Influence of coronary artery diameter on eNOS protein content

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Am J Physiol Heart Circ Physiol 284: H1307–H1312, 2003. First published December 19, 2002; 10.1152/ajpheart.00792.2002.

Influence of coronary artery diameter on eNOS protein content M. H. Laughlin, J. R. Turk, W. G. Schrage, C. R. Woodman, and E. M. Price Departments of Veterinary Biomedical Sciences and Medical Physiology, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211 Submitted 13 September 2002; accepted in final form 10 December 2002

Laughlin, M. H., J. R. Turk, W. G. Schrage, C. R. Woodman, and E. M. Price. Influence of coronary artery diameter on eNOS protein content. Am J Physiol Heart Circ Physiol 284: H1307–H1312, 2003. First published December 19, 2002; 10.1152/ajpheart.00792.2002.—The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-␮m internal diameter (ID)], small arteries (201- to 300-␮m ID), resistance arteries (151- to 200-␮m ID), large arterioles (101- to 150-␮m ID), intermediate arterioles (51- to 100-␮m ID), and small arterioles(⬍50-␮m ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1–2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles. arteries; blood flow; coronary disease; endothelium; endothelial-derived factors; capillary endothelium

that fundamental differences exist among arteries of different tissues. (13). Indeed, there is evidence for inherent diversity of phenotype of vascular smooth muscle (12) and endothelium (1) within and among arteries in the same tissue. Consistent with the notion of variable expression of endothelial nitric oxide synthase (eNOS) in endothelium of different sized arteries, Ando et al. (1) report that eNOS expression and eNOS enzyme activity are greater in cultured aortic endothelial cells than in cultured microvascular endothelial cells. Furthermore, a recent study of the effects of exercise training on

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eNOS content throughout the coronary arterial tree of pigs revealed that exercise training induced nonuniform increases in eNOS protein content along the coronary tree (10). Therefore, available evidence suggests that eNOS protein content differs among coronary arteries of differing size. The endothelium is a single cell layer throughout the coronary arterial tree (9). If all coronary endothelial cells contain a similar amount of eNOS protein, one would expect the amount of eNOS per gram of artery to decrease as artery size increases because the relative amounts of vascular smooth muscle protein and connective tissue protein increase with increasing diameter of arteries. Therefore, on the basis of the assumptions that the relative amount of total endothelial cell protein decreases with increasing artery diameter and that endothelial cells contain similar amounts of eNOS protein, we formulated the hypothesis that eNOS protein content increases with decreasing artery diameter in the coronary arterial tree. The following study was designed to test this hypothesis. We examined eNOS expression in six specific sizes of coronary arteries: large coronary arteries [internal diameter (ID) ⫽ 301–2,500 ␮m], small coronary arteries (ID ⫽ 201–300 ␮m), resistance arteries (ID ⫽ 151–200 ␮m), large arterioles (ID ⫽ 101–150 ␮m), intermediate arterioles (ID ⫽ 51–100 ␮m), and small arterioles (ID ⬍ 50 ␮m). We selected these specific sizes of arteries because these size categories encompass the size range of arteries used in previous work showing increased endothelium-mediated dilation (14) and increased eNOS expression in some coronary arteries after exercise training (10). Also, these size categories encompass the size range of coronary arterioles in which differential sensitivity to shear stress was observed in arteries of different size (7). Content of eNOS protein (amount of eNOS protein/g total protein) was measured with standard immunoblot techniques, and the distribution of eNOS in the different cell types of the arteries was assessed with immunohistochemistry. MATERIALS AND METHODS

Experimental animals. Fourteen female Yucatan miniature swine (25–40 kg) were used for these experiments. Animal care and use procedures were approved by the UniThe 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.

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versity of Missouri, Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 80-23, Revised 1996). Isolation of coronary arteries. Pigs were anesthetized and hearts were removed and maintained at 0–4°C during vessel isolation as described previously (10). Segments of large coronary artery (ID ⬎ 300 ␮m), small coronary artery (ID ⫽ 201–300 ␮m), and resistance arteries (ID ⫽ 151–200 ␮m) were carefully dissected from left ventricular myocardium, fat, and connective tissue. Arterioles were isolated from myocardium (left ventricular free wall) by dissecting along the length of 300-␮m-ID arteries to the smallest branches. Single arterioles 1–2 mm in length (or 2–5 arterioles with equal diameters, sufficient to have a total length of 1–2 mm) were isolated, diameters and lengths were recorded, and anterioles were placed in a microcentrifuge tube (⫺70°C). This approach to isolation of arteries resulted in most samples coming from the epicardium except for the resistance arteries and arterioles, which were collected from the epicardium and midmyocardium of the left ventricle. Amount of eNOS protein in arterioles of similar size. To determine whether there is significant variation of eNOS content among arterioles of similar size, we compared eNOS content among arterioles of the same diameter. We completed three experiments in which we compared eNOS content of arterioles of equal size that were isolated from different locations in the left ventricle: at the base of the heart, near the circumflex coronary artery, at the apex of the heart near the terminal branch of the left anterior descending coronary artery, and two or three arterioles from different locations in the left ventricular free wall. Thus we made the following comparisons of eNOS content in 1) five arterioles that had an ID of 85 ␮m and a length of 1,530 ␮m, 2) four arterioles that had an ID of 119 ␮m and a length of 1,700 ␮m, and 3) four arterioles that had an ID of 170 ␮m and a length of 1,700 ␮m. Arterioles with similar lengths and diameters were selected so that samples would contain similar amounts of total arteriolar protein, and eNOS was measured in single arterioles as described (4). Data from the single arteriole experiments are presented as eNOS protein per arteriole in relative densitometric units (Fig. 4). eNOS protein content. eNOS protein content of arteries was determined from immunoblots by using standard procedures of loading equal amounts of total artery or arteriole protein on different lanes of the same gel allowing comparisons among arteries and arterioles of different sizes on the same gel (10). Total protein was measured with NanoOrange Protein Quantitation kits (Molecular Probes), which allow measurement of total protein in small samples. Because there is not sufficient protein in a single arteriole to allow measurement of protein content and have a sufficient sample to run on an SDS gel, it was necessary to pool samples of five arterioles. In preliminary experiments, we determined that five arterioles of a length of 1–2 mm provided enough protein to measure total protein content and to have a sufficient amount of sample remaining for immunoblot analysis. All samples of arteries and arterioles were solubilized in 20 ␮l of Laemmli buffer (8), boiled, and sonicated for 2 min and subjected to SDS-PAGE under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes (Hybond-ECL, Amersham) and blocked (1 h at 25°C) with 5% nonfat milk in Tris-buffered saline-Tween (20 mM Tris 䡠 HCl, 137 mM NaCl, and 0.1% Tween 20). Blots were incubated overnight (25°C) with primary antibody against eNOS (1: 1,600; Transduction Laboratories) and, in some cases, GAPDH (1:10,000; Chemicon), followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidaseAJP-Heart Circ Physiol • VOL

conjugated anti-mouse). Protein content was determined with chemiluminescence (enhanced chemiluminescence, Amersham) and quantified with densitometry by using NIH Image software (National Institutes of Health, Bethesda, MD). To verify that cellular-derived proteins were not underrepresented in some sizes of artery due to contamination with connective tissue, we did immunoblot analysis by using anti-collagen antibodies. This experiment demonstrated that our solubilized samples do not contain measurable amounts of collagen (data not shown), i.e., the solubilization conditions used do not extract extracellular connective tissue. Therefore, the material loaded onto each lane in our gels is representative of cellular-derived proteins. Vascular histology. In eight hearts, the distal portion of the anterior descending branch of the left coronary artery was cannulated and perfusion fixed as described previously (11). Cardiac and smooth muscle were relaxed with infusion of 10–20 ml of Lock’s solution containing 10 ml/100 ml procaine. Without interruption of perfusion, the perfusion solution was switched to neutral buffered formalin fixative. Perfusion pressure was held at 120 mmHg. The myocardium was then stored in fixative until time for processing. The heart was sliced serially at 5-mm intervals from apex toward the base orthogonal to the anterior descending branch of the left coronary artery. Samples of the artery and ⬃2 cm of the adjacent myocardium were processed routinely to paraffin, sectioned at a thickness of 5 ␮m, and stained with hematoxylin and eosin. Images of coronary arteries and arterioles in cross section were captured with a Spot Insight digital camera (Diagnostic Instruments, Sterling Heights, MI). Digital images were analyzed with Image Pro Plus (Version 4.1, Media Cybernetics, Silver Spring, MD) to determine external diameter and counts of nuclei within the intima (endothelium) and media (vascular smooth muscle). To confirm the localization of eNOS to the endothelium, sections from four hearts were floated onto positively charged slides (Fisher, St. Louis, MO). Sections were deparaffinized and steamed in citrate buffer at pH 6.0 (Dako target retrieval solution S1699, Carpintera, CA) for 20 min to achieve antigen retrieval and then cooled for 20 min. The Dako autostainer (Dako DC34000) was used for immunohistochemical staining. Sequential Tris buffer and water wash steps were performed after each step in the automated staining protocol. Sections were incubated with avidin biotin two-step blocking solution (Dako X590) to inhibit background staining and in 3% hydrogen peroxide to inhibit endogenous peroxidase. Nonserum protein block (Dako X909) was applied to inhibit nonspecific protein binding, and slides were incubated in primary mouse monoclonal IgG1 anti-eNOS antibody (Transduction Laboratories N30020) at 1:800 dilution for 60 min. After the appropriate washing steps were completed, slides were incubated with biotinylated anti-mouse link secondary antibody in PBS containing 15 mM sodium azide and peroxidase-labeled streptavidin (Dako LSAB⫹ kit, peroxidase, K0690). Diaminobenzidine (Dako) applied for 5 min allowed visualization of eNOS antibody staining. Slides were counterstained with Mayer’s Hematoxylin stain for 1 min, dehydrated, and coverslipped as described previously (5). Data analysis. All values are means ⫾ SE. Between-group differences were assessed by using repeated-measures ANOVA or Student’s t-tests where appropriate. Differences of P ⬍ 0.05 were considered significant. RESULTS

