Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wildtype (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naive and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naive WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-beta (TGF-beta) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/ in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions

Fibrotic remodeling of colonic tunica muscularis is associated with vascular network activation in ulcerative colitis

Intestinal fibrosis in inflammatory bowel disease is a dynamic, multifactorial process, which involve multiple cell types and interconnected events [1]. Angiogenesis is a hallmark of active gut disease and closely related to fibrogenetic processes. Endothelial cells and pericytes of neovessels have been found to be able to differentiate into fibroblasts and can be considered good candidates for fibrogenesis also in the intestinal tract [2]. This study was aimed to study whether the fibrotic processes occurring within the tunica muscularis of UC patients are associated with vascular remodeling. Full-thickness left colonic samples were obtained from patients with established, severe and pharmacologically unresponsive UC, who underwent bowel resection. Routine histology, histochemistry and immunohistochemistry were conducted in paraffin cross-sections. Collagen and elastic fiber distribution was evaluated within the tunica muscularis by both histochemical and immunohistochemical assays. The vascular network pattern was analyzed by revealing the expression of CD31, CD105 and nestin by immunofluorescence applied to laser confocal microscopy. A significant increase in collagen fibers and a decrease in elastin content were detected in the tonaca muscularis of UC inflamed colon, as compared with controls. In particular, enhanced collagen deposition were found at level of the longitudinal muscle and circular muscle layer, and in perivascular spaces. By contrast, elastic fiber pattern was significantly decreased throughout the whole muscle compartment. Increased blood vessel density was observed in the colonic tunica muscularis of UC samples compared with samples from healthy control individuals. In particular, the neovessels of inflamed colon showed the activation of both endothelial cells and pericytes, which overexpressed CD105 and nestin, respectively. A significant vascular remodelling (i.e., angiogenesis, endothelial proliferation and pericyte activation) has been observed in the fibrotic tunica muscularis of colon from UC patients. On the basis of the present findings, it is possible to argue that cells of newly formed vessels within the tunica muscularis may contribute to the UC-associated fibrosis by cell transition to mesenchymal phenotype

