AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H1362-H1368, 2004. First published May 6, 2004; doi:10.1152/ajpheart.00193.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/3/H1362    most recent
00193.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sasse, A.
Right arrow Articles by Schrader, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sasse, A.
Right arrow Articles by Schrader, J.

Vascular transfer of adenovirus is augmented by nitric oxide in the rat heart

Alexander Sasse, Zhaoping Ding, Martina Wallich, Axel Gödecke, and Jürgen Schrader

Institut für Herz und Kreislaufphysiologie, Heinrich Heine University, 40225 Duesseldorf, Germany

Submitted 26 January 2003 ; accepted in final form 16 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reversible opening of the endothelial barrier remains a major obstacle when hearts are transfected via the coronary system. Our aim was to establish an experimental system permitting the continuous analysis of vascular transfer of virus in the intact heart. Isolated saline-perfused rat hearts were inverted and covered with a latex cap to collect interstitial transudate (IT) on the pericardial surface. Adenovirus (109 pfu/ml) was stably labeled with rhodamine fluorescent dye. Analysis of IT and coronary perfusate revealed that under baseline conditions, adenovirus in the IT reached 75% of its vascular concentration within 3 min. The nitric oxide-donors S-nitroso-N-acetyl penicillamine (SNAP) and bradykinin (BK) were the most effective substances to increase total IT volume and adenoviral interstitial concentration. Perfusion with 9% serum markedly reduced IT volume flow and delayed the SNAP/BK effect. Our findings demonstrate that SNAP and BK effectively increased coronary transfer of adenovirus suggesting that the inverted isolated heart is a suitable model to optimize vascular transfer of virus under standardized conditions.

endothelial permeability; transvascular and transcoronary gene transfer; S-nitroso-N-acetyl penicillamine; interstitial transudate

IN THE TREATMENT OF HEART diseases, such as heart failure, ischemia, and arrhythmias, gene transfer among other modalities has evolved as a major therapeutic option. In myocardial gene transfer experiments, the adenovirus is the preferred vector because of its large cloning capacity, the nonintegration into the host DNA, an early gene expression, and the availability of high titers (31). Whereas the adenovirus represents the most commonly used vector system, the modalities of local gene delivery to the heart vary substantially and include local intramyocardial injection (19), antegrade (5, 8, 15, 26) and retrograde coronary perfusion (6), and intrapericardial injection techniques (13, 21, 25), as well as high-pressure instillation into the left ventricle (17). Furthermore, ex vivo approaches have been explored with virus delivery via the coronary circulation and transplantation of the transfected hearts (4, 16, 20). Although almost all protocols show some degree of myocardial transfection, the overall transfection rate is generally low and often inhomogeneous. Moreover, little is known on the route of adenovirus transfer from the vascular space to the cardiac interstitium. This includes mechanisms by which the adenovirus passes the endothelial barrier in the heart, which is known to be of the nonporous and continuous form.

Little is known of the factors that influence the extent and kinetics of the viral transport process. Whereas perfusion pressure remains one of the most important driving forces for the shift of adenovirus into the interstitial space (17), several substances, such as bradykinin (9, 33), serotonin (9), rmp-7 (11), histamine (24), VEGF (8), nitric oxide (NO) (8), the cGMP-specific phosphodiesterase inhibitor zaprinast (33), increased calcium (9), {alpha}-thrombin (30), protamine (22), and mannitol (10) have been used in gene transfer protocols with varying success. There are presently also no data available on the kinetics of the pharmacologically stimulated virus uptake (10, 11, 22).

