| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Eur J Cardiothorac Surg 2002;21:331-336
© 2002 Elsevier Science NL
a Department of Cardiac surgery, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, ROC
b Department of Physiology, College of Medicine, National Taiwan University, No. 1,Section 1, Jen-Ai Rd., Taipei, Taiwan, ROC
Received 16 October 2001; received in revised form 21 November 2001; accepted 24 November 2001.
* Corresponding author. Tel./fax: 886-2-3222954
e-mail: chfochen{at}ha.mc.ntu.edu.tw
 |
Abstract |
|---|
 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
Key Words: Arteriovenous shunt • Pulmonary hypertension • Shearing stress • Nitric oxide synthase
|
1. Introduction |
|---|
|
TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
Nitric oxide (NO) is an endogenous endothelium-derived dilator contributing to the regulation of vasomotor tone. NOS is the enzyme responsible for generation of NO by a variety of diverse cell types. NOS is a family of enzymes which currently consists of three major isoforms. The inducible form (Type II, iNOS) generates NO in large amounts for longer periods. It is induced in inflammatory cells but also in other cells such as endothelial and smooth muscle cells, in response to endotoxin and cell injury. The endothelial isoform (Type III, eNOS) is found exclusively in endothelial cells. It should be stressed that endothelium-dependent dilation and contraction have been demonstrated in pulmonary vessels, including human, and that circulating vasoactive materials are present in abnormal concentrations in humans with pulmonary hypertension [6,7].
Increase in protein and gene levels of eNOS in the aortic wall were found after 3 days [8] or long-term [9] arteriovenous shunt in rats. Similar findings showed that total lung eNOS activity were increased in young lambs pretreated with utero aortopulmonary shunt [10]; however, Everett et al. [11] reported that eNOS expression was not altered after 6 weeks of arteriovenous shunt lungs. This discrepancy prompted up to hypothesize that the animal condition and the time course of the shunt might make the difference. In the present study, a common carotid artery-to-jugular vein shunt (CJS) was created in rats to mimic the chronically increased pulmonary artery flow state of a congenital heart defect. Our goal was to characterize the potential alterations in eNOS expression induced by increased pulmonary blood flow.
|
2. Materials and methods |
|---|
|
TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
After the surgical procedure, the rats were allowed to awaken. Lincocin (15 mg/kg/twice per day) and Karamycin (15 mg/kg/day) were administered intramuscularly for 3 days. All rats were housed in constant-temperature facilities, exposed to a 12-h light–12-h dark cycle, and given standard lab chow and water. All the animal experiments and care were performed according to the Guide for the Care and Use of Laboratory Animals (published by the National Academy Press, Washington, DC, 1996). The ‘Laboratory Animal Care Committee’ of the National Taiwan University College of Medicine has approved all protocols used in this study.
2.2. Measurements of pulmonary arterial blood pressure, flow and oxygen content
The acute effects of the arteriovenous shunt on pulmonary circulation were immediately measured. The changes of pulmonary blood flow, pressure and oxygen content after the shunt operation were obtained. The rat was incubated and ventilated with a respirator (tidal volume 3 ml/100 mg body weight, respiratory rate 60 breaths/min). After the midline sternotomy, the pulmonary blood flow was measured with a transonic flowmeter (Transonic Systems Inc. T106, New York, USA). The blood flow change in the pulmonary artery was recorded before and after the shunt was clumped. Then, the pulmonary artery pressure was measured by direct puncture into the pulmonary artery from the outflow tract of the right ventricle. The pressure change of pulmonary artery was recorded before and after the shunt was clumped. Immediately after measuring the pulmonary artery pressure, a blood sample (0.3 ml) was taken from the pulmonary artery. This sample was utilized to determine blood oxygen content (PO2, mmHg) using a blood gas analyzer (Rapidlab800, Chiron Diagnostics Corporation, East Walpole, MA, USA).
