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10.1245/ASO.2006.03.038
Annals of Surgical Oncology 13:125-133 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Interferon {gamma}–Inducible Protein 10 Selectively Inhibits Proliferation and Induces Apoptosis in Endothelial Cells

Elizabeth D. Feldman, MD1, David M. Weinreich, MD1, Nancy M. Carroll, MD1, Monika L. Burness, MD1, Andrew L. Feldman, MD2, Ewa Turner, BS1, Hui Xu, BS1 and H. Richard Alexander, Jr., MD1

1 Surgical Metabolism Section, Surgery Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 2B07, Bethesda, Maryland 20892-1502
2 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Building 10, Room 2N06, Bethesda, Maryland 20892-1502

Correspondence: Address correspondence and reprint requests to: H. Richard Alexander, Jr., MD; E-mail: richard_alexander{at}nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Interferon {gamma} –inducible protein 10 (IP-10) has antitumor effects in various murine models. The IP-10 receptor has two distinct splice variants, CXCR3A and CXCR3B, that have paradoxical effects after ligand-receptor interaction.

Methods: To characterize the putative antiangiogenic effects of IP-10, we measured proliferation rates and apoptosis in human umbilical vein endothelial cells (HUVECs), fibroblasts, and A375 melanoma or WIDR adenocarcinoma cell lines after exposure to the recombinant protein. CXCR3A (activating) and CXCR3B (inhibitory/proapoptotic) messenger RNA (mRNA) expression levels in fibroblasts, 2 human tumor cell lines, T lymphocytes, and HUVECs of varying cell densities were characterized.

Results: IP-10 resulted in dose-dependent and selective inhibition of proliferation and countered the proliferative effects of vascular endothelial growth factor in HUVECs but did not affect fibroblasts or 2 human tumor cell lines. In addition, IP-10 resulted in potent and selective induction of apoptosis in HUVECS but had no effect on fibroblasts or A375 melanoma. Confluent HUVECs had a predominance of mRNA for the CXCR3B splice variant by reverse transcriptase-polymerase chain reaction, and the ratio of CXCR3B to CXCR3A mRNA was >40 in HUVECs, compared with ≤ 10 in the other cell types. Moreover, CXCR3B mRNA levels were significantly higher in proliferating compared with confluent HUVECs. In vivo, systemic IP-10 administration resulted in slower A375 xenograft growth rates compared with control-treated animals, and immunohistochemical staining showed decreased microvessel density in xenografts of IP-10–treated mice.

Conclusions: IP-10 has antiangiogenic properties and selective effects on endothelial tissue that may be secondary to higher levels of the CXCR3B inhibitory/proapoptotic receptor in that cell type, particularly in its actively proliferating state.

Key Words: Angiogenesis • Interferon {gamma}–inducible protein 10 • CXCR3 • A375 melanoma • Endothelium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis is defined as the growth of neovasculature from preexisting vessels and is an essential biological event encountered in several physiological and pathological processes, including the growth of malignant solid-organ tumors.1 Tumor angiogenesis is characterized by an imbalance that favors overexpression of angiogenic factors and underexpression of angiostatic substances, thus resulting in endothelial cell migration, proliferation, and organization into functional luminal structures.2,3

Investigations of tumor angiogenesis have primarily focused on the role of angiogenic factors, such as vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basic fibroblast growth factor.4–7 More recently, studies have focused on the increasingly important role of endogenous angiostatic factors that may regulate the balance of neovascularization.8 One such putative endogenous angiostatic molecule is the CXC chemokine interferon {gamma} –inducible protein 10 (IP-10).9 IP-10 is a 10-kDa secreted protein produced by activated monocytes, fibroblasts, endothelial cells, and keratinocytes in response to stimulation with interferon {gamma}.10,11 It has in vivo antitumor effects in various experimental models1,12–14; these effects have been proposed to be mediated via a T cell–dependent mechanism.15 However, Feldman et al.12 demonstrated significant growth inhibition of A375 human melanoma cells transduced with the human IP-10 gene in a subcutaneous tumor model in nude athymic mice which was associated with a marked reduction in tumor microvessel density.

