Analysis of the Sinusoidal Endothelial Cell Organization During The

Analysis of the Sinusoidal Endothelial Cell Organization during the Develop- mental Stage of the Fetal Rat Liver Using a Sinusoidal Endothelial Cell Specific Antibody, SE-1 Mamoru Morita1, Wang Qun2, Hitoshi Watanabe1, Yuko Doi1, Michio Mori3, and Katsuhiko Enomoto1'* department of Pathology, Akita University School of Medicine, Hondo, Akita 010-8543, Japan, department of Pathology, School of Basic Medicine, NormanBethune University of Medical Science, Jilin, P.R. of China, and *Department of Pathology, Sapporo Medical University School of Medicine, S-l, W-17, Chuo-ku, Sapporo 060, Japan

Keywords: sinusoidal endothelial cells/SE-1 antibody/fetal rat liver

ABSTRACT.The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membraneand was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid maystart at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possi- bly regulated by the intimate association with hepatocytes.

The liver sinusoid is a specialized vascular system which changes in SEC during development of the fetal rat liv- consists of endothelial cells with characteristic morpho- er (1, 2, ll). Barbera-Guillem et al. (2) reported that the logical features, such as open fenestration and lack of number and size of the fenestration of SECdiffer great- basal lamina (10, 16). These features are thought to fa- ly depending on their zonal location in the liver and cilitate the active exchangeof various metabolic sub- stage of development. However, the details in the sinu- stances between blood and hepatocytes through the soidal organization in fetal livers have yet to be well es- space of Disse. Although the mechanisms of appear- tablished. ance and maintenance of these morphological features Wepreviously established a novel monoclonal anti- are not yet known, recent studies have suggested that body, SE-1, which specifically recognized an antigen these structures are regulated by hepatocyte related fac- (M.W. 45 kD) expressed only in rat liver SEC and in no tors or by the extracellular matrix (8, 9). other type of endothelial cell (7, 12). Although the na- Structure and function of SEC have been well stud- ture of SE-1 antigen has yet to be elucidated, the ex- ied in adult rat livers as described above. However, pression of SE-1 antigen has been considered to be re- only a few studies have reported on the structural lated to some specific function of SEC. In this study, in order to elucidate the structural organization and the * Corresponding author: Katsuhiko Enomoto Ph.D, Department functional maturation of SECin the fetal rat liver, we of Pathology, Akita University School of Medicine, Hongo 1-1-1, examined the expression of SE-1 antigen during devel- Akita 010-8543, Japan opment of the fetal rat liver immunohistochemically Tel: +81-18-884-6059, Fax: +81-18-836-2601 and immunoelectron microscopically. E-mail: [email protected]

341 M. Morita et al.

MATERIALS AND METHODS For the quantitative analysis of the length of SE-1 positive SEC, fluorescent microphotographs of the fetal livers (15th, Animals 17th, 19th and 21st day of age) were randomly taken and the Pregnant Fischer 344 (F344) rats at 13 days of gestation were length of positive sinusoids (more than 200 sinusoids/each purchased from Charles River Japan Co. (Kanagawa, Japan) age) was measured. or obtained by mating in the animal facility of Akita Univer- sity, School of Medicine. The rats were maintained in rooms Immunoelectronmicroscopicexamination with temperature and light control. Theywere anesthetized The liver tissues were fixed in PLPsolution for 12 hr at 4°C. with ehter and fetuses from 13 to 21 days of gestation were After washing in TBS, approximately 50 //m liver slices were obtained after hysterectomy. Livers of the fetus at each gesta- prepared with a Micro-Slicer DTK-2000 (Dosaka EM, Kyoto, tional day were carefully removed and processed for the fol- Japan), and 10 times-diluted normal goat serum was applied lowing histological examinations. A part of the livers was fro- to them to block the nonspecific reactions. The liver slices zen in liquid nitrogen and stored at -80°C for immunofluo- were then incubated with SE-1 antibody for 12 hr at room rescent staining, and the remaining part of the livers was temperature, washed again in TBSand incubated with peroxi- fixed in freshly prepared periodate-lysine-paraformaldehyde dase-conjugated goat anti-mouse IgG (DAKOJapan, Kyoto, (PLP) solution for immunoelectron microscopic analysis. For Japan, 1:100 dilution) as a second antibody for 1hr at the control experiment, livers of the newborn and 10-week- roomtemperature. Specific reaction products were detected old rats were also processed. in 0.02% diaminobenzidine-4HCl (Wako Pure Chemical, Osaka, Japan) and 0.0045% H2O2 solution as previously Immunofluorescentstaining described (12). They were post-fixed in 1% osmium solution, For immunofluorescent staining, frozen sections (approxi- dehydrated in a series of concentrated ethanol, and embed- mately 5 fim thick) of the fetal liver were placed on silane- ded in epoxy resin using propylene oxide. Ultrathin sections coated slides and fixed in cold acetone for 15 min. The sec- counterstained with lead citrate were examined with a JEM tions were dried and incubated with SE-1 monoclonal anti- 1200 EXelectron microscope. body or rabbit antibody against Factor VHI-related antigen (DAKOJapan, Kyoto, Japan) for 1 hr at room temperature. RESULT S After several washes with Tris buffered saline (0.05 MTris, 0.83% NaCl: TBS), the second antibody, a nuorescein isothi- In the adult rat liver, as previously reported (12), ex- ocyanate (FITC)-conjugated goat anti-mouse IgG or a rho- pression of SE-1 antigen was observed only in SECbut damine (TRITC)-conjugated swine anti-rabbit IgG (DAKO not in the endothelial cells of portal or hepatic veins Japan, Kyoto, Japan) diluted to 1:100 with TBS was ap- (Fig. la). Onthe contrary, the Factor vin related anti- plied. Stained sections were examined with a fluorescence mi- gen (F vm) was positive in the endothelial cells of hepa- croscope (Nikon Co., Tokyo, Japan). tic veins but negative in SEC(Fig. lb).

