Gene Transfer to Carotid Sinus In Vivo
A Novel Approach to Investigation of Baroreceptors
Abstract Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli β-galactosidase (β-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. β-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.
Baroreceptor sensory nerves terminate in the adventitia of the carotid sinuses and aortic arch and play a major role in the reflex regulation of the circulation.1 2 Baroreceptor activity is influenced by mechanical coupling of the nerve endings to the vascular wall and by the local concentration of chemical factors released from nearby cells or circulating in the blood.2 3 4 5 6 7 8 9 10
Investigation into local mechanisms that modulate baroreceptor activity has relied almost exclusively on short-term experiments. We reasoned that manipulation of gene expression through gene transfer might provide a novel method to investigate both acute and chronic influences on baroreceptor function. Recent advances in gene transfer technology have provided new insights into cellular and molecular mechanisms and have led to novel therapeutic approaches in many areas of biology and medicine.11 12 13 We have recently demonstrated gene transfer into cultured sensory neurons isolated from nodose ganglia (site of aortic baroreceptor cell soma) using adenoviral vectors.14 Several groups including our own have used adenovirus to transfer genes to blood vessels.15 16 17 18 19 20 21 Particularly relevant to the present study is the finding that perivascular administration of adenoviral vectors results in expression of the transgene only in the adventitia and not the media.15 16 Thus, topical application of adenoviral vectors to carotid sinuses may selectively transduce cells in the region of baroreceptor endings in the adventitia without directly influencing vascular function. The method may also transduce the baroreceptor neurons. Previous studies have demonstrated that adenoviral vectors can be taken up by nerve endings and retrogradely transported to the cell somata to alter gene expression.22 23
The major goal of the present study was to develop a method to transfer genes to the carotid sinus adventitia. We used a reporter gene that encodes for the enzyme β-galactosidase (β-Gal),13 which is generally not present in mammalian cells, and that can be detected with a histochemical stain and quantified by a chemiluminescence assay.13 15 16 17 18 19 20 21 An additional aim of the study was to determine whether exposure of the carotid sinus to the adenovirus vector adversely influences baroreceptor sensitivity.
A total of 47 carotid sinuses from 30 New Zealand White rabbits of either sex were studied. The experimental procedures were carried out in accordance with institutional guidelines and the Guiding Principles for the Care and Use of Animals approved by the Council of the American Physiological Society.
An adenovirus vector containing the gene encoding for β-Gal was constructed in the University of Iowa Gene Transfer Vector Core Laboratory. Construction of the recombinant adenoviral vector has been described in detail elsewhere.13 In brief, the cDNA for β-Gal was cloned into a shuttle vector containing sequences from serotype 5 human adenovirus, the Rous sarcoma virus (RSV) promoter to drive transcription, and a simian virus 40 polyadenylation signal. The resulting plasmid and a plasmid containing the serotype 5 human adenovirus genome with deletions in the E1A, E1B, and a portion of the E3 regions were cotransfected into human embryonic kidney (293) cells. Homologous recombination within the 293 cells produced the replication-deficient vector (AdRSVβ-Gal). We chose to use the RSV promoter on the basis of our previous study that compared RSV and cytomegalovirus promoters for gene transfer into cultured nodose sensory neurons.14 AdRSVβ-Gal was subjected to double cesium gradient purification, suspended in 3% sucrose solution (1012 particles/mL, or ≈1010 plaque forming units [pfu]/mL), and stored at −70°C.
Application of Adenovirus to Carotid Sinuses
Rabbits were anesthetized with xylazine (20 mg/kg) and ketamine (55 mg/kg) administered intramuscularly. Under sterile surgical technique, either one or both carotid sinuses were exposed via a small incision in the cervical region. The carotid sheath was opened, and either adenovirus suspension or vehicle solution (3% sucrose) was applied topically to the carotid sinus region within the sheath by using a pipette. The sheath restricted the solutions to the region of the carotid bifurcation and prevented spread of the solutions to adjacent tissues. The dose of adenovirus administered was either 5×107, 2.5×108, or 5×108 pfu suspended in either 25 or 50 μL of sucrose solution. No attempt was made to remove the small volume of viral suspension. The incisions were closed and the rabbits were observed until they regained consciousness. Penicillin (600 000 U) was administered before surgery.