Smooth muscle content and artery size. A set of representive photomicrographs used in our examina-

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Fig. 1. Effects of coronary artery size on the number of smooth muscle cells (SMC)/endothelial cells (EC). A: cross section of a 670-␮mdiameter coronary artery branch of the left anterior descending coronary artery. Hematoxylin (stains in nuclei, basophilic) and eosin (stains in the cytoplasm) were used. Inset: high magnification immunohistochemistry for endothelial nitric oxide synthase (eNOS) (to identify the endothelial cells). B: cross section of a intramyocardial, 180-␮m-diameter resistance artery immunohistochemistry for eNOS. C: cross section of an intramyocardial, 32-␮m-diameter arteriole immunohistochemistry for eNOS. D: negative control immunohistochemistry without eNOS antibody. Note the positive eNOS staining in endothelium of arteries (A and B), arteriole (C), venules (arrows in B and C), and capillaries (B and C).

tion of the relationship between coronary artery diameter and number of smooth muscle cells/endothelial cells in the arterial wall are presented in Fig. 1 for a large artery (670-␮m outer diameter), a resistance artery (180-␮m outer diameter), and two small arterioles (32- and 42-␮m outer diameter). As shown in Fig. 2, our results indicate that the smallest coronary arterioles consist of one layer of endothelial cells and one layer of smooth muscle cells, whereas the large coronary arteries have 10–15 smooth muscle cells per endothelial cell (Figs. 1 and 2). Because our eNOS protein content is examined in coronary artery size categories, we have also averaged the data presented in Fig. 2 to reflect the number of smooth muscle cells per endothelial cell in

Fig. 2. Relationship between coronary artery diameter and the ratio of the number of SMC to the number of EC across the wall of the artery. Scatter plot of the SMC-to-EC ratio in coronary arteries of increasing external diameter in micrometers is shown. Each dot represents data from one artery/arteriole. AJP-Heart Circ Physiol • VOL

these size categories (coronary arteries ⬎ 301 ␮m: 5.42 ⫾ 0.68; 201–300 ␮m: 2.71 ⫾ 0.43; 151–200 ␮m: 2.5 ⫾ 0.32; 101–150 ␮m: 2.06 ⫾ 1.58; 51–100 ␮m: 1.58 ⫾ 0.07; and 50 ␮m: 0.85 ⫾ 0.04). In all sized coronary arteries throughout the coronary arterial tree, eNOS was found to be localized in coronary endothelium (Fig. 1, A–C). Also, results indicate that the immunoreactivity of the endothelium appears similar in all sizes of coronary artery. Importantly, we consistently observed positive eNOS staining in the endothelium of capillaries throughout the myocardium (Fig. 3). Positive eNOS staining of capillary endothelium is also apparent in Fig. 1, B and C. Although we did not design our study to examine eNOS expression in coronary veins, coronary veins were apparent in some of our specimens. For example, two small veins can be seen in the lower right hand corner of Fig. 1B and one in the lower right hand corner of Fig. 1C, as indicated by the arrows. When veins were apparent in our samples, the results consistently suggested that venous endothelium has similar levels of eNOS as capillaries and that venous endothelium has lower levels of eNOS than arterial endothelium. Similar amount of eNOS protein in arterioles of similar size. Figure 4A presents a sample immunoblot that compares eNOS protein in arterioles isolated from different locations in the left ventricle. As can be seen from these data, arterioles of similar diameter have similar amounts of eNOS protein. Average results for two different sizes of arteriole are presented in Fig. 4B. These results indicate that it is acceptable to pool arterioles of similar size as is done in the next sets of data on eNOS protein content of small- and intermediate-sized arterioles. Amount of eNOS protein in coronary arteries. Figure 5 presents two sample immunoblots for eNOS protein.

