The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

La néphropathie diabétique est l’une des complications les plus fréquentes chez les patients diabétiques à long terme, et est la première cause du besoin de dialyse. Les lésions glomérulaires semblent jouer un rôle clé dans le développement de la néphropathie diabétique. L’épaississement de la membrane basale glomérulaire, l’hypertrophie des cellules glomérulaires et la perte de podocytes font partie des principaux changements pathologiques survenant au cours de la néphropathie diabétique et peuvent conduire à une protéinurie. Il a été suggéré que le dysfonctionnement endothélial joue un rôle important dans la pathogenèse des lésions glomérulaires au cours de la maladie rénale diabétique. En effet, l’altération de la fonction et de l’intégrité des cellules endothéliales glomérulaires est l’une des principales causes de la microalbuminurie observée dans l’insuffisance rénale diabétique précoce. Les lésions des cellules endothéliales glomérulaires peuvent endommager les podocytes et même induire une perte podocitaire ce qui aggrave d’avantage les liaisons des cellules endothéliales glomérulaires et ainsi de suite. Actuellement, les traitements de la néphropathie diabétique visent le contrôle de la glycémie et de la pression artérielle dans le but de maintenir un bon débit de filtration glomérulaire. Cependant, l’étude de traitements pouvant cibler les lésions endothéliales ou les interactions podocyte-cellule endothéliales, qui jouent un rôle essentiel dans la progression de la maladie rénale diabétique, est nécessaire. Les traitements ciblant l’endothélium glomérulaire pourraient offrir des avantages thérapeutiques pour la néphropathie diabétique. En effet, des facteurs anti-angiogéniques tels que les inhibiteurs du VEGF pourraient prévenir les lésions rénales et les altérations glomérulaires sur des modèles de souris diabétiques. Cependant, d’autres données ont montré que des injections d’inhibiteurs du VEGF pouvaient être néfastes pour les cellules endothéliales et podocytaires. Ainsi, de nouvelles molécules ciblant l’endothélium vasculaire pourraient améliorer le pronostic et la qualité de vie chez les patients présentant une insuffisance rénale diabétique à un stade précoce. Nous avons précédemment montré que Alk1, avec son ligand BMP9, joue un rôle important dans le maintien de l’intégrité vasculaire chez les animaux diabétiques. En effet, 4 la perte de signalisation d’Alk1 chez les animaux diabétiques conduit à la dissociation des jonctions vasculaires et à une augmentation des fuites vasculaires dans la rétine. Compte tenu de son rôle dans le maintien de la quiescence et de l’intégrité de l’endothélium, nous avons évalué les effets de la surpression d’Alk1 sur l’intégrité de l’endothélium glomérulaire et la fonction rénale chez la souris diabétique. Nous avons utilisé des souris avec délétion conditionnelle de Alk1 dans l’endothélium (Alk1ΔEC) pour évaluer le rôle de Alk1 dans la filtration glomérulaire chez des souris diabétiques induits par le STZ. Les souris ont été euthanasiées quatre mois après le début du diabète et des analyses sérologiques et urinaires ont été effectuées, ainsi que des études immunohistochimiques. Nous avons démontré que l’haplo-insuffisance d’Alk1 aggrave la microalbuminurie et induit une perte de podocytes chez des souris diabétiques. De plus, une augmentation significative de l’apoptose glomérulaire a été observée chez les souris Alk1ΔEC hétérozygotes diabétiques. L’analyse de souris Alk1ΔEC homozygotes non diabétiques a également révélé une perte importante de cellules endothéliales glomérulaires. Ensemble, ces données suggèrent que la signalisation du récepteur Alk1 joue un rôle essentiel dans le maintien des cellules endothéliales glomérulaires et participe au maintien de l’intégrité glomérulaire à travers un mécanisme de podocyte-endothelial cross-talk. / Diabetic kidney disease one of the most frequent microvascular long-term complications in diabetic patients and is the first cause for the need for dialysis. The glomerular damage seems to play a key role in the development of diabetic nephropathy. Thickening of the glomerular basement membrane, glomerular cell hypertrophy, and podocyte loss is among the main pathological changes occurring during diabetic nephropathy and can lead to proteinuria. Endothelial dysfunction has been suggested to play an important role in the pathogenesis of glomerular damage during diabetic kidney disease. Indeed, alteration of the glomerular endothelial cell function and integrity is a leading cause of microalbuminuria observed in early diabetic kidney disease. Injury to glomerular endothelial cells may lead to podocyte damage, while podocyte loss further exacerbates glomerular endothelial cell injury, forming a vicious cycle. Currently, therapies in diabetic nephropathy are focusing on glycemia control and adequate arterial pressure levels in order to maintain an adequate glomerular filtration rate. However, the study of some treatments that may target endothelial lesions or podocyte-endothelial cell interactions, which play a vital role in the progression of diabetic kidney disease is necessary. It has been suggested that antiangiogenic treatments for diabetic kidney disease could provide therapeutic benefits. Indeed, anti-angiogenic factors such as VEGF inhibitors have been demonstrated to suppress renal damage and glomerular alterations in a diabetic mouse model. However, some other data have shown that anti-VEGF injections could be detrimental for podocytes and endothelial cells. Thus, new molecules targeting the vascular endothelium could possibly improve prognosis and quality of life in patients with early stages of diabetic kidney disease. We have previously shown that Alk1, along with its ligand BMP9, plays an important function to maintain vascular integrity in diabetic animals. Loss of Alk1 signaling in diabetic animals led to dissociation of vascular junctions and increased vascular leakage. Given its role in the maintenance of endothelial quiescence and integrity, we evaluated the effects of Alk1 suppression on kidney integrity and renal function in diabetic mice. 6 We used mice with conditional deletion of Alk1 in the endothelium (Alk1ΔEC) to evaluate the role of Alk1 in glomerular filtration in STZ-induced diabetic mice. Mice were euthanized four months after the onset of diabetes and urine, and serological analyzes were performed, along with immunohistochemical studies. We demonstrated that Alk1 haploinsufficiency worsens microalbuminuria and induces podocyte loss. Furthermore, a significant increase in glomerular apoptosis was observed in Alk1ΔEC mice. Analysis of homozygous Alk1ΔEC mice also revealed a significant loss of glomerular endothelial cells. Together, these data suggest that Alk1/BMP9 signaling plays a critical role in the maintenance of glomerular endothelial cells and has important functions to maintain glomerular integrity through a crosstalk podocyte-endothelial mechanism.

Indiana University-Purdue University Indianapolis (IUPUI) / Adenosine is widely thought to elicit coronary vasodilation and attenuate smooth muscle cell (SMC) proliferation, thereby providing cardioprotection. We cloned the porcine adenosine A1 receptor (A1R) subtype and found that it paradoxically stimulated proliferation of cultured coronary SMC by the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathways, thus suggesting A1R dysregulation could play a role in coronary artery disease (CAD), restenosis, and regulation of coronary blood flow (CBF). We utilized the Ossabaw swine model of metabolic syndrome (MetS) to test the hypothesis that A1R activation contributes to development of CAD, in-stent stenosis, and CBF regulation. Swine were fed standard chow (Lean) or excess calorie atherogenic diet for over 20 weeks, which elicited MetS characteristics and coronary atherosclerosis compared to Lean. We observed increased A1R in native CAD in MetS, which was reversed by exercise training, and upregulation of A1R expression and A1R-ERK1/2 activation in an in vitro organ culture model of CAD. Intracoronary stent deployment followed by different durations of recovery showed A1R upregulation occurred before maximal in-stent stenosis in vi vivo. More importantly, selective A1R antagonism with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX)-eluting stents decreased coronary ERK1/2 activation and reduced in-stent stenosis comparable to Taxus® (paclitaxel-eluting stents). A1R antagonism potentiated vasodilatory effects of some vasodilators other than adenosine in porcine coronary microcirculation under basal conditions. Short-term exercise training around stenting prevented stent-induced microvascular dysfunction and attenuated native atheroma in the genetically lean Yucatan swine. Conclusions: A1R upregulation and activation contributes to coronary in-stent stenosis in vivo in MetS, plays a role in the development of coronary atherosclerosis in vitro, and might involve in CBF dysregulation in dyslipidemia and stenting. Exercise training decreased A1R expression in atherosclerosis, reduced native atheroma, and prevented stent-induced microvascular dysfunction. Selective pharmacological antagonism of A1R holds promise for treatment of CAD.

