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ARTICLE |
Cellular Distribution of Endothelin-converting Enzyme-1 in Human Tissues
Petra Kortha, Rainer M. Bohleb, Pierre Corvola, and Florence Pinetaa INSERM U36, Collège de France, Paris, France
b Institute of Pathology, University of Giessen, Giessen, Germany
Correspondence to: Florence Pinet, INSERM U36, Collège de France, 3 rue d'Ulm, 7505 Paris, France. Fax number: 1 44 27 16 91.
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Summary |
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Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447–461, 1999)
Key Words: endothelin-converting enzyme, human, in situ hybridization, immunocytochemistry
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Endothelin-1 (ET-1), the most potent vasoconstrictor peptide known, was identified by 
ETs are initially synthesized as large precursor polypeptides, called preproETs, which are cleaved at two pairs of basic amino acids, generating intermediate peptides, the bigETs. The bigETs are then cleaved by endothelin-converting enzyme (ECE), producing the mature ETs. ECE is a key enzyme in the biosynthesis of the ETs because the biological activities of bigETs are negligible (
The distribution of ECE-1 mRNA in bovine and rat tissues has been investigated by Northern blot and RT/PCR analyses (
The distribution of human ECE-1 has been analyzed by Northern blotting (
Targeted disruption of ECE-1 showed that it is the activating protease for both bigET-1 and bigET-3 in vivo (
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Human Tissues
Normal human tissues were obtained at surgery from tumor-free or disease-free resection margins from patients (n = 39) undergoing biopsy or resections for various reasons (e.g., localized tumor resection) or less than 24 hr after death from cadavers (n = 7) at the Institute of Pathology, Justus-Liebig-University (Giessen, Germany) or the Hôpital Tenon (Paris, France). The ages of the patients undergoing biopsy/resection ranged from 1 week to 80 years; 20 were male and 19 were female. The age of the deceased individuals ranged from 1 week to 49 years; five were male and two were female. We examined only tissue areas that appeared macroscopically and microscopically completely normal after routine histological staining. In postmortem tissues, autolytic tissue damage was excluded by hematoxylin–eosin-stained sections. In addition, protein integrity was checked by performing CD31 (1:10 in paraffin sections and 1:50 in frozen sections, clone JC/70A; DAKO, Hamburg, Germany) and vimentin (1:50 in paraffin sections and 1:100 in frozen sections, clone V9; DAKO) immunolabeling. Only positive stained specimens were used in the study. Tissues were either snap-frozen in liquid nitrogen, embedded in OCT Compound (Miles; Elkhart, IN), and stored at -80C until use or fixed in 4.5% formalin, pH 7.0, or 4% paraformaldehyde in PBS for at least 24 hr, routinely processed, and embedded in paraffin.
Immunohistochemistry (IHC) with CHO Cells
A stable CHO cell line that overproduces human ECE-1a (
Immunohistochemistry with Human Tissue Specimens
Cryostat or microtome sections (5 µm) of the specimens were mounted on slides, air-dried, frozen, and stored at -20C until required. Hematoxylin–eosin staining was used to exclude autolytic tissue damage in postmortem tissues. ECE-1 was detected (
Preparation of Riboprobes
The human ECE-1a partial cDNA, corresponding to nucleotides 304–1666, was generated by RT/PCR using mRNA from primary cultures of human umbilical vein endothelial cells with the sense primer 5'-TCCATGGACCCCACGGTGGACCCC-3' and the reverse primer 5'-AGGCGTTCACCATGGGCGGGGTCA-3'. It was subcloned into the pCRII vector using the TA cloning kit (Invitrogen; Leek, Netherlands). The sequence of the insert was checked using a Sequenase kit (US Biochemicals; Cleveland, OH). The recombinant plasmid was linearized by digestion with Hind III to obtain the anti-sense or Xba I to obtain the sense RNA. In vitro transcription and labeling with [35S]-UTP (Amersham; Les Ulis, France) were carried out together with T7 or SP6 RNA polymerase (Boehringer; Mannheim, Germany). Probes were precipitated with ammonium acetate and ethanol, dried by speed-vac centrifugation, and dissolved in TE-DTT (10 mM Tris, 1 mM EDTA, 20 mM DTT).
In Situ Hybridization (ISH)
We used an ISH protocol involving microwave pretreatment to enhance the hybridization signal, as described in detail elsewhere (
IHC and ISH labeling was evaluated by different investigators (RMB, PK), each blind to the other's assessment. Results were classified on a 4-point scale, as shown in Table 1.
