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Am J Physiol Heart Circ Physiol 283: H1439-H1445, 2002. First published June 6, 2002; doi:10.1152/ajpheart.01090.2001
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Vol. 283, Issue 4, H1439-H1445, October 2002

Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

Margot C. LaPointe, Xiao-Ping Yang, Oscar A. Carretero, and Quan He

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1beta , phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.

gene transfer; cardiac-specific promoter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIOVASCULAR DISEASE IS the leading cause of death in the United States. The primary causes of cardiovascular disease, such as hypertension, coronary artery disease, cardiomyopathy, and diabetes, can be treated, but often the treatment only slows the progression of disease, and heart failure ultimately results (2). With aging of the population and increased mortality from heart failure, great interest has been focused on novel therapies such as gene transfer. For therapeutic efficacy, it is important that a gene be delivered directly to the heart and be expressed at high enough levels over a prescribed period of time to effect a response. In animal models, methods for in vivo cardiac gene transfer include direct injection of DNA into the myocardial wall, perfusion of the heart via the coronary arteries, intrapericardial injection, and catheter-based injection into the heart coupled with cross-clamping of either the aorta alone or both the aorta and pulmonary artery (6).

Presently, adenoviruses represent the best-characterized vector system for expressing recombinant proteins in cardiac myocytes in vitro and in vivo (3, 7, 16, 20). One way to improve the delivery capability of the adenovirus would be inclusion of a cardiac myocyte-specific promoter that would allow expression of the transgene only in myocytes, thus eliminating the problem of inappropriate transgene expression in other organs and avoiding a more widespread inflammatory response. The brain natriuretic peptide (BNP) gene is constitutively expressed in the ventricle and is induced by ischemic injury (21). In fact, BNP gene expression is a good marker for left ventricular (LV) dysfunction (1, 23). We have studied regulation of the human BNP (hBNP) promoter and identified some of the elements involved in cardiac myocyte-specific expression (8, 9, 11). We have also used the proximal hBNP promoter coupled to a luciferase reporter gene to generate transgenic mice and have shown that the proximal promoter confers high-level cardiac-specific expression, primarily in the ventricles. Expression of the reporter gene is virtually absent in all other tissues. In addition, this proximal promoter region responds to ischemic injury caused by coronary artery ligation, resulting in chronic expression of the reporter gene for at least 3 wk in the mouse, reflecting endogenous BNP mRNA (10). On the basis of these studies, we hypothesized that administration of an adenovirus in which the luciferase reporter gene was regulated by the proximal hBNP promoter would allow for 1) cell-specific and inducible regulation of the reporter gene in myocytes in vitro and 2) selective expression in the mouse heart in vivo.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of adenovirus vector. We previously described (18) the construction of the hBNP promoter coupled to a luciferase reporter gene. The proximal promoter from -408 to +100 coupled to firefly luciferase cDNA was subcloned into the HinDIII and PvuII sites of the Ad5mcspA shuttle vector. A recombinant, replication-defective adenovirus was constructed by homologous recombination in HEK293 cells, resulting in viral particles that we refer to as Ad.hBNPLuc. All steps of the process (construction of the shuttle vector, homologous recombination, and plaque isolation and purification) were carried out by the University of Iowa Gene Vector Core (supported in part by the National Institutes of Health and the Ray J. Carver Foundation). The viral titer was 1 × 1012 particles/ml, equivalent to ~1010 plaque-forming units (PFU)/ml. The virus was diluted in PBS-3% sucrose and stored at -70°C. A second, replication-defective adenovirus, Ad.CMVLuc, was obtained from the University of Pittsburgh Pre-Clinical Vector Core Facility. The viral titer was 2 × 1010 PFU/ml and was stored in 20 mM Tris, 75 mM NaCl, 2 mM MgCl2, 5% trehalose, and 0.0025% Tween 80 at -70°C.

