Name: vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers
Uploaded: 2014-08-12T13:00:00.000+00:00
Duration: 12 min 30 s
Description: Watch this Scientific Journal Video about Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers at JoVE.com

The authors do not have competing financial interests to disclose.

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Protocol development and studies presented in this paper were supported in part by American Heart Association Scientist Development grants (I.F. 0735110N and M.C. 10SDG4020003), U.S. National Heart, Lung, and Blood Institute grant HL 72108 (R.J.L.), and U.S. National Heart, Lung, and Blood Institute T32 007915 (S.P.F.). Angioplasty catheters were kindly donated by NuMED (Hopkinton, NY). The authors wish to acknowledge secretarial assistance of Ms. Susan Kerns.

<html> 제시된 프로토콜은 스테인레스 스틸 표면 무 코팅하는 아데노 바이러스 벡터를 통해 달성 가역적 부착 기판 매개 된 유전자 전달에 대한 동작 방법을 설명한다. 혈관 재 협착의 스텐트 기반 유전자 치료의 특정 목적을 위해 개발 된 반면,이 기술은 생체 재료, 바이오 메디컬 임플란트 및 유전자 치료 등의 분야에서의 더 넓은 응용을 갖는다. 제시된 연구는 유일하게 원형...

치료 적 양상과 같은 유전자 치료의 효과는 유전자 치료 벡터의 1,2 나쁨 타겟팅 용량에 의해 방해된다. 목표 위치에 유전자 발현의 하위 치료 수준에서 적절한 타겟팅 결과 부족하고, 벡터 및 부호화 치료 제품 4 모두에 대하여 면역 반응을 마운팅 할 책임을 포함한 비 - 표적 기관 03 행 벡터의 넓은 보급에 이르게 5. 하나의 가능성은 전달의 성행위를 상쇄하고 타겟팅 혈액과 림프를 통해 자신의 무료 보급을 배제 형태로 원하는 위치에 유전자 벡터를 소개하는 홍보하는 것을 의미한다. 전형적으로, 이러한 노력들은 물리적 번째 포획하여 주사 부위에서 일시적으로 유지하는 유전자 벡터 수있는 피브린, 콜라겐이나 히알루 론산 하이드로 젤 매트릭스 6-10과 혼합 바이러스 성 또는 비 바이러스 벡터 중 구성된 국소 주사 전달 시스템에 의존고분자 네트워크의 그들. 지역화 된 유전자 치료를위한 또 다른 일반적으로 인정 패러다임 주입 보철 장치 (11, 12)의 표면에 유전자 벡터의 고정화를 이용한다. 영구 이식 의료 (혈관, 기관지, 비뇨기과 및 위장 스텐트, 심장 박동기, 인공 관절, 수술 및 부인과 메시, 등.) 환자 13 수천만에서 연간 사용됩니다. 일반적으로 효과가 있지만,이 장치는 부적절 현재의 의료 관행 14-17에 의해 제어되는 합병증하는 경향이 있습니다. 이식 보철 장치는 현지화 된 유전자 치료 치료를위한 프록시 플랫폼 역할을 할 수있는 특별한 기회를 제공하고 있습니다. 약동학 관점 파묻혀의 동력학을 임플란트 / 조직 인터페이스 유전자 벡터의 높은 지방 농도 모두를 달성하고 둔화 유전자 벡터 결과 비교적 낮은 입력 용량 의료 임플란트 표면에서 유도화이 위치에서 R 제거. 대상 세포 인구에 의해 연장 된 거주지의 결과 및 향상된 흡수으로, 벡터 고정화는 유전자 벡터의 확산을 최소화 할 수 있습니다. 따라서, 비 표적 조직의 부주의 접종이 감소된다. (또한 기판 매개 유전자 전달 또는 고상 유전자 전달 되나) 이식 생체 재료에 유전자 벡터의 표면 테 더링 사용 세포 배양 및 동물 실험에서 구현되었습니다 모두 특정 (항원 - 항체 18-20, 아비딘 - 비오틴 21, 22) 그리고 23 ~ 26 (요금, 반 데르 발스) 상호 작용 비특이적. 표면이 지나치게 강한 결합이 표적 세포로 내재화 벡터 배제 이후 주입 장치의 표면에 벡터의 공유 결합은 이전에 비 기능적으로 간주되어왔다. 최근이 제한 TET로서 사용 자발적 가수 분해성 가교제의 사용을 통해 극복 될 수 있다는 것을 입증했다아데노 바이러스 벡터의 27,28 스텐트 및 캡시드 단백질의 변성 금속 표면 사이 그녀. 또한, 벡터 방출률 및 생체 외 및 생체 내에서 유전자 발현의 시간 경과 (28)의 가수 분해 반응 속도를 나타내는 다른 가수 분해성 가교제를 이용하여 변조 될 수있다. 본 논문은 활성화 된 금속 표면에 아데노 바이러스 벡터의 가역적 공유 결합에 대한 상세한 프로토콜을 제공하고 스텐트 혈관 성형술의 쥐 경동맥 모델에서 배양 평활근 및 내피 세포 및 생체 내에서 시험 관내에서 이어지는 전달 사건을 연구하는데 유용한 실험 구성 소개 .

