116

Lymphology 37 (2004) 116-126

THE PATTERN OF FmRILLIN DEPOSITION CORRELATES WITH MICROFmRIL-ASSOCIATED GLYCOPROTEIN 1 (MAGP-l) EXPRESSION IN CULTURED BLOOD AND LYMPHATIC ENDOTHELIAL CELLS

E. Weber, A. Rossi, R. Solito, M. Agliano, G. Sacchi, R. Gerli

Molecular Medicine Section, Department of Neuroscience, University of Siena, Siena, Italy

ABSTRACT could in turn account for the specificity of elastic fibers in compliance with the specific constitute the major structural functional requirements of the tissue. components of lO-12nm of the of several elastic and non Fibrillins are large glycoproteins of about elastic tissues and of initial lymphatic vessel 350 kDa with a high cysteine content, which anchoring filaments. -Associated constitute the major structural components Glycoprotein-l (MAGP-l) binds to of 10-12 nm microfibrils of the extracellular tropoelastin during elastogenesis. We recently matrix. There are two isoforms of fibrillin: reported that cultured blood endothelial cells fibrillin-1 (1) and fibrillin-2 (2,3), encoded by deposit fibrillin in a honeycomb pattern, different genes. The two isoforms have signi­ whereas lymphatic endothelial cells form an ficant similarities in primary sequence and irregular web. The aim of this immunohisto­ predicted protein domains, but differ in a chemical study was to verify whether the region near the aminoterminal domain which deposition pattern of fibrillin is related to the is rich in proline in fibrillin-1 and glycine in expression of MAGP-l in confluent and post­ fibrillin 2. Fibrillins have a multidomain confluent cultures of bovine aortic (AEC), structure comprising 47 epidermal growth pulmonary artery (PAEC) and lymphatic factor-like domains, 43 of which are of the endothelial cells (LEC). In AEC and PAEC, calcium binding type, interspersed with 8 MAGP-l andfibrillin co-localized and their cysteine motifs with homology to the deposition increased with time in culture. In transforming growth factor B binding protein. AEC, both proteins formed a honeycomb Fibrillin 1 and 2 both contain an RGD pattern. In LEC, MAGP-l deposition was still sequence that may bind

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 117 endothelial cells from blood and lymphatic findings in literature, we noticed a deposition vessels (12). In the pericellular space they pattern of fibrillin resembling that of polymerize by head-to-tail alignment and lymphatic endothelial cells in fetal bovine undergo conformational maturation that chondroblasts (16) and a honeycomb pattern presumably renders the molecule extensible resembling that of blood endothelial cells in (7). The next step is assemblage with other immortalized ciliary body pigmented molecules onto thread-like filaments which epithelial cells. Interestingly, the authors, in turn give rise to macroaggregates with or who do not mention this morphology, report without (5). that the expression of microfibril-associated Fibrillin-containing microfibrils are glycoprotein-l (MAGP-l) was much lower in distributed in a wide variety of tissues and fetal bovine chondroblasts than in ciliary organs, either associated with elastin in body pigmented epithelial cells (16). elastic fibers or as elastin-free bundles (the MAGPs, originally identified in bovine latter are also known as oxytalan fibers). nuchal , are a family of small Elastin-associated microfibrils are found in structurally related glycoproteins that include elastic tissues, subjected to stretching and two molecules specifically associated with expansible forces, where they form the fibrillin-containing microfibrils: MAGP-l, external coating of elastic fibers. They are 31-kDa, and MAGP-2, 25-kDa (17-19). These thought to act as a scaffold for tropoelastin molecules undergo posttranslational modifi­ deposition and alignment. Both fibrillins cations that may influence their interaction guide elastogenesis. Indeed microfibrils with microfibrillar proteins. It has been appear before tropoelastin deposition, shown that MAGP-l is covalently bound by however they intervene sequentially: fibrillin- disulfide bonds to fibrillin beads (19) and that 2 is presumably involved in morphogenesis its N-terminus may also bind the C-terminus of elastic fibers during development since it of tropoelastin (20,21). Binding of MAGP-l to appears before elastogenesis, whereas tropoelastin may serve during development to fibrillin-l is probably involved in the homeo­ guide elastogenesis in relation to the functional stasis of elastic fibers throughout life (13). requirements of the tissue and its exposure to Microfibrils devoid of elastin are found stress and, at more mature stages, to stabilize in elastic and non elastic tissues, especially in the interactions between microfibrils and the those subjected to mechanical stress. In elastic elastic core of elastin (22). MAGP-2 has an tissues, they connect elastic fibers among {Xv6rintegrin-binding RGD motif near the N themselves and to other structural and cellular terminus. This raises the possibility that components (5); in non-elastic tissues, like MAGP-2 may playa unique function, within ciliary zonules (14), they have an anchoring microfibrils, as a molecular bridge between function. microfibrils and the cell surface (22). We previously used immunohistochemical The aim of this study was to verify techniques (15) to show that the microfibrils whether differences in MAGP-l production of anchoring filaments of human skin initial might relate to the different fibrillin lymphatic vessels, contain fibrillin. We also deposition patterns of cultured bovine aortic recently reported that fibrillin deposition (AEC) and lymphatic (LEC) endothelial cells. differs in cultured blood and lymphatic Pulmonary artery endothelial cells (PAEC) endothelial cells (12). Blood endothelial cells were also investigated under the assumption deposit fibrillin in a honeycomb pattern with that their deposition pattern of fibrillin and empty spaces through which endothelial cells MAGP-l production might have intermediate are visible; in lymphatic endothelial cells characteristics between those of AEC and fibrillin covers the entire monolayer forming LEe. The pulmonary artery is a low pressure a thick, irregular web. Looking for similar vessel with the structure of an artery. Its

