The authors do not have competing financial interests to disclose.

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+adenovirus+gene+delivery+using+antiviral+IgG+complexation.">Localized adenovirus gene delivery using antiviral IgG complexation.</a> Gene Ther. 8, 659-667 (2001).</li><li>Ma, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchoring+of+self-assembled+plasmid+DNA/anti-DNA+antibody/cationic+lipid+micelles+on+bisphosphonate-modified+stent+for+cardiovascular+gene+delivery.">Anchoring of self-assembled plasmid DNA/anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery.</a> Int J Nanomedicine. 8, 1029-1035 (2013).</li><li>Hu, W. W., Lang, M. W., Krebsbach, P. 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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.

The presented protocol describes an operational method for substrate mediated gene delivery achieved through reversible attachment of adenoviral vectors to coatless stainless steel surfaces. While developed for the specific purpose of stent-based gene therapy of vascular restenosis, this technique has much broader applications in the areas of biomaterials, biomedical implants and gene therapy.
Although presented studies have solely utilized stainless steel as a prototypical metal substrate, PABT binds other metal alloys such as cobalt/chromium and nitinol with comparable affinity (not published). The indiscriminant interaction of PABT with metals significantly expands the number of potential biomedical applications for this technology31. Moreover, the use of HC-tethered gene vectors, albeit requiring other methods of thiol installation onto material surface, is feasible with other types of biomaterials.
While Ad gene vectors achieve robust transduction in many human cell types and tissues, their clinical significance is limited by strong inflammatory and immune responses elicited by Adenoviridae4. To this end, prolonged (4 months) arterial expression with gene eluting stents formulated using HC modification of luciferase-expressing adeno-associated viral vectors was recently shown (not published).
The methodology is robust. Small deviations from the main protocol do not generally lead to significant alterations of gene expression in vitro and in vivo. Nevertheless, several critical issues that may affect outcomes were identified.
First, as the name suggests, hydrolysable cross-linkers are cleavable upon reaction with water due to hydrolysis of the in-chain ester. Moreover, the amine-reactive moiety, N-sulfosuccinimidyl is hydrolysable as well. Cross-linkers should be stored under an argon atmosphere at -20 &#176;C to preserve their activity. For the same reasons, after preparing solutions of HC (protocol section 3.2) proceed immediately with the addition of HC solutions to the virus preps.
Second, thiol groups that are formed on the metal surface during several intermediate steps of the presented bioconjugation sequence are rapidly oxidized by ambient air oxygen. To prevent this, keep the vulnerable metal samples submerged in degassed water at all times.
Third, a perfect alignment of the mesh with the bottom of the well is required for successful transduction. Do not bend mesh disks during handling.
Fourth, avoid excessive rubbing of gene-eluting stents against the Teflon sheath and vessel wall during stent insertion to prevent dislodging of surface-associated Ad vectors.
Covalent attachment of gene vector to the surface of implantable biomaterials has an apparent advantage of better control over the vector release kinetics than realizable with non-covalent tethering of the vectors. In the setting of gene delivery from stent platform most studies report complete release of viral and non-viral vectors within 1-7 days after stent deployment32-34 . On the other hand, Ad vector immobilization via HC demonstrated release of only 20-45% of the vector load in the first month (Figures&#160;4A and 4B). Moreover, a partial modulation of release and transduction kinetics is achievable with the use of different HC exhibiting dissimilar hydrolysis kinetics or by varying the concentration of HC employed for virus surface modification. Additionally, a concurrent co-modification of the vector with different cross-linker molecules, although not explored in the present study, provides another opportunity for fine-tuning vector release and transduction.