Analysis of β-Gal Expression
Qualitative analysis and localization of β-Gal expression were performed on 19 carotid sinuses from 12 rabbits by using a histochemical procedure.13 15 16 17 Twelve carotid sinuses exposed to AdRSVβ-Gal and 7 control sinuses (5 exposed to sucrose vehicle and 2 from intact rabbits) were analyzed. In brief, the carotid sinus region was removed, rinsed with PBS, and fixed in 0.5% glutaraldehyde for 30 minutes at room temperature. The blood vessel segments were then washed with PBS containing MgCl2 (1 mmol/L) and exposed to K3Fe(CN)6 (4.9 mmol/L), K4Fe(CN)6 (4.7 mmol/L), and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal, 2.4 mmol/L) in PBS containing MgCl2 for 1 hour at 37°C. X-Gal is hydrolyzed by β-Gal to produce an indolyl that is oxidized to an indigo blue derivative.13 The X-Gal solution was then replaced with PBS, and the blood vessels were stored at 4°C. Photographs were taken of the carotid sinus region and cross sections of 20-μm thickness.
In separate experiments (20 carotid sinuses from 10 rabbits), β-Gal activity in the carotid sinus region was measured quantitatively by chemiluminescence (Galacto-Light Plus, Tropix). Sixteen sinuses exposed to AdRSVβ-Gal and 4 sinuses exposed to sucrose vehicle were analyzed. Each carotid sinus region was minced and placed in lysis buffer (0.2% Triton X-100 and 100 mmol/L potassium phosphate in 175 μL, pH 7.8) for 20 minutes. The samples were centrifuged at 12 000 rpm for 10 minutes and the supernatant removed. Supernatants were analyzed for β-Gal activity with 3-(4-methoxyspirol-[1,2-dioxetane-3,21-tricyclo-[126.96.36.199,7]decan]-4-yl)-phenyl-β-d-galactopyranoside. The reaction produces light that can be detected by a Monolight 2010 luminometer (Analytical Luminescence Laboratory Inc). Standards of purified Escherichia coli β-Gal (Boehringer Mannheim) were used to generate a standard curve. β-Gal activity was expressed in milliunits per milligram protein (Bio-Rad DC protein assay).
Analysis of Baroreceptor Sensitivity
Baroreceptor sensitivity was evaluated in 3 rabbits 4 to 5 days after topical application of AdRSVβ-Gal (5×108 pfu) to the carotid sinus and in 8 control rabbits. The 3 carotid sinuses exposed to adenovirus were stained with X-Gal after each experiment to confirm gene transfer. Rabbits were anesthetized with sodium pentobarbital (30 to 35 mg/kg IV) and ventilated through a tracheotomy with room air supplemented with O2. Arterial blood gases and pH were kept within normal ranges by adjusting the ventilation. Catheters were introduced into the femoral artery for measurement of arterial pressure and in the femoral vein for administration of supplemental anesthetic when needed.
In each rabbit, one carotid sinus was vascularly isolated as described previously.5 6 8 9 10 Catheters were placed in the common carotid and lingual arteries, and the carotid sinus was filled with oxygenated Krebs-Henseleit buffer. Carotid sinus pressure was controlled by varying the inflow of air into a pressure bottle attached to the common carotid artery catheter and was measured with a transducer (model P23ID, Statham) connected to the lingual artery catheter.5 6 8 9 10 The cervical sympathetic, aortic depressor, and vagus nerves were sectioned. Decamethonium bromide (0.3 mg/kg IV) was administered to eliminate skeletal muscle contractions before recording nerve activity.
The carotid sinus nerve was isolated, sectioned, and placed on a unipolar electrode. The electrode and nerve were insulated either by covering the area with warm paraffin oil (37°C) and/or by encasing them in Wackers silicone gel.8 9 10 Nerve activity was recorded using a high-impedance probe (model HIP511J, Grass Instrument Co) and a Grass band-pass amplifier (model P511J, 100 to 300 Hz to 3 kHz bandwidth). The neurogram was displayed on a dual-beam storage oscilloscope (model 5113, Tektronix). The frequency of action potentials exceeding a selected voltage level just above the electrical noise was counted with a nerve traffic analyzer (model 706C, Department of Bioengineering, University of Iowa, Iowa City).5 6 8 9 10 Systemic arterial pressure, carotid sinus pressure, and the output of the nerve traffic analyzer were continuously recorded on a chart recorder (model 11-1202-25, Gould Inc).