No description available.

Made available in DSpace on 2014-08-20T14:37:57Z (GMT). No. of bitstreams: 1 dissertacao_matheus_folgearin_silveira.pdf: 274999 bytes, checksum: 17765fa39ad1a46f221283537ac0adae (MD5) Previous issue date: 2009-05-20 / Soft tissue sarcomas are mesenchymal origin neoplasms collectively classified according to histological characteristics and biological behaviour similiarities. Various neoplasms are included in this major group, as fibrosarcoma, hemangiosarcoma, peripheral nerve sheath tumor, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomiosarcoma, malignant fibrous histiocytoma, synovial cell sarcoma and undifferentiated sarcoma. The microvessel density measure has been applied to investigate tumor angiogenesis in many neoplasms. The objectives of this study were to evaluate de microvessel density in canine and feline soft tissue sarcomas and compare the general vessel measurement area and intense vascular areas hot spot. Those data were compared to usually applied prognostic factors like mitotic index, necroses presence and amount and cellular differentiation. Soft tissue sarcomas were collected in Laboratório Regional de Diagnóstico da Universidade Federal de Pelotas from 1978 to 2008 among canine and feline necropsies and biopsies. In a total of 1668 neoplasms cases during this period, 100 were soft tissue sarcomas, 87 canine and 13 feline. Sex prevalence was not observed in the analyzed species. The major prevalence in canine were hemangiosarcomas (n=34) and fibrosarcomas (n=20), being the mongrel dogs (n=35) most. The large breeds presented major frequence (n=22), followed by medium size (n=16) and small sized breeds (n=9). . In feline population, there were not accentuated prevalence, exciding hemangiosarcomas (n=4) and fibrosarcomas (n=4). In those 100 cases, 39 paraffin blocks were obtained, being 36 canine and 3 feline, mostly mongrel specimens, with 9,21(±2,99) medium age. The general vascular and hot spot areas were significantly correlated (r2=0,98; p<0,01). The mitotic index between areas were in those areas (r2=0,95, p<0,01). Hemangiosarcomas presented major vascular media in the techniques, followed by muscular sarcomas and fibrosarcomas (p<0,05). The microvessel density in soft tissue sarcomas do not present correlation to other prognostic factor usually applied. Hot spot areas can be utilized to determine the vascular degree and mitotic index in soft tissue sarcomas. / Os sarcomas de tecidos moles são neoplasmas de origem mesenquimal classificados coletivamente devido a características histológicas e comportamento biológico similares. Vários neoplasmas estão incluídos neste grande grupo, como fibrossarcoma, hemangiossarcoma, tumor de bainha de nervo periférico, mixossarcoma, lipossarcoma, leiomiossarcoma, rabdomiossarcoma, histiocitoma fibroso maligno, sarcoma sinovial e sarcoma indiferenciado. A mensuração da densidade vascular tem sido utilizada para investigar a angiogênese tumoral em diferentes neoplasmas. Este estudo teve como objetivo a determinação da microdensidade vascular de sarcomas de tecidos moles caninos e felinos através da comparação entre a mensuração geral vascular e áreas de intensa proliferação hot spot. Estes dados foram comparados a fatores prognósticos usualmente empregados, como índice mitótico, presença e quantidade de necrose e diferenciação celular. Os sarcomas de tecidos moles foram resgatados dos arquivos do Laboratório Regional de Diagnóstico da Universidade Federal de Pelotas entre 1978 a 2008 dentre necropsias e biópsias caninas e felinas. Num total de 1668 neoplasmas deste período, 100 eram sarcomas de tecidos moles, sendo 87 caninos e 13 felinos. Não se observou prevalência de sexo nas espécies analisadas. Em caninos, a maior prevalência foi de hemangiossarcomas (n=34) e fibrossarcomas (n=20), sendo os sem raça definida (n=35) os mais acometidos. Os animais de porte