Immunohistochemical Localization of Vascular Endothelial Growth Factor in the Endocrine Glands of the Rat*
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(2) 18. L. FAN and S. ISEKI;. paraformaldehyde in 0.1 M phosphate buffer. The following organs and tissues were excised out: the brain, pineal body, pituitary gland, eye, submandibular gland, thyroid gland, parathyroid gland, trachea, lung, heart, lymph node, thymus, spleen, liver, bile duct, pancreas, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, adrenal gland, kidney, urinary bladder, ovary, uterus, oviduct, testis, epididymis, prostata, soleus muscle and skin. They were further fixed by immersion in the same fixative for 4 h at 4 °C, and then each was divided into several specimens. The specimens for cryosectioning were rinsed in 30 sucrose/0.1 M phosphate buffer overnight at 4 °C for cryo-protection. The specimens for paraffin sectioning were dehydrated in a graded series of ethanol/ xylene and embedded in paraffin at 60°C . The specimens for immuno-electron microscopy were cut into small pieces, dehydrated in a graded ethanol series and embedded in LR-White resin (London Resin Co., London, UK) in the presence of 1% benzoyl peroxide. They were then polymerized for 24 h at room temperature with an ultraviolet polymerizer. Some organs of special interest were subjected to Western blot analysis. For this purpose, following sacrifice of the animals by perfusion with cold physiological saline, the kidney, liver, pituitary gland, thyroid gland, pancreas, adrenal gland and stomach were excised out, frozen immediately in liquid nitrogen and kept at -80°C until homogenization .. Fig. 1. Western blot analysis of rat organ homogenates. Purified VEGF (lane 1, 0.3pg) and homegenate samples (20 ,ig) from the kidney (lane 2), liver (lane 3), pituitary body (lane 4), thyroid gland (lane 5), pancreas (lane 6), adrenal gland (lane 7) and stomach (lane 8) were electrophoresed, blotted onto PVDF membranes and immunostained with the anti-VEGF antibody (A) and anti-VEGF antibody preabsorbed with the VEGF peptide (B). The molecular weight of the immunopositive protein (kDa) is indicated.. Antibodies Anti-VEGF (V3) antibody, a purified IgG fraction of rabbit antiserum raised against a synthetic pentadecapeptide (SERRKHLFVQDPQTC) corresponding to amino acids 121-134 of the mature human and rat VEGF (CoNN et al., 1990), was purchased from Immuno Biological Laboratories (IBL), Fujioka, Japan. For the negative control, the antibody was absorbed by the same synthetic VEGF peptide (kindly provided by IBL) at a concentration of l0,ug/ml for 8 h at 4°C. Antisera against growth hormone (GH), prolactin, thyroid stimulating hormone (TSH) and luteinizing hormone (LH) were donated by Dr. K. WAKABAYASHI, Institute for Molecular and Cellular Regulation, Gunma University. Antisera or antibodies against the following peptides were obtained from commercial sources: adrenocorticotropic hormone (ACTH), S-100 protein, calcitonin gene-related peptide (CGRP), insulin, glucagon, somatostatin and phenyl-ethanolamineN-methyl transferase (PNMT). Western blot analysis Purified recombinant human VEGF was purchased from PeproTech, Inc, Rocky Hill, NJ. The frozen organ specimens were homogenized in ice-cold 10 mM potassium phosphate buffer (pH 7.4) containing 150 mM KC1 using an Ultra-Dispensor (Yamato, Tokyo, Japan). They were then centrifuged at 3000 rpm for 10 min and the supernatants were used as homogenate samples after measurement of their protein content using BCA Protein Assay Reagent (Pierce, Rockf ord, Ill). The purified VEGF, 0.3 jig in amount, and the homogenate samples, 20 ,ug in amount, were separated under reducing conditions by electrophoresis in a 15% polyacrylamide slab gel at pH 8.8 in the presence of 0.1°c SDS, according to LAEMMLI (1970). They were then transferred electrophoretically to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) by the method of TOWBIN et al. (1979). The membranes were incubated overnight at room temperature with the anti-VEGF antibody at a concentration of 5 pg IgG/ ml and with the same antibody preabsorbed by the VEGF peptide. After washing in PBS/0.1% Tween 20, the immunoreaction was visualized by incubating the membranes successively with biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) at 1: 200 dilution for 1 h, streptavidin-conjugated horseradish peroxidase (Dakopatts, Glostrup, Denmark) at 1: 300 dilution for 1 h, and with 3'3'diaminobenzidin tetrahydrochloride in the presence of 0.01% hydrogen peroxide in 0.05 M Tris-HC1 (pH 7.5)..