Fig. 1. Immunofluorescent localization of SE-1 antigen (a) and Factor vmrelated antigen (b) in the contiguous sections of the adult rat liver. The SE-1 antigen is expressed only in the sinusoidal endothelial cells but not in the endothelial cells of hepatic vein. Factor vm related antigen is positive in endothelial cells of the hepatic vein. ( x 90)

342 Sinusoidal Endothelial Cell Organization in the Fetal Rat Liver

More than 20 fetal rat livers at each gestational day immature hepatocytes and hematopoietic cells were ob- were examined. As shown in Fig. 2a, the dilated vascu- served in the liver of 13th day fetuses. However, no lar lumens corresponding to the fetal vitelline vein with positive staining for SE-1 antigen was seen in the fetal

Fig. 2. Changes in histology and expression of the SE-1 antigen in livers at 13th day (a, b), 15th day (c, d), 17th day (e, f) and 21st day (g, h) fetuses. At 13th day, the dilated vascular lumens derived from the vitelline veins are observed (a). Expression of the SE-1 antigen (b, d, f and h) increases during the development of fetal liver. H&Estaining; a, c, e, g. ( x 110)

343 M. Morita et al. liver at this age (Fig. 2b). At 15th day, the liver paren- ducts were found along the surface of plasma mem- chymamassively proliferated and a few vascular lu- brane of SEC(Fig. 3a), but the positive reaction at this mens were present (Fig. 2c). Although it is difficult to stage was weaker than that of the adult rat liver as re- identify the organization of sinusoidal structure in the ported previously (12) and sometimes showed segmen- liver by hematoxylin and eosin stained sections, immu- tal distribution on the plasma membrane of SEC. The nostaining for fibronectin revealed organized sinusoid- SE-1 positive SEC (Fig. 3a) showed relatively thin cyto- like structures delineated by fibronectin deposits (data plasm which extended along the sinusoidal lumen as not shown). Presence of the organized sinusoid-like compared with the negative SEC (Fig. 3b). Interesting- structures in the liver of 15-day-old fetus was also con- ly, the hepatic stellate-like cell (S) was localized at the firmed by electron microscopical examination. perisinusoidal space and seemed to contact with both Immunofluorescent staining of the livers of 15-day- SECand hematopoietic cells (Fig. 3a). These cells were old fetus revealed sporadic positive reactions in the si- often observed in the space of Disse at the later stage of nusoid-like lumens (Fig. 2d). The length of the positive development. However, characteristic lipid droplets as lumens was short. The majority of lumens was not seen in stellate cells of the adult liver were not seen in completely delineated by the positive reactions. There- their cytoplasm. after, the positive staining for SE-1 increased in num- The relationship between the expression of SE-1 anti- ber and length, and connected with each other, result- gen and the appearance of fenestration in SECwas not ing in the formation of the mature sinusoidal struc- clear. The SE-1 antigen expressed-SEC increased in tures during development of the fetal liver (Fig. 2d, f, number along with the course of development of the fe- h). The lining of sinusoidal lumens at the later stages of tal liver. Pinocytotic vesicles which are positive for the development (after 18th day) showed completely posi- SE-1 antigen at their inner surface were often seen (Fig. tive for SE-1. During the development, expression of 4). At the 21st day of gestation, foci of hematopoietic the SE-1 antigen in the fetal livers seemed to occur ran- cells diminished in number and hepatocytes showed domly in the liver parenchyma. There was no intimate the more mature morphology with glycogen rich cyto- relation to the preexisting vitelline veins which stained plasm (Fig. 2g). Thus, SEC and hepatocytes closely as- positively for F vm. sociated each other and formed the space of Disse at the Table I showed the changes in the percentage of late stage of development. The arrangement of sinu- short (<32^m), medium (32-64/mi) and long (64 soids at this period was almost similar to that seen in the /im<) sinusoids which expressed SE-1 antigen during adult liver. All SEC were strongly positive for the SE-1 fetal liver development. The percentage of SE-1 posi- antigen at this stage. With respect to the expression of tive short sinusoids in the liver decreased from 89.9% to SE-1 antigen, a clear difference was seen at the junc- 43.1% at 15 and 21-day-old fetuses, respectively. On tion between sinusoids and hepatic veins. SECassoci- the contrary, that of the long sinusoids increased from ated directly with hepatocytes (without basal lamina) 0.4% to 24.1%. Thus, it is clearly indicated that the expressed SE-1 antigen, whereas no SE-1 antigen ex- SE-1 positive SECextends to form organized sinusoid- pression was observed in endothelial cells of the hepa- al structures during fetal hepatogenesis. However,it tic vein which had basal lamina (Fig. 5). is not yet clear whether all endothelial cells comprising the sinusoids of the fetal livers express SE-1 antigen or DISCUSSION not. To elucidate this issue, we have performed immunoelectron microscopic analysis. As mentioned above, A number of studies have been reported on the fine electron microscopic observation revealed the presence structure of the mammalian fetal liver (3, 5, 10, ll, 14). of immature sinusoid structures surrounded by hemato- Most of these studies have focused on hepatic hematopoietic cells in the liver of 15-day-old fetus. Immuno- poiesis and/or development of Kupffer cells. Only a few electron microscopically, the SE-1 antigen positive as reports have described the changes in SECof the fetal well as negative SECwere observed. The reaction pro- liver. Bankston and Pino (1) reported the presence of