Baroreceptor activity was recorded during slow ramp increases in nonpulsatile carotid sinus pressure from 0 to 160 mm Hg. Pressure ramps were repeated once approximately every 5 minutes until baroreceptor responses were consistent and reproducible. Carotid sinus pressure was held at 60 mm Hg in between pressure ramps in all experiments. The rate of pressure rise (dP/dt) was equivalent during all pressure ramps within each experiment and was similar in virus-treated (2.9±0.3 mm Hg/s) and control (2.7±0.2 mm Hg/s) carotid sinuses.
Baroreceptor activity was measured at 20–mm Hg increments in pressure over a range of 20 to 160 mm Hg. The data from three or four pressure ramps with consistent baroreceptor responses were analyzed and the average responses calculated for each experiment. Because the absolute level of multifiber baroreceptor activity (in spikes per second) varies from preparation to preparation, baroreceptor activity was expressed as a percentage of the maximum activity recorded at high carotid sinus pressure for analysis of differences between virus-treated and control carotid sinuses. The slope of the linear region of the baroreceptor pressure-activity (%) curve was calculated using linear regression analysis and served as a measure of baroreceptor sensitivity.8 9 The linear region of the curve occurred between 40 and 100 mm Hg (r=.989±.004). The position of the curve along the pressure axis was also determined directly from the experimental traces by measuring the carotid sinus pressure corresponding to 50% of the maximum baroreceptor activity (EP50).
Statistical Analysis of Data
All data are expressed as mean±SEM. The levels of β-Gal activity in carotid sinuses from the various experimental groups were analyzed by one-factor ANOVA and the Tukey-Kramer post hoc test (GB-Stat 6.0 software). The effects of changes in carotid sinus pressure and treatment group (virus-treated versus control carotid sinuses) on normalized baroreceptor activity were analyzed by two-factor ANOVA and the Tukey-Kramer post hoc test (GB-Stat 6.0 software). Differences in baroreceptor slope and EP50 between the groups were determined by unpaired t test. Statistical significance of differences was determined at P<.05.
Gene Transfer to the Carotid Sinus
β-Gal expression, as indicated by positive X-Gal staining, was observed in each carotid sinus exposed to AdRSVβ-Gal (12 of 12 carotid sinuses; Fig 1⇓). Expression of β-Gal was not observed in carotid sinuses exposed to sucrose vehicle (n=5) or in untreated carotid sinuses (n=2; Fig 1⇓). Expression was observed both 1 (n=6) and 4 (n=6) days after virus application, and the intensity of staining appeared greater after 4 days than after 1 day.
Cross sections of the carotid sinus region exposed to the adenovirus vector indicated that β-Gal expression was restricted to the adventitia of the blood vessel wall (n=3, Fig 2⇓). Staining for β-Gal was never observed in the media or intima.
β-Gal activity was measured by chemiluminescence assay. AdRSVβ-Gal applied topically to carotid sinuses produced dose-dependent increases in β-Gal activity 1 day after application of the virus (n=12, Fig 3⇓). Four days after virus application, β-Gal activity was more than 25-fold greater than that measured 1 day after the same dose of virus was applied (n=4, Fig 3⇓). β-Gal activity was absent in carotid sinuses exposed to sucrose vehicle (n=4, Fig 3⇓).
Baroreceptor activity was recorded from the vascularly isolated carotid sinus to evaluate the effects of exposure to the adenovirus. The experiments were conducted 4 to 5 days after application of the virus (5×108 pfu AdRSVβ-Gal) to the carotid sinus at a time when β-Gal expression was pronounced (Fig 3⇑). There were no significant differences in the levels of normalized baroreceptor activity at equivalent pressures measured from virus-treated and control carotid sinuses (Fig 4⇓). In addition, neither the slope of the pressure-activity curve nor the EP50 values were significantly different between the groups. The slope averaged 1.24±0.09 and 1.21±0.25%/mm Hg for control and virus-treated sinuses, respectively. There was a tendency for the virus-treated carotid sinuses to exhibit a leftward shift in the pressure-activity curve; EP50 values averaged 77±5 and 62±7 mm Hg for control and virus-treated carotid sinuses, respectively.
