SPECIAL COMMUNICATION
Nitric oxide modulates capillary formation at the endothelial cell-tumor cell interface

Patricia G. Phillips1,2,3, Linda M. Birnby1, Amithi Narendran4, and Wendy L. Milonovich1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Diagram of in vitro angiogenesis model systems. A: Transwell cell culture chamber used in tumor-induced angiogenesis. Each well contains a Transwell insert (T) with a polycarbonate membrane (M) 10-µm thick with 8-µm pores. Insert (T) delineates upper chamber (U) and lower chamber (L), with the levels of media equal in both chambers. B: endothelial cell (EC)-cancer (CA) cell conditioned media (CM) model. Magnification of the separation between both chambers. Arrows point to human umbilical vein endothelial cells (HUVEC) seeded on reduced growth factor (RGF)-Matrigel and lower chamber that contains either serum-free RPMI (control) or CA cell CM. C: EC-CA cell coculture model. Magnification of the separation between both chambers. Arrows point to HUVEC seeded on RGF-Matrigel and CA cells seeded on collagen type I.

For model 1 (Fig. 1B), used for testing of CM from CA cells, HUVEC were seeded at confluent density into the upper compartment at 1 × 105 cells/well in 0.1 ml of HUVEC complete medium. The lower compartment received 0.6 ml of either serum-free RPMI 1640 medium or CM from CA cells, prepared by incubating these cells in serum-free RPMI for 24 h. EC were grown in this CM for 3 days when the cultures were then fixed and stained. For model 2 (Fig. 1C), HUVEC-CA cell cocultures, individual CA cells were first cultured on the inverted undersurface of the wells. For HUVEC control cocultures, HUVEC were cultured on the upper and lower surfaces of Transwells. Cell suspensions (50 µl) containing 1 × 106 cells/ml were pipetted on the collagen-coated surface, and the cells were allowed to adhere in a CO2 incubator for 2-5 h at 37°C. After cell adherence, cocultures were incubated for 1-3 days, depending on the types of studies to be performed.

Because cell lines vary with respect to their ability to adhere to collagen surfaces, optimum conditions for adherence of CA cells were first determined. For these preliminary experiments, attached cells were treated with a combination of 0.25% trypsin-1 mM EDTA-0.05% collagenase type 2 solution to remove them from coated membranes, and the number of cells that adhered at each time period was recorded. Phase-microscopic examination of wells at various time intervals was used to confirm removal of the cells. SQ and AD cells are small round cells that remain rounded throughout the adherence period. These two cell types showed similar adherence characteristics. At 3 h, 31,550 and 28,635 cells, respectively, had adhered to the undersurface of the Transwell. After adherence for the predetermined time period, individual inserts were then washed gently to remove nonadherent cells and then were placed in wells of the 24-well culture dish, each of which contained 0.6 ml of serum-free RPMI 1640 medium. Next, HUVEC were seeded in upper chambers of the Transwell inserts at confluent density in complete medium as described above for model 1. All cultures were incubated at 37°C in a CO2 incubator for 2-3 days to evaluate capillary sprouting. Although vigorous capillary formation was observed at 2 days with AD cells, final readings were made at 3 days. In preliminary studies, comparison of AD wells harvested at either 2 or 3 days showed similar values.

To confirm the identity of capillary-forming cells in cocultures as EC, and to quantify the capillary networks by digital image analysis, cultures were also stained for the EC-specific marker CD31/PECAM. No staining of lung CA cells was observed with either DiI-LDL or CD31. For CD31 staining, medium bathing the cells was removed, both surfaces of the inserts were washed with DPBS with Ca2+/Mg2+ (DPBS+), and cells were fixed with 10% buffered formalin solution for 30 min at room temperature. Normal goat serum blocker (2%) diluted with Tris-Cl buffer, pH 7.6, was applied for 10 min and then primary antibody monoclonal anti-human PECAM/CD31 was applied at a 1:40 dilution. Wells were incubated for 1 h in a moist chamber at 37°C. After washing with Tris-Cl, pH 7.6, to remove primary antibody, secondary antibody in the form of DAKO linker solution (cocktail of biotinylated anti-mouse and anti-rabbit IgG) was applied for 10 min followed by washing with Tris-Cl. To quench endogenous peroxidase activity before color development with streptavidin-conjugated horseradish peroxidase (Vectastain ABC kit), samples were treated with freshly prepared 0.3% hydrogen peroxide for 5 min. Diaminobenzidine solution was applied to samples, and color development in the dark was performed for at least 20 min until adequate intensity was obtained by microscopy examination. Rinsed samples were stored in the refrigerator.

