Mental chronometry using latency-resolved functional MRI

Vascular responses to neural activity are exploited as the basis of a number of brain imaging techniques. The vascular response is thought to be too slow to resolve the temporal sequence of events involved in cognitive tasks, and hence, imaging studies of mental chronometry have relied on techniques such as the evoked potential. Using rapid functional MRI (fMRI) of single trials of two simple behavioral tasks, we demonstrate that while the microvascular response to the onset of neural activity is delayed consistently by several seconds, the relative timing between the onset of the fMRI responses in different brain areas appears preserved. We examined a number of parameters that characterize the fMRI response and determined that its onset time is best defined by the inflection point from the resting baseline. We have found that fMRI onset latencies determined in this manner correlate well with independently measurable parameters of the tasks such as reaction time or stimulus presentation time and can be used to determine the origin of processing delays during cognitive or perceptual tasks with a temporal accuracy of tens of milliseconds and spatial resolution of millimeters.

Nuclear localization of prostaglandin E2 receptors

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1 and α2β1 integrins—through induction of mRNAs encoding the α1 and α2 subunits. In contrast, VEGF did not induce increased expression of the α3β1 integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1 and α2β1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs. In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1 and α2β1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1 and α2β1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules >20 Å and that transcytosis is an N-ethylmaleimide–sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein–lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.

Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.

The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression.

Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.

Expressão gênica de metaloproteinases e de seus reguladores em neoplasias mieloproliferativas: associação com biomarcadores de angiogênese e status mutacional

As neoplasias mieloproliferativas (NMPs) BCR-ABL1 negativas compreendem a mielofibrose primária (PMF), trombocitemia essencial (TE) e a policitemia vera (PV). A patogênese e progressão dessas NMPs não estão completamente elucidadas. As metaloproteinases de matriz (MMPs) degradam a matriz extracelular, ativando citocinas e fatores de crescimento que, por sua vez, participam da tumorigênese e angiogênese. O objetivo deste estudo foi avaliar a relação da expressão gênica das MMPs, TIMPs, HIF1-α e SPARC com os marcadores angiogênicos bFGF e VEGFA em pacientes com MF e TE, considerando o status mutacional; bem como avaliar a regulação desses genes em camundongos submetidos à hipóxia, e em modelos HIF1-α(-/-) e VHL(-/-). Foram incluídos 21 pacientes com MF, 21 com MF pós-TE, 6 com MF pós-PV, 23 com TE e 78 indivíduos controle. As análises realizadas foram: dosagem sérica e expressão de RNAm de MMP2, MMP9, TIMP1, TIMP2 e SPARC, hemograma, determinação da proteína C reativa ultrassensível, determinação das concentrações de VEGFA e bFGF e avaliação das mutações nos genes JAK2, cMPL e CALR. A avaliação da densidade microvascular da medula óssea foi feita em 30 dos pacientes incluídos. Os pacientes com MFP, MFPTE e TE apresentaram maior expressão de MMP2, SPARC, TIMP1, TIMP2 e bFGF quando comparados aos seus controles (P<0,05), enquanto MMP9 foi mais expressa nos pacientes com MFPTE e TE (P= 0,011 e P=0,047, respectivamente). Os pacientes com TE apresentaram maior expressão de HIF1-α e VEGFA em relação ao grupo controle (P<0,05). Pacientes com MF JAK2V617F positivos apresentaram maiores concentrações de MMP9, TIMP2, bFGF e VEGFA quando comparados aos pacientes portadores de mutações na CALR (P<0,05). Os pacientes com TE JAK2V617F positivos apresentaram maiores concentrações de MMP2 e TIMP2 (P=0,049 e P=0,020, respectivamente). As concentrações das proteínas estudadas não apresentaram correlação com a carga alélica de JAK2V617F e nem com a densidade microvascular da medula óssea. Células de medula óssea de camundongos submetidos à hipóxia apresentaram maior expressão de MMP2 e TIMP1 comparados aos camundongos em normóxia. Camundongos VHL(-/-) apresentaram aumento na expressão dos genes MMP2, MMP9, TIMP1, TIMP2 e VEGFA. Diferentemente, embriões HIF1-α(-/-) não foram considerados um bom modelo para este estudo devido ao envolvimento das MMPs na embriogênese/organogênese. Frente aos resultados encontrados, pode-se sugerir que a maior expressão de MMP2, SPARC e de bFGF estão associadas às NMPs. A mutação JAK2V617F foi associada a maiores concentrações de MMPs, TIMP2 VEGFA e bFGF. HIF1-α foi mais expresso na PV e na TE, sugerindo uma possível regulação da expressão das MMPs e TIMPs nessas doenças.

