ARTICLE |
Correspondence to: Sandra E. Reznik, PhD; Dept of Pathology, Montefiore Medical Center, Forchheimer Bldg., 1300 Morris Park Avenue, Bronx, NY, 104612373.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins. (J Histochem Cytochem 46:13591367, 1998)
Key Words: carboxypeptidase E (CPE), carboxypeptidase D (CPD), placenta, umbilical cord, immunohistochemistry, immunoelectron microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carboxypeptidase E (CPE) was originally discovered as an enzyme associated with enkephalin biosynthesis in the adrenal medulla, and was named "enkephalin convertase" (
CPD has been localized to the trans-Golgi network (
Although a variety of both growth factors and neuroendocrine peptides have been identified and localized in the human placenta and umbilical cord (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
Two rabbit polyclonal antisera were used for immunostaining of each enzyme, one directed against the C-terminal portion of the enzyme and a second antiserum directed against the N-terminal portion of the enzyme. The specificities of the anti-CPE antisera and of the anti-CPD antisera were previously characterized by Western blotting (
Human Tissue
Frozen tissue sections taken from both the fetal and maternal sides of the central portion of placentas were retrieved from the human placental frozen tissue bank, which stores placental tissues at -80C from over 2000 patients who delivered at Georgetown University Hospital. Tissue from five patients with normal spontaneous vaginal deliveries without induction at 40 weeks of gestation, with no history of gestational diabetes mellitus, pregnancy-induced hypertension, or pre-eclampsia, were examined for chorionic plate, chorionic villous, and basal plate staining. Segments of fresh umbilical cords were collected in the delivery room from patients at Jack D. Weiler Hospital, sectioned, snap-frozen with optimal cutting compound and liquid nitrogen, and stored at -80C. All studies involving human tissue were conducted with IRB approval (IRB Protocol Number 1199707276.)
Immunohistochemistry
Tissue blocks were warmed to -20C, mounted on a microtome chuck with optimal cutting compound, and sectioned at 4 µm. After air-drying for at least 1 hr and desiccating for at least 24 hr, sections were fixed in PBS4% paraformaldehyde for 20 min. After extensive washing in PBS, sections were permeabilized with 0.2% Triton X-100 for 15 min. After subsequent washing in PBS, sections were incubated in a humidified chamber with PBS5% bovine serum albumin (BSA) for 30 min to reduce nonspecific binding. Excess buffer was blotted off and the primary antiserum, or preimmune serum for negative controls, was added, diluted 1~1000 for placental staining and 1~200 for umbilical cord staining, in PBS containing 1% BSA and 0.1% Tween 20. After a 1-hr incubation, the sections were washed in 0.2% Tween 20 in PBS and immunostaining was achieved using an avidinbiotin complex developer kit (Dako; Carpinteria, CA) and 3,3'-diaminobenzidine as substrate. Sections were counterstained with Mayer's hematoxylin.
Double Labeling
Sections of chorionic villous parenchyma were incubated sequentially with anti-CPD antiserum and anti-CD34 mouse monoclonal antibodies (Dako). Staining was developed with fluorescein-labeled anti-rabbit Ig antibodies and Texas red-labeled anti-mouse Ig antibodies (Vector; Burlingame, CA) according to the manufacturer's instructions. Tissue sections were examined with a Bio-Rad (Richmond, CA) confocal microscope.
Immunoelectron Microscopy
Immunohistochemistry was performed as described above, but tissue sections were cut by hand, incubated with 10% sucrose in PBS for 30 min, and kept floating in buffer throughout the staining protocol. After immunoperoxidaseDAB staining the sections were washed in PBS and postfixed in 1% osmium tetroxide with 0.01% potassium ferrocyanide for 1 hr. The tissue was dehydrated in a series of ascending ethanols from 50 to 100%, cleared in acetonitrile, infiltrated, and then embedded with a mixture of aralditeepon resin. Thin sections were examined unstained with a JEOL 100S electron microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For each enzyme, the same pattern of immunostaining was obtained with both the antiserum directed against the C-terminal end and the antiserum directed against the N-terminal portion of the protein. Furthermore, the staining patterns were essentially the same in tissues from five different patients. CPD and CPE appear to be co-localized in some placental cell types and in the umbilical cord. Whereas intense CPD immunostaining is observed in both the placenta and umbilical cord, CPE staining is intense in the umbilical cord but relatively weak in the placenta.