The effectiveness of gene therapy as a therapeutic modality is hampered by the poor targeting capacity of gene therapy vectors1,2. The lack of proper targeting results in sub-therapeutic levels of transgene expression at the target location and leads to a wide dissemination of vectors to non-target organs3, including those responsible for mounting immune responses against both the vector and encoded therapeutic product4,5. One potential means to offset the promiscuity of transduction and to promote targeting is to introduce gene vectors at the desired location in a form that precludes their free dissemination via blood and lymph. Typically, such efforts rely on a locally injectable delivery systems comprising of either viral or non-viral vectors admixed with fibrin, collagen or hyaluronic acid hydrogel matrices6-10 that are capable of transiently sustaining gene vectors at the injection site by physically entrapping them in a polymeric network.
Another generally accepted paradigm for localized gene therapy utilizes immobilization of gene vectors on the surface of implanted prosthetic devices11,12. Permanent medical implants (endovascular, bronchial, urological and gastrointestinal stents, pacemakers, artificial joints, surgical and gynecological meshes,&#160;etc.) are used yearly in tens of millions of patients13. While generally effective, these devices are prone to complications that are inadequately controlled for by current medical practices14-17. Implantable prosthetic devices present a unique opportunity to serve as proxy platforms for localized gene therapy treatment. From the pharmacokinetic standpoint, surface derivatization of medical implants with relatively low input doses of gene vectors results in achieving both high local concentrations of gene vectors on the implant/tissue interface and slowing the kinetics of their elimination from this location. As a consequence of protracted residence and enhanced uptake by the targeted cell population, vector immobilization minimizes spread of the gene vector. Thus the inadvertent inoculation of non-target tissues is reduced.
Surface tethering of gene vectors on implantable biomaterials (also termed as substrate-mediated gene delivery or solid phase gene delivery) has been implemented in cell culture and animal experiments using both specific (antigen-antibody18-20, avidin-biotin21,22) and non-specific23-26 (charge, van der Waals) interactions. The covalent attachment of vectors to the surface of the implanted device has been previously considered as non-functional since excessively strong bonds with the surface preclude vector internalization by target cells. Recently it was demonstrated that this limitation can be overcome through the use of spontaneously hydrolysable cross-linker used as the tethers between the modified metallic surface of the stent and capsid proteins of the adenoviral vector27,28. Moreover, the vector release rate and time course of transgene expression in vitro and in vivo can be modulated with the use of hydrolysable cross-linkers exhibiting different kinetics of hydrolysis28.
The present paper provides a detailed protocol for the reversible covalent attachment of adenoviral vectors to activated metal surface and introduces a useful experimental setup for studying ensuing transduction events in vitro in cultured smooth muscle and endothelial cells and in vivo in the rat carotid model of stent angioplasty.