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Fig. 3. Positive eNOS staining in myocardial capillary endothelium. Cross section of myocardial capillaries and hematoxylin (stains in nuclei, basophilic) and eosin (stains in the cytoplasm) immunohistochemistry for eNOS are shown.

Note that each gel was loaded with equal amounts of total protein for arteries of all six sizes. When equal amounts of total protein for all sizes of interest from one heart are loaded, it serves to control for variations in background, primary and secondary antibody reactivity, and film processing in the data. This strategy allows comparison of the relative eNOS immunoreactivity (optical density) in arteries of different sizes from the heart of each pig. Data for eNOS content of the coronary arteries from the 14 hearts included in this study are presented in Fig. 6. One heart exhibited the increase in eNOS con-

Fig. 4. Levels of eNOS and GAPDH expression are similar in equalsized coronary arteries. A: sample immunoblot for 4 arterioles with internal diameter of 170 ␮m and length of 1,700 ␮m isolated from one heart. Proteins were electrophoretically separated and probed for immunoreactivity as described in the text. B: bar graphs presenting means ⫾ SE of eNOS content for 2 different groups of arterioles with diameters of 85 ␮m (n ⫽ 5 arterioles from the same heart) and 119 ␮m (n ⫽ 4 arterioles from the same heart). AJP-Heart Circ Physiol • VOL

tent with decreasing artery size predicted by our hypothesis, and another exhibited increasing eNOS content with decreasing diameter from 201- to 300-␮m ID through the smallest arterioles. Three hearts exhibited a pattern of increased eNOS content with decreasing size through resistance sized arteries, and then eNOS content decreased with size in the smaller arteries and arterioles. In six hearts, eNOS content decreased with decreasing artery diameter, a pattern opposite of that predicted by our hypothesis. In four hearts, the data suggested no difference in eNOS protein content across coronary artery diameters. Figure 7 illustrates the relationship between eNOS protein content and coronary artery size reflected in means ⫾ SE. Note that, except for the smallest coronary arterioles, which have significantly less eNOS protein than the largest coronary arteries, eNOS protein content is relatively constant across coronary artery size.

Fig. 5. Sample immunoblots showing eNOS protein content of the 6 different sized arteries isolated from 2 different hearts. Gels were loaded with 2 ␮g of total protein on each lane. HEL, human endothelial cells lysates, used as an internal standard.

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Fig. 6. eNOS protein content throughout the coronary arterial tree. Data sets are for 6 arteries each from 14 individual pig hearts. eNOS protein content was quantified by scanning densitometry of individual autoradiograms for each pig. DISCUSSION

This study was designed to test the hypothesis that eNOS protein content of coronary arteries increases with decreasing artery diameter. The major findings of this study are that 1) the relative amount of vascular smooth muscle decreases with decreasing diameter in the coronary arterial tree from 10 to 15 smooth muscle cells per endothelial cell in large arteries to 1 smooth muscle cell per endothelial cell in small arterioles; 2) coronary arterioles of the same diameter isolated from different regions of the left ventricular free wall have similar eNOS content; 3) the amount of eNOS protein, relative to total arterial protein, is similar across different sized coronary arteries (ID of 1,000 through 51 ␮m) and arterioles of ⬍50-␮m ID have significantly less eNOS protein per gram protein than large coronary arteries; 4) these results indicate that the amount of eNOS in endothelial cells of porcine coronary arteries is not the same throughout the coronary arterial tree. Indeed, endothelial cells of larger coronary arteries appear to contain substantially greater amounts of eNOS than do endothelial cells of coronary arterioles. Differences in eNOS protein content of arteries throughout the coronary arteriolar tree may reflect the prevailing hemodynamic conditions in these arteries (i.e., shear stress, pressure, etc.). On the other hand, the eNOS content of endothelial cells in conduit arteries may be greater than in coronary arterioles to compensate for the extra NO needed to relax the smooth muscle cells in the outer ring of these larger arteries. Additionally, it is possible that the extra NO released into the bloodstream by larger coronary arteries participates in control of structure and/or function of downstream arteries and arterioles so that eNOS protein content is not required to be as great in small coronary arterioles (3, 19). Ando et al. (1) reported that eNOS expression and eNOS enzyme activity are greater in cultured aortic endothelial cells than in cultured microvascular endothelial cells. It would be reasonable to expect that our conduit coronary artery endothelial cells would be similar to aortic endothelial cells and that our arteriolar endothelial cells would be similar to the “microvascuAJP-Heart Circ Physiol • VOL