Cell culture. Neonatal rat ventricular myocyte-enriched cultures were generated from Sprague-Dawley rat pups (Charles River) as described previously (17). A mouse embryonic fibroblast (MEF) cell line was obtained from Clontech. Cells were transduced with 10 PFU/cell Ad.hBNPLuc or Ad.CMVLuc and then lysed and assayed for luciferase activity at selected time points.

In vivo injection of Ad.hBNPLuc and Ad.CMVLuc. C57BL/6J mice (Jackson Laboratory) weighing at least 22 g were anesthetized with pentobarbital sodium (50 mg/kg ip). They were placed supine on a heating pad, intubated, and ventilated with a volume of 0.2 ml at a rate of 95-110 min-1. A thoracotomy was performed, and the heart was exposed. With a 30-gauge 0.5-in. needle attached to a 0.5-ml syringe, 108 PFU of adenovirus in a total volume of 50 µl of PBS-3% sucrose or 50 µl of vehicle was directly injected into the free wall of the LV from the apex upward and distributed among three or four adjacent sites of the LV free wall. Direct injection of Ad.hBNPLuc into the quadriceps muscle served as a control for cardiac muscle-specific expression. Background luciferase activity was measured in vehicle-injected tissue. The LV, right ventricle, septum, atria, lung, liver, kidney, and skeletal muscle were either removed 1-28 days after injection and assayed for luciferase activity or fixed and subjected to immunocytochemistry. We also introduced either Ad.hBNPLuc or Ad.CMVLuc directly into the LV chamber for systemic distribution throughout the mouse. Four days later, tissues were removed and examined for luciferase activity. These studies were approved by the Henry Ford Hospital Institutional Animal Care and Use Committee in compliance with Public Health Service guidelines.

Luciferase assay. Tissues were homogenized with a Polytron in 10 mM Tris (pH 7.5). After homogenization, one-fifth volume of 5× reporter lysis buffer (Promega) was added. Samples were processed further, and duplicate aliquots of each sample assayed as described previously (10). Luciferase activity (relative light units, RLU) was normalized to milligrams of protein. Data are expressed as means ± SE. Where necessary, statistical significance was determined with Student's t-test.

Immunocytochemistry. Hearts injected with either Ad.hBNPLuc or vehicle were fixed in formalin, embedded in paraffin, and cut into 5-µm sections. Sections were stained for luciferase protein with a rabbit anti-luciferase antibody (1:250 dilution; Cortex, San Leandro, CA) and an immuno-alkaline phosphatase staining kit (Biomeda) with fast red (Sigma) as the substrate (10).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell-specific and inducible expression of Ad.hBNPLuc. We compared the activity and cell type specificity of the hBNP promoter with the cytomegalovirus (CMV) enhancer/promoter in neonatal ventricular myocytes (NVM) and a MEF cell line (Table 1). The ratio of activity of Ad.CMVLuc in myocytes to that in fibroblasts was ~20 [luciferase activity in NVM: 9.2 ± 0.96 × 109 RLU/mg protein (n = 6); luciferase activity in MEF cells: 5.2 ± 1.5 × 108 RLU/mg protein (n = 6)]. The ratio of activity of Ad.BNPLuc in myocytes to that in fibroblasts was ~1,000 [luciferase activity in NVM: 13.4 ± 7.2 × 106; luciferase activity in MEF cells: 10,000 ± 5,000 (n = 6 for each)]. Thus the CMV enhancer/promoter was more active in both cell types, but it was much less cardiac specific than the hBNP promoter.

                              
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  		Table 1.   Ad.BNPLuc and Ad.CMVLuc activity in vitro

In a separate experiment, we examined time-dependent changes in transgene expression in myocytes. Luciferase activity was high 24 h after transduction and persisted for at least 96 h (Fig. 1A). Because expression was high over a 4-day period, we examined whether the proinflammatory cytokine IL-1beta and the hypertrophic growth factors phenylephrine (PE, an alpha -adrenergic agonist) and isoproterenol (Iso, a beta -adrenergic agonist) would activate the proximal promoter in the context of the adenoviral expression vector. Myocytes were transduced with virus and 24 h later were treated with IL-1, PE, or Iso for 24 h, resulting in stimulation of luciferase activity by 2.2-, 10.1-, and 2.5-fold versus control, respectively (Fig. 1B).