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 118 endothelium is exposed to intraluminal 5 minutes in acetone at -20°C and washed systolic pressure, much lower than that of again extensively. Unspecific binding sites arteries but higher than that of lymphatic were blocked with 3% BSA in PBS. Cells vessels in the absence of obstruction to lymph were then incubated overnight at 4°C either flow. We provide evidence that differences in with a monoclonal antibody to fibrillin MAGP-1 do correlate with differences in the (Chemicon, MAb 1919) or a polyclonal deposition pattern of fibrillin. antibody to MAGP-1 (EPC Elastin Products Company, Owensville, Missouri U.S.A.) MATERIALS AND METHODS diluted 1:50 and 1:100 respectively in PBS containing 0.5% BSA. After incubation with Cell Cultures the primary antibody, cells were washed and incubated for 2 h with fluorescein All cells were bovine: LEC were obtained isothiocyanate-Iabeled goat anti- mouse or from the thoracic duct (23), AEC from the anti-rabbit IgG secondary antibodies (Sigma) thoracic aorta and P AEC from the pulmonary diluted 1:100 in PBS with 0.5% BSA, washed trunk and left and right pulmonary arteries. again and mounted upside-down on glass Cells were detached by enzyme digestion with slides with Vectashield mounting media 0.05% collagenase type 2 (Worthington, (Vector). Controls were obtained omitting the Lakewood, NJ, USA, 239 U1mg) and seeded primary antibodies. into four-well Nunc plates (1.5 cm diameter), Double immunolabeling was also containing round glass cover slips coated with performed on cells at day 4 after confluence. 0.1 % gelatin (Difco, Detroit, Michigan, USA, After fixation and blockade of unspecific in phosphate buffered saline, PBS, without binding sites, cells were incubated overnight calcium and magnesium). Culture medium at 4°C with the polyclonal antibody to was Dulbecco's modified Eagle's MEM MAGP-1, then washed and incubated with a (DMEM, GIBCO, Life Technologies Ltd, goat anti-rabbit FITC-conjugated secondary Paisley, Scotland, UK) containing 20% fetal antibody for 2 hours at room temperature. bovine serum (FBS, GIBCO), 100 JIg/ml After thorough washing, unspecific binding endothelial cell growth supplement (ECGS, sites were blocked with 3% BSA and 5% goat Sigma, St. Louis, MO, USA), 100 JIg/ml serum in PBS for 40 min., then the cells were kanamycin (Sigma) and 2 mM glutamine incubated for 2 hours at room temperature (Sigma). FBS was reduced to 10% the day with the monoclonal antibody to fibrillin, before confluence, and to 2% the next day to after which they were washed and incubated minimize outgrowth of contaminating smooth for 2h at room temperature with a rabbit muscle cells. For the same reason, ECGS was anti-mouse TRITC-conjugated secondary no longer added to culture medium as soon as antibody. cells reached confluence (24). Fibrillin and MAGP-1 distribution was evaluated in ex Quantitative Analysis vivo cells (freshly isolated) and in primary culture cells at confluence and at days 2 and MAGP-1 deposition was evaluated with 4 after confluence, since it is known that a morphometric program, "Lucia G," by fibrillin deposition progressively increases Nikon, on at least 500 cells/condition. The after confluence (25). amount of MAGP-1 deposited was expressed as Integral Density (LD.) which is the sum of Immunohistochemistry individual optical densities of each pixel in the area being measured. In this program, Cultures were washed in PBS containing Optical Density (O.D.), the negative 0.5% bovine serum albumin (BSA), fixed for logarithm of the transmittance is evaluated

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 119 according to the formula: 0.0. = -log((Pixel Gray Value+ 0.5)/ 62.5). The program automatically selected positive areas and calculated their integral density. The data were corrected for the percentage of the field covered by the positive area.