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Specificity of Anti-ECE-1 Antiserum and ECE-1 Riboprobe
The specificity of the polyclonal anti-ECE-1 antiserum 473-17-A (
These sections were also used to ascertain the specificity of the ECE-1 RNA probe. The anti-sense ECE-1 probe (Figure 1A) and antiserum 473-17-A (Figure 1C) strongly labeled about half the cells, the proportion of cells producing ECE-1a. Therefore, antiserum 473-17-A and the anti-sense riboprobe for human ECE-1a were specific in paraffin-embedded specimens. Furthermore, the sense probe (Figure 1B) and preimmune serum (Figure 1D) did not react nonspecifically with transfected CHO cells overproducing ECE-1.
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To exclude a crossreaction of anti-ECE-1 antiserum with ECE-2, we performed IHC with peptide-blocked antiserum on frozen sections of adrenal gland. When anti-ECE-1 antiserum was preincubated with the corresponding ECE-1 peptide, labeling was absent for a peptide concentration of 1 mg/ml and 100 µg/ml, whereas the same concentration of the corresponding ECE-2 peptide did not affect the labeling intensity (not shown). As a positive control, a serial section was labeled with untreated antiserum. This experiment demonstrated that the new anti-ECE-1 antiserum 473-17-A does not crossreact with ECE-2, despite the high homology between the two enzymes within the peptide region used for ECE-1 antiserum generation.
In a previous study, crossreaction of anti-ECE-1 antiserum with NEP was checked by immunoblotting of purified NEP (
Synthesis of ECE-1 mRNA and Protein in Human Tissues
We compared the immunostaining pattern of freshly fixed cryostat sections with that of formalin-fixed, paraffin-embedded sections of the same material. The distribution and intensity of labeling were similar in each case (not shown). ISH and IHC were performed for at least two paraffin-embedded specimens of each tissue examined. We looked for the presence of ECE-1 mRNA and protein in the same cells, by performing ISH and IHC on serial sections for some tissues (Figure 3 and Figure 6).
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The distribution patterns of ECE-1 mRNA and protein in normal human tissues are summarized in Table 1. The average staining intensity values, as estimated by two independent investigators in two to seven tissue sections from different patients, are given. Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 show photomicrographs of typical examples of ECE-1 mRNA or protein synthesis in all major organs. In almost all tissues examined, ECE-1 mRNA and ECE-1 protein had identical distributions. Their relative staining intensities were equivalent except for some epithelia (e.g., those of the bladder), in which there were differences between mRNA and protein levels. ECE-1 mRNA and protein were widely distributed in epithelial and nonepithelial tissues and in almost all major organs. However, neither ECE-1 mRNA nor protein was detected in certain nonvascular mesenchymal tissues, such as supporting/connective tissue, white adipose tissue, striated muscles, and hyaline cartilage.
Circulatory System
A high level of ECE-1 mRNA and protein was detected in almost all vascular endothelial cells, regardless of the organ of origin. There were only minor differences in the ECE-1 labeling intensities of endothelial cells from conducting (Figure 2A–D) or resistance vessels (Figure 2E, Figure 3A, and Figure 3B). Moreover, ECE-1 labeling intensity was higher in the endothelial cells of microvascular arteries than in the venous endothelial cells. However, in some specialized endothelial cells from the glomeruli of kidney and the sinusoidal cells of the liver and spleen, the level of ECE-1 mRNA and protein synthesis was less predictable (Table 1). Moderate levels of ECE-1 mRNA and protein were also detected in the endocardium (Table 1). In contrast to the situation in the vascular endothelial cells, ECE-1 mRNA and protein levels in vascular smooth muscle cells differed greatly, from undetectable to moderate within the same vessel or within several vessels from the same specimen, whereas the myocardium produced no ECE-1 (Table 1).
Respiratory System
ECE-1 mRNA and protein are synthesized throughout the respiratory system. The respiratory epithelium, from the trachea (Figure 4A) down to the bronchioli, was moderately to strongly labeled for ECE-1 mRNA and its protein. The smooth muscle cells of the tunica muscularis in the conducting airways were irregularly labeled for ECE-1 protein, whereas its mRNA was not detectable (Table 1). ECE-1 mRNA was absent from hyaline cartilage, whereas the seromucous glands of the trachea and main bronchus synthesized both mRNA and protein (Figure 4A). There was strong labeling for ECE-1 LmRNA in the alveoli of lung (Figure 4B) and moderate staining for ECE-1 protein (Figure 5A) in almost all cells. No staining was observed when preimmune serum was used (Figure 5B). We were unable to distinguish accurately between capillary endothelial cells and pneumocytes. However, ECE-1 was synthesized in almost all the cells of the alveolar region, so it is probable that, in addition to endothelial cells, at least one type of pneumocyte also synthesizes ECE-1.
