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Objective.Analyze the performance of electrical impedance tomography (EIT) in an innovative porcine model of subclinical hemorrhage and investigate associations between EIT and hemodynamic trends.Approach. Twenty-five swine were bled at slow rates to create an extended period of subclinical hemorrhage during which the animal's heart rate (HR) and blood pressure (BP) remained stable from before hemodynamic deterioration, where stable was defined as <15% decrease in BP and <20% increase in HR-i.e.hemorrhages were hidden from standard vital signs of HR and BP. Continuous vital signs, photo-plethysmography, and continuous non-invasive EIT data were recorded and analyzed with the objective of developing an improved means of detecting subclinical hemorrhage-ideally as early as possible.Main results. Best area-under-the-curve (AUC) values from comparing bleed to no-bleed epochs were 0.96 at a 80 ml bleed (â¼15.4 min) using an EIT-data-based metric and 0.79 at a 120 ml bleed (â¼23.1 min) from invasively measured BP-i.e.the EIT-data-based metric achieved higher AUCs at earlier points compared to standard clinical metrics without requiring image reconstructions.Significance.In this clinically relevant porcine model of subclinical hemorrhage, EIT appears to be superior to standard clinical metrics in early detection of hemorrhage.
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Hemorragia , Tomografia , Animais , Impedância Elétrica , Hemorragia/diagnóstico por imagem , Processamento de Imagem Assistida por Computador , Suínos , Tomografia/métodos , Tomografia Computadorizada por Raios XRESUMO
AIM: Pseudo-pulseless electrical activity (pseudo-PEA) is a global hypotensive ischemic state with retained coordinated myocardial contractile activity and an organized ECG with no clinically detectable pulses. The role of standard external chest compressions (CPR) and its associated intrinsic hemodynamics remains unclear in the setting of pseudo-PEA. We undertook an experimental trial to compare epinephrine alone versus epinephrine with CPR in the treatment of pseudo-PEA. METHODS: Using a porcine model of hypoxic pseudo-PEA, we randomized 12 Yorkshire male swine to resuscitation with epinephrine only (control) (0.0015 mg/kg) versus epinephrine plus standard CPR (intervention). Animals who achieved return of spontaneous circulation (ROSC) were stabilized, fully recovered to hemodynamic and respiratory baseline, and rearrested up to 6 times. Primary outcome was ROSC defined as a sustained systolic blood pressure (SBP) of 60 mmHg for 2 min. Secondary outcomes included time to ROSC, coronary perfusion pressure (CoPP), and end-tidal carbon dioxide (ETCO2). RESULTS: Among 47 events of pseudo-PEA in 12 animals, we observed significantly higher proportion of ROSC when treatment included CPR (14/21 - 67%) compared to epinephrine alone (4/26 - 15%) (p = 0.0007). CoPP, aortic pressures and ETCO2 were significantly higher, and right atrial pressures were lower in the intervention group. CONCLUSIONS: In a swine model of hypoxia-induced pseudo-PEA, epinephrine plus CPR was associated with improved intra-arrest hemodynamics and higher probability of ROSC. Thus, epinephrine plus CPR may be superior to epinephrine alone in the treatment of patients with pseudo-PEA.
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BACKGROUND: Pseudo-pulseless electrical activity (pseudo-PEA) is a lifeless form of profound cardiac shock characterized by measurable cardiac mechanical activity without clinically detectable pulses. Pseudo-PEA may constitute up to 40% of reported cases of cardiac arrest. Resuscitation from pseudo-PEA is often associated with hypotension refractory to catecholamine pressors. We hypothesized that this post-resuscitation state may be associated with hypocalcemic hypotension responsive to intravenous calcium. METHODS: Using pre-existing data from our hypoxic swine pseudo-PEA model, we measured blood pressure, hemodynamics, and electrolytes. Physiological data were analyzed on a heartbeat by heartbeat basis. The midpoint of the calcium response was defined using change of curvature feature detection. Hemodynamic parameters were shifted such that the value at the midpoint was equal to zero. RESULTS: In 9 animals with refractory hypotension, we administered 37 boluses of intravenous calcium in the dosage range of 5-20 mg. Comparisons were made between the average values in the time period 40-37 s before the midpoint and 35-40 s after the midpoint. Of the 37 administered boluses, 34 manifested a change in the blood pressure, with mean aortic pressure, systolic and diastolic pressures all increasing post bolus administration. CONCLUSIONS: Administration of intravenous calcium may be associated with a pressor-like response in refractory hypotension after resuscitation from pseudo-PEA. Relative ionized hypocalcemia may cause hypotension after resuscitation from pseudo-PEA. Therapy with intravenous calcium should be further investigated in this setting.