The purpose of the present study was to establish a model permitting the continuous evaluation of the uptake of fluorescence-labeled adenovirus particles from the vascular into the interstitial space by using the isolated inverted rat heart. In this system, the interstitial transudate (IT) was continuously collected, and this enabled us to investigate the role, optimal concentration, and kinetics of various vasodilators on fluid flow and their effect on virus passage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Latex MR Revultex was a gift of H. D. Cotterell (Hamburg, Germany). Rotilabo tube (model 9557.1) was from Roth (Karlsruhe, Germany). Donor horse serum, heat inactivated at 55°C, S-9135 was from Biochrome Seromed (Berlin, Germany). HES 200/0.5 was from Fresenius Kabi (Hamburg, Germany). Histamine, bradykinin, serotonin, and penicillin/streptomycin (cat. no. P 0781) were from Sigma (St. Louis, MO). Adenosine was from Boehringer-Mannheim (Mannheim, Germany), synaptosomal-associated protein (SNAP) was from Biomol (Hamburg, Germany), and RMP-7 (Cereport) was a gift from Preclinical Research and Development (Alkermes; Cambridge, MA). Glycine was from Degussa. Sephadex G-50 fine column was from Amersham-Pharmacia-Biotech. Centrifugal filter devices (model YM-10, 10,000 mol/wt) were from Centricon. Human embryonic kidney (HEK)-293 cells were from DSMZ (Braunschweig, Germany); HEK-293 N3S suspension cells were from Microbix Biosystems (Ontario, Canada). Jokliks medium (cat. no. 22300–107) was from GIBCO-BRL. Rhodamine (C-1171) and nanoparticles fluospheres (0.044 µm; orange; 540/560 nm) were from Molecular Probes (Eugene, Oregon).

Isolated perfused heart: reverse heart model. All animal experiments were conducted in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" (3a). Male Wistar rats (280–300 g, heart weight: {approx}1 g) were anesthetized with ether, and after opening of the thorax, the hearts were quickly removed, and the aorta was cannulated. Perfusion was initiated in a nonrecirculating manner using Krebs-Henseleit buffer equilibrated with 95% O2-5% CO2 at 37°C. Hearts were perfused at a constant pressure of 65 mmHg. Proximal to the aortic cannula coronary flow (ultrasonic flowmeter; Transonic) and pressure (Statham transducer model P23 XL) were recorded. To permit measurement of left ventricular pressure development, a latex balloon mounted on a catheter was inserted in the left ventricle via the left atrium. After an equilibration period of 20 min, the Langendorff-perfused heart was changed to the reverse heart model.

In the reverse heart model, the heart was fixed to a rigid catheter via the left ventricular balloon and inverted according to published protocols (7, 32). The ventricular surface was covered with a latex cap (thickness: {approx}0.2 mm), which was previously cast as appropriate for the average shape of the heart. Thereafter the apex of the latex cap was connected to a silicon-tube, which ended 4 cm below the base of the heart, allowing for minimal suction. The IT, continuously produced and excreted on the epicardial surface into the pericardial space, was collected in 2-ml Eppendorff caps, and flow was determined by weighing the collected fluid. The coronary venous effluent perfusate was sampled separately by collecting the outflow from the inverted heart via pulmonary artery and right atrium. Heart rate, developed left ventricular pressure, coronary flow, and coronary pressure remained unchanged after inversion of the heart and covering of the ventricles with the latex cap (Fig. 1).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Schematic drawing of the reverse heart experimental setup. 1, heart is inverted and perfused via aortic cannula. 2, interstitial transudate (IT) is collected on the epicardial surface of the heart with a latex cap and gathered separately. 3, venous effluent perfusate is collected. 4, to permit measurement of left ventricular pressure development, a latex balloon, mounted on a catheter, was inserted in the left ventricle via the left atrium.

 
Unless indicated otherwise, hearts were perfused with Krebs-Henseleit buffer containing (in mM) 116 NaCl, 4.6 KCl, 1.1 MgSO4, 1.18 KH2PO4, 24.9 NaHCO3, 8.32 glucose, 2.0 pyruvate, and 2.52 CaCl (salts from Merck). In specified experiments, hearts were perfused with 2.5–6.5% poly(O-2-hydroxyethyl) starch (HES) or serum (heat-inactivated horse serum, added at a rate of 3 and 5% of the continuously measured coronary flow). Total protein concentration of the effluent perfusate in serum-perfused hearts was determined by using the Lowry method; perfused buffer with 3 and 5% serum contained 4.8 ± 2.4 (n = 12) and 8.8 ± 2.8 mg/ml BSA total protein (n = 8), respectively. Compared with the normal reference range in rats of representative gender and age (56.0 ± 3.0 g protein/l) the detected protein concentrations in our model are comparable to a concentration of 8.5% (in 3% serum perfusion) and 15.7% (in 5% serum perfusion) found in the in vivo situation (35).