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">316 stainless steel mesh disks</td>
			<td style="width:183px;">Electon Microscopy Sciences</td>
			<td style="width:104px;">E200-SS</td>
			<td style="width:75px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Generic 304-grade stainless steel stents</td>
			<td>Laserage</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AdeGFP</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td>AD-5-PV0504</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AdLuc</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td>AD-5-PV1028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">AdEMPTY</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td style="width:104px;">A858</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:263px;">Cy3(NHS)2</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:104px;">PA23000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Sepharose 6B</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">6B100-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">UV 96-well plates</td>
			<td style="width:183px;">Costar</td>
			<td style="width:104px;">3635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fluorometry 96-well plates</td>
			<td style="width:183px;">Costar</td>
			<td style="width:104px;">3915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cell culture 96-well plates</td>
			<td style="width:183px;">Falcon</td>
			<td style="width:104px;">353072</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Tris(2-carboxyethyl)phosphine hydrochloride (TCEP )</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">20490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">dithiothreitol (DTT)</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">20290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">sulfo-LC-SPDP</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">21650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Spectrophotometer</td>
			<td style="width:183px;">Molecular Devices&#160;</td>
			<td style="width:104px;">SpectraMax 190</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Spectrofluorometer</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:104px;">SpectraMax Gemini EM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Orbital shaker incubator</td>
			<td style="width:183px;">VWR</td>
			<td style="width:104px;">1575R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Horizontal airflow oven</td>
			<td style="width:183px;">Shel Lab</td>
			<td style="width:104px;">1350 FM</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Centra-CL2 centrifuge&#160;</td>
			<td style="width:183px;">International Equipment Company</td>
			<td style="width:104px;">426</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Digital vortex mixerer</td>
			<td style="width:183px;">Fisher Thermo Scientific</td>
			<td style="width:104px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse TE300 fluorescence microscope</td>
			<td>Nikon&#160;</td>
			<td>TE300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">DC 500 CCD camera</td>
			<td style="width:183px;">Leica</td>
			<td style="width:104px;">DC-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">7500 Real-Time PCR system</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IVIS Spectrum bioluminescence station</td>
			<td>Perkins-Elmer</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">EDTA dipotassium salt</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">ED2P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin fraction V (BSA)</td>
			<td>Fisher Thermo Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Tween-20</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Dumont forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11255-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">A10 cell line&#160;</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:104px;">CRL-1476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Bovine aortic endothelial cells</td>
			<td style="width:183px;">Lonza</td>
			<td style="width:104px;">BW-6002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Luciferin, potassium salt</td>
			<td style="width:183px;">Gold Biotechnology</td>
			<td style="width:104px;">LUCK-1Ge</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Pluronic F-127</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">P2443-250G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">PBS&#160; without calcium and magnesium</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fetal bovine serum</td>
			<td style="width:183px;">Gemini Bio-Products</td>
			<td style="width:104px;">100-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Penicillin/Streptomycin solution</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">11540-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">DMEM, high glucose</td>
			<td style="width:183px;">Corning cellgro</td>
			<td style="width:104px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">0.25% Trypsin/EDTA</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">QIAamp DNA micro kit</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:104px;">56304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Power Sybr Green PCR Master Mix</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">MicroAmp Optical 96-well Reaction Plate</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">N8010560</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">4360954</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cephazolin&#160;</td>
			<td style="width:183px;">Apotex</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Loxicom (Meloxicam)</td>
