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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2479-2488

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2479-2488.)
© 1997 American Heart Association, Inc.

Articles

Expression of Multiple Isoforms of Nitric Oxide Synthase in Normal and Atherosclerotic Vessels

Josiah N. Wilcox; Romesh R. Subramanian; Cynthia L. Sundell; W. Ross Tracey; Jennifer S. Pollock; David G. Harrison; ; Philip A. Marsden

From the Department of Medicine, Emory University, Atlanta, Ga (J.N.W., R.R.S., C.L.S., D.G.H.); Abbott Laboratories, Chicago, Ill (W.R.T.); the Vascular Biology Center, Medical College of Georgia, Augusta, Ga (J.S.P.); and St Michael's Hospital and University of Toronto, Toronto, Ontario, Canada (P.A.M.).

Correspondence to Josiah N. Wilcox, PhD, Emory University, Department of Medicine, Division of Hematology/Oncology, 1639 Pierce Dr, Room 1115 WMRB, Atlanta, GA 30322. E-mail medjnw{at}emory.edu


*    Abstract
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Abstract Atherosclerosis is associated with reduced endothelium-derived relaxing factor bioactivity. To determine whether this is due to decreased synthesis of nitric oxide synthase (NOS), we examined normal and atherosclerotic human vessels by in situ hybridization and immunocytochemistry by using probes specific for endothelial (ecNOS), inducible (iNOS), and neuronal (nNOS) NOS isoforms. ecNOS was detected in endothelial cells overlying normal human aortas, fatty streaks, and advanced atherosclerotic lesions. A comparison of the relative expression of ecNOS to von Willebrand factor on serial sections of normal and atherosclerotic vessels indicated that there was a decrease in the number of endothelial cells expressing ecNOS in advanced lesions. iNOS and nNOS were not detected in normal vessels, but widespread production of these isoforms was found in early and advanced lesions associated with macrophages, endothelial cells, and mesenchymal-appearing intimal cells. These data suggest that there is (1) a loss of ecNOS expression by endothelial cells over advanced atherosclerotic lesions and (2) a significant increase in overall NOS synthesis by other cell types in advanced lesions composed of the ecNOS, nNOS, and iNOS isoforms. We hypothesize that the increased expression of NOS and presumably NO in atherosclerotic plaques may be related to cell death and necrosis in these tissues.


Key Words: atherosclerosis • nitric oxide synthase • endothelium-derived relaxing factor • endothelium


*    Introduction
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 *Introduction
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The endothelium modulates vascular tone by the synthesis and release of NO or a closely related compound.1 2 Endothelium-derived NO is synthesized from one of the two chemically equivalent guanidino nitrogens of l-arginine via the enzyme NOS.3 4 NOS exists as a family of related but distinct isoforms, including neuronal (nNOS),5 6 inducible (iNOS),7 8 9 10 and endothelial constitutive (ecNOS)11 12 13 isoforms. The nNOS and ecNOS enzymes are considered to be constitutively expressed, although recent data indicate that expression of ecNOS may be modulated by increased shear stress and cytokines.11 12 Constitutive expression of nNOS and ecNOS contrasts with that of iNOS, for which enzymatic activity is not detected under basal conditions but is induced by a variety of proinflammatory cytokines and endotoxin.7 Expression of iNOS activity has been found in a variety of cell types, including macrophages, vascular SMCs, mesangial cells, hepatocytes, Kupffer cells, and cardiac myocytes.

It is well established that endothelium-dependent vascular relaxation is abnormal in the setting of atherosclerosis and hypercholesterolemia. The molecular mechanisms responsible for this abnormality have been the subject of substantial investigation.14 15 16 17 There is evidence for deficiencies of substrate (arginine) availability,18 19 20 21 alterations of membrane signaling,22 23 and enhanced degradation of endothelium-derived NO.24 25 26 It is also possible that impaired endothelium-dependent vascular relaxation in atherosclerosis could occur as a result of decreased NOS expression or function. Multiple causes of altered endothelium-dependent vascular relaxation in hypercholesterolemia may exist, the expression of which may depend on the severity and duration of hypercholesterolemia or the vascular tissue in question. In the present experiments, we sought to examine NOS isoform expression in human blood vessels with various degrees of atherosclerosis by using the combined approach of in situ hybridization and immunohistochemistry.


*    Methods
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 *Methods
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Tissue Preparation
Human tissues were collected at Emory University Hospital. Aortas were obtained at autopsy within 4 hours of death or from transplant donors at the time of organ procurement and were fixed within 2 hours of surgical removal. These vessels were divided into three stages of atherosclerotic development based on the degree of intimal lesion formation, the presence of inflammatory cells, and regions of necrosis. Normal vessel segments (n=4; age range, 7 to 16 years old) had almost no intimal development and no inflammatory cells as defined by CD68 immunohistochemistry; early atherosclerotic aortas (n=4; age range, 21 to 29 years old) had only minor intimal development with scattered macrophage staining just under the luminal surface; and advanced atherosclerotic aortas (n=2; age range, 42 to 53 years old) had a thickened intima, numerous CD68-positive macrophages in the intima and media, and regions of necrosis and cholesterol deposits in the lesion. Human carotid endarterectomy specimens (n=3; age range, 68 to 87 years old) were obtained within 60 minutes of surgical removal and were used as additional representative samples of advanced human atheromas. Tissue collection was performed in accordance with Emory University guidelines and was approved by the Emory University Hospital Human Investigations Committee.

The tissues were fixed in 4% paraformaldehyde buffered with 0.1 mol/L NaPO4 (pH 7.4) for 3 to 4 hours at 4°C, cryoprotected in 15% sucrose–PBS overnight, embedded in optimal cutting temperature compound (OCT, Miles Laboratories), frozen in liquid N2, and stored at -70°C. Cryosections (7 to 10 µm) were thaw-mounted onto Fisher SuperFrost Plus slides (Fisher Scientific), immediately refrozen, and stored with desiccant at -70°C until use.

In Situ Hybridization
In situ hybridization using antisense 35S-labeled riboprobes was performed as previously described.27 28 29 (Also see www.emory.edu/wilcox/)In brief, cryosections were pretreated with paraformaldehyde and proteinase K (Sigma Chemical Co) and prehybridized in 100 µL hybridization buffer (50% formamide; 0.3 mol/L NaCl; 20 mmol/L Tris, pH 8.0; 5 mmol/L EDTA; 0.02% polyvinylpyrrolidone; 0.02% Ficoll; 0.02% BSA; 10% dextran sulfate; and 10 mmol/L DTT) at 42°C. Serial sections were hybridized with 6x105 cpm of 35S-labeled riboprobes at 55°C. After hybridization, the sections were washed with 2x SSC (1x SSC=150 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.0) with 10 mmol/L ß-mercaptoethanol and 1 mmol/L EDTA, treated with RNase A (Sigma), and washed in the same buffer followed by a high-stringency wash in 0.1x SSC with 10 mmol/L ß-mercaptoethanol and 1 mmol/L EDTA at 55°C. The slides were then washed in 0.5x SSC and dehydrated in graded alcohols containing 0.3 mol/L ammonium acetate. The sections were dried, coated with NTB2 nuclear track emulsion (International Biotechnologies), and exposed in the dark at 4°C for 4 to 12 weeks. After development, the sections were counterstained with hematoxylin and eosin to aid in cell identification.

The ecNOS probe was PM7, a 1.2-kb cDNA from the coding region of the human endothelial calcium/calmodulin–dependent NOS12 subcloned into the EcoRI site of pBluescript I SK(-) (Stratagene). The iNOS probe was pHuNOS.ind, a 0.6-kb cDNA subcloned into the pCR II vector (Invitrogen), and represents amino acids 47 to 241 from exons 3 to 8 of the human inducible calcium-independent NOS that was cloned from cytokine-treated human mesangial cells.30 The cDNA sequence of pHuNOS.ind was identical to the 5' end of an iNOS cDNA isolated from cytokine-treated human hepatocytes.31 The nNOS probe was pHuNOS.neur, a 1.4-kb cDNA cloned into the BamH1 site of pBluescript I SK (-) and represents the 5' untranslated region and amino acids 1 to 423 (5' region) from exons 2 to 6 of the human neuronal NOS that was cloned from human fetal brain tissue.32 These cDNAs were transcribed33 using RNA polymerases in the presence of 35S-UTP (Amersham; specific activity, 1200 Ci/mmol). Full-length antisense transcripts were used for hybridizations. The likelihood of cross-hybridization of specific probes to other NOS isoforms was very low, since the nucleotide sequence of these NOS isoform probes showed only 65% homology, with the longest region of identity being 17 bases.

In situ hybridization experiments were controlled by hybridizing serial sections with the same cDNA probes transcribed in the sense orientation or with riboprobes directed against human vWF. The vWF cDNA was a 0.8-kb Pst I fragment subcloned into pGEM3Z (Promega), which originated from a larger cDNA obtained from the American Type Culture Collection (clone No. 59787 deposited by Evan Sadler, Washington University School of Medicine, St Louis, Mo). vWF in situ hybridizations were also used as a positive control, since every tissue had ECs, either on the luminal surface or in the adventitial vasa vasorum, that hybridized with this probe. Each in situ hybridization experiment was performed in triplicate on serial sections with the four 35S-UTP–labeled riboprobes (ecNOS, iNOS, nNOS, and vWF) and developed after 4, 8, or 12 weeks of exposure. This allowed direct comparison of hybridization results obtained with these probes for each tissue. In addition, experiments were replicated a minimum of three times for each tissue specimen over a period of 24 months with essentially identical results.

Immunohistochemistry
Immunohistochemistry was performed using monoclonal antibody H-3234 directed against purified bovine endothelial NOS (1/100 dilution of tissue culture supernatant),35 a polyclonal antibody directed against mouse iNOS (antibody 8196, 1/500 dilution),36 or a polyclonal antibody directed against rat nNOS (1/4000 dilution).37 Another polyclonal anti-peptide antibody was developed and used for immunohistochemistry directed against amino acid sequence 117 to 128 of mouse iNOS (antibody P1225; used at a concentration of 40 µg/mL).38 Recently, three additional monoclonal antibodies directed against peptide fragments of the NOS isoforms have become commercially available and were also tested in these studies including anti-bNOS (directed against amino acids 1095 to 1289 of human nNOS and used at a concentration of 5 µg/mL), anti-ECNOS (directed against amino acids 1030 to 1203 of human ecNOS and used at a concentration of 5 µg/mL), and anti-macNOS (directed against amino acids 961 to 1144 of mouse iNOS and used at a concentration of 5 µg/mL) (Transduction Laboratories).

In brief, frozen, paraformaldehyde-fixed tissue sections were thawed and fixed in acetone for 5 minutes, dried, and rehydrated in PBS. The primary antibodies were applied at the indicated dilutions in 1.0% BSA in PBS and incubated in a humidified chamber for 60 minutes at room temperature. The sections were washed in PBS and then incubated with a biotinylated secondary antibody (horse anti-mouse IgG at a 1/400 dilution or goat anti-rabbit IgG at a 1/200 dilution for the monoclonal or polyclonal antibodies, respectively; Vector Laboratories) in PBS containing 1.0% BSA and 2.0% normal serum (horse or goat) for 30 minutes at room temperature. This was followed by washing the sections in PBS and incubation with the avidin-biotin enzyme complex and chromogenic substrate as described by the manufacturer. NOS proteins were visualized using the Vectastain Elite ABC peroxidase system or the Vectastain ABC alkaline phosphatase system with substrate kit III (blue reaction product; Vector Laboratories). Serial sections treated with secondary antibodies only or with nonimmune IgG did not show any staining. For cell identification, serial sections were stained with markers for ECs (Ulex lectin; Vector Laboratories),39 40 SMCs (HHF35; Enzo Diagnostics),41 or macrophages (CD68; DakoPatts) as previously described27 using the Vectastain ABC alkaline phosphatase system and Vector substrate kit I (red reaction product).

Statistics
The numbers of cells hybridizing to the ecNOS- or vWF-specific riboprobes were compared on serial sections. The luminal surface was examined with polarized light epiluminescence (Leitz) and positive cells scored at 250x magnification. Luminal cells were considered positive if there were >5 silver grains clustered around a hematoxylin-stained nucleus. The total numbers of cells scored positive by hybridization to the vWF or ecNOS probes on representative sections were determined and the data expressed as the mean percent of vWF-positive cells containing ecNOS mRNA (±SEM). Statistical comparison was made by using a two-tailed t test.


*    Results
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 *Results
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Normal Tissue Distribution
The normal distributions of ecNOS, iNOS, and nNOS were determined by in situ hybridization and immunohistochemistry from samples of nonatherosclerotic human aorta obtained at the time of organ procurement from heart transplant donors. In general, ecNOS mRNA and protein were found in the endothelium overlying normal nonatherosclerotic aortas (Fig 1Down) as well as in the endothelium lining the vasa vasorum vessels in the adventitia and media (data not shown). A comparison of the results obtained from hybridization of the ecNOS and vWF riboprobes indicated that not all ECs contained ecNOS; this was especially evident in the vasa vasorum. A comparison of Ulex lectin staining and/or vWF in situ hybridization with results of hybridizations obtained using the ecNOS riboprobe suggested that ecNOS expression appeared to be confined to larger arteries and vasa vasorum venules, with little or no expression detected in small arterioles. Immunohistochemistry using antibodies directed against ecNOS (H-32 or anti-ECNOS) confirmed the presence of ecNOS protein in the endothelium of those vessels that contained ecNOS mRNA (Fig 1ADown versus 1D). ecNOS mRNA was not detected in any other cell types in these normal tissues.



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  		Figure 1. Localization of NOS protein and mRNA in normal human aorta by in situ hybridization and immunohistochemistry. Serial sections from normal human aortas were hybridized using an antisense 35S-labeled riboprobe directed against ecNOS (A), vWF (B), or nNOS (C) or stained with antibody H32 directed against bovine ecNOS (D). Luminal ECs contained both ecNOS mRNA (A) and protein (D) but did not show any positive hybridization to nNOS- (C) or iNOS- (data not shown) specific probes. These vessels did not contain iNOS or nNOS protein as determined by immunohistochemistry with any of the antibody preparations, nor were any macrophages detected in these tissues by CD68 staining. Immunohistochemistry was performed using the Vector ABC peroxidase kit with diaminobenzidine, so the specific reaction appears brown. All in situ hybridizations shown were exposed for 8 weeks prior to development and staining with hematoxylin and eosin (original magnification for A through C. x100; for D, x80).

iNOS and nNOS mRNAs or proteins were not detected in the intima or media of normal vessels (Fig 1CUp). iNOS protein staining (with antibody 8196, anti- macNOS, or P1225) was very strong in the adventitia surrounding some normal aortas but was localized to neutrophils and monocytes trapped in thrombi surrounding these vessels (data not shown). iNOS mRNA was not detected in these cells by in situ hybridization. These aortas had been classified as normal vessels, since they had no intimal thickening and no CD68-positive macrophages in the intima or media. The inflammation associated with the adventitia of these vessels may have been stimulated by thrombus formation caused by traumatic injury at the time of death, or it could have formed postmortem during surgery to remove vital organs for transplantation. These results may suggest a role for iNOS in acute inflammatory processes.

Fatty Streaks/Early Atherosclerotic Lesions
To determine whether NOS expression was altered during development of atherosclerosis, we examined early atherosclerotic lesions in human aortas by immunohistochemistry and in situ hybridization (Fig 2Down). ecNOS protein staining with the H-32 antibody was localized to the endothelium overlying these lesions as well as in macrophages in the upper intima. In general, H-32 staining in ECs over the early lesions appeared weaker than that seen in normal arteries. Strong iNOS protein staining was also often found localized to CD68-positive macrophages in early lesions. The results of staining with both the polyclonal anti-mouse iNOS (antibody 8196) or the anti-human peptide iNOS antibodies (P1225 or anti-macNOS) were essentially the same. nNOS protein staining was also found in the inflammatory cells in these early lesion sites by the polyclonal rat nNOS antibody as well as the monoclonal anti-bNOS human peptide antibody. Occasional staining of the luminal cells overlying these early lesions was sometimes seen with the iNOS and nNOS antibodies.