Specifically, in the basal plate, CPE immunostaining is present in extravillous invasive trophoblasts (Figure 1A, Figure T) and in vascular endothelial cells (Figure 1A, Figure E). The trophoblast staining is intracellular, with the brown DAB reaction product consistently localized to the perinuclear region of the cell (Figure 1A, Figure T). The endothelial cells show a uniform cytoplasmic immunoreactivity for CPE (Figure 1A, Figure E). No staining is seen in the basal plate when preimmune serum is substituted for immune serum (Figure 1B). CPD immunostaining is present in the same cell types of the basal plate that contain CPE immunoreactivity (Figure 1C). CPD staining is also perinuclear in the trophoblast (Figure 1C, Figure T) and uniform in the cytoplasm of the maternal endothelial cell (Figure 1C, Figure E).
|
In the chorionic villi, weak CPE staining is consistently observed in the fetal endothelial cells (Figure 2A, E; negative control Figure 2B), but not in the trophoblast. Intense chorionic villous immunostaining with CPD antisera is also consistently observed in the fetal endothelial cells (Figure 2C, Figure E). To confirm that the chorionic villous cells containing this intense CPD immunoreactivity are endothelial cells, the same tissue sections incubated with anti-CPD antiserum were double stained with monoclonal CD34 antibodies. Figure 3A shows labeling with fluorescein-tagged anti-rabbit antiserum, which represents CPD immunoreactivity. Figure 3B contains the same microscopic field as Figure 3A, but the filter is changed to reveal the Texas red-labeled anti-mouse antiserum, which represents CD34 immunoreactivity. By double labeling, CPD co-localizes with the endothelial cell marker CD34.
|
In the chorionic plate, focal CPE immunostaining was obtained in the amnion (Figure 4A; negative control, Figure 4B). CPD immunoreactivity, on the other hand, is present in many amniotic epithelial cells (Figure 4C). Both CPD and CPE amnion staining appear perinuclear.
|
CPD and CPE appear to be co-localized in the umbilical cord to vascular smooth muscle cells of both the umbilical vein (Figure 5) and the umbilical arteries (Figure 6). Both CPD and CPE antisera produce intense perinuclear staining in cells of the umbilical venous wall (Figure 5A and Figure 5C). All staining in the umbilical vein is eliminated with the substitution of preimmune serum for immune serum (Figure 5B). For both enzymes, the immunostaining is consistently more intense in the cells closest to the lumen, the smooth muscle cells just subjacent to the endothelial cells (Figure 5A and Figure 5C, double arrows). The identity of the stained cells as smooth muscle cells is confirmed by electron microscopy (see below).
CPD and CPE umbilical artery staining is similar to immunostaining patterns of these enzymes seen in the umbilical vein. Intense perinuclear staining of CPE is present throughout the artery wall (Figure 6A). A very similar staining pattern is obtained with CPD antisera (Figure 6C; negative control Figure 6B), whereas no staining is obtained with preimmune serum (Figure 6B). For both enzymes, staining is particularly intense in the cells closest to the artery lumen, just subjacent to the endothelial cells (Figure 6A and Figure 6C, double arrows).
In addition, intense immunoreactivity for both enzymes is focally present in Wharton's jelly cells (Figure 7). Umbilical cord sections stained with CPE antisera show dark perinuclear staining in focal Wharton's jelly myofibroblasts (Figure 7A, Figure M). No staining is present in these cells after incubation with preimmune serum (Figure 7B). Reaction with CPD antisera, however, also produces intense perinuclear staining in focal umbilical myofibroblasts (Figure 7C, Figure M).