In model 1 cultures, because only those cells that have migrated through the pores to form capillaries on the lower collagen surface were of interest, cells growing in the upper compartment were removed with a cotton swab and underwent several DPBS+ washes before formalin fixation. For model 2 cultures, capillaries formed on the upper surface of the wells were separated from the lung CA cells by a 10-µm-thick membrane. Because the two cell types remained essentially segregated, at least for the first 24 h of coculture (see below), separate populations could be isolated for further biochemical analyses of cells in the early phase of capillary formation, as described below for substrate zymography and immunocytochemistry studies.

Examination and photodocumentation of capillary formation were performed using an Olympus IMT2 inverted microscope equipped for light, phase, and fluorescence microscopy. For microscopy examination using an inverted microscope, wells were examined in situ in the 24-well plates where they were stored or placed directly on a coverslip above the objective below. DPBS or distilled water was used to hydrate samples for viewing. Quantification of capillary networks was performed using the SAMBA 4000 image analysis system (Imaging Products International) equipped with a Sony 3CCD color video camera interfaced with a Zeiss Axioskop upright microscope. For visualization with the upright microscope, the upper portion of the Transwell insert was rapidly sliced off using a heated scalpel, leaving the stained membrane and 3-4 mm of plastic insert attached to it. This preparation was then placed membrane side down on a drop of PBS-glycerol (1:1) on a microscope slide and sealed with clear nail polish around the outer rim of the insert base. A typical example of staining of cultures for EC-specific markers DiI-LDL and CD31/PECAM is shown in Fig. 2.


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Fig. 2.   Capillary formation in EC-CA cell cocultures. Typical example of capillary formation induced in cocultures of EC and AD cells is shown. Double-stained preparations of these cultures show EC cultured either alone (A and B) or in coculture with AD cells (C and D). Photomicrograph shows positive staining for both EC-specific markers as described in Methods. A and C: CD31/PECAM; B and D: DiI-acetylated low-density lipoprotein (LDL). Arrows in C and D point to networks of capillaries that have formed over the course of 2 days of coculture. Network formation involves organization of EC into ropelike structures accompanied by denuding of the previously confluent monolayer. Although clusters of EC remain associated with these structures, with time, fusion of adjacent cells becomes more apparent as tubelike segments emerge. Note that CD31 stain intensity is markedly increased in capillary-forming cells compared with that in EC monolayer (A). Small open circles are 8-µm pores in the membrane. Magnification, ×200.

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× PBS-, 1% Nonidet P-40 (igepal), 0.5% sodium deoxycholate, and 0.1% SDS]. Protease inhibitors phenylmethylsulfonyl fluoride and aprotinin were added fresh in final concentrations of 0.1 and 0.3 mg/ml RIPA buffer, respectively. Equal amounts of cell protein from each cell line (40-60 µg) were loaded in each lane of a 7.5% SDS-PAGE gel. Peptides were separated, transferred to polyvinylidene difluoride membranes, and probed for either rabbit anti-eNOS at 1:200 (Santa Cruz Biotechnology) or rabbit anti-iNOS at 1:10,000 dilution (Transduction Laboratories). Bands were detected using biotinylated donkey anti-rabbit IgG and the tertiary reagent strepavidin-horseradish peroxidase followed by enhanced chemiluminescence.

Studies 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Quantification of secreted VEGF or bFGF from CA cell lines



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Fig. 3.   Western blotting of total cell lysates from each lung CA cell line for basic fibroblast growth factor (bFGF) protein on a 12.5% SDS-PAGE gel. Molecular mass markers (kDa) at right indicate approximate sizes of bFGF species produced. Recombinant human bFGF standard shows a major band at 18 kDa. Adenocarcinoma (AD) cells produce multiple forms of bFGF (filled arrows, left), with predominant high molecular mass forms at 22 and 24 kDa. Both cell lines show bFGF bands as dimers and trimers (open arrows). Blot shown is representative of 3 experiments. SQ, squamous; STD, standard.