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  		Figure 2. Localization of NOS mRNAs and proteins in early atherosclerotic lesions from human aortas by in situ hybridization and immunohistochemistry. Tissue sections of human aortas obtained from organ transplant donors were hybridized with 35S-labeled riboprobes specific for ecNOS (A), iNOS (B), or nNOS (C) or immunostained with antibodies directed against ecNOS (H32; D), iNOS (antibody 8196; E), or nNOS (rat polyclonal antibody; F). Serial section immunohistochemistry revealed an intact endothelium and the presence of scattered macrophages in the intima by Ulex lectin binding or CD68 staining, respectively (data not shown). ecNOS mRNA tended to be localized over ECs on the luminal surface or in macrophages in the neointima (A, small arrows) but was much stronger in adventitial vessels (not shown). No mRNA was detected with the iNOS probe (B), but an increased number of silver grains were found associated with intimal cells on sections hybridized with the nNOS probe (C, small arrows). Immunohistochemistry indicated that ecNOS, iNOS, and nNOS proteins were localized to inflammatory cells in the neointima (D through F). The localization of ECs in this lesion was also confirmed by hybridization of a serial section with a vWF-specific riboprobe, which suggested a continuous endothelium in this region without hybridization to mononuclear-appearing cells in the neointima (data not shown). Serial sections hybridized with an 35S-labeled sense ecNOS riboprobe were negative (data not shown). All in situ hybridizations shown were exposed for 8 weeks prior to development and counterstaining with hematoxylin and eosin. Immunostaining was as described in "Methods" using Vector blue as the chromogen, and the tissue was counterstained with methyl green (original magnification for A through C, x125; for D through F, x80). Large arrows indicate border of internal elastic lamina.

ecNOS mRNA was detected in ECs by in situ hybridization overlying early aortic lesions, although this hybridization often appeared to be weaker than that seen in normal vessels (Fig 2Up). A surprising finding was that nNOS mRNA was often localized to the intima of the early lesions in macrophages and/or ECs at sites consistent with the localization of nNOS protein by nNOS antibody staining. In contrast, iNOS mRNA was not detected in early lesions by in situ hybridization, even though the presence of the protein was confirmed with two separate antibodies.

Advanced Atherosclerotic Lesions
The expression of the various NOS isoforms was also examined in advanced atherosclerotic lesions in the aorta and carotid arteries. ecNOS mRNA and protein were localized to luminal ECs and macrophages in the advanced lesions (Figs 3Down and 4Down). Many ECs lining the vasa vasorum in the adventitia or intima of the plaque also contained ecNOS mRNA and protein (data not shown).



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  		Figure 3. Localization of ecNOS in macrophages and ECs of advanced atherosclerotic lesions. Human carotid endarterectomy specimens were stained using antibody H-32 directed against bovine endothelial NOS (A and C) or with the CD68 antibody to identify macrophages (B and D). ecNOS immunoreactivity could be detected in ECs on the luminal surface of advanced lesions (C) as well as in mononuclear-appearing cells, which also stained with the CD68 antibody. Tissue was counterstained with methyl green (original magnification for A and B, x5; for C and D, x40).



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  		Figure 4. Localization of NOS mRNA and protein in advanced human carotid atherosclerotic plaques. In situ hybridization and immunohistochemistry were performed on serial sections from human carotid endarterectomy specimens using antisense 35S-labeled ecNOS (A), iNOS (B), nNOS (C), or vWF (D) riboprobes or stained with antibodies against ecNOS (H-32; E), iNOS (8196; F), nNOS (rat polyclonal antibody; G), or CD68 to identify macrophages (H). ecNOS mRNA was found in luminal ECs as well as inflammatory cells in the upper intima (A). A few silver grains were found over the inflammatory cells after hybridization with the iNOS riboprobe (B), but this was judged to be negative, since there was no specific clustering of silver grains around cell nuclei in this regions and the signal was very weak. Longer exposures of the slides hybridized with the iNOS probe (12 weeks) did not improve the hybridization signal and confirmed our interpretation that this was a negative hybridization result. nNOS mRNA was found in both ECs and macrophages in this lesion (C). Serial sections stained with the NOS isoform antibodies using diaminobenzidine as the chromogen (brown reaction product) confirmed localization of ecNOS (E), iNOS (F), and nNOS (G) staining in CD68-positive macrophages in this lesion (H). Similar results were obtained by immunohistochemistry using the anti-peptide NOS antibodies (not shown). All in situ hybridizations shown were exposed for 8 weeks. Tissue sections were counterstained with hematoxylin and eosin (A through D) or hematoxylin alone (E through H). Original magnification for A through H, x80. Small arrows in all panels indicate positive ECs; arrowheads, inflammatory zones.

