The purpose of this manuscript is to establish a unified theory of porohyperelasticity with transport and growth and to demonstrate the capability of this theory using a finite element model developed in MATLAB. We combine the theories of volumetric growth and mixed porohyperelasticity with transport and swelling (MPHETS) to derive a new method that models growth of biological soft tissues. The conservation equations and constitutive equations are developed for both solid-only growth and solid/fluid growth. An axisymmetric finite element framework is introduced for the new theory of growing MPHETS (GMPHETS). To illustrate the capabilities of this model, several example finite element test problems are considered using model geometry and material parameters based on experimental data from a porcine coronary artery. Multiple growth laws are considered, including time-driven, concentrationdriven, and stress-driven growth. Time-driven growth is compared against an exact analytical solution to validate the model. For concentration-dependent growth, changing the diffusivity (representing a change in drug) fundamentally changes growth behavior. We further demonstrate that for stress-dependent, solid-only growth of an artery, growth of an MPHETS model results in a more uniform hoop stress than growth in a hyperelastic model for the same amount of growth time using the same growth law. This may have implications in the context of developing residual stresses in soft tissues under intraluminal pressure. To our knowledge, this manuscript provides the first full description of an MPHETS model with growth. The developed computational framework can be used in concert with novel in-vitro and in-vivo experimental approaches to identify the governing growth laws for various soft tissues.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and the limiting factor for the enzyme activity. The expression of TERT and telomerase activity is increased in atherosclerotic plaques. However, the role of TERT dysregulation during atherosclerosis formation remains unknown. The work herein first identified a multi-tiered regulation of TERT expression in smooth muscle cells (SMC) through histone deacetylase (HDAC) inhibition. HDAC inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Furthermore, during vascular remodeling in vivo, TERT protein expression in the neointima is prevented by HDAC inhibition. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition. TERT is highly expressed in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, neointima formation was reduced in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program which could not be reversed by ectopic overexpression of E2F1. Chromatin immunoprecipitation and accessibility assays revealed that TERT was recruited to E2F1 target sites to increase chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a mitogenic effect of TERT on SMC growth and neointima formation through epigenetic regulation of proliferative gene expression. Furthermore, TERT expression is induced in activated macrophages during experimental and human atherosclerosis formation. To investigate the role for TERT in lesional macrophages and the subsequent effect on atherosclerosis formation, TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wild-type controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, gene ontology (GO) enrichment analysis of silenced inflammatory genes indicated that TERT positively regulates signal transducer and activator of transcription (STAT) cascade, which was confirmed by the decreased tyrosine phosphorylation of STAT3 protein resulting from TERT deletion. These findings indicate genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway in activated macrophages. In conclusion, the dysregulation of TERT expression within atherosclerotic plaques plays a causative role for vascular remodeling, including injury-induced neointima formation and hypercholesterolemia-induced atherosclerosis, through inducing SMC proliferation and a pro-inflammatory phenotype in infiltrating macrophages. These findings unveil a mechanism of TERT exacerbating the pathological vascular remodeling, which may provide a novel therapeutic target to combating vascular diseases.

Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation.

Nous avons étudié l’expression et la régulation de la lipogenèse dans les aortes et CMLV et déterminé si elle est modifiée par l’insulino-résistance et le diabète. Les rats Zucker obèses (ZO), diabétiques et Psammomys obesus accumulent plus de lipides dans leurs aortes que leurs contrôles. Cependant l’expression des gènes de la lipogenèse et ceux impliqués dans la captation des acides gras, n’est pas augmentée. Un résultat similaire a été retrouvé dans des pièces d’endartériectomies chez l’homme. Le milieu adipogénique, le glucose ou l’insuline seule stimulent modérément la lipogenèse uniquement dans les CML de Zucker contrôles, aucun effet n’a été observé dans les CML de ZO. Nous avons montré que les effets du TO901317 sur la lipogenèse dans les CMLV sont dus uniquement à l’activation du récepteur nucléaire LXRα, PXR n’a aucun effet. En conclusion, la lipogenèse n’est pas augmentée dans la paroi artérielle durant l’insulino-résistance et le diabète. / We investigated the expression and regulation of lipogenesis in aortas and VSMC and determined if it is modified during metabolic abnormalities. Zucker obese (ZO), diabetic (ZDF) rats, and the high fat diet fed Psammomys obesus accumulated more triglycerides in their aortas than control rats. However the expression of lipogenic genes, or of genes involved in fatty acids uptake, was not increased. Lipogenesis was not increased in human carotid endarterectomy of diabetic compared to non-diabetic patients. The adipogenic medium (ADM), glucose or insulin stimulated moderately lipogenesis but only in VSMC from control rats. No effect was observed in VSMC from ZO. We showed that the lipogenic effects of TO901317observed in VSMC from Zucker control rats are due solely to the nuclear receptor LXRα, PXR agonist had no effect. Conclusion: Lipogenesis is not increased in arterial wall during insulin-resistance and diabetes.