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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Fig. 4.   Top: lung CA cells constitutively produce endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Immunocytochemistry was used to demonstrate eNOS and iNOS in individual CA cell types growing on coverslips. Cells were formalin-fixed, permeabilized with 1% Triton X-100, and stained with either rabbit anti-eNOS or rabbit anti-iNOS, as described in Methods. Cells were viewed by immunofluorescence microscopy at ×200 magnification. A and B: eNOS. C and D: iNOS. A and C: SQ cells. B and D: AD cells. Bottom: Western blot demonstrating nitric oxide synthase (NOS) isoforms in lung CA cells. Equal amounts of protein from total cell lysates from each CA cell line were separated on 10% SDS-PAGE, and blots were probed for either eNOS or iNOS as described in Methods. A single band for each isoform was obtained as indicated by arrows at 137 and 130 kDa. iNOS standard was mouse peritoneal macrophage iNOS. HU, human umbilical vein EC (HUVEC). Gels shown are representative of 3-4 experiments.

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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Fig. 5.   Capillary formation: conditioned medium, coculture with CA cells, and effect of NOS inhibition. For CM studies HUVEC (HU) growing in the top chamber of Transwells were exposed to either serum-free RPMI (controls) or serum-free CA CM. For coculture studies (Co-C), HUVEC were cocultured in Transwells with either HUVEC (HU controls) or individual CA cell lines (HU/CA) on the lower membrane surface. Cultures were either untreated (Co-C), treated with 100 µM aminoguanidine (Co-C + AG), or cotreated with 100 µM AG + 400 µM L-arginine (Co-C + AG + LA). HUVEC were stained for CD31/PECAM to identify EC forming capillary networks. Capillary lengths were derived for each individual well by digital image analysis of the lengths of capillaries in 3 representative fields at ×100 magnification. Bars represent capillary lengths (mean ± SE) from 3-6 experiments performed in duplicate. None of the HU groups was significantly different from each other. *For SQ, P < 0.001 vs. means for the other 3 SQ groups. For AD, P < 0.01 vs. CM (#), = 0.04 vs. Co-C (*), and < 0.001 vs. Co-C or Co-C + AG (**).

Studies using CM to induce capillary formation were performed here by adding the CM only one time at the beginning of the experiment, as has been described by others (15, 38). Growth factors such as bFGF and VEGF are labile in culture and without renewal should be rate limiting with respect to initiation and maintenance of capillary formation. We anticipated that cocultures of HUVEC and lung CA cells would provide a continuous source of stimulatory growth factors, resulting in increased capillary formation. It was also possible that labile inhibitory factors would diminish in CM, and so their influence might not be evident in the model 1-type cultures. The next series of experiments was performed in EC-CA cell cocultures to evaluate these possibilities.

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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Fig. 6.   Lung CA cells constitutively produce NO: modulation of NO production by AG or AG + LA. Total nitrate/nitrite (NOx) concentrations were measured in lung CA CM using nitrate reductase and Griess reagents as described in Methods. Cells were either untreated (control) or incubated with 100 µM AG or 100 µM AG + 400 µM LA for 6 h before harvesting CM for NOx determinations. Data are expressed as NOx concentration in nmol/105 cells based on cell number determined in parallel from each well (n = 3). For SQ, P = 0.037 (*) and 0.001 (**) vs. AG. **For AD, P < 0.03 vs. control or AG.

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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Fig. 7.   Effect of NO concentration on HUVEC matrix metalloproteinase (MMP) expression/activities. Gelatin substrate zymography was used to evaluate MMP activities in total cell extracts from 16-h cultures as described in Methods. A: single cell-type cultures, as indicated at the top. B: lane 1, left, standard, rat skin collagenase/gelatinase. Bracketed pairs indicate HUVEC either alone or in coculture with SQ or AD cells. Cultures were either untreated (-) or treated (+) with 100 µM AG. Arrows point to 72-kDa (MMP-2) or 96-kDa (MMP-9) gelatinase activities. Decreasing NO concentrations with AG resulted in increased production and activation of EC-associated MMP-2 and -9. Gels shown are representative of 3 experiments.