Gastrointestinal Tract
ECE-1 was detected in the secretory and collecting parts of the submandibular gland. ECE-1 mRNA synthesis was moderate in intercalated and large duct epithelial cells but was low in acinar cells, whereas ECE-1 protein synthesis was variable in the acini and moderate in all ducts (Figure 4C). The squamous epithelium of the esophagus contained a low level of ECE-1 in its basal cell layers. The smooth muscle cells of the muscularis produced small quantities of ECE-1 protein, but no mRNA was detected. However, both mRNA and protein were detected in the submucosal glands (Table 1). The intensity of ECE-1 mRNA and protein labeling in the tunica muscularis was low (Table 1). There was low to moderate labeling of ECE-1 mRNA in the intestine (Figure 4D), with no consistent difference between the basal and luminal parts of the intestinal epithelium. Little or no ECE-1 was detected in the intestinal muscularis. Lymphoid tissue (known as Peyer's patches) in the small intestine was weakly labeled for ECE-1 mRNA and protein in a diffuse pattern, whereas ECE-1 was absent from the lymphoid aggregations of the large intestine (Table 1). The strongest labeling was detected in the ganglia of the mesenteric plexus (Figure 4E) and in the submucosal plexus. In the liver, little or no ECE-1 was present in hepatocytes. There was low labeling in the endothelial cells of the portal vein and no mRNA was detected in the bile ducts, whereas the intensity of ECE-1 protein labeling was absent to moderate in the various specimens (Table 1). The secretory acini of the exocrine pancreas were strongly labeled for ECE-1 mRNA and moderately stained for the protein (Figure 5C). The ECE-1 protein was also detected in the duct epithelium, but its mRNA was undetectable. In the endocrine pancreas, there was weak to moderate staining for ECE-1 (Table 1).
Urinary System
The levels of ECE-1 mRNA and protein detected in the urinary system differed. In the kidney, there was strong staining for ECE-1 protein in the glomerulus (Figure 5D), whereas the staining intensity was weak to moderate in the distal and collecting tubules (Table 1). In contrast, ECE-1 mRNA levels were low in the glomerulus and undetectable in the proximal tubules (Figure 5D). No mRNA but moderate levels of ECE-1 were detected in the bladder epithelium, whereas there was no staining in the smooth muscle layers (Table 1).
Reproductive System
The hormone-secreting Leydig cells of the testis were strongly labeled for ECE-1 mRNA and protein, whereas the seminiferous tubules were only weakly labeled (Figure 4K and Figure 5F). In the epididymis, moderate amounts of ECE-1 were detected in the epithelium, but ECE-1 was not detected in the smooth muscle cells (Table 1). In the prostate gland, ECE-1 was restricted to the glandular epithelium (Figure 5E). In the female reproductive system, there was a clear ECE-1 distribution pattern in the ovary. The follicular granulosa and theca cells were intensely labeled, whereas the surrounding stromal cells were only moderately or weakly labeled (Figure 3C and Figure 3D). There was less ECE-1 in the oviduct than in the ovary, and it was restricted to the epithelium (Table 1). In this study, we examined breast tissue from nonpregnant nonlactating women only. Moderate levels of ECE-1 were detected in the duct and lobular epithelium, whereas the myoepithelial cells produced less ECE-1 (Figure 4I).
Endocrine Glands
ECE-1 labeling was strongest in endocrine tissue. In the adrenal gland, the strongest staining for ECE-1 was in the zone glomerulosa and fasciculata, with slightly less staining in the zona reticularis (Figure 6A and Figure 6B). In the medulla, there was scattered but strong labeling (Figure 6C and Figure 6D). The follicular epithelium of the thyroid was moderately labeled for ECE-1 (Figure 6E and Figure 6F).
Lymphoid Tissues
In the thymus, there was weak ECE-1 labeling in the medulla, but no ECE-1 mRNA was detected in Hassall's corpuscules from the medullar region and the thymic cortex (Figure 4F). In the lymph nodes, high endothelial venules were strongly labeled for ECE-1 mRNA and protein, whereas sinus cells produced very little. The germinal centers in follicules were moderately labeled. In contrast, ECE-1 was absent from the T-cell region of the lymph nodes (Figure 4G and Figure 4H), but small amounts were detected in the red and white pulp of the spleen (Table 1). The follicles of the lymphoid tissue of the tonsil were weakly labeled, whereas the surrounding lymphoid tissue was unstained. As in the esophagus, the basal cell layers of the stratified squamous epithelium were weakly labeled for ECE-1 mRNA and protein (Table 1).
