Name: in vivo gene transfer to the rabbit common carotid artery endothelium
Uploaded: 2018-05-06T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

Agradecemos AdVec, Inc. permissão usar HDAd reagentes, Julia Feyk para assistência administrativa e os serviços veterinários do departamento de medicina comparativa para cirúrgico conselhos e apoio. Este trabalho foi financiado pelo HL114541 e o John L. Locke, Jr. Charitable Trust.

Os autores não têm nada para divulgar.

Certos aspectos da técnica cirúrgica merecem particular atenção. Exposição completa e mobilização da artéria carótida comum através de dissecção cuidadosa vai facilitar a transferência e arteriotomia reparação de gene. No entanto, durante a dissecção, manipulação direta da artéria carótida deve ser minimizada para evitar o vasoespasmo. Além disso, qualquer sangramento adjacente à artéria deve ser interrompida, aplicando uma ligeira pressão com gaze e sangue extravasado deve ser limpa imediatamen...

O objetivo desse método é introduzir um transgene no endotélio das artérias carótidas comuns de coelho. Introdução do transgene permite a avaliação da função biológica do produto do transgene em artérias normais e em modelos de coelho de doença arterial humana. Superexpressão do transgene em modelos de doença pode revelar se o transgene (e seu produto proteico) mostram a promessa como agentes terapêuticos1,2,3,4. Inclusão de elementos reguladores de cis-agindo na gaveta do transgene expressão permite a avaliação da actividade destes elementos no endotélio arterial na vivo5,6. Conhecimento da atividade de elementos reguladores de cis-agindo de forma específicas pode ser usado para projetar cassettes de expressão mais ativo e para sondar os mecanismos de regulação gênica em grande artéria endotélio na vivo7.
Os coelhos são um modelo valioso para vários aspectos da fisiologia humana vascular e doença. Coelhos partilham muitas das características vasculares com seres humanos. Por exemplo, valores hematológicos basais, regulamento hemostático e tensão longitudinal vascular são semelhantes entre os coelhos e os humanos8. Modelos de coelho de doenças vasculares replicam características-chave de muitas doenças humanas, incluindo: aneurismas (semelhante geométricas e características de fluxo)9, vasoespasmo (resposta semelhante ao tratamento endovascular)10,11, e aterosclerose (placas da íntima com características semelhantes, incluindo um núcleo rico em células de músculo liso, macrófagos e lipídios em uma tampa fibrosa)12,13. Nesse sentido, foram desenvolvidos modelos de coelho para muitas doenças vasculares, como trombose, vasoespasmo, aneurisma, estenose de enxerto vascular, diabetes e aterosclerose8,13,14, 15,16.
Para os investigadores a escolher entre os modelos animais de Fisiologia vascular e doença, o coelho tem várias vantagens. Em comparação com roedores, os maiores navios de coelhos permitam fácil manipulação cirúrgica, uso de dispositivos endovasculares e uma maior quantidade de tecido para medições quantitativas. Coelhos são muito mais próximos filogeneticamente primatas que são roedores17, e a maior diversidade genética de coelhos outbreds melhor se aproxima da variabilidade genética dos seres humanos. Diversidade genética é particularmente importante para estudos pré-clínicos, que-por sua natureza-visam desenvolver terapias que podem ser aplicadas para a população humana geneticamente diversa. Como com muitas se não todas as outras espécies de modelo, genes do coelho são facilmente clonados ou sintetizados porque foi sequenciado o genoma de coelho com cobertura alta (7,48 x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]. Comparado a outros grandes modelos animais (tais como cães, porcos ou ovelhas), os coelhos são relativamente baratos comprar e casa e eles são mais fáceis de criar e manipular. Modelos específicos de doença vascular em coelhos cada tem suas próprias vantagens e deficiências como modelos de doenças humanas que estão além do escopo deste manuscrito8,12,18. Um investigador deve rever essas vantagens e deficiências para determinar se o coelho é o melhor modelo para responder a uma pergunta específica e experimental.
Introdução de sequências regulamentar de Ácido desoxirribonucleico (DNA) em células endoteliais na vivo permite a investigação da actividade destas sequências em um ambiente fisiológico complexo. Estudos in vitro em células endoteliais transfectadas podem ser útil para a avaliação inicial de sequências reguladoras de DNA; no entanto, os níveis de expressão em cultura de tecidos modelos às vezes não são reproduzidos quando os estudos são repetidos na vivo19,5,20. Sistemas in vitro também podem ser útil para explorar caminhos básicos de proteínas de sinalização e fisiologia endotelial, bem como comunicação entre culturas de células vasculares; no entanto, caminhos mais complexos ou redes reguladoras que são influenciadas pelas populações complexas de células vasculares vizinhas ou o sistema imunológico são mais bem estudadas na vivo sistema6,20. O método descrito aqui fornece uma plataforma para explorar a regulação da expressão do transgene no endotélio dentro do contexto de uma nave intacta, com ou sem doença. O sistema in vivo também permite investigação do crosstalk celular fisiológico e patológico e a identificação das contribuições do sistema imunológico para regulamento de gene expressão6.
