1 Research Service, Samuel S. Stratton Veterans Affairs Medical Center, 2 Department of Medicine and 3 Centers for Cardiovascular Sciences and 4 Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Nitric oxide synthase expression has been documented in lung tumors, but a potential role for nitric oxide (NO) in induction of capillary formation remains to be elucidated. The purpose of this report was to characterize the direct effects of NO at the level of the tumor-endothelium interface with respect to angiogenesis. A Transwell two-compartment culture system, human endothelial cells (EC), and two human non-small cell lung cancer (CA) lines that constitutively produce NO were used to simulate the EC-tumor cell interface. Both histological types of lung CA, squamous and adenocarcinoma, induced baseline capillary formation by EC within 3 days. This process was inhibited by NO in the microenvironment because decreasing NO production with 100 µM aminoguanidine (AG) significantly increased capillary formation, whereas coincubation with 100 µM AG plus 400 µM L-arginine returned angiogenesis to baseline values. We demonstrate further that NO may exert its inhibitory effects by influencing matrix metalloproteinase expression/activity and tyrosine phoshorylation of proteins in the sprouting tips of nascent capillaries.
angiogenesis; CD31/platelet endothelial cell adhesion molecule; matrix metalloproteinases; focal adhesion kinase; protein tyrosine phosphorylation
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INTRODUCTION |
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SOLID TUMORS cannot grow beyond a few millimeters in diameter unless they recruit a blood supply to furnish the rapidly growing cells with nutrients and oxygen (9). Cancer (CA) cells stimulate new vessel development through the process of angiogenesis by inducing migration of endothelial cells (EC) from nearby vessels to form capillary networks within the tumor. A number of recent studies demonstrate that microvessel density within a tumor, an endpoint reflecting the extent of angiogenesis, is an important prognostic indicator for solid tumors, including lung non-small cell carcinomas (9, 16, 20). Nitric oxide (NO) has been shown to possess potent angiogenesis-modulating activities (33, 34, 57). However, there is controversy as to whether it stimulates or inhibits neovascularization in tumors, and conflicting observations have been made for different tissues and tumor types (50, 51). Solid tumors from various locations show elevated constitutive levels of nitric oxide synthase (NOS), as reflected in increased RNA expression and/or enzyme activities. The localization of this activity varies from tumor to tumor, found either in the tumor cells themselves or associated with tumor stroma in mononuclear cells or EC (46).
Reports from some laboratories associate increased inducible nitric oxide synthase (iNOS) expression with a setting favorable to tumor progression, at least in part as a result of the suppressive effect of elevated NO concentrations on the tumoricidal activity of macrophages and lymphocytes (7). NO-associated vasodilatory activity would also favor tumor growth and metastasis by augmenting blood flow and providing a route for metastatic invasion (12, 43). However, elevated concentrations of NOS are not always associated with tumor growth promotion and invasion (45). Indeed, successful suppression of these components has been obtained using systemic NO donors (42, 54), in animals transfected to overexpress iNOS within their tumors (53), or by targeted induction of iNOS expression with lipopeptide-containing liposomes (52). Recent studies have suggested that NO donor compounds may improve the efficacy of both radiotherapy and chemotherapy, as reviewed previously (50). Given the complex chemistry of the NO molecule and the large number of potential cellular targets, it is not surprising that diametrically opposing roles have been reported in CA and in angiogenesis (reviewed in Ref. 51).
Blood vessels within a tumor mass show numerous abnormalities; they are tortuous, saccular, and dilated (43). Their responsiveness to pharmacological stimulation by vasoconstrictors and vasodilators is compromised (36). Therefore, it is feasible that localized concentrations of NO could impact on the initiation of angiogenesis in such a microenvironment. Indeed, data obtained for microvessel counts, as an index of angiogenesis in human tumors, are derived from microscopic analyses of "hot spots" of focally concentrated angiogenic activity (48).
The current literature supports a proangiogenic role for NO in breast, head, neck, and colon CAs (1, 13, 14), and mechanistic studies have begun to define the likely biochemical targets of NO that might influence tumor invasion/metastasis (24, 25, 52). In contrast, a role for NO in lung CA growth and progression remains to be elucidated. Studies have reported NOS expression in lung tumors (11, 55) and documented the presence of increased exhaled NO from lung CA patients (27), but there have been no studies that correlate expression of NOS with the potential for inducing or inhibiting new vessel formation in lung CA. The presence of histological subtypes for adenocarcinoma (AD) and squamous (SQ) carcinoma likely contributes to some of the difficulty in assigning a single role for NO in lung CA. Of equal importance is the fact that the labile and highly reactive NO molecule itself shows biphasic concentration dependence in a number of physiological processes (51). Given the focal nature of new vessel development, it is essential to begin to characterize the direct effects of NO concentration at the level of the tumor-endothelium interface.