La cellule musculaire lisse (CML) a un rôle pivot dans la physiopathologie de l’asthme. Dans ce travail de thèse nous avons pu mettre en avant l’implication du récepteur de type 2 activé par les protéases (PAR-2) dans une composante majeure du remodelage bronchique : la prolifération musculaire lisse. Dans le premier travail, nous avons montré une augmentation de l’expression du PAR-2 au niveau des CML bronchiques d’asthmatiques in vitro. La réponse calcique est dépendante du niveau d’expression du récepteur, mais n’influence pas la réponse proliférante. La stimulation répétée du PAR-2 augmente la prolifération des seules CML d’asthmatiques, par un mécanisme dépendant de la voie ERK. Dans le second travail, nous avons montré que la production basale d’un épithélium reconstitué entraine une prolifération plus importante des CML d’asthmatiques comparée aux CML de témoins. Une augmentation supplémentaire de la prolifération des seules CML d’asthmatiques a été observée, après activation par le surnageant d’épithélium stimulé par des acariens de maison comparé au surnageant épithélial non stimulé. Ce mécanisme est dépendant du PAR-2 épithélial, qui induit la production de leucotriènes C4, sur des CML dont l’expression du récepteur (CysLTR1) est augmentée chez l’asthmatique. Ces résultats apportent de nouvelles connaissances dans le remodelage musculaire lisse bronchique de l’asthmatique et met en avant le PAR-2 comme cible thérapeutique potentielle. / Smooth muscle cells (SMC) play an important role in asthma pathophysiology. In this thesis, we have highlighted the involvement of protease activated receptor type-2 (PAR-2) in SMC proliferation, which is a major component of airway remodeling. In the first study, we have shown an increased expression of PAR-2 in asthmatic bronchial SMC in vitro. Calcium response is dependent on the expression level of PAR-2, which does not affect the proliferative response. Repeated stimulation of PAR-2 increases the proliferation of asthmatics SMC only, by an ERK-dependent mechanism. In the second study, we have demonstrated that the basal production of reconstituted epithelium leads to a greater proliferation of asthmatics SMC compared to controls. Increased proliferation of asthmatics SMC only was observed, after stimulation with supernatant of the epithelium stimulated by house dust mites (HDM) compared to unstimulated epithelial supernatant. This mechanism is epithelial PAR-2-dependent, which induces the production of leukotrienes C4, whose receptor expression (CysLTR1) is increased in asthmatics SMC. These results provide new insights into bronchial smooth muscle remodeling in asthma and highlights the PAR-2 as a potential therapeutic target.