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Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.
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Materiais Biocompatíveis , Biopolímeros/química , Celulose , Nanopartículas/química , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Materiais Biocompatíveis/química , Bovinos , Celulose/química , Quitina/química , Colágeno/química , Reação a Corpo Estranho , Liofilização , Congelamento , Sistema Imunitário , Teste de Materiais , Camundongos , Camundongos Endogâmicos C3H , Modelos Teóricos , Polímeros/química , Porosidade , RegeneraçãoRESUMO
Current FDA-approved permanent female sterilization procedures are invasive and/or require the implantation of non-biodegradable materials. These techniques pose risks and complications, such as device migration, fracture, and tubal perforation. We propose a safe, non-invasive biodegradable tissue scaffold to effectively occlude the Fallopian tubes within 30 days of implantation. Specifically, the Fallopian tubes are mechanically de-epithelialized, and a tissue scaffold is placed into each tube. It is anticipated that this procedure can be performed in less than 30 minutes by an experienced obstetrics and gynaecology practitioner. Advantages of this method include the use of a fully bio-resorbable polymer, low costs, lower risks, and the lack of general anaesthesia. The scaffold devices are freeze-cast allowing for the custom-design of structural, mechanical, and chemical cues through material composition, processing parameters, and functionalization. The performance of the biomaterial and de-epithelialization procedure was tested in an in vivo rat uterine horn model. The scaffold response and tissue-biomaterial interactions were characterized microscopically post-implantation. Overall, the study resulted in the successful fabrication of resilient, easy-to-handle devices with an anisotropic scaffold architecture that encouraged rapid bio-integration through notable angiogenesis, cell infiltration, and native collagen deposition. Successful tubal occlusion was demonstrated at 30 days, revealing the great promise of a sterilization biomaterial.
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A novel freeze-cast porous chitosan conduit for peripheral nerve repair with highly-aligned, double layered porosity, which provides the ideal mechanical and chemical properties was designed, manufactured, and assessed in vivo. Efficacies of the conduit and the control inverted nerve autograft were evaluated in bridging 10-mm Lewis rat sciatic nerve gap at 12 weeks post-implantation. Biocompatibility and regenerative efficacy of the porous chitosan conduit were evaluated through the histomorphometric analysis of longitudinal and transverse sections. The porous chitosan conduit was found to have promising regenerative characteristics, promoting the desired neovascularization, and axonal ingrowth and alignment through a combination of structural, mechanical and chemical cues.
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The presence and benefit of a radiation therapy-associated immune reaction is of great interest as the overall interest in cancer immunotherapy expands. The pathological assessment of irradiated tumors rarely demonstrates consistent immune or inflammatory response. More recent information, primarily associated with the "abscopal effect", suggests a subtle radiation-based systemic immune response may be more common and have more therapeutic potential than previously believed. However, to be of consistent value, the immune stimulatory potential of radiation therapy (RT) will clearly need to be supported by combination with other immunotherapy efforts. In this study, using a spontaneous canine oral melanoma model, we have assessed the efficacy and tumor immunopathology of two nanotechnology-based immune adjuvants combined with RT. The immune adjuvants were administered intratumorally, in an approach termed "in situ vaccination", that puts immunostimulatory reagents into a recognized tumor and utilizes the endogenous antigens in the tumor as the antigens in the antigen/adjuvant combination that constitutes a vaccine. The radiation treatment consisted of a local 6 × 6 Gy tumor regimen given over a 12 day period. The immune adjuvants were a plant-based virus-like nanoparticle (VLP) and a 110 nm diameter magnetic iron oxide nanoparticle (mNPH) that was activated with an alternating magnetic field (AMF) to produce moderate heat (43 °C/60 min). The RT was used alone or combined with one or both adjuvants. The VLP (4 × 200 µg) and mNPH (2 × 7.5 mg/gram tumor) were delivered intratumorally respectively during the RT regimen. All patients received a diagnostic biopsy and CT-based 3-D radiation treatment plan prior to initiating therapy. Patients were assessed clinically 14-21 days post-treatment, monthly for 3 months following treatment, and bimonthly, thereafter. Immunohistopathologic assessment of the tumors was performed before and 14-21 days following treatment. Results suggest that addition of VLPs and/or mNPH to a hypofractionated radiation regimen increases the immune cell infiltration in the tumor, extends the tumor control interval, and has important systemic therapeutic potential.