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  		Fig. 1.   Regulation of Ad.hBNPLuc. The y-axis is luciferase activity expressed as relative light units (RLU) per milligram of protein (A) or as the fold increase vs. control (arbitrarily set to 1; B); the x-axis expresses time (A) or treatment (B). A: time course of transgene expression; n = 6 for 24-h time point and n = 3 for other times. B: regulation of transgene expression. Virus [10 plaque-forming units (PFU)/cell] was added to cells cultured in serum-free medium. After 24 h, IL-1beta (IL, 5 ng/ml; n = 8), isoproterenol (Iso, 100 µM; n = 8), or phenylephrine (PE, 50 µM; n = 8) was added for 24 h. Each bar represents the mean ± SE. P < 0.05 for PE and IL vs. control (Cont).

In vivo injection of Ad.hBNPLuc. To test whether the proximal hBNP promoter would confer cardiac-specific expression on the luciferase reporter gene in an adenoviral vector in vivo, we directly injected either vehicle alone or 108 PFU of virus in 50 µl PBS-3% sucrose into the apex of the mouse LV. At 1, 4, 7, 14, and 28 days after injection, tissues were removed and assayed for luciferase activity. We averaged the luciferase activity from tissues removed from mice injected with PBS-3% sucrose and determined that background luciferase activity was 300-900 RLU/mg protein. Luciferase activity in the LV increased from 2,000 ± 1,000 RLU/mg protein (n = 5) on day 1 to 126,840 ± 45,181 RLU/mg protein (n = 10) on day 4 (Fig. 2A) and then declined by day 7 to 27,000 ± 5,000 RLU/mg protein (n = 5). Luciferase activity remained stable at 7,000 RLU/mg protein for 14-28 days after injection. For each time point, luciferase activity in virus-injected LVs was significantly higher than in LVs injected with vehicle (P < 0.001 by t-test).


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  		Fig. 2.   Time course of Ad.hBNPLuc expression in vivo after injection into mouse hearts. A: luciferase activity in the left ventricle (LV). The y-axis is luciferase activity expressed as RLU/mg protein; the x-axis is days after injection (A) or tissue (B). Each point is the mean ± SE for 5-10 mice. B: tissue-specific luciferase activity. Luciferase activity was measured 4 days after adenovirus injection into the LV (n = 7). Other tissues assayed included the ventricular septum (SPT), right ventricle (RV), right and left atria (AT), lung (LU), liver (LIV), kidney (KID), and skeletal muscle (SKM). The PBS control represents background luciferase activity and is the average of values from the different organs from 5 mice injected with PBS in the heart (1,013 ± 198 RLU/mg protein). Only activity in the LV was statistically different from the PBS control (**P < 0.01).

We examined all areas of the mouse heart for luciferase expression and found no luciferase activity above background level in the septum, right ventricle, or atria (Fig. 2B) at 4 days after injection. Because we were injecting the virus into the LV free wall, we considered the possibility that some virus could enter the circulation and be distributed to other tissues. We assayed luciferase activity in the lung, liver, kidney, and skeletal muscle and found no activity above background levels at the 4 day time point (Fig. 2B). Of all of these extracardiac sites, luciferase activity in the liver is of greatest importance, because the liver is a major site of adenovirus uptake and transgene expression, especially with vectors that use viral enhancers/promoters like CMV. Nonetheless, when Ad.hBNPLuc was injected into the LV free wall, luciferase activity in the liver was 573 ± 187 RLU/mg protein (n = 10), a value not statistically different from background (400 ± 121 RLU/mg protein; n = 5).