The major findings of the present study are that (1) gene transfer to the carotid sinus of intact rabbits can be accomplished by using an adenovirus vector; (2) after perivascular application of adenovirus, expression of the transgene is restricted to the region of baroreceptor innervation in the adventitia; (3) transgene expression driven by the RSV promoter occurs within 1 day of virus application and is markedly enhanced after 4 days; and (4) exposure of the carotid sinus to the adenovirus vector does not appear to adversely influence baroreceptor sensitivity.
Applications of Gene Transfer to the Carotid Sinus
Gene transfer has been used to gain insight into fundamental biological mechanisms and to provide novel therapies for disease.11 12 An advantage of gene transfer over a pharmacological approach is the potential for longer-term manipulation of physiological systems. Gene transfer to the carotid sinus may enable sustained delivery of neuroactive substances such as neurotrophic factors and prostanoids to the baroreceptors. Baroreceptor responses to short-term administration of paracrine and vasoactive factors have been investigated,3 4 5 6 7 8 9 10 whereas little is known concerning longer-term responses and potential trophic effects on baroreceptor structure and function.
We demonstrated in the present study that β-Gal expression was not only maintained but also markedly enhanced between 1 and 4 days after applying adenovirus to the carotid sinus. Although additional studies are needed to determine the maximum duration of altered gene expression in the carotid sinus, our results both in vivo and in isolated sensory neurons14 suggest that transgene expression can be sustained for at least several days. The time course and duration of expression depends in part on the promoter used to drive gene expression.11 12 13 14 15 16 Therefore, the time course of transgene expression can be modified to some extent to meet the needs of a particular experiment by using different promoters.
The importance of baroreceptors in chronic pathologic states such as hypertension and atherosclerosis remains controversial. Decreased baroreflex sensitivity predicts the occurrence of sudden cardiac death in patients with coronary artery disease and myocardial infarction and in animal models.24 25 Baroreflex impairment may be caused by decreased baroreceptor sensitivity or altered central mediation of the reflex. The impact of provoking chronic changes in baroreceptor sensitivity by using gene transfer on the development of hypertension and occurrence of sudden cardiac death would provide new insights into the importance of baroreceptors in those states. Furthermore, restoration of baroreceptor sensitivity by gene transfer would suggest a possible new therapeutic approach.
A second advantage of gene transfer is the ability to specifically alter expression of individual molecules in a regionally selective manner. This is not easily accomplished with a pharmacological approach, particularly during systemic administration of drugs. We found that β-Gal expression was confined to the adventitia of the carotid sinus after topical application of AdRSVβ-Gal. The lack of expression in the media likely reflects a diffusion barrier that prevents the adenovirus vector from crossing the external elastic lamina.18 Rios et al16 have also observed that trans-gene expression following topical application of adenovirus vector to carotid arteries is restricted to the adventitia. We did not investigate whether the adenovirus vector may have reached the systemic circulation by way of absorption into the vasa vasorum in carotid sinus adventitia but believe it is very unlikely. Adenovirus does not easily cross the endothelium,18 20 and intraluminal administration of virus to isolated vessel segments does not generally lead to reporter gene expression at distant sites despite significant expression in the vasa.26 27
Baroreceptor nerve endings are present in the adventitia of carotid sinuses.1 2 Selective targeting of gene transfer to the adventitia may enable modulation of baroreceptor sensitivity by neuroactive factors without interfering with normal gene expression in vascular muscle in the media. In addition, baroreceptor activity may be modulated indirectly by changes in vascular distensibility elicited by delivery of vasoactive substances.2
A third advantage of the gene transfer approach is that the transferred molecule (eg, an enzyme or ion channel) may remain under physiological regulation and therefore may vary its activity in response to appropriate stimuli. For example, paracrine factors such as prostacyclin and nitric oxide are produced intermittently in response to elevations in cytosolic Ca2+ concentration. Mechanical deformation of various types of cells, including baroreceptor neurons, increase cytosolic Ca2+.28 29 30 31 Therefore, gene transfer of prostaglandin H synthase or nitric oxide synthase (NOS) may enable regulated production of prostacyclin and nitric oxide, which may be more physiologically relevant than continuous exposure to exogenously administered factors.