			<td style="width:183px;">Norbrook</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heparin sodium</td>
			<td style="width:183px;">APP Pharmaceuticals</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Ketavet (Ketamine)</td>
			<td style="width:183px;">VEDCO</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Anased (Xylazine)&#160;</td>
			<td style="width:183px;">Lloid</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Forane (Isoflurane)&#160;</td>
			<td style="width:183px;">Baxter</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Curved Moria iris forceps</td>
			<td style="width:183px;">Fine Science tools</td>
			<td style="width:104px;">11370-31</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">&#160;Curved extra-fine Graefe forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11152-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Dumont #5 forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Vannas spring scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">15018-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fine scissors - ToughCut</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">14058-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">14101-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Vicryl suture (5-0)</td>
			<td style="width:183px;">Ethicon</td>
			<td style="width:104px;">J385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Suture thread (4/0 silk)&#160;</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">18020-40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Michel suture clips</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">12040-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wound dilator (Lancaster eye specula)</td>
			<td>KLS Martin</td>
			<td>34-149-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Michel suture clip applicator</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">112028-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Insyte Autoguard 24G IV catheter</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">381412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">2F Fogarty catheter</td>
			<td style="width:183px;">Edwards Lifesciences</td>
			<td style="width:104px;">120602F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Teflon tubing</td>
			<td style="width:183px;">Vention</td>
			<td style="width:104px;">041100BST</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">PTA catheter</td>
			<td style="width:183px;">NuMed</td>
			<td style="width:104px;">custom order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Gauze pads</td>
			<td style="width:183px;">Kendall Healthcare</td>
			<td style="width:104px;">9024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cotton applicators</td>
			<td style="width:183px;">Solon Manufacturing</td>
			<td style="width:104px;">WOD1003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td style="width:104px;">281321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">10 ml syringe (Luer-Lok)</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">1 ml syringe (Luer-Lok)</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Clippers with #40 blade</td>
			<td style="width:183px;">Oster&#160;</td>
			<td style="width:104px;">78005-314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Transpore surgical tape</td>
			<td style="width:183px;">3M</td>
			<td style="width:104px;">MM 15271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube vet ointment</td>
			<td style="width:183px;">Pharmaderm</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Vector Release Experiments
Tethering of adenoviral vectors to the surface of implants, including interventional devices such as endovascular stents, approximates the vector to the disease site, partially obviating the lack of vectors&#8217; physical targeting. However, to be able to achieve therapeutic effects via the transduction of target tissue, the vector must be released from the surface (Figure&#160;2). The use of hydrolysable cross-linkers was hypothesized to allow 1) effective attachment of vectors to polybisphosphonate-modified, thiol-installed metal implants and 2) sustained release mediated by hydrolysis of the cross-linker.
The number of genomic copies of AdeGFP vector associated with the mesh disks by the end of the derivatization procedure is determined by PCR of viral DNA with eGFP-specific primers (Figure&#160;3). Depending on the specific cross-linker used, the amount of bound Ad vector should vary between 3.5 x 109 and 5.7 x 109 particles/mesh. This amount is in fair correspondence with the estimated maximal capacity (2.5 x 109) of a single mesh disk with a total area of 0.25 cm2 to accommodate 100-nm Ad particles in a monolayer arrangement. Since the starting amount of Ad vector at the step of cross-linker modification is 5 x 1011 particles, only ~1% of the modified vector is eventually immobilized on the PABT/PEIPDT-derivatized stainless steel surface.
Both the rate of in-chain ester hydrolysis and the number of links between the vector and the biomaterial are expected to affect the overall kinetics of Ad vector release from the surface.
To facilitate the release experiments, adenoviral particles can be pre-labeled with Cy3 fluorophore (Protocol; section 1) prior to cross-linker modification and surface immobilization (Protocol; section 3). The decrease of surface-associated fluorescence over time assessed concurrently by fluorometry (Figure&#160;4A) and fluorescence microscopy (Figure&#160;4B) can then be used as an indirect method for monitoring vector release into physiological buffer. Ad vectors attached through RHC and IHC linkages should demonstrate significantly faster release in comparison with their SHC- and NHC-attached counterparts. In the given example, a 45% and a 39% loss of surface-associated vector was observed with RHC and IHC-tethered vector by day 30, respectively, while less than 20% of tethered vector was observed to be released in SHC and NHC-immobilized samples (Figure&#160;4A).