We next determined whether a higher titer of Ad. hBNPLuc (109 PFU in 100 µl of PBS-sucrose) would result in expression of the luciferase transgene in the liver. We injected Ad.hBNPLuc into the LV free wall and then measured luciferase activity in all regions of the heart and in the liver. Luciferase activity was 2.1 ± 0.6 × 105 RLU/mg protein in the LV free wall, whereas it was only 719 ± 265 RLU/mg in the liver. Activity in the atria, right ventricle, and septum was 11,434 ± 7,861, 10,678 ± 4,539, and 25,720 ± 11,260 RLU/mg protein, respectively. Thus luciferase activity is restricted to the heart, even when a high titer of Ad. hBNPLuc is used.

To further confirm luciferase gene expression in the heart, we performed immunocytochemistry on sections of mouse hearts taken 4 days after injection of either Ad.hBNPLuc or vehicle. Luciferase protein (red staining) was evident in the LV free wall in the area where the virus was injected but was absent in the control hearts (Fig. 3).


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  		Fig. 3.   Immunocytochemical detection of luciferase in the mouse heart 4 days after virus injection. A: section of PBS-injected mouse heart. B: section of Ad.hBNPLuc-injected mouse heart. Red color indicates luciferase staining. Sections have been counterstained with hematoxylin; ×400 magnification.

Finally, Ad.hBNPLuc or vehicle was injected directly into the skeletal muscle of the mouse. Four days later, luciferase activity in virus-injected skeletal muscle was not different from muscle injected with vehicle (Fig. 4). There was only background luciferase activity in the LV of mice injected with virus in the skeletal muscle (329 ± 74 RLU/mg protein, n = 5).


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  		Fig. 4.   Ad.408hBNPLuc is not expressed in mouse skeletal muscle. Either virus or PBS was injected into mouse quadriceps muscle. After 4 days, skeletal muscle was removed and assayed for luciferase activity; n = 6-7.

In vivo injection of Ad.CMVLuc. To confirm the tissue specificity of Ad.BNPLuc, we repeated the experiments described above using Ad.CMVLuc, which contains a very strong viral enhancer/promoter that is expressed in all cell types. Injection of Ad.CMVLuc into the LV free wall resulted in very high luciferase activity in the LV (108 RLU/mg protein), ~1,000 times the activity of the hBNP promoter. In other tissues, luciferase activity ranged from 10% (in the liver, septum and RV) to 0.1% (in the atria, lung, kidney, and skeletal muscle) of that of the LV (Fig. 5A). Moreover, Ad.CMVLuc injected into the skeletal muscle also demonstrated widespread luciferase activity in tissues throughout the mouse, with activity in the liver being ~1% that of the skeletal muscle (Fig. 5B). When Ad. CMVLuc was injected into the LV cavity to distribute it to the general circulation, the highest activity was detected in the liver (Fig. 6A). In contrast, when Ad. hBNPLuc was injected into the circulation through the LV cavity, low-level activity was detected only in the heart (Fig. 6B). Thus the CMV enhancer/promoter results in widespread expression of reporter gene activity, in contrast to the cardiac-specific nature of the hBNP promoter.


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  		Fig. 5.   Expression of Ad.CMVLuc in mouse tissues after direct injection into LV free wall and skeletal muscle. Axes are identical to Fig. 2B. A: luciferase activity after injection into LV free wall; n = 5. B: luciferase activity after injection into skeletal muscle; n = 5.



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  		Fig. 6.   Luciferase activity after systemic administration of virus. Axes are identical to Fig. 2B. A: luciferase activity in tissues after injection of Ad.CMVLuc into LV chamber; n = 4. B: luciferase activity after injection of Ad.hBNPLuc into LV chamber; n = 6.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our findings demonstrate that the hBNP promoter in the context of an adenoviral expression vector confers cardiac-specific transgene expression both in vitro and in vivo. When luciferase activity was measured in vitro in both cardiac myocytes and fibroblasts, the CMV enhancer/promoter generated much more luciferase activity than the BNP promoter; however, CMV was almost as active in fibroblasts as in myocytes (ratio of activity in myocytes to fibroblasts was 20), whereas the hBNP promoter was preferentially active in myocytes (ratio of activity in myocytes to fibroblasts was 1,000).