The aim of the present study was to study lung CA cell-induced capillary formation using cell lines that constitutively produce NO. We hypothesized that NO is an important component of the EC/tumor cell microenvironment that regulates the development of a migratory phenotype essential for capillary formation. Two complementary new culture models were used for these studies, one that evaluates angiogenic potential of diffusible factors in CA cell-conditioned medium (CM) and another that permits evaluation of the effects of cell-cell communication and continuously generated or labile molecules in the formation of capillaries. Both models provide for the basolateral stimulation of EC essential for angiogenesis (30) and simulate some of the crucial early steps involved in this process. These models show lung CA cell-induced capillary development within 3 days, a time frame that closely parallels initiation of angiogenesis in animal models (6). Because EC and CA cells remain segregated from each other in individual compartments for 24 h, the model system was also used to study cellular/biochemical mechanisms involved in the initiation of tumor cell-induced angiogenesis.
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MATERIALS AND METHODS |
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Materials
The Transwell culture system used for angiogenesis studies consisted of 6.5-mm diameter polycarbonate inserts with 8-µm pores (Costar, Cambridge, MA). RPMI 1640 and Hams F-12-K+ (F-12-K) medium, penicillin, streptomycin, gentamicin, trypsin-EDTA, Dulbecco's PBS (DPBS), Hanks' balanced salt solution, trypsin-EDTA (both 0.05 and 0.25% trypsin-1 mM EDTA), and amphotericin B (Fungizone) were obtained from GIBCO BRL (Grand Island, NY). Medium for growth of human umbilical vein endothelial cells (HUVEC) was obtained from Vec Technologies (Rensselaer, NY). FCS was obtained from HyClone (Logan, UT). H-Neurext, a hypothalamus-derived microvessel EC growth supplement, was purchased from UBI (Lake Placid, NY). Costar Transwell culture wells were obtained from Fisher Scientific (Springfield, NJ). Sterile DiI-conjugated acetylated low-density lipoprotein (DiI-LDL) was obtained from Biomedical Technologies (Stroughton, MA). Monoclonal antibody to human platelet endothelial cell adhesion molecule (PECAM)/CD31 (clone JC/70A) was obtained from DAKO (Carpinteria, CA). Reagents for visualization of CD31 were the DAKO linker solution biotinylated anti-mouse and anti-rabbit immunoglobulins, Vectastain avidin-biotin complex (ABC) kit, and diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA). Reagents for visualization of basic fibroblast growth factor (bFGF) protein were mouse anti-bovine bFGF type I (UBI) and FITC-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA). Reagents for Western blotting for endothelial cell nitric oxide synthase (eNOS) and iNOS were rabbit anti-eNOS (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-iNOS (Transduction Laboratories, Lexington, KY). Analysis of lung CA cell cultures for constitutively produced NO was performed using the Cayman nitrite/nitrate colorimetric assay kit (Alexis Biochemicals, San Diego, CA). Sterile solutions of rat tail collagen type I and Matrigel [reduced growth factor (RGF) formulation] for coating surfaces of the Transwells were obtained from Collaborative Research (Bedford, MA). Determination of growth factors vascular endothelial growth factor (VEGF) and bFGF in conditioned medium of lung CA cell lines was performed using Quantikine ELISA kits purchased from R&D Systems (Minneapolis, MN).Methods
Cell culture. HUVEC were obtained at passage 2 from Vec Technologies. Two human lung non-small cell carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA). The tumor cell lines were NCI-H157 (SQ) and NCI-H522 (AD). HUVEC were cultured in the following HUVEC complete medium: F-12-K supplemented with final concentrations of 20% FCS, 30 µg/ml Neurext, 0.1 mg/ml heparin, 100 U/ml penicillin-streptomycin (PS), 50 µg/ml gentamicin, and 0.5 µg/ml amphotericin B (Fungizone). Lung CA cell lines were cultured in the following lung CA complete medium: RPMI 1640 supplemented with final concentrations of 5% FCS, 100 U/ml PS, 50 µg/ml gentamicin, and 0.5 µg/ml Fungizone. All cells were routinely cultured in T25 or T75 flasks and subcultured by trypsinization (0.05% for HUVEC and 0.25% for CA cells). For angiogenesis studies, cells were subcultured in Transwells as described below, with all morphology studies performed on the Transwell filter insert membranes unless stated otherwise. For other studies, cells were cultured in T25 flasks or 35-mm dishes to provide conditioned medium (CM) for determination of secreted growth factors and 24-mm dishes to provide CM for NO measurements.
Angiogenesis model systems.
The method for coating of Transwell membranes for cell culture was a
modification of a technique previously described by Garrido et al.
(15). The upper surface was coated with basement
membrane-like RGF-Matrigel, and the lower surface was coated with
collagen type I to simulate the composition of the in vivo
microenvironment for EC and CA growth. Each Transwell insert was
suspended in an individual well of a 24-well cluster dish, with 1 mm
clearance between the bottom of the well and the culture dish.
Preparation of matrix protein solutions was done on ice, using ice-cold
solutions and chilled pipettes to prevent gelling. RGF-Matrigel stock
was diluted to a final concentration of 250 µg/ml with
DPBS (without Ca2+ or Mg2+), and
then 50 µl were pipetted into the upper compartment of each well.
Cluster dishes were tapped gently to ensure even distribution of the
solution, and the Matrigel was allowed to dry on the membrane in a
bacteriological hood (3-4 h). Individual Transwell inserts were
then inverted in a 150-mm sterile culture dish for coating of the lower
membrane surface with collagen. Rat tail collagen type I stock was
diluted to 250 µg/ml with 0.02 N acetic acid, and a 50-µl drop was
placed on the lower membrane with care to ensure even coverage. These
wells were allowed to dry as described above for 1-2 h. Coated
wells were returned to the cluster dishes and kept refrigerated until
used (usually overnight). To prepare for cell culture, the coated wells
were equilibrated with F-12-K medium for 1 h before seeding of
cells. For some studies, HUVEC growing in flasks were labeled live with
DiI-LDL, final concentration 5 µg/ml in HUVEC complete medium, for
2 h and then removed by trypsinization for counting and
subculture. Labeling with fluorescent DiI-LDL enabled monitoring of the
time course of capillary formation in live cultures. A diagram of
the angiogenesis model systems is shown in Fig.
1.
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Quantification of capillary formation. Values were derived from digital image analysis of capillaries in three representative fields for each well at ×100 magnification. Capillary formation is not homogeneous, so the following considerations were taken into account in quantifying capillary lengths. 1) Each well was scanned visually to evaluate the extent and pattern of capillary formation before any attempt at quantification was made. 2) Certain constraints were imposed by the optical properties of the Transwells and the imaging system used. The area of the filter immediately adjacent to the sides of the wells showed some distortion because of the meniscus properties of the plastic well. Because of this distortion, these areas were omitted from the evaluations, and capillary formation was assessed in the remaining center surface area of the Transwell. 3) Care was taken to prevent the quantification of overlapping microscope fields by selecting fields that were not adjacent to each other. This eliminated the possibility of quantifying the entire surface of the filter. 4) Capillary formation was extensive in some cultures, simplifying the choice of three fields. In contrast, under control conditions or in some treatment groups, capillary sprouting was minimal and heterogeneous. In these cultures, even though many fields contain no structures, fields that contain some rudimentary structures are always included in the quantification so that no cultures show zero sprout formation. We believe that these concessions allow reasonable evaluation of capillary sprouting in our system.
Manipulation of NO concentrations. In experiments to study the role of NO in capillary formation, 100 µM aminoguanidine (AG), either with or without 400 µM L-arginine (LA), was added to RPMI in the lower chamber at the time of coculture. In some studies, these reagents were also added 24 h later. Results of multiple experiments demonstrated that identical effects on angiogenesis were obtained with either single or multiple doses of AG.
Quantification of secreted growth factor in tumor cell CM.
CM from each lung CA cell line was obtained from cells grown in
complete medium in 35-mm dishes. Before preparation of CM, complete
medium was removed, and serum-free RPMI was added. Dishes were
incubated for 24 h at 37°C. CM was harvested and centrifuged, and 1% FCS was added to stabilize growth factors. Samples were analyzed immediately or frozen at 80°C until used (<1 mo).
Analysis was performed using Quantikine EIA kits specific for either
bFGF or VEGF. Cells remaining in individual dishes after removal of CM
were counted, and growth factor concentration is expressed per number
of cells in each dish.
Analysis of lung CA cell cultures for constitutively produced NO. total nitrate/nitrite concentrations. Total nitrate/nitrite (NOx) concentrations were evaluated in lung CA cell CM using nitrate reductase and Griess reagents. Values were determined by spectrometric analysis, reading absorbance at 540 nm. Individual cell lines were initially seeded in complete medium containing 5% serum to permit attachment and spreading. For preparation of CM, cells were washed with PBS and then incubated in F-12-K-3% serum for 6 h. NOx values were determined in CM from each of the lung CA cell types using sodium nitrite in F-12-K-3% serum as the standard. After removal of CM for NOx determinations, cells in the culture wells were removed by trypsinization and counted. Data are expressed on a per cell basis as nanomoles per 105 cells. In some experiments, NO concentrations were manipulated by addition of 100 µM AG or 100 µM AG plus 400 µM LA to confirm arginine dependence of the inhibition.
