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J Appl Physiol 95: 1688-1694, 2003. First published July 3, 2003; doi:10.1152/japplphysiol.00896.2002
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INNOVATIVE METHODOLOGY

Adeno-associated virus mediated gene delivery into coronary microvessels of chronically instrumented dogs

Heiner Post,1 Jan Kajstura,2 Biao Lei,1 William C. Sessa,3 Barry Byrne,4 Piero Anversa,2 Thomas H. Hintze,1 and Fabio A. Recchia1

Departments of1Physiology and 2Medicine, New York Medical College, Valhalla, New York 10595;3Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; and 4Department of Molecular Genetics and Microbiology, Centers for Gene Therapy and Mammalian Genetics, University of Florida College of Medicine, Gainesville, Florida 32610

Submitted 30 September 2002 ; accepted in final form 27 May 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The objective of this study was to assess the potential of adeno-associated virus (AAV)-mediated gene delivery into coronary microvessels in vivo in a large animal. Ten mongrel dogs were chronically instrumented and allowed to recover for 10 days. Dogs were reanesthetized, and the aorta was constricted by a hydraulic occluder, whereby left ventricular (LV) pressure increased by 30% and left circumflex coronary artery blood flow by 50%. Recombinant AAV (serotype 2, CMV enhancer/chicken {beta}-actin promoter) encoding for green fluorescent protein (GFP) was injected as a bolus into the left atrium during aortic constriction at total titers of 1010 or 1012 infectious units. Dogs were followed for 2 (n = 4)or4wk(n = 6). Hemodynamics or body weight did not change. In LV tissue slices, a fluorescein-labeled antibody to GFP stained endothelial and smooth muscle cells but was absent in myocytes. To quantify transduction, slices were then stained with antibodies against {alpha}-smooth muscle actin or von Willebrand factor. Approximately 4% of arterioles and 2% of microvessels stained positive for anti-GFP independent from viral titer or duration. By regression analyses, the percent of vessels transfected was proportional to the increase in LV systolic pressure during occlusion. AAV is a potential vector for gene transfer into the coronary microcirculation in large animals, including perhaps humans.

white cells; hemodynamics; green fluorescent protein; von Willebrand factor

THE ENDOTHELIAL CELL LAYER plays a key role in maintaining vascular homeostasis by regulating vasomotion, leukocyte adherence, platelet activation, thrombosis, and vascular inflammation (24). In the coronary circulation, microvascular endothelial dysfunction underlies and limits vascular function in hypertension (4), diabetes (16), and heart failure (4) and predicts a poor prognosis in patients with coronary heart disease (22).

Gene therapy with its potential for long-term correction of changes in protein expression in transduced cells represents a potential approach for treatment of vascular diseases (12), particularly if methods are developed to transduce microvessels, the site of vascular resistance and exchange, in large animals including humans. The use of adeno-associated viruses (AAV) has many advantages. Wild-type AAV infection does not produce any symptoms in humans (2). Gene expression in skeletal muscle after AAV-mediated gene transfer has been demonstrated for up to a year in mice (11, 27) and dogs (8), indicating the potential use of AAV for long-term effective gene transfer.

Arterial gene transfer using AAV vectors has been demonstrated in normal rats (1, 6, 19), rabbits (18), and hypercholesterolemic monkeys (13). Although providing proof of principle, these studies applied vascular occlusion of 20- to 60-min duration to arrest blood flow and to provide a greater time to incubate the vessels with the vector, which, in the coronary circulation, is hardly feasible as a clinical approach. Kaplitt et al. (9) reported a small portion of transduced cardiomyocytes after intracoronary administration of AAV; however, that study did not determine the extent of coronary microvascular transduction. This is important because at least part of the cardiac dysfunction occurring in many cardiac diseases may be attributed to microcirculatory dysfunction including altered eNOS gene expression, and a number of therapies directed toward correcting coronary vascular dysfunction are effective in the treatment of heart disease.