Compresiones neurovasculares de la fosa craneal posterior en la neuralgia del trigémino. Estudio anatomoclínico

La neuralgia del trigémino (NT) es un síndrome doloroso facial caracterizado por un dolor neuropático paroxístico irradiado al territorio sensitivo del nervio trigémino. A lo largo de las últimas décadas la creciente evidencia experimental y clínica ha contribuido a establecer la teoría de una compresión neurovascular (CNV) del nervio trigémino como causa fundamental de este trastorno. Esta teoría apoya el tratamiento quirúrgico de la NT mediante una descompresión microvascular (DMV). Actualmente la DMV es aceptada como la primera opción de tratamiento quirúrgico de la NT refractaria a tratamiento médico. Pese a la cantidad de trabajos que analizan la utilidad de la RM preoperatoria con secuencias de alta resolución volumétricas potenciadas en T2 (RM 3DT2), no hay unanimidad respecto a la precisión de estas secuencias de RM para el diagnóstico y la caracterización de una posible CNV en pacientes con NT refractaria candidatos a tratamiento mediante DMV...

Transferrina e colagénio tipo IV : novos marcadores glomerulares de lesão renal

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Rôle des cellules endothéliales dans l’immunité innée précoce induite lors d’infections par des coronavirus murins

Les cellules endothéliales (EC) constituent une première barrière physique à la dissémination de virus pléiotropiques circulant par voie hématogène mais leur contribution à la défense innée anti-virale est peu connue. Des dysfonctions des EC de la barrière hémato-encéphalique (BMEC) et des sinusoïdes hépatiques (LSEC) ont été rapportées dans des neuropathologies et des hépatites aiguës ou chroniques d’origine virale, suggérant que des atteintes à leur intégrité contribuent à la pathogenèse. Les sérotypes de coronavirus de l’hépatite murine (MHV), se différenciant par leur capacité à induire des hépatites et des maladies neurologiques de sévérité variable et/ou leur tropisme pour les EC, représentent des modèles viraux privilégiés pour déterminer les conséquences de l’infection des EC sur la pathogenèse virale. Lors d’infection par voie hématogène, le sérotype MHV3, le plus virulent des MHV, induit une hépatite fulminante, caractérisée par une réponse inflammatoire sévère, et des lésions neurologiques secondaires alors que le sérotype moins virulent, MHV-A59, induit une hépatite modérée sans atteintes secondaires du système nerveux central (SNC). Par ailleurs, le sérotype MHV3, à la différence du MHV-A59, démontre une capacité à stimuler la production de cytokines par la voie TLR2. Les variants atténués du MHV3, les virus 51.6-MHV3 et YAC-MHV3, sont caractérisés par un faible tropisme pour les LSEC et induisent respectivement une hépatite modérée et subclinique. Compte tenu de l’importance des LSEC dans le maintien de la tolérance hépatique et de l’élimination des pathogènes circulants, il a été postulé que la sévérité de l’hépatite et de la réponse inflammatoire lors d’infections par les MHV est associée à la réplication virale et à l’altération des propriétés tolérogéniques et vasculaires des LSEC. Les désordres inflammatoires hépatiques pourraient résulter d’une activation différentielle du TLR2, plutôt que des autres TLR et des hélicases, selon les sérotypes. D’autre part, compte tenu du rôle des BMEC dans la prévention des infections du SNC, il a été postulé que l’invasion cérébrale secondaire par les coronavirus est reliée à l’infection des BMEC et le bris subséquent de la barrière hémato-encéphalique (BHE). À l’aide d’infections in vivo et in vitro par les différents sérotypes MHV, chez des souris ou des cultures de BMEC et de LSEC, nous avons démontré, d’une part, que l’infection in vitro des LSEC par le sétotype MHV3, à la