Adenoviral vectors can enter sensory nerve endings and be transported retrogradely to the neuronal soma to alter gene expression.22 23 Therefore, gene transfer to the carotid sinus may enable alteration of gene expression in the baroreceptors themselves and provide a method to modulate electrophysiological properties of nerve endings. Adenovirus-mediated gene transfer of a K+ channel has been used to modify membrane excitability in cardiac myocytes.32 In addition, gene transfer of the endothelial isoform of NOS into hippocampal neurons modifies synaptic transmission.33 Transfer of truncated eNOS, a putative dominant negative inhibitor of endogenous eNOS, prevented long-term potentiation in a subpopulation of hippocampal neurons.33 These studies illustrate that gene transfer may be used to overexpress a functional gene to alter neural function or to block endogenous gene expression.
Limitations of the Study
Several questions remain to be answered before the full potential of gene transfer can be realized. A major concern with the use of adenoviral vectors is the potential for the vector itself to cause biological responses not related to the function of the transferred gene. Adenovirus triggers immune and inflammatory responses that can contribute to loss of expression of the transgene.20 21 In previous studies, the extent of inflammation and the duration of gene expression have been variable, depending on the type of vector, the viral titer, the site of virus application, and the species of animal studied.13 15 16 17 18 19 20 21 The inflammatory response after gene transfer was not evaluated in the present study but was investigated by us in a recent study using similar techniques.16 In that study, no infiltration of inflammatory cells was observed 1 day after adenovirus vector containing the β-Gal gene (0.3 to 1.5×1010 pfu) was applied topically to carotid and femoral arteries.16 We used a lower dose of virus (5×107 to 5×108 pfu) in the present study, which may have further limited the inflammation. It is possible that the inflammatory response to adenovirus is less severe when it is applied to the adventitia instead of intravascularly. Our finding that baroreceptor sensitivity was not significantly altered 4 to 5 days after virus application suggests that inflammation, if it did occur, did not markedly influence function. The results also suggest that adenovirus itself does not adversely influence baroreceptor sensitivity, which will be important when investigating responses in carotid sinuses transduced with functional genes. Nevertheless, further studies are needed to characterize the duration of gene expression and the impact of immune and inflammatory responses in adenovirus-treated carotid sinuses.
The absolute amount of activity recorded from multiple baroreceptor fibers cannot be easily compared among separate groups of animals because the measured activity varies depending on the contact between the electrode and carotid sinus nerve. Therefore, we expressed activity as a percentage of the maximum activity in each experiment. The analysis allows the sensitivity to changes in pressure and possible shifts in the pressure-activity curve along the pressure axis to be evaluated but does not enable a comparison of maximum baroreceptor activity. One way we plan to overcome this limitation in future experiments is to investigate effects of carotid sinus gene transfer on baroreflex control of systemic arterial pressure and heart rate, the measurements of which are not dependent on the recording conditions and exhibit much less interanimal variability.
The results of the present study do not define which types of cell in the carotid sinus adventitia expressed β-Gal after gene transfer. The type of cell transduced may not be critically important in studies attempting to deliver paracrine factors to the region of baroreceptor innervation. In contrast, it will be essential to transduce the baroreceptor neurons in studies investigating the neural mechanisms of mechanoelectrical transduction and determinants of membrane excitability.
The results of the present study demonstrate that gene transfer to the site of baroreceptor innervation in the carotid sinus can be accomplished by using an adenovirus vector. Exposure of the carotid sinus to the vector containing the reporter gene β-Gal does not appear to adversely influence baroreceptor sensitivity. The technique provides a novel approach for future studies of baroreceptor mechanisms.
The work was supported by a grant from the Iowa Affiliate of the American Heart Association and research funds from the Department of Veterans Affairs awarded to M.W. Chapleau and funds available to M.W. Chapleau through a program project grant (PO1-HL-14388) from the National Institutes of Health. Silvana S. Meyrelles was a graduate student enrolled in the Department of Physiological Sciences, Federal University of Espirito Santo Biomedical Center, Vitoria, ES, Brazil, and was supported by funds from CNPq, Brazil during the time that the work was being carried out. The authors thank Drs Beverly L. Davidson and Richard D. Anderson and the Gene Transfer Vector Core at the University of Iowa for providing the adenovirus vector. The Vector Core is supported in part by funds from the University of Iowa Carver Trust. The authors also thank Dr Donald Lund for assistance with the β-galactosidase chemiluminescence assay, Aaron T. Holley and Melissa Mora for technical assistance, and Jackie Schneider for typing the manuscript.
- Received March 18, 1997.
- Revision received April 17, 1997.
- Accepted May 7, 1997.
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