Essays in macroeconomics

Esta tesis consta de tres capítulos, cada uno de los cuales analiza temas relevantes en el campo de la Macroeconomía. La investigación realizada en los tres capítulos se basa en la metodología de los modelos dinámicos de equilibrio general. Los fundamentos microeconómicos se encuentran en el núcleo de este tipo de modelos, en los que los agentes forman sus expectativas sobre el futuro de manera racional. Esta metodología ha demostrado ser una herramienta poderosa para abordar un gran conjunto de problemas económicos. El primer capítulo, ``Risk Aversion, Entrepreneurship and Wealth Distribution'', explora el efecto de la heterogeneidad en la aversión al riesgo sobre el emprendimiento, el comportamiento del ahorro y la distribución de la riqueza. La distribución de la riqueza es uno de los temas más debatidos no solo en la política contemporánea, sino también en los medios de comunicación y en ámbitos académicos. En particular, la gran concentración de riqueza en la parte superior de la distribución, que se observa en muchas economías avanzadas, se ha situado en el centro del debate. La relevancia de este problema no se ha visto socavada a pesar de la dificultad que supone estimar los niveles de riqueza acumulada en manos de los hogares más ricos. Apoyado por razones empíricas, el marco teórico para analizar la distribución de la riqueza debe incluir tanto el emprendimiento como la heterogeneidad de las preferencias. Con este objetivo, en el primer capítulo se desarrolla un modelo de elección ocupacional con agentes heterogéneos en el que los hogares varían en su aversión al riesgo, se enfrentan a perturbaciones idiosincrásicas no asegurables, tienen restricciones financieras y deben decidir si convertirse en trabajadores o en emprendedores. Esta decisión se basa en una combinación de la aversión al riesgo individual, las habilidades específicas en cada ocupación y los niveles de riqueza acumulados. Como resultado se obtiene que la aversión al riesgo tiene un efecto directo e indirecto en la decisión de ahorro y en la ocupación de los agentes. Por un lado, una mayor aversión al riesgo disuade a los agentes de convertirse en emprendedores y, por lo tanto, los ahorros destinados a superar las restricciones financieras son menores. Por otro lado, una mayor aversión al riesgo también conduce a un mayor ahorro por motivo de precaución que, indirectamente, atenúa las restricciones financieras y hace que la opción de convertirse en emprendedor sea más atractiva. En el modelo desarrollado, el último efecto domina, y los agentes con mayor aversión al riesgo acumulan mayores niveles de riqueza y también se convierten en emprendedores. La contribución más importante de este modelo es que permite desentrañar los efectos que la aversión al riesgo y las fricciones financieras tienen sobre el emprendimiento, que es un factor clave para explicar la desigualdad en la acumulación de la riqueza. El segundo capítulo se titula ``Long-term business relationships, bargaining and monetary policy'' y es un trabajo conjunto con Mirko Abbritti y Tommaso Trani. Su motivación se deriva de la creciente literatura empírica que documenta la importancia de las relaciones comerciales a largo plazo y los procesos de negociación sobre la rigidez de los precios y las dinámicas empresariales. Este artículo introduce relaciones comerciales entre empresas (B2B) a largo plazo y procesos de negociación de precios en un modelo monetario de equilibrio general dinámico estocástico (DSGE) estándar. El modelo se basa en dos suposiciones. Primero, tanto los productores mayoristas como los minoristas necesitan gastar recursos para formar nuevas relaciones comerciales. Segundo, una vez que se forma una relación comercial, el precio se establece en una negociación bilateral entre las empresas. El modelo proporciona un marco riguroso para estudiar el efecto de las relaciones comerciales a largo plazo y los procesos de negociación sobre la política monetaria y la dinámica del ciclo económico. Además, se demuestra que estas relaciones comerciales reducen tanto el papel de asignación de los precios intermedios como los efectos reales de las perturbaciones de la política monetaria. También encontramos que el modelo hace un gran trabajo al replicar los segundos momentos y las correlaciones cruzadas de los datos, y que mejora con respecto al modelo de referencia Neo Keynesiano en explicar dichos estadísticos. El tercer y último capítulo de esta tesis, ``On Staggered Prices and Optimal Inflation'', es coautoreado con uno de mis directores, Miguel Casares. En este artículo se calcula la tasa de inflación óptima en estado estacionario bajo dos especificaciones de rigidez de precios diferentes, Calvo (1983) y Taylor (1980), en un modelo con competencia monopolística. Encontramos que la tasa óptima de inflación en estado estacionario es siempre positiva. Este resultado es robusto a los cambios en el grado de rigidez de los precios. En ambos casos con rigidez de precios, la tasa de inflación óptima es aproximadamente igual al cociente entre la tasa de descuento y la elasticidad de Dixit-Stiglitz. Para calibraciones estándar, el coste de bienestar de la inflación es cuantitativamente pequeño, pero significativamente más alto si los precios son rígidos a la Calvo que si son rígidos a la Taylor.Fundación Banco Sabadell, Fundación Bancaria Caja Navarra y Universidad Pública de NavarraPrograma de Doctorado en Economía, Empresa y Derecho (RD 99/2011)Ekonomiako, Enpresako eta Zuzenbideko Doktoretza Programa (ED 99/2011

Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood-brain barrier: Therapeutic avenues

: Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mimicking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood-brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of control in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and functional integrity of the BBB results in enhanced transmigration of leukocytes across the BBB that could contribute to an initiation of a neuroinflammatory response through activation of microglia. Using a humanized in vitro model of the BBB and T2DM patient post-mortem brains, we show the translatable applicability of our results. We find a leaky BBB phenotype in T2DM patients can be attributed to a loss of junctional proteins through changes in inflammatory mediators and MMP/TIMP levels, resulting in increased leukocyte extravasation into the brain parenchyma. We further investigated therapeutic avenues to reduce and restore the BBB damage caused by HFHS-feeding. Pharmacological treatment with recombinant annexin A1 (hrANXA1) or reversion from a high-fat high-sugar diet to a control chow diet (dietary intervention), attenuated T2DM development, reduced inflammation, and restored BBB integrity in the animals. Given the rising incidence of diabetes worldwide, understanding metabolic-disease-associated brain microvessel damage is vital and the proposed therapeutic avenues could help alleviate the burden of these diseases

Vascular Wall-Resident CD44+ Multipotent Stem Cells Give Rise to Pericytes and Smooth Muscle Cells and Contribute to New Vessel Maturation

Here, we identify CD44(+)CD90(+)CD73(+)CD34(−)CD45(−) cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs). VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes

Characterization of TEM1/endosialin in human and murine brain tumors

<p>Abstract</p> <p>Background</p> <p><it>TEM1/endosialin </it>is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of <it>TEM1/endosialin </it>in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models.</p> <p>Methods</p> <p><it>In situ </it>hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize <it>TEM1/endosialin </it>expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays. Changes in <it>TEM1/endosialin </it>expression in response to pro-angiogenic conditions were assessed in human endothelial cells grown <it>in vitro</it>. Intracranial U87MG glioblastoma (GBM) xenografts were analyzed in nude <it>TEM1/endosialin </it>knockout (KO) and wildtype (WT) mice.</p> <p>Results</p> <p><it>TEM1/endosialin </it>was upregulated in primary and metastatic human brain tumors, where it localized primarily to the tumor vasculature and a subset of tumor stromal cells. Analysis of 275 arrayed grade II-IV astrocytomas demonstrated <it>TEM1/endosialin </it>expression in 79% of tumors. Robust <it>TEM1/endosialin </it>expression occurred in 31% of glioblastomas (grade IV astroctyomas). <it>TEM1/endosialin </it>expression was inversely correlated with patient age. TEM1/endosialin showed limited co-localization with CD31, αSMA and fibronectin in clinical specimens. <it>In vitro</it>, <it>TEM1/endosialin </it>was upregulated in human endothelial cells cultured in matrigel. Vascular <it>Tem1/endosialin </it>was induced in intracranial U87MG GBM xenografts grown in mice. <it>Tem1/endosialin </it>KO vs WT mice demonstrated equivalent survival and tumor growth when implanted with intracranial GBM xenografts, although <it>Tem1/endosialin </it>KO tumors were significantly more vascular than the WT counterparts.</p> <p>Conclusion</p> <p><it>TEM1/endosialin </it>was induced in the vasculature of high-grade brain tumors where its expression was inversely correlated with patient age. Although lack of <it>TEM1/endosialin </it>did not suppress growth of intracranial GBM xenografts, it did increase tumor vascularity. The cellular localization of <it>TEM1/endosialin </it>and its expression profile in primary and metastatic brain tumors support efforts to therapeutically target this protein, potentially via antibody mediated drug delivery strategies.</p

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance

Theories of schizophrenia: a genetic-inflammatory-vascular synthesis

BACKGROUND: Schizophrenia, a relatively common psychiatric syndrome, affects virtually all brain functions yet has eluded explanation for more than 100 years. Whether by developmental and/or degenerative processes, abnormalities of neurons and their synaptic connections have been the recent focus of attention. However, our inability to fathom the pathophysiology of schizophrenia forces us to challenge our theoretical models and beliefs. A search for a more satisfying model to explain aspects of schizophrenia uncovers clues pointing to genetically mediated CNS microvascular inflammatory disease. DISCUSSION: A vascular component to a theory of schizophrenia posits that the physiologic abnormalities leading to illness involve disruption of the exquisitely precise regulation of the delivery of energy and oxygen required for normal brain function. The theory further proposes that abnormalities of CNS metabolism arise because genetically modulated inflammatory reactions damage the microvascular system of the brain in reaction to environmental agents, including infections, hypoxia, and physical trauma. Damage may accumulate with repeated exposure to triggering agents resulting in exacerbation and deterioration, or healing with their removal. There are clear examples of genetic polymorphisms in inflammatory regulators leading to exaggerated inflammatory responses. There is also ample evidence that inflammatory vascular disease of the brain can lead to psychosis, often waxing and waning, and exhibiting a fluctuating course, as seen in schizophrenia. Disturbances of CNS blood flow have repeatedly been observed in people with schizophrenia using old and new technologies. To account for the myriad of behavioral and other curious findings in schizophrenia such as minor physical anomalies, or reported decreased rates of rheumatoid arthritis and highly visible nail fold capillaries, we would have to evoke a process that is systemic such as the vascular and immune/inflammatory systems. SUMMARY: A vascular-inflammatory theory of schizophrenia brings together environmental and genetic factors in a way that can explain the diversity of symptoms and outcomes observed. If these ideas are confirmed, they would lead in new directions for treatments or preventions by avoiding inducers of inflammation or by way of inflammatory modulating agents, thus preventing exaggerated inflammation and consequent triggering of a psychotic episode in genetically predisposed persons

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16–27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17–P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60–70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4th ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity