Impact du stress hyperoxique en période néonatale sur la structure vasculaire : implication des phénomènes de sénescence et rôle possible dans la programmation développementale de l'hypertension artérielle

Réalisé en cotutelle avec l'Université de Lorraine (France)

Hypertension et régulation de l'expression moléculaire de l'angiotensinogène par la ribonucléoprotéine hétérogène nucléaire K

Le diabète est une maladie chronique dont la principale caractéristique est un niveau plasmatique élevé de glucose, qui est causé soit par un défaut dans la production d’insuline, l’action de l’insuline, ou les deux à la fois. Plusieurs études ont démontré que l’hyperglycémie chronique peut mener à la dysfonction et même la défaillance de plusieurs organes, dont le coeur, le système vasculaire, les yeux et les reins, se traduisant par des infarctus du myocarde, des accidents cérébro-vasculaires et des complications rétinales et rénales, respectivement. La néphropathie diabétique (DN) est la principale cause de déficience rénale et affecte près de 25-40% des patients diabétiques. La DN est invariablement associée à un risque élevé d’accident cérébrovasculaire et de dysfonction cardivasculaire. L’angiotensinogène (Agt) est l’unique précurseur de tous les types d’angiotensines. En plus du système rénine-angiotensine (RAS) sytémique, le rein possède son propre système intrarénal et exprime tous les composants du RAS. L’Agt est fortement exprimé dans les cellules du tubule proximal rénal (RPTC) et y est converti en angiotensine II (AngII), le peptide biologiquement actif du RAS. Les patients diabétiques présentent de hauts niveaux d’AngII et une augmentation de l’expression des gènes du RAS, suggérant que l’activation du RAS intrarénal joue un rôle important dans la progression de la DN. Les mécanismes qui contrôlent la régulation du niveau rénal d’Agt par l’hyperglycémie et l’insuline demeurent mal compris. Le but global de cette thèse est de mieux comprendre les mécanismes moléculaires qui contrôlent l’expression du gène Agt chez la souris Akita (un modèle murin de diabète de type 1). Dans cette optique, la première partie de la thèse se concentre sur deux facteurs de transcription de la famille des ribonucléoprotéines nucléaires hétérogènes (hnRNP). Chan et collaborateurs ont déjà identifié 2 protéines nucléaires hnRNP F et hnRNP K, de 48kD et 70kD respectivement. HnRNP F et hnRNP K forment un hétérodimère et se lient à l’élément de réponse à l’insuline (IRE) présent dans le promoteur du gène Agt du rat et inhibent la transcription du gène Agt in vitro. Afin de déterminer si hnRNP F / K sont responsables de l’inhibition de l’expression rénale de Agt par l’insuline in vivo, nous avons étudié des souris Akita males traités ou non avec des implants d’insuline pour une période de 4 semaines. Des souris non-Akita males ont été employées comme contrôles. Les souris Akita développent de l’hypertension et de l’hypertrophie rénale. Le traitement à l’insuline rétablit les niveaux de glucose plasmatiques et la pression systolique (SBP), et atténue l’hypertrophie rénale, l’albuminurie (ratio albumine/créatinine urinaire, ACR) et les niveaux urinaires d’Agt et AngII chez les souris Akita. De plus, le traitement à l’insuline inhibe l’expression rénale du gène Agt, tout en augmentant l’expression des gènes hnRNP F, hnRNP K et ACE2 (enzyme de conversion de l’angiotensine-2). Dans des RPTC in vitro, l’insuline inhibe Agt, mais stimule l’expression de hnRNP F et hnRNP K en présence de hautes concentrations de glucose, et ce via la voie de signalisation MAPK p44/42 (protéine kinase activée par un mitogène). La transfection avec des petits ARN interférents (siRNA) contre hnRNP F et hnRNP K prévient l’inhibition de l’expression d’Agt par l’insuline dans les RPTC. Cette étude démontre bien que l’insuline prévient l’hypertension et atténue les dommages rénaux observés chez les souris Akita diabétiques, en partie grâce à la suppression de la transcription rénale de Agt, via une augmentation de l’expression de hnRNP F et hnRNP K. La seconde partie de cette thèse change de focus et se tourne vers le facteur Nrf2 (nuclear factor erythroid 2-related factor 2). Nrf2 est un facteur de transcription qui contrôle les gènes de la réponse antioxydante cellulaire en réponse au stress oxydant ou aux électrophiles. Le but de cette étude est d’examiner l’impact de la surexpression de la catalase (Cat) dans les RPTC sur l’expression du gène Agt via Nrf2 et sur le développement de l’hypertension et des dommages rénaux résultants chez les souris diabétiques Akita transgéniques (Tg). Nos études ont démontré que la surexpression de Cat dans les souris Akita Cat-Tg normalise la SBP, atténue les dommages rénaux et inhibe l’expression des gènes Nrf2 et Agt dans les RPTC. In vitro, le glucose élevé (HG) et l’oltipraz (un activateur de Nrf2) stimulent l’expression de Nrf2 et Agt, et cet effet peut être bloqué par la trigonelline (inhibiteur de Nrf2), des siRNA contre Nrf2, des antioxydants ou des inhibiteurs pharmacologiques NF-κB et MAPK p38. La suppression de sites de réponse à Nrf2 présents dans le promoteur du gène Agt du rat abolit la stimulation par l’oltipraz. Finalement, des souris males adultes non-transgéniques traitées avec l’oltipraz montrent une augmentation de l’expression de Nrf2 et Agt dans leurs RPTC et cette augmentation peut être normalisée par la trigonelline. Ces données permettent d’identifier un nouveau mécanisme d’action de Nrf2, par la stimulation du gène Agt intrarénal et l’activation du RAS, qui induisent l’hypertension et les dommages rénaux par le glucose élevé et les espèces réactives de l’oxygène chez les souris diabétiques. Nos conclusions permettent de démontrer que l’insuline induit l’expression de hnRNP F et hnRNP K, qui jouent ensuite un rôle protecteur en prévenant l’hypertension. La surexpression de la catalase dans les RPTC vient quant à elle atténuer l’activation de Nrf2 et ainsi réduit la SBP chez les souris Akita.

Non-invasive Studies on Age Related Parameters Using a Blood Volume Pulse Sensor

A non-invasive technique is implemented to measure a parameter which is closely related to the distensibility of large arteries, using the second derivative of the infrared photoplethysmographic waveform. Thirty subjects within the age group of 20-61 years were involved in this pilot study. Two new parameters, namely the area of the photoplethysmographic waveform under the systolic peak, and the ratio of the time delay between the systolic and the diastolic peaks and the time period of the waveform ( T/T) were studied as a function of age. It was found that while the parameter which is supposed to be a marker of distensibility of large arteries and T /T values correlate negatively with age, the area under the systolic peak correlates positively with age. The results suggest that the derived parameters could provide a simple, non-invasive means for studying the changes in the elastic properties of the vascular system as a function of age.

GABAA and GABAB Receptor Gene Expression and Functional Regulation During Pancreatic Regeneration and Insulin Secretion in Rats

The present study demonstrate the functional alterations of the GABAA and GABAB receptors and the gene expression during the regeneration of pancreas following partial pancreatectomy. The role of these receptors in insulin secretion and pancreatic DNA synthesis using the specific agonists and antagonists also are studied in vitro. The alterations of GABAA and GABAR receptor function and gene expression in the brain stem, crebellum and hypothalamus play an important role in the sympathetic regulation of insulin secretion during pancreatic regeneration. Previous studies have given much information linking functional interaction between GABA and the peripheral nervous system. The involvement of specific receptor subtypes functional regulation during pancreatic regeneration has not given emphasis and research in this area seems to be scarce. We have observed a decreased GABA content, down regulation of GABAA receptors and an up regulation of GABAB receptors in the cerebral cortex, brain stem and hypothalamus. Real Time-PCR analysis confirmed the receptor data in the brain regions. These alterations in the GABAA and GABAB receptors of the brain are suggested to govern the regenerative response and growth regulation of the pancreas through sympathetic innervation. In addition, receptor binding studies and Real Time-PCR analysis revealed that during pancreatic regeneration GABAA receptors were down regulated and GABAB receptors were up regulated in pancreatic islets. This suggests an inhibitory role for GABAA receptors in islet cell proliferation i.e., the down regulation of this receptor facilitates proliferation. Insulin secretion study during 1 hour showed GABA has inhibited the insulin secretion in a dose dependent manner in normal and hyperglycaemic conditions. Bicuculline did not antagonize this effect. GABAA agonist, muscimol inhibited glucose stimulated insulin secretion from pancreatic islets except in the lowest concentration of 1O-9M in presence of 4mM glucose.Musclmol enhanced insulin secretion at 10-7 and 10-4M muscimol in presence of 20mM glucose- 4mM glucose represents normal and 20mM represent hyperglycaemic conditions. GABAB agonist, baclofen also inhibited glucose induced insulin secretion and enhanced at the concentration of 1O-5M at 4mM glucose and at 10-9M baclofen in presence of 20mM glucose. This shows a differential control of the GABAA and GABAB receptors over insulin release from the pancreatic islets. During 24 hours in vitro insulin secretion study it showed that low concentration of GABA has inhibited glucose stimulated insulin secretion from pancreatic islets. Muscimol, the GABAA agonist, inhibited the insulin secretion but, gave an enhanced secretion of insulin in presence of 4mM glucose at 10-7 , 10-5 and 1O-4M muscimol. But in presence of 20mM glucose muscimol significantly inhibited the insulin secretion. GABAB agonist, baclofen also inhibited glucose induced insulin secretion in presence of both 4mM and 20mM glucose. This shows the inhibitory role of GABA and its specific receptor subtypes over insulin synthesis from pancreatic bete-islets. In vitro DNA synthesis studies showed that activation of GABAA receptor by adding muscimol, a specific agonist, inhibited islet DNA synthesis. Also, the addition of baclofen, a specific agonist of GABAB receptor resulted in the stimulation of DNA synthesis.Thus the brain and pancreatic GABAA and GABAB receptor gene expression differentially regulates pancreatic insulin secretion and islet cell proliferation during pancreatic regeneration. This will have immense clinical significance in therapeutic applications in the management of Diabetes mellitus.

Molecular mechanisms controlling the precision of chemokine guided cell migration

By the end of the first day of embryonic development, zebrafish primordial germ cells (PGCs) arrive at the site where the gonad develops. In our study we investigated the mechanisms controlling the precision of primordial germ cell arrival at their target. We found that in contrast with our expectations which were based on findings in Drosophila and mouse, the endoderm does not constitute a preferred migration substrate for the PGCs. Rather, endoderm derivatives are important for later stages of organogenesis keeping the PGC clusters separated. It would be interesting to investigate the precise mechanism by which endoderm controls germ cell position in the gonad. In their migration towards the gonad, zebrafish germ cells follow the gradient of chemokine SDF-1a, which they detect using the receptor CXCR4b that is expressed on their membrane. Here we show that the C-terminal region of CXCR4b is responsible for down-regulation of receptor activity as well as for receptor internalization. We demonstrate that receptor molecules unable to internalize are less potent in guiding germ cells to the site where the gonad develops, thereby implicating chemokine receptor internalization in facilitating precision of migration during chemotaxis in vivo. We demonstrate that while CXCR4b activity positively regulates the duration of the active migration phases, the down-regulation of CXCR4b signalling by internalization limits the duration of this phase. This way, receptor signalling contributes to the persistence of germ cell migration, whereas receptor down-regulation enables the cells to stop and correct their migration path close to the target where germ cells encounter the highest chemokine signal. Chemokine receptors are involved in directing cell migration in different processes such as lymphocyte trafficking, cancer and in the development of the vascular system. The C-terminal domain of many chemokine receptors was shown to be essential for controlling receptor signalling and internalization. It would therefore be important to determine whether the role for receptor internalization in vivo as described here (allowing periodical corrections to the migration route) and the mechanisms involved (reducing the level of signalling) apply for those other events, too.

Complicaciones del uso del angioseal vs compresión manual en cateterismo cardíaco mediante punción femoral

Objetivo: Comparar las complicaciones del uso de Angioseal® versus compresión manual en los pacientes llevados a cateterismo cardíaco en el Servicio de Hemodinamia de la Fundación Santa Fe de Bogotá, del 1º de enero de 2005 al 31 de diciembre de 2010, mediante punción arterial femoral percutánea. Metodología: Se realizó un Estudio Observacional, Analítico, de tipo Cohorte Retrospectiva. Partiendo de dos grupos de personas con indicación de cateterismo cardíaco por cualquier causa, uno expuesto al procedimiento con Angioseal® y el otro con compresión manual. Resultados: Con el uso de Angioseal® versus compresión manual la aparición de complicaciones fue 7,3% vs 4,1%, estas diferencias no fueron significativas (OR 1,81 IC95 0,96-3,40; RR 1,75 IC95 0,96-3,18) . La enfermedad coronaria (OR 2,27 IC95 1,07-4,79; RR 2,18 IC95 1,06-4,46) y a la colocación de stent (OR 3,49 IC95 1,82-6,69; RR 3,25 IC95 1,75-6,02 si se relacionaron significativamente con la aparición de complicaciones menores. Conclusión: No encontramos soporte para aprobar o desaprobar el uso de Angioseal® o compresión manual como manejo de la hemostasia, con respecto a las complicaciones. Sin embargo, se encontró que la colocación de stents está fuertemente relacionada con el desarrollo de complicaciones menores, lo cual hace que estos pacientes deban ser objeto de monitorización estrecha.

Factores de riesgo para el desarrollo de complicaciones: infecciones en varicosafenectomia

La enfermedad venosa periférica de miembros inferiores se considera una de las afecciones más prevalentes, con morbilidad considerable, deterioro de la calidad de vida, e importante demanda de recursos de salud, que en la mayoría de casos requiere manejo quirúrgico como parte del tratamiento. Este estudio busca analizar si la técnica quirúrgica es un factor de riesgo, que incrementa la aparición de infección de sitio quirúrgico en 257 pacientes a quienes se les realizó varicosafenectomia. Metodología: Se realizó un estudio de cohorte retrospectiva; se recolectaron los datos a partir de los registros clínicos y quirúrgicos de pacientes que cumplieron con los criterios de inclusión, registrando factores de riesgo, descripción quirúrgica, complicaciones, y evolución post operatoria hasta 8 semanas. El análisis estadístico se realizó mediante el cálculo de riesgo relativo y regresión logística binomial. Resultados: Se demostró asociación entre la presencia de infección de sitio quirúrgico y tipo de cirugía con un RR = 3,05 (P= 0,01), clasificación CEAP con 29% menos riesgo en varicosafenectomia total (P= 0,008). Conclusiones: Existen variables clínicas y quirúrgicas que influyen en la probabilidad de desarrollar infección del sitio quirúrgico.

Validación diagnóstica del test neural 1 para el síndrome de túnel del carpo

El objetivo de este estudio fue realizar una prueba de validez diagnostica del test neural 1 para el diagnóstico del Síndrome de Túnel del Carpo (STC) utilizando como prueba de referencia o de oro el test de conducción nerviosa. En este estudio participaron 115 sujetos, 230 manos con sospecha clínica de STC quienes fueron evaluados con el test de conducción nerviosa y el test neural 1. Se encontró una sensibilidad del 93.0% (IC 95%:88,21-96,79) y una especificidad del 6,67% (IC 95%:0,0-33,59), razón de verosimilitud positiva fue de 1,00 y razón de verosimilitud negativa de 1,05. Valor predictivo positivo de 86,9% y un valor predictivo negativo de 12,5%. Se concluye que el test neural 1 es una prueba clínica de alta sensibilidad y baja especificidad de gran utilidad para el monitoreo e identificación del STC. Es un procedimiento para el diagnóstico clínico de bajo costo que puede incluirse en los exámenes de rutina de los trabajadores como complemento a las pruebas clínicas sugeridas por las Gatiso para dar mayor precisión a la identificación temprana del STC. Se sugiere combinarla con otros test de mayor especificidad para ser aplicada en trabajadores en condiciones de riesgo o que presenten síntomas en miembros superiores y realizar otros estudios en donde participen sujetos sin diagnóstico clínico del STC.

Auditory difference limen for duration in deaf and hearing listeners

This paper reviews a study to determine if deaf children can discriminate fine durational changes in acoustic signals or whether the impairment of the peripheral auditory system interferes with the temporal precision necessary for such tasks.

Tachykinins regulate the function of platelets

Evidence has been mounting for peripheral functions for tachykinins, a family of neuropeptides including substance P (SP), neurokinin A, and neurokinin B, which are recognized for their roles in the central and peripheral nervous system. The recent discovery of 4 new members of this family, the endokinins (EKA, B, C, and 13), which are distributed peripherally, adds support to the notion that tachykinins have physiologic/endocrine roles in the periphery. In the present study we report a fundamental new function for tachykinins in the regulation of platelet function. We show that SP stimulates platelet aggregation, and underlying this is the intracellular mobilization of calcium and degranulation. We demonstrate the presence of the tachykinin receptors NK1 and NK3 in platelets and present evidence for the involvement of NK1 in SP-mediated platelet aggregation. Platelets were found to contain SP-like immunoreactivity that is secreted upon activation implicating SP-like substances in the autocrine/paracrine regulation of these cells. Indeed, NK1-blocking antibodies inhibited aggregation in response to other agonists. Of particular note is the observation that EKA/B cross-react in the SP immunoassay and are also able to stimulate platelet activation. Together our data implicate tachykinins, specifically SP and EKA/B, in the regulation of platelet function. (C) 2004 by The American Society of Hematology.

The neuroprotective potential of flavonoids: a multiplicity of effects

Flavonoids exert a multiplicity of neuroprotective actions within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. These effects appear to be underpinned by two common processes. Firstly, they interact with critical protein and lipid kinase signalling cascades in the brain leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and synaptic plasticity. Secondly, they induce beneficial effects on the vascular system leading to changes in cerebrovascular blood flow capable of causing angiogenesis, neurogenesis and changes in neuronal morphology. Through these mechanisms, the consumption of flavonoid-rich foods throughout life holds the potential to limit neurodegeneration and to prevent or reverse age-dependent loses in cognitive performance. The intense interest in the development of drugs capable of enhancing brain function means that flavonoids may represent important precursor molecules in the quest to develop of a new generation of brain enhancing drugs.

Protease-activated receptor 2: activation, signalling and function

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Compliant Multi-electrode Micro-channel Array for Recording Afferent Nerve Activity

We have fabricated a compliant neural interface to record afferent nerve activity. Stretchable gold electrodes were evaporated on a polydimethylsiloxane (PDMS) substrate and were encapsulated using photo-patternable PDMS. The built-in microstructure of the gold film on PDMS allows the electrodes to twist and flex repeatedly, without loss of electrical conductivity. PDMS microchannels (5mm long, 100μm wide, 100μm deep) were then plasma bonded irreversibly on top of the electrode array to define five parallel-conduit implants. The soft gold microelectrodes have a low impedance of ~200kΩ at the 1kHz frequency range. Teased nerves from the L6 dorsal root of an anaesthetized Sprague Dawley rat were threaded through the microchannels. Acute tripolar recordings of cutaneous activity are demonstrated, from multiple nerve rootlets simultaneously. Confinement of the axons within narrow microchannels allows for reliable recordings of low amplitude afferents. This electrode technology promises exciting applications in neuroprosthetic devices including bladder fullness monitors and peripheral nervous system implants.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Morpho-anatomical variations during stem development in some epiphytic Cactaceae

LEMOS, R. C. C. AND G. F. A. MELO-DE-PINNA (Departamento de Botanica, Instituto de Biociencias, Universidade de Sao Paulo, Rua do Matao 277, Travessa 14, Cidade Universitaria, Butanta, Caixa Postal 11461, 05422-970, Sao Paulo, SP, Brasil). Morpho-anatomical variations during stem development in some epiphytic Cactaceae. J. Torrey Bot. Soc. 138: 16-25. 2011. In this study, the morpho-anatomical features of Hatiora salicornioides (Harworth) Britton & Rose, Rhipsalis floccosa Salm-Dyck Pfeiffer, Rhipsalis elliptica G. Lindb. ex K. Schum. and Epiphyllum phyllanthus (L.) Haworth. were studied during different phases of stem development. Primary (more developed) and terminal (less developed) segments showed variations of anatomical features as exhibited by the epidermal cells in surface view and transverse section. Features of the vascular system, e.g., the occurrence of non-lignified parenchyma in bands (H. salicornioides) or in small groups (R. floccosa and R. elliptica), as well as pericycle fibers and lignified cells in the medullar region, were only observed on the primary segments. Nevertheless, based on our anatomical analysis of stem segments in different developmental phases, we conclude that some characters described and used in systematic interpretations should be revised, mainly in the vascular (secondary xylem; non-xylematic vascular fibers) and dermal systems (epidermis in surface view and transverse section).