JOURNALOF

Dermatological Science

Journal of Derrnatological Science 12 (1996) 36-43

CDw49b/CD29 integrin complex mediates the differentiation of human endothelial cells into capillary-like structures in vitro

Yasuo Kubota*, Yoko Kawa, Masako Mizoguchi St. Marianna University School of Medicine, Department of Dermatology, Z-16-1, Sugao, Miyamae, Kawasaki, Japan 216

Received 14 February 1995; revision received 17 July 1995; accepted 17 July 1995

Abstract

We have investigated different p-1 integrins (CDw49/CD29) on human umbilical vein endothelial cells (HUVEC) with regard to their roles in modifying the morphological structure of these cells on/in matrigel. The inhibition of matrigel-induced capillary formation by antibodies against subunits of p-1 integrins was examined quantitatively using a digital analyzer, Antibodies to CDw49b and CD29 (common beta chain) caused a marked inhibition of capillary formation (up to 70%) in a dose-dependent manner, whereas antibodies to CDw49d, CDw49e and CDw49f were less inhibitory. We also examined the appearance of HWEC cultured in matrigel. HUVEC suspended in matrigel for 24 h formed extended cell processes which connected, resulting in the formation of a capillary network. In contrast, fibroblasts cultured in matrigel showed only bipolar extensions without cell-cell contact. After 48 h in culture in matrigel, some HUVEC showed the capillary-unit of a lumen encircled by EC which may mimic the basic putative unit in the formation of capillaries. However, HUVEC pretreated with antibodies to CDw49b and CD29 failed to form significant processes and a hollow lumen. These phenomena may illustrate the importance of endothelial cell-basement membrane matrix interaction (through integrins, especially CDw49b/CD29 complex) occurring during differentiation of endothelial cells in angiogenesis.

Keywords: Endothelial cells; Angiogenesis; Extracellular matrix; Tube formation; Integrin

1. Introduction

The formation of new blood vessels (angiogene- sis) is fundamental in wound healing and in dis- eases such as thrombosis and arteriosclerosis [l], and is thought to be a complex process. It re-

* Corresponding author. Tel.: 044-977-8111; Fax: 044-977- 3540.

quires the sprouting, migration, and proliferation of endothelial cells, their differentiation into tube- like structures and finally the formation of base- ment membrane [2,3]. Endothelial cells aggregate and form capillary-like structures in culture. This phenomenon may be related to the differentiation of the endothelial cells and their ability to form capillaries in vivo [4]. It is well known that inter- action with extracellular matrix (ECM) or its components can trigger migration, proliferation,

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Y. Kubota et al. 1 Journal of Dermatological Scie.vc;e 12 (1996) 36-43 37

and differentiation of a variety of cell types in- cluding endothelial cells [5,6]. Generally, initial ECM binding to endothelial cells is mediated through adhesion receptors of the integrin family. The integrins are a family of at least 19 different cell-surface heterodimers composed of at least 8 distinct beta subunits and 13 alpha subunits [7,8]. Members of the p-1 integrin family (CDw49/ CD29 integrins) mediate binding to a variety of substances present in the extracellular matrix, such as collagen, fibronectin and laminin [9]. It has been shown that vascular tube formation involves endothelial cell-surface receptors of the integrin superfamily [lo- 121. We observed previ- ously that a substrate of reconstituted basement membrane proteins (matrigel) induces rapid for- mation of tubes by endothelial cells [13,14]. In the present study, we examined the roles of integrins in the morphological differentiation of human endothelial cells into capillary-like structures cul- tured on/in matrigel and showed that integrins containing the CDw49b/CD29 subunit are criti- cally involved in matrigel-induced tube formation. We also observed that HUVEC cultured in ma- trigel formed numerous branching processes and tubular structures with a hollow lumen.

2. Materials and methods

2.1. Isolation and culture of human umbilical vein endothelial cells (HUVEC)

HUVEC were isolated from fresh umbilical cords obtained by caesarean section by a modifi- cation of the technique of Jaffe et al. [15]. All cells were used at passages 2-6. Cells were character- ized as endothelial cells based on their cobble- stone morphology and uptake of Dil-Ac-LDL [161.