Table I. Changes in the length of SE-1 positive sinusoid during fetal rat liver development. L en gt h o f si n us oi d Ag e of f et us No . of si n us o id e xa m in e d < 3 2 ｫ m 3 2- 64 ur n 6 4 u m < 15 th 2 4 7 2 2 2 (8 9 .9 % ) 2 4 ( 9 .1 % ) 1 ( 0 .4 % ) 1 7th 2 0 0 1 6 4 ( 82 . 0% ) 2 9 (14 .5 % ) 7 ( 3 .5 % ) 1 9th 34 9 2 0 1 (5 7 .6 % ) 9 9 (2 8 .4 % ) 4 9 ( 14 .0 % ) 2 1th 35 3 1 5 2 (4 3 .1% ) 1 16 (2 9 .5 % ) 8 5 (2 4 .1 % )

344 Sinusoidal Endothelial Cell Organization in the Fetal Rat Liver

Fig. 3. An endothelial cell with extended thin cytoplasm along sinusoid-like structure stain positively for the SE-1 antigen (a), whereas a relatively round-shaped endothelial cell stain negatively (b) in the liver of 15-day-old fetus. A stellate-like cell with one fat droplet (S) is also observed at the perisinusoidal portion (a). Bar; 5 /am.

345 M. Morita et al.

Fig. 4. Pinocytotic vesicles (arrows) which are positive for SE-1 antigen at their inner surface are seen in the cytoplasm of the endothelial cell. The liver of 17-day-old fetus. Bar; 1 //m. the diaphragmed fenestration in SECbefore the onset red in a multi-focal manner and were independent of of hepatic hematopoiesis and the switching to open the vitelline veins which showed the positive staining fenestration at aroung 17 days of gestation. Barbera- for F vm in fetal livers. Weinitially assumed that the Guillem et al. (2) reported alternations in number and organization of sinusoids occurred around the perivas- size of the fenestration during the developmental stage cular area of the preexisting vitellin veins by extension of fetal liver. They speculated that the alternation might of endothelial cells from these vessels to the liver paren- be related to either differentiation of hepatocytes or ac- chyma. Unexpectedly, an immunofluorescent staining tivity of hepatic hematopoiesis. Although these studies for the SE-1 antigen together with the fibronectin stain- reported the ultrastructural changes in SECof the fetal ing revealed small sinusoid-like lumens distributed ran- liver, the process of sinusoidal organization in the fetal domly throughout the fetal livers at the early stage of liver, especially its relation to the preexisted vitelline liver development. These lumens elongated and con- vein, is still unclear. nected with each other during the development (Table The monoclonal antibody, SE-1, has been estab- I), resulting in the formation of the sinusoidal struc- lished as an antibody that reacts especially with SECof ture seen at the late stage of liver development (Fig. 2). the adult rat liver but not with any other type of vascu- There seemed to be no intimate relationship between lar endothelial cell (12). While the nature and function the SE-1 antigen positive small sinusoidal lumens and of SE-1 antigen have yet to be clarified, the antigen is the F vmpositive vascular lumens. These results indi- considered to be related to the specific function of SEC cate that the organization of sinusoidal structures oc- in adult rat livers. Judging from its cellular localiza- curs randomly in the parenchyma of fetal liver, which tion (surface of plasma membraneand inner surface of consisted mostly of hematopoietic cells and immature pinocytotic vesicles), it is postulated to be a kind of re- hepatocytes. ceptor molecule which is specifically expressed in SEC. The hepatic stellate cells are knownas an important Therefore, in this study, we used SE-1 antigen as a constituent of liver sinusoid and play a role in the me- marker for the phenotypic maturation of SEC. tabolism of vitamin A (15). Enzan et al (4) reported In the present study, we demonstrated that both the that the appearance of the primitive stellate cell with organization and the maturation of the sinusoid occur- few fat droplets at the early developmental stage of the 346 Sinusoidal Endothelial Cell Organization in the Fetal Rat Liver

