A vasculopatia crônica do transplante constitui um dos principais obstáculos para o sucesso do transplante de órgãos a longo prazo. Caracteriza-se pela formação de uma camada neoíntima, que culmina no estreitamento da luz do vaso com consequente isquemia e falência do órgão transplantado. Embora diversos mecanismos imunológicos tenham sido descritos, a patogênese da vasculopatia crônica do transplante ainda não foi esclarecida e continua sem tratamento específico. Neste contexto, estratégias com alvos imunomodulatórios são de extrema importância. Dentre estas possíveis estratégias destaca-se a talidomida, uma droga que apresentada potentes propriedades anti-inflamatórias e imunomodulatórias e que recentemente voltou a ter indicações clínicas. Assim, os objetivos do presente estudo foram: 1) Padronizar o modelo experimental de vasculopatia crônica do transplante no modelo experimental de transplante de aorta, pois este modelo mimetiza as principais características observadas na parede do vaso durante o processo de rejeição; 2) Analisar o efeito da talidomida sobre as alterações morfológicas vasculares (histologia para Verhoeff), componentes celulares (expressão de a-actina, atividade proliferativa, número de macrófagos e linfócitos T, através de imuno-histoquímica), além da participação da apoptose (técnica de TUNEL); 3) Avaliar o potencial efeito anti-inflamatório e imunomodulador da talidomida neste modelo, através da análise da expressão de mediadores da resposta imune (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), bem como a atividade do NFkB e o envolvimento das subunidades p50 e p65 no enxerto vascular. Foram utilizados ratos machos das linhagens isogênicas específicas, Fisher 344 e Lewis, distribuídos em 3 diferentes grupos (32 por grupo): ISO (transplante isogênico de aorta, Fisher para Fisher); ALO (transplante alogênico de aorta, Fisher para Lewis); ALO+TALID (transplante alogênico de aorta tratado com talidomida (200mg/Kg/dia/gavagem)). Os animais foram acompanhados por um período de 30 dias e, em seguida, sacrificados. Os resultados obtidos demonstraram que a padronização do modelo experimental de transplante de aorta foi viabilizada dentro de uma sequência metodológica extremamente precisa e reprodutível. Como esperado, os animais do grupo ISO não apresentaram sinais de rejeição no enxerto vascular, mantendo íntegra a arquitetura das camadas do vaso. Por outro lado, animais do grupo ALO desenvolveram vasculopatia crônica do transplante caracterizada pelo espessamento das camadas íntima e adventícia. Na camada média evidenciou-se diminuição das VSMC e acentuado rompimento das fibras elásticas. Além disso, os animais do grupo ALO apresentaram aumento da expressão de a-actina acompanhada de intensa atividade proliferativa celular, particularmente nas camadas neoíntima e adventícia, provavelmente pela migração das VSMC para a camada íntima. A análise por IH revelou intenso infiltrado de macrófagos, linfócitos T, além de apoptose presente em todas as camadas do aloenxerto. A expressão gênica e a concentração tecidual dos mediadores da resposta imune TNF-a, IL-1b, IL-6, IL-2, INF-g e IL-10 também foram significativamente aumentadas no grupo ALO. Esses achados associaram-se com a ativação do NFkB, bem como o envolvimento dos dímeros p50 e p65. O tratamento com talidomida promoveu um efeito vasculoprotetor através da redução da camada neoíntima e da inflamação local. Além disso, induziu diminuição da expressão gênica e da concentração tecidual dos mediadores TNF-a, IL-1b, IL-6, IL-2, INF-g, bem como aumento da IL-4 e IL-10. A talidomida também diminuiu a ativação do NFkB. Conclusão: os resultados do presente estudo indicam que o modelo experimental de vasculopatia crônica do transplante constitui um modelo adequado para estudar as alterações nas camadas do vaso durante o processo de rejeição ao aloenxerto. A talidomida exerceu um efeito vasculoprotetor, atenuando a formação da neoíntima e do processo inflamatório local, bem como dos mediadores da resposta imune, provavelmente devido as suas propriedades anti-inflamatórias e imunomodulatórias / Chronic transplant vasculopathy is a major obstacle for the long-term success of organ transplantation. It is characterized by the formation of neointimal layer that culminate in the narrowing of the vessel lumen with consequent ischemia and failure of the transplanted organ. Although several immunological mechanisms have been described, the pathogenesis of chronic transplant vasculopathy remains unclear and continues without specific treatment. In this context, immunomodulatory strategies are extremely important. Among these possible strategies stands out thalidomide, a drug that display a potent antiinflammatory and immunomodulatory properties and recently have returned to clinical indications. The aims of this study were: 1) To standardize the experimental model of chronic transplant vasculopathy, induced in an experimental model of aortic transplantation, because this model mimics the key features observed in the vessel wall during the rejection process; 2) To analyze the effects of thalidomide on vascular morphological (Verhoeff staining), cellular components (a-actin expression, proliferative activity, number of macrophages and T lymphocytes by immunohistochemistry), and the participation of apoptosis (TUNEL assay); 3) To evaluate the antiinflammatory and immunomodulatory potential effects of thalidomide in this model by analyzing the mediator expression of the immune response (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), as well as the NFkB activity and involvement of p50 and p65 subunits in the vascular graft. We used male rats from the specific inbred strains, Fisher 344 and Lewis, divided into 3 different groups (32 per group). ISO (isogeneic aortic transplantation, Fisher to Fisher); ALO (allogeneic aortic transplantation, Fisher to Lewis); ALO+THALID (allogeneic aortic transplantation treated with thalidomide (200mg/Kg/day/gavage)). The animals were followed during a period of 30 days and then sacrificed. The results showed that the standardizing of the experimental model of aortic transplantation was possible within a sequence highly accurate and reproducible methodology. As expected, the animals of the ISO showed no sign of graft rejection keeping the entire layered architecture of the vessel. On the other hand, the ALO group developed chronic transplant vasculopathy characterized by thickness of the intima and adventitia. Furthermore, ALO group animals showed increase of a-actin expression accompanied by an intense cellular proliferative activity, particularly in the neointima and adventitia layers, probably due to VSMC migration to the intima. Analysis by IH revealed intense infiltration of macrophages and T lymphocytes, as well as apoptosis in all layers of the allograft. The gene expression and tissue concentrations of the mediators of immune response TNF-a, IL-1b, IL-6, IL-2, INF-g, and IL-10 were also significantly higher in the ALO group. These findings were associated with the NFkB activation, as well as the involvement of p50 and p65 dimers. Treatment with thalidomide promoted a vascular protective effect by reducing the neointimal formation and local inflammation. Moreover, induced a decrease in the gene expression and tissue concentration of the inflammatory mediators TNF-a, IL-1b, IL-6, IL-2, INF-g, as well as increase of IL-4 and IL-10. Thalidomide also decreased the NFkB activation. Conclusion: the results of this study indicate that experimental model of chronic transplant vasculopathy is an appropriate model to study changes in the vessel layers during the process of allograft rejection. Thalidomide promoted a vascular protective effect, attenuating neointimal formation and local inflammation, as well as mediators of immune response, probably due to its anti-inflammatory and immunomodulatory properties

Despite advancements in percutaneous coronary intervention, stents are still limited by a 2% annual rate of in-stent restenosis (ISR) related to neointimal (NI) tissue proliferation. Efforts to prevent ISR formation remain the focus of ongoing work. Adenosine (ADO) is a purine nucleoside with integral roles in vascular homeostasis, though it has limited clinical application. ADO signals primarily via four receptors with ADO receptor-A2B (ADOR-A2B) considered to play an integral role in vascular healing. Dipyridamole (DP) is a commercially approved therapy known to improve vascular events and modulate adenosine biology. Our objectives with this study included (i) assessing whether ADO could serve as a biomarker of cardiac events; (ii) determine if DP could mitigate NI formation in a pre-clinical stent model; and, (iii) quantify the mechanisms of DP-related vasculoprotection, specifically related to ADOR-A2B. We assessed the analytic and biologic variability of circulating ADO levels in humans and demonstrated that circulating ADO was not predictive of cardiac events at one year following invasive coronary angiography. We then assessed whether modulation of adenosine biology with DP had therapeutic efficacy in a pre-clinical model. Utilizing meta-analysis, we confirmed the sustained effects of DP on vascular patency rates in both pre-clinical and clinical studies. We refined a pre-clinical rabbit model of stent implantation with assessment of stent healing by intravascular optical coherence tomography – with excellent translation to clinical observations. We then assessed DP in a pre-clinical model, demonstrating reduction in ISR and improved stent healing with DP compared to control. Last, we sought to elucidate the mechanisms behind the observed DP effects, specifically related to ADOR-A2B. In vivo, DP therapy demonstrated reduced NI smooth muscle cell (SMC) content. In vitro assessment of DP demonstrated dose-dependent inhibition of SMC proliferation and migration with alteration of SMC phenotypic switching, while selective modulation of ADOR-A2B and ADOR-A2B knockdown support an ADOR-A2B-mediated component to the observed DP effects. Adenosine biology is integral to vascular homeostasis. In humans, circulating adenosine levels in humans are not predictive of one year cardiovascular events. However, DP may improve vascular healing post stent implantation and warrants clinical evaluation for stent healing. The observed DP benefits may, in part, stem from ADOR-A2B modulation. ADOR-A2B is a viable target for assessment of small molecule modulation as a novel therapeutic target to improve vascular outcomes.

EINLEITUNG: Die externe Stabilisierung von Venengrafts soll die Scherkräfte auf die Venenwand vermindern und dadurch die Ausbildung einer Neointimaproliferation reduzieren. In experimentellen Modellen wurde diese Hypothese im Kurzzeitversuch überprüft. Es fanden sich in diesem Zeitraum widersprüchliche Ergebnisse. Ziel unserer Untersuchung war es, in einem neuen Modell der arterialisierten segmentalen Vena-jugularis-Transposition auf die infrarenale Aorta den Einfluss einer externen Fibrinkleberstabilisierung auf die Neointimabildung des Venengrafts im Langzeitversuch darzustellen. MATERIAL UND METHODEN: Männlichen Wistar-Ratten wurden Segmente der Vena jugularis entnommen und in Flussrichtung, nach Entfernen eines Aortensegmentes, in die infrarenale Aorta eingebracht. Somit entspricht dieses einem Venenbypassmodell mit komplett arterialisiertem Venengraft. Vor

Fundamentos: A caracterização precisa da interação da placa aterosclerótica no momento do implante do stent é crucial para o entendimento da complacência e da cicatrização vasculares. Objetivamos investigar se a composição da placa avaliada pela tomografia de coerência óptica (OCT), influencia as alterações agudas no procedimento índice do implante do stent e na cicatrização vascular no seguimento tardio. Métodos: Os pacientes tratados com um único tipo de stent eluidor de fármaco (cromo cobalto, eluidor de sirolimus e polímero bioabsorvível) foram incluídos prospectivamente, seguindo um protocolo com etapas de dilatações progressivas do vaso. As imagens de OCT sequenciais foram realizadas no procedimento índice (basal e a cada etapa do protocolo) e no seguimento tardio, co-registradas e analisadas a cada 0,6mm. A avaliação semiquantitativa da placa foi realizada dividindo-se secções transversas em 4 quadrantes, com cada quadrante rotulado de acordo com o seu componente mais prevalente (fibrótico, calcificado, lipídico, normal). A interação stent-vaso avaliada pela OCT foi utilizada como indicador substituto para lesão e cicatrização vasculares após o implante do stent. Resultados: Um total de 22 lesões (1stent/lesão) de 20 pacientes e 2298 seções transversas de OCT foram analisadas no procedimento índice. O reestudo com OCT foi realizado em 17 pacientes e 19 lesões (86%). O componente de placa predominante foi fibrótico (fibrótico = 46.84 ± 16%; lipídico = 17.63 ± 10.72%; calcificado = 4.63 ± 5.9%; normal = 29.16 ± 12.24; não analizável=1.74 ± 5.35%). Houve um aumento nas áreas da luz (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) e do stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001), com um aumento na área do prolapso tecidual (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segmentos com muito tecido fibrocalcificado tiveram áreas luminais menores ao longo das etapas da intervenção. Por outro lado, placas com muito conteúdo lipídico ou vaso normal tiveram maiores ganhos nas medidas das áreas luminais mínimas ao longo das dilatações sequenciais. Além disso, placas com muito tecido fibrocalcificado no momento basal apresentaram menor crescimento neointimal no seguimento tardio, enquanto que o grau de conteúdo lipídico e de vaso normal não tiveram impacto sobre a formação do tecido neointimal. Os indicadores substitutos de lesão vascular após o implante do stent correlacionaram-se significativamente com o crescimento neointimal no seguimento tardio. Conclusões: A composição tecidual das placas subjacentes influencia significativamente o comportamento mecânico agudo e a longo prazo dos vasos coronarianos submetidos ao implante de stent. Além disso, a lesão vascular após o implante do stent está potencialmente ligada ao futuro crescimento neointimal no seguimento tardio / Background Accurate characterization of atherosclerotic plaque interaction during stent deployment is crucial to understand vascular compliance and healing. We sought to determine whether plaque composition assessed by optical coherence tomography (OCT), influences acute changes at index procedure and vascular healing at follow up. Methods Patients treated with a single drug-eluting stent type (cobalt chromium with bioabsorbable polymer eluting sirolimus stent) were prospectively included, following a pre-defined step-by-step progressive vessel dilatation. Sequential OCT imaging were performed at the index procedure (baseline and at each time point of the protocol) and at follow up, co-registered and analyzed every 0.6mm for quantitative measurements. Semi-quantitative plaque assessment was performed at baseline by dividing cross-sections into 4 quadrants, with each quadrant labeled according to its most prevalent component (fibrotic, calcific, lipid). OCT assessments of stent-vessel interactions were used as a surrogate for vessel injury and healing after stent implantation. Results A total of 22 lesions (1stent/lesion) of 20 patients and 2298 OCT crosssections were analyzed at the index procedure. For an average of 19.7 months (591.88 ± 60.52 days), 17 of the patients and 19 lesions (86%) underwent OCT imaging at follow up. The predominant percentage plaque component was fibrotic (fibrotic = 46.84 ± 16%; lipid = 17.63 ± 10.72%; calcific = 4.63 ± 5.9%; normal = 29.16 ± 12.24; non-analyzable = 1.74 ± 5.35%). There was an increase in lumen (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) and stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001) areas, with an increase in tissue prolapse area (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segments with high fibrocalcific content tended to have decreased minimal luminal areas along the intervention time-points. Conversely, plaques with high lipid content had increased minimal luminal areas during sequential dilatations. Moreover, plaques with high fibrocalcific tissue at baseline had significantly smaller neointimal growth at follow-up, whereas the degree of lipid content or normal tri-layered vessel had no impact on neointimal formation. OCT surrogates of vessel injury after coronary stenting significantly correlated with neointimal growth at follow-up. Conclusions: Tissue composition of underlying plaques significantly influences the acute mechanical and the long-term behavior of coronary vessels undergoing stent implantation. In addition, vessel injury after coronary stenting is potentially linked to future neointimal growth at follow-up

Estudamos se a sobrecarga salina leva ao aumento de neoíntima em vasos submetidos à lesão e avaliamos a participação do sistema renina-angiotensina neste processo. Usamos camundongos C57Bl6 machos tratados por 2 ou 12 semanas com NaCl 1% para beber ou água (grupos cont, sal2 e sal12), submetidos à lesão vascular. O grupo sal12 aumentou a lesão vascular e alterou a relação de fibras colágenas, com exacerbação de deposição colágena na lesão vascular, e a relação entre as angiotensinas II e (1-7) vasculares, com aumento de Ang (1-7) e sem redução de ang II após a lesão. Os resultados obtidos permitem concluir que a sobrecarga salina aumenta a neoíntima nas artérias com lesão e altera a relação entre angiotensinas II e (1-7) de forma possivelmente propiciar o aumento de relação íntima/média observado. / We studied if salt overload leads to an increase in neointima in injured arteries, and evaluate the involvement of the renin-angiotensin in this process. Were used C57Bl6 male mice treated for 2 or 12 weeks with 1% NaCl to drink or water (cont, sal2 sal12 groups) submitted to vascular injury. The group sal12 increased vascular injury and altered the relationship of collagen fibers, with exacerbation of collagen deposition in the vascular injury, and the relationship between vascular angiotensin II and (1-7), with an increase of Ang (1-7) and no reduction of Ang II after injury. The results indicate that salt overload increases neointima in injured arteries, and modifies the relationship between angiotensin II and (1-7), possibly providing the increase in intima/media ratio observed.

Estudamos se a sobrecarga salina leva ao aumento de neoíntima em vasos submetidos à lesão e avaliamos a participação do sistema renina-angiotensina neste processo. Usamos camundongos C57Bl6 machos tratados por 2 ou 12 semanas com NaCl 1% para beber ou água (grupos cont, sal2 e sal12), submetidos à lesão vascular. O grupo sal12 aumentou a lesão vascular e alterou a relação de fibras colágenas, com exacerbação de deposição colágena na lesão vascular, e a relação entre as angiotensinas II e (1-7) vasculares, com aumento de Ang (1-7) e sem redução de ang II após a lesão. Os resultados obtidos permitem concluir que a sobrecarga salina aumenta a neoíntima nas artérias com lesão e altera a relação entre angiotensinas II e (1-7) de forma possivelmente propiciar o aumento de relação íntima/média observado. / We studied if salt overload leads to an increase in neointima in injured arteries, and evaluate the involvement of the renin-angiotensin in this process. Were used C57Bl6 male mice treated for 2 or 12 weeks with 1% NaCl to drink or water (cont, sal2 sal12 groups) submitted to vascular injury. The group sal12 increased vascular injury and altered the relationship of collagen fibers, with exacerbation of collagen deposition in the vascular injury, and the relationship between vascular angiotensin II and (1-7), with an increase of Ang (1-7) and no reduction of Ang II after injury. The results indicate that salt overload increases neointima in injured arteries, and modifies the relationship between angiotensin II and (1-7), possibly providing the increase in intima/media ratio observed.

Fundamentos: A caracterização precisa da interação da placa aterosclerótica no momento do implante do stent é crucial para o entendimento da complacência e da cicatrização vasculares. Objetivamos investigar se a composição da placa avaliada pela tomografia de coerência óptica (OCT), influencia as alterações agudas no procedimento índice do implante do stent e na cicatrização vascular no seguimento tardio. Métodos: Os pacientes tratados com um único tipo de stent eluidor de fármaco (cromo cobalto, eluidor de sirolimus e polímero bioabsorvível) foram incluídos prospectivamente, seguindo um protocolo com etapas de dilatações progressivas do vaso. As imagens de OCT sequenciais foram realizadas no procedimento índice (basal e a cada etapa do protocolo) e no seguimento tardio, co-registradas e analisadas a cada 0,6mm. A avaliação semiquantitativa da placa foi realizada dividindo-se secções transversas em 4 quadrantes, com cada quadrante rotulado de acordo com o seu componente mais prevalente (fibrótico, calcificado, lipídico, normal). A interação stent-vaso avaliada pela OCT foi utilizada como indicador substituto para lesão e cicatrização vasculares após o implante do stent. Resultados: Um total de 22 lesões (1stent/lesão) de 20 pacientes e 2298 seções transversas de OCT foram analisadas no procedimento índice. O reestudo com OCT foi realizado em 17 pacientes e 19 lesões (86%). O componente de placa predominante foi fibrótico (fibrótico = 46.84 ± 16%; lipídico = 17.63 ± 10.72%; calcificado = 4.63 ± 5.9%; normal = 29.16 ± 12.24; não analizável=1.74 ± 5.35%). Houve um aumento nas áreas da luz (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) e do stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001), com um aumento na área do prolapso tecidual (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segmentos com muito tecido fibrocalcificado tiveram áreas luminais menores ao longo das etapas da intervenção. Por outro lado, placas com muito conteúdo lipídico ou vaso normal tiveram maiores ganhos nas medidas das áreas luminais mínimas ao longo das dilatações sequenciais. Além disso, placas com muito tecido fibrocalcificado no momento basal apresentaram menor crescimento neointimal no seguimento tardio, enquanto que o grau de conteúdo lipídico e de vaso normal não tiveram impacto sobre a formação do tecido neointimal. Os indicadores substitutos de lesão vascular após o implante do stent correlacionaram-se significativamente com o crescimento neointimal no seguimento tardio. Conclusões: A composição tecidual das placas subjacentes influencia significativamente o comportamento mecânico agudo e a longo prazo dos vasos coronarianos submetidos ao implante de stent. Além disso, a lesão vascular após o implante do stent está potencialmente ligada ao futuro crescimento neointimal no seguimento tardio / Background Accurate characterization of atherosclerotic plaque interaction during stent deployment is crucial to understand vascular compliance and healing. We sought to determine whether plaque composition assessed by optical coherence tomography (OCT), influences acute changes at index procedure and vascular healing at follow up. Methods Patients treated with a single drug-eluting stent type (cobalt chromium with bioabsorbable polymer eluting sirolimus stent) were prospectively included, following a pre-defined step-by-step progressive vessel dilatation. Sequential OCT imaging were performed at the index procedure (baseline and at each time point of the protocol) and at follow up, co-registered and analyzed every 0.6mm for quantitative measurements. Semi-quantitative plaque assessment was performed at baseline by dividing cross-sections into 4 quadrants, with each quadrant labeled according to its most prevalent component (fibrotic, calcific, lipid). OCT assessments of stent-vessel interactions were used as a surrogate for vessel injury and healing after stent implantation. Results A total of 22 lesions (1stent/lesion) of 20 patients and 2298 OCT crosssections were analyzed at the index procedure. For an average of 19.7 months (591.88 ± 60.52 days), 17 of the patients and 19 lesions (86%) underwent OCT imaging at follow up. The predominant percentage plaque component was fibrotic (fibrotic = 46.84 ± 16%; lipid = 17.63 ± 10.72%; calcific = 4.63 ± 5.9%; normal = 29.16 ± 12.24; non-analyzable = 1.74 ± 5.35%). There was an increase in lumen (10atm = 5.5 (4.5 - 7.4) mm2, 14-16atm = 6.0 (4.7 - 7.70) mm2, 20atm = 6.7 (5.5 - 8.2) mm2; P < 0.001) and stent (10atm = 5.2 (4.3 - 7.0) mm2, 14-16atm = 5.7 (4.5 - 7.5) mm2, 20atm = 6.5 (5.3 - 7.9) mm2; P < 0.001) areas, with an increase in tissue prolapse area (10atm =0.09 (0.06 - 0.12) mm2, 14-16atm =0.10 (0.06 - 0.15) mm2, 20atm =0.15 (0.08 - 0.20) mm2; P < 0.01). Segments with high fibrocalcific content tended to have decreased minimal luminal areas along the intervention time-points. Conversely, plaques with high lipid content had increased minimal luminal areas during sequential dilatations. Moreover, plaques with high fibrocalcific tissue at baseline had significantly smaller neointimal growth at follow-up, whereas the degree of lipid content or normal tri-layered vessel had no impact on neointimal formation. OCT surrogates of vessel injury after coronary stenting significantly correlated with neointimal growth at follow-up. Conclusions: Tissue composition of underlying plaques significantly influences the acute mechanical and the long-term behavior of coronary vessels undergoing stent implantation. In addition, vessel injury after coronary stenting is potentially linked to future neointimal growth at follow-up

Orientador: Claudio Chrysostomo Werneck / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T00:56:21Z (GMT). No. of bitstreams: 1 Vassequi-Silva_Tallita_M.pdf: 17060353 bytes, checksum: 04c3d8b469909adce6cf7a0f8977283d (MD5) Previous issue date: 2011 / Resumo: As fibras elásticas são responsáveis por darem sustentação a tecidos, como artéria aorta, pele, pulmão. São compostas por dois componentes distintos, elastina e microfibrila, quando analisadas por microscopia eletrônica. Anteriormente, os pesquisadores acreditavam que a principal função da rede de microfibrila, composta principalmente de fibrilinas e MAGPs, era na formação das fibras elásticas. Mas, sabe-se hoje, que a rede de microfibrila também é essencial na sinalização molecular. As MAGPs são proteínas aparentemente importantes para a função estrutural das microfibrilas e seu desenvolvimento. Embora sua função biológica ainda não seja conhecida, muitos estudos têm demonstrado sua interação com várias moléculas in vitro, como as fibrilinas 1 e 2, tropoelastina, biglican, decorina e colágeno VI. Até o momento nenhuma patologia foi relacionada à deficiência ou mutações no gene da MAGP1. Entretanto, dados preliminares demonstram que camundongos deficientes em MAGP1, quando submetidos à angioplastia, desenvolvem mais neoíntima quando comparados com animais selvagens. O procedimento de angioplastia resulta muitas vezes no remodelamento vascular, levando a reestenose ou formação de neoíntima. Muitos estudos têm relacionado o TGF-ß com o processo de reestenose após angioplastia, justamente pelo fato desse fator de crescimento ser capaz de modular o desenvolvimento e remodelamento vascular. Levando em consideração dados recentes que demonstram que a MAGP1 tem a capacidade de interagir com o TGF-ß ativo, foi de nosso interesse tratar os camundongos deficientes em MAGP1 e selvagens com losartan (antagonista de TGF-ß) para verificar a função da MAGP1 e a possível participação de TGF-na formação de neoíntima. Os animais foram tratados com losartan ou placebo por 4 semanas e então submetidos ao ensaio de angioplastia, aferição da pressão sanguínea e dosagem de TGF-ß plasmático. Observou-se que todos os animais são normotensos e, que o tratamento com losartan resultou em uma redução na formação de neoíntima em comparação com os animais não tratados e que não houve diferença na formação de neoíntima entre os animais deficientes em MAGP1 comparados com selvagens. Não houve alteração dos níveis de TGF-ß total. Os dados sugerem que a MAGP1 parece não ser importante no processo de formação de neoíntima e que o tratamento com losartan é eficaz na redução da formação da neoíntima, sem alterar a pressão sanguínea e os níveis de TGF-ß / Abstract: Elastic fibers are responsible for providing support to tissues such as aorta, skin and lung. They are composed of two distinct components, elastin and microfibrils, when analyzed by electron microscopy. Previously, researchers believed that the main function of the microfibrils network, mainly composed of fibrilin and MAGPs, was the formation of the elastic fiber. But, today, we know that the microfibril network is essential for molecular signaling also. The MAGPs are proteins apparently important for the structural functions of the microfibrils and development. Although its biological function is not known yet, many studies have demonstrated its interaction with various molecules in vitro, such as fibrilin 1 and 2, tropoelastin, biglycan, decorin and type VI collagen. So far no pathology was related to MAGP1 deficiency or mutations. However, preliminary data show that MAGP1 deficient mice when assayed using angioplasty model they developed more neointima than wild-type animals. The angioplasty procedure often results in vascular remodeling, leading to restenosis. Many studies have shown that TGF-ß is related to this process mainly because this growth factor is able to modulate the development and vascular remodeling. Take into account recent data showing that MAGP1 has the ability to interact with active TGF-ß, it was our interest to treat the MAGP1 deficient and wild-type mice with losartan (antagonist of TGF-ß) to verify the MAGP1 function and possible involvement of TGF-? in the neointima formation. Mice were treated with losartan or placebo for 4 weeks and then tested on angioplasty, blood pressure measurement and serum TGF-ß. It was observed that all animals are normotensive, and that losartan treatment resulted in a neointima reduction compared with untreated animals. No difference in neointima formation was observed between the MAGP1 deficient and wild-type mice. There was no change in total serum TGF-ß levels. Data suggest that MAGP1, apparently is not important in neointima formation and the losartan treatment is effective in reducing neointima formation without affecting blood pressure and levels of TGF-ß / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

L’apoptose endothéliale initie le processus menant au remodelage vasculaire et au développement de la néointima dans la vasculopathie du greffon. La formation de néointima résulte de l’accumulation de leucocytes, de matrice extracellulaire et de cellules positives pour l’actine musculaire lisse alpha (αSMA+) dans l’intima des artères, artérioles et capillaires du greffon. Les cellules αSMA+ dans la néointima sont des cellules musculaires lisses vasculaires (CMLV) dérivées du donneur ainsi que des cellules souches dérivées du receveur, dont des cellules souches mésenchymateuses (CSM). L’acquisition d’un phénotype anti-apoptotique chez ces cellules est déterminante pour le développement de la néointima. Le laboratoire de Dre Hébert a démontré que les cellules endothéliales (CE) apoptotiques libèrent des médiateurs induisant une résistance à l’apoptose chez les CMLV et les fibroblastes. Notamment, les CE apoptotiques relâchent la cathepsine L qui clive le perlécan et ainsi libère un fragment C-terminal correspondant au troisième motif laminine G du domaine V du perlécan (LG3). Le LG3 est anti-apoptotique pour les fibroblastes. Nous avons donc émis l’hypothèse que le LG3 est un des médiateurs clés libéré par les CE apoptotiques favorisant le développement de la néointima via l’induction d’un phénotype anti-apoptotique chez les cellules néointimales αSMA+. Nous avons démontré que les médiateurs libérés par les CE apoptotiques induisent un phénotype anti-apoptotique chez les CSM dépendant de l’activation de la voie ERK1/2. De plus, le LG3 active la voie ERK1/2 via son interaction avec les intégrines beta 1 et induit une réponse anti-apoptotique chez ces cellules. Cependant l’activation de ERK1/2 par le LG3 est plus faible en comparaison de son activation par le milieu conditionné par des CE apoptotiques. Nos résultats suggèrent que les CE apoptotiques libèrent aussi de l’EGF qui agit de façon paracrine sur les CSM en coopération avec le LG3 pour induire un phénotype anti-apoptotique chez les CSM. Nous avons poursuivi l’étude de l’effet du LG3 in vivo sur le remodelage vasculaire en transplantation. Nous avons pour cela développé un modèle murin de rejet vasculaire qui consiste en une transplantation aortique entre des souris alloincompatibles. Nous avons ensuite injecté du LG3 chez les souris receveuses en post-transplantation. Nous avons observé dans ce modèle que des niveaux augmentés de LG3 sérique augmentent la formation de néointima, favorisent l’accumulation de cellules néointimales αSMA+ et diminuent le nombre de cellules CD31+ au niveau du greffon aortique. Parallèlement nous avons vérifié que le LG3 induit aussi un phénotype anti-apoptotique chez les CMLV et nous avons démontré un nouvel effet du LG3, soit une activité pro-migratoire, qui dépend de l’activation de la voie ERK1/2 chez les CMLV. Nous avons complété cette étude par l’analyse des niveaux de LG3 sérique dans une cohorte de patients receveurs d’allogreffe rénale. Nous avons observé chez ces patients, une association entre des niveaux élevés de LG3 sérique et un rejet vasculaire. Le LG3 contribue à la formation de néointima par son activité pro-migratoire et pro-survie chez les cellules néointimales et aussi de par son activité angiostatique. Nos résultats suggèrent que le LG3 est un nouveau médiateur important dans le remodelage vasculaire en transplantation / In allogeneic transplanted organs, endothelial apoptosis is associated with vascular remodeling and neointima formation which in turn leads to allograft vasculopathy, a maladaptive form of vascular repair. In allograft vasculopathy, neointima results from the accumulation of leukocytes, extracellular matrix and alpha-smooth muscle actin positive (αSMA+) cells in the intima of allogeneic arteries, arterioles and capillaries. Neointimal αSMA+ cells comprise vascular smooth muscle cells (VSMC) derived from the donor and stem cells derived from the recipient, including mesenchymal stem cells (MSC). Acquisition of an anti-apoptotic phenotype of neointimal cells is central to the development of vascular obliterative changes. Dr Hébert’s team demonstrated that apoptotic endothelial cells release mediators which in turn induce a state of resistance to apoptosis of VMSC and fibroblasts. Apoptotic endothelial cells release cathepsin-L which cleaves perlecan therefore releasing a C-terminal fragment harbouring a laminin G motif and referred to as LG3. LG3 is anti-apoptotic for fibroblasts. We hypothesized that LG3 is a key mediator produced by endothelial apoptosis of importance in favoring neointima formation via the induction of an anti-apoptotic phenotype in αSMA+ neointimal cells We demonstrated that mediators released by endothelial apoptosis induce an ERK1/2-dependent anti-apoptotic phenotype in MSC. We identified LG3 as one of the mediators implicated in the induction of this anti-apoptotic response. Interactions between LG3 and beta 1 integrins expressed on MSC trigger ERK1/2 activation albeit to a lesser degree than medium conditioned by apoptotic endothelial cells. We showed that apoptotic endothelial cells also release EGF which cooperates with LG3 to induce an anti-apoptotic phenotype on MSC through cross-talk between EGF receptor and integrin-dependent pathways. Next, we characterized the impact of LG3 on allogeneic vascular remodeling in vivo. We developed a murine model of vascular rejection based on orthotopic transplantation of an aortic segment between two fully MHC-incompatible mice in absence of immunosuppression. Recombinant LG3 was injected intravenously post-transplantation in recipients resulting in higher circulating levels of LG3. In LG3-injected mice, accumulation of αSMA+ neointimal cells was enhanced resulting in significantly increased intima/media ratios in the allogeneic aortic graft. Aortic grafts of LG3-injected allografts also showed decreased CD31+ cells. We also demonstrated, using cell-based approaches, that LG3 exerts a pro-migratory activity on VSMC through beta 1-integrin and ERK1/2 -dependent pathways. In line with these observations we also reported augmented serum LG3 in human renal transplant patients in association with acute vascular rejection episodes. Collectively these results suggest that the pro-migratory, pro-survival and angiostatic activities of LG3 contribute to neointima formation. Our results suggest that LG3 is a novel mediator of importance in the development of obliterative vascular remodeling associated with rejection of allogeneic organs.