To determine whether ecNOS mRNA synthesis was downregulated in ECs overlying atherosclerotic lesions, a direct comparison was made of ecNOS and vWF in situ hybridizations on serial sections from normal and atherosclerotic arteries (the TableDown). There was no significant difference in the percent of ecNOS to vWF mRNA–containing cells in sections from normal and early atherosclerotic samples (45.8±14.1% versus 41.1±16.1%, respectively; P=.84). There was, however, a significant decrease in the percent of ecNOS to vWF mRNA–positive cells in advanced atherosclerotic lesions compared with the combined cell counts derived from normal and early atherosclerotic vessels (the TableDown; 13.5±7.6% versus 43.4±9.6%, respectively; P<.05).


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  		Table 1. Comparison of ecNOS and vWF In Situ Hybridizations on Serial Sections From Normal and Atherosclerotic Arteries

iNOS protein was detected in advanced lesions by immunohistochemistry in areas of inflammation and in the necrotic cores where iNOS was localized primarily in macrophages (Fig 4Up). Staining using all three iNOS antibodies was essentially the same, thus confirming the presence of iNOS protein at these sites. Serial sections stained with secondary antibodies alone or nonimmune IgG were negative, indicating the specificity of staining (data not shown). However, we were unable to detect iNOS mRNA by in situ hybridization in any advanced lesions. The best example of a potential iNOS mRNA signal in macrophages is shown in Fig 4Up, but even this was a relatively weak signal and was considered negative in comparison with the vWF or other NOS hybridizations. Parallel studies were conducted with human SMCs stimulated with cytokines, which have previously been shown to express iNOS mRNA.30 31 These cells were hybridized in the same experiments with the human vascular tissues as a positive control for iNOS mRNA localization. These cells showed appropriate induction of iNOS mRNA as detected by in situ hybridization, indicating that there was nothing wrong with our iNOS probe or the detection system (data not shown). In the same experiments, no iNOS mRNA was detected by in situ hybridization in the advanced atherosclerotic plaques in areas of strong iNOS immunohistochemical staining (data not shown).

nNOS was detected in macrophages in inflammatory zones of advanced atherosclerotic lesions, ECs, and mesenchymal-appearing intimal cells (Figs 4Up and 5Down). The staining pattern with the nNOS antibodies was similar to that seen with the iNOS antibodies but was more restricted and less robust. nNOS mRNA was also found in macrophages in the inflammatory zones of these lesions in a pattern consistent with nNOS immunohistochemical localization (Fig 4Up). In some cases, nNOS mRNA and protein could be found in ECs and mesenchymal-appearing intimal cells in the advanced lesions (Fig 5Down). Mesenchymal-appearing intimal cells were identified as those that did not stain with macrophage, EC, or SMC markers (CD68, Ulex lectin, or HHF35, respectively). These cells are thought to be derived from vascular SMCs and may be a population of dedifferentiated or modulated SMCs that lack {alpha}-smooth muscle actin.27 The localization of nNOS in mesenchymal-appearing intimal cells may reflect production of NOS by SMCs in the plaque.



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  		Figure 5. Localization of nNOS mRNA and protein in ECs and mesenchymal-appearing intimal cells of advanced human carotid atherosclerotic lesions. In situ hybridization was performed on serial sections from human carotid endarterectomy specimens using 35S-labeled ecNOS (A), iNOS (B), nNOS (C), or vWF (D) riboprobes. ECs were observed to hybridize with the ecNOS-, nNOS-, and vWF-specific riboprobes. Mesenchymal-appearing intimal cells containing nNOS mRNA (C) were present in the fibrous cap/upper intima of this lesion. The conclusion that these are mesenchymal-appearing intimal cells is based on the lack of staining seen in this region with CD68, Ulex lectin, or HHF35 antibodies on serial sections (data not shown). No ecNOS protein was detected with the H-32 antibody in this region of the lesion (E). Immunohistochemistry indicated that nNOS protein was colocalized with nNOS mRNA in the ECs and mesenchymal-appearing intimal cells (F). Immunohistochemistry was performed as described in "Methods." Vector blue was used as the final chromogen, and the tissue was counterstained with methyl green. All in situ hybridizations shown were exposed for 8 weeks and were counterstained with hematoxylin and eosin (original magnification for all panels, x100). In all panels, small arrows represent ECs; arrowheads, mesenchymal-appearing intimal cells.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Endothelium-dependent vascular relaxation is abnormal in both hypercholesterolemia and atherosclerosis.14 15 16 17 In the present study, we sought to determine whether this might be explained by a loss of NOS within ECs overlying early or advanced atherosclerotic lesions by examining the distribution of NOS mRNA and protein by in situ hybridization and immunohistochemistry. Our results indicate that although ecNOS mRNA and protein are present in ECs overlying fatty streaks and advanced atherosclerotic lesions, fewer ECs appear to be expressing ecNOS in advanced lesions compared with normal vessels. Thus, our results indicate a downregulation of ecNOS in ECs overlying advanced lesions. These findings suggest a molecular explanation for impairments in endothelium-dependent vascular relaxation in these vessels.