The hBNP promoter in the adenoviral vector was also inducible in cardiac myocytes by hypertrophic and proinflammatory stimuli shown to affect BNP promoter activity in transient transfection studies. Ad. BNPLuc responded to the hypertrophic stimuli PE and Iso as well as the proinflammatory cytokine IL-1beta . Stimulation of the -408hBNP promoter in the present studies was similar in magnitude to that in transient transfection studies using the -1818hBNP promoter (8, 11) and the -408hBNP promoter (Q. He and M. C. LaPointe, unpublished observations) in plasmid expression vectors. These data suggest that regulatory elements in the adenoviral DNA sequences are not influencing hBNP promoter activity. Because the hBNP promoter in the vector can respond to pathophysiological stimuli, we believe it could be used to deliver genes to the diseased heart in vivo, where these stimuli would foster expression of therapeutic transgenes. Given a viral delivery system with long-term expression, such as adeno-associated virus, it is conceivable that the hBNP promoter would foster low-level, constitutive expression of a transgene. Changes in the structure/function of the heart, such as in hypertrophy or heart failure, would induce the promoter to be expressed at even higher levels. A transgene with the ability to oppose the signals stimulated by hypertrophy and heart failure thus could elicit a therapeutic response in the heart. We have indirect evidence of the inducibility of the hBNP promoter in vivo. In our studies (10) of 408hBNPLuc transgenic mice, ischemic injury caused persistent upregulation of the luciferase transgene for at least 3 wk.

In our in vivo studies, the CMV enhancer/promoter was much more active than the hBNP promoter, as expected. When Ad.CMVLuc was injected into the LV free wall, its activity was 1,000 times greater than the hBNP promoter, similar to the difference in transduced myocytes in vitro. However, using the CMV promoter/enhancer, we detected luciferase activity in every other tissue tested, with the highest extracardiac activity in the liver (10% of the activity of the heart), presumably because at least 10% of the virus leaked out of the heart into the general circulation. The difference in liver expression between Ad.CMVLuc and Ad.hBNPLuc did not result from the difference in promoter strengths or problems with detecting low-level luciferase activity in tissues in Ad.hBNPLuc-injected mice. When Ad.hBNPLuc was injected into the heart, luciferase activity at day 4 was 1.3 × 105 RLU/mg. Given that we can expect the same amount of virus leakage for Ad.BNP as Ad.CMV and that in other similar experiments (15), liver expression was >= 10% of heart expression, we would expect to find at least 104 RLU/mg in the liver if the hBNP promoter were nonspecific. Instead, the value was 573 ± 187 RLU/mg (n = 10), which is not different from background. When Ad.CMV was introduced into the circulation by injection into the LV cavity, activity in the liver was higher than in the heart (20 × 106 vs. 10 × 106 RLU/mg). In contrast, when Ad.hBNP was injected into the circulation, activity in the heart was 6 × 103 RLU/mg. Again assuming the same scenario as with Ad.CMV, we would expect to find at least 6 × 103 RLU in the liver if the hBNP promoter were nonspecific. Instead, only background levels of activity were detected, suggesting cardiac specificity. Finally, when Ad.CMVLuc was directly injected into skeletal muscle, luciferase activity was 5 × 106 RLU. Given that the CMV promoter is 1,000 times stronger in vitro and in vivo than the hBNP promoter, one might expect that we would detect 5 × 103 RLU in skeletal muscle injected with Ad.hBNP if the difference in activity were solely based on promoter strength. Instead, we detected fewer than 300 RLU. In all of our protocols, our assay would have allowed us to detect Ad.hBNPLuc activity in the liver and skeletal muscle if the promoter were nonspecific, but because of its cardiac specificity, the promoter was essentially inactive and luciferase activity was background. Thus the higher activity of the CMV enhancer/promoter in vivo is likely a combination of two factors, 1) high-level constitutive activity of the promoter and 2) the ability of the promoter to function in many different cell types in the heart, such as fibroblasts, endothelial cells, and smooth muscle cells, and many cell types in other tissues.