The IP-10 receptor, CXCR3, has 2 distinct splice variants, CXCR3A and CXCR3B, which have paradoxical effects after ligand-receptor interaction.16 Lasagni et al.16 transfected human microvascular endothelial cell lines (HMVECs) with either variant and noted that overexpression of CXCR3A induced an increase in cell survival, whereas overexpression of CXCR3B dramatically reduced DNA synthesis and upregulated apoptotic cell death. Therefore, CXCR3A may be considered a mediator of cellular activation and endothelial cell angiogenesis, whereas CXCR3B acts as a cellular inhibitor and a promoter of apoptosis.

These experiments were performed to characterize the putative antiangiogenic effects of IP-10. We propose, on the basis of our studies with recombinant IP-10, that tumor growth inhibition by IP-10 is mediated via selective effects of IP-10 on endothelial tissue and that the specificity of these effects may be due to the predominance of the CXCR3B receptor on activated or proliferating endothelium present in the tumor microenvironment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Human umbilical vein endothelial cells (HUVECs; Clonetics, San Diego, CA) were maintained at 37° C in a 5% carbon dioxide incubator in basal endothelial cell media (EGM-2; Clonetics) enriched with fetal bovine serum, hydrocortisone, human recombinant basic fibroblast growth factor, insulin-like growth factor 1, ascorbic acid, epidermal growth factor, GA-1000 (gentamicin/amphotericin), and heparin (Clonetics). They were passaged for 2 generations for the experiments described below. Human fibroblasts (donated by Dr. J. Wunderlich, Surgery Branch, National Cancer Institute) were maintained at 37° C in a 5% carbon dioxide incubator in RPMI complete media (Life Technologies, Carlsbad, CA) enriched with fetal bovine serum (Gemini Bio-products, Woodland, CA), L-glutamine, and penicillin-streptomycin (Life Technologies). A375 human melanoma cells (American Type Culture Collection, Frederick, MD) were maintained at 37° C in a 5% carbon dioxide incubator in Dulbecco’s modified Eagle medium media (Dulbecco’s modified Eagle medium enriched with 10% non–heat-inactivated fetal bovine serum; American Type Culture Collection). WIDR, a human colon cancer cell line, was maintained at 37° C in a 5% carbon dioxide incubator in RPMI complete media.

Proliferation Assays
Various cell types were plated at a density of 1000 cells per well in 96-well plates treated with nonpyrogenic polystyrene (Corning Incorporated, Corning, NY) and incubated at 37° C in complete medium for 24 hours. Cell types included HUVECs, human fibroblasts, A375 melanoma, and WIDR adenocarcinoma. After 24 hours, each well was aspirated, and cells were treated with the appropriate complete media alone or IP-10 1, 5, or 10 µg/mL in complete media. Proliferation was analyzed at 30 minutes and 24, 48, and 72 hours by tetrazolium salt assay (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Six samples in each group were tested. The absorbance of the samples was measured against a background control, basal medium, by using a Multiskan MCC/340 plate reader (Titertek, Huntsville, AL) at 450 nm with a reference wavelength of 650 nm. The degree of proliferation was expressed as the absorbance at 450 nm.

Inhibition of VEGF Stimulation With Exposure to IP-10
HUVECs were plated at a density of 1000 cells per well in 96-well plates (Corning Incorporated) and incubated at 37° C in complete medium for 24 hours. The medium was then aspirated, and new complete media were added that contained no additional additives, IP-10 10 µg/mL, VEGF .1 µg/mL (R&D Systems, Minneapolis, MN), or IP-10 10 µg/mL plus VEGF .1 µg/mL, all in complete media. Proliferation was analyzed at 30 minutes and 24, 48, and 72 hours by tetrazolium salt assay (Boehringer Mannheim) according to the manufacturer’s instructions. Six samples in each group were tested. The absorbance of the samples was measured against a background control, basal medium, by using a Multiskan MCC/340 plate reader (Titertek) at 450 nm with a reference wavelength of 650 nm. The degree of proliferation was expressed as the absorbance at 450 nm.