Virus production and labeling. Adenovirus stocks were prepared by infection of human embryonic kidney (HEK)-293 cells with {Delta}E1/{Delta}E3-recombinant Ad5.GFP (Qbiogene; Carlsbad, CA) at a multiplicity of infection of 25 pfu/cell. Cells and medium were harvested 48 h after infection, centrifuged at 2,000 rpm for 5 min, and resuspended in sterile PBS. The virus was released by four cycles of freezing and thawing. Viral preparations were purified by CsCl density centrifugation and virus band was extracted and dialyzed at 4°C in 4 liters of solution [3.16 liters H2O, 400 ml PBS, 40 ml 10 mM MgCl2, 400 ml 87% glycerol (Sigma), 40 ml of 50 µM CaCl2] for 4 h. This was repeated twice. Virus was then stored at 4°C for immediate processing or at –80°C for later preparation.

HEK-293 cells for virus assays and HEK-293 N3S suspension cells for virus production were grown in Jokliks medium supplemented with 10% horse serum and 1% penicillin/streptomycin. Viral titer was estimated by measuring the absorbance of the viral DNA at 260 nm (A260) after incubation in lysis buffer (1:20 in 0.1% SDS, 10 mM Tris·HCl pH 7.4, 1 mM EDTA; 56°C, 10 min). The titer was calculated from these data assuming a molecular weight of 2.5·107 g·mol for the adenoviral genomic DNA and a DNA concentration of 50 µg/ml at an A260 = 1. Virus titer was also determined by plaque assay with HEK-293 cells as described elsewhere (29).

Adenovirus was stably labeled with rhodamine fluorescence dye according to published protocols (23). In brief, rhodamine was dissolved in DMSO at a concentration of 10 mg/ml and stored at –20°C. Buffer (490 µl buffer; 0.2 M NaHCO3, pH 8.3) and adenovirus (500 µl; 109 – 1010 pfu/ml) were mixed; 10 µl rhodamine solution was added and after mixing were incubated for 30 min at room temperature. Glycine (110 µl; 2% dissolved in PBS) are added. Free rhodamine and rhodamine attached to adenovirus were separated by using a Sephadex G-50 column. Fluorescence was detected with Fluoromax 3 (Jobin-Yvon Horiba; Edison, NY). The virus titer of this solution was determined in parallel to fluorescence to enable the calculation of a standard curve for the assessment of virus titer from fluorescence measurement.

To determine the amount of rhodamine attached to the adenovirus, the virus preparation was centrifuged over a filter (cutoff 10,000 mol/wt) at 3,000 g for 30 min. Fluorescence (93.3 ± 4.2%; n = 4) remained above the filter representing rhodamine attached to adenovirus. The rhodamine-labeled adenovirus retained its infectious properties, because infected HEK-293 cells displayed positive green fluorescent protein (GFP) staining after 48 h.

Statistics. Results were analyzed by one-way ANOVA and unpaired t-test using GraphPad Prism version 3.0 (GraphPad Software). Data are expressed as means ± SD. In all experiments with measurement of IT flow or fluorescence, baseline values were subtracted from results.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mean IT fluid collected under baseline conditions via the pericardial cover within the first hour at a coronary perfusion pressure of 65 mmHg was 41.5 ± 2.3 µl/min (n = 6). Mean coronary flow equaled 14.9 ± 2.3 ml/min (n = 6). Therefore, the IT-flow represents 0.43% of the fluid perfused through the coronary system. IT-flow remained stable over the first 2 h of perfusion; thereafter, IT-flow continuously increased in a linear fashion (slope 1.03 ± 0.07, r2 = 0.56). All further experiments were conducted within the first 120 min of heart perfusion.

To evaluate the effect of changes in oncotic pressure on IT flow, the perfusion medium was supplemented with heat-inactivated horse serum (9 and 16% of normal total protein in vivo) and HES (2.5, 5.0, 6.5%). From the results shown in Fig. 2, it can be seen that 9 and 16% serum decreased IT flow to 30 ± 10.1 and 13.8 ± 5.3%, respectively. Similarly, HES, a substance known to increase the intravascular oncotic pressure (1) also dose dependently decreased IT. At 2.5 and 5% HES, IT flow was ~50%, whereas 6.5% HES further reduced IT flow to 20%.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2. Effect of the heat-inactivated serum and O-2-hydroxyethyl (HES) on the IT fluid collected when perfusing in the reverse heart model. n, Number of experiments. Percents are relative share of serum and HES, respectively.

