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J. Biol. Chem., Vol. 278, Issue 36, 34598-34604, September 5, 2003
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¶
From the
Donald W. Reynolds Cardiovascular
Clinical Research Center, Division of Cardiovascular Medicine, Stanford
University School of Medicine, Stanford, California 94305 and
Division of Cardiovascular and Respiratory
Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho,
Chuo-ku, Kobe 650-0017, Japan
Received for publication, May 9, 2003 , and in revised form, June 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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One complex group of molecules that are essential for all aspects of endothelial cell structure and morphogenesis are those cell surface proteins found at the cell-cell junctions. These molecules are organized in two primary structures, the adherens junctions and tight junctions. Both play critical roles in endothelial permeability and cell polarity. Multiple different protein types reside in these structures and mediate physical adhesion and are also well known to transmit signals to the cellular cytoskeleton and to the nucleus. Vascular endothelial cadherin molecules are endothelial cell-specific and localized in adherens junctions, where they have been shown to interact with structural proteins and signaling molecules including catenins (7). Targeted disruption of vascular endothelial cadherin reveals an early embryonic role for this molecule in endothelial cell differentiation, endothelial cell organization into vessels, and angiogenesis (8). Endothelial specific molecules in the tight junctions include junctional adhesion molecule (JAM)1 family members. Platelet endothelial cell adhesion molecule (PECAM-1) or CD31 and melanoma cell adhesion mole (MCAM) or CD146/MUC18 are not restricted to one type of junctional structure, and this broad localization appears to be important to their vascular functions. PECAM-1 function has been linked to angiogenesis (9–11).
A novel endothelial cell surface protein of the immunoglobulin superfamily has been cloned recently in this laboratory and named endothelial cell-selective adhesion molecule (ESAM) (12). Previous studies have determined that ESAM expression is restricted to endothelial cells in the embryonic and adult vasculature (12, 13). ESAM has been shown to mediate homophilic and calcium-independent adhesion of expressing cells (12). These data suggest that ESAM has specific endothelial cell functions and thus may play a role in maintaining vascular integrity or blood vessel formation. To gain better understanding of the angiogenic functions of ESAM in vivo, knockout mice functionally lacking ESAM have been generated. These mice have been evaluated with in vivo and in vitro models of angiogenesis. In vivo tumor growth in ESAM knockout (ESAM–/–) mice was significantly retarded compared with that in wild type (WT) mice. The reduced tumor volume in ESAM knockout mice was associated with less vascular density. We also documented that ESAM null vascular endothelial cells have less migratory and angiogenic activity. These data therefore provide novel insights into mechanisms of angiogenesis and suggest that ESAM may play a critical role in pathological blood vessel assembly.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A mouse ESAM cDNA probe (EcoRI-SacI fragment)
was employed for screening a 
-phage 129/SvJ genomic library
(Stratagene), providing two overlapping clones 18 and 13.5 kb in size. These
phage clones were restriction mapped, and the exon/intron structure was
partially determined by mapping and nucleotide sequence analysis. The
replacement targeting vector was constructed in the pKO Scrambler NTKV-1901
vector (Stratagene) using a 3.4-kb ScaI-ScaI fragment for
the 5' arm and a 6.4-kb PstI-PstI fragment for the
3' arm (see Fig.
1A). After homologous recombination, a 3' portion
(from 20 bp upstream of the translation initiation codon) of exon I of
ESAM was thus replaced by a 
-galactosidase (lacZ) and neomycin
phosphotransferase (neo) cassette. Also, a cassette for herpes simplex
virus-thymidine kinase was provided outside the region of homology to allow
negative selection.