2.2. Matrix proteins and antibodies Matrigel (Collaborative Research, Inc.,

Waltham, MA), an extract containing basement membrane components, was applied to either 35 mm tissue culture dishes or 16 mm multiwell culture plates (Coster, Cambridge, MA) and incu- bated at 37°C for 60 min, which induced gelation. The protein concentration of matrigel is approxi- mately 10 mg/ml, and 50%-60% of matrigel is

composed of laminin. Mouse laminin was ob- tained from GIBCO BRL (Gaithersburg, MD) and mouse types I and IV collagen and human fibronectin were from Becton Dickinson Labware (Bedford, MA).

Mouse monoclonal antibodies (MAbs) utilized were as follows: anti-CDw49b (clone Gig), anti- CDw49d (clone HP2/1), anti-CDw49f (clone GoH3) were all obtained from Japan Tanner Inc. (Kobe, Japan); antiCDw49e (clone SAM-l) and anti-CD29 (common beta chain, K20) were ob- tained from IMMUNOTECH S.A. (Marseille, France); antiCDw49c (clone M-KID2) was ob- tained from Cosmo Bio. Inc. (Tokyo, Japan); anti-/?-3-integrin (CD61, clone BBlO) was ob- tained from BIOHIT OY (Helsinki, Finland). Rabbit polyclonal antiserum to laminin or to collagen type IV was obtained from E-Y Lab., Inc. (San Mateo, CA) and from Becton Dickinson Lab. (Bedford, MA), respectively. Irrelevant mouse monoclonal antibody or normal rabbit serum were used as controls.

2.3. Cultured HUVEC on/in matrigel Matrigel was applied to 16 mm multiwell cul-

ture plates and incubated at 37°C for 60 min, which induced gelation. Routinely, 300 ~1 of ma- trigel was used to coat each 16 mm dish, and l-2 x 10’ HUVEC in culture media per well were then pipetted on top of the gel. The total volume of medium in each well was always adjusted to 1 ml.

To measure the total length of tube-like struc- tures per well, a computerized digitizer (LUZEX 3, Nireco Corp., Tokyo) was used at a magnifica- tion of x 40. At least four areas were examined at this magnification under each experimental condition tested. The results are shown as per- centage of tube formation over control. The per- centage was averaged and plotted. Standard deviation was less than 15% in all experiments. Each experiment was repeated at least three times and results from one representative experiment are shown.

In other experiments, 2 x 10’ HUVEC in 300 ~1 of culture medium were mixed well with 100 ~1 of undiluted matrigel and then pipetted into 16 mm culture wells immediately. The morphology

38 Y. Kubota et al. 1 Journal of‘Dermatologica1 Science 12 (1996) 36-43

and reorganization of HUVEC were monitored at intervals by phase contrast microscopy (model IMT-2, Olympus).

2.4. Flow cytofluorometric analysis of integrin M- and p-subunit expression

Expression of integrin alpha and beta subunits on HUVEC was quantified by flow cytometry on a Becton Dickinson FACScan device. Briefly, sub- confluent monolayer HUVEC cultures were har- vested by washing with phosphate buffered saline (PBS) and then adding 5 mM ethylenediamine tetraacetic acid (EDTA) in PBS containing 0.5% bovine serum albumin (BSA). After detachment from the substrate, 2 volumes of serum-free Dul- becco’s modified Eagle’s medium (DMEM) was added and the cells were pelleted. Cells were washed once with PBS containing 0.5% BSA and then resuspended at IO6 cells per ml in PBS con- taining 0.5% BSA. Antibodies were added accord- ing to the standard flow cytometry protocol.

2.5. Antibody inhibition studies To further assess the role of p-1 integrin sub-

units in the formation of tubes, HUVEC in sus- pension were preincubated with either rabbit anti-laminin serum or MAbs against integrin sub- units as described above at concentrations from 0.2 pg/ml to 20 @g/ml for 30 min at room temper- ature and then plated on/in matrigel and cultured overnight in the continuous presence of the anti- body. Normal rabbit serum and irrelevant mouse monoclonal antibodies were used as controls. Cell morphology was observed at intervals, and the formation of tube-like structures was calculated as described above.