Deoxyuride-5'-Triphosphate Biotin Nick End Labeling Assays for Apoptosis: HUVECs
A375 human melanoma cells and fibroblasts were plated in six-well plates treated with nonpyrogenic polystyrene (Corning Incorporated) at a density of 200,000 cells per well at 37° C in complete medium for 24 hours. The medium was aspirated, and fresh media were applied that contained IP-1001 or 10 µg/mL in complete media. Cells were trypsinized at 24 or 48 hours, washed in phosphate-buffered saline (PBS), fixed for 1 hour with 1% paraformaldehyde, permeabilized with 70% ethanol at –20° C for several days, and subjected to analysis for the presence of apoptosis by using the APO-BRDU kit (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instructions via flow cytometry within 2 hours of staining. The assay was run on a single laser flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, CA) at 488 nm and analyzed with the CellQuest program (Becton Dickinson Immunocytometry Systems). The gate for detection of apoptosis was set with reference to the positive and negative controls provided by the manufacturer.

Reverse Transcriptase-Polymerase Chain Reaction for CXCR3 Messenger RNA Expression
HUVECs were plated at known cell densities from 50,000 to 800,000 cells per well in 6-well plates (Corning Incorporated) in complete media for 48 hours at 37° C in 5% carbon dioxide. Total RNA was harvested from HUVECs, human fibroblasts, A375, WIDR, and IL-2–stimulated T lymphocytes (provided by Dr. R. Morgan, Surgery Branch, National Cancer Institute) by using the Qiagen RNAeasy mini kit (Qiagen, Hilden, Germany), and 1 µg of total RNA was transcribed to complementary DNA by using Superscript II reverse transcriptase (Life Technologies).

ß-Actin was used as a housekeeping gene to standardize quantification of CXCR3A and CXCR3B copy number and to account for varying amounts of initial complementary DNA in the reaction. All primers and probes were obtained from Biosource International (Camarillo, CA). Polymerase chain reaction amplification of ß-actin was performed with the primers 5'-GCGAGAAGATGAC CCAGATC-3'(forward primer) and 5'-CCAGTGGTACGGCCA GAGG-3' (reverse primer) by using the Platinum Taq DNA polymerase kit (Life Technologies). The quantity and quality of the amplified products were determined by spectrophotometry and gel electrophoresis, and then progressive 10-fold dilutions were conducted to generate the standards for use in an ABI Prism 7700 Sequence Detector (Applied Biosciences). The probe sequence was 5'-TCAAGATCA TTGCTCCTCCTGAGCGC-3'.

Standards were generated for both CXCR3A and CXCR3B by using the same conditions as described previously. The primers and probe sequences for CXCR3A were 5'-CAGGTGCCCTCTTC AACATCA-3' (forward primer), 5'-ATGTTCAGGTAGCGG TCAAAGC-3' (reverse primer), and 5'-CCCTCCTG CTGGCCT GCATCA-3' (probe). The primers and probe sequences for CXCR3B were 5'-TGCCAGGC CTTTACACAGC-3' (forward primer), 5'-TCGGCG TCATTTAGCACTTG-3' (reverse primer), and 5'-CCCGTTCCCGCCCTCACAGG-3' (probe). Gene copy numbers of CXCR3A and CXCR3B were assessed in each cell line and expressed as a ratio to 1 x 105 copies of ß-actin.

In Vivo Studies
Animal experiments were conducted according to protocols approved by the National Institutes of Health Animal Care and Use Committee. Twenty 7-to 8-week-old female nude athymic mice (Taconic, Germantown, NY, and Frederick Cancer Research Center, Frederick, MD) were inoculated with 2 x 106 A375 human melanoma cells in 100 µL of PBS subcutaneously in the right flank. Subjects were then divided into 2 groups and randomized to receive peritumoral subcutaneous injections with either recombinant IP-10 10 µg/100 µL (R&D Systems) or carrier, .1% bovine serum albumin. IP-10 was received in lyophilized form and was reconstituted by using .1% bovine serum albumin. The peritumoral injections were initiated on the same day as tumor inoculation. Tumors were measured in two dimensions by using calipers at regular intervals by a blinded observer (M.B.), and tumor areas were calculated as the product of the greatest perpendicular diameters.