Processos celulares que governam as NADPH oxidases vasculares em condições patológicas não estão claros ainda. Como os processos redox são parte intrínseca da resposta da célula ao estresse, temos investigado se o estresse oxidativo pode convergir com outros tipos de estresse via Nox(es). No presente estudo, focamos na inibição do proteasoma como uma condição relevante de estresse. A incubação de células musculares lisas com concentrações não apoptóticas de inibidores do proteasoma, MG132 e lactacistina, promoveu aumento na produção basal de superóxido e na atividade da NADPH oxidase, diminuição da atividade da SOD e da razão GSH/GSSG. Por outro lado, a inibição do proteasoma diminui a atividade da Nox após estímulo com Angiotensina II ou Tunicamicina, conhecido estressor do retículo endoplasmático. Em condições basais, MG132 induz a expressão de mRNA da Nox1, entretanto o aumento de Nox1 induzido por Angiotensina II foi diminuído na presença de MG132. O mesmo efeito ocorre com a indução de Nox4 pela Tunicamicina, que nesse caso foi drasticamente reduzida na presença de MG132. Além disso, tanto Angiotensina II quanto Tunicamicina induziram a atividade lítica do proteasoma 20S. A seguir, investigamos as conseqüências fisiológicas do MG132 na sinalização do estresse do RE, uma conhecida resposta mediada por Nox4. Células vasculares incubadas com MG132 induzem a expressão de marcadores do estresse do RE, GRP78 e XBP1, e também os marcadores mais tardios ATF4 e o próapoptótico CHOP/GADD153. Resultados similares ocorrem também com a Tunicamicina. Entretanto, a co-incubação de Tunicamicina e MG132 diminui e a sinalização do estresse do RE. AKT e p38 MAPK foram ativados por MG132, possivelmente como resposta ao estresse induzido pela inibição do proteasoma. Assim, a inibição do proteasoma bloqueia a NADPH oxidase, com aumento da atividade basal e expressão da Nox1 versus forte inibição da ativação e expressão da Nox4 frente ao estímulo. A inibição da Nox4 associa-se e pode contribuir para a inibição pelo MG132 da sinalização do estresse do RE. Portanto, o proteasoma parece exercer papel na integração de estresses celulares envolvendo a NADPH oxidase. A inibição do proteasoma pode ter papel na terapia de doenças associadas a estresse do RE. / Cellular processes governing vascular Nox family NADPH oxidases in disease conditions are unclear. Since redox processes are intrinsic to cell stress response, we asked whether oxidative stress merges with other types of stress via Nox(es). We focused on proteasome inhibition as a relevant stress condition. Vascular smooth muscle cells (VSMC) incubation with non-apoptotic concentrations of proteasome inhibitors MG132 or lactacystin promoted increased baseline superoxide generation (HPLC/DHE products) and NADPH oxidase activity, decreased SOD activity and GSH/GSSG ratio. Conversely, proteasome inhibitors decreased by Nox response to Angiotensin II (AngII) and abrogated Nox response to endoplasmic reticulum (ER) stressor tunicamycin. With MG132, basal Nox1 mRNA levels were increased, while Nox1 response to AngII was blunted. Moreover, MG132 abolished Nox4 mRNA levels TN-induced. Both AngII and TN (at 2 and 4 hs) promoted increased 20S proteasome lytic activity. We next assessed physiological consequences of MG132 in ER stress signaling, a known Nox4- mediated response. VSMC incubation with MG132 alone enhanced expression of the ER stress markers Grp78 and XBP1 and late markers such as ATF4 and proapoptotic CHOP/GADD153. Similar results occurred with the known ER stressor TN. However, co-incubation of TN and MG132 decreased Grp78, Grp94 and CHOP/GADD153, indicating that proteasome inhibition interrupts ER stress. AKT and p38 are activated by MG132 as response to stress and recover to survival. Thus, proteasome inhibition disrupts NADPH oxidase, with increased baseline activity and Nox1 expression vs. strong inhibition of stimulated Nox1 and Nox4 activation/expression. The later effect may underlie MG132-mediated inhibition of ER stress signaling. (Support: FAPESP, CNPq Milênio Redoxoma)