Additionally, Ad vectors immobilized by using different concentrations of the same HC and thus presumably having differing numbers of tethers between the vector and metal substrate should have dissimilar kinetics of vector release. Indeed, vector modification using 0.1 mM RHC resulted in significantly faster release rates than observed for vector immobilized with 0.5 mM RHC (Figure&#160;4A). The fluorescence microscopy data (Figure&#160;4B) correlates well with the fluorometry-based quantitative vector release analysis, demonstrating a faster and more profound decrease of surface-associated fluorescence with mesh samples formulated using IHC-, RHC- and especially low concentration RHC-tethered vectors in comparison with their NHC- and SHC-tethered counterparts.
In vitro&#160;Transduction with Mesh Immobilized Ad Vectors
By being compatible with both quantitative expression analysis (fluorometry) and spatial observations (fluorescence microscopy), AdeGFP is the preferred reporter vector to monitor transduction in SMC and endothelial cell cultures treated with both free and mesh-immobilized Ad vectors. Chemical modification of Ad capsid with RHC (Figure&#160;5) as well as with other cross-linkers (not shown) at concentrations exceeding 75 &#181;M significantly impairs transduction effectiveness of non-immobilized vector in vitro, most probably due to masking and reconfiguring of fiber knob domains utilized by Ad for binding to the cells through the Coxsackie-Adenovirus receptors (CAR). However, the role of knob-CAR interactions is less vital for the transduction with substrate immobilized vector since the vector is retained in the vicinity of targeted cells by tethering to substrate. In cell culture experiments regardless of the HC type, mesh-associated Ad vectors are consistently capable of spatially restricting transduction events to cells located within 300-500 &#181;m from the mesh borders (Figures&#160;6A and 6C). Typically the peak levels of transgene expression are achieved 3-5 days after the placement of AdeGFP-loaded meshes into SMC (Figures&#160;6A and 6B) or BAEC (Figures&#160;6C and 6D) cultures. At the same vector load, peak eGFP expression levels increase in the following sequence: NHC-, SHC-, IHC- and RHC-tethered vector, reflecting the differences in their respective release kinetics profiles (Figure&#160;4). Furthermore, more durable transgene expression is observed in cells treated with IHC-formulated meshes in comparison to cells treated with the RHC-formulated counterparts (Figure&#160;6D).
The utilized cell culture system presents a convenient set-up for the investigation of delayed transduction events brought about by vector particles released from the metal surface at or later than 48 hr after mesh placement. In this application, after determining eGFP expression by fluorometry and fluorescence microscopy at 48 hr after commencement of transduction, BAEC are trypsinized and aspirated out of the wells without disturbing the meshes. Freshly passaged non-transduced BAEC are then re-plated over the partially released meshes. &#8220;New&#8221; transduction events caused by the virus particles released from the mesh carrier after the 2 day time point are then assessed by fluorescence microscopy and fluorometry (Figures&#160;7A and 7B). Robust eGFP expression demonstrating the characteristic spatial restriction to a mesh locale is typically observed in these &#8220;new&#8221; cultures (Figure&#160;7), confirming a well-preserved transduction capacity of the mesh-bound Ad vectors even after 48 hr exposure to complete cell culture medium at 37 &#176;C.
In vivo&#160;Studies
To examine potential effects related to the use of different HC on arterial transduction, animals implanted with AdLuc-eluting stents prepared with RHC-, IHC-, SHC- and NHC-modified vector underwent bioluminescent imaging 1 and 8 days after stent deployment (Figure&#160;8). The imaging was carried out following local perivascular administration of luciferin (2 mg) co-formulated with Pluronic gel. This formulation is a viscous fluid when chilled on ice, but undergoes immediate phase transition to gel when brought in contact with tissue at 37 &#176;C. Preliminary experiments (not shown) demonstrated that perivascular delivery of luciferin results in more stable and reproducible luminescence signals in AdLuc-transduced vascular tissue when compared to systemic (intraperitoneal or intravenous) luciferin administration.
If virus tethering to the stent and the stent deployment surgery were technically sound, a well-defined signal corresponding to the stented segment of rat carotid artery is emitted and recorded. One day after stent implantation, animals treated with RHC-formulated AdLuc stents typically exhibit the highest luminescence signal, followed by the rats receiving IHC- and SHC- formulated stents (Figures&#160;8A and 8B). No perceptible signal is observed in the group of rats implanted with stents prepared using NHC-tethered AdLuc, underscoring the importance of unimpeded vector release from the stent for effective transduction of vascular tissue (Figures&#160;8A and 8B). By day 8, the average intensity of luciferase expression with RHC-formulated stents drops several-fold, while it increases 1.4- and 1.8-fold with IHC- and SHC-formulated stents, respectively (Figures&#160;8A and 8B). This finding is consistent with the hypothesis of the more durable vascular transduction with gene-eluting stents exhibiting a slower kinetics of vector release from the stent surface.