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Imunoterapia/métodos , Melanoma/tratamento farmacológico , Melanoma/terapia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/terapia , Nanopartículas/química , Nanotecnologia/métodos , Animais , Antineoplásicos/uso terapêutico , Terapia Combinada , Modelos Animais de Doenças , Cães , Feminino , Campos MagnéticosRESUMO
It has recently been shown that cancer treatments such as radiation and hyperthermia, which have conventionally been viewed to have modest immune based anti-cancer effects, may, if used appropriately stimulate a significant and potentially effective local and systemic anti-cancer immune effect (abscopal effect) and improved prognosis. Using eight spontaneous canine cancers (2 oral melanoma, 3 oral amelioblastomas and 1 carcinomas), we have shown that hypofractionated radiation (6 x 6 Gy) and/or magnetic nanoparticle hyperthermia (2 X 43°C / 45 minutes) and/or an immunogenic virus-like nanoparticle (VLP, 2 x 200 µg) are capable of delivering a highly effective cancer treatment that includes an immunogenic component. Two tumors received all three therapeutic modalities, one tumor received radiation and hyperthermia, two tumors received radiation and VLP, and three tumors received only mNP hyperthermia. The treatment regimen is conducted over a 14-day period. All patients tolerated the treatments without complication and have had local and distant tumor responses that significantly exceed responses observed following conventional therapy (surgery and/or radiation). The results suggest that both hypofractionated radiation and hyperthermia have effective immune responses that are enhanced by the intratumoral VLP treatment. Molecular data from these tumors suggest Heat Shock Protein (HSP) 70/90, calreticulin and CD47 are targets that can be exploited to enhance the local and systemic (abscopal effect) immune potential of radiation and hyperthermia cancer treatment.
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Cerebral tissue oxygenation (oxygen tension, pO2) is a critical parameter that is closely linked to brain metabolism, function, and pathophysiology. In this work, we have used electron paramagnetic resonance oximetry with a deep-tissue multi-site oxygen-sensing probe, called implantable resonator, to monitor temporal changes in cerebral pO2 simultaneously at four sites in a rabbit model of ischemic stroke induced by embolic clot. The pO2 values in healthy brain were not significantly different among the four sites measured over a period of 4 weeks. During exposure to 15% O2 (hypoxia), a sudden and significant decrease in pO2 was observed in all four sites. On the other hand, brief exposure to breathing carbogen gas (95% O2 + 5% CO2) showed a significant increase in the cerebral pO2 from baseline value. During ischemic stroke, induced by embolic clot in the left brain, a significant decline in the pO2 of the left cortex (ischemic core) was observed without any change in the contralateral sites. While the pO2 in the non-infarct regions returned to baseline at 24-h post-stroke, pO2 in the infarct core was consistently lower compared to the baseline and other regions of the brain. The results demonstrated that electron paramagnetic resonance oximetry with the implantable resonator can repeatedly and simultaneously report temporal changes in cerebral pO2 at multiple sites. This oximetry approach can be used to develop interventions to rescue hypoxic/ischemic tissue by modulating cerebral pO2 during hypoxic and stroke injury.