(3) VEGF in Endocrine. Glands. 19. Fig. 2. Light micrographs of the pituitary gland immunostained with the anti-VEGF antibody (a) and antiVEGF antibody preabsorbed with the VEGF peptide (b). a. A minority of cells in the anterior lobe are immunopositive for VEGF. b. No cells are immunostained. a, b: x 155 Fig. 3. Light micrographs of the serial paraffin sections of the pituitary gland immunostained with the anti-VEGF antibody (a) and anti-TSH antiserum (b). Note that most VEGF-immunopositive cells (arrows) also react with the anti-TSH antiserum, whereas only a part of TSH-positive cells are VEGF-positive. x 460 Fig. 4. Electron micrograph of the anterior lobe of the pituitary gland immunostained for VEGF. Gold particles are localized in most of the secretory granules of the TSH cell, whereas the growth hormone cells (GH) are immunonegative. x 18,000.
(4) 20. L. FAN and S. ISEKI:. Immunohistochemistry For light-microscopic immunohistochemistry, the cryoprotected organ specimens were frozen and cut into 15 ,um sections using a cryostat, and the paraffinembedded tissue blocks were cut into 3 ,um sections using a microscme. Both sections were mounted onto gelatin-coated glass slides, and the paraffin sections were deparaffinized with xylene/graded ethanol series. They were then subjected to the routine light immunohistochemical procedure as described previously (YAMAMOTOet al., 1995). Briefly, the sections were first treated successively with 0.3% Tween 20 in PBS, 0.3% hydrogen peroxide/methanol and normal swine serum (Dakopatts) in 1: 20 dilution. Subsequently, the sections were incubated overnight at room temperature with the anti-VEGF antibody at concentrations of 2.5-5,ug/ml. The site of immunoreaction was then visualized with the same procedure as in Western blotting. To identify VEGF-immunoreactive cell types, serial paraffin sections were alternately immunostained with the antibodies to VEGF and various marker peptides. For electron-microscopic immunocytochemistry, ultrathin sections were cut from the LR-White-embedded tissue blocks and mounted onto nickel grids. They were first treated with 1% bovine serum albumin in PBS for 15 min and then incubated with the antiVEGF antibody (5 jig/ml) overnight at room temperature. After washing with PBS, the sections were subjected to the reaction with colloidal gold (20 nm)conjugated anti-rabbit IgG (British BioCell, Cardiff, UK) at 1: 20 dilution in PBS containing 0.1% BSA for 2 h at room temperature. They were then contrasted with uranyl acetate and lead citrate, and examined with a Hitachi H-700 electron microscope.. present anti-VEGF antibody and also demonstrated the presence of VEGF immunoreactivity in some endocrine glands.. Immunohistochemistry First, the cryosections from all organs and tissues were examined with light-microscopic immunohistochemistry for VEGF. Strong to moderate immunoreactivities were found in the pituitary gland, thyroid gland, pancreas, adrenal gland and stomach. A weak immunoreactivity was also found in the kidney, trachea, intestines and ovary. In all these sites, the specificity of the immunoreaction was confirmed using the control reaction with preabsorbed antibody. The rest of the organs and tissues showed no specific immunoreactivity for VEGF. Subsequently, identification of VEGF-positive cell types using consecutive paraffin sections as well as subcellular location of the immunoreactivity using electron microscopy were performed, and the following results were obtained.. Pituitary. RESULTS Western. an approximate size of 23 kD, which corresponded to the molecular weight of the VEGF monomer in a reducing condition (FERRARAand HENZEL,1989) (Fig. 1A). The immunopositive band with the same size was also detected in the homogenates from major endocrine glands. The intensity of immunoreaction decreased in order from the thyroid gland, pancreas, pituitary body to the adrenal gland. The stomach also showed the immunoreactivity. In contrast, only a faint reactivity in the kidney and scarcely any reactivity in the liver were detected. Control reactions with the anti-VEGF antibody preabsorbed by the VEGF peptide produced no immunopositive 23 kD band either in purified VEGF or in any organ homegenate (Fig. 1B). These results confirmed the specificity of the. blot. analysis. The recombinant antibody to form. VEGF reacted with the anti-VEGF a clear immunopositive band with. gland. VEGF immunoreactivity with moderate intensity was present in a small number of cells scattered throughout the anterior lobe, whereas no reactivity was observed in the intermediate and posterior lobes (Fig.. Fig. 5. Light micrographs of the thyroid gland immunostained with the anti-VEGF antibody (a) and anti-VEGF antibody preabsorbed with the VEGF peptide (b). a. An intense immunoreactivity for VEGF is present in the parafollicular cells, whereas the follicular cells are immunonegative. b. No cells are immunostained. a, b: x 80 Fig. 6. Light micrographs of the serial paraffin sections of thyroid gland immunostained with anti-VEGF (a) and anti-CGRP (b) antibodies. Almost all parafollicular cells react with both antibodies. x 310 Fig. 7. Electron micrograph of the thyroid gland immunostained for VEGF. Gold particles are localizaed in part of the secretory granules of the parafollicular cell (F), whereas the follicular epithelial cell (F) is immunonegative. x 14000. Inset. Higher magnification of the secretory granules of the parafollicular cell is shown. x 25,000.
(5) VEGF. Figs.. 5-7.. in Endocrine. Legends. on. Glands. the opposite. 21. page..
(6) 22. L. FAN and S. ISEKI:. 2a). This immunoreactivity was abolished when the primary antibody was absorbed by the VEGF peptide (Fig. 2b). The immunopositive cells appeared angular in shape and middle in size (Fig. 3a). To identify the type of VEGF-positive cell population, serial sections were alternately immunostained with antibodies against VEGF and one of the pituitary-specific hormones (GH, prolactin, TSH, ACTH and LH) and S-100 protein, a marker of the f olliculo-stellate cell. The results demonstrated that the VEGF-positive cell population was essentially coincident with the TSH-positive cell population, although the former was smaller than the latter (Fig. 3b). In three pairs of sections from different pituitaries, an average of 92% of the total VEGF-positive cells were simultaneously positive for TSH. On the other hand, only 31% of all TSHpositive cells were positive for VEGF. No other types of cells coincided with VEGF-positive cells (data not shown). By immuno-electron microscopy, the gold particles representing VEGF immunoreactivity were localized strictly to the secretory granules in all immunopositive TSH cells, which were characterized by well-developed rough endoplasmic reticulum and small round secretory granules 120-140 nm in diameter (KURosuMI, 1968) (Fig. 4).. Thyroid. gland. Numerous cells with very strong VEGF immunoreactivity were observed throughout the gland (Fig. 5a, b). They were scattered in the follicular epithelium and also present in groups in the interf ollicular space. In serial sections, this VEGF-positive cell population was identified as the paraf ollicular cell with its simultaneous immunoreactivity for CGRP, a marker of calcitoninproducing cells (KAMEDA,1987) (Fig. 6a, b). Virtually all paraf ollicular cells identified with anti-CGRP antibody appeared immunopositive for VEGF. Electron microscopy demonstrated the immunoreaction to be localized exclusively in the secretory granules of parafollicular cells (Fig. 7). No VEGF immunoreactivity was detected in the follicular epithelial cells at either light- or electron-microscopic levels.. Pancreas Intense VEGF immunoreactivity was localized to the endocrine pancreas, i. e., the islet of Langerhans, where the immunopositive cells occupied a majority of the area (Fig. 8a, b). In contrast, the exocrine pancreas was free of the immunoreaction both in acinar and ductal areas. Consecutive sections incubated with antiVEGF and anti-insulin antibodies clearly demonstrated the staining of identical cell populations (Fig. 9a, b), whereas the immunoreactivity for glucagon or somatostatin did not coincide with VEGF immunoreactivity (data not shown). These results indicated that the VEGF-positive cell type is the B cell of the endocrine pancreas. In electron microscopy, the immunoreactivity was confined to the secretory granules characteristic of B cells (ERLANDSEN,1980) (Fig. 10).. Adrenal. gland. VEGF immunoreactivity with moderate intensity was restricted to the medulla, where the immunopositive cells scattered in clusters constituted the minor cell population (Fig. ha, b). In contrast, much weaker reactivity was