Other Tissues
There was little or no ECE-1 mRNA and protein in the skin and its appendages, with the exception of the acinar cells and ducts of skin sweat glands, which were moderately stained for ECE-1 (Table 1). In contrast to white adipose tissue, brown adipose tissue had scattered patches of ECE-1 synthesis (Table 1).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Endothelins (ETs) have a variety of biological effects in noncardiovascular tissues, and there is increasing evidence that ETs act locally rather than as circulating peptides. Endothelial cells in culture release ET-1, predominantly into the basolateral compartment (
We developed specific tools for detecting ECE-1 and its mRNA with high sensitivity in cells rather than merely in tissues. For immunohistochemistry, an antiserum was raised against a synthetic peptide containing amino acids 473–489 of the extracellular part of human ECE-1, which recognizes the monomeric and dimeric forms of ECE-1 (
Three human ECE-1 isoforms (ECE-1a, ECE-1b, and ECE-1c) have been identified (
There are known differences in the distribution patterns of ETs between rodent species and humans (see below), which led us to study ECE-1 expression in human tissues. This study aimed to (a) determine the distribution of ECE-1 in most tissues; (b) compare the distribution of ECE-1 mRNA with that of ECE-1 protein; (c) compare the level of ECE-1 produced in the cardiovascular system with that of other tissues; and (d) clarify whether the distribution of ECE-1 is the same as that of its substrates (bigETs) and their corresponding receptors (ET-A and ET-B).
The ECE-1 gene was expressed in diverse human tissues, suggesting a major biological role for the enzyme. ECE-1 was not strictly limited to endothelial cells in the organs examined but was also detected in mesenchymal and epithelial cells. In almost all organs, the cellular distributions of ECE-1 mRNA and ECE-1 protein were identical, demonstrating that ECE-1 mRNA synthesis and protein expression occur at the same site. This result is not surprising because ECE-1 is a membrane-anchored zinc metalloprotease and no soluble secreted form has yet been described that could potentially be detected in a cell compartment other than that in which mRNA was localized. However, the relative ECE-1 protein and mRNA contents differed in some epithelia. In the bladder, ECE-1 mRNA labeling was lower than that of protein staining, in contrast to the respiratory epithelium, in which mRNA and protein were present at equivalent intensities. This may reflect different stabilities of ECE-1 protein between tissues and suggests that there may be different functions of ECE-1 in different tissues.
The strongest staining was associated with the vascular endothelial cells of most organs examined. This is consistent with the work of
Although ECE-1 has been cloned from cultured human endothelial cells, ECE-1 mRNA and protein were only very recently detected for the first time in endothelial cells in human kidney sections by in situ hybridization and immunohistochemistry (
Strong staining intensity of ECE-1 was observed in a variety of epithelial tissues. As expected, epithelial cells of the respiratory system, such as those of trachea and main bronchus, were labeled. This is not surprising because ECE was initially purified from rat lung (
The reproductive system has among the highest staining intensity of ECE-1. In the testis, ECE-1 staining was higher in the Leydig cells than in the seminiferous tubules, which were only weakly stained.
ECE-1 was also investigated in the female reproductive system. ECE-1 mRNA and protein labeling were strong in the granulosa and theca cells of the ovary.
Strong staining intensity for ECE-1 was detected in the adrenal gland. There was an ECE-1 gradient accross the zones of the adrenal gland, with the zona glomerulosa having the highest staining. Autoradiography has shown that ET-A receptors are limited exclusively to the zona glomerulosa, whereas ET-B receptors are found throughout the adrenal cortex (
The use of a sensitive, specific RNA probe and a specific polyclonal antibody made it possible to map in detail the cellular distribution of ECE-1 mRNA and protein in the cells of a variety of human tissues. This approach could be used for retrospective analysis of a large number of paraffin-embedded autopsy and surgical archive specimens. ECE-1 mRNA and its protein were abundantly synthesized in all endothelial cells and in a number of nonvascular cell types, consistent with the ET system being mainly paracrine in nature. The high level of ECE-1 in endocrine cells (e.g., zona glomerulosa and zona fasciculata of the adrenal gland, granulosa and theca cells of the ovary, and Leydig cells of the testis), suggests that ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. This idea is supported by a recent study showing the hydrolysis of bradykinin by ECE-1 (
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Acknowledgments |
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Supported in part by the Ministère de la Recherche et de l'Enseignement (ACC-SV9). Dr P. Korth received funding from the Fondation pour la Recherche Medicale.
We would like to thank H. Wallenfels, M.-T. Morin, and F. Mongiat for technical expertise, Dr J.-M. LeMoullec for providing ECE-1 cDNA, and Dr J.-M. Gasc for helpful discussion.
Received for publication September 18, 1998; accepted October 28, 1998.
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