Transgênese germinal (especialmente em ratos) é uma abordagem alternativa para dirigir a expressão do transgene para células endoteliais. Esta abordagem pode fornecer a expressão do transgene ao longo da vida, com direcionamento endotelial mediada pelo promotor específico ou regiões reguladoras21,22. No entanto, a geração de ratos transgénicos é demorado e caro, várias linhas de transgênicas devem ser frequentemente testadas para garantir direcionamento do transgene para o tipo de célula desejada e a conquista de níveis de expressão do transgene adequada e experimental resultados em sistemas de murino podem ser dependente da tensão. Murino modelos transgénicos com transgenes endotelial-alvo têm muitas vantagens: não há necessidade de realizar a cirurgia em cada animal experimental a fim de alcançar a transgênese, ratos experimentais podem ser criados com numerosos outros ratos transgênicos disponíveis no a fim de testar interações genéticas e fenotípicas, e há uma grande variedade de anticorpos que reagem com proteínas murino, facilitando a caracterização de fenótipos. No entanto, direcionamento de transgenes para o endotélio através da linha de germe normalmente resulta na expressão do transgene em todo o sistema vascular,22 , tornando-se difícil determinar o local em que o produto do transgene está agindo. Isto é especialmente verdadeiro quando o produto do transgene é secretado, porque um transgene produto secretado pelas células endoteliais em todo o sistema vascular pode ter atividade biológica em qualquer número de sites dentro de um animal. Embora o método descrito neste manuscrito requer conhecimento técnico e instalações especializadas, pode ser menos demorado e menos caro do que desenvolver uma linha de rato transgénico endoteliais específicas. Permite a avaliação da função de uma proteína seletivamente nas células endoteliais de um segmento de grande artéria, e que permite o uso da carótida contralateral comum como um controle pareado (eliminando os fatores sistêmicos que podem variar entre experimental animais-por exemplo, a pressão arterial ou os níveis de colesterol-como variáveis não controladas).
Terapia gênica é uma abordagem promissora para o tratamento das doenças vasculares, doenças crônicas particularmente, porque um único aplicativo pode fornecer sustentada ou possivelmente ao longo da vida a expressão de um gene terapêutico23. A promessa terapêutica da terapia genética tem sido explorada em modelos animais de transferência de genes somáticos, muitas vezes direcionando o fígado24,,25, que é um alvo relativamente fácil, porque muitos vetores virais transmitidas pelo sangue são hepatotropic. No entanto, para ter efeito na doença vascular, terapia genética direcionada para o fígado deve alcançar sistêmica superexpressão de proteínas. Isso normalmente requer grandes doses de vetor, que pode ser tóxico ou mesmo fatal26. Além disso, aumentou os níveis sistêmicos de um aumento de proteínas, o risco de efeitos secundários fora do alvo, o que pode complicar ou mesmo obscurecer a interpretação dos resultados experimentais. Terapia de gene locais visando o endotélio vascular, conforme descrito neste manuscrito poderia evitar efeitos secundários sistêmicos porque o vetor infundido não é amplamente divulgado além do segmento arterial transduzido e efeitos vasculares locais podem ser obtidos sem alterações nos níveis plasmáticos sistêmicos de proteína. 27 -além disso, uma quantidade muito menor de vetor é necessária para transduce um segmento arterial do que é necessário para alcançar a transdução hepática robusta. Expressão do transgene do fígado relatou a diminuir ao longo do tempo, provavelmente devido a renovação celular, que exigem dosagem repetida se expressão do transgene de alto nível é para ser mantida. 28 em contraste, a baixa rotatividade do endotélio fornece expressão estável durante pelo menos 48 semanas em coelhos alimentados com comida e pelo menos 24 semanas em lesões ateroscleróticas de coelhos alimentados com colesterol. 1 , 27
Para determinar se este método de transferência de genes de endotélio de carótida comum coelho é apropriado, as vantagens e desvantagens (tabela 1) devem ser consideradas no contexto dos objetivos específicos de investigação. As vantagens deste método incluem: outbreds coelhos são melhor representante da diversidade genética humana do que são puras ratos (importantes para trabalhos pré-clínicos); coelhos de fornecer maiores navios para manipulação mais fácil e mais tecido para análise; o método pode atingir a expressão do transgene endotélio-alvo muito mais rapidamente do que o germe-linha endotelial de direcionamento em ratos transgénicos; dose de vetor pode ser facilmente ajustada aos níveis variáveis de modelo da expressão do transgene; processos específicos de grande artéria endotélio podem ser investigados; e transgênese vascular local permite a carótida oposta no mesmo animal para ser usado como um controle, eliminando fatores sistêmicos como variáveis não controladas. As desvantagens incluem: instalações especiais e conhecimentos são necessários; menos fundos geneticamente modificados no qual a experiência estão disponíveis em coelhos do que em ratos; e há uma menos extensa seleção de anticorpos de coelho contra proteínas do mouse (para immunodetection de proteína do transgene e outros antígenos que podem ser importantes na interpretação de resultados experimentais).

<table><tbody><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3mL syringe with 24G needle</td><td>Becton Dickinson</td><td>309571</td><td>2x for gene transfer surgery; 3x for harvest surgery</td></tr><tr><td>1mL syringe with 27G needle</td><td>Becton Dickinson</td><td>309623</td><td>6x for gene transfer surgery; 1x for harvest surgery</td></tr><tr><td>20mL syringe, luer lock</td><td>Nipro Medical Corp</td><td>JD+20L</td><td></td></tr><tr><td>Catheters, 24G x 3/4&quot;</td><td>Terumo Medical Products</td><td>SROX2419V</td><td></td></tr><tr><td>19G needle</td><td>Becton Dickinson</td><td>305187</td><td>Gene transfer surgery only</td></tr><tr><td>21G needle</td><td>Becton Dickinson</td><td>305165</td><td>For 20 mL syringe of saline</td></tr><tr><td>Gauze 4&quot; x 4&quot;</td><td>Dynarex</td><td>3242</td><td>~10-15 per surgery</td></tr><tr><td>3-0 silk suture</td><td>Covidien Ltd.</td><td>S-244</td><td></td></tr><tr><td>5-0 silk suture</td><td>Covidien Ltd.</td><td>S-182</td><td>Gene transfer surgery only</td></tr><tr><td>7-0 polypropylene suture</td><td>CP Medical</td><td>8648P</td><td>Gene transfer surgery only</td></tr><tr><td>5-0 polyglycolic acid suture</td><td>CP Medical</td><td>421A</td><td>Gene transfer surgery only</td></tr><tr><td>3-0 polyglycolic acid suture</td><td>CP Medical</td><td>398A</td><td>Gene transfer surgery only</td></tr><tr><td>Alcohol swabs</td><td>Covidien Ltd.</td><td>6818</td><td>For placement of I.V. line</td></tr><tr><td>Catheter plug</td><td>Vetoquinol</td><td>411498</td><td>Gene transfer surgery only</td></tr><tr><td>Ketamine HCl, 100 mg/mL</td><td>Vedco Inc.</td><td>05098916106</td><td></td></tr><tr><td>Xylazine, 100 mg/mL</td><td>Akorn Inc.</td><td>4821</td><td></td></tr><tr><td>Lidocaine HCl, 2%</td><td>Pfizer</td><td>00409427702</td><td></td></tr><tr><td>Bupivacaine HCl, 0.5%</td><td>Pfizer</td><td>00409161050</td><td></td></tr><tr><td>Beuthanasia D-Special</td><td>Intervet Inc.</td><td>NDC 00061047305</td><td>Harvest surgery only</td></tr><tr><td>Buprenorphine HCl, 0.3 mg/mL&amp;nbsp;</td><td>Patterson Veterinary</td><td>12496075705</td><td>Gene transfer surgery only</td></tr><tr><td>Saline IV bag, 0.9% sodium chloride</td><td>Baxter</td><td>2B1309</td><td>2x for gene transfer surgery; can use vial of sterile saline in place of one</td></tr><tr><td>Heparin&amp;nbsp; (5000 U/mL)</td><td>APP Pharmaceuticals</td><td>NDC 63323-047-10</td><td>Gene transfer surgery only</td></tr><tr><td>Fentanyl patch, 25 mcg/hr&amp;nbsp;</td><td>Apotex Corp.</td><td>NDC 60505-7006-2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Gene transfer vector</td><td></td><td></td><td>Dilute 350 &amp;micro;L per artery; 2 x 10&lt;sup&gt;11&lt;/sup&gt; vp/mL for adenovirus; gene transfer surgery only</td></tr><tr><td>&lt;strong&gt;Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metzenbaum needle holder 7&quot; straight</td><td>Roboz</td><td>RS-7900</td><td>Gene transfer surgery only</td></tr><tr><td>Operating scissors 6.5&quot; straight blunt/blunt</td><td>Roboz</td><td>RS-6828</td><td></td></tr><tr><td>Needle