Multiple isoforms of NOS have been identified, including ecNOS,11 12 13 nNOS,5 6 and iNOS.8 9 10 The structural organization of the three NOS genes has been reported and the endothelial, neuronal, and inducible isoforms localized to human chromosomes 7, 12, and 17 respectively.30 32 Overall there appears to be a shift away from endothelial expression of ecNOS in the normal vessel to increased expression of all three NOS isoforms in inflammatory cells of advanced atherosclerotic lesions. ecNOS, iNOS, and nNOS protein staining patterns were consistently found in inflammatory zones or areas of necrosis in macrophages. Consistent with the localization of protein, ecNOS and nNOS mRNAs were also found in similar sites by in situ hybridization. In contrast, virtually no iNOS mRNA was detected in the present series of experiments using sense or antisense riboprobes directed against exons 2 to 8 of the iNOS gene.

The original full-length human iNOS sequence was cloned from cytokine-treated human hepatocytes and had been previously used to localize iNOS mRNA in cytokine-stimulated SMCs.31 The iNOS cDNA used in the present series of experiments was derived from cultured human mesangial cells treated with multiple cytokines30 and was identical to the 5' end of the hepatocyte iNOS cDNA. Despite the fact that we were able to easily detect iNOS mRNA by in situ hybridization of cytokine-stimulated SMCs by using mesangial cell iNOS cDNA, in parallel experiments we were unable to detect iNOS mRNA in human vascular tissues by in situ hybridization. This is surprising, given the extensive protein staining observed with multiple iNOS antibodies in these tissues. There are two possible explanations for this: either there is no iNOS mRNA present in these tissues or it is present at levels below the level of detection by in situ hybridization. This may suggest that iNOS mRNA is very labile and/or that the protein is very stable in these tissues, thus maintaining high protein levels with very little mRNA present. Recent evidence indicates that iNOS mRNA has a short half-life, a finding consistent with this hypothesis.42

We believe that the hybridization results and immunohistochemical staining are specific for each isoform in these tissues. It is unlikely that there were significant problems with cross-reactivity of the riboprobes used for in situ hybridization. In the present studies, the antisense riboprobes directed against the ecNOS, iNOS, and nNOS used for in situ hybridization were 65% homologous, with <17 bases of continuous identity. The posthybridization washes used in the in situ hybridization procedure included both an RNase digestion and a high-stringency wash, which would be expected to differentiate between 17-base or full-length hybridizations. It is unlikely that there was significant cross-reactivity of the antibodies used for immunohistochemistry, since ecNOS, iNOS, and nNOS are only 47% to 66% homologous at the amino acid level. The specificity of the ecNOS antibody H-32 has been confirmed by Western blots, which indicate that this antibody does not cross-react with either iNOS or nNOS.34 36 The specificity of the anti-iNOS polyclonal antibody 8196 has also been demonstrated by Western blotting, which also confirms that this antibody does not cross-react with either ecNOS or nNOS.36 43 Although the anti-peptide NOS antibody (P1225) has been used previously in immunohistochemical analyses,38 no Western blot data are available for this antibody or for the antibodies from Transduction Laboratories (anti-ECNOS, anti-macNOS, or anti-bNOS ). There is recent evidence suggesting that there is more than one iNOS isoform with high homology on the 3' but not the 5' end of the gene.44 45 If further work supports these findings, then perhaps it is possible that we detected this iNOS isoform by antibody staining in the present study but were unable to detect its mRNA by in situ hybridization because we used a probe that corresponded to the 5' end of the previously cloned cDNA. Additional work is now under way to identify the source of the iNOS protein staining in advanced atherosclerotic lesions.