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Encéfalo/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Oximetria , Oxigênio/análise , Tromboembolia/patologia , Angiografia , Animais , Encéfalo/patologia , Artérias Carótidas/diagnóstico por imagem , Feminino , Hiperóxia , Hipóxia , Coelhos , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Tromboembolia/complicações , Tromboembolia/metabolismoRESUMO
BACKGROUND: Endoscopy is useful in assessing conduit ischemia and anastomotic leaks after esophagectomy but poses a theoretical threat of anastomotic disruption. We used a porcine model to evaluate the safety of endoscopy after esophagectomy. METHODS: We performed esophagectomies in 10 live pigs and performed endoscopy with progressive air insufflation and continuous intraluminal pressure monitoring. We stopped insufflation when the intraluminal pressure reached a plateau. We assessed the integrity of the conduit and anastomosis via endoscopy. We also performed pulse oximetry of the stomach and Doppler velocimetry of the right gastroepiploic artery on 5 live pigs to study the effects of endoscopic gastric insufflation. RESULTS: With gentle air insufflation, there was no measurable increase in intraluminal pressure, disruption of the conduit or anastomosis, or significant gastric distension. With progressive insufflation, the intraluminal pressure reached a plateau at a maximum of 8.7 ± 2.1 cm H2O (95% confidence interval, 7.2-10.2). At this plateau, air leaked retrograde via the mouth, which prevented further gastric distension. There were no significant changes in oxyhemoglobin saturation along various regions in the stomach even with maximal insufflation sustained for 10 minutes. There was a momentary reduction in gastroepiploic flow from 12.0 ± 1.0 [95% confidence interval, 10.8-13.2] mL/min/100 g to 9.6 ± 1.5 [95% confidence interval, 7.8-11.4] mL/min/100 g immediately after maximal insufflation, but flow recovered to 11 ± 1.3 [9.6, 12.8] mL/min/100 g after 10 minutes of sustained insufflation. CONCLUSIONS: Endoscopy after esophagectomy with gentle or maximal air insufflation results in safe endoluminal pressures and minimal disturbance of blood flow and oxygenation.
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Esofagectomia , Esôfago/cirurgia , Anastomose Cirúrgica , Animais , Endoscopia , Metais , Stents , Estômago/cirurgia , SuínosRESUMO
PURPOSE: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. PROCEDURES: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. RESULTS: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. CONCLUSION: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.
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Receptores ErbB/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/toxicidade , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/toxicidade , Proteína Estafilocócica A/farmacologia , Proteína Estafilocócica A/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Fluorescência , Humanos , Injeções , Luz , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/administração & dosagem , Proteína Estafilocócica A/administração & dosagem , Distribuição Tecidual/efeitos dos fármacosRESUMO
Recent failures in hysteroscopic female sterilization procedures have brought into question the implantation of non-resorbable metal devices into the fallopian tubes due to long-term risks such as migration, fragmentation, and tubal perforation. The goal of this study is to assess whether a porous, biodegradable implant can be deposited into the fallopian tube lumen with or without a local mild heat treatment to generate a safe and permanent fallopian tube occlusion/sterilization event. The technologies investigated included freeze-cast collagen-based scaffolds and magnetic nanoparticle (MNP) based scaffolds. In vitro assessment of iron oxide MNP-based scaffolds was performed to determine the absorption rate density (ARD); subsequent computational modeling quantified the thermal in vivo steady state temperature as a function of tubal radius for treatment planning. For collagen-based scaffolds, in vivo testing was performed to study the biocompatibility in a mouse flank model, followed by implantation into an in vivo anestrus feline uterine horn (animal model for the fallopian tube). Biological responses were studied histopathologically. Uterine horn patency was assessed via radiographic imaging. Preliminary studies suggest the MNP-impregnated scaffold and a safe, noninvasive AMF excitation field have potential to generate a sufficient focal fallopian tube thermal dose to create a fibrotic healing event and ultimately, permanent tubal occlusion.
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BACKGROUND: Barium esophagograms have poor sensitivity in detecting leaks. We hypothesized that heating barium would decrease viscosity, facilitate extravasation, and enhance its sensitivity in detecting esophageal leaks. METHODS: We characterized the viscosity of barium at increasing temperatures. We measured the radiopacity of barium at 25°C and 50°C. We determined the smallest diameter defect in esophagus that barium can detect by perforating a porcine esophageal segment with angiocatheters of various diameters, injecting barium at 25°C, and observing extravasation of contrast. We repeated this with barium heated to 30°C, 40°C, 50°C, and 70°C. To determine the ability of barium to detect a staple line leak, we perforated a stapled esophageal segment by air insufflation, injected barium at different temperatures, and monitored extravasation. We used Visipaque, a water-soluble contrast agent, for comparison in all experiments. RESULTS: The viscosity of barium decreased with increasing temperature. The radiopacity of barium did not change with increasing temperature and was higher than that of Visipaque (P < 0.001). The size of the smallest detectable leak decreased from 2.1 mm with barium at 25°C to 1.3 mm at 40°C and 1.1 mm with Visipaque (P < 0.0001). The sensitivity of staple line leak detection increased from 0% for barium at 25°C to 80% (P = 0.02) with barium at 40°C. There was no significant difference in sensitivity between barium at 40°C and Visipaque. CONCLUSIONS: Barium warmed to 40°C offers the best sensitivity of esophageal leak detection without compromising radiopacity. Barium at 40°C may be the optimum choice for swallow study to detect esophageal leaks.