 
In a further experimental series, influence of substances known to alter capillary permeability were analyzed. From the data summarized in Fig. 3, it can be seen that in the absence of serum, serotonin (5 µM), potent bradykinin-2 receptor agonist RMP-7 (25 nM) (10), bradykinin (1 µM), and the NO-donor SNAP (1 µM) significantly increased IT. The concentration chosen for each substance maximally increased IT as was derived from separate dose-finding studies. The most potent substance was SNAP, which doubled IT flow. Histamine (5 µM) and adenosine (3 µM), despite substantially increasing coronary flow, proved to be ineffective. The combined application of bradykinin (1 µM) and serotonin (5 µM) increased IT by 67 ± 29% (n = 2), an effect similar to a single application of bradykinin or serotonin. In the presence of 9% serum (Fig. 3B), only the bradykinin- and SNAP-mediated increase of IT flow reached the level of significance. The vasodilator adenosine, which maximally increased coronary flow, did not change IT-flow. In the histamine experiments, we noted that the hearts became severely edematous with an increase of heart weight from 1.05 ± 0.08 to 1.32 ± 0.1 g wet wt (n = 7, P < 0.001, t-test).



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. Changes of IT relative to baseline conditions (100%) in percent mean ± SD. Hearts are perfused with Krebs-Henseleit buffer in the reverse heart model and histamine (5 µM), serotonin (5 µM), RMP-7 (25 nm), bradykinin (1 µM), synaptosomal-associated protein (SNAP; 1 µM), and adenosine (3 µM). A: data of saline-perfused hearts. B: results from hearts perfused with 9% heat-inactivated serum additive. Displayed flow rates are measured after steady-state conditions were reached depending on kinetics of individual substances (see Table 1). *P < 0.05, **P < 0.01 compared with controls; n, number of experiments.

 
Substances tested not only differed in their ability to increase IT, they also differed in the time required to reach maximum IT. From the data summarized in Table 1, it is evident that SNAP is the most rapid-acting substance reaching its maximum effect after 23 s. In contrast, serotonin required >3 min to reach its maximal effect on IT.


View this table:
[in this window]
[in a new window]
  		Table 1. Interstitial transudate time kinetics of analyzed substances

 
To investigate the passage of the adenovirus from the vascular space to the IT, rhodamine-labeled adenovirus was perfused through the coronary system and the IT, and the coronary effluent perfusate was collected. Fig. 4 shows representative data from a single experiment; the adenovirus reached the IT with a time delay of 2 min, arrived at its peak concentration level after ~7 min, and nearly approximated the virus concentration in the effluent perfusate compared with the respective virus concentration in the vascular space. After cessation of virus perfusion, there was, as expected, a delayed washout from IT. In all of the experiments, concentration of the adenovirus in interstitial fluid reached 74.9 ± 35.3% (n = 4) compared with concentration found in effluent perfusate. Additionally, the total amount of virus reaching the pericardial space and perfused via the coronary system was calculated. Of the total virus perfused, 0.404 ± 0.027% (n = 4) reached the epicardial space; the effluent perfusate contained 99.6 ± 1.9% of the perfused virus. After a washout phase, no relevant amounts of virus are detected in the effluent perfusate. Therefore, <1% of perfused adenovirus actually penetrated the myocardial space under experimental conditions.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. Reverse heart model in the absence of serum, rhodamine-labeled adenovirus, or fluorescent nanoparticles are detected in the IT fluid or effluent perfusate. Time 0 represents the start of adenovirus/nanoparticle per fusion. Top boxes, duration. Data shown represent one typical experiment, respectively. A: perfusion with rhodamine-labeled adenovirus. B: perfusion with 44-nm nanoparticles detected by fluorescence measurement. Note that in contrast to virus experiments, nanoparticles were recirculated leading to a gradual loss in concentration.

 
To prove that fluorescence detected in IT on the epicardial surface did in fact represent rhodamine-labeled adenovirus, the IT was centrifuged for 30 min at 3,000 g through Centricon filters (cutoff 10,000 mol wt; see Materials). Of detected fluorescence, 94.25 ± 3.4% (n = 6) was found to be still associated with the virus. In separate experiments, IT containing rhodamine-labeled virus was added to HEK-293 cells in culture, and GFP-positive staining was found after 48 h.

To investigate whether the transendothelial migration of the adenovirus was solely dependant on the size of the perfused virus particles (73.1 nm OD, Ref. 12) experiments with fluorescent nanoparticles (44 nm) were performed. As shown in Fig. 4B, the concentration of the nanoparticles detected in the IT was far from reaching the equilibrium with the effluent perfusate. The average nanoparticle concentration reached in the interstitial fluid in four experiments was 23.5 ± 2.8%.