To confirm the identity of the stained umbilical venous cells as smooth muscle cells and to examine the subcellular localization of CPD and CPE in these cells, immunoelectron microscopy was performed. The location of the stained cells subjacent to the internal elastic lamina and the presence of dense bodies in the stained cells (Figure 8D) indicate that they are smooth muscle cells. In tissue reacted with CPE antiserum, DAB reaction product is consistently associated with the rough endoplasmic reticulum that is in close proximity to the cell nucleus (Figure 8A, RER). The same staining pattern is obtained in tissue allowed to react with CPD antiserum (Figure 8C, RER), whereas the substitution of preimmune serum for immune serum eliminates staining (Figure 8B). Although CPD and CPE co-localize to the same cells in the umbilical vein at the light microscopic level, analysis by electron microscopy reveals distinct subcellular distributions. In addition to staining perinuclear rough endoplasmic reticulum, CPE antisera produce clusters of DAB reaction product subjacent to the cell membrane, following the entire perimeter of the cell (Figure 8A, Mb; Figure 9A, double arrows). This staining is not seen with either CPD antiserum (Figure 8C) or preimmune serum (Figure 8B and Figure 9B).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present work we provide immunohistochemical and immunoelectron microscopic evidence for the expression of two related carboxypeptidases in various cell types in the placenta and umbilical cord. Previous work had shown that CPD is highly expressed in human placenta (
Both the placenta and umbilical cord lack innervation and therefore must effect cell-to-cell communication under the influence of a variety of autocrine and paracrine factors. Many of these peptides are pro-cessed by the removal of C-terminal basic amino acid residues from intermediate peptide precursors. One example is insulin growth factor II (IGF-II), a single-chain polypeptide that shares 62% homology with proinsulin, a known CPE substrate (
CPE and CPD may also be involved in the processing of the precursor to ET-1, a potent vasoconstrictor peptide produced by endothelial cells (
In the present work, CPD and CPE are co-localized to vascular smooth muscle cells and the myofibroblasts of Wharton's jelly in the umbilical cord. Both of these cell types contain immunoreactivity for epidermal growth factor and transforming growth factor- (
At the ultrastructural level, the presence of immunoreactivity for both enzymes in the rough endoplasmic reticulum is consistent with their role in peptide and protein processing in umbilical venous smooth muscle cells. Although the two enzymes were co-localized at the light microscopic level, localization at the ultrastructural level also showed an important difference between CPE and CPD, i.e., the presence of CPE immunoreactivity in an additional site subjacent to the smooth muscle cell membrane. The difference in subcellular localization underlines the difference in function these two enzymes may have in peptide processing. CPE is probably involved in processing in the latter parts of the secretory pathway and CPD is more likely involved in processing that occurs in the Golgi/trans-Golgi network. The endogenous subcellular localization seen in umbilical venous smooth muscle cells in the present work is consistent with the immunocytochemical localization of both CPE and CPD previously seen in NIT cell lines, derived from mouse pancreatic ß-cells (
![]() |
Acknowledgments |
---|
Supported by National Institutes of Health Grants 1K08HD01209-01 (to SR) and DK-51271, DA-04494, and RSDA DA-00194 (to LDF).
We gratefully acknowledge Dr Yvonne Kress for her assistance with electron microscopy and Rachel Tucker for expert technical advice on immunohistochemistry.