<table><tbody><tr><td>316 stainless steel mesh disks</td><td>Electon Microscopy Sciences</td><td>E200-SS</td><td></td></tr><tr><td>Generic 304-grade stainless steel stents</td><td>Laserage</td><td>custom order</td><td></td></tr><tr><td>Ad&lt;sub&gt;eGFP&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>AD-5-PV0504</td><td></td></tr><tr><td>Ad&lt;sub&gt;Luc&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>AD-5-PV1028</td><td></td></tr><tr><td>Ad&lt;sub&gt;EMPTY&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>A858</td><td></td></tr><tr><td>Cy3(NHS)&lt;sub&gt;2&lt;/sub&gt;</td><td>GE Healthcare</td><td>PA23000</td><td></td></tr><tr><td>Sepharose 6B</td><td>Sigma-Aldrich</td><td>6B100-500ML</td><td></td></tr><tr><td>UV 96-well plates</td><td>Costar</td><td>3635</td><td></td></tr><tr><td>Fluorometry 96-well plates</td><td>Costar</td><td>3915</td><td></td></tr><tr><td>Cell culture 96-well plates</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Pierce Thermo Scientific</td><td>20490</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Pierce Thermo Scientific</td><td>20290</td><td></td></tr><tr><td>sulfo-LC-SPDP</td><td>Pierce Thermo Scientific</td><td>21650</td><td></td></tr><tr><td>Spectrophotometer</td><td>Molecular Devices&amp;nbsp;</td><td>SpectraMax 190</td><td></td></tr><tr><td>Spectrofluorometer</td><td>Molecular Devices</td><td>SpectraMax Gemini EM</td><td></td></tr><tr><td>Orbital shaker incubator</td><td>VWR</td><td>1575R</td><td></td></tr><tr><td>Horizontal airflow oven</td><td>Shel Lab</td><td>1350 FM</td><td></td></tr><tr><td>Centra-CL2 centrifuge&amp;nbsp;</td><td>International Equipment Company</td><td>426</td><td></td></tr><tr><td>Digital vortex mixerer</td><td>Fisher Thermo Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Eclipse TE300 fluorescence microscope</td><td>Nikon&amp;nbsp;</td><td>TE300</td><td></td></tr><tr><td>DC 500 CCD camera</td><td>Leica</td><td>DC-500</td><td></td></tr><tr><td>7500 Real-Time PCR system</td><td>Applied Biosystems</td><td>not available</td><td></td></tr><tr><td>IVIS Spectrum bioluminescence station</td><td>Perkins-Elmer</td><td>not available</td><td></td></tr><tr><td>EDTA dipotassium salt</td><td>Sigma-Aldrich</td><td>ED2P</td><td></td></tr><tr><td>Bovine serum albumin fraction V (BSA)</td><td>Fisher Thermo Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dumont forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>A10 cell line&amp;nbsp;</td><td>ATCC</td><td>CRL-1476</td><td></td></tr><tr><td>Bovine aortic endothelial cells</td><td>Lonza</td><td>BW-6002</td><td></td></tr><tr><td>Luciferin, potassium salt</td><td>Gold Biotechnology</td><td>LUCK-1Ge</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>PBS without calcium and magnesium</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Penicillin/Streptomycin solution</td><td>Gibco</td><td>11540-122</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Corning cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>QIAamp DNA micro kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>Power Sybr Green PCR Master Mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>MicroAmp Optical 96-well Reaction Plate</td><td>Applied Biosystems</td><td>N8010560</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied Biosystems</td><td>4360954</td><td></td></tr><tr><td>Cephazolin&amp;nbsp;</td><td>Apotex</td><td>not available</td><td></td></tr><tr><td>Loxicom (Meloxicam)</td><td>Norbrook</td><td>not available</td><td></td></tr><tr><td>Heparin sodium</td><td>APP Pharmaceuticals</td><td>not available</td><td></td></tr><tr><td>Ketavet (Ketamine)</td><td>VEDCO</td><td>not available</td><td></td></tr><tr><td>Anased (Xylazine)&amp;nbsp;</td><td>Lloid</td><td>not available</td><td></td></tr><tr><td>Forane (Isoflurane)&amp;nbsp;</td><td>Baxter</td><td>not available</td><td></td></tr><tr><td>Curved Moria iris forceps</td><td>Fine Science tools</td><td>11370-31</td><td></td></tr><tr><td>Curved extra-fine Graefe forceps</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>Fine scissors - ToughCut</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14101-14</td><td></td></tr><tr><td>Vicryl suture (5-0)</td><td>Ethicon</td><td>J385</td><td></td></tr><tr><td>Suture thread (4/0 silk)&amp;nbsp;</td><td>Fine Science Tools</td><td>18020-40</td><td></td></tr><tr><td>Michel suture clips</td><td>Fine Science Tools</td><td>12040-02</td><td></td></tr><tr><td>Wound dilator (Lancaster eye specula)</td><td>KLS Martin</td><td>34-149-07</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Michel suture clip applicator</td><td>Fine Science Tools</td><td>112028-12</td><td></td></tr><tr><td>Insyte Autoguard 24 G IV catheter</td><td>Beckton-Dickinson</td><td>381412</td><td></td></tr><tr><td>2F Fogarty catheter</td><td>Edwards Lifesciences</td><td>120602F</td><td></td></tr><tr><td>Teflon tubing</td><td>Vention</td><td>041100BST</td><td></td></tr><tr><td>PTA catheter</td><td>NuMed</td><td>custom order</td><td></td></tr><tr><td>Gauze pads</td><td>Kendall Healthcare</td><td>9024</td><td></td></tr><tr><td>Cotton applicators</td><td>Solon Manufacturing</td><td>WOD1003</td><td></td></tr><tr><td>Saline</td><td>Baxter</td><td>281321</td><td></td></tr><tr><td>10 ml syringe (Luer-Lok)</td><td>Beckton-Dickinson</td><td>309604</td><td></td></tr><tr><td>1 ml syringe (Luer-Lok)</td><td>Beckton-Dickinson</td><td>309628</td><td></td></tr><tr><td>Clippers with #40 blade</td><td>Oster&amp;nbsp;</td><td>78005-314</td><td></td></tr><tr><td>Transpore surgical tape</td><td>3M</td><td>MM 15271</td><td></td></tr><tr><td>Puralube vet ointment</td><td>Pharmaderm</td><td>not available</td><td></td></tr></tbody></table>

vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers

스텐트 재 협착 널리 관상 동맥 및 말초 동맥의 매우 좁은 부분을 통해 다시 설정 혈액의 흐름에 사용되는 스텐트 기반 재 시술 절차의 주요 합병증을 선물한다. 대안 전략을 제시 할 수있다 방지 재 협착 활성 유전자의 조정 릴리스 할 수있는 혈관 내 스텐트는 현재 약물 방출 스텐트를 사용합니다. 일반적으로 벡터의 물리적 포획을 위해 사용되는 폴리머 코팅과 관련된 과도한 염증 반응을 피하면서 임상 변환을 달성하기 위하여, 유전자 스텐트는 스텐트 고정화 유전자 벡터 방출 및 혈관의 부위 - 특이 적 전달의 예측 동력학을 나타낸다한다. 이 논문은 비스포스포네이트 (PABT)이 가수 분해 가교제 (HC)를 통해 스테인리스 표면을 개질 된 폴리 알릴 아민하는 아데노 바이러스 입자의 가역적 인 결합을 기반으로 스텐트에 아데노 바이러스 유전자 벡터의 무 코팅 테 더링에 대한 자세한 방법을 설명합니다. 의 가족5 내지 오십일 레인 징의 쇄상 에스테르 가수 분해의 평균 t와 2 관능 성 (아민 - 및 티올 - 반응성) HC는 스텐트 벡터를 연결하기 위해 사용되었다. 벡터 고정화 절차는 일반적으로 9 시간 이내에 수행하고 여러 단계로 구성된다 : PABT (4 시간)의 수용액에 금속 샘플 1) 배양; 트리스 (2 - 카르복시 에틸) 포스 핀 (20 분 간격)으로 설치 PABT 티올 기, 2) 탈; 피리 딜 디티 (PDT) 그룹 (2 시간)과 폴리에틸렌 이민 유도체와 샘플을 반응시켜 금속 표면의 반응성 티올 용량 3) 확장; 디티 오 트레이 톨 (10 분)에 PDT와 티올 기의 4) 변환; HC (1 시간)와 아데노 바이러스의 5) 수정; 크기 배제 칼럼 크로마토 그래피 (15 분) 및 티올 철강 표면 (1 시간)에 티올 반응성 아데노 바이러스 입자의 7)에 의해 수정 고정화 아데노 바이러스 입자의 6)의 정제. 이 기술은 스텐트를 넘어 폭 넓은 잠재적 인 응용 성을 가지고주입 된 이물질 인터페이싱 세포 기질 - 매개 유전자 전달을 통해 자신의 생체 적합성을 향상시키기 위해 판막 장치의 표면 공학을 촉진시켜.

