Transforming-Growth-Factor-f31-Stimulated - Europe PMC

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Guillermo Garcia-Cardenia,* Joseph A. Madri,§ and William ..... 1838 Papapetropoulos et al. AJPMay 1997, Vol. 150, No. 5 were purchased from Life Technologies, Inc. (Grand. Island, NY). .... NOS Inhibitors Attenuate TGF-X31-Stimulated Capillary Organization 1839 ..... Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang.
American Journal of Pathology, Vol. 150, No. 5, May 1997 Copyright © American Society for Investigative Pathology

Nitric Oxide Synthase Inhibitors Attenuate Transform ing-G rowth-Factor-f31 -Stimulated Capillary Organization in Vitro

Andreas Papapetropoulos,* Kaushik M. Desai,* R. Daniel Rudic,* Bernd Mayer,t Rong Zhang,* Maria P. Ruiz-Torres,t Guillermo Garcia-Cardenia,* Joseph A. Madri,§ and William C. Sessa* From the Department ofPharmacology and Molecular Cardiobiology Division,* Boyer Center for Molecular Medicine, and Departments of Pathology and Biology,5 Yale University School of Medicine, New Haven, Connecticut; Institut fur Pharmakologie and Toxikologie,t Karl-FranzensUniversitat Graz, Graz, Austria; and Department of

Physiology and Pharmacology,t University de Alcala de Henares, Madnid, Spain

Angiogenesis is a complex process involving endothelial ceUl (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as weU as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capiUary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) coUagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-,81. Addition of the NO synthase (NOS) inhibitors Lnitro-arginine methylester (L-NAME, I mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor- 43 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of aU three

NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for aU NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capiUary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capiUary tube organization of RFCs. (Am JPathol 1997, 150:1835-1844)

Angiogenesis, the process of new capillary vessel formation from preexisting vessels, is a highly regulated, multistep process observed during a limited number of physiological processes, including vasculogenesis, wound healing, and the reproductive cycle.1'2 On the other hand, many pathological conditions, such as solid tumor growth and metastasis, chronic inflammatory diseases, diabetic retinopathy, and atherosclerosis, are associated with an increase in neovascularization.34 Endothelial cells (ECs), the major cell type constituting the neovessel, are under the influence of many endogenous factors, some of which stimulate whereas others inhibit angiogenesis.1 These proangiogenic and This work was supported by National Institutes of Health grants R29-HL51948 and RO1-HL57665 and American Heart Association (Connecticut Affiliate) grant (W. C. Sessa), National Institutes of Health grants RO-1 HL28373 and PO-1 DK38979 and American Heart Association grant 92006500 (J. A. Madri), Wellcome International Prize traveling research fellowship 038282/Z93 by the Welcome Trust (K. M. Desai), and Patrick and Catherine Weldon Donaghue Medical Research Foundation grant DF96-133 (A. Papapetropoulos). W. C. Sessa is an established investigator of the American Heart Association. Accepted for publication February 5, 1997. Address reprint requests to Dr. William C. Sessa, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812.