<img alt="Figure 1" src="/files/ftp_upload/51653/51653fig1highres.jpg" /> 
Figure 1. Structural formulas of cross-linkers (NHC, SHC, IHC and RHC) used throughout the described studies (modified with permission28).
<img alt="Figure 2" src="/files/ftp_upload/51653/51653fig2highres.jpg" /> 
Figure 2. A scheme representing Ad vector tethering to a PABT/PEIPDT-modified stainless steel surface via a HC and subsequent release of the vector upon cross-linker hydrolysis (modified with permission28).
<img alt="Figure 3" src="/files/ftp_upload/51653/51653fig3highres.jpg" /> 
Figure 3. PCR-based quantification of AdeGFP immobilized via cross-linker tethering on the surface of PABT/PEIPDT-modified stainless steel meshes.&#160;The stainless steel meshes were formulated with AdeGFP immobilized via RHC, IHC, SHC, or NHC (n = 3 for each condition). After proteinase K treatment, viral DNA was eluted and purified using MinElute columns. Amplification of viral DNA using eGFP-specific primers was carried out in a 7500 Real-Time PCR engine and was detected with Sybr Green. Data normalization was based on a calibration curve prepared with a known amount of non-immobilized AdeGFP(modified with permission28).
<img alt="Figure 4" src="/files/ftp_upload/51653/51653fig4highres.jpg" /> 
Figure 4. Release kinetics of Ad vector tethered to metal substrates with HC. Stainless steel meshes were derivatized with ~2 x 109 Cy3-labeled Ad particles attached to the PABT/PEIPDT-modified surface after modification with 500 &#181;M NHC, SHC, IHC, RHC and 100 &#181;M RHC (designated as RHC-L). The release of fluorescently labeled Ad-particles was studied at the indicated time points by (A) well-scan fluorometry at 550/570 nm and (B) fluorescent microscopy (rhodamine filter set; original magnification 200X). Fluorometry results are presented as means &#177; SEM, n = 8-10; p &#60; 0.001 for all comparisons between the NHC and SHC vs IHC, RHC and RHC-L groups were determined by Anova with a post-hoc Tukey test (modified with permission28).
<img alt="Figure 5" src="/files/ftp_upload/51653/51653fig5highres.jpg" /> 
Figure 5. Transduction effectiveness of non-immobilized AdeGFP following modification with RHC. Rat embryonic aorta-derived SMC (A10 line) were transduced at a MOI of 1,000 with either unmodified AdeGFP or vector modified with RHC as indicated. A reporter expression was determined fluorometrically (485/535 nm) 48 hr&#160;after transduction (modified with permission27).
<img alt="Figure 6" src="/files/ftp_upload/51653/51653fig6highres.jpg" /> 
Figure 6. Transduction of cultured SMC and BAEC with Ad vector immobilized to stainless steel meshes with HC. Meshes were derivatized with ~2 x 109 AdeGFP particles appended via NHC, SHC, IHC, and RHC. Meshes were then individually placed on top of sub-confluent A10 (A, B) and BAEC (C, D) monolayers. Transduction (expressed as eGFP expression levels) of cells treated with Ad vector-tethered meshes was assessed by fluorescence microscopy (A, C; FITC filter set; original magnification 100X) and well-scan fluorometry (B, D). Fluorometry results are presented as means &#177; SEM, n = 4 (modified with permission28). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51653/51653fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/51653/51653fig7highres.jpg" /> 
Figure 7. Transduction competence of substrate immobilized Ad vectors at delayed time points. AdeGFP was immobilized on steel meshes through NHC, SHC, IHC or RHC tethers. The meshes were placed on the subconfluent BAEC monolayers. Two days after placement, BAEC were trypsinized and removed without disturbing the meshes. New, non-transduced BAEC were then seeded over the meshes. AdeGFP transduction competency was measured by eGFP expression using fluorescent microscopy (A; FITC filter set; original magnification 100X) and fluorometry (B). Measurements were taken at indicated days, representative images were used and fluorometry results are means &#177; SEM, n = 4 (modified from with permission28). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51653/51653fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/51653/51653fig8highres.jpg" /> 
Figure 8.Transduction efficiency of stent-immobilized AdLuc in vivo. AdLuc (1.3 x 1010 particles) was tethered to endovascular stents via NHC, SHC, IHC or RHC (n = 3-4 for all groups). Transduction efficiency of AdLuc eluted from stents was measured by bioluminescence imaging (IVIS Spectrum) 1 and 8 days post stent deployment (A), and plotted using a logarithmic scale (B) (modified from with permission28). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51653/51653fig8highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K Views.  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. 

Video: 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.

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

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

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.

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.

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

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

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.

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 ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...