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TL-1 129 embryonic stem cells (Brigid Hogan, Vanderbilt University, Nashville, TN) were used for transfection for the targeting vector. Cell culture, transfection, and positive and negative selection were performed as described previously (14). Genomic DNA was isolated from each clone and evaluated by Southern blot with 5' and 3' probes outside the homology domains (see Fig. 1A). Three of the correctly targeted embryonic stem cell clones were injected into C57Bl/6 blastocysts to generate chimeric animals. Three chimeric male mice were obtained and bred to C57Bl/6 females (Jackson Laboratories, Bar Harbor, ME) to obtain heterozygous pups. Male heterozygous mice (ESAM+/–) were bred to C57Bl/6 females five times before homozygous animals (ESAM–/–) were generated. WT littermates served as controls for all lines studied. Genotyping of knockout animals was performed with DNA isolated from tail tissue that was digested with EcoRV and separated on 0.65% agarose gels. After transfer to nylon membranes, blots were hybridized with radiolabeled probes synthesized utilizing 5'(XbaI-SalI) or 3'(PstI-EcoRV) fragments by random priming (see Fig. 1). Genotypes of mice were verified by detection of the fragments that differ in size between the wild type and the targeted locus. For detection of ESAM expression in the mutants, total RNA was isolated from mouse tissues, and Northern blotting were performed as described previously (12). A mouse ESAM (1.2 kb) cDNA was used as a probe.
Retinal Fluorescein Angiogram—Mice were anesthetized with pentobarbital, and a median laparotomy was performed. One milliliter of PBS containing 25 mg of 2 x 106 molecular weight fluorescein isothiocyanate-dextran (FD-2000S; Sigma) dye was injected into the portal vein. Eyes were subsequently removed and fixed in 4% paraformaldehyde for 3 h. The cornea was removed, the sclera was cut sagittally, and the retina was mounted on a glass slide. Fluorescent micrographs were taken.
In Vivo Tumor Model and Immunofluorescence Microscopy— ESAM–/– mice, 2 to 3 months of age, of both sexes, and age-matched WT mice were used in this study. Two tumor cell lines, B16F10 melanoma and LL/2 Lewis lung carcinoma (American Type Culture Collection), were used as in vivo tumor growth models in mice. The cells were grown according to the manufacturer's instruction. 1 x 106 tumor cells in 500 µl of Dulbecco's modified Eagle's medium were inoculated subcutaneously into the mouse flank, and tumor growth was monitored every other day with calipers. Tumor size was calculated as length x width x height (mm3) (15).
For immunofluorescence analysis, mice were perfused with 3% paraformaldehyde/PBS under anesthesia. Tumors were excised and fixed further with 3% paraformaldehyde/PBS, and frozen sections were made according to standard methodology. The cryosections were blocked with 1% bovine serum albumin, 0.5% Triton X-100/PBS for 30 min. Anti-mouse CD31 antibody (MEC13.3; BD Biosciences) was used as a primary antibody and incubated for1hat room temperature. After washing three times with PBS, sections were incubated with Cy3-conjugated mouse anti-rat IgG antibody (Jackson Laboratories, Bar Harbor, ME) for 30 min at room temperature. Sections were washed three times with PBS and then mounted in the Vectashield with DAPI (Vector Laboratory, South San Francisco, CA). Specimens were observed using a Zeiss fluorescence microscope equipped with a cooled CCD camera for digital image acquisition. For quantitation of vascular density, stained luminal structures were identified at low power microscopy and counted at x20 magnification in five random fields from each tumor section. An average of vessel numbers from five fields was regarded as n = 1, and a total of 10–12 tumors (50–60 sections) were evaluated.
Matrigel Plug Assay—This assay was performed as described previously (16). Briefly, 500 µl of Matrigel (BD Biosciences) containing 100 ng/ml basic fibroblast growth factor (bFGF) was injected subcutaneously into the frank of WT and ESAM–/– mice. Seven days later, Matrigel plugs were excised under anesthesia, weighed, and homogenized in the Drabkin's reagent (Sigma). After centrifugation, the supernatants were filtered through 0.45-µm filters, and hemoglobin content was measured at the wavelength of 540 nm following the manufacturer's instruction.
In Vitro Two-dimensional Tube Formation Assay on Matrigel—Aortic endothelial cells were isolated from the thoracic aorta of WT and ESAM–/– mice by an explant method (17). The cells were propagated in M199 medium, with 10% fetal bovine serum, 30 µg/ml endothelial cell growth supplement, 20 units/ml heparin, 2 mM L-glutamine, and penicillin/streptomycin. Cells were verified as endothelial by DiI-acetyl-LDL uptake prior to the assay. Third or forth passage cells were used for tube-forming assays. In vitro angiogenesis assays on Matrigel were conducted in 24-well plates coated with 100 µl of Matrigel (18). The cells were plated at a density of 5 x 104 cells/well, and tube formation was observed every 3 h.