In a final experimental series, we explored the influence of bradykinin and SNAP on adenoviral migration into the IT. As shown in Fig. 5, it can be seen that SNAP and bradykinin almost doubled adenovirus concentration in the IT compared with baseline conditions. This was associated with an increase of virus concentration in the IT above concentrations measured in the effluent perfusate; in the SNAP and bradykinin experiments, the adenovirus concentration in the IT rose by 47.1 ± 34.1 and 41.9 ± 5.4%, respectively, above concentrations detected in effluent perfusate (both P < 0.001) (Fig. 6). Surprisingly, this effect was almost completely blunted by the application of 9% serum. Note, however, that the maximum values for controls were not different after the addition of 9% serum. Although less effective, serotonin significantly increased adenovirus concentration in IT to 108% compared with effluent perfusate.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Reverse heart model in the absence of serum. Rhodamine-labeled adenovirus is detected in the IT fluid or effluent perfusate. Time 0 represents start of adenovirus perfusion. Top boxes, duration. Data shown represent one typical experiment, respectively. Virus titer is calculated via standard curve from fluorescence data. A: perfusion with 1 µM bradykinin. B: perfusion with 1 µM of the NO-donor SNAP.

 


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 6. Concentration of rhodamine-labeled adenovirus in the IT measured in the reverse heart model. Adenovirus concentration is expressed as mean concentration in the IT relative to the virus concentration in the perfusate mean ± SD. Perfusion with Krebs-Henseleit buffer with and without 9% heat-inactivated serum. Hearts are also perfused with SNAP (1 µM), bradykinin (BK; 1 µM), and serotonin (5 µM). ***P < 0.001, **P < 0.01, respectively, compared with controls.

 
Similar to the time-to-peak measurement of IT (Table 1), there were also pronounced differences in the time required for the virus to reach maximum concentrations in the IT as shown in Table 2. SNAP reduced the time-to-peak value substantially; under control conditions the time to peak was almost doubled if perfused with 3% serum. Again, bradykinin and SNAP almost halved this time interval.


View this table:
[in this window]
[in a new window]
  		Table 2. Perfusion of the reverse heart model with rhodamine-labeled adenovirus

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reports that the isolated reverse heart presents a suitable model that permits the continuous measurement of two important parameters: 1) determination of water flux from the vascular space to the interstitial space as influenced by various vasodilators and in parallel and 2) measurement of the kinetics of adenoviral particle migration across the vascular barrier and its concentration in the interstitial relative to vascular space. We found that substances increasing local NO, such as SNAP and bradykinin, significantly increased the interstitial virus concentration, which is relevant for future gene therapy protocols.

Previous publications (32) have validated the isolated reverse heart model showing that interstitial substrate concentrations could be reliably determined. Lactate and glucose concentrations were measured in this model under conditions of anoxia. Moreover, interstitial adenosine and inosine kinetics under various states of oxygenation were determined by using the reverse heart model (7). The present study extends the utility of this model to the analysis and characterization of adenoviral particle and water flux across the endothelial barrier. With the use of the oncotically active components serum and HES, in the reverse heart model, both were associated with a decrease of interstitial fluid volume, underscoring the ability to accurately assess endothelial permeability in the intact heart. However, the presented model due to limited temporal stability is not suited to study transgenic expression.

In the saline-perfused heart, the adenovirus in the interstitial fluid reaches a concentration equal to ~75% of the concentration in the perfusion medium. However, when hearts were stimulated with SNAP or bradykinin, the interstitial virus concentration significantly increased above the respective vascular concentration despite the fact that interstitial fluid flow also increased. The most straightforward interpretation of this surprising finding is that SNAP and bradykinin by some unknown mechanism caused an active virus uptake across the vasculature. A recent ultrastructural study suggested that adenovirus vectors can traverse the continuous endothelium of the heart via a transcellular vesicular transport pathway (14). Alternatively, it is conceivable that there was more active water reabsorption induced by SNAP and bradykinin that would, as a result, increase interstitial virus concentration. NO has been reported to increase the permeability of individual microvessels (36); however, the mechanism is not well understood (28). Vasodilatation as such does not seem to play a role, because adenosine, which maximally increased interstitial fluid flow, did not increase the interstitial virus concentration.