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antiangiogenic factors are produced (basally or in response to cytokine stimulation) by the ECs, vascular smooth muscle, fibroblasts, and blood-borne cells. Because angiogenesis is a complex in vivo phenomenon, simpler in vitro models have been developed to study discrete aspects of the angiogenic process and to investigate the role of endogenously produced factors in angiogenesis."8 A well-characterized in vitro model for the study of the later phases of angiogenesis (EC organization and differentiation into capillary networks) is the culturing of ECs into three-dimensional (3D) collagen gels.7'9'10 Placement of microvascular ECs (RFCs) into 3D collagen gels results in a phenotypic switch in EC behavior from a proliferative to a highly organized, differentiated state.7'11 ECs seeded into such gels exhibit almost no proliferation and organize into patent capillary tubes.7'10 Nitric oxide (NO), a short-lived free radical gas derived from the amino acid L-arginine, has been implicated in a variety of paracrine functions. NO released by ECs can elicit relaxation of vascular smooth muscle, inhibition of platelet aggregation, and inhibition of leukocyte adherence to the endothelium.12-14 In an autocrine manner, NO can influence the barrier function of the endothelium and influence the release of endothelin-1 and plateletderived growth factor15'16. Recently, NO was shown to play a role in various phenomena that contribute to the angiogenic process. NO produced by NO donors or through the inducible NO synthase (iNOS) activates the 72-kd gelatinase in mesangial cells.17 Similar results were also reported in articular cartilage, in which NO stimulates the activity of collagenase and stromelysin.18 The mitogenic action of vascular endothelial growth factor in postcapillary venular ECs is mediated by NO.19 Substance P, an agent that stimulates NO release through elevations of intracellular calcium, promotes EC migration in an NO-dependent manner.20 Endogenously produced NO is also required for the growth arrest and differentiation of PC-12 cells following nerve growth factor treatment.21 Thus, NO has the propensity to influence extracellular matrix degradation, cellular proliferation, migration, and differentiation, all requisite events for angiogenesis to proceed. Recently, there were conflicting reports about the role of NO in in vivo models of angiogenesis. NOS inhibitors attenuate [Sar9]substance P- sulfone and prostaglandin-El- but not basic fibroblast-growth-factor-induced angiogenesis in a rabbit corneal model.20 In contrast, NOS inhibitors promote neovessel formation, whereas NO donors attenuated this process in the chick chorioallantoic membrane model.22'23 Thus, the present study was undertaken to investigate the role of

NO in transforming growth factor-081 capillary organization in vitro.

(TGF-f31)-driven

Materials and Methods Culture of RFCs RFCs were isolated from rat epidydymal fat pads, as previously described.24 RFCs were characterized by indirect immunofluorescent staining for CD31, von Willebrand factor, and uptake of acetylated low-density lipoprotein. RFCs were maintained in culture in gelatincoated flasks and grown in medium consisting of four parts Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and one part bovine aortic EC (BAEC)-conditioned medium. RFCs were split once a week at a 1:3 ratio and fed twice weekly. Cells up to passage 8 were used in this study. Cells were cultured in either two-dimensional (2D) or 3D collagen gels. For the 2D cultures, cells were seeded on type collagen (30 mg/ml), a concentration previously shown to saturate the surface of the tissue culture flask.25 For 3D cultures, bovine skin acid-soluble type collagen was prepared as previously described26 and dissolved at 40C in 10 mmol/L acetic acid to a concentration of 2.5 mg/ml. To establish the 3D cultures, the collagen was then mixed with 1 OX Earle's balanced salt solution and neutralized with 1 N NaOH. RFCs were added immediately to achieve a final concentration of 0.8 x 106 cells/ml collagen. Ten drops (0.1 ml each) of the cell-collagen mixture were added to a 100-mm plate. Plates were then placed in a humidified incubator (370C), and the cell-collagen mixtures were allowed to gel for 10 minutes, after which 20 ml of growth medium was added to each plate. RFCs were allowed to form capillary-like tubes over a 5-day period. TGF-31 (0.5 ng/ml) and test substances (nitro-L-arginine methyl ester (L-NAME), 1 mmol/L; NG-monomethyl-L-arginine (L-NMMA), 1 mmol/L; sodium nitroprusside, 100 ixmol/L; 8-bromo-cGMP, 1 mmol/L; and methylene blue 500 ,umol/L) were added on the day of seeding (day 0) and on day 3 when fresh media were added to the cultures. To evaluate tube formation in 3D cultures morphologically, cells were washed three times with phosphate-buffered saline and snap frozen in O.C.T. embedding compound. Cryostat sections of the gels (6 ,um) were then placed on poly-L-lysine-coated glass slides, fixed with acetone for 10 minutes at -20°C, and air dried. The sections were examined directly by phase-contrast microscopy, and the total vessel area (in two or three fields per slide in each experiment) was quantified using the National Institutes of Health (Bethesda, MD) Image program.