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lar” endothelial cells studied by Ando et al. (1). If endothelial cells in the smallest coronary arterioles reflect the microvascular endothelial cells of Ando et al., then our results confirm those of Ando et al., indicating that endothelial cells of large conduit arteries have greater eNOS protein content than endothelial cells of microvessels. However, because the microvascular endothelial cells of Ando et al. were likely from various sized coronary arteries, veins, and perhaps capillaries, it is difficult to know whether these cultured cells reflect properties of small coronary arteries, or large or small coronary arterioles. Joyce et al. (6) reported that endothelial cells of the first three branch orders of sheep uterine artery exhibit a decreasing gradient of eNOS protein content during the luteal phase of the ovarian cycle. Our porcine coronary artery tree did not exhibit such a pattern. Although the conduit coronary arteries have greater eNOS content than the smallest diameter arterioles, the eNOS content of the other four sizes of coronary arteries were similar to those of the large coronary arteries (Fig. 7). These results do not support the hypothesis that eNOS protein content of coronary arteries increases with decreasing artery diameter in the coronary tree. Positive eNOS staining was observed in the endothelium of capillaries (Figs. 1 and 3) of every heart examined. Similarly, Segal et al. (18) observed eNOS immunoreactivity in the capillaries as well as throughout the skeletal muscle vascular beds in hamster retractor and cremaster muscles. Although our study was not designed to examine eNOS expression in coronary veins, we observed positive eNOS staining in the veins contained in our specimens. Thus our results indicate that eNOS protein is present in the endothelial cells throughout the porcine coronary vascular tree. These results are consistent with the report of Andries et al. (2), showing that eNOS staining is present in all coronary endothelial cells of rat heart. Andries et al. (2) also reported that eNOS staining was more intense in coronary arterial endothelium than in venous or capillary endothelial cells. We did not measure eNOS staining intensity in our study, but our results are consis-

Fig. 7. Comparison of average eNOS protein content in coronary arteries and arterioles. eNOS protein content was quantified by scanning densitometry of individual autoradiograms. * eNOS content of the smallest arterioles is significantly less than the large arteries.

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tent with the observations of Andries et al., as it can be seen in Fig. 1, B and C, that arterial endothelial cell staining appears darker than in capillaries or veins. Our hypothesis that eNOS protein content would increase with decreasing coronary artery diameter was based on two assumptions: 1) that endothelial cells lining all coronary arteries contain a similar amount (concentration) of eNOS protein; and 2) that as coronary artery diameter increases, the amount of vascular smooth muscle relative to the amount of endothelium increases because the endothelium is a single cell layer throughout the coronary arterial tree. If the amount of connective tissue and smooth muscle proteins relative to total arterial protein do increase with increasing coronary artery diameter as proposed, then the relative eNOS protein content of coronary arteries would decrease with increasing artery diameter. Present results confirm that the number of smooth muscle cells per the number of endothelial cells across the arterial wall decreases with diameter (Fig. 1). However, our results also indicate that eNOS protein content/total protein content is similar across coronary arteries of differing diameters (Fig. 7). These results do not support our hypothesis. Furthermore, these results indicate that endothelial cells lining coronary arteries do not contain similar amounts of eNOS protein. Indeed, these data suggest that eNOS protein content is greater in endothelial cells of large arteries than in endothelial cells of small coronary arterioles. In conclusion, the results of this study indicate that eNOS protein content does not increase with decreasing artery diameter in the coronary arterial tree. We conclude from these results that eNOS protein content is not equal among endothelial cells throughout the coronary arterial tree. Indeed, similar eNOS protein content in arteries with increasing smooth muscle cellto-endothelial cell ratios suggests that eNOS expression per endothelial cell increases with increasing diameter of the artery in the coronary tree. The authors thank Pam Thorne, Tammy Strawn, and Denise Holiman for excellent technical contributions to this work. This work was supported by National Heart, Lung, and Blood Institute Grants HL-52490 (to M. H. Laughlin) and HL-09739 (to C. R. Woodman) and National Aeronautics and Space Association Grant 00-GSRP 045 (to W. G. Schrage). REFERENCES 1. Ando H, Kubin T, Schaper W, and Schaper J. Cardiac microvascular endothelial cells express alpha-smooth muscle actin and show low NOS III activity. Am J Physiol Heart Circ Physiol 276: H1755–H1768, 1999.

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