La vasculopathie du greffon est une pathologie caractérisée par un rétrécissement progressif et oblitérant des vaisseaux sanguins menant à une ischémie et une perte de fonction du greffon. Le rétrécissement vasculaire est dû à une accumulation de matrice extracellulaire (MEC) et de cellules mononuclées positives pour l’actine musculaire lisse alpha (alphaSMA) dont les cellules souches mésenchymateuses, le tout formant une néointima oblitérante. Cette pathologie est la cause principale de la perte des greffons rénaux et cardiaques à long terme. Le rejet vasculaire aigu est un prédicteur de la vasculopathie du greffon. L’équipe du Dr Hébert a démontré que l’apoptose endothéliale, qui joue un rôle important dans le développement du rejet vasculaire, initie la libération de LG3, un fragment du protéoglycan perlécan. Les taux sanguins et urinaires de LG3 sont augmentés chez les receveurs d’allogreffe rénale avec rejet vasculaire et vasculopathie du greffon. Les résultats obtenus en laboratoire durant ma maîtrise ont permis de mieux caractériser l’impact du LG3 sur un type cellulaire important participant à la formation de néointima : les cellules souches mésenchymateuses. Mes travaux ont démontré que le LG3 induit à la fois la migration horizontale des MSC et la transmigration des MSC. Cette migration est dépendante de la voie de signalisation d’ERK1/2, précédemment identifiée comme voie centrale dans la formation de néointima. De plus, nos résultats démontrent que la kinase Src est activée en amont de l’activation de la voie MAPK. La migration horizontale et la transmigration induites par le LG3 sont aussi dépendantes des intégrines alpha2beta1, ainsi que l’activation de la voie MAPK. Dans un modèle de transplantation murin, nous avons également démontré que l’injection sérique de LG3 recombinant favorise l’accumulation de cellules positives pour alphaSMA dans la néointima. En outre, lorsque le receveur est déficient pour l’intégrine alpha2, mais que le greffon est sauvage, la formation de néointima induite par l’injection de LG3 est diminuée dans le greffon suggérant que les cellules du receveur jouent un rôle important dans la formation de la néointima. Enfin, nous avons démontré que l’injection de LG3 augmente aussi le nombre de cellules positives pour la forme phosphorylée d’ERK1/2 (p-ERK1/2) dans la néointima du greffon et que cette accumulation est dépendante de la présence des intégrines 2 1 chez les cellules du receveur.Lorsque le receveur est sauvage, il y a une augmentation du nombre de cellules positives pour p-ERK1/2. L’investigation de ces mécanismes dans le remodelage vasculaire expose de nouvelles opportunités pour inhiber la réponse cellulaire qui mène au remodelage inadapté lors d’un dommage vasculaire chronique et ainsi prolonger la survie du greffon. / Graft vasculopathy is diseases characterized by a progressive and obliterate narrowing of the blood vessels leading to ischemia and loss of graft function. This vascular narrowing is due to an accumulation of extracellular matrix and mononuclear cells positive for alpha smooth muscle actin (alphaSMA) including mesenchymal stem cells, thus forming an occlusive neointima. This condition is the leading cause of long term loss of kidney and heart transplants. Acute vascular rejection is a predictor of graft vasculopathy. Dr. Hébert’s team has demonstrated that endothelial apoptosis, which plays an important role in the development of vascular rejection, initiates the release of LG3, a fragment of the proteoglycan perlecan. Blood and urine levels of LG3 are increased in renal allograft recipients with vascular rejection and graft vasculopathy. The results obtained in the laboratory during my Master have helped to better characterize the impact of LG3 on an important cell type involved in neointima formation: the mesenchymal stem cells. My work has shown that the LG3 induces both the horizontal migration and the transmigration of MSC. This migration is ERK1/2-dependent, previously identified as a key molecule involved in neointima formation. In addition, our results demonstrate that Src kinase is activated by upstream activation of the MAPK pathway. Horizontal migration and transmigration induced by LG3 are also dependent on alpha2beta1 integrins, and the activation of the MAPK pathway. In a murine transplantation model, we also demonstrated that intravenous injection of recombinant LG3 promotes the accumulation of alphaSMA positive cells in the neointima. In addition, when the recipient is deficient for the alpha2 integrin but the graft is wild type, LG3 fails to induce neointima formation in the graft suggesting that recipient cells play an important role in the neointima formation. Finally, we demonstrated that intravenous injection of LG3 also increases the number of positive cells for the phosphorylated form of ERK1/2 (p-ERK1/2) in the neointima. This accumulation is dependent on the presence of alpha2beta1 integrins on recipient cells: when the recipient is wild type, there is an increase in the number of cells positive for p-ERK1/2.The investigation of these mechanisms in vascular remodeling presents new opportunities to inhibit the cellular response that leads to inadequate remodeling during chronic vascular damage and prolong graft survival.