Fig. 5. The junction of sinusoid and hepatic vein in the 2-day-old of neonatal rat liver. The sinusoidal endothelial cells associated intimately with hepatocytes (without basal lamina) strongly express the SE-1 antigen, whereas endothelial cells of the hepatic vein (with basal lamina) are negative for the SE-1 antigen. Bar; 5 /mi. mouse, rat and human livers. As shown in Fig. 3a, at cific functions are characteristic features at this time pe- 15-day-old fetal liver, we also observed the stellate-like riod of the fetal liver (1), the phenotypic maturation of cell containing one fat droplet in the cytoplasm at the SECseen in this study maybe in good agreement with perisinusoidal space. This suggests a possible impor- hepatic differentiation. tant role of the stellate cell in the fetal sinusoid organi- A close association between the SE-1 antigen ex- zation. A further analysis of the association of the expressed SECand the hepatocytes was evident at the late pression of SE-1 antigen in endothelial cells and the stage of development (Fig. 4). The direct contact of stellate cell-endothelial cell interaction is required. SECto hepatocytes seems to be an important factor for Recently, it was reported that the process of vasculo- the expression of SE-1. We assumed that the contact genesis, in which primitive angioblasts form a vascular between the primitive SECand hepatocytes may be cru- network, is distinct from that of angiogenesis which is cial in the first step of sinusoid organization, although dependent on preexisting vessels (6). Poole and Coffine wehave not yet confirmed such clear association at the (13) showed that both vasculogenesis and angiogenesis early developmental stage because of the presence of are involved in the formation of the embryonic vascu- numeroushematopoietic cells as well as immaturity of lar network. Our results thus suggest a possibility that hepatocytes. However, the recent report by Modis and the sinusoid organization in fetal rat liver may occur in Martinez-Hernandez (9), showing the importance of he- a manner similar to vasculogenesis. patocytes in modulating endothelial cells toward sinu- Expression of SE-1 antigen became pronounced with soid-type morphology, also supports our hypothesis the elongation of sinusoidal lumens (Fig. 2). At the late of sinusoid organization. More recently, it has been stage of fetal liver development (21-day-old fetus), the shown that vascular endothelial growth factor (VEGF), pattern of SE-1 expression was similar to that seen in which is produced by hepatocytes, regulates the prolif- the adult liver. This indicates that the phenotypic matu- eration and survival of the cultured SECvia tyrosine ration of sinusoid has nearly been completed at the late kinase type receptors, Flt-1 and Flk-1 (17). It is impor- stage of liver development. Since the cessation of hepa- tant to examine the role of such paracrine factors for tic hematopoiesis and the appearance of adult liver spe- understanding the mechanisms of sinusoid organiza- 347 M. Morita et al. tion in the fetal liver. 8. McGuire, R., Bissell, D.M., Boyles, J., and Roll, F.J. 1992. Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver. Hepatol- Acknowledgment. Wethank Ms. K. Takeda for her help in manu- ogy, 15: 989-997. script preparation. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports 9. Modis, L. and Martinez-Hernandez, A. 1991. Hepatocytes modulate the hepatic microvascular phenotype. Lab Invest. , 65: and Culture of Japan (to M. Morita and to K. Enomoto). 661-670. 10. Motta, P.M. 1984. The three-dimensional microanatomy of REFEREN CES the liver. Arch. Histol Jpn., 47: 1-30. ll. Naito, M. and Wisse, E. 1977. 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(Received for publication, September 1 7, 1998 and accepted, October s, 1998)

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