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Nitrite Determination Nitrite, a stable breakdown product of NO, was measured in the supernatants of cells cultured in 3D gels. Aliquots (150 ml of media) were removed after 1, 2, 3, 4, or 5 days, and nitrite concentration was determined by the Griess reaction. Media were combined with an equal volume of the Griess reagent (1% sulfanilamide and 0.1% napthylethylenediamide in 5% phosphoric acid), and the optical density was measured at 550 nm using a Molecular Dynamics (Sunnyvale, CA) microplate reader. Nitrite concentration was calculated by comparison with the optical density measured at 550 nm of standard solutions of sodium nitrite prepared in cultured medium.

Transcriptase-Polymerase Chain Reaction (PCR) of NOS Isoforms Reverse

RNA was isolated using a commercially available kit (Trizol). Collagen gels were completely dissolved in the RNA isolation reagent by incubating for 30 to 45 minutes at room temperature with gentle agitation. RNA (0.5 ,ug/reaction) was reverse transcribed with 20 U of Moloney murine leukemia virus reverse transcriptase in 50 mmol/L Tris-HCI (pH 8.3), 3 mmol/L MgCI2, 75 mmol/L KCI, 1 mmol/L deoxynucleotide triphosphates, 20 U of ribonuclease inhibitor, and 200 ng of oligodeoxythymidylic acid primers to prime cDNA synthesis, in a reaction volume of 20 ,ul, for 50 minutes at 42°C. Samples were then heated for 15 minutes at 700C to destroy reverse transcriptase activity before the PCR reaction. Rat-specific primers for all three isoforms were designed based on previously published sequences.27 29 Primer sequences (5' to 3', sense and antisense, respectively) were CTGGCGGCGGAAGAGAAAGGAGTC and GGGGCTGGGTGGGGAGGTGATGTC for endothelial NOS (eNOS; expected PCR product, 739 bp), CCCTTCCGAAGTTTCTGGCAGCAG and GGGCTCCTCCAAGGTGTTGCCC for iNOS (expected PCR product, 474 bp), and GAATACCAGCCTGATCCATGGAA and TCCTCCAGGAGGGTGTCCACCGCATG for neuronal NOS (nNOS; expected PCR product, 602 bp). cDNA was amplified for 35 cycles at 940C for 1 minute, 55°C for 1 minute, and 720C for 1 minute (melting, annealing, and extension temperatures, respectively); 2.5 U of Taq polymerase, 0.2 ,umol/L primer, and 2 mmol/L MgCI2 were used per reaction. To test for primer specificity, isoform-specific primers were used with rat nNOS, partial rat eNOS, and murine iNOS cDNA. Murine and rat iNOS are 92% identical at the nucleotide level.30 All primers were specific under the conditions tested. The plasmids

containing the rat nNOS, murine iNOS, and partial rat eNOS cDNAs were kindly provided by Drs. D. Bredt, (University of California, San Francisco, CA), S. Snyder, (Johns Hopkins University, Baltimore, MD), J. Cunningham, (Harvard Medical School, Boston, MA), and B. Kone (University of Florida, Gainesville, FL). After amplification, PCR reaction mixtures (10 ,ul) were electrophoresed on 1% agarose/Tris-acetate-EDTA gels containing ethidium bromide, and PCR products were visualized on an ultraviolet transilluminator and photographed. A molecular-weightstandard DNA ladder was used to confirm the predicted PCR product size.