Introdução: A disfunção neurogênica do trato urinário inferior (DNTUI) é comum, tendo como causa diversas doenças neurológicas e se apresentando de maneira diversa e heterogênea. O tratamento da DNTUI pode incluir medicamentos, utilização de toxina botulínica intravesical e tratamento cirúrgico. A cirurgia de ampliação vesical é uma alternativa consagrada, usada principalmente no caso de bexigas fibrosadas ou em pacientes refratários ao tratamento conservador ou minimamente invasivo. Os mecanismos que levam à progressão da fibrose vesical e à refratariedade aos tratamentos conservadores nessa população de pacientes não são bem conhecidos. Objetivos: Avaliar a expressão gênica dos fatores relacionados à hipóxia e fibrose nos pacientes com DNTUI e correlacionar o padrão de expressão gênica com as características urodinâmicas dos pacientes estudados. Métodos: Foram avaliados prospectivamente 17 pacientes com DNTUI que foram submetidos à cirurgia de ampliação vesical pelo Departamento de Urologia do HCFMUSP. Os pacientes foram avaliados de acordo com os sintomas clínicos, exames laboratoriais e avaliação urodinâmica completa. Durante a cirurgia foi obtido um fragmento vesical interessando todas as camadas da bexiga para análise de expressão gênica com utilização de qRT-PCR de MMP-1, TIMP-1, HIF1alfa, HIF2alfa, HIF3alfa, iNOS, eNOS, VEGF e CTGF. Amostras vesicais de 9 doadores cadavéricos foram utilizados como controles. Resultados: A média dos pacientes estudados foi de 37,5 anos, com complacência vesical variando entre 3,8 e 50 ml/cmH20, com média de 11,16ml/cmH20. Os pacientes apresentaram superexpressão estatisticamente significativa de TIMP-1 e HIF3alfa e subexpressão estatisticamente significativa de MMP-1. A expressão gênica de HIF1alfa e HIF2alfa também apresentou uma tendência à superexpressão, apesar de não ser estatisticamente significativa (p = 0,14 e p = 0,11). Os outros genes avaliados foram expressos de forma heterogênea. Conclusão: Em pacientes com DNTUI associados a fibrose vesical e/ou refratariedade ao tratamento conservador, que foram submetidos à cirurgia de ampliação vesical, os fatores relacionados ao aumento da deposição da matriz extracelular e à hipóxia parecem ser relevantes na progressão da disfunção vesical. Esse achados são compatíveis com estudos em modelos animais de obstrução infravesical e hipóxia e reforçam a necessidade de estudos complementares para o melhor entendimento da fisiopatologia da progressão da disfunção vesical na DNTUI / Introduction: The neurogenic lower urinary tract dysfunction (NLUTD) is a common complication of a variety of neurological diseases and can present itself in a diverse and heterogeneous way. NLUTD can be treated with anticholinergic or alfa3 agonists drugs, intravesical botulinum toxin and/or surgical treatment. The bladder augmentation surgery is a classic alternative, used mainly in cases of fibrotic bladders or in patients with refractory symptoms with conservative treatment. The mechanisms that lead to the progression of bladder fibrosis and to conservative treatments failure are not well known. Objectives: To evaluate the expression of fibrosis and hypoxia related genes in patients with NLTUD and correlate the pattern of gene expression with urodynamic data of the population studied. Methods: We analyzed bladder specimens of 17 patients with NLUTD undergoing bladder augmentation surgery due to bladder fibrosis or conservative treatment failure. Urodynamic tests were performed in all patients. A bladder fragment was obtained during surgery for relative gene epression with quantitative real-time polymerase chain reaction (qRT-PCR) of MMP-1, TIMP-1, HIF1alfa, HIF2alfa, HIF3alfa, iNOS, eNOS, VEGF e CTGF. 9 cadaveric organ donors composed the control group. Results: The mean age of the patients was 37.5 years. Bladder compliance ranged form 3,8 to 50 ml/cmH20, with a mean of 11,16 ml/cmH20. Patients undergoing bladder augmentation surgery presented a statistically significant overexpression of TIMP-1 and HIF3alfa. MMP-1 was statistically significant underexpressed. The gene expression of HIF1alfa and HIF2alfa also showed a tendency to overexpression, although it was not statistically significant (p = 0,14 and p = 0,11). The other genes were expressed heterogenously. Discussion: In patients with NLUTD associated with bladder fibrosis and/or failure to conservative treatment, the gene expression of factors related to increased extracellular matrix deposition and hypoxia appears to be relevant in the progression of bladder dysfunction. These findings are comparable with studies in animal models of bladder oulet obstrucion and hypoxia and reinforces the need for complentary studies to better understand the pathophysiology of the progression of bladder dysfunction in the NLTUD