The chronic effect of the shunt operation on pulmonary circulation was measured 4 weeks after shunt surgery. The rat was anesthetized and on respirator ventilation and a midline sternotomy performed. The blood flow and pressure of the pulmonary artery were recorded before and after the shunt was clumped as previously described. After the hemodynamic studies were finished, the hearts were removed and the right ventricle and the left ventricle with the septum were individually weighed to calculate the ratio of right ventricle weight to left ventricle plus septum weight. Approximately one-half of each lung was flash-frozen in liquid nitrogen and stored at -70°C for subsequent Western blot analysis. At the same time, 1- to 2-mm-thick pieces of lung from different lobes were cut and immersed in fixative (4% paraformaldehyde in phosphate-buffered saline) for immunocytochemical analysis.
2.3. Western blotting
The lung fragments were homogenized on ice in 10 mM Tris–HCl buffer (pH 7.4) containing 255 mM sucrose, 2 mM EDTA (ethylene diamine tetraacetic acid), 12 µM leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Tissue homogenates were centrifuged at 1500g at 4°C for 10 min to remove insoluble debris. Protein concentrations of samples were determined by the Bradford method (Bio-Rad Protein Assay). The lung homogenates were separated under denaturing conditions in a 7.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophorsis) gel, followed by blotting of the proteins onto polyvinylidene difluoride (PVDF) sheets (Immobillon-P, Millipore Corp. Bedford, MA, USA) from polyacrylamide gels at 400 mA for 6 h at 4°C. Blots were blocked in Blotto (5% non-fat dry milk in TBS, 0.1% Tween, pH 7.6) at 4°C overnight. Subsequently, blots were immunoblotted with mouse anti-eNOS monoclonal antibody (dilution 1:2500, Transduction Laboratories, Lexington, KY, USA) for 2 h at room temperature. Then, membranes were washed repeatedly in Blotto and incubated with HRP-conjugated goat anti-mouse IgG (1:2000, Leinco, St. Louis, Mo, USA) for 1 h. After an additional wash for 1 h, the immunoreactive protein was detected with enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK) and exposed to Kodak X-film. Signal bands were quantified by laser densitometry.
2.4. Immunocytochemistry
Five micron-thick sections were cut from paraffin-fixed rat lung blocks. After the sections were dewaxed and rehydrated, endogenous peroxidase activity was quenched by 3% H2O2 in methanol for 30 min. Non-specific binding was blocked by 0.5% normal horse serum. The sections were then incubated at room temperature for 1 h with three monoclonal anti-eNOS antibodies N30020, directed against the amino acids 1030–1209 of human eNOS (Transduction Laboratories, Lexington, KY, USA); H32, directed against the particulate eNOS purified from bovine aortic endothelial cells (Biomol Research Laboratories, Plymouth Meeting, PA, USA); 6C6, directed against the N-terminal amino acids 1–35 of bovine eNOS (Zymed Laboratories, So. San Francisco, CA, USA) at a dilution of 1:100, respectively. After the section was washed in 10 mM Tris-buffered saline (10 mM Tris pH 7.5, 100 mM NaCl), biotinylated goat anti-mouse IgG at a dilution of 1:100 and then avidin–biotin-peroxidase (Vector Labs. Burlingame, KY, USA) were added sequentially. After development in DAB, sections were counterstained with hematoxylin. All slides were mounted and examined using an Olympus microscope for bright-field microscopy.
2.5. Statistics
The results are expressed as means±SD. Newman–Keuls test was used to determine statistical significance after one-way ANOVA. P values less than 0.05 were considered to be statistically significant.
|
3. Results |
|---|
|
TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
|
|
|
|
|
4. Discussion |
|---|
|
TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
The pulmonary vasculature is relatively tolerant to increased pulmonary blood flow. The pulmonary artery pressure did not significantly enhance long time after arteriovenous shunt in dogs [12] or pigs [13]. In rats, Everett et al. [11] reported that Sprague-Dawley rats with abdominal aortocaval shunts resulted in a significant increase in heart- and lung-to-body weight ratios without significant alteration of pulmonary blood pressure. Passing a needle through the inferior vena cava and into the aorta created their fistula. However, the percentages of increased pulmonary blood flow and oxygenation were completely unknown. On the contrary, significant elevations in pulmonary artery pressures were seen in our study, suggesting that pulmonary vascular resistance was increased. Age might be one of the important factor [14], the rats were young in our study, and 42 days of age at time of shunting in their study. The other reason might be due to the location of the shunt that was close to the heart in our rats.