recognized in the major cell population of the medulla, with no reactivity in the cortex. When two consecutive sections were immunostained for VEGF and PNMT, a marker enzyme specific for the adrenaline (A) cell, the distribution patterns of the two immunopositive cell populations were completely opposite (Fig. 12a, b). These results indicated that VEGF immunoreactivity occurs preferentially in the noradrenaline (N) cell. Electron microscopy further demonstrated the localization of immunoreactivity to the secretory granules characteristic of N cells, with slightly larger sizes and darker cores as compared with those of A cells (CoUPLAND and KoBAYASHI, 1976) (Fig. 13).. Stomach Moderate. VEGF. immunoreactivity. was. localized. Fig. 8. Light micrographs of the pancreas immunostained with the anti-VEGF antibody (a) and anti-VEGF antibody preabsorbed with the VEGF peptide (b). a. An intense immunoreactivity for VEGF is present in most cells of the pancreatic islet (I), whereas the exocrine pancreas (E) is immunonegative. b. No cells are immunostained. x 83 Fig. 9. Light micrographs of the serial paraffin sections of the pancreas immunostained with the anti-VEGF antibody (a) and anti-insulin antiserum (b). The identical cells in the islet are immunostained. x 165 Fig. 10. Electron micrograph of the pancreatic islet immunostained localized in many of the secretory granules of the B cell (a), whereas immunonegative. E exocrine cell. x 26,700. for VEGF. Abundant gold particles are those of the A cell (b) and D cell (c) are. to.
(7) VEGF. Figs.. 5-10.. in Endocrine. Legends. Glands. on the opposite. 23. page..
(8) 24. Figs.. L. FAN and S. ISEKI:. 11-15.. Legends. on the. opposite. page..
(9) VEGF. the surface mucous cells in all areas of the stomach, with slightly higher intensity in the pyloric region, whereas the cells of mucosal glands, including the mucous neck cells, were free of the immunoreaction (Fig. 14a, b). Electron microscopy further confirmed the localization of VEGF immunoreactivity in the apical secretory granules of surface mucous cells (Fig. 15).. Other organs. and tissues. No VEGF immunoreactivity was recognized in the peptide-amine endocrine cells other than those described above, including the neurons of the hypothalamus, chief cells of pineal body and parathyroid gland, as well as various enteroendocrine cells. Weak VEGF immnoreactivity was demonstrated in the glomerular epithelial cell (podocyte) of the kidney, luteal cell of the ovary and a certain subset of mesenchymal cells in the trachea and digestive tract. Further analysis of these cell populations was not performed in the present study.. DISCUSSION. Unlike other growth factors, which target a wide range of different cell types, VEGF is thought to act selectively on endothelial cells through binding its specific receptors, fit-1 and KDR/fik-1 (DEVRIES et al., 1992; TERMAN et al., 1992). Also, VEGF is a unique growth factor in that it induces not only the proliferation of endothelial cells but also the permeabilization of microvessels, mainly of venules (CoLLINs et al., 1993). In past studies, VEGF has been investigated mostly in the tumor and embryonal tissues, where VEGF has been implicated in the neoangiogenesis (BREIERet al., 1992; JAKEMAN et al., 1993; PLATE et al., 1994). The. in Endocrine. Glands. 25. present study has provided immunohistochemical evidence of the occurrence of VEGF in subsets of peptideamine endocrine and in an exocrine cell type of normal adult rats, namely, the TSH cell of pituitary gland, paraf ollicular cell of the thyroid gland, B cell of the pancreatic islet, N cell of adrenal medulla and surface mucous cell of the stomach. Furthermore, immuno-electron microscopy has here demonstrated that VEGF is localized in the secretory granules, suggesting that, except for the surface mucous cell, these cell types secrete VEGF together with their hormones into the interstitial space. In regard to the occurrence of VEGF in normal adult organs and tissues, there is significant inconsistency between the mRNA expression revealed by previous studies and the immunoreactivity revealed by the present study. The abundant expression of VEGF mRNA has been reported for a variety of cell types in organs rich in vasculature, namely, the neurons of the brain, hepatocytes of the liver, alveolar cells of the lung, podocytes