Introdução: As células do músculo liso vascular (CMLV) são quiescentes nos vasos adultos, com baixa capacidade de migração e de secreção de matriz extracelular, caracterizando fenótipo contrátil. Evidências apontam para a heterogeneidade fenotípica das CMLV ao longo da árvore arterial: há distribuição heterogênea de doenças e de resposta a determinadas drogas nos diferentes vasos, além de variabilidade de expressão dos genes de proteínas contráteis de músculo liso entre eles. O papel das CMLV, em fase adulta, é classicamente descrito como restrito à regulação do tônus de pequenos vasos, sendo insignificante a contribuição da mecânica das CMLV para a complacência das artérias elásticas. Existe a hipótese de que a viscoelasticidade das CMLV contribua para a mecânica final das artérias, sendo o enrijecimento dessas células associado à rigidez arterial. Objetivo: Estudar a variabilidade das propriedades mecânicas e de expressão proteica das CMLV, ao longo da árvore arterial, buscando identificar moduladores regionais para esse fenótipo. Avaliar se situações clínicas sabidamente associadas à rigidez arterial (envelhecimento, sexo feminino pós-menopausa, ancestralidade genética africana, diabetes mellitus e tabagismo) cursam com enrijecimento de CMLV. Métodos: 1) Estudou-se a composição e a organização da camada média de diferentes artérias. As CMLV desses vasos foram avaliadas quanto à viscoelasticidade de citoplasma (G), por meio do ensaio de Citometria Magnético Ótica de Oscilação e, quanto à expressão proteica global, usando cromatografia multidimensional e espectrometria de massas em tandem de alta resolução (Proteômica Shotgun). Os dados mecânicos obtidos foram correlacionados com as características da matriz extracelular (MEC) dos vasos de origem (porcentagem de elastina e quantidade de MEC). Em paralelo, foi realizado experimento de estiramento cíclico (10%/1Hz) das CMLV das diferentes artérias por 24 e 48h, seguido pela mensuração de rigidez de citoplasma. 2) Foram isoladas as CMLV de fragmentos de artéria mamária de 80 pacientes submetidos à cirurgia de revascularização do miocárdio, células essas que foram avaliadas quanto à viscoelasticidade de citoplasma (G, G\' e G\'\'). Elaborou-se modelo estatístico para avaliar se as variáveis clínicas idade, sexo feminino, ancestralidade africana, tabagismo e diabetes mellitus estavam associadas a alterações de mecânica celular. Resultados: 1) A viscoelasticidade das CMLV variou significativamente entre as artérias. As CMLV provenientes de artérias distais (artérias femoral e renal) mostraram-se significativamente mais rígidas que as CMLV de aorta torácica (p < 0,001). Identificou-se correlação negativa entre rigidez de CMLV e quantidade de MEC / elastina na camada média vascular. O regime de estiramento cíclico por 48h reduziu globalmente a rigidez das CMLV. As CMLV provenientes da aorta torácica expressaram maior quantidade de proteínas relacionadas com a estrutura e a organização do citoesqueleto em relação às CMLV da artéria femoral. 2) Constatou-se variabilidade interindividual de viscoelasticidade de CMLV e associação entre tabagismo e sexo feminino com enrijecimento de CMLV. Conclusões: As CMLV são heterogêneas quanto às propriedades mecânicas, à organização do citoesqueleto e à expressão proteica ao longo da árvore arterial, reforçando o conceito de plasticidade fenotípica das CMLV. A mecânica das CMLV é modulada pelas características da MEC e pela tensão circunferencial cíclica aplicada às paredes vasculares pelo fluxo sanguíneo. Mulheres pós-menopausa e tabagistas exibem enrijecimento de CMLV, sendo esse fato um provável contribuinte para a rigidez arterial associada a essas condições e um possível alvo terapêutico a ser avaliado futuramente / Rational: Vascular smooth muscle cells (VSMC) lose their ability to migrate and secrete extracellular matrix (ECM) with the end of vascular development, condition known as contractile phenotype and reversible in the presence of vascular injury. There is evidence of heterogeneity of VSMC phenotype along arterial tree, as the distribution of diseases (atherosclerosis) and the response to drugs vary between different vessels, as well as the expression of smooth muscle-contractile protein genes. The role played by VSMC mechanics on determining large arteries\' compliance was always considered irrelevant. It has been hypothesized that the VSMC mechanical properties are important for vascular mechanics, especially in the pathological scenario, where VSMC stiffening may be associated with arterial rigidity. Goals: Study the variation of VSMC mechanics and protein expression along arterial tree, identifying regional modulators of this phenotype. Evaluate if clinical situations associated with arterial rigidity (ageing, post-menopausal women, African ancestry, diabetes mellitus and smoking) concur with VSMC stiffening. Methods: 1) Different arteries were studied in terms of composition and organization of their media layer. VSMC isolated from these arteries were evaluated regarding cytoplasm viscoelasticity, measured using Optical Magnetic Twisting Cytometry Assay (OMTC), and protein expression, using two-dimensional liquid chromatography and tandem mass spectrometry (Shotgun Proteomics). Mechanical data were correlated with ECM characteristics (percentage of elastin and ECM amount) of the vessels of origin. In parallel, VSMC of different arteries were subjected to cyclic stretching (10%/1Hz) during 24 and 48h, followed by the measurement of their cytoplasm rigidity. 2) VSMC were isolated from fragments of mammary artery of 80 patients subjected to coronary bypass surgery and evaluated regarding their viscoelasticity (G, G\' e G\'\'). A statistic model was elaborated to address if the clinical variables age, female sex, African ancestry, smoking and diabetes mellitus were associated with changes of VSMC mechanics. Results: 1) VSMC viscoelasticity varied significantly amongst the studied arteries. VSMC from heart-distant arteries (femoral and renal arteries) were stiffer than VSMC from thoracic aorta (p < 0,001). There was a negative correlation between VSMC rigidity and the amount of ECM / percentage of elastin within the media layer. 48h-cyclic stretching was associated with a global reduction of VSMC rigidity. VSMC of thoracic aorta expressed significantly more proteins associated with cytoskeleton structure and organization than VSMC of femoral artery. 2) There was a significant inter-individual variation of VSMC viscoelasticity. Smoking and female sex were associated with VSMC stiffening. Conclusion: VSMC mechanics, cytoskeleton organization and protein expression are heterogeneous along arterial tree. VSMC mechanical properties are modulated by ECM characteristics and by regional mechanical forces. This reinforces the concept of phenotypic heterogeneity of VSMC. Post-menopausal women and smokers exhibit stiffer VSMC, representing an important factor for the understanding of the arterial rigidity associated with these conditions and also a possible future therapeutic target

Die Migration und Proliferation vaskulärer Zellen spielt eine entscheidende Rolle in der Pathogenese atherosklerotischer Gefäßwandveränderungen und trägt zudem in hohem Maße zu restenosebedingten Komplikationen interventioneller Therapien der Atherosklerose bei. Dies bedingt ein großes Interesse an pharmakologischen Interventionsmöglichkeiten zur Prävention / Therapie atherosklerotischer und restenotischer vaskulärer Läsionen. Dabei kommen neben lokal applizierbaren Substanzgruppen insbesondere Pharmaka in Betracht, die bereits Anwendung zur Behandlung metabolischer Risikofaktoren kardiovaskulärer Erkrankungen finden. Hierzu gehören insbesondere die oralen Antidiabetika vom Typ der Thiazolidindeone, die als Liganden für den "Peroxisome Proliferator-Activated Receptor gamma" (PPARg) agieren. PPARs sind eine Gruppe neuer Regulatoren der Genexpression, für die in den vergangenen Jahren eine Reihe vaskulärer Wirkungen nachgewiesen wurden. Wir konnten zeigen, daß die Proliferation und Migration von Gefäßmuskelzellen durch PPARg-Liganden gehemmt wird. Untersuchungen zu den beteiligten Signalübertragungsschritten ergaben, daß die pharmakologische Aktivierung von PPARg in Gefäßmuskelzellen insbesondere die durch die Mitogen-aktivierten Protein Kinasen ERK1/2 vermittelte Signaltransduktion beeinflußt. Diesbezüglich haben wir nachgewiesen, daß PPARg in Gefäßmuskelzellen die mitogene Signaltransduktion via ERK1/2 MAPK -> Elk-1 -> c-fos und die chemotaktische Signalübertragung via ERK1/2 MAPK -> Ets-1 -> Matrixmetalloproteinase-9 hemmt. Wir konnten ferner zeigen, daß PPARg-Liganden die Endothelzellmigration hemmen, die durch die Neovaskularisation atherosklerotischer Plaques und der damit verbundenen erhöhten Vulnerabilität einer Plaqueruptur eine Rolle in der Pathobiologie der Atherosklerose spielt. Diese migrationshemmende Wirkung der PPARg-Liganden basiert vermutlich auf einer Inhibition der für die Endothelzellmigration erforderlichen Signaltransduktion über den PI3 Kinase -> Akt -> eNOS Pathway. Die Inhibition dieses Signalwegs könnte die Folge der von uns beobachteten PPARg-Ligand-induzierten Expression der Phosphatase PTEN sein, die den PI3K -> Akt Signalweg negativ reguliert und die Aktivierung und Phosphorylierung von Akt inhibiert. Somit haben PPARg-aktivierende Liganden eine wichtige Funktion in der Behandlung der metabolischen Hauptrisikofaktoren kardiovaskulärer Erkrankungen, spielen aber gleichzeitig eine vermutlich ebenso wichtige Rolle in der Protektion atherosklerotischer und restenosebedingter Gefäßwandveränderungen durch direkte vaskuläre Effekte. / Migration and proliferation of vascular cells not only play an important role in the pathogenesis of atherosclerotic lesion formation, but also contribute to restenosis after therapeutic angioplasty. Therefore, pharmacological strategies for the prevention and/or treatment of atherosclerotic and restenotic vascular lesions are of great clinical interest. This involves substances that can be locally administered via stents, as well as agents that are already in clinical use for the treatment of metabolic risk factors. Among the latter, antidiabetic thiazolidinediones which function as ligands for the "peroxisome proliferator-activated receptor gamma" (PPARg), have been identified as promising drugs to target vascular lesion formation. PPARs constitute a group of novel regulators of gene expression, that exert several vascular effects. We report that vascular smooth muscle cell proliferation and migration is inhibited by PPARg-ligands. Investigating the signalling steps that are involved, we find that pharmacological activation of PPARg interferes with signal transduction through the mitogen-activated protein kinases ERK1/2 in vascular smooth muscle cells. We demonstrate that PPARg inhibits mitogenic signal transduction via ERK1/2 MAPK -> Elk-1 -> c-fos and also blocks chemotactic signalling through the ERK1/2 MAPK -> Ets-1 -> matrix metalloproteinase-9 pathway in vascular smooth muscle cells. We also showed that PPARg-ligands inhibit endothelial cell migration, which participates in the neovascularization of atherosclerotic plaques, thereby contributing to plaque destabilization and increased risk of plaque hemorrhage. This antimigratory action of PPARg-ligands results from an inhibition of signal transduction via PI3 Kinase -> Akt -> eNOS, a pathway that is crucial for endothelial cell migration. Since we observed a PPARg-ligand-induced upregulation of PTEN, a phosphatase that negatively regulates the PI3K -> Akt signalling pathway, this might constitute the mechanism by which PPARg-ligands inhibit endothelial cell migration. In conclusion, PPARg-activating ligands may provide a dual benefit in cardiovascular disease by ameliorating metabolic risk factors, as well as protecting the vasculature from atherosclerotic and restenotic alterations through direct vascular effects.

As metaloproteinases, abundantes em venenos de serpentes da família Viperidae, apresentam homologia estrutural e funcional com as metaloproteinases de mamíferos (MMPs), cujos níveis estão elevados em doenças de natureza inflamatória, como a aterosclerose. A metaloproteinase BaP1, do veneno da serpente Bothrops asper, apresenta potente atividade inflamatória e constitui ferramenta científica importante para o estudo das ações das MMPs. Durante a aterosclerose, as células de músculo liso vascular (CMLVs) mudam do fenótipo contrátil para sintético, migram para a camada subendotelial do vaso, liberam mediadores inflamatórios e expressam MMPs. No entanto, o papel destas enzimas na resposta inflamatória das CMLVs e a potencial relação deste efeito com a migração das mesmas, não foi esclarecida. Neste estudo, investigou-se os efeitos da BaP1 em CMLVs, quanto à 1) indução da migração; 2) liberação de diferentes classes de mediadores inflamatórios e expressão de moléculas de adesão; 3) indução da mudança fenotípica das CMLVs; 4) expressão e participação de enzimas de síntese de prostaglandinas e de receptores de PGE2 na liberação deste eicosanoide; 5) participação de eicosanoides e da IL-1&#946 na migração e mudança de fenótipo das CMLVs. Os resultados obtidos, a partir dos ensaios de transwell e wound healing, mostraram que a BaP1(50nM) induziu a migração das CMLVs, após 48 h, mas não a proliferação celular, observada pelo ensaio de ciclo celular. Além disso, a metaloproteinase induziu aumento da liberação de PGE2 (1-48h), LTB4 (1-3h), IL-1&#946 (12-24h), MCP-1 (24-48h) e fractalcina (24-48h), mas não de PGI2 e nem TXA2, analisados por ensaios de EIA e multiplex. Ainda, a BaP1 aumentou a expressão proteica de COX-2 (1° h) e de PGESm-1 (4° h), analisada por Western blotting, e expressão gênica das sFLA2-IIA (30 min) e cFLA2-IVA (30 min), verificada por PCR em tempo real, sem alterar os níveis de COX-1, dos receptores EP1, EP2, EP3 e EP4, de ICAM-1 e VCAM-1 e da iFLA2. A intervenção farmacológica com os inibidores de COX-2 ou de FLA2 intracelulares reduziu a liberação de PGE2 induzida pela BaP1. Além disso, o pré-tratamento das células com o inibidor da FLAP e com os antagonistas do receptor de IL-1&#946 ou do receptor EP3 reduziu a migração celular induzida pela BaP1. Esta metaloproteinase também induziu a mudança de fenótipo contrátil para o sintético, das CMLVs, verificada pela diminuição da expressão de &#945-actina pelo ensaio de citometria de fluxo. A inibição da COX-2 e da FLAP não alterou este efeito. Este conjunto de dados demonstra a capacidade da BaP1 estimular diretamente as CMLVs para migração, liberação de mediadores inflamatórios e a expressão de COX-2, PGESm-1, sFLA2-IIA e cFLA2-IVA. A produção de PGE2 induzida pela BaP1 depende das FLA2s intracelulares, com ativação das vias da COX-1 e -2. A migração das CMLVs, induzida pela BaP1, depende da PGE2 via ativação do receptor EP3, do LTB4 e da IL-1&#946. Ainda, esta metaloproteinase estimula a mudança fenotípica das CMLVs para o estágio sintético, em que as CMLVs migram e proliferam. Os dados deste estudo, ao demonstrarem que as metaloproteinases contribuem para o desenvolvimento de eventos inflamatórios, em CMLVs, apontam um papel adicional desta classe de enzimas em doenças de natureza inflamatória, como a aterosclerose. / Metalloproteinases are abundant enzymes in Viperidae family snake venoms and exhibit structural and functional homology with mammalian matrix metalloproteinases (MMPs). The levels of these enzymes are incresead in inflammatory diseases, such as atherosclerosis. The BaP1 metalloproteinase from Bothrops asper snake venom presents potent inflammatory activity and constitutes important scientific tool for the study of the actions of MMPs. During atherosclerosis, vascular smooth muscle cells (VSMCs) switch their phenotype from a contractile to a synthetic state, migrate into the subendothelial vessel layer, release inflammatory mediators and express high levels of MMPs. However, the role of these enzymes in the inflammatory response of VSMCs and the potential relationship of this effect with cell migration have not been clarified. In this study, we investigated the effects of BaP1 on CMLVs with focus on: 1) induction of cell migration; 2) release of different classes of inflammatory mediators and protein expression of adhesion molecules; 3) induction of VSMCs phenotype switching; 4) expression and participation of prostaglandin synthesis enzymes and PGE2 receptors in the release of this eicosanoid; 5) participation of eicosanoids and IL-1&#946 in migration and phenotype switching of VSMCs. Results obtained from the transwell and wound healing assays showed that BaP1 (50nM) induced VSMCs migration after 48 h, but not cell proliferation, observed by the cell cycle assay. In addition, this metalloproteinase caused release of PGE2 (1-48h), LTB4 (1-3h), IL-1 (12-24h), MCP-1 (24-48h) and fractalkine (24-48h), but not PGI2 and TXA2, analyzed by EIA and multiplex assays. Furthermore, BaP1 increased protein expression of COX-2 (1 h) and PGESm-1 (4 h), analyzed by western blotting and gene expression of sFLA2-IIA (30 min) and cFLA2-IVA (30 min), evaluated by real-time PCR, without altering COX-1, EP1, EP2, EP3 and EP4, ICAM-1 and VCAM-1 and iFLA2 levels. Pharmacological intervention with COX-2 or intracellular FLA2 inhibitors reduced PGE2 release induced by BaP1. In addition, pretreatment of cells with either a FLAP inhibitor, or IL-1&#946 receptor, or EP3 receptor antagonist reduced cell migration induced by BaP1. This metalloproteinase also induced conversion of contractile VSMCs to an synthetic phenotype, as evidenced by decrease of -actin expression, analyzed by flow cytometry assay. Inhibition of COX-2 and FLAP did not alter this effect. Altogether, these data demonstrate the ability of BaP1 to directly stimulate VSMCs for migration, release of inflammatory mediators and expression of COX-2, PGESm-1, sFLA2-IIA and cFLA2-IVA. PGE2 production induced by BaP1 depends on the intracellular FLA2s, with activation of COX-1 and -2 pathways. VSMCs migration induced by BaP1 depends on PGE2 via EP3 receptor engagement, LTB4 and IL-1&#946. Furthermore, this metalloproteinase stimulates VSMCs phenotypic switching to a synthetic phenotype, in which these cells migrate and proliferate. These data demonstrate that metalloproteinases can contribute to the development of inflammatory events in VSMCs, evidencing an additional role of this class of enzymes in inflammatory diseases, such as atherosclerosis.