For a gain-of-function experiment with ESAM, an embryonic yolk sac cell
line (Pro5) was engineered to express ESAM tagged with the FLAG epitope at the
C terminus. The Pro5 cells were cultured in minimum essential 
medium
containing 20% fetal bovine serum and 0.002% -mercaptoethanol and used
for transfection. An ESAM-pcDNA3 plasmid
(12) was introduced into the
cells, and stable transfectants were selected in the presence of 1000 µg/ml
G418. Clones were evaluated for ESAM mRNA expression by Northern
blotting and then evaluated for protein expression by Western blotting with
the anti-FLAG M2 monoclonal antibody (Sigma). Mock (pcDNA3 vector)-transfected
clones were used as controls. For in vitro angiogenesis assays,
transfectants were plated on Matrigel at a density of 5 x 104
cells/well, and tube formation was monitored every 3 h.
Cell Migration Assay—Cell migration assays were performed using modified Boyden chambers (Corning Coaster) containing polycarbonate membranes (19). The bottom side of the membrane of the upper chamber was coated with 10 µg/ml collagen type I for2hat37 °C, rinsed once with PBS, and then placed into the lower chamber containing 500 µl of migration buffer (Dulbecco's modified Eagle's medium with 0.5% bovine serum albumin). Endothelial cells from WT or ESAM–/– mice were removed from culture dishes with PBS containing 5 mM EDTA, washed twice with migration buffer, and then resuspended in migration buffer at the density of 1 x 106 cells/ml. 20,000 cells were then added to the top chamber. Migration buffer alone (negative control), 50 ng/ml bFGF, or 3% fetal bovine serum (positive control) in migration buffer was added to the bottom chamber to stimulate cell migration. Cells were incubated at 37 °C for 4 h. Non-migratory cells on the upper membrane surface were removed with a cotton swab, and the migratory cells attached to the bottom surface of the membrane were stained with 0.1% crystal violet in 0.1 M borate (pH 9.0) and 2% ethanol for 1 h at room temperature. The number of migratory cells per membrane was counted with an inverted microscope. Each measurement was the average of three or four individual wells.
Statistics—Differences between groups were analyzed using one-way analysis of variance. Data are expressed as means ± S.E. When statistically significant differences were found (p < 0.05), individual comparisons were made using the Bonferroni/Dann's test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Physiological Vessel Density Was Normal in ESAM–/– Mice—It has been shown that ESAM is expressed in endothelial cells in a variety of organs including the skin and eye. To examine the vascular network in ESAM–/– mice, fluorescein angiography was performed by injection of fluorescein isothiocyanate-dextran in vivo (Fig. 2). Similar blood vessel density was observed between WT and ESAM–/– mice. There was no difference in the number of large vessels originating from the optic nerve head, and small branching vessels were similar between WT and ESAM–/– mice. Similarly, there was no difference in the skin vessel character or density between WT and ESAM–/– mice (data not shown).
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ESAM–/– Mice Showed Decreased Tumor Growth and in Vivo Angiogenesis—It has been recognized that tumor growth is dependent on vascularization. To explore a role for ESAM in pathological angiogenesis, we employed an in vivo angiogenesis model using two different types of murine tumors, the B16F10 melanoma and LL/2 Lewis lung carcinoma. Representative sets of tumors in each group of mice at day 14 are shown (Fig. 3, A and B, top panels). Solid tumors that developed after injecting tumor cells were smaller in the ESAM–/– mice compared with those in WT mice. The delayed tumor growth in ESAM–/– mice was observed at all time points until tumor excision was performed 14 days after injection (Fig. 3, A and B, bottom panels). By 12 days after injection, the mean B16F10 tumor size was 48% smaller in ESAM null mice than WT mice (p < 0.01). The mean LL/2 tumor size was 37% smaller in ESAM–/– mice than WT mice by 14 days after injection (p < 0.05). We confirmed before conducting these experiments that neither tumor expressed ESAM in vivo under normal circumstances (data not shown).