2.6. Cell adhesion assay HUVEC were dispersed by trypsinization and

then allowed to attach at 37°C overnight to tissue culture wells coated with different purified ECM molecules (e.g., 20 pg/ml of laminin, fibronectin, and collagen IV ). Cell adhesion was monitored by thoroughly rinsing the cultures and counting the attached cells by either Alamar Blue (Iwaki Glass Co., Chiba) assay [17] or using a hemocy- tometer followed by trypsinization. Each experi- ment was repeated at least. three times and results

from one representative experiment are shown. Antibodies against alpha and beta chains of p-1 integrins were preincubated with HUVEC at a variety of concentrations (up to 100 pug/ml) for 15 min on ice prior to plating and were present continuously during the assay.

3. Results

3.1. Morphology of HUVEC on/in matrigel HUVEC assume a ‘cobblestone’ pattern when

cultured on either gelatin- or fibronectin-coated tissue culture dishes in media supplemented with endothelial cell growth factor (ECGF) and hep- arin. As described previously [13,14], when HU- VEC were plated onto matrigel, they attached rapidly (0.5-l h) and proceeded to form a capil- lary network over the next few hours. Due to this ability of the basement membrane to stimulate differentiation, when HUVEC were cast and incu- bated in matrigel for 48 h at 37°C they showed numerous copiously branching processes followed by abundant networks of branching and anasto- mosing of cells. Interestingly, some elongated cells (20% of the total) appeared as ring-like structures surrounding a hollow lumen (Fig. 1). The forma- tion of capillary-like structures in matrigel ap- peared to be relatively specific for endothelial cells since human dermal fibroblasts showed only bipo- lar cytoplasmic elongation without branching and

Fig. 1. HUVEC form tube-like structures in matrigel. HUVEC were cast in matrigel and incubated for 48h at 37°C. Cells showed numerous branching processes and some appeared in a ring-like formation. Magnification 300 x

Y. Kubota et al. /Journal of Dermatological Science 12 (1996) 36-43 39

Expression of the integrin a and p snbunits on cultured human endothelial cells

200

CD29 CDw49e :.

t t

f

2

loo 10' lo2 lo3 FL1

Log Fluorescence Intensity I)

Fig. 2. Expression of the integrin a and /I subunits on cultured endothelial cells. HUVEC were stained in suspension for the presence of one beta and four alpha subunits with monoclonal antibodies Gi9 (CDw49b), HP2/1 (CDw49d), SAM-l (CDw49e), GoH3 (CDw49f), K20 (CD29), analyzed by flow cytometry. The results are expressed as histograms with cell number (y-axis) plotted as a function of relative fluorescence intensity (x-axis) on a log scale. Background fluorescence with irrelevant mouse monoclonal antibody control is also shown.

did not form a lumen when cultured in matrigel. On the other hand, when HUVEC were cast and incubated in collagen I gel, they neither aligned themselves end to end nor elongated (data not shown).

3.2. Expression of integrin subunits on HUVEC The integrin surface phenotype of HUVEC was

examined by staining in suspension for the pres- ence of integrin subunits with the monoclonal antibodies listed above and were analyzed by flow cytometry. High levels of CDw49b and CDw49e were found, and CDw49f was present at a low level, while CDw49d was detected only in trace amounts. CD29 was expressed at a high level (Fig. 2), and expression of CD61 was also ob- served significant (data not shown).