A major finding of the present work is the increased expression of nNOS mRNA in association with atherosclerosis. In contrast to the relatively restricted expression of ecNOS in ECs, the mRNA and protein for nNOS have been detected in a wide variety of cell types and tissues37 46 47 : neurons of the central and peripheral nervous system, the macula densa, skeletal muscle, adrenal gland, pancreatic islets, and the uterus, among others. The finding in the present study that nNOS mRNA and protein are expressed in human atherosclerotic vessels is of great biological interest. In situ hybridization indicated that nNOS mRNA and protein were localized to ECs, macrophages, and mesenchymal- appearing intimal cells in advanced atherosclerotic lesions. Mesenchymal-appearing intimal cells exhibit a stellate shape, display variable amounts of cytoplasm, and have large, pale, hematoxylin-staining nuclei. These cells do not stain well with cell type–specific antibodies directed against SMCs, ECs, macrophages, or T cells.27 By light microscopy these cells have sometimes been referred to as intimal SMCs,48 stellate cells,49 or synthetic-state SMCs.50 Platelet-derived growth factor-A and -B chains, as well as the platelet-derived growth factor-ß receptor,27 tissue factor,28 and monocyte chemotactic protein-151 mRNAs have been detected previously in mesenchymal-appearing intimal cells in advanced atherosclerotic plaques. Previous work indicated that NO production by SMCs may be inhibitory to cell proliferation,52 and iNOS mRNA has been identified from cytokine-stimulated SMCs.10 31 53 Our data suggest that local generation of NO in SMCs in atherosclerotic lesions may be accounted for by nNOS.

These studies suggest that the amount of NOS present in the atherosclerotic vessel wall overall is substantially increased compared with that in normal vessels. On the basis of antibody staining, this increase is composed of the ecNOS, nNOS, and iNOS isoforms. The finding of increased NOS mRNA and protein in atherosclerosis is compatible with earlier experiments showing that aortas of hypercholesterolemic rabbits release larger quantities of nitrogen oxides than do normal vessels.24 Recent studies using pharmacological probes have suggested that there is increased production of NO in nonendothelial layers of vessels from hypercholesterolemic animals.54 With respect to this issue, it is interesting to speculate that increased NO production within the intima may have adverse biological effects on the blood vessel wall. iNOS produces substantially larger quantities of nitrogen oxides than does the ecNOS isoform.55 Increased release of NO into the intima of the atherosclerotic plaque may increase cellular damage, potentially by forming Fe-S-NO clusters within the mitochondria.56 The interaction of NO with superoxide anions, which may be produced in excess by vessels in the setting of hypercholesterolemia, may also yield peroxynitrite radicals. Peroxynitrite radicals are known to induce cellular damage via their strong oxidant properties and release of hydroxyl radicals.57 58 It is conceivable that these toxic, cytolytic effects of excess NO may contribute to cell death and tissue necrosis commonly observed within advanced atherosclerotic lesions. In support of this hypothesis, it is interesting to note that nNOS has been implicated in the generation of tissue damage and necrosis associated with cerebral ischemia in mice59 while ecNOS was thought to play a protective role.59 60 Thus, the shift from ecNOS expression in normal arteries to increased expression of other NOS isoforms in the mature lesions of atherosclerosis may contribute to the overall process of atherogenesis by increasing cell death and necrosis. In this regard, the excess production of NO within atherosclerotic lesions may be deleterious to the function of the vessel wall.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
ecNOS = endothelial constitutive NOS
iNOS = inducible NOS
nNOS = neuronal NOS
NOS = nitric oxide synthase
SMC = smooth muscle cell
vWF = von Willebrand factor


*    Acknowledgments
 
This work was supported by grants HL48667-02 (to J.N.W. and D.G.H.) and HL49743-02 (to J.N.W.) from the National Institutes of Health, Bethesda, Md, and a research grant from The Medical Research Council of Canada (to P.A.M.) The authors wish to thank Cheryl Ross for her expert technical assistance.

Received February 26, 1997; accepted July 29, 1997.


*    References
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