grande apresentaram maior freqüência (n=22), seguidos pelos de porte médio (n=16) e de porte pequeno (n=9). Em felinos não houve uma prevalência acentuada, destacando-se os hemangiossarcomas (n=4) e fibrossarcomas (n=4). Destes 100, obtiveram-se blocos de 39 casos, sendo 36 caninos e 3 felinos, em sua maioria sem raça definida nas duas espécies, com idade média observada foi de 9,21(±2,99 anos). A mensuração vascular geral e de áreas hot spot apresentaram correlação significativa (r2=0,98; p<0,01). O índice mitótico nas duas áreas observadas foram significativas (r2=0,95, p<0,01). Os hemangiossarcomas apresentaram maior média vascular nas duas técnicas, seguidos pelos sarcomas musculares e fibrossarcomas (p<0,05). Houve diferença entre os hemangiossarcomas e os fibrossarcomas, e os sarcomas musculares não diferiram desses (p<0,05). A microdensidade vascular em sarcomas de tecidos moles não apresentou correlação com outros fatores empregados usualmente. A visualização de campos hot spot pode ser utilizada para determinar o grau vascular e índice mitótico.

Microglia and brain-resident macrophages are the sentinel immune cells of the central nervous system (CNS), and are ideally situated to respond to any damage to the brain parenchyma or vasculature. Circulating leukocytes are generally excluded from the CNS environment under homeostatic conditions but can gain access to this region in diseases that disrupt immune system function and blood-brain barrier integrity. Although these diverse immune cells exhibit properties that may engender them to be well-suited to resolve microcirculatory insults, their relative contributions to the recanalization of capillary rupture in the cortex, known as cerebral microbleeds (CMBs), has yet to be described. CMBs are particularly concerning in conditions, such as diabetes mellitus (DM), in which these insults occur more frequently and potentially underlie the onset and progression of cognitive decline. Using in vivo 2-photon microscopy and confocal imaging, here I highlight the compromised repair of CMBs in a mouse model of type 1 DM and characterize the robust, heterogeneous macrophage response to these insults. Specifically, 20% of damaged capillaries were eliminated from the circulation in the diabetic cortex and chronic insulin treatment failed to prevent this microvascular loss. Administration of interferon-α or interferon-γ neutralizing antibodies to dampen inflammatory signalling, or dexamethasone to reduce global inflammation, also failed to improve repair rates of damaged microvessels in diabetic mice. In contrast, CMBs in nondiabetic mice repaired without exception. Interestingly, depletion of CNS macrophages using the colony stimulating factor-1 receptor antagonist PLX5622 resulted in microvascular elimination in nondiabetic mice. Given the robust depletion of brain macrophage populations with this treatment, at first these data suggested that these cells were necessary for microvascular repair since their elimination produced vessel loss. However, by parsing the data I identified that microvessels repaired in all cases where macrophages were not identified at the CMB; when CX3CR1+ aggregate was localized to the injury, ~20% of microvessels were eliminated. These findings show that CNS macrophages are not required for microvascular repair following CMB. Immunofluorescent co-labelling of various microglial and macrophage markers within the diabetic CMB milieu revealed a novel population of Mac2+/TMEM119- cells, distinct from homeostatic TMEM119+ microglia. These cells reliably localized to CMBs that failed to repair and rarely associated with vessels that recanalized; Mac2+/TMEM119- cells were not found within nondiabetic CMBs. Treatment of diabetic mice with clodronate liposomes (CLR) to deplete circulating phagocytic leukocytes prevented aggregation of Mac2+/TMEM119- cells to CMBs and improved capillary