벡터 출시 실험 이러한 혈관 내 스텐트 같은 중재 장치를 포함하는 임플란트의 표면에 아데노 바이러스 벡터의 테 더링 부분적 벡터 &#39;물리적 타겟팅의 부족을 미연에 방지, 질병 사이트 벡터 가깝다. 그러나, 표적 조직의 형질 도입을 통해 치료 효과를 달성 할 수 있도록, 벡터는 표면 (도 2)로부터 방출되어야한다. 가수 분해성 가교제의 사용은 가...

Watch this Scientific Journal Video about Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers at JoVE.com

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

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.

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐​​트 표면에서 혈관 유전자 전송

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

vascular-gene-transfer-from-metallic-stent-surfaces-using-adenoviral

Research

JoVE Journal

Medicine

9.8K 조회수.  The Children's Hospital of Philadelphia, University of Pennsylvania. 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. 

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

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.

잠복기 모델에서 허혈성 심장 마비에 대한 유전자 전송

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

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의학

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

당뇨병 각막에 대한 아데노 바이러스 유전자 치료 : 인간 장기 배양 각막 및 윤부 상피 세포에 상처 치유에 효과 및 세포 마커의 발현 줄기

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

발생학

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

생체공학

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

마우스 동정맥루 내피에 관내 약물 전달

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

전기 방사 및 풍선 확장 베어 메탈 스텐트를 사용하여 작은 구경 스텐트 - 이식의 제작

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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 focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Vivo에서 토끼 일반적인 경 동맥 내 피에 유전자 이동

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 ...

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.

일반적인 경 동맥 중재 이식 정 맥 경에서에서 튼튼한 Transgene 식의 토끼 모델

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.

당뇨병 토끼에서의 뒷 사지의 전 임상 모델

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

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.

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐트 표면에서 혈관 유전자 전송

요약

더 많은 비디오 탐색

이 비디오의 챕터

잠복기 모델에서 허혈성 심장 마비에 대한 유전자 전송

당뇨병 각막에 대한 아데노 바이러스 유전자 치료 : 인간 장기 배양 각막 및 윤부 상피 세포에 상처 치유에 효과 및 세포 마커의 발현 줄기

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

마우스 동정맥루 내피에 관내 약물 전달

전기 방사 및 풍선 확장 베어 메탈 스텐트를 사용하여 작은 구경 스텐트 - 이식의 제작

Vivo에서 토끼 일반적인 경 동맥 내 피에 유전자 이동

일반적인 경 동맥 중재 이식 정 맥 경에서에서 튼튼한 Transgene 식의 토끼 모델

당뇨병 토끼에서의 뒷 사지의 전 임상 모델

커프 기법을 통한 마우스 경동맥에 조직 공학 혈관 이식편 이식

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐​​트 표면에서 혈관 유전자 전송

요약

더 많은 비디오 탐색

이 비디오의 챕터

잠복기 모델에서 허혈성 심장 마비에 대한 유전자 전송

당뇨병 각막에 대한 아데노 바이러스 유전자 치료 : 인간 장기 배양 각막 및 윤부 상피 세포에 상처 치유에 효과 및 세포 마커의 발현 줄기

생분해 성 고분자 나노 입자를 사용하여 치료 혈관 신생에 대한 프로그래밍 줄기 세포

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

마우스 동정맥루 내피에 관내 약물 전달

전기 방사 및 풍선 확장 베어 메탈 스텐트를 사용하여 작은 구경 스텐트 - 이식의 제작

Vivo에서 토끼 일반적인 경 동맥 내 피에 유전자 이동

일반적인 경 동맥 중재 이식 정 맥 경에서에서 튼튼한 Transgene 식의 토끼 모델

당뇨병 토끼에서의 뒷 사지의 전 임상 모델

커프 기법을 통한 마우스 경동맥에 조직 공학 혈관 이식편 이식

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐트 표면에서 혈관 유전자 전송