Western Blotting of NOS Isoforms Three-dimensional cultures were washed three times with phosphate-buffered saline, and proteins were extracted with a buffer containing Tris-HCI (50 mmol/L, pH 7.5), NaCI (0.15 mol/L), EGTA (0.1 mmol/ L), EDTA (0.1 mmol/L), Nonidet P-40 (1%, v/v), ,B-mercaptoethanol (0.1%, v/v), and protease inhibitors (10 ,ug/ml leupeptin, 10 ,g/ml pepstatin A, 10 ,ug/ml aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride). The type collagen pellets are not soluble under these conditions, whereas capillary ECs are lysed, and proteins are solubilized. To enrich the amount of NOS proteins in the lysate, samples used for detection of eNOS or nNOS were partially purified with 2',5'-ADP-Sepharose and electrophoresed.31 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 7.5% polyacrylamide gels, and the proteins were then transferred to a nitrocellulose membrane at 100 V for 2 hours at 4°C in a buffer containing 25 mmol/L Tris and 200 mmol/L glycine. After the transfer, membranes were incubated overnight at 4°C with 5% dry milk in buffer containing 0.1% Tween 20 in Tris-buffered saline to block nonspecific binding. The following day, membranes were incubated with isoform-specific polyclonal anti-NOS antisera in 5% milk in Tween 20 and Tris-buffered saline for 2 hours at room temperature, washed three times with Tween 20 and Tris-buffered saline for 5 minutes each time, and finally incubated for 1 hour with a horseradish-peroxidase-conjugated anti-rabbit IgG. Immunoreactive protein bands were visualized using the enhanced chemiluminescence system (Amersham Corp., Arlington Heights, IL).

Materials Tissue culture plastic ware was from Becton Dickinson (Franklin Lakes, NJ). Growth medium, fetal calf serum, human recombinant TGF-31, the Trizol reagent and SUPERSCRIPT 11 reverse transcriptase

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were purchased from Life Technologies, Inc. (Grand Island, NY). Recombinant RNasin ribonuclease inhibitor was obtained from Promega (Madison, WI). The enhanced chemiluminescence detection system and the horseradish-peroxidase-conjugated antirabbit antibody were obtained from Amersham Corp. (Arlington Heights, IL). O.C.T. embedding medium was purchased from Miles, Inc. (Elkhart, IN). Nitrocellulose membranes were purchased from Gelman Sciences (Ann Arbor, MI). X-ray film was obtained from Eastman Kodak Co. (Rochester, NY), and all other chemicals, including penicillin, streptomycin, bovine serum albumin, Nonidet P-40, phenylmethylsulfonyl fluoride, aprotinin, EDTA, and others, were purchased from Sigma Chemical Co. (St. Louis, MO). 2',5'-ADP-Sepharose was from Pharmacia Biotech (Uppsala, Sweden). The anti-iNOS polyclonal antibody was obtained from Affinity Bioreagents (Golden, CO). The anti-nNOS polyclonal antibody was made and used as previously described.32 The anti-eNOS polyclonal antibody was made in our laboratory against the amino-terminal fragment of bovine eNOS (amino acids 131 to 586) fused to glutathione S-transferase. Sera from antigen-challenged rabbits were isolated and affinity purified using the eNOS-glutathione S-transferase fusion protein coupled to Affi-Gel resin (Pierce, Rockford, IL). eNOS antiserum did not cross-react with the other forms of NOS, as determined by Western blot analysis of lysates of HEK 293 cells expressing rat nNOS. The iNOS and nNOS antisera were also tested with HEK 293 cell lysates and were found to be isoform specific at the dilutions used.

Data Analysis and Statistics Data are presented as means + SEM of the indicated numbers of individual observations. Statistical comparisons between groups were performed using the one-way analysis of variance or Student's t-test, as appropriate. Differences among means were considered significant when P < 0.05.