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Sulfato de Bário , Perfuração Esofágica/diagnóstico por imagem , Animais , Temperatura Alta , Modelos Animais , Radiografia , Distribuição Aleatória , Suínos , ViscosidadeRESUMO
SU-8 negative photoresist is a high tensile strength polymer that has been used for a number of biomedical applications that include cell encapsulation and neuronal probes. Chemically, SU-8 comprises, among other components, an epoxy based monomer and antimony salts, the latter being a potential source of cytotoxicity. We report on the in vitro and in vivo evaluation of SU-8 biocompatibility based on leachates from various solvents, at varying temperatures and pH, and upon subcutaneous implantation of SU-8 substrates in mice. MTT cell viability assay did not exhibit any cytotoxic effects from the leachates. The hemolytic activity of SU-8 is comparable to that of FDA approved implant materials such as silicone elastomer, Buna-S and medical steel. In vivo histocompatibility study in mice indicates a muted immune response to subcutaneous SU-8 implants.
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Materiais Biocompatíveis/farmacologia , Compostos de Epóxi/farmacologia , Teste de Materiais , Polímeros/farmacologia , Ágar , Animais , Antimônio/análise , Sobrevivência Celular/efeitos dos fármacos , Compostos de Epóxi/química , Hemólise/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Implantes Experimentais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/efeitos dos fármacos , Polímeros/química , Implantação de Prótese , Ratos , Espectrofotometria Atômica , Coloração e RotulagemRESUMO
BACKGROUND: Electrical impedance tomography (EIT) is a method that can render continuous graphical cross-sectional images of the brain's electrical properties. Because these properties can be altered by variations in water content, shifts in sodium concentration, bleeding, and mass deformation, EIT has promise as a sensitive instrument for head injury monitoring to improve early recognition of deterioration and to observe the benefits of therapeutic intervention. This study presents a swine model of head injury used to determine the detection capabilities of an inexpensive bedside EIT monitoring system with a novel intracranial pressure (ICP)/EIT electrode combination sensor on induced intraparenchymal mass effect, intraparenchymal hemorrhage, and cessation of brain blood flow. Conductivity difference images are shown in conjunction with ICP data, confirming the effects. METHODS: Eight domestic piglets (3-4 weeks of age, mean 10 kg), under general anesthesia, were subjected to 4 injuries: induced intraparenchymal mass effect using an inflated, and later, deflated 0.15-mL Fogarty catheter; hemorrhage by intraparenchymal injection of 1-mL arterial blood; and ischemia/infarction by euthanasia. EIT and ICP data were recorded 10 minutes before inducing the injury until 10 minutes after injury. Continuous EIT and ICP monitoring were facilitated by a ring of circumferentially disposed cranial Ag/AgCl electrodes and 1 intraparenchymal ICP/EIT sensor electrode combination. Data were recorded at 100 Hz. Two-dimensional tomographic conductivity difference (Δσ) images, rendered using data before and after an injury, were displayed in real time on an axial circular mesh. Regions of interest (ROI) within the images were automatically selected as the upper or lower 5% of conductivity data depending on the nature of the injury. Mean Δσ within the ROIs and background were statistically analyzed. ROI Δσ was compared with the background Δσ after an injury event using an unpaired, unequal variance t test. Conductivity change within an ROI after injury was likewise compared with the same ROI before the injury making use of unpaired t tests with unequal variance. RESULTS: Eight animal subjects were studied, each undergoing 4 injury events including euthanasia. Changes in conductivity due to injury showed expected pathophysiologic effects in an ROI identified within the middle of the left hemisphere; this localization is reasonable given the actual site of injury (left hemisphere) and spatial warping associated with estimating a 3-dimensional conductivity distribution in 2-dimensional space. Results are shown as mean ± 1 SD. When averaged across all 8 animals, balloon inflation caused the mean Δσ within the ROI to shift by -11.4 ± 10.9 mS/m; balloon deflation by +9.4 ± 8.8 mS/m; blood injection by +19.5 ± 11.5 mS/m; death by -12.6 ± 13.2 mS/m. All induced injuries were detectable to statistical significance (P < 0.0001). CONCLUSION: This study confirms that the bedside EIT system with ICP/EIT combination sensor can detect induced trauma. Such a technique may hold promise for further research in the monitoring and management of traumatically brain-injured individuals.