The adequate mixing of viral particles used therapeutically on injection into the flowing blood, arterial or venous, is likely to result in a more even distribution of particles especially to blood vessels compared with intramyocardial injection. This is particularly true if the gene to be used encodes for a diffusible substance such as a local hormone, i.e., nitric oxide, which itself diffuses to adjacent cells. Endothelial cells produce a number of substances such as nitric oxide or prostaglandins that can diffuse over some distance and whose production may be decreased during disease states. With this in mind and to develop methods in large animals to address the potential mechanisms and therapies needed to correct endothelial coronary dysfunction in humans, we assessed the potential of AAV to transfer a gene into the coronary microvasculature after direct injection of the vector into the arterial circulation. This study was designed to respond to the call (14) to extend studies in small animals, particularly mice and rats, to large animal models to develop methods potentially applicable to treatment of humans. These sentiments were also expressed in an editorial (7) indicating that making the connections between basic investigative studies and gene therapy remains to be done. Studies in rodents need to be extended to large-animal models.

To demonstrate successful transduction of coronary microvessels, the vector was designed to encode for green fluorescent protein (GFP). This is important because the endothelium is the first structure encountered by a virus particle after intra-arterial or intravenous injection. In addition, because endothelial dysfunction is characteristic or pathonomic for many cardiovascular diseases, the endothelium may be an ideal target for gene transfer.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The protocols were approved by the Institutional Animal Care and Use and Biosafety Committees of the New York Medical College and conform to the Guiding Principles for the Care and Use of Animals published by the National Institutes of Health and the American Physiological Society.

Surgical procedure and instrumentation. Ten male mongrel dogs were anesthetized (25 mg/kg iv pentobarbital sodium) and instrumented with fluid-filled catheters in the left atrium and aorta, with a hydraulic occluder around the ascending aorta, and for measurement of left ventricular (LV) pressure and left circumflex coronary artery blood flow as described previously (21, 25). Dogs were allowed to recover for 10-14 days and trained to lie quietly on the laboratory table.

AAV preparation. Recombinant AAV type 2 with a cytomegalus virus enhancer/chicken {beta}-actin promoter, encoding for GFP, was generated and purified as reported previously (5, 28, 29). AAV was suspended in 1 ml of phosphate buffer, stored at -70°C, and thawed immediately before use.

Experimental protocols. The dogs were weighed, body temperature was measured, and a venous blood sample was taken from a foreleg. Baseline hemodynamics were measured while the animal was lying quietly on the laboratory table, followed by anesthesia (pentobarbital, 25 mg/kg iv) and intubation. The dog was ventilated (Harvard Instruments) at a positive end-expiratory pressure of 7 cmH2O for 30 min by using a water bottle containing 1% bleach to destroy all exhaled virus. The aortic constrictor was inflated to raise LV pressure by ~50 mmHg for 5-10 min. At steady state, the virus suspension was injected into the left atrial catheter as a bolus. The dog was kept in a separate cage for 24 h, after which feces, urine, and the cage were thoroughly disinfected with bleach. Four animals received a virus concentration of 1010 infectious units (iu) and were followed for 14 days; six animals received a virus concentration of either 1010 (n = 3) or 1012 iu (n = 3) and were followed for 28 days. Body temperature and weight were monitored daily, and venous blood sampling and hemodynamic measurements were repeated at days 3, 7, 14, 21, and 28. The animals were killed with an overdose of pentobarbital (60 mg/kg), and LV tissue samples (1 cm3) were stored in either liquid nitrogen or phosphate-buffered formalin solution (10%).