IMMUNOCYTOCHEMISTRY TO DEMONSTRATE ENOS AND INOS. To determine the source(s) of NO production in the cells used for our experiments, lung CA cells were first examined morphologically for expression of eNOS or iNOS protein. Cells growing on coverslips were formalin fixed, permeabilized with 1% Triton X-100, and stained with either rabbit anti-eNOS or rabbit anti-iNOS (Alexis Laboratories, Carpinteria, CA) followed by staining with FITC-conjugated goat anti-rabbit IgG and visualization by fluorescence microscopy as described previously (39). WESTERN BLOTTING FOR ENOS AND INOS. To corroborate immunocytochemical evidence of these proteins in lung CA cells, Western blotting was performed using total cell lysates obtained by extraction of cells with RIPA buffer [1× PBSStudies to evaluate cellular and biochemical aspects of capillary formation. determination of the cell-type composition on each side of the transwell membrane. Studies of the early crucial steps leading to capillary formation in cocultures would be facilitated by biochemical analyses of the individual cell populations involved. We performed the following experiments to assess the transmigration of individual cell types into the adjacent compartment across the Transwell membrane and thus the extent of comixing of populations within the first 24 h of coculture. First, we evaluated the migration of EC from the upper to the lower compartment as follows. Unlabeled EC or CA cells were seeded on the lower collagen-coated surface of the Transwell. Next, EC that had been labeled with DiI-LDL before culture were seeded in the upper compartment, and cocultures were allowed to grow for 24 h. The cells growing in the upper compartment were removed with cotton swabs and PBS washes, and the lower surface was carefully examined for the presence of DiI-LDL-positive cells using immunofluorescence microscopy. DiI-LDL-positive EC were not observed on the lower membrane surface, demonstrating that capillary formation occurred on the upper compartment of the Transwell in these cocultures.
We also determined whether CA cells migrated from the lower to upper compartment by performing the following two complementary sets of experiments. 1) Cocultures were set up as described above using DiI-LDL-labeled EC. At 24 h, CA cells were thoroughly scraped from the lower surface with cotton swabs and PBS washes. The cells in the upper compartment were fixed and then stained using an antibody for epidermal growth factor (EGF) followed by FITC-labeled secondary antibody. EGF protein is present in abundance in the two CA cell types but shows faint staining in EC with a characteristic distribution different from that of the CA cells. EC in the upper chamber would therefore appear as strongly positive for DiI-LDL (red) and faintly positive for EGF (green). CA cells that had migrated into to the upper chamber would be seen as strongly positive for EGF and negative for DiI-LDL. With the use of immunofluorescence microscopy, the number of EGF-positive/DiI-LDL-negative cells was counted on the upper surface of each well, and the value was expressed as a percentage of the total number of EC that grew on the upper membrane surface of Transwells at 24 h postseeding. This latter number was obtained by counting, in replicate wells, the number of EC removed from the upper surface by trypsin treatment (29,130 ± 2,858, mean ± SE, n = 4). Counting of cells in the upper chamber from HUVEC-SQ cocultures showed that all of the cells were DiI-LDL positive, with no cells exhibiting EGF staining typical of SQ cells. Counting of cells in the upper chamber from HUVEC-AD cocultures showed 231 and 242 DiI-LDL-negative cells in the upper compartments in two replicate wells. These DiI-LDL-negative cells showed intense green fluorescence consistent with the appearance of EGF in the CA cell lines. This number, expressed as a percentage of total HUVEC in the upper compartment, was 0.8% based on the EC cell number above. 2) In the second parallel set of experiments, cultures were set up as described above for EC labeled with DiI-LDL before culture. After scraping off CA cells from the lower surface of the well, the remaining cells in the upper chamber were stained with acridine orange, a fluorescent compound that stains the whole cell body of all cell types green (40). This was done to facilitate assessment of complete removal of cells by trypsin. Because acridine orange stained all of the cells whether they were DiI-LDL positive or not, we were able to confirm the first series of studies with respect to the presence of DiI-LDL-negative cells in the upper chamber. We were also able to ensure that all of the cells were included in the total cell count. The number of cells from the upper compartment of Transwells containing cocultures was compared with those containing EC alone. Cell numbers were as follows: HUVEC alone, 29,130 ± 2,858; cells from the upper well of HUVEC-SQ cocultures: 32,267 ± 4,878; cells from the upper well of HUVEC-AD cocultures: 28,901 ± 180. None of these values was significantly different from the other by one-way ANOVA. These data correlate with the results of the first series of experiments and demonstrate that <1% of the cells in the upper well are not HUVEC. We conclude that the HUVEC compartment is composed almost entirely of HUVEC, with only minor contamination by CA cells after 24 h of coculture. EVALUATION OF EC MATRIX METALLOPROTEINASE ACTIVITY BY SUBSTRATE ZYMOGRAPHY. Model 2 HUVEC-CA cell cocultures were used to