holder /w suture scissors</td><td>Miltex</td><td>8-14-IMC</td><td>Gene transfer surgery only</td></tr><tr><td>Castroviejo scissors</td><td>Roboz</td><td>RS-5658</td><td></td></tr><tr><td>Castroviejo needle holder, 5.75&quot; straight with lock</td><td>Roboz</td><td>RS-6412</td><td>Gene transfer surgery only</td></tr><tr><td>Stevens scissors 4.25&quot; curved blunt/blunt</td><td>Roboz</td><td>RS-5943</td><td></td></tr><tr><td>Alm retractor 4&quot; 4X4 5mm blunt prongs</td><td>Roboz</td><td>RS-6514</td><td>2x</td></tr><tr><td>Backhaus towel clamp 3.5&quot;</td><td>Roboz</td><td></td><td>4x</td></tr><tr><td>Micro clip setting forceps 4.75&quot;</td><td>Roboz</td><td>RS-6496</td><td>Gene transfer surgery only</td></tr><tr><td>Micro vascular clips, 11 mm</td><td>Roboz</td><td></td><td>2x for gene transfer surgery only</td></tr><tr><td>Surg-I-Loop</td><td>Scanlan International</td><td>1001-81M</td><td>5 cm length</td></tr><tr><td>Bonaccolto forceps, 4&amp;rdquo; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td><td>Roboz</td><td>RS-5210</td><td></td></tr><tr><td>Dumont #3 forceps Inox tip size .17 X .10mm</td><td>Roboz</td><td>RS-5042</td><td></td></tr><tr><td>Graefe forceps, 4&amp;rdquo; (10 cm) long serrated straight, 0.8mm tip</td><td>Roboz</td><td>RS-5280</td><td></td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; straight, 1.3mm tips</td><td>Roboz</td><td>RS-7110</td><td>2x</td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; curved, 1.3mm tips</td><td>Roboz</td><td>RS-7111</td><td></td></tr><tr><td>Jacobson mosquito forceps 5&quot; curved extra delicate, 0.9 mm tips</td><td>Roboz</td><td>RS-7117</td><td></td></tr><tr><td>Kantrowitz forceps, 7.25&quot; 90 degree delicate, 1.7 mm tips</td><td>Roboz</td><td>RS-7305</td><td></td></tr><tr><td>Tissue forceps 5&quot;, 1X2 teeth, 2 mm tip width</td><td>Roboz</td><td>RS-8162</td><td></td></tr><tr><td>Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td><td>Fine Science Tools</td><td>11092-12</td><td>2x</td></tr><tr><td>Adson forceps, 12 cm, serrated, straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Veterinary electrosurgery handpiece and electrode</td><td>MACAN Manufacturing</td><td>HPAC-1; R-F11</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Suite Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Circulating warm water blanket and pump</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Forced air warming unit</td><td>3M</td><td>Bair Hugger Model 505</td><td>Gene transfer surgery only</td></tr><tr><td>IV infusion pump</td><td>Heska</td><td>Vet IV 2.2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane vaporizer and scavenger</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Veterinary multi-parameter monitor</td><td>Surgivet</td><td>Surgivet Advisor</td><td></td></tr><tr><td>Veterinary electrosurgery unit</td><td>MACAN Manufacturing</td><td>MV-9</td><td></td></tr><tr><td>Surgical microscope</td><td>D.F. Vasconcellos</td><td>M900</td><td>Needs ~16x magnification</td></tr></tbody></table>