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To investigate whether decreased tumor growth in ESAM–/– mice was attributable to decreased host angiogenesis, we compared vessel density and morphology between the different genotypes. Immunofluorescence analysis with antibody to the accepted endothelial cell marker CD31 revealed that tumor sections in ESAM–/– mice displayed decreased vascularization compared with tumors in WT mice (Fig. 4). In particular, there appeared to be a decrease in larger vessels in ESAM–/– mice, although the number of all size vessels was lower in the null mice. To quantitate the overall degree of vascularization, vessels were counted in sections stained with anti-CD31 antibody (Fig. 5). Vascular density in the ESAM–/– mice was decreased for B16F10 tumors (6.7 ± 3.4 versus 13.2 ± 1.3 microvessels/field, p < 0.01) and LL/2 tumors (5.3 ± 1.2 versus 8.0 ± 1.1 microvessels/field, p < 0.05). Thus, retarded tumor growth in ESAM–/– animals correlated with decreased vascular formation.
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ESAM–/– Mice Showed a Decrease in Neovascularization in Matrigel Plugs—To explore the angiogenic potential of ESAM–/– mice, we implanted Matrigel plugs in the flank region of mice. In this model, the host endothelial cells migrate and form a capillary network in the Matrigel implants (16). The degree of vessel formation within the implants can be quantified by measuring the hemoglobin content of the excised implants. At 7 days after implantation, Matrigel plugs excised from ESAM–/– mice showed a significant decrease in hemoglobin content compared with those from wild type mice, 53% of wild type levels (p < 0.05, n = 5; see Fig. 6). Therefore, ESAM–/– mice had less ability to vascularize the Matrigel plug.
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ESAM-deficient Endothelial Cells Showed Attenuated in Vitro Tube Formation—To address whether the decreased vascularization of Matrigel plugs and tumors in ESAM–/– mice was an autonomous defect of the endothelial cells, we isolated aortic endothelial cells from WT and ESAM–/– mice and compared their tube forming capacity in vitro. Cells were verified as endothelial by DiI-acetyl-LDL uptake (Fig. 7A). These cells were plated on Matrigel, and tube-like network formation was observed (Fig. 7B). The endothelial cells displayed morphological changes such as elongation and alignment on Matrigel to form two-dimensional tube-like structures. However, this activity was markedly attenuated with ESAM–/– endothelial cells. The network formation of endothelial cells on the Matrigel was quantified by measuring the tube number. As depicted in Fig. 7C, ESAM–/– endothelial cells showed a significant 32% decrease in the number of tubes compared with wild type mice (p < 0.05, n = 5). These finding suggest that ESAM–/– endothelial cells per se have less angiogenic potential.
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ESAM Expression Promotes Tube Formation in Yolk Sac Cells—To further explore the role of ESAM through in vitro studies, a gain-of-function experiment was performed using a yolk sac cell line that stably overexpressed ESAM. This yolk sac cell line was documented to express low levels of ESAM, and transfectants were documented to express high levels of ESAM mRNA (data not shown). When mock- and ESAM-transfected Pro5 cells were plated on Matrigel, ESAM-Pro5 cells showed more rapid and complete tube formation than mock-Pro5 cells (Fig. 8). At 3 h after plating, some ESAM-Pro5 cells were already elongated and aligned to form a tube-like network, whereas mock-Pro5 cells did not show this behavior. At 6 h, network formation was observed in ESAM-Pro5 cells but not in mock-transfected Pro5 cells. At 12 h, network formation was observed with both cell types but with thinner tubes and a more complete pattern in ESAM-Pro5 than in mock-Pro5. This accelerated tube formation in ESAM-Pro5 cells was observed at all the time points but more prominent before 12 h (Fig. 8). In combination with the data on ESAM–/– endothelial cells, therefore, a dose-dependent effect of ESAM was observed in this model of in vitro tube formation. When ESAM was overexpressed in an endothelial cell line that normally expresses ESAM, no increase in tube formation was observed (data not shown). Expression of ESAM in a fibroblast cell line did not induce any morphological change or tube formation (data not shown).