In the systemic vascular bed, increases in pressure augment vascular resistance (autoregulation) and this is restrained by flow-induced vasodilatation [15,16]. On the contrary, increase in pressure in the pulmonary circulation actually decreases vascular resistance due to pressure-induced vessel distension or vessel recruitment. The response of the pulmonary endothelium to increased flow may function to reduce or modulate the vascular response to increasing pressure.
Recent evidence suggests that normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances that are produced locally by the vascular endothelium [17]. Because the mechanisms by which flow regulates pulmonary vascular remodeling are largely unknown. Evidence that NO mediates normal pulmonary vascular tone has led to the hypothesis that endothelial stress induced by congenital heart disease with increased pulmonary blood flow disrupts these regulatory mechanisms and participates in the development of pulmonary hypertension and its associated altered vascular reactivity. eNOS could modulate the pulmonary vascular response to increase pulmonary blood flow. Except a direct vasodilator effect, NO was also involved in the inhibition of smooth muscle cells proliferation [18,19], therefore may serve to the vascular remodeling.
Increased shear stress exerted on the endothelial surface by blood flow may upregulate eNOS. It was found that acute elevation of shear stress in endothelial cells in vitro of increase blood flow by arteriovenous fistulas chronically, or, enhanced mRNA and protein of eNOS [9]. Recently, Jeon et al [8] reported that 3 days after a creation of femoral arteriovenous shunt increased the expression of eNOS but not iNOS in the aorta of rats. Everett et al [11] argued that pulmonary and systemic vascular eNOS are regulated differently by flow. In the present study, we found that eNOS proteins were mildly upregulated in the lungs of rats 72 h after shunt surgery. eNOS might play a role in modulating the pulmonary vascular response to increased pulmonary blood flow and pressure in the early stage of shunt. This hypothesis is supported by a recent report that a model of pulmonary hypertension with increased pulmonary blood flow in the lamb after in utero placement of an aorta-to-pulmonary vascular graft demonstrated that eNOS gene expression and activity were upregulated at 4 week of life [10].
In addition, it is unclear what effect the increased pulmonary arterial oxygen content produced by the creation of the shunt has on eNOS regulation. However, Le Cras et al. [20], strongly suggested that upregulation of eNOS in chronic hypoxia is related to hypoxia per se. Pulmonary blood oxygenation may play a factor because the vascular response to NO can be inhibited or abolished by hemoproteins and superoxide radicals [21]. Heller et al. [22], reported that the reduction of the Hb-induced pulmonary hypertension by NO-donors points toward the inactivation of NO by free hemoglobin. Liu et al. [23], reported that in small, porcine, isolated pulmonary arteries, intralumenal flow increases the production of NO but this is obscured by the generation of superoxide, which causes vasoconstriction. The current study in combination with the report by Black et al. [10], strongly suggests that the upregulation of eNOS in the increased pulmonary blood flow model, the vasodilator effect may be transient. The increase of oxygen content, vasoconstrictors of endothelium like endothelin, may be more important long-term in modulating the vascular tone and growth promotor vascular remodeling in this model [24,25]. Finally, iNOS cannot be detected either in the sham or in the shunt lungs. This indicated that creation of arteriovenous shunt in our rats did not cause an inflammatory response.
In summary, in this model, we demonstrated mild early upregulation of eNOS protein in rats 72 h after shunt and this returned to the control level 1 week later. This implicates that eNOS might be mediating the early pulmonary vascular remodeling in this model of increased pulmonary blood flow and pressure.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Zhang, S. Wu, X. Lu, M. Wang, and M. Liu Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in Rabbits Ann. Thorac. Surg., February 1, 2008; 85(2): 581 - 585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jain, H. Ventura, and B. deBoisblanc Pathophysiology of Pulmonary Arterial Hypertension Seminars in Cardiothoracic and Vascular Anesthesia, June 1, 2007; 11(2): 104 - 109. [Abstract] [PDF]
|
|
|
|
 |
|
 C.-F. Lam, T. E. Peterson, A. J. Croatt, K. A. Nath, and Z. S. Katusic Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2334 - H2341. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