The nonfenestrated and continuous coronary endothelium is likely to be the major barrier for fluid transport and virus uptake into the myocardium. However, very little is known about the mechanisms of virus transport through endothelial cells. The pathway of the adenovirus does not appear to be through gaps between adjacent endothelial cells, which average ~20 nm, although this space might be widened through endothelial contractions (3, 34). Preliminary evidence was recently provided showing that adenovirus (73 nm) most likely passes across the endothelial barrier through a vesicular transport (transcytosis) (14). Vesicles of endothelial cells have a mean diameter of 70 nm continually fusing and separating, leading to the formation of channels (28). Whether NO can stimulate transcytosis of virus particles through endothelial cells as suggested by our study remains to be investigated. Whereas the relatively large adenovirus particle is quite efficiently transported across the endothelial barrier, the permeability for considerably smaller nanoparticles (44 nm) is significantly lower (Fig. 4). Although this might, in part, be due to a different surface charge of the nanoparticles, our data point to a potentially specific adenoviral transport mechanism across the endothelium.

Transfection efficiency for intracoronally applied adenovirus is determined by the following factors: 1) the interstitial virus concentration, which is a direct function of endothelial permeability; 2) the duration of exposure of the cells with the virus; and 3) the density of the coxsackie and adenovirus receptor on cardiomyocytes (18, 27). The present experimental model is the first permitting in a beating heart the continuous measurement of the interstitial virus concentrations and the kinetics of these changes. Again the NO-donors SNAP and bradykinin were the most potent substances to rapidly increase the kinetics of virus transport. Whereas under serum-free control conditions the adenovirus required 7.4 min to reach its peak value in the interstitial fluid, this value was reached after 3.3 min when treated with SNAP. The addition of serum (9%) generally doubled the time-to-peak value interval, which is relevant for in vivo conditions in which serum is usually present. Our findings are consistent with the observation that plasma proteins are adsorbed to the endothelial cell glycocalyx and form part of the structure making up the molecular filter at the cell surface tighter (2). More importantly, serum reversed the NO-induced effect on interstitial virus concentration, thereby reducing the utility of NO for practical application in vivo. Due to the presence of hemoglobin as an important NO scavenger, the situation in vivo will be even less favorable for the use of NO. However, in a recent study (7a), we succeeded in transfecting 43% of the in vivo rat heart by flushing the coronary system with serum-free medium before the application of virus.

Histamine has been often used in the literature to augment the viral transfection efficiency in the heart (24). In our experiments, however, histamine did not significantly increase the IT volume. Moreover, we found that histamine delayed the time-to-peak interstitial fluid levels and caused tachyphylaxia and visible edema of the heart. Histamine, due to its side effects, therefore appears not to be the preferred agent to facilitate gene transfer into the rat heart.

In conclusion, the present study has used a novel approach to study transvascular gene transfer into the rat heart. We made the unexpected observation that vasodilators such as SNAP and bradykinin, which most likely work through the formation of NO, substantially augment the kinetics and extent of interstitial accumulation of adenovirus in the heart. Inclusion of NO donors into a future cardiac gene therapy protocol should therefore facilitate the transfection rate of the heart.


   ACKNOWLEDGMENTS
 
Research presented in this study was supported by a grant form the Forschungskommission of the Medical Faculty of the Heinrich-Heine University Duesseldorf (to A. Goedecke).