Results Effects of Endogenous NO on EC Proliferation in 2D Culture To examine whether endogenous NO influenced the growth patterns of RFCs in 2D tissue culture, a 5-day proliferation assay was performed in the absence and presence of the NOS inhibitor L-NAME (1 mmol/L) and TGF-f31 (0.5 ng/ml). RFCs proliferated

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Figure 1. NOS inhibition does niot influence RFCgrotwth in 2D clulture. RFCs uere seeded at 1.5 X 103 cellslcm2 in the presence of senum and allowed to proliferate for 1, 3, or 5 days. The cell number was determined after incubation? with or without TGF-,31 (0.5 ng/mb) in the absence orpresence of L- or D-NAME ( 1 mmol/L) at the indicated times using a Coulter Electronics (Hialeab, FL) counter. Results arepresented as averages + SEM; n = 3 in duiplicates.

over the course of 5 days at comparable rates in the absence or presence of the L-NAME or the inactive D-isomer (1 mmol/L; Figure 1). Consistent with previously published results,10 addition of TGF-13, (0.5 ng/ml) to the culture medium led to a decrease in the rate of RFC proliferation (1.07 x 105 versus 0.55 x 105 cells on day 3 and 3.5 x 105 and 1.3 x 105 cells on day 5 for control and TGF-,31, respectively). Coincubation of the cells with L- or D-NAME had no effect on the ability of TGF-f31 to inhibit RFC growth, indicating that the antiproliferative effects of TGF-13, are not mediated by NO.

Effects of Endogenous NO on EC Organization and Differentiation in 3D Cultures Culturing of RFCs in 3D collagen gels in the presence of TGF-f31 has been previously shown to promote the organization of ECs into differentiated, capillary-tube-like structures, a process reminiscent of the late stages of angiogenesis.33 As shown in Figure 2A, complex, branching capillary-tube-like structures form in a random fashion and anastomose, causing the gels to contract. This dynamic process of in vitro angiogenesis was inhibited by both L-NAME and L-NMMA (Figure 2A). Addition of excess L- but not D-arginine (data not shown) into the culture medium partially reversed the effects of the

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Figure 2. A: Culture o?f FC's in 3D collagen gels induces angiogenesis in a NO-dependenit mannier. RFCs were grown in 3D cultures (0.8 16 0`ml of collagen) for 5 days in the presetnce of TGF-,3 (0 5 uig/ml). Phase-contrast photounicrographs oj control RFCs in 3D culture, RFCs treated u'ith L-NAME ( 1 mIolIL), RFCs treated with .-NMMA (1 nimoll L), and RFCs treated with L-NAME anzd L-arginine (1 mmol L) are sho'n. Data arefrom a representative expeninient. Similar results were obtained in at least five experiments. Magnification, X 400. B: Morphomietnic quatntification of the inhibitory effects ofNOS blockade onl in vitro anigiogeniesis. Results presented are froni multiple fields (tuo orthree per slide; magnification, 20) in three different experiments. X

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NOS inhibitors. Morphometric quantification of vessel length per square millimeter by image analysis (Figure 2B) revealed that L-NAME and L-NMMA significantly inhibited tube formation by 52.9 and 70.3%, respectively. Similar results were obtained using the inhibitor of the NO-cGMP pathway, methylene blue (500 ,tmol/L; data not shown). In addition, in the face of NOS blockade, supplementation of either NO in the form of the NO donor sodium nitro-

RFCs cultured in type collagen gels produced NO in a time-dependent manner, as reflected by the accumulation of the stable degradation product nitrite in the culture medium. Nitrite levels increased over the course of 5 days, reaching 148.2 + 5.2 ,umol/L per 1 million cells in control 3D cultures (Figure 3). Addition of L-NAME (1 mmol/L) to the culture medium significantly attenuated nitrite accumulation at all time points tested by approximately 40%. This level of inhibition is consistent with previously reported effects of L-NAME in vitro, in which the concentration of L-arginine in tissue culture media was 400 ,mol/L.34 To investigate the expression of NOS isoforms in RFCs, specific primers were designed for each isoform. Primer specificity was tested using plasmids containing the rat NOS isoforms as positive controls. Following the isolation of total RNA, mRNA was reverse transcribed using oligodeoxythymidylic acid primers, and cDNA was amplified for 35 cycles. As shown in Figure 4, RFCs expressed all three isoforms of NOS when placed in 2D and 3D culture for 5 days,

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