Immunohistochemistry
Tumors from animals treated with IP-10 or carrier were harvested immediately after mice were sacrificed after cessation of the subcutaneous injections, as described previously. Tumors were snap-frozen in liquid nitrogen and stored at –80° C. Frozen sections (7 µm) were cut on a cryostat and stained with hematoxylin and eosin or antibodies specific for CD31 (Pharmingen, San Diego, CA). For immunostaining, sections were fixed in acetone for 10 minutes. After endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes, sections were incubated for 1 hour in a blocking solution that contained 10% normal goat serum. Sections were incubated with primary antibody at 4° C overnight at a 1/50 dilution. Slides were then washed three times in PBS, incubated in biotinylated species-specific appropriate secondary antibody for 1 hour, and exposed to avidinbiotin-peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). Sections were reacted with .06% 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and counterstained with hematoxylin.

Hematoxylin and eosin–stained and immunostained sections were analyzed by a pathologist (A.L. Feldman) who was blinded to the identity of the groups. Only good-quality sections with uniform, well-demarcated staining and low background were analyzed. Microvascular density of two tumors from each group was assessed by using a scoring system based on the number of CD31-positive cells per high-power field (x400).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proliferation
We first evaluated the effect of IP-10 on the proliferation rates of HUVECs, fibroblasts, and two human tumor cell lines, A375 and WIDR. IP-10 had a dose-dependent selective inhibition of the proliferation of HUVECs that was evident 24 hours after exposure to the protein (Fig. 1AGo). Application of IP-10 decreased the HUVEC proliferation rate by 30% and 47% at doses of 5 and 10 µg/mL, respectively, at 72 hours. However, treatment with IP-10 1, 5, or 10 µg/mL did not affect the proliferation rates of fibroblasts, A375 human melanoma, or WIDR human colon carcinoma at any given time point (Fig. 1B–DGo). Exposure to VEGF .1 µg/mL resulted in augmented HUVEC proliferation as compared with untreated control HU-VECs (Fig. 2Go). IP-10 inhibited the proliferative effects of VEGF in HUVECs such that proliferation was equivalent to that in untreated HUVECs.


Figure 1
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  		FIG. 1. Results of proliferation assays in various cell lines with and without exposure to interferon {gamma} –inducible protein 10 (IP-10). (A) IP-10 dose-dependent inhibition of human umbilical vein endothelial cell (HUVEC) proliferation curves normalized to complete media (black circle, control; down-pointing triangle, IP-10 5 µg/mL; upward-pointing triangle, IP-10 10 µg/mL). (B) Lack of effect of IP-10 on fibroblast proliferation normalized to RPMI (black circle, control; down-pointing triangle, IP-10 5 µg/mL; upward-pointing triangle, IP-10 10 µg/ mL). (C) Lack of effect of IP-10 on WIDR proliferation rate normalized to RPMI (black circle, control; down-pointing triangle, IP-10 1 µg/mL; upward-pointing triangle, IP-10 10 µg/mL). (D) Lack of effect of IP-10 on the A375 proliferation rate normalized to Dulbecco’s modified Eagle medium (black circle, control; down-pointing triangle, IP-10 5 µg/mL; upward-pointing triangle, IP-10 10 µg/mL). OD, optical density.

 

Figure 2
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  		FIG. 2. Results of proliferation assay for human umbilical vein endothelial cells (HUVECs) exposed to vascular endothelial growth factor (VEGF) with or without interferon {gamma} –inducible protein 10 (IP-10) at 72 hours. Untreated cells represented 100% proliferation. Cells exposed to IP-10 10 µg/mL had proliferation decreased to 82% of control, whereas those treated with VEGF .1 µg/mL had proliferation rates of 117% of control. Cells treated with the combination of VEGF and IP-10 at the given doses had proliferation rates similar to those of untreated cells. There was a statistically significant difference in the percentage control proliferation in comparing HUVECs treated with VEGF alone and those treated with IP-10 and VEGF (*P < .05; Student’s t-test).

 
Deoxyuride-5'-Triphosphate Biotin Nick End Labeling Assays for Apoptosis
We next examined the effect of IP-10 on apoptosis in HUVECs, fibroblasts, and A375 cells at 24 and 48 hours as compared with cells treated with complete media alone. IP-10 caused apoptosis in HUVECs selectively but did not affect the percentage of apoptotic cells in treated fibroblasts or A375 human melanoma cells (Fig. 3Go). The percentage of cells that were gated for apoptotic activity in HUVECs increased from 2.