Present address of A. Sasse: University Clinic Aachen, Medical Clinic I, Dept. Cardiology, Pauwelsstr. 30, 52057 Aachen, Germany (E-mail: asasse@ukaachen.de).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Schrader, Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Univ. Duesseldorf, Germany, Universitaetsstr. 1, 40225 Duesseldorf, Germany (E-mail: Schrader{at}uni-duesseldorf.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adams HA, Piepenbrock S, and Hempelmann G. Volume replacement solutions–pharmacology and clinical use. Anasthesiol Intensivmed Notfallmed Schmerzther 33: 2–17, 1998.[Web of Science][Medline]
  2. Adamson RH and Clough G. Plasma proteins modify the endothelial cell glycocalyx of frog mesenteric microvessels. J Physiol 445: 473–486, 1992.[Abstract/Free Full Text]
  3. Adamson RH and Michel CC. Pathways through the intercellular clefts of frog mesenteric capillaries. J Physiol 466: 303–327, 1993.[Abstract/Free Full Text]
  4. American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 283: R281–R283, 2002.[Free Full Text]
  5. Asfour B, Byrne BJ, Baba HA, Hammel D, Hruban RH, Weyand M, Deng M, and Scheld HH. Effective gene transfer in the rat myocardium via adenovirus vectors using a coronary recirculation model. Thorac Cardiovasc Surg 47: 311–316, 1999.[Web of Science][Medline]
  6. Barr E, Carroll J, Kalynych A, Tripathy S, Kozarsky K, and Wilson JM. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther 1: 51–58, 1994.[Web of Science][Medline]
  7. Boekstegers P, von Degenfeld G, Giehrl W, Heinrich D, Hullin R, Kupatt C, Steinbeck G, Baretton G, Middeler G, Katus H, and Franz WM. Myocardial gene transfer by selective pressure-regulated retroinfusion of coronary veins. Gene Ther 7: 232–240, 2000.[CrossRef][Web of Science][Medline]
  8. Decking UK, Juengling E, and Kammermeier H. Interstitial transudate concentration of adenosine and inosine in rat and guinea pig hearts. Am J Physiol Heart Circ Physiol 254: H1125–H1132, 1988.[Abstract/Free Full Text]
  9. Ding Z, Fach C, Sasse A, Godecke A, and Schrader J. A minimally invasive approach for efficient gene delivery to rodent hearts. Gene Ther 11: 260–265, 2004.[CrossRef][Web of Science][Medline]
  10. Donahue JK, Heldman AW, Fraser H, McDonald AD, Miller JM, Rade JJ, Eschenhagen T, and Marban E. Focal modification of electrical conduction in the heart by viral gene transfer. Nat Med 6: 1395–1398, 2000.[CrossRef][Web of Science][Medline]
  11. Donahue JK, Kikkawa K, Thomas AD, Marban E, and Lawrence JH. Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin. Gene Ther 5: 630–634, 1998.[CrossRef][Web of Science][Medline]
  12. Doran S, Ren XD, Betz AL, Pagel MA, Neuwelt EA, Roessler BJ, and Davidson BL. Gene expression from recombinant viral vectors in the central nervous system after blood-brain barrier disruption. Neurosurgery 36: 965–970, 1995.[Web of Science][Medline]
  13. Elliott PJ, Hayward NJ, Huff MR, Nagle TL, Black KL, and Bartus RT. Unlocking the blood-brain barrier: a role for rmp-7 in brain tumor therapy. Exp Neurol 141: 214–224, 1996.[CrossRef][Web of Science][Medline]
  14. Fields BN, Howley PM, and Griffin DE. Fields Virology. Philadelphia, PA: Lippincott Williams & Wilkins, p. 2111–2148, 2001.
  15. Fromes Y, Salmon A, Wang X, Collin H, Rouche A, Hagège A, Schwartz K, and Fiszman MY. Gene delivery to the myocardium by intrapericardial injection. Gene Ther 6: 683–688, 1999.[CrossRef][Web of Science][Medline]
  16. Giordano F, Toman J, Huang Y, Nath A, Hickey R, and Coven D. Can adenovirus vectors traverse the endothelium by transcytosis (Abstract)? Mol Ther 3: S6–S7, 2001.
  17. Giordano FJ, Ping P, McKirnan MD, Nozaki S, DeMaria AN, Dillmann WH, Mathieu-Costello O, and Hammond HK. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med 2: 534–539, 1996.[CrossRef][Web of Science][Medline]
  18. Gojo S, Niwaya K, Taniguchi S, Nishizaki K, and Kitamura S. Gene transfer into the donor heart during cold preservation for heart transplantation. Ann Thorac Surg 65: 647–652, 1998.[Abstract/Free Full Text]
  19. Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey JK, Dec GW, Semigran MJ, and Rosenzweig A. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci USA 95: 5251–5256, 1998.[Abstract/Free Full Text]
  20. Ito M, Kodama M, Masuko M, Yamaura M, Fuse K, Uesugi Y, Hirono S, Okura Y, Kato K, Hotta Y, Honda T, Kuwano R, and Aizawa Y. Expression of coxsackie virus and adenovirus receptor in hearts of rats with experimental autoimmune myocarditis. Circ Res 86: 275–280, 2000.[Abstract/Free Full Text]
  21. Kass-Eisler A and Leinwand LA. DNA- and adenovirus-mediated gene transfer into cardiac muscle. Methods Cell Biol 52: 423–437, 1997.[Web of Science][Medline]
  22. Kypson AP, Peppel K, Akhter SA, Lilly RE, Glower DD, Lefkowitz RJ, and Koch WJ. Ex vivo adenovirus-mediated gene transfer to the adult rat heart. J Thorac Cardiovasc Surg 115: 623–630, 1998.[Abstract/Free Full Text]
  23. Lamping KG, Rios CD, Chun JA, Ooboshi H, Davidson BL, and Heistad DD. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol Heart Circ Physiol 272: H310–H317, 1997.[Abstract/Free Full Text]
  24. Lanuti M, Kouri CE, Force S, Chang M, Amin K, Xu K, Blair I, Kaiser L, and Albelda S. Use of protamine to augment adenovirus-mediated cancer gene therapy. Gene Ther 6: 1600–1610, 1999.[CrossRef][Web of Science][Medline]
  25. Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, Rodriguez-Boulan E, and Crystal RG. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 11: 151–165, 2000.[CrossRef][Web of Science][Medline]
  26. Logeart D, Hatem SN, Heimburger M, Le Roux A, Michel JB, and Mercadier JJ. How to optimize in vivo gene transfer to cardiac myocytes: mechanical or pharmacological procedures? Hum Gene Ther 12: 1601–1610, 2001.[CrossRef][Web of Science][Medline]
  27. March KL, Woody M, Mehdi K, Zipes DP, Brantly M, and Trapnell BC. Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors. Clin Cardiol 22: I23–I29, 1999.[Web of Science][Medline]
  28. Maurice JP, Hata JA, Shah AS, White DC, McDonald PH, Dolber PC, Wilson KH, Lefkowitz RJ, Glower DD, and Koch WJ. Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary {beta}2-adrenergic receptor gene delivery. J Clin Invest 104: 21–29, 1999.[Web of Science][Medline]
  29. McDonald D, Stockwin L, Matzow T, Zajdel MB, and Blair G. Coxsackie and adenovirus receptor (CAR)-dependent and major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses into human tumour cells. Gene Ther 6: 1512–1519, 1999.[CrossRef][Web of Science][Medline]
  30. Michel CC and Curry FE. Microvascular permeability. Physiol Rev 79: 703–761, 1999.[Abstract/Free Full Text]
  31. Mittereder N, March KL, and Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 70: 7498–7509, 1996.[Abstract]
  32. Nevo N, Chossat N, Gosgnach W, Logeart D, Mercadier JJ, and Michel JB. Increasing endothelial cell permeability improves the efficiency of myocyte adenoviral vector infection. J Gene Med 3: 42–50, 2001.[CrossRef][Web of Science][Medline]
  33. Pfeifer A and Verma I. Gene therapy: promises and problems. Annu Rev Genomics Hum Genet 2: 177–211, 2001.[CrossRef][Web of Science][Medline]
  34. Strupp M and Kammermeier H. Interstitial lactate and glucose concentrations of the isolated perfused rat heart before, during and after anoxia. Pflügers Arch 423: 232–237, 1993.[CrossRef][Web of Science][Medline]
  35. Sugita M and Black KL. Cyclic GMP-specific phosphodiesterase inhibition and intracarotid bradykinin infusion enhances permeability into brain tumors. Cancer Res 58: 914–920, 1998.[Abstract/Free Full Text]
  36. Weselcouch EO, Lucchesi KJ, Luneau CJ, and Gosselin RE. Transcapillary transport of solutes in the myocardium: effects of nitroglycerin, isoproterenol and histamine on permeability-surface area products of inulin and sucrose. J Pharmacol Exp Ther 234: 19–24, 1985.[Abstract/Free Full Text]
  37. Wolford ST, Schroer RA, Gohs FX, Gallo PP, Brodeck M, Falk HB, and Ruhren R. Reference range data base for serum chemistry and hematology values in laboratory animals. J Toxicol Environ Health 18: 161–188, 1986.[Web of Science][Medline]
  38. Yuan Y, Granger HJ, Zawieja DC, and Chilian WM. Flow modulates coronary venular permeability by a nitric oxide-related mechanism. Am J Physiol Heart Circ Physiol 263: H641–H646, 1992.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/3/H1362    most recent
00193.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sasse, A.
Right arrow Articles by Schrader, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sasse, A.
Right arrow Articles by Schrader, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.