RESULTS

Immunohistochemistry

Fibrillin deposition

We failed to detect fibrillin in ex vivo AEC, LEC and PAEC (data not shown). In culture, fibrillin deposition increased as a function of time in all cell types. At day 4 after confluence of AEC (Fig. Ia) fibrillin formed a honeycomb pattern of filaments arranged in strands surrounding fibrillin-free spaces. Fibrillin deposition in LEC was less regular: fibrillin microfibrils arranged in fila­ ments of different size that eventually formed a sort of web covering the cell monolayer (Fig. Ib). Cultured PAEC produced fibrillin with a deposition pattern which was intermediate between that of AEC and LEC (Fig. Ic): in some areas filament bundles of various dimensions formed a web covering the culture like in LEC, in other areas of the same culture, fibrillin filaments formed a honeycomb pattern with thick fibrillin microfibrils surrounding fibrillin-free spaces as in AEC.

MAGP-I deposition

In AEC, MAGP-l deposition mimicked that of fibrillin. At confluence MAGP-l positive thin filaments could be detected in some areas of the culture (Fig. 2a). MAGP-l deposition increased gradually with time and Fig.I. Fibril/in deposition at day 4 after confluence. eventually, 4 days after confluence, a honey­ a) AEC deposit fibril/in in a honeycomb pattern; comb pattern was formed (Fig. 2b). Through b) LEC in an irregular web; c) PAEC with an inter­ MAGP-l free spaces in the wide mesh of mediate pattern between AEC and LEC: honeycomb MAGP-l positive strands, the cell monolayer pattern (*) and irregular web (arrowheads). Orig. could still be recognized. Some of these spaces mag. x40.

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Fig. 2. MAGP-l deposition in AEC (a and b), LEC (c and d) and PAEC (e and!) at confluence (a, c and e) and at day 4 after confluence (b, d and!). MAGP-l deposition mimics that offibrillin in AEC (a and b) and PAEC (e and !), whereas LEC do not deposit MAGP-l either at confluence (c) or 4 days later (d). Honeycomb pattern (*) and irregular web (arrowheads). Orig. mag. x40. were further divided into a closer mesh by filaments and thicker strands. Some meshes finer filaments sprouting from the wider were formed, but thin filaments also formed mesh, creating a complex mesh structure. a delicate web. At day 4 after confluence PAEC also deposited MAGP-l with a (Fig. 21) some wide meshes including MAGP-l pattern resembling that of fibrillin. At free spaces, like in AEC, were formed. These confluence MAGP-l was only detected in meshes alternated with the web-like deposi­ some areas of the culture (Fig. 2e). In these tion characteristic of LEe. areas, MAGP-l was deposited as thin No MAGP-l was ever detected in

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Fig. 3. A cryostat section of bovine thoracic duct: a) the endothelium is labeled by the polyclonal antibody to MAGP-l, b) nuclei are stained with Hoechst solution. Orig. mag. x40.

cultured LEC at confluence (Fig. 2e). In most Quantitative analysis of integral density cases there was still no detectable MAGP-1 at (Fig. 5) showed that MAGP-1 production day 4 after confluence (Fig. 2d). In three cases significantly increased from confluence to day out of eight, a few small areas containing 4 in AEC and PAEC (p

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Fig. 4. Double labeling with monoclonal antibody to fibril/in (a, c and e) and antiserum to MAGP-l (b, d and 1) at day 4 after confluence. Fibrillin and MAGP-l co-localize in AEC (a and b) and PAEC (e and 1). LEC stain for fibril/in (c) but not for MAGP-l (d). Orig. mag. x40. a honeycomb pattern. In P AEC, areas with MAGP-1 used even at day 4 when fibrillin is a honeycomb pattern coexisted with areas abundant all over the culture. where fibrillin and MAGP-1 formed an It is not clear why LEC behave so irregular web. This is in line with the obser­ differently from the other endothelial cells vation that MAGP-1 associates with fibrillin tested. Since small amounts of MAGP-1 are molecules, specifically the beads (19). On the present 4 days after confluence, when fibrillin other hand, LEC, that also deposit fibrillin in deposition has already taken place, we vitro (12), were mostly negative to immuno­ hypothesize that LEC require a longer time labeling with the polyclonal antibody to to express MAGP-l. Alternatively, culture