Kass-Eisler et al. (14) showed that direct injection of an adenoviral vector into adult rat hearts resulted in at least 5,000-fold higher transgene expression than injection of plasmid DNA. Expression was highest at the injection site and persisted for at least 55 days, but because they used a CMV viral enhancer/promoter, expression was seen in nonmyocytes as well as myocytes. In a separate study, they found that there was widespread transgene expression in all tissues tested when virus was administered either directly to the LV free wall or to the LV chamber (15). Thus our results with Ad.CMVLuc in the mouse confirm these studies in the rat and point to the utility of a cardiac-specific promoter to localize gene expression to the heart.

Rothmann et al. (24) showed that 800 bp of the myosin light chain-2v (MLC-2v) promoter allowed for heart-specific transgene expression after virus was injected into the cardiac cavity of neonatal rats. This method of delivery resulted in spillover of the virus into the circulation, likely reducing expression in the heart. In contrast to our studies, they did not test inducible regulation of the MLC-2v promoter in the adenoviral expression vector either in vitro or in vivo. In other studies using the MLC-2v promoter (either 2,100 or 250 bp) to drive expression of a luciferase transgene in mice, ventricle-specific expression was shown; however, the promoter was not tested to see whether it responded to pathophysiological stimuli in vivo (5, 12, 19). Our studies extend these previous observations, indicating that the hBNP promoter can produce localized transgene expression in cardiac myocytes for at least 28 days while at the same time minimizing expression in other tissues. In light of this finding, coupled with our transgenic mouse data, we believe that the hBNP promoter would be inducible in vivo when included in an adenoviral vector.

There are several advantages to viral delivery by direct injection into the LV free wall, including high-level, localized expression and reduced viral spillover. A potential drawback to this method is that most of the myocardium would not be affected, which might limit the ability of a transgene to impact global myocardial function. However, Okubo et al. (22) indicated that direct injection of an adenovirus expressing heat shock protein 70 into the rabbit heart reduced infarct size in an ischemia-reperfusion injury model. Also, direct injection of adenoviruses encoding two therapeutic genes [sarco(endo)plasmic reticulum Ca2+-ATPase 1 and Kir2.1] into a normal guinea pig heart was able to modulate cardiac function in vivo (4). In addition, phase I clinical trials with adenoviruses encoding secreted angiogenic factors have used direct intramyocardial injection for ischemic heart disease and direct skeletal muscle injection for peripheral vascular disease. Although most of these trials were uncontrolled studies to test the safety of the viral vector, improvement in the function of the ischemic tissue was noted (13), thus establishing the efficacy of this delivery method.

Other means of effecting global transgene expression in the heart have been developed, most notably catheterization of the LV coupled with cross-clamping of vessels, allowing for modification of cardiac function as a result of transgene overexpression. Despite the enhanced degree of expression of transgenes in the heart, a substantial amount of virus is localized and expressed in the liver (7, 20). Percutaneous delivery of adenovirus through the left circumflex artery has also been effective (25-27). With this method, genes have been transferred to the infarcted and failing heart to improve cardiac function. Use of either delivery method coupled with an adenoviral vector employing the hBNP promoter or another cardiac-specific promoter could conceivably have even greater beneficial effects, especially in light of the inducible regulation of the promoter in the heart during ischemia.

In conclusion, our studies indicate that the proximal hBNP promoter results in inducible and cardiac-specific transgene expression. Because some forms of cardiovascular disease resulting from ischemic injury are amenable to short-term treatment by gene transfer, the hBNP promoter may prove effective in regulating expression of selected therapeutic gene products.


    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Grant P01-HL-28982.


    FOOTNOTES

Address for reprint requests and other correspondence: M. C. LaPointe, Hypertension and Vascular Research Div., Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689 (E-mail: mclapointe{at}aol.com).

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.

June 6, 2002;10.1152/ajpheart.01090.2001

Received 30 December 2001; accepted in final form 3 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(4):H1439-H1445
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