3.3. Efiects of antibodies to integrin subunits on the capillary-like structure formation of HUVEC on/in matrigel

Antibodies to laminin or MAbs to the subunits of integrin, as well as pre-immune rabbit serum or irrelevant MAbs, were incubated with 2 x 10 s HUVEC for 30 min at 22°C at a variety of concentrations (0.2 pg/ml-20 pg/ml). Then these cells were plated on matrigel-coated dishes to examine the roles of integrin in formation of capillary-like structures. Pre-immune serum or ir- relevant MAbs did not alter capillary formation. When the antibody to laminin was added, how- ever, HUVEC still attached to the matrigel but did not form tubes, as described previously [13]. Furthermore, MAbs to CDw49b and CD29 caused marked inhibition of tube formation in a dose-dependent manner (up to 20 pg/ml), whereas MAbs to CDw49d, CDw49e and CDw49f were all less inhibitory. The inhibitory effects of anti- CDw49b and CD29 MAbs were almost the same as those of the antibody to laminin. This inhibi- tion of matrigel-induced capillary formation by MAbs against subunits of B-1 integrin and by polyclonal antibodies to laminin was examined quantitatively using a LUZEX 3 digital analyzer (Fig. 3a,b.c,d). Additionally, MAb had the same inhibitory effect on CDw49c as on CDw49d, and MAb to B-3 integrin showed only a limited inhi- bition effect (data not shown).

Although the antibody against CD29 inhibited HUVEC adhesion to laminin-coated plastic dishes at concentrations in excess of 40 pg/ml (20%- 30% inhibition), the anti-CD29 antibody did not inhibit adhesion to fibronectin or collagen type IV (Fig. 4a). Antibodies against alpha and beta sub- units of p-1 integrin at concentrations of less than 20 pgg/ml did not show any inhibitory effect on HUVEC adhesion to laminin (Fig. 4b). The anti- body concentrations required to inhibit cell adhe- sion to the purified matrix component laminin were much higher than those necessary to block capillary-like formation of HUVEC cultured on matrigel. HUVEC seemed to be somehow more sensitive to antibody-mediated inhibition of tube formation than to cell attachment on purified basement membrane matrix components (laminin and collagen type IV are the major constituents of matrigel).

In other experiments, capillary-like tube forma- tion in matrigel was inhibited markedly by pre-

40 Y. Kubora et al. /Journal of Dermarological Science 12 (1996) 36-43

treatment of HUVEC with MAbs to CDw49b with MAbs to CDw49b or to CD29 did not form and CD29, however MAbs to CDw49d and tube-like structures in matrigel and showed appar- CDw49f showed lesser effects. HUVEC treated ent perturbation of capillary formation in that a

(4 (b)

Effect of MAbs to integrins on tube formation by matrigel

( Tube area was measured at a maanlflcatlon of 40x using LUZEX Jd~gital analyzer

100

80

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3--O---,- ----o -----__ -----o

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Fig. 3. HUVEC were plated on matrigel-coated surfaces and allowed to attach and form tubes for 18 h in the presence of various antibodies, all diluted to a concentration of 20 pg/ml in media. (a) Untreated control. (b) Pretreated with antibody to CD29. Note that HUVEC in (a) formed an anastomosing capillary-like network, while cells in (b) were inhibited from forming tubes. Magnification x 150. (c) Quantification of tube formation. Total lengths of the tubes were measured using a computerized digitizer LUZEX 3 (Nireco). Results shown are averages. Standard deviation was less than 15% in all experiments. The results are shown as percentages over control value. Antibodies to CDw49b and CD29 subunit decreased total lengths by about 60%-70%, while antibodies to CDw49d, CDw49e and CDw49f were all less inhibitory. (d) MAbs to CDw49b and CD29 caused marked inhibition of tube formation by matrigel in a dose-dependent manner (up to 20 pg/ml).

Y. Kubota et al. /Journal of Dermatological Science 12 (1996) 36-43

600

+ 100’ f B 60.

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a C.otrol-

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n CD29

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*control=mouse IgG

o- , 0 0 2 4 20 40 100 (rglml) 0 I 5 10 20

ANTIBODY CONCENTRATION Concentrations of antibody (up/ml)

(4 (b)

Fig. 4. (a) Inhibition of HUVEC adhesion to purified ECM (laminin, fibronectin, collagen IV) using anti-CD29 antibody. The results are shown as percentages over control values. Results are averaged and plotted. Standard deviation was less than 10% in all experiments. (b) A variety of antibodies against alpha and beta subunits of B-1 integrin at concentrations of less than 20 pg/ml showed no inhibition of HUVEC adhesion to laminin as demonstrated by Alamar Blue assay (O.D. 570-620).

complete network was absent and each cell was rounded instead of elongated in shape (Fig. 5).