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ESAM Null Endothelial Cells Showed Decreased Migratory Response to bFGF—To further characterize the angiogenic function of ESAM, in vitro migration assays were performed with endothelial cells isolated from WT and ESAM–/– mice. As shown in Fig. 9, a decrease (30%) in migration was observed with 50 ng/ml bFGF-treated ESAM–/– cells compared with bFGF-treated WT cells, whereas equivalent migration was seen with serum-treated ESAM–/– and WT endothelial cells. These data suggest the ESAM may be important for endothelial cell migration under certain circumstances, especially when cells are exposed to bFGF.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In the context of in vitro tube formation studies using Matrigel, the expression level of ESAM was directly related to the endothelial potential to form luminal structures. When plated on Matrigel, endothelial cells derived from ESAM–/– mice exhibited significantly less tube formation than WT endothelial cells, suggesting that cell-cell contacts mediated by ESAM directly contribute to the formation and function of blood vessels. The idea was confirmed by expression studies using the yolk sac cells. The Pro5 cell has a very low endogenous expression of ESAM and a much lower capacity for forming tube-like networks than native endothelial cells. Expression of ESAM in this cell type resulted in augmented tube-forming capability. ESAM-Pro5 cells showed more cell-cell contacts, organization, and alignment to form tubes compared with control, suggesting that ESAM promoted intercellular contacts and associated cytoskeletal changes as well. Overexpression of ESAM in endothelial cell lines that already expressed ESAM did not augment tube formation, suggesting that functional levels of ESAM are present in most endothelial cells.
In vitro migration assays suggested that ESAM is involved in endothelial migration. Although equivalent migration was seen with serum-treated ESAM–/– and WT endothelial cells, a lower level was observed with bFGF-treated ESAM–/– cells compared with bFGF-treated WT cells. Thus ESAM may be a component of the bFGF signaling pathway regulating endothelial cell motility. Although the addition of serum to the medium completely compensated for the lack of ESAM in these assays, this is not surprising, because serum contains a variety of migratory stimuli, and other factors likely compensated for the loss of functional ESAM. It is suggested, therefore, that ESAM-mediated processes are a prominent part of migration under specific conditions, primarily those associated with bFGF stimulation of endothelial cells. Although the loss of ESAM resulted in a 30% decrease in bFGF-stimulated migration, the absence of ESAM resulted in a greater than 70% decrease in tube formation, suggesting that ESAM may have additional roles in morphogenesis.
Findings from the in vitro experiments are relevant to those obtained with the in vivo model of tumor angiogenesis. Tumors require adequate blood supply for expansive growth and are impaired in this regard if vascularization is attenuated (20, 21). Tumors implanted in ESAM–/– mice showed markedly retarded tumor growth and decreased vascular density. The findings were consistent between two different types of tumor cell lines. The growth differential was greater for the B16F10 tumors, which are more vascular than those produced with LL/2 cells, in keeping with tumor growth dependence on the degree of vascularity. Interestingly, although the absence of ESAM was associated with decreased vessels of all sizes in the tumors, the decrease in larger vessels rather than capillaries appeared more prominent. The retarded tumor growth in ESAM–/– mice was likely attributable to less angiogenic activity of the endothelial cells, through loss of homophilic interactions. However, loss of heterophilic interaction of ESAM with a tumor molecule might also be the mechanism of the observed slower tumor growth rate in knockout animals.
ESAM co-localizes with several tight junction proteins in native endothelial cells, suggesting that ESAM is a component of the tight junction complex in this cell type (13). Tight junctions seal the intercellular space and regulate paracellular permeability across endothelial or epithelial cell layers. In addition to the mechanical adhesion properties of adhesion molecules, however, there is accumulating evidence that immunoglobulin superfamily adhesion molecules modulate angiogenic processes both in vitro and in vivo by activation of cytoplasmic signaling cascades, by interaction with cytoskeletal proteins, and by functional interaction with other types of cells such as bone marrow-derived cells and tumor cells (8, 22). It has been shown that ESAM has putative interaction domains, including an Src homology 3 binding domain (12) and possibly a PDZ binding domain (13). Further studies are required to address these issues.