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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Fig. 8.   NO concentration influences protein tyrosine phosphorylation in sprouting tips of nascent capillaries. Transwell cocultures were used for immunocytochemical studies of HUVEC during the early phase of EC sprouting as described in Methods. HUVEC were stained with antibody to phosphotyrosine (C-E) or to focal adhesion kinase (FAK; F). Phase microscopy enhanced by CD31/PECAM staining was used to visualize the EC monolayer with no capillary formation evident (A) or with tubelike sprouts visible in relief (arrows in B). Small even holes are pores in Transwell membrane. C: phosphotyrosine staining. Faint staining in only a few cells is seen. This view 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 (D) and HUVEC from HUVEC-AD AG-treated cocultures (E) showed positive fluorescence signals for phosphotyrosine in focal areas of the monolayer and tips of nascent capillaries (arrows). For FAK staining, HUVEC either alone or in coculture with SQ or AD were stained for FAK. Fluorescence microscopy of HUVEC stained for FAK showed lack of staining similar to C in all cultures except HUVEC from HUVEC-AD coculture treated with AG (F, arrow). Magnification, ×400.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This work was supported by the Department of Veterans Affairs Medical Research and National Heart, Lung, and Blood Institute Grant HL-51360.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ambs, S, Merriam WG, Bennett WP, Felley-Bosco E, Orgunfusika MO, Oser SM, Klein S, Shields PG, Billiar TR, and Harris CC. Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res 58: 334-341, 1998[Abstract].

2.   Aoudjit, F, Potworowski EF, and St.-Pierre Y. Bi-directional induction of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 during T lymphoma/endothelial cell contact: implication of ICAM-1. J Immunol 160: 2967-2973, 1998[Abstract/Free Full Text].

3.   Calmels, TP, Mattot V, Wernert N, Vandenbunder B, and Stehelin D. Invasive tumors induce c-ets1 transcription factor expression in adjacent stroma. Biol Cell 84: 53-61, 1995[ISI][Medline].

4.   Cao, M, Stefanovic-Racic M, Georgescu HI, Miller LA, and Evans CH. Generation of nitric oxide by lapine meniscal cells and its effect on matrix metabolism: stimulation of collagen production by arginine. J Orthop Res 16: 104-111, 1998[ISI][Medline].

5.   Clancy, RM, Rediske J, Tang X, Nijher N, Frenkel S, Philips M, and Abramson SB. Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/Rho/focal adhesion kinase signaling complex. J Clin Invest 100: 1789-1796, 1997[Abstract/Free Full Text].

6.   Culp, LA, Lin WC, and Kleinman NR. Tagged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis. Histol Histopathol 14: 897-886, 1999.

7.   Dong, Z, Qi X, Xie K, and Fidler IJ. Protein tyrosine kinase inhibitors decrease induction of nitric oxide synthase activity in lipopolysaccharide-responsive and lipopolysaccharide-nonresponsive murine macrophages. J Immunol 151: 2717-2724, 1993[Abstract/Free Full Text].

8.   Ellis, SM, Nabeshima K, and Biswas C. Monoclonal antibody preparation of a tumor cell collagenase-stimulatory factor. Cancer Res 49: 3385-3391, 1989[Abstract].

9.   Folkman, J. Clinical applications of research on angiogenesis. N Engl J Med 333: 1757-1763, 1995[Free Full Text].

10.   Fontanini, G, Lucchi M, Mussi A, Calcinai A, Boldrini L, Chiné S, Silvestri V, Angeletti CA, Basolo F, and Bevilacqua G. Neoangiogenesis and p53 protein in lung cancer: prognostic role and their relation with vascular endothelial growth factor (VEGF) expression. Br J Cancer 75: 1295-1301, 1997[ISI][Medline].

11.   Fujimoto, H, Ando Y, Yamashita T, Terazaki H, Tanaka Y, Sasaki J, Matsumoto M, Suga M, and Ando M. Nitric oxide synthase activity in human lung cancer. Jpn J Cancer Res 88: 1190-1198, 1997[ISI][Medline].

12.   Fukumura, D, and Jain RK. Role of nitric oxide in angiogenesis and microcirculation in tumors. Cancer Metastasis Rev 17: 77-89, 1998[ISI][Medline].

13.   Gallo, O, Masini E, Morbidelli L, Franchi A, Fini-Storchi I, Vergari WA, and Ziche M. Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer. J Natl Cancer Inst 90: 567-596, 1998[Free Full Text].

14.   Garcia-Cardena, G, and Folkman J. Is there a role for nitric oxide in tumor angiogenesis? J Natl Cancer Inst 90: 12-13, 1998[Free Full Text].

15.   Garrido, T, Riese HH, Aracil M, and Perez-Aranda A. Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay. Br J Cancer 71: 770-775, 1995[ISI][Medline].