Received for publication April 9, 1998; accepted July 22, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anfossi G, Cavalot F, Massucco P, Mattiello L, Mularoni E, Hahn A, Trovati M (1993) Insulin influences immunoreactive endothelin release by human vascular smooth muscle cells. Metab Clin Exp 42:1081-1083[Medline]
Arnqvist HJ, Bornfeldt KE, Chen Y, Lindstrom T (1995) The insulin-like growth factor system in vascular smooth muscle: interaction with insulin and growth factors. Metab Clin Exp 44:58-66[Medline]
Bogoni G, Rizzi A, Calo G, Campobasso C (1996) Characterization of endothelin receptors in the human umbilical artery and vein. Br J Pharmacol 119:1600-1604[Abstract]
Challis JR, Matthews SG, Van Meir C, Ramirez MM (1995) Current topic: the placental corticotrophin-releasing hormone-adrenocorticotrophin axis. Placenta 16:481-502[Medline]
Cohick WS, Gockerman A, Clemmons DR (1993) Vascular smooth muscle cells synthesize two forms of insulin-like growth factor binding proteins which are regulated differently by the insulin-like growth factors. J Cell Physiol 157:52-60[Medline]
Cooper ES, Greer IA, Brooks AN (1996) Placental proopiomelanocortin gene expression, adrenocorticotropin tissue concentrations, and immunostaining increase throughout gestation and are unaffected by prostaglandins, antiprogestins, or labor. J Clin Endocrinol Metab 81:4462-4469[Abstract]
Delafontaine P (1995) Insulin-like growth factor I and its binding proteins in the cardiovascular system. Cardiovasc Res 30:825-834[Medline]
Docherty K, Hutton JC (1983) Carboxypeptidase activity in the insulin secretory granule. FEBS Lett 162:137-141[Medline]
Fant M, Munro H, Moses AC (1986) An autocrine/paracrine role for insulin-like growth factors in the regulation of human placental growth. J Clin Endocrinol Metab 63:499-505[Abstract]
Fricker LD (1988) Carboxypeptidase E. Annu Rev Physiol 50:309-321[Medline]
Fricker LD (1998a) Carboxypeptidase E/H. In Barrett AJ, Rawlings ND, Woessner JFJ, eds. Handbook of Proteolytic Enzymes. London, Academic Press, 1341-1344
Fricker LD (1998b) Carboxypeptidase D. In Barrett AJ, Rawlings ND, Woessner JFJ, eds. London, Academic Press, 13491351
Fricker LD, Snyder SH (1982) Enkephalin convertase: Purification and characterization of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal chromaffin granules. Proc Natl Acad Sci USA 79:3886-3890[Abstract]
Graf AH, Hutter W, Hacker GW, Steiner H, Anderson V, Staudach A, Dietze O (1996) Localization and distribution of vasoactive neuropeptides in the human placenta. Placenta 17:413-421[Medline]
Grino M, Chrousos GP, Margioris AN (1987) The corticotropin releasing hormone gene is expressed in human placenta. Biochem Biophys Res Commun 148:1208-1214[Medline]
Hill DJ, Clemmons DR, Riley SC, Bassett N, Challis JR (1993) Immunohistochemical localization of insulin-like growth factors (IGFs) and IGF binding proteins -1, -2 and -3 in human placenta and fetal membranes. Placenta 14:1-12[Medline]
Hook VYH, Loh YP (1984) Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary. Proc Natl Acad Sci USA 81:2776-2780[Abstract]
Kamoi K, Sudo N, Ishibashi M, Yamaji T (1990) Plasma endothelin levels in patients with pregnancy induced hypertension. N Engl J Med 323:1486-1487[Medline]
Kanzaki M, Nobusawa R, Mogami H, Yasuda H, Kawamura N, Kojima I (1995) Production of activin A and follistatin in cultured rat smooth muscle cells. Mol Cell Endocrinol 108:11-16[Medline]
Laatikainen T, Saijonmaa O, Salminen K, Wahlstrom T (1987) Localization and concentration of beta-endorphin and beta-lipotrophin in human placenta. Placenta 8:381-387[Medline]
Margioris AN, Grino M, Rabin D, Chrousos GP (1988) Human placenta and the hypothalamic-pituitary-adrenal axis. Adv Exp Med Biol 245:389-398[Medline]