of the kidney, cortical cells of the adrenal gland, Leydig cells of the testis, luteal cells of the ovary and endometrial cells of the uterus (PHILLIPS et al., 1990; MONACCIet al., 1993; SHWEIKI et al., 1993; SIMON et al., 1995). However, the present study shows only weak VEGF immunoreactivity in the podocyte and luteal cell, and no reactivity in the rest of above cell populations. This discrepancy may be interpreted as follows: the cells described in the previous studies lack secretory granules and release VEGF rapidly in a constitutive manner resulting in low intracellular levels of VEGF, whereas the cells described in the present study accumulate VEGF in their secretory granules and release it in a regulated manner. This notion is supported by the present Western blot analysis demonstrating the presence of VEGF immunoreactivity in the endocrine organs in question and its absence in the liver, an organ known. Fig. 11. Light micrographs of the adrenal gland immunostained with the anti-VEGF antibody (a) and anti-VEGF antibody preabsorbed with the VEGF peptide (b). a. An intense immunoreaction for VEGF is present in some cells of the medulla (M), whereas the cortex (C) is immunonegative. b. No cells are immunostained. a, b: x85 Fig. 12. Light micrographs of the serial paraffin sections of the adrenal gland immunostained with the anti-VEGF antibody (a) and anti-PNMT antiserum (b). The opposite distribution patterns of two immunopositive cell populations in the medulla (M) indicate that VEGF immunoreactivity belongs to the noradrenaline cell. The nuclear staining in b is presumably nonspecific. C cortex. x 280 Fig. 13. Electron micrograph part of the secretory granules. of the adrenal medulla immunostained of the noradrenaline cell. x 32,000. for VEGF. Gold particles. Fig. 14. Light micrographs of the stomach immunostained with the anti-VEGF antibody preabsorbed with the VEGF peptide (b). a. An intense immunoreaction surface mucous cells. b. No cells are immunostained. a, b: x 260 Fig. 15. localized. Electron micrograph of the surface mucous cell immunostained in most of the secretory granules. x 13,200. are located in. antibody (a) and anti-VEGF for VEGF is present in the. for VEGF. Abundant. gold particles. are.
(10) 26. L. FAN and S. ISEKI:. to express abundant VEGF mRNA. The biological significance of the occurrence of VEGF in distinct endocrine cell types should be discussed in terms of their relation with the blood vascular system. Since neoangiogenesis in normal adult animals is restricted to certain areas such as the ovarian corpora, the role of VEGF released by the present endocrine cell types is most likely vascular permeabilization rather than angiogenesis. An abundant supply of blood vessels containing f enestrated capillary endothelium is a characteristic of most endocrine glands. The permeability of local microvessels is considered crucial in endocrine cells for their release of hormone products into the blood stream. Furthermore, all of the VEGF-positive endocrine glands, i.e., pituitary gland, thyroid gland, endocrine pancreas and adrenal gland, are composed of more than two types of endocrine cells. Taken together, the present endocrine cell types, by secreting VEGF in a regulated manner, may increase the permeabilization of local microvessels and thereby facilitate their own endocrine functions and also those of adjacent endocrine cell types. It should be mentioned that VEGF was originally isolated from the cultured folliculo-stellate (FS) cell of the bovine pituitary (GoSPODAROWICZet al., 1989; FERRARA and HENZEL, 1989), whereas the present study shows the absence of VEGF immunoreactivity in FS cells. Two explanations are possible for this discrepancy. One is that FS cells do not synthesize VEGF in vivo but become capable to do so after being growth in a culture system. Alternatively, FS cells, without secretory granules, synthesize VEGF but secrete it constitutively, as hepatocytes do, so that the routine immunohistochemical procedure is unable to detect VEGF in the FS cell cytoplasm. Also, it is of interest that, unlike other VEGFimmunoreactive endocrine cell types found in this study, which are immunopositive in almost all of the population, only a part of the total TSH cell population was immunopositive for VEGF. Whether this result indicates that VEGF-positive