Immunohistochemistry. Frozen LV tissue sections were fixed in 3.7% formaldehyde. Fluorescence of anti-GFP anti-body was detected by confocal laser microscopy (MRC-1000, Bio-Rad) with an optical section thickness of 0.57 µm. The green fluorescence of GFP was detected but not used for quantitation because of potential nonspecific tissue autofluorescence. For additional analyses, samples of myocardium were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections 4 µm thick were deparaffinized and heated in a microwave oven in 10 mM citric buffer, pH 6.0. Subsequently, GFP was stained with goat anti-GFP (Rockland), nuclei with propidium iodide (10 mg/ml), smooth muscle cells with mouse anti-{alpha}-smooth muscle actin (clone 1A4, Sigma Chemical), endothelial cells with rabbit polyclonal anti-human von Willebrand factor (vWF; Sigma Chemical), and myocytes with mouse monoclonal anti-{alpha}-sarcomeric actin (clone 5C5, Sigma Chemical). To quantify GFP expression, two or three paraffin-embedded LV tissue sections per sample were stained with anti-GFP and either anti-{alpha}-smooth muscle actin as an indicator of arterioles, or anti-human vWF as an indicative of microvessels. The number of arterioles antibody-positive for GFP was divided by the number of arterioles counted (the same for microvessels). The number of arterioles sampled was 178 ± 10 per animal; the corresponding value for microvessels was 703 ± 18. Quantification was done by fluorescent microscopy at a magnification of x1,000. Further LV tissue sections were stained with hematoxylineosin. Tissues were saved from almost all organs in the body, but, because of the extensive time commitment to perform morphometrics on all those tissues, those studies are not included in this report.

Statistical analysis. All data are mean values ± SE. Expression of GFP was not different between dogs followed for 2 or 4 wk, or between dogs injected with 1010 or 1012 iu of AAV. Therefore, for calculation of transduction efficiency, data from dogs with 1010 and 1012 iu of AAV were combined, and data on hemodynamics, body weight, body temperature, and cell counts were pooled among all dogs. Mean values were compared by one-way ANOVA for repeated measurements, followed by Tukey's post hoc test when an overall significance was detected. Linear least squares regression analysis was performed to determine the relationship between percent of vessels transfected and LV systolic pressure during occlusion.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Hemodynamics. During pentobarbital anesthesia, heart rate increased, whereas dP/dtmax was reduced (Table 1). With aortic constriction, LV peak pressure and left circumflex coronary artery blood flow increased. None of the hemodynamics changed significantly during 2-4 wk compared with baseline values before virus administration in the conscious state.


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  		Table 1. Hemodynamics

 

Body weight and temperature. Body weight and temperature remained unchanged during 2-4 wk. For instance, body weight and temperature were 25.9 ± 0.7 kg and 39 ± 0.1°C and 26.0 ± 0.7 kg and 38.6 ± 0.1°C at control and day 28, respectively.

Differential cell counts. Hemoglobin content and the number of red and white blood cells remained unchanged during 2-4 wk (Table 2). The percentage of neutrophils was significantly decreased at days 21 and 28. The percentage of lymphocytes, eosinophils, and monocytes was unchanged.


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  		Table 2. Blood cell count

 

Standard histology. With hematoxylin-eosin staining (not shown), the LV myocardium appeared normal. No white cell infiltration was found.

Distribution of GFP. GFP was detected in vascular structures by an antibody (Figs. 1 and 2) but was absent in nonvascular structures, i.e., cardiomyocytes. In blood vessels, the antibody to GFP stained endothelial and smooth muscle cells and to a lesser degree the adventitial layer.



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  		Fig. 1. Confocal microscopy of left ventricular (LV) free wall tissue sections. Tissues were stained with anti-green fluorescent protein (GFP) antibody (green), anti {alpha}-smooth muscle actin (red) to identify smooth muscle cells, and propidium iodide (blue) to identify nuclei (A). A: nuclei of several nonsmooth muscle cells (i.e., cardiomyocytes), which did not stain with anti-GFP antibody. B: spontaneous green fluorescence is shown alone. Green fluorescence of GFP is demonstrated in endothelial and smooth muscle cells of small and large arterioles and also in the adventitial layer of the large arteriole (B).

 


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  		Fig. 2. Confocal microscopy of LV free wall tissue sections. Of 8 capillaries, identified by anti-human von Willebrand factor (vWF) (endothelial cells, red), two are positive for GFP (A, top). A, bottom: a capillary and an arteriole are positive for using the antibody to GFP. Myocytes (purple), nuclei (dark blue), endothelial cells (light blue), and smooth muscle cells (red) are shown. B: antibody staining for GFP confirms GFP expression (green) in vascular structures.

 

Quantification of GFP. The frequency of transduction of antibody-positive GFP was ~4% (range: 1.0-11.5%) in arterioles (Fig. 3) and 2% (range: 1.3-2.3%) in microvessels (Fig. 4). Transduction efficiency was not different in samples from LV septum or free wall, with transfection by either 1010 or 1012 iu (data not shown), or at 14 or 28 days. There was a correlation between the percent vessels transfected and the LV systolic pressure during occlusion of the aorta (Fig. 5).



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  		Fig. 3. Percentage of arterioles as identified by anti-{alpha}-smooth muscle actin staining that were positive for GFP using the antibody. Transduction was not different at 14 or 28 days after adeno-associated virus (AAV) administration. Data are means ± SE. fw, LV free wall; sept, LV septum.

 


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  		Fig. 4. Percentage of microvessels identified as anti-human vWF positive by use of anti-GFP antibody at 28 days after AAV administration. Data are means ± SE.

 


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  		Fig. 5. There was a linear correlation between LV systolic pressure during the period of occlusion during pentobarbital (PB) anesthesia and the transduction of coronary arterioles in the canine heart.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
We demonstrated for the first time that AAV-mediated gene delivery with a clinically feasible route of administration is able to significantly transduce coronary microvessels in a large animal species without affecting myocardial function or provoking deleterious side effects. This is the first step to answer the challenge (7, 14) to develop methods to bridge the gap between studies in rodents and humans.

Previous studies demonstrating AAV-mediated arterial gene transfer used incubation of clamped carotid artery segments with an AAV suspension, interrupting the bloodstream for 20 (19), 30 (1, 13, 18), or 60 (18) min. Whereas such procedure may be performed in the peripheral vasculature or in vein grafts before implantation during bypass surgery, it is clearly not feasible during coronary catheterization. In pigs, intracoronary infusion of an AAV preparation encoding for {beta}-galactosidase transduced cardiomyocytes up to 6 mo at a rate of 0.2% (9). Surprisingly, these authors did not report the extent of transduction of vascular structures; however, the dose of AAV used may have been too low for detectable gene delivery into vascular cells (107-108 vs. 1010-1012 iu in the present study). Whereas intramyocardial injection of AAV can transduce cardiomyocytes in rabbits (26) and rats (15), we, after injection of AAV into the atrium, did not observe expression of GFP in cardiomyocytes, even when increasing the dose of AAV from 1010 to 1012 iu. We consistently found expression of GFP in endothelial and smooth muscle cells and, although to a lesser degree, in the adventitial layer. It appears that, at a particle size of ~20 nm, vascular layers do not represent an anatomical barrier for AAV distribution as was demonstrated for adenoviruses with a particle size of 70-100 nm (20). This may indicate a considerably higher tropism of AAV, at least the type 2 serotype we used, for endothelial and smooth muscle cells rather than for cardiomyocytes in dogs.

AAV-mediated gene transfer to carotid arteries was reported to transduce all vascular layers in rat (1, 19) but exhibited a preference for smooth cells in rabbits (14) and for microvascular endothelial cells of vasa vasorum in monkeys (13). In the present study in dogs, we did not observe a preference for either endothelial or smooth muscle cells, further pointing to potential species differences. We demonstrated transduction of ~4% of arterioles both at 14 and 28 days after AAV administration, confirming stability of AAV-mediated gene transfer over time. Such transduction rate may already be pathophysiologically relevant, given that the encoded protein would release paracrine mediators that impact on vascular and cardiomyocyte function (17).

None of the dogs investigated in this study showed any clinical sign of disease, compromised myocardial function, myocardial damage, or inflammatory white cell infiltration in standard histology, in line with previous reports (20). We observed no change in the lymphocyte fraction in differential blood cell counts at days 21 and 28 after AAV administration, similar to the changes observed after intramuscular administration of AAV in dogs (8). Indeed, mice after intravenous but not after intramuscular delivery of AAV encoding for ovalbumin developed cytotoxic lymphocytes against ovalbumin (3).

The method we used to administer AAV was taken from studies validating the distribution of radioactive microspheres in the coronary circulation in the 1970s (21). This method takes advantage of the mixing in the left atrium and left ventricle before expulsion into the aorta and distribution into the coronary circulation. Additionally, partial occlusion of the aorta serves to increase coronary blood flow, as we found, via metabolic dilation and increased perfusion pressure. There was a linear relationship between LV systolic pressure during occlusion and transduction, suggesting that partial occlusion of the aorta facilitates transfection of coronary microvessels. Finally, anesthetizing and ventilating the dog into a bottle containing bleach eliminates the possibility that the vector will be exhaled to endanger laboratory workers, regardless of the infectivity. These precautions may not have been necessary under the conditions of our study, but similar precautions may be needed in the future if the gene contained within the AAV has activity in humans. Lastly, counterpulsation techniques and balloon catheters, depending on the state of contractile function, could be used to "direct" virus into the coronary circulation in humans. Although the number of endothelial cells positive for anti-GFP seems small, a few percent, the production of the gene product may be much higher. This could be altered by changing the promoter and by designing strategies for incorporation of multiple copies of the gene in the same endothelial cell.

The 2-4% of microvessels transduced after injection of AAV containing GFP may seem like a small number; however, there are literally millions of microvessels, a difficult number to calculate, in each heart. If we assume that all the blood vessels are represented by the length of the capillaries (an underestimate), then we can calculate the total capillary length in an average 72-g LV free wall. From previous studies, we have found that there are 3,011 capillaries/mm2 dog heart. If we assume that each capillary is 1 mm long, then this results in 3,011 mm of capillary/mm3 dog tissue. Times 1,000 mm3/cm3, this equals 3,011,000 mm capillary/cm3 dog heart. If dog tissue has a density of 1.06 g/cm3, then a 72-g dog LV free wall has a volume of 67.9 cm3. 3,011,000 mm capillary/cm3 x 67.9 cm3 = 204,446,900 mm capillary/dog heart. If we take an average 3% transduction, then 0.03 times 204,446,900 equals 6,133,407 mm capillary were infected by the virus and expressed GFP. We injected 1010 particles (the injection of 1012 did not increase the infection-expression index), and 10% of cardiac output goes to the coronary circulation (excluding recirculation). Then a total of 109 particles went through the coronary circulation to get 6 x 106 mm capillary transduced. This indicates that millions of blood vessels were transduced. Furthermore, because more than one virus particle may have infected each cell (which we cannot determine), because other cell types expressed the GFP (i.e., smooth muscle and white blood cells), and because not all the cells containing GFP may have been measured (the transduction rate unknown), the actual number of cells transfected after our injection of AAV is undoubtedly even greater than six million. Finally, it should be remembered that each cardiac myocyte is surrounded by three capillaries, located at the angles of an equilateral triangle, so that infection of only one in three capillaries would be needed to influence all the cardiac myocytes.

Because of the potential problem of nonspecific tissue autofluorescence when using the fluorescence properties of GFP, we used an antibody to GFP to quantitate the expression of GFP. Using these techniques, we found staining in blood vessels and not in cardiac myocytes. These conclusions would not have been different when the fluorescence of GFP was used because the nonspecific fluorescence would if anything lead to an overestimate of GFP expression.

In conclusion, we demonstrated AAV-mediated gene transfer to the coronary microcirculation in dogs using a clinically relevant route of administration and a clinically relevant vector. AAV-mediated gene transfer may prove to be a valuable tool to study coronary pathophysiology in a large animal species relevant for human disease. Furthermore, depending on the gene to be transferred, the promoter to be used, the biological and therapeutic properties of the gene product, and the number of copies of the gene expressed in each cell, our data indicate that blood vessels may be a therapeutic target in disease states associated with endothelial dysfunction.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
H. Post was supported by a grant from the German Research Foundation (Po 672/1-1). This study was supported by National Institutes of Health Grants PO1 HL-43023, RO1 HL-50142, HL-61290 (to T. H. Hintze), HL-62573 (to F. A. Recchia), HL-65577 (to J. Kajstura), PO-1 HL-59412 (to B. Byrne), and HL-39902 and AG-17042 (to P. Anversa).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, Basic Sciences Bldg., Rm. 636, New York Medical College, Valhalla, NY 10595 (E-mail: thomas_hintze{at}nymc.edu).

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
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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