Marinoni E, Picca A, Scucchi I, Cosmi EV, DiIorio R (1995) Immunohistochemical localization of endothelin-1 in placenta and fetal membranes in term and preterm human pregnancy. Am J Reprod Immunol 34:213-218[Medline]
Masaki T, Yanagisawa M (1992) Endothelins. Essays Biochem 27:79-89[Medline]
Matsumara Y, Ikegawa R, Tsukahara Y, Takaoka M, Morimoto S (1990) Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells. FEBS Lett 272:166-170[Medline]
Naggert JK, Fricker L, Varlamov O, Nishina PM, Rouille Y, Steiner DF, Carroll RJ, Paigen BJ, Leiter EH (1995) Hyperproinsulinemia in obese fat/fat mice associated with a point mutation in the carboxypeptidase E gene and reduced carboxypeptidase E activity in the pancreatic islets. Nature Genet 10:135-142[Medline]
Petraglia F, Pasquale F, Nappi C, Genazzani AR (1996) Peptide signaling in human placenta and membranes: autocrine, paracrine, and endocrine mechanisms. Endocrine Rev 17:156-186[Medline]
Rao CV, Li X, Toth P, Lei ZM (1995) Expression of epidermal growth factor, transforming growth factor-, and their common receptor genes in human umbilical cords. J Clin Endocrinol Metab 80:1012-1020[Abstract]
Romero R, Avila C, Edwin SS, Mitchell MD (1992) Endothelin-1,2 levels are increased in the amniotic fluid of women with preterm labor and microbial invasion of the amniotic cavity. Obstet Gynecol 166:95-99
Schafer MK-H, Day R, Cullinan WE, Chretien M, Seidah NG, Watson SJ (1993) Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in-situ hybridization analysis. J Neurosci 13:1258-1279[Abstract]
Shen SJ, Wang CY, Nelson KR, Jansen M, Ilan J (1986) Expression of insulin-like growth factors in the regulation of human placental growth. J Clin Endocrinol Metab 63:499-505[Abstract]
Song L, Fricker LD (1995) Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J Biol Chem 270:25007-25013
Song L, Fricker LD (1996) Tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase D. J Biol Chem 271:28884-28889
Stewart AA, Haley JD, Qu GY, Stam K, Fenyo D, Chait BT, Marshak DR, Ng AY, Marley G, Iwata KK (1996) Umbilical cord transforming growth factor-beta 3: isolation, comparison with recombinant TGF-beta 3 and cellular localization. Growth Factors 13:87-98[Medline]
Sudo N, Kamoi K, Ishibashi M, Yamaji T (1993) Plasma endothelin-1 and big endothelin-1 levels in women with pre-eclampsia. Acta Endocrinol 129:114-120[Medline]
Takahashi M, Matsishita Y, Iijima Y, Tanzawa K (1993) Purification and characterization of endothelin-converting enzyme from rat lung. J Biol Chem 268:21394-21398
Turner AJ, Murphy LJ (1996) Molecular pharmacology of endothelin converting enzymes. Biochem Pharmacol 51:91-102[Medline]
Varlamov O, Fricker LD, Furukawa H, Steiner DF, Langley SH, Leiter EH (1997) ß-Cell lines derived from transgenic Cpefat/Cpefat mice are defective in carboxypeptidase E and proinsulin processing. Endocrinology 138:4883-4892
Varlamov O, Leiter EH, Fricker LD (1996) Induced and spontaneous mutations at Ser202 of carboxypeptidase E: effect on enzyme expression, activity, and intracellular routing. J Biol Chem 271:13981-13986
Varlamov O, Song L, Fricker LD (in press) Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: Localization to the trans-Golgi network and recycling from the cell surface. J Cell Sci
Xin X, Varlamov O, Day R, Dong W, Bridgett MM, Leiter EH, Fricker LD (1997) Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D. DNA Cell Biol 16:897-909[Medline]
Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayishi Y, Mitsui Y, Yazaki Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415[Medline]
Zhou J, Bondy C (1992) Insulin-like growth factor II and its binding proteins in placental development. Endocrinology 131:1230-1240[Abstract]