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conditions and particularly the addition of 16 serum to the medium, may better mimic the -AEC 14 * ~ PAEC normal situation of blood than of lymphatic 'Uj = c 12 ==::-:1 LEC * endothelial cells which are in vivo exposed G) 10 to lymph. Q 8 We think it unlikely that LEe do not express MAGP-1 at all. MAGP-1 is a key -f! 6 molecule for elastogenesis and the endothe­ =G) 4 ..c lium of all lymphatic vessels, particularly - 2 that of initial lymphatic vessels, has close 0 connections with elastic fibers (15,29-32). o 4 Transmission electron micrographs often Days after confluence catch elastic fibers contained in small "pocket-like" invaginations of the abluminal Fig.5. Integral density, expressing MAGP-l deposition. side of lymphatic endothelial cells. This MAGP-l deposition increased from confluence to finding strongly suggests that the cells day 4 in AEC and PAEC (p

ECM

ENDOTHELIAL CELL

Fig. 6. Schematic representation of a possible molecular mechanism of elastogenesis in endothelial cells. Fibrillin microfibrils may attach to a.,B3 integrins on the plasma membrane of endothelial cells either via an RGD sequence of their own or via an RGD sequence of MAGP-2. The latter may correlate elastogenesis with cell specificity. MAGP-l regulates the polymerization oftropoelastin molecules on the scaffold offibrillin microfibrils.

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY . 124 unites endothelial cells with fibers We suggest that elastogenesis is an (33) and the latter with the elastic fibers of earlier event in the aorta than in the thoracic the extracellular matrix by means of duct. The aorta has an intrinsic functional anchoring filaments (15,30). Blood capillaries need for elastic fibers and resilience in an are never surrounded by elastic fibers (29) early phase of development. In line with this, and their endothelium therefore presumably MAGP-1 deposition in AEC is perfectly does not produce elastic fibers. A profound correlated with that of fibrillin from the first difference exists between the endothelium of days of culture. Early elastogenesis is not a blood capillaries and lymphatic initial vessels requirement for the thoracic duct, the wall of in this respect. It may have repercussions on which can form and develop more slowly. the deposition pattern of fibrillin, the time Hence, MAGP-1 is not expressed in LEC at required for its secretion, the morphology of early times of culture and is not correlated its network and also on MAGP-1 production with fibrillin deposition. P AEC, which are by the two types of cells. derived from a vessel that has intermediate The cells that we used in this study are structural and functional characteristics not microvascular, they were taken from between the aorta and the thoracic duct and large vessels, where other factors may be an intermediate exposure to oxygen and relevant during elastogenesis. The walls of intraluminal pressure, also have intermediate the thoracic duct and large arteries contain deposition patterns of fibrillin and MAGPs. smooth muscle cells and fibroblasts, which In our opinion, the timing of MAGP-1 are also known to produce fibrillin and elastic deposition may influence the pattern of fibers. Culture conditions deprive cells of the fibrillin deposition in the three endothelial possibility to interact with other cell types cell types tested. Different fibrillin scaffolds and to respond to signals and stimulations of could in turn account for the specificity of the extracellular matrix and a dynamic envi­ elastic fibers in compliance with the specific ronment. Lymphatic endothelial cells may functional requirements of the aorta, the depend more than blood endothelial cells on thoracic duct and the pulmonary artery. these external stimuli and interactions. It is even feasible that the elastic fibers ACKNOWLEDGMENTS surrounding lymphatic vessels are mostly produced by cells other than endothelial. This work was financed by the University It might be interesting to evaluate of Siena Research Plan (PAR). whether cultured LEC deposit tropoelastin and whether other microfibril-associated REFERENCES glycoproteins, like MAGP-2, which also specifically associates with fibrillin-containing 1. Sakai, LY, DR Keene, E Engvall: Fibrillin, a microfibrils, but has a more restricted pattern new 350-kD glycoprotein, is a component of of tissue localization than MAGP-1 (34), is extracellular microfibrils. J. Cell BioI. 103 expressed by cultured LEC earlier and more (1986),2499-2509. abundantly. Lymphatic (12,35) and blood 2. Lee, B, M Godfrey, E Vitale, et al: Linkage of and a phenotypically endothelial cells (36) possess CJ.v integrins. related disorder to two different fibrillin Gibson et al (22) suggest that MAGP-2 may genes. Nature 352 (1991), 330-334. be relevant in modulating the interactions of 3. Zhang, H, SD Apfeiroth, W Hu, et al: microfibrils with cell surfaces in particular Structure and expression of fibrillin-2, a novel tissue environments. Whether lymphatic microfibrillar component preferentially located in elastic matrices. J. Cell BioI. 124 vessels belong to these environments is (1994), 855-863. unknown. Unfortunately, the antibody to 4. Sherratt, MJ, T J Wess, C Baldock, et al: MAGP-2 is not yet commercially available. Fibrillin-rich microfibrils of the extracellular

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