différence des variants 51.6- et YAC-MHV3, altérait la production du facteur vasodilatant NO et renversait leur phénotype tolérogénique en favorisant la production de cytokines et de chimiokines inflammatoires. Ces dysfonctions se traduisaient in vivo par une réponse inflammatoire incontrôlée et une dérégulation du recrutement intrahépatique de leucocytes, favorisant la réplication virale et les dommages hépatiques. Nous avons aussi démontré, à l’aide de souris TLR2 KO et de LSEC dont l’expression du TLR2 a été abrogée par des siRNA, que la sévérité de l’hépatite et de la réponse inflammatoire induite par le sérotype MHV3, dépendait en partie de l’induction et de l’activation préférentielle du TLR2 par le virus dans le foie. D’autre part, la sévérité de la réplication virale au foie et des désordres dans le recrutement leucocytaire intrahépatique induits par le MHV3, et non par le MHV-A59 et le 51.6-MHV3, corrélaient avec une invasion virale subséquente du SNC, au niveau de la BHE. Nous avons démontré que l’invasion cérébrale du MHV3 était associée à une infection productive des BMEC et l’altération subséquente des protéines de jonctions serrées occludine, VE-cadhérine et ZO-1 se traduisant par une augmentation de la perméabilité de la BHE et l’entrée consécutive du virus dans le cerveau. Dans l’ensemble, les résultats de cette étude mettent en lumière l’importance du maintien de l’intégrité structurale et fonctionnelle des LSEC et des BMEC lors d’infections virales aigües par des MHV afin de limiter les dommages hépatiques associés à l’induction d’une réponse inflammatoire exagérée et de prévenir le passage des virus au cerveau suite à une dissémination par voie hématogène. Ils révèlent en outre un nouveau rôle aggravant pour le TLR2 dans l’évolution de l’hépatite virale aigüe ouvrant la voie à de nouvelles avenues thérapeutiques visant à moduler l’activité inflammatoire du TLR2.

Lung Structure and the Intrinsic Challenges of Gas Exchange

Structural and functional complexities of the mammalian lung evolved to meet a unique set of challenges, namely, the provision of efficient delivery of inspired air to all lung units within a confined thoracic space, to build a large gas exchange surface associated with minimal barrier thickness and a microvascular network to accommodate the entire right ventricular cardiac output while withstanding cyclic mechanical stresses that increase several folds from rest to exercise. Intricate regulatory mechanisms at every level ensure that the dynamic capacities of ventilation, perfusion, diffusion, and chemical binding to hemoglobin are commensurate with usual metabolic demands and periodic extreme needs for activity and survival. This article reviews the structural design of mammalian and human lung, its functional challenges, limitations, and potential for adaptation. We discuss (i) the evolutionary origin of alveolar lungs and its advantages and compromises, (ii) structural determinants of alveolar gas exchange, including architecture of conducting bronchovascular trees that converge in gas exchange units, (iii) the challenges of matching ventilation, perfusion, and diffusion and tissue-erythrocyte and thoracopulmonary interactions. The notion of erythrocytes as an integral component of the gas exchanger is emphasized. We further discuss the signals, sources, and limits of structural plasticity of the lung in alveolar hypoxia and following a loss of lung units, and the promise and caveats of interventions aimed at augmenting endogenous adaptive responses. Our objective is to understand how individual components are matched at multiple levels to optimize organ function in the face of physiological demands or pathological constraints. © 2016 American Physiological Society. Compr Physiol 6:827-895, 2016.