repair rates. The efficacy of CLR in excluding these cells from the CMB aggregate, coincident with eradication of monocytes from circulation, indicated that these cells likely arose from the periphery. In vivo 2-photon imaging revealed significant increases in lipofuscin at the site of diabetic CMBs relative to the nondiabetic context; other phagocytic markers including CD68 and TREM2 were also upregulated. Mac2+/TMEM119- cells showed elevated lipofuscin content relative to homeostatic microglia; their association with CMBs may thus signal an increase in phagocytosis that contributes to capillary pruning. Taken together, these data identify a novel Mac2+/TMEM119- macrophage associated with pathological microvascular elimination following CMB in the diabetic neocortex. These findings highlight the diversity of immune cell responses to CNS injury and provide insights into the cellular mechanisms of capillary pruning. Furthermore, these advances in our understanding of the regulation of microvascular elimination in the diabetic brain may have clinical implications for patients with DM as they provide evidence for putative adjuvant anti-inflammatory treatments, such as CLR, in mitigating cerebrovascular pathology. / Graduate / 2022-05-06

3 Abstract The doctor thesis is composed of two major studies, both of them focused on the mantle cell lymphoma (MCL). The first part deals with the verification of the prognostic influence of Mantle Cell Lymphoma International Prognostic Index (MIPI) and of the proliferative activity in 235 patients with MCL diagnosed in 1996-2008 in the Czech Republic. This population study was performed in the collaboration with the Czech Lymphoma Study Group. The clinical data of patients were completed in April 2012. The diagnosis of MCL was confirmed by our central histopathologic examination of pretherapeutic histological samples. The median overall survival (OS) was 47 months, median progression free survival (PFS) was 22 months. We demonstrated the influence of proliferative activity, MIPI and of the therapy type (intensive/non-intensive) on OS and PFS in univariate and multivariate analysis. Using univariate analysis we showed the prognostic influence of aggressive/other cytomorphological variants of MCL, nodal/extranodal localization of primary sample and also of the variants of MIPI - s-MIPI, MIPIb and a completely new variant of MIPI - combined MIPI. The prognostic influence of growth pattern and of the results of immunohistochemical reaction with CD23, CD5 and cyclin D1 antibodies were not confirmed. The other...

3 Abstract The doctor thesis is composed of two major studies, both of them focused on the mantle cell lymphoma (MCL). The first part deals with the verification of the prognostic influence of Mantle Cell Lymphoma International Prognostic Index (MIPI) and of the proliferative activity in 235 patients with MCL diagnosed in 1996-2008 in the Czech Republic. This population study was performed in the collaboration with the Czech Lymphoma Study Group. The clinical data of patients were completed in April 2012. The diagnosis of MCL was confirmed by our central histopathologic examination of pretherapeutic histological samples. The median overall survival (OS) was 47 months, median progression free survival (PFS) was 22 months. We demonstrated the influence of proliferative activity, MIPI and of the therapy type (intensive/non-intensive) on OS and PFS in univariate and multivariate analysis. Using univariate analysis we showed the prognostic influence of aggressive/other cytomorphological variants of MCL, nodal/extranodal localization of primary sample and also of the variants of MIPI - s-MIPI, MIPIb and a completely new variant of MIPI - combined MIPI. The prognostic influence of growth pattern and of the results of immunohistochemical reaction with CD23, CD5 and cyclin D1 antibodies were not confirmed. The other...