TSH cells constitute a distinct subtype of TSH cells or they represent a different functional state of the same TSH cell population will require further investigation. In the thyroid gland, networks of numerous anastomosing capillaries tightly surround individual follicles like baskets (FUIITA and MURAKAMI, 1974). Paraf ollicular cells are situated in the periphery of the follicles and also in the interf ollicular connective tissue, and hence in the close vicinity of the microvessels. The present study raises the possibility that the Paraf ollicular cell, besides producing calcitonin, plays an important role in regulating vascular permeability. through the action of VEGF, and thereby facilitates the endocrine functions of both parafollicular and follicular epithelial cells. In the endocrine pancreas, the expression and localization of VEGF in islet cells-more accurately the B cells-has previously been reported by other investigators (KURODAet al., 1995, CHRISTOFORIet al., 1995), although the subcellular localization of VEGF is not mentioned by these authors. We have demonstrated with immuno-electron microscopy that VEGF is localized exclusively in the secretory granules of B cells, suggesting that this cell type produces and secretes VEGF into the interstitial space in a regulated manner. The unique characteristic of islet blood flow is that some of the arteries entering the pancreatic lobule first terminate in the capillary network of the islet, where the blood receives the islet hormones and then carries them into another network of capillaries in the acinar tissue (FUIITA, 1973). The previous and present results suggest that the B cell regulates the vascular permeability in islets through VEGF, and thereby influences the functions not only of endocrine cells but also of exocrine cells. In the adrenal medulla, two chromaffin cell populations, i.e., A and N cells, have been distinguished by a variety of criteria at the light-and electron-microscopic levels (WooD,1963; COUPLANDand KOBAYASHI,1976), including PNMT as a marker of A cell (WURTMAN and AXELROD, 1966). The present study indicates VEGF to be a histochemical marker of the N cell. Also, chromaffin cells have been known to localize not only monoamines but also peptides such as enkephalin in their secretory granules (KOBAYASHIet al., 1983). The present study provides evidence that VEGF is also secreted by chromaffin cells together with monoamines in a regulated pathway. Finally, the surface mucous cell of stomach is exceptional among the present VEGF-immunopositive cell types in that it belongs to the exocrine cell population. VEGF, located in the secretory granules of surface mucous cells, is most likely released into the gastric lumen together with mucus. The recent observation of VEGF mRNA expression in the epithelia of prostata and the seminal vesicle, as well as the occurrence of VEGF protein in semen, is also indicative of VEGF production by exocrine cells (BROWN et al., 1995). The pathway by which gastric luminal VEGF reaches mucosal blood vessels is not clear. Possibly, VEGF penetrates into the lamina propria through some discontinuity in the tight junctions between the epithelial cells. In connection with this, the microvessels just beneath the surface epithelium are known to carry not only oxygen and nutritients but also parietal cell-derived bicarbonate ions, which.
(11) VEGF. may neutralize the back diffusion of luminal gastric acid to the mucosal surface (CANNON et al., 1982). It is tempting to assume that VEGF secreted by surface mucous cells takes part in the gastric mucosal protection by increasing the permeability of subepithelial microvessels. Alternatively, luminal VEGF may act directly on the mucosal epithelium. Such a possibility is suggested by recent evidence of the occurrence of flk-1, a VEGF receptor, on the pancreatic ductal epithelium (ROOMANet al., 1997). Further attempts to search for VEGF receptors on the epithelial cells of gastrointestinal Acknowledgments.. tract will be necessary. We. thank. tographic work. The kind gift BAYASHI is also acknowledged.. Mr.. S. YAMAZAKI for pho-. of antisera. by. K. WAKA-. REFERENCES BREIER, G., U. ALBRECHT, S. STERRER and W. RISAU : Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 114: 521-532 (1992). BROWN, L. 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