ESAM is one of a number of immunoglobulin domain cell surface molecules that are specifically expressed in the endothelial cell, localize to the cell-cell junction, and may have overlapping functional roles. Members of the JAM family are most relevant, as they are also found to localize to the tight junction. This family of proteins includes JAM, which is expressed in endothelial cells and epithelial cells, JAM-2 (23, 24), JAM-3 (25, 26), and vascular endothelial JAM (27). It has been shown that JAM plays a role in paracellular permeability and leukocyte transmigration, but a potential role in angiogenesis has not been elucidated (28). Although ESAM subcellular localization is similar to JAM-1 at tight junctions, ESAM does not bind the PDZ domain proteins that associate with JAM-1 (13), suggesting that ESAM may have different functions from JAM-1 or that ESAM might bind this molecule or other members of JAM family. Another immunoglobulin superfamily adhesion molecule MCAM/MUC18/CD146 is a transmembrane glycoprotein that is constitutively expressed by vascular endothelial cells, smooth muscle cells, and melanoma cells and localizes at cell-cell interfaces outside of the adherens junctions, as well as the apical cell surface (29–31). MCAM can mediate homophilic cell-cell adhesion, as well as heterophilic adhesions, through interaction with an unknown ligand. MCAM has been shown to activate intracellular signaling pathways and to regulate cell growth (32, 33). MCAM inhibition attenuated tumor growth and metastasis by inhibition of interaction between melanoma cells and endothelial cells (34, 35). JAM family members or MCAM may have functionally overlapping roles with ESAM.
The best studied member of this family of endothelial cell immunoglobulin superfamily adhesion receptor family is PECAM-1 (CD31). PECAM-1 mediates homophilic and heterophilic interactions and modulates leukocyte trafficking through the endothelial layer (36, 37). Interendothelial cell adhesion by homophilic PECAM-1 interactions promotes endothelial cell survival (38), and PECAM-1 has been reported to regulate endothelial cell motility (39–41). Inhibition of PECAM-1 function attenuated both in vitro and in vivo angiogenic processes (10, 40, 42, 43). Interestingly, initial study of targeted disruption of PECAM-1 revealed no overt abnormality in blood vessel morphology during embryogenesis (44), indicating significant complexity and redundancy of the functions of endothelial cell adhesion molecules of this class. Thus, the data from in vitro and in vivo studies of PECAM-1 function in angiogenesis were similar to those reported here for ESAM, and PECAM-1 is one molecule that might have overlapping functions with ESAM and compensate for its loss in vivo.
ESAM expression has been reported to be highly restricted to endothelial cells in embryonic and adult vasculature (12). In the process of targeting ESAM, the lacZ reporter gene was inserted into the locus to produce a chimeric transcript. Unfortunately, expression of the lacZ reporter gene did not reflect the cell and developmental-specific expression pattern of the native ESAM locus (data not shown). Fidelity of the chimeric transcript was indicated by extraneous expression of the reporter gene. This result was felt most likely to reflect the loss of critical endothelial cell regulatory domains in the ESAM locus, because a portion of exon I and the first intron were deleted during targeting. Both of these regions have been noted to contain regulatory sites in other endothelial cell genes, including tie2, flk-1, and von Willebrand factor (45–47). This fortuitous result implicates a 3-kb region of the ESAM locus as a critical transcriptional control sequence for expression of this gene.
In conclusion, in vitro studies reported here reveal that the endothelial cell-specific adhesion molecule ESAM has a role in endothelial cell migration and tube formation. Functional loss of ESAM does not interfere with physiological angiogenesis associated with development and reproduction, suggesting that other immunoglobulin adhesion receptors may compensate for its loss. However, in one setting of pathological angiogenesis, which associated with tumor growth, ESAM appears to have a significant role.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Division of Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Dr., Falk CVRC, Stanford, CA 94305. Tel.: 650-723-5013; Fax: 650-725-2178; E-mail: tomq1{at}stanford.edu.
1 The abbreviations used are: JAM, junctional adhesion molecule; PECAM,
platelet endothelial cell adhesion molecule; MCAM, melanoma cell adhesion
mole; ESAM, endothelial cell-selective adhesion molecule; WT, wild type; PBS,
phosphate-buffered saline; bFGF, basic fibroblast growth factor; LDL, low
density lipoprotein; DAPI, 4',6'-diamidino-2-phenylindole
hydrochloride.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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