provide total cell extracts for evaluation of matrix metalloproteinase (MMP) activity after 16 h of culture in the early sprouting phase of capillary formation. Cultures were set up to evaluate activity in HUVEC alone, CA cells alone, or HUVEC cocultured with CA cells. Cultures were either untreated or treated with 100 µM AG to inhibit NO production. For preparation of samples in the EC-CA cell cocultures, CA cells were scraped off the bottom of Transwells, and EC in the upper compartment were washed extensively before extraction. Cells were extracted with electrophoresis sample buffer without mercaptoethanol, and the peptides were separated on a 10% SDS-PAGE gel copolymerized with 1% gelatin as substrate for MMP activity, as described previously (35, 39). Areas of proteolytic activity were visualized as light bands in dark Coomassie-stained gels. Metallo dependence of proteolytic activity was confirmed by lack of proteolysis in gels incubated in 10 mM sodium EDTA in 50 mM Tris-Cl, pH 8.5, before Coomassie staining. IMMUNOCYTOCHEMISTRY: TYROSINE-PHOSPHORYLATED PROTEINS AND FOCAL ADHESION KINASE. Model 2 cultures were used to study immunolocalization of phosphotyrosine in HUVEC either alone or grown in the presence of lung CA cells. Studies were performed for 16 h in the early sprouting phase of capillary formation. Cultures were either untreated or treated with 100 µM AG to inhibit NO production. Before staining of the cocultures, CA cells were scraped off the lower surface of Transwells so the stained cells visualized were EC. Cells were washed with 1 mM sodium orthovanadate in DPBS+ before fixation and were stained to demonstrate protein tyrosine phosphorylation. Cells were stained with antibody to either phosphotyrosine, rabbit anti-phosphotyrosine (5 µg/ml), or focal adhesion kinase (FAK), mouse anti-FAK, at a 1:50 dilution of stock antibody (Transduction Laboratories) followed by FITC-conjugated secondary antibody at 1:40 dilution.Statistics
Where appropriate, data are expressed as means ± SE. Mean values were compared for statistical significance using ANOVA and were followed by Student's t-test. A value of P < 0.05 was considered to be statistically significant. ![]() |
RESULTS |
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Lung CA Cell Lines Produce Angiogenic Growth Factors VEGF and bFGF
A number of recent studies have documented angiogenic growth factor expression in human lung non-small cell carcinomas. In particular, VEGF has been shown to correlate with increased microvessel density and progression of these CAs (10). To characterize the lung CA cell lines used in this study, we focused on two growth factors, bFGF and VEGF. These factors have been shown to play major roles in the process of angiogenesis both in vitro and in vivo (9), and the effects of NO on angiogenesis have been shown by some laboratories to be different, depending on which of these growth factors is used to induce it (31, 34, 57).VEGF or bFGF protein concentrations were determined in CM obtained from
lung CA cells by ELISA as described in Methods and shown in
Table 1. Lung CA cells secrete nanogram
quantities of VEGF into the medium but only picogram quantities of
bFGF. This was not unexpected because all forms of bFGF lack a
hydrophobic signal sequence required for secretion via the vesicular
pathway and in the absence of membrane injury are not found in the
medium (29, 41). Both cell types, however, produce bFGF
protein in readily detectable amounts in total cell lysates, as shown
by Western blotting in Fig. 3. Although
AD cells produce multiple forms of this growth factor, including low
(18 kDa) and high molecular mass forms (22 and 24 kDa) associated with
cell proliferation (41), SQ cells show only dimer and
trimer forms.
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NO Production by Lung CA Cell Lines
Constitutive production of NO in CM of lung CA cells was evaluated by analysis of stable end products nitrite and nitrate. Although both cell lines produced relatively high amounts of NO, SQ cells produced the highest amount on a per-cell basis [SQ: 31.9 ± 5.1 and AD: 9.8 ± 0.53 (SE) nmol/105 cells; n = 4].To determine the source of NO production in these cells, lung CA cells
were first stained to demonstrate NOS isoforms by immunocytochemistry (Fig. 4, top). Both cell lines
express both eNOS and iNOS protein. Western blotting of total cell
extracts confirmed the production of these NOS isoforms in both cell
types (Fig. 4, bottom).
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Effect of NO on Capillary Formation
To investigate the effect of NO in the EC-tumor cell microenvironment on the development of capillaries, we used the two angiogenesis models described in Methods and Fig. 1.Capillary formation in response to lung CA cell CM.
We first determined the angiogenic potential of each type of lung CA
cell CM without the influence of continuously produced NO from live
lung CA cells. Twenty-four-hour serum-free CM prepared from each CA
cell line was used as a source of angiogenic stimulation. Capillary
networks were quantified by digital image analysis. Results are shown
in Fig. 5, open bars. Qualitatively,
capillaries varied from short segments to elongated branching networks
(data not shown). Quantitatively, values for CA cell CM were
significantly higher than those for HUVEC controls that form only short
rudimentary segments. However, the angiogenic potentials of individual
CA cell CM were not statistically different from each other.