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Traumatismos Craniocerebrais/diagnóstico por imagem , Traumatismos Craniocerebrais/fisiopatologia , Modelos Animais de Doenças , Tomografia Computadorizada por Raios X/métodos , Animais , Impedância Elétrica , Eletrodos , Pressão Intracraniana/fisiologia , Monitorização Fisiológica , SuínosRESUMO
Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser(806), which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx(-/-) mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function.
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Angiopoietina-1/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Junções Intercelulares/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Permeabilidade Capilar , Proteínas de Transporte/metabolismo , Cães , Forminas , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Volume Sistólico , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
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Proteínas Sanguíneas/metabolismo , Capilares/fisiologia , Capilares/ultraestrutura , Permeabilidade Capilar , Proteínas de Transporte/metabolismo , Endotélio Vascular/fisiologia , Endotélio Vascular/ultraestrutura , Proteínas de Membrana/metabolismo , Animais , Proteínas de Transporte/genética , Cavéolas/fisiologia , Membrana Celular/metabolismo , Endotélio Vascular/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Enteropatias Perdedoras de Proteínas/fisiopatologiaRESUMO
BACKGROUND: Obesity is a growing worldwide problem with genetic and environmental causes, and it is an underlying basis for many diseases. Studies have shown that the toxicant-activated aryl hydrocarbon receptor (AHR) may disrupt fat metabolism and contribute to obesity. The AHR is a nuclear receptor/transcription factor that is best known for responding to environmental toxicant exposures to induce a battery of xenobiotic-metabolizing genes. OBJECTIVES: The intent of the work reported here was to test more directly the role of the AHR in obesity and fat metabolism in lieu of exogenous toxicants. METHODS: We used two congenic mouse models that differ at the Ahr gene and encode AHRs with a 10-fold difference in signaling activity. The two mouse strains were fed either a low-fat (regular) diet or a high-fat (Western) diet. RESULTS: The Western diet differentially affected body size, body fat:body mass ratios, liver size and liver metabolism, and liver mRNA and miRNA profiles. The regular diet had no significant differential effects. CONCLUSIONS: The results suggest that the AHR plays a large and broad role in obesity and associated complications, and importantly, may provide a simple and effective therapeutic strategy to combat obesity, heart disease, and other obesity-associated illnesses.
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Gorduras na Dieta/metabolismo , Fígado/metabolismo , Obesidade/genética , Receptores de Hidrocarboneto Arílico/genética , Tecido Adiposo/metabolismo , Animais , Peso Corporal , Dieta , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Modelos Animais , Obesidade/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Little is known of the environmental factors that initiate and promote disease. The aryl hydrocarbon receptor (AHR) is a key regulator of xenobiotic metabolism and plays a major role in gene/environment interactions. The AHR has also been demonstrated to carry out critical functions in development and disease. A qualitative investigation into the contribution by the AHR when stimulated to different levels of activity was undertaken to determine whether AHR-regulated gene/environment interactions are an underlying cause of cardiovascular disease. We used two congenic mouse models differing at the Ahr gene, which encodes AHRs with a 10-fold difference in signaling potencies. Benzo[a]pyrene (BaP), a pervasive environmental toxicant, atherogen, and potent agonist for the AHR, was used as the environmental agent for AHR activation. We tested the hypothesis that activation of the AHR of different signaling potencies by BaP would have differential effects on the physiology and pathology of the mouse cardiovascular system. We found that differential AHR signaling from an exposure to BaP caused lethality in mice with the low-affinity AHR, altered the growth rates of the body and several organs, induced atherosclerosis to a greater extent in mice with the high-affinity AHR, and had a huge impact on gene expression of the aorta. Our studies also demonstrated an endogenous role for AHR signaling in regulating heart size. We report a gene/environment interaction linking differential AHR signaling in the mouse to altered aorta gene expression profiles, changes in body and organ growth rates, and atherosclerosis.