16.   Giatromanolaki, A, Koukourakis M, O'Byrne K, Fox S, Whitehouse R, Talbot DC, Harris AL, and Gatter KC. Prognostic value of angiogenesis in operable non-small cell lung cancer. J Pathol 179: 80-88, 1996[ISI][Medline].

17.   Goligorsky, MS, Abedi H, Noiri E, Takhtajan A, Lense S, Romanov V, and Zachary I. Nitric oxide modulation of focal adhesions in endothelial cells. Am J Physiol Cell Physiol 276: C1271-C1281, 1999[Abstract/Free Full Text].

18.   Goto, F, Goto K, Weindel K, and Folkman J. Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Invest 69: 508-517, 1993[ISI][Medline].

19.   Hanahan, D, and Folkman J. Mechanisms of angiogenic switching. Cell 86: 353-364, 1996[ISI][Medline].

20.   Harpole, DH, Richards WG, Herndon JE, II, and Sugarbaker DJ. Angiogenesis and molecular biologic substaging in patients with stage I non-small cell lung cancer. Ann Thorac Surg 61: 1470-1476, 1996[Abstract/Free Full Text].

21.   Heppner, KJ, Matrisian LM, Jensen RA, and Rodgers WH. Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response. Am J Pathol 149: 273-282, 1996[Abstract].

22.   Ingber, DE. Extracellular matrix as a solid-state regulator in angiogenesis: identification of new targets for anti-cancer therapy. Cancer 3: 57-63, 1992.

23.   Kaur, K, Yao J, Pan X, Matthews C, and Hassid A. NO decreases phosphorylation of focal adhesion proteins via reduction of Ca in rat aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 274: H1613-H1619, 1998[Abstract/Free Full Text].

24.   Lala, PK. Significance of nitric oxide in carcinogenesis, tumor progression and cancer therapy. Cancer Metastasis Rev 17: 1-6, 1998[ISI][Medline].

25.   Lala, PK, and Orucevic A. Role of nitric oxide in tumor progression: lessons from experimental tumors. Cancer Metastasis Rev 17: 91-106, 1998[ISI][Medline].

26.   Laterra, J, Guerin C, and Goldstein GW. Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro. J Cell Physiol 144: 204-215, 1990[ISI][Medline].

27.   Liu, CY, Wang CH, Chen TC, Lin HC, Yu CT, and Kuo HP. Increased level of exhaled nitric oxide and up-regulation of inducible nitric oxide synthase in patients with primary lung disease. Br J Cancer 78: 534-541, 1998[ISI][Medline].

28.   Maeda, H, Okamoto T, and Akaike T. Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals. Biol Chem 379: 193-200, 1998[ISI][Medline].

29.   Mignatti, P, Morimoto T, and Rifkin DB. Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-golgi complex. J Cell Physiol 151: 81-93, 1992[ISI][Medline].

30.   Montesano, R, Pepper MS, and Orci L. Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts. J Cell Sci 105: 1013-1024, 1993[Abstract/Free Full Text].

31.   Montrucchio, G, Lupia E, De Martino A, Battaglia E, Arese M, Tizzani A, Bussolino F, and Camussi G. Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha . Am J Pathol 151: 557-563, 1997[Abstract].

32.   Owens, MW, Milligan SA, Jourd'heuil D, and Grisham MB. Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity. Am J Physiol Lung Cell Mol Physiol 273: L445-L450, 1997[Abstract/Free Full Text].

33.   Papapetropoulos, A, Garcie-Cardena G, Madri JA, and Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 100: 3131-3139, 1997[Abstract/Free Full Text].

34.   Parenti, A, Morbidelli L, Cui X-L, Douglas JG, Hood JD, Granger HJ, Ledda F, and Ziche M. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase 1/2 activation in postcapillary endothelium. J Biol Chem 273: 4220-4226, 1998[Abstract/Free Full Text].

35.   Partridge, CA, Phillips PG, Neidbala MJ, and Jeffrey JJ. Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts. Am J Physiol Lung Cell Mol Physiol 272: L813-L822, 1997[Abstract/Free Full Text].

36.   Paweletz, N, and Knierim M. Tumor related angiogenesis. Crit Rev Oncol Hematol 9: 197-242, 1989[ISI][Medline].

37.   Pelletier, JP, Jovanovic D, Fernandes JC, Manning P, Connor JR, Currie MG, DiBattista JA, and Martel-Pelletier J. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum 41: 1275-1286, 1998[ISI][Medline].