4. Discussion

When HUVEC are plated on matrigel, they cease DNA synthesis and the cells rapidly align and form capillary-like networks within 18 h [13,14]. In this present study, we examined the role of integrins in the morphological differentia- tion of HUVEC into capillary-like structures in culture on matrigel. As matrigel is a mixture of

Fig. 5. Integrin antibody treated-HUVEC did not form tube- like structures in matrigel. Note that pretreatment with MAb to CDw49b appeared to perturb capillary formation in that a complete network was absent and cells appeared rounded and not elongated (Fig. 1). Magnification x 200.

basement membrane proteins such as laminin and collagen type VI, our attention was directed towards the role of p-1 integrins in HUVEC differentiation, some of which have been sug- gested to be laminin and/or collagen receptors [10,18]. It has been shown that endothelial cell- surface receptors of the integrin superfamily play an important role in a variety of endothelial cell functions [12,19] and that vascular tube forma- tion also involves these endothelial cell-surface receptors [lo, 11,201. Endothelial cells are known to express CDw49a, 49b, 49e and CD29, CD61 at high levels, while CDw49d, Cdw49f are ex- pressed only at trace levels, as shown by Lan- guino et al. (1989) [21]. The results of the present study confirmed their findings partially, however significant CDw49f expression was found in our study. Albelda (1992) also deter- mined that both HUVEC and fat capillaries ex- pressed very similar complements of integrins including CDw49b, 49c, 49e, 49f, and CD51 and CD61 by immunoprecipitation and immunohis- tochemical methods [22]. The differences between our results and those of other studies may be related to differences in culture conditions, clones of monoclonal antibodies used or detec- tion methods (flow cytometry vs. immunoprecip- itation or immunohistochemistry).

Tube formation by HUVEC on matrigel was blocked markedly by preincubating the cells with

42 Y. Kubota et al. /Journal of Dermatological Science 12 (1996) 36-43

anti-CDw49b and -CD29 antibodies in a dose-de- pendent manner. However, anti-CDw49d, -49e and -49f showed less effects on tube formation. Interestingly, although the level of expression of CDw49e on HUVEC was high, the MAb to CDw49e showed little inhibitory effect on tube formation by matrigel. One of the p-1 integrins, CDw49b/CD29 complex, is known to be a major laminin receptor on endothelial cells [21]. There- fore, these results suggest that the interaction of p-1 integrins with basement membrane compo- nents, mainly laminin, results in the formation of capillary-like structures in culture on matrigel. Bauer et al. (1992) [23] demonstrated that the process of matrigel-induced tube formation of a human endothelium-derived permanent cell line involved integrins since antibodies to integrin sub- units (CDw49f or CD29) blocked the process. Furthermore, Davis et al. (1993) [24] reported recently that MAbs to CD51, CD61 and CD29 integrin subunits inhibited tube formation of HU- VEC cultured on matrigel, and thus the vit- ronectin receptor (CD51/CD61) may play a very important role in this process. Although the pre- cise mechanisms underlying the results of the present study and the observations of other groups remain to be defined, several integrin sub- units may mediate crucial angiogenic signals from the basement membrane matrix to endothelial cells.

The process of new capillary formation in vivo (angiogenesis) is extremely complex, and quantita- tive analysis of angiogenesis in both in vitro and in vivo systems is still complicated and such stud- ies have been limited. Although the process of matrigel-induced morphological differentiation of HUVEC is unlikely to be pre- cisely the same as the formation of new vessels which occurs during angiogenesis in vivo, our in vitro matrigel culture system in combina- tion with the use of a digital analyzer allowed both rapid and easy quantitative determination of tube formation induced by matrigel. This model could also be used to define other fac- tors or molecular events affecting endothelial cell differentiation and new blood vessel forma- tion.

Acknowledgment

This work was supported in part by a Grant-in- Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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