in vivo gene transfer to the rabbit common carotid artery endothelium

O objetivo desse método é introduzir um transgene no endotélio de segmentos isolados de ambas as artérias carótidas comuns do coelho. O método alcança focal transgênese endotelial-seletivo, permitindo assim que um investigador para determinar as funções biológicas de transgenes endotelial-expresso e quantificar a actividade transcricional in vivo de sequências de DNA em grande artéria células endoteliais. O método usa isolamento cirúrgico das artérias carótidas comuns de coelho e uma arteriotomia para entregar um vetor viral do transgene-expressando no lúmen arterial. Um período de incubação curto do vetor no lúmen, com subsequente aspiração do conteúdo do lúmen, é suficiente para alcançar expressão eficiente e durável do transgene no endotélio, sem transdução detectável ou expressão fora da segmento arterial isolado. O método permite avaliação das atividades biológicas dos produtos do transgene em artérias normais e em modelos de doenças humanas e vascular, evitando efeitos sistêmicos que possam ser causados ou mediante a entrega do gene para outros sites (por exemplo. o fígado) ou pela abordagem alternativa de entregar o endotélio por germe linha transgênese construções genéticas. Aplicação do método é limitada pela necessidade de um cirurgião e anestesista, uma bem equipada sala de operações, os custos de aquisição e coelhos de habitação e a necessidade de experiência em construção de vetor de transferência de genes e uso. Resultados obtidos com este método incluem: transgene relacionadas a alterações na estrutura arterial, celularidade, matriz extracelular ou função vasomotora; aumentos ou reduções na inflamação arterial; alterações na apoptose celular vascular; e progressão, retardo ou regressão de doenças como a hiperplasia da íntima ou aterosclerose. O método também permite que a medição da capacidade nativas e sintéticas normativas de sequências de ADN para alterar a expressão do transgene em células endoteliais, fornecendo resultados que incluem: níveis de transgene mRNA, níveis de proteína do transgene e níveis de transgene atividade enzimática.

Para implementar esse método com confiança, experimentos preliminares são necessários para estabelecer que o operador realize transferência de genes eficiente e reprodutíveis, com expressão do transgene principalmente em células endoteliais luminal. Em nossa experiência, isto é mais facilmente avaliado utilizando um vetor que expressa a β-galactosidase. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) dos segmentos de carótida comuns coloração removido 3 dias após ...

Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

Este método é introduzir um transgene no endotélio de artérias carótidas de coelho. Introdução do transgene permite a avaliação da função biológica do produto do transgene em artérias normais ou modelos de doença. O método também é útil para medir a atividade de sequências reguladoras de DNA.

Na Vivo Transferência de genes para o endotélio da artéria carótida comum de coelho

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

in-vivo-gene-transfer-to-the-rabbit-common-carotid-artery-endothelium

Research

JoVE Journal

Medicine

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

9.0K visualizações.  University of Washington. Este método é introduzir um transgene no endotélio de artérias carótidas de coelho. Introdução do transgene permite a avaliação da função biológica do produto do transgene em artérias normais ou modelos de doença. O método também é útil para medir a atividade de sequências reguladoras de DNA.

Vídeo: In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Um modelo de Disturbed Aterosclerose Flow-induzido em rato artéria carótida pela ligadura parcial e um método simples de isolamento de RNA a partir do endotélio Carótidas

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Medicina

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

A estratégia para identificar mutações de novo em Distúrbios comuns, tais como autismo e esquizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Vascular transferência de genes de Metallic Stent superfícies usando Adenoviral Vetores Tethered através hidrolisável reticulantes

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

O modelo de coelho da aterosclerose acelerada: uma perspectiva metodológica da artéria ilíaca balão lesão

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Um modelo de coelho da expressão do Transgene durável na veia Jugular para enxertos de interposição da artéria carótida comum

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Modelo pré-clínico de isquemia de membros posteriores em coelhos diabéticos

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Um modelo de interposição venosa de coelho imitando cirurgia de revascularização usando enxertos de veia para avaliar hiperplasia intimal sob pressão arterial

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

Implante de enxerto vascular de engenharia de tecidos na artéria carótida de camundongo via técnica de manguito

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Bioengenharia

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www.jove.com/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

Interposição de Artéria Carótida e Veia Jugular: Técnica para Induzir Hiperplasia Intimal Venosa em Modelo de Coelho

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...