38.   Pepper, MS, Ferrara N, Orci L, and Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 189: 824-831, 1992[ISI][Medline].

39.   Phillips, PG, Birnby LM, and Narendran A. Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel endothelial cells. Am J Physiol Lung Cell Mol Physiol 268: L789-L800, 1995[Abstract/Free Full Text].

40.   Phillips, PG, and Tsan M-F. Direct staining and visualization of endothelial monolayers cultured on synthetic polycarbonate filters. J Histochem Cytochem 36: 551-554, 1988[Abstract].

41.   Piotrowicz, RS, Maher PA, and Levin EG. Dual activities of 22-24 kDA basic fibroblast growth factor: inhibition of migration and stimulation of proliferation. J Cell Physiol 178: 144-153, 1999[ISI][Medline].

42.   Pipili-Synetos, E, Papageorgiou A, Sakkoula E, Sotiropoulou G, Fotsis T, Karakiulakis G, and Maragoudakis ME. Inhibition of angiogenesis, tumour growth and metastasis by the NO-releasing vasodilators, isosorbide mononitrate and dinitrate. Br J Pharmacol 116: 1829-1834, 1995[Abstract].

43.   Rak, JW, Hegemann EJ, and Kerbel RS. The role of angiogenesis in tumor progression and metastasis. Adv Mol Cell Biol 7: 205-251, 1993.

44.   Sasaki, K, Hattori T, Fujisawa T, Takahashi K, Inoue H, and Takigawa M. Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes. J Biochem (Tokyo) 123: 431-439, 1998[Abstract].

45.   Sumitani, K, Kajima R, and Nagumo M. Cytotoxic effect of sodium nitroprusside on cancer cells: involvement of apoptosis and suppression of c-myc and c-myb proto-oncogene expression. Anticancer Res 17: 865-871, 1997[ISI][Medline].

46.   Thomsen, LL, and Miles DW. Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev 17: 107-118, 1998[ISI][Medline].

47.   Tsujii, M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93: 705-716, 1988.

48.   Vartanian, RK, and Weidner N. Endothelial cell proliferation in prostatic carcinoma and prostatic hyperplasia: correlation with Gleason's score, microvessel density, and epithelial cell proliferation. Lab Invest 73: 844-850, 1995[ISI][Medline].

49.   Werb, Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91: 439-442, 1997[ISI][Medline].

50.   Wink, DA, Vodovotz Y, Cook JA, Krishna MC, Kim S, Coffin D, DeGraff W, Deluca AM, Liebmann J, and Mitchell JB. The role of nitric oxide chemistry in cancer treatment. Biochemistry (Mosc) 63: 802-809, 1998[ISI][Medline].

51.   Wink, DA, Vodovotz Y, Laval J, Laval F, Dewhirst MW, and Mitchell JB. The multifaceted roles of nitric oxide in cancer. Carcinogenesis 19: 711-721, 1998[Abstract].

52.   Xie, K, and Fidler IJ. Therapy of cancer metastasis by activation of the inducible nitric oxide synthase. Cancer Metastasis Rev 17: 55-75, 1998[ISI][Medline].

53.   Xie, K, Huang S, Dong Z, Juang SH, Gutman M, Xie Q, Nathan C, and Fidler IJ. Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J Exp Med 181: 1333-1343, 1995[Abstract].

54.   Yamamoto, T, Terada N, Nishisawa Y, Tanaka H, Akedo H, Seiyama HA, Shiga T, and Kosaka H. Effects of NG-nitro-L-arginine and or L-arginine on experimental pulmonary metastasis in mice. Cancer Lett 87: 115-120, 1995[ISI].

55.   Yuan, A, Yang P, Yu C, Lee Y, Yao Y, Chen C, Lee L, Kuo S, and Luh K. Tumor angiogenesis correlates with histologic type and metastasis in non-small-cell lung cancer. Am J Respir Crit Care Med 152: 2157-2162, 1995[Abstract].

56.   Zhang, J, Li YD, Patel JM, and Block ER. Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells. Am J Physiol Lung Cell Mol Physiol 275: L288-L293, 1998[Abstract/Free Full Text].

57.   Ziche, M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, and Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 94: 2036-2044, 1994[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 281(1):L278-L290