducts with a similar select group of proteins rich in reactive thiols. In this study, we treated human pulmonary artery endothelial cells (HPAEC) with NQ, MCTP, or preformed NQ or MCTP adducts to the protein galectin-1 (gal-1) and examined indicators of reactive oxygen species (ROS) oxidative injury, markers of apoptosis (caspase-3 and annexin V), and gene responses of cellular stress. ROS production was assayed fluorescently using CM-H(2)DCFDA. NQ adducts to gal-1 (NQ-gal) produced 183% more intracellular ROS than gal-1 alone (p < 0.0001). Caspase-3 activity and annexin V staining of phosphatidylserine were used to assess apoptotic activity in treated cells. HPAEC exposed to MCTP-gal had increases in both caspase-3 activation and membrane translocation of annexin V relative to gal-1 alone (p < 0.0001). Direct application of NQ produced significantly more ROS and induced significant caspase-3 activation, whereas MCTP did not. Human bronchial epithelial cells were also exposed to MCTP-gal and found to have significant increases in both caspase-3 activation and annexin V staining in comparison to that of gal-1 (p < 0.05). Western blot analysis showed that both NQ and MCTP significantly induced the Nrf2 mediated stress response pathway despite differences in ROS generation. ER stress was not induced by either adducts or parent compounds as seen by quantitative RT-PCR, but HOX-1 expression was significantly induced by NQ-gal and MCTP alone. Electrophile adduction to gal-1 produces different cytotoxic effects specific to each reactive intermediate.

CONN SYNDROME PRESENTING AS CENTRAL RETINAL VEIN OCCLUSION.

PURPOSE: To describe a case of central retinal vein occlusion in a young patient presenting with symptomatic malignant hypertension because of Conn syndrome. METHODS: Single interventional case report. RESULTS: A 44-year-old man presented with a 1-day history of headache and vision loss in his right eye on a background of malignant hypertension. He was diagnosed with right central retinal vein occlusion. Further investigation of his malignant hypertension revealed Conn syndrome because of an aldosterone-secreting adenoma in the left adrenal gland. CONCLUSION: This is the first reported case of Conn syndrome presenting as central retinal vein occlusion. Conn syndrome should be suspected in young patients with central retinal vein occlusion because it is a common cause of hypertension and carries significant cardiovascular risk if left untreated.

Uptake and inflammatory effects of nanoparticles in a human vascular endothelial cell line.

The mechanisms governing the correlation between exposure to nanoparticles and the increased incidence of cardiovascular disease remain unknown. Nanoparticles appear to cross the pulmonary epithelial barrier into the bloodstream, raising the possibility of direct contact with the vascular endothelium. Because endothelial inflammation is critical for the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs*) to nanoparticles induces an inflammatory response and that this response depends on the composition of the particles. To test this hypothesis, we incubated HAECs for 1 to 8 hours with different concentrations (0.001-50 microg/mL) of iron oxide (Fe2O3), yttrium oxide (Y2O3), cerium oxide (CeO2), and zinc oxide (ZnO) nanoparticles. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we subsequently measured messenger RNA (mRNA) levels of three markers of inflammation: intercellular cell adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1). The particles were well characterized in terms of size, surface area, composition, and crystal structure. To determine the interactions of nanoparticles with HAECs, we used inductively coupled plasma-mass spectrometry (ICP-MS) to measure the concentration of internalized particles. Our data indicate that the delivery of nanoparticles to the HAEC surface and their uptake within the cells correlate directly with the concentration of particles in the cell culture medium. Transmission electron microscopy (TEM) revealed that the Fe2O3, Y2O3, and ZnO nanoparticles are internalized by HAECs and are often found within intracellular vesicles (the CeO2 particles were not imaged). Fe2O3 nanoparticles did not provoke an inflammatory response in HAECs at any of the concentrations tested, CeO2 particles elicited no response at low concentrations and a weak response above 10 microg/mL, and Y2O3 and ZnO nanoparticles elicited a pronounced inflammatory response above a threshold concentration of 10 microg/mL. We used fluorescent markers to identify the production of reactive oxygen species (ROS) in cells; the results showed that Y2O3 and ZnO particles at the highest concentrations may lead to the production of ROS. At the highest concentration, ZnO nanoparticles caused significant loss of cell adherence. These results demonstrate that inflammation in HAECs after acute exposure to metal oxide nanoparticles depends on the concentration and composition of the particles.

Triboelectric charging and dissipation characteristics of the Maquet Quadrox-D membrane oxygenator.

Triboelectric charging is commonly detected during cardiopulmonary bypass in circuits using roller pumps and PVC tubing. Dissipation of this charging is needed to prevent a spontaneous discharge from occurring. We evaluated the ability of the Quadrox-D (Maquet) to effectively remove the electrostatic charge accumulation, with and without a heater/cooler connection (H/C). A Quadrox-D oxygenator was evaluated using a Stockert SIII pump head, Cincinnati subzero H/E, and a custom adult extracorporeal membrane oxygenation tubing pack with 1/2" PVC raceway. The circuit was primed and evaluated for triboelectric accumulation with and without H/C use. The results showed a linear relationship between increasing pump flow and static charge buildup when an H/C was not applied. The calculated r2 value was .95. Incorporation of the H/C effectively eliminated charge accumulation. Increasing pump speed increases the amount of static charge created without the use of an H/C. Incorporation of an H/C effectively eliminates charge build-up in the Quadrox-D and is recommended while priming the circuit.

Mitochondrial oxidative stress-induced transcript variants of ATF3 mediate lipotoxic brain microvascular injury.

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

Smad signaling in the rat model of monocrotaline pulmonary hypertension.

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.

Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size.

Our lab previously demonstrated that triglyceride-rich lipoprotein (TGRL) lipolysis products induce lipid droplet formation and pro-inflammatory gene expression in monocytes. We hypothesized that the inhibition of perilipin 2 expression in THP-1 monocytes would reduce lipid droplet formation and suppress pro-inflammatory gene expression induced by TGRL lipolysis products. In the current study, we use microarray analysis to identify gene expression altered by TGRL lipolysis products in THP-1 monocytes. We confirmed the expression of selected genes by quantitative reverse transcription PCR and characterized lipid droplet formation in these cells after exposure to TGRL lipolysis products. Using siRNA inhibition of perilipin 2 expression, we examined the role of perilipin 2 in the response of THP-1 monocytes to TGRL lipolysis products. We found that perilipin 2 siRNA increased the intracellular triglyceride content, increased the size of lipid droplets, and reduced pro-atherogenic and pro-inflammatory gene expression. We saw a reduction of serum/glucocorticoid kinase 1, v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian), chemokine (C-C motif) ligand 3, and interleukin 8 gene expression induced by TGRL lipolysis products. This study supports previous findings that reduction of perilipin 2 expression is protective against atherogenesis, while finding an unexpected increase in lipid droplet size with reduced perilipin 2 expression.

Ultrafine Particulate Matter Combined With Ozone Exacerbates Lung Injury in Mature Adult Rats With Cardiovascular Disease.

Particulate matter (PM) and ozone (O3) are dominant air pollutants that contribute to development and exacerbation of multiple cardiopulmonary diseases. Mature adults with cardiovascular disease (CVD) are particularly susceptible to air pollution-related cardiopulmonary morbidities and mortalities. The aim was to investigate the biologic potency of ultrafine particulate matter (UFPM) combined with O3 in the lungs of mature adult normotensive and spontaneously hypertensive (SH) Wistar-Kyoto rats. Conscious, mature adult male normal Wistar-Kyoto (NW) and SH rats were exposed to one of the following atmospheres: filtered air (FA); UFPM (∼ 250 µg/m3); O3 (1.0 ppm); or UFPM + O3 (∼ 250 µg/m3 + 1.0 ppm) combined for 6 h, followed by an 8 h FA recovery period. Lung sections were evaluated for lesions in the large airways, terminal bronchiolar/alveolar duct regions, alveolar parenchyma, and vasculature. NW and SH rats were similarly affected by the combined-pollutant exposure, displaying severe injury in both large and small airways. SH rats were particularly susceptible to O3 exposure, exhibiting increased injury scores in terminal bronchioles and epithelial degeneration in large airways. UFPM-exposure groups had minimal histologic changes. The chemical composition of UFPM was altered by the addition of O3, indicating that ozonolysis promoted compound degradation. O3 increased the biologic potency of UFPM, resulting in greater lung injury following exposure. Pathologic manifestations of CVD may confer susceptibility to air pollution by impairing normal lung defenses and responses to exposure.

Reduced cognitive function, increased blood-brain-barrier transport and inflammatory responses, and altered brain metabolites in LDLr -/-and C57BL/6 mice fed a western diet.

Recent work suggests that diet affects brain metabolism thereby impacting cognitive function. Our objective was to determine if a western diet altered brain metabolism, increased blood-brain barrier (BBB) transport and inflammation, and induced cognitive impairment in C57BL/6 (WT) mice and low-density lipoprotein receptor null (LDLr -/-) mice, a model of hyperlipidemia and cognitive decline. We show that a western diet and LDLr -/- moderately influence cognitive processes as assessed by Y-maze and radial arm water maze. Also, western diet significantly increased BBB transport, as well as microvessel factor VIII in LDLr -/- and microglia IBA1 staining in WT, both indicators of activation and neuroinflammation. Interestingly, LDLr -/- mice had a significant increase in 18F- fluorodeoxyglucose uptake irrespective of diet and brain 1H-magnetic resonance spectroscopy showed increased lactate and lipid moieties. Metabolic assessments of whole mouse brain by GC/MS and LC/MS/MS showed that a western diet altered brain TCA cycle and ß-oxidation intermediates, levels of amino acids, and complex lipid levels and elevated proinflammatory lipid mediators. Our study reveals that the western diet has multiple impacts on brain metabolism, physiology, and altered cognitive function that likely manifest via multiple cellular pathways.