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Capillary formation in EC-CA cell cocultures. In these experiments, the effect of continuously generated NO on capillary formation was evaluated by manipulating NO production in cocultures. Quantification of capillary formation in cocultures of HUVEC and lung CA cells untreated or incubated with 100 µM AG or 100 µM AG plus 400 µM LA is shown in Fig. 5. In AD cocultures, capillary formation was significantly higher than that observed in EC-CM cultures. However, in EC-SQ cocultures, capillary formation was not increased above CM-stimulated levels. Qualitatively, examination of DiI-LDL-labeled live cocultures revealed that angiogenesis stimulated by AD cells was both rapid and aggressive, so that capillary networks formed by 48 h were equivalent to those formed at 72 h for these cells.
To investigate the role of NO in modulating angiogenesis at the EC-tumor cell interface, NO production was inhibited with the NOS inhibitor AG. Capillary formation in SQ cocultures was nearly doubled by this treatment, although this process was increased by a smaller, but statistically significant, amount (24%) in AD cocultures. Furthermore, when LA was coadministered with AG, providing substrate for NO production, capillary formation decreased to values comparable to, in HUVEC-SQ cocultures, or significantly less than, in HUVEC-AD cocultures, that for untreated cocultures. Figure 6 demonstrates the effects of treatment with AG or AG plus LA on NOx production by the two lung CA cell lines. AG produced significant inhibition of NO production in SQ cells that was reversed by coadministration of LA. AG inhibited NO production to a lesser extent in AD cells, whereas LA coadministration significantly enhanced NO production. These observations parallel the effects seen in Fig. 5 with respect to NO concentration and capillary formation.
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Effect of NO Concentration on HUVEC MMP Activities
The coculture experiments above clearly demonstrated that NO concentrations at the HUVEC-CA cell interface modulated the process of capillary formation. We were interested in investigating the possible mechanisms involved in these effects. Increased expression and/or activation of MMP occurs early in the process of EC sprouting to facilitate release of attached cells from their constraining matrix (49). We studied MMP expression using substrate gel zymography to evaluate gelatinase activities in total cell extracts obtained from 1) HUVEC or individual CA cells alone or 2) HUVEC cocultured with SQ cells or AD cells. NO concentrations in the cocultures were manipulated by NOS inhibition with AG. Results are shown in Fig. 7. In Fig. 7A, total cell extracts obtained from HUVEC cultured alone showed a major band at 72 kDa that is consistent with the latent form of MMP-2 (gelatinase A). Individual lung CA cell lines also showed the presence of major gelatinase activity at 72 kDa, with faint bands at 64 and 66 kDa, representing the activated forms of MMP-2. AD cells cultured alone showed an additional faint band of activity at 96 kDa, consistent with the latent form of MMP-9 (gelatinase B). In Fig. 7B, addition of AG to HUVEC cultures resulted in the appearance of two faint bands at ~64 and 66 kDa as shown in Fig. 7A for CA cell lines. HUVEC extracts obtained from cocultures with SQ cells showed predominantly MMP-2 activity. However, HUVEC cocultured with SQ cells in the presence of 100 µM AG showed increased 72-kDa activity. MMP-9 activity was also observed in the form of additional bands at ~96 kDa and several bands with decreased molecular mass that represent the activated form of MMP-9. HUVEC cocultured with AD cells showed a more complex pattern of MMP activity than that observed with HUVEC-SQ cocultures, consistent with the more aggressive angiogenic profile of AD cells. Although the intensity of both MMP-2 and MMP-9 activities was greater than observed in the HUVEC-SQ cell cocultures, addition of AG to HUVEC-AD cell cultures augmented this activity above the levels of untreated HUVEC-AD cocultures. These data demonstrate that NO concentration modulates the expression and/or the activation of two MMPs in EC during the early phase of capillary formation.
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NO Concentration Influences Protein Tyrosine Phosphorylation in Sprouting Tips of Nascent Capillaries
Model 2 cocultures were used to study immunolocalization of tyrosine-phosphorylated proteins at 16 h in the early sprouting phase of capillary formation. Cultures were either untreated or treated with 100 µM AG to inhibit NO production. Before staining of cocultures, CA cells were scraped off the lower surface of Transwells so the stained cells observed were EC (Fig. 8). Phase microscopy enhanced by CD31 staining was used to show the appearance of the EC monolayer that was not forming capillaries (Fig. 8A) or the presence of EC sprouts at this early time period in the process of capillary formation (Fig. 8B). Fluorescence microscopy of the monolayer stained for phosphotyrosine (Fig. 8C) showed only faint staining in a few cells. Lack of staining shown in Fig. 8C was representative of what was observed for HUVEC either untreated or AG treated, untreated SQ, and untreated AD. Only HUVEC from HUVEC-SQ AG-treated cocultures (Fig. 8D) and HUVEC from HUVEC-AD AG-treated cocultures (Fig. 8E) showed positive staining for the presence of tyrosine-phosphorylated proteins. HUVEC either alone or in coculture with SQ or AD were stained for FAK. Fluorescence microscopy showed lack of staining for FAK in all cultures except HUVEC from HUVEC-AD cocultures treated with AG (Fig. 8F). These cultures show intense positive staining in focal areas of capillary sprouting.
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DISCUSSION |
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Microvessel density within a tumor mass is an important prognostic indicator for a number of solid tumors, including lung non-small cell carcinomas (9, 16, 20). Although recent reports demonstrate that the levels of total NOS are significantly elevated in many tumors, the role of NO in tumor biology remains to be clarified. Many tumors also express high levels of angiogenic molecules, such as VEGF, that cause increased vascular permeability, thereby superimposing hemodynamic and angiogenic components within the tumor-EC microenvironment. The process of angiogenesis is a focal phenomenon as demonstrated by the presence of "hot spots" of localized angiogenic activity in tumor tissues in animals and humans (48). In recent years, considerable emphasis has been placed on manipulation of soluble growth factors as a means to inhibit tumorigenesis. However, it has been shown that even in the presence of saturating concentrations of growth factors, only certain EC undergo sprouting to form vessel segments, whereas EC a few micrometers away remain quiescent (22). This observation supports the concept that local microenvironmental factors may be equally important in determining the initiation of new vessel formation. Data from human and animal models that show these principles were used to formulate the concept of the "angiogenic switch," namely that alteration in the balance of positive and negative factors that maintain quiescent behavior of the endothelium within a given tissue results in initiation of angiogenesis (19).
In this report, we have used a culture system that simulates many features of the tumor-endothelium interface as a model for studying the angiogenic microenvironment of a tumor. Here, EC and CA cells interact in close proximity (10 µm) to each other in a setting that permits the ready exchange of continuously generated angiogenesis-modulating molecules. When coculture is initiated, the EC are growing in a monolayer conformation on a basement membrane-like substrate (Matrigel). The tumor cells are attached to interstitial collagen type I on the underside of the membrane providing basolateral stimulation for angiogenesis, settings similar to their in vivo milieu. Within 48-72 h, the EC undergo a series of cellular and biochemical changes culminating in the formation of capillary networks. Although a few investigators have successfully cocultured EC and CA cells in monolayer systems (26, 47), there are inherent problems with cocultures that contain CA cells (30). Rapid growth of CA cell populations, particularly in collagen gels, results in significant changes in culture pH that are toxic to EC. The coculture model described here successfully allows EC and CA cells to grow in close proximity, providing for continuous generation of angiogenesis-modulating molecules and resulting in the development of capillary networks in 2-3 days. This time frame closely simulates that for initiation of angiogenesis in animal models (6).
To characterize the proangiogenic properties of the CA cell lines used in these studies, we determined their expression of two factors, bFGF and VEGF, that have been shown to be involved in angiogenesis in vitro and in vivo (9). Our data demonstrate that both cell types produce both of these angiogenic growth factors, although in different amounts, in unstimulated cultures of a single cell type. Although bFGF was not detected in the medium in amounts equivalent to VEGF, coculture with EC could result in changes in growth factor expression by either or both cell types and changes in the pattern of release into the medium. VEGF and bFGF have been shown to have different potencies with respect to stimulation of angiogenesis, with bFGF being severalfold more stimulatory than VEGF (38). These two growth factors also exhibit marked synergy in the stimulation of the angiogenic response (18, 38). Capillary network formation in our system likely results from angiogenic stimulation provided by at least these two growth factors, a situation analogous to the microenvironment of tumor-induced angiogenesis in vivo. Both EC and CA cells constitutively produce NO, and NO production at the EC-tumor cell interface represents the physiological concentrations and fluxes of this labile molecule that occur during the interaction of these two cell types. In a proangiogenic climate provided by these two growth factors, we demonstrate that endogenously generated NO is inhibitory to the capillary formation.
The two lung CA cell lines chosen for study were representative of two histological types of lung CA, each with specific characteristics with respect to invasiveness and predicted angiogenic potential (55). Quantification of capillary formation in the coculture setting showed that the proangiogenic behaviors parallel observed characteristics of the tumor types in vivo, with AD exhibiting a more aggressive angiogenic phenotype than SQ carcinoma (55). The effect of NO on capillary formation was most pronounced in cocultures of EC with the cell line that produced the highest amount of NO on a per cell basis, SQ cells. Here, capillary formation was not increased above that observed with CM stimulation alone. In contrast, cocultures of EC with AD cells showed significantly increased angiogenesis compared with that stimulated by their CM. We have shown that there is a baseline component of capillary formation at the EC-tumor cell interface, even in the presence of NO generated by each of these two cell types. This finding is consistent with the observations of others that EC-associated NO/NOS is essential for angiogenesis (17, 33, 34, 57). However, elevated NO concentrations in the EC-tumor cell microenvironment attenuate capillary formation, with the extent of inhibition dependent on the concentration and flux of NO produced in this milieu. This conclusion was supported by studies performed to manipulate NO concentrations in the cocultures. In both cases, inhibition of NO production with AG increased capillary formation. Furthermore, if excess LA was added with AG, providing a source of substrate for NOS, angiogenesis decreased to untreated coculture levels. With this regimen in HUVEC-AD cocultures, capillary formation was significantly below that in baseline cocultures, supporting the notion that increased NO concentration impairs new vessel formation. Elevated exogenous NO concentrations have been shown to induce posttranscriptional modulation of lung eNOS that results in loss of enzymatic activity. Possible mechanisms include direct action of NO with active-site cysteines in this protein or interaction with allosteric thiols of proteins that modulate expression of redox regulatory proteins thioredoxin and thioredoxin reductase (56). We acknowledge that the model used for these studies lacks the contribution of inflammatory cells that would be present in a tumor microenvironment. The presence of these cells, with concomitant production of reactive oxidant species and NO, would markedly influence the capillary-forming ability of the resident EC population, perhaps to an even greater extent than shown in this report.
We performed studies to investigate the possible mechanisms involved in NO-induced inhibition of angiogenesis. Characterization of the coculture system with respect to potential intermixing of the EC and CA cell population demonstrated that there was minimal cross-contamination with either cell type in the individual monolayers within the first 24 h of coculture. Therefore, the model was used to study the effect of NO on the following two proangiogenic properties: MMP expression and focal contact protein distribution/phosphorylation status of EC in this early sprouting phase of angiogenesis.
Because extracellular matrix remodeling is essential for the development of a migratory phenotype, the effect of NO concentrations on MMP expression was examined. Gelatin substrate zymography demonstrated that these lung CA cells induced increased expression and/or activity of MMP-2 and MMP-9 in EC within 16 h of coculture, possibly via a soluble mediator. CA cells have been shown to produce an extracellular MMP inducer (EMMPRIN) that is present on the surface of the tumor cell and is shed by the tumor (8). Tumors also upregulate expression of MMP in EC within the stromal environment in vivo (2, 3, 21). Both endogenous and exogenous NO have been shown to selectively inhibit expression of MMP but not its inhibitor (tissue inhibitor of MMP) in one system (53). However, in another experimental tumor model, tumor-derived NO upregulated MMP and downregulated MMP inhibitors (25). In general, elevated levels of NO have been shown to influence the expression of MMPs, with the concentration of NO and the system studied determining whether MMP expression is increased (37, 44) and/or decreased (4). In addition to regulation of MMP gene expression, these enzymes can be regulated by posttranscriptional mechanisms that result in their activation or inactivation. For example, processing of human MMPs by bacterial proteases from the proenzyme form to active forms depended on free radical generation by activated neutrophils, involving peroyxnitrite or nitrogen dioxide radical (28). In contrast, in a system in which NO and peroxynitrite were generated chemically, only peroxynitrite modulated gelatinase A activity, causing inhibition of activation (32). Further evaluation of the effects of NO at the tumor cell-EC interface with respect to MMP induction and/or activation should be the subject of future studies.
We also performed studies to evaluate the effect of NO concentrations on sprouting EC in the early phase of capillary formation. This molecule clearly inhibited protein tyrosine phosphorylation in the sprouting tips of nascent capillaries because inhibition of NO production resulted in strong positive fluorescence for phosphotyrosine at these sites. These observations implicate activation of one or more EC protein tyrosine kinase(s) that mediate phosphorylation of adhesion proteins such as FAK or paxillin. These proteins localize in advancing tips of invading cells and participate in signaling pathways that regulate the state of focal adhesion assembly and cell-substrate adhesion. Our results with FAK in AD cells suggest that this adhesion molecule is a possible candidate for NO modulation during the early process of capillary formation. NO donors have been shown to significantly diminish the de novo formation of focal adhesions in HUVEC by affecting the state of tyrosine phosphorylation of focal adhesion complexes (17). NO decreases phosphorylation of focal adhesion proteins via activation of a protein tyrosine phosphatase in smooth muscle cells (23). It also blocks the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins in chondrocytes (5). Further characterization of NO-induced modulation of these changes in adhesion proteins may be the subject of future studies.
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ACKNOWLEDGEMENTS |
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This work was supported by the Department of Veterans Affairs Medical Research and National Heart, Lung, and Blood Institute Grant HL-51360.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. G. Phillips (151D), Research Service, Samuel S. Stratton VA Medical Center, 113 Holland Ave., Albany, NY 12208 (E-mail: Patricia.Phillips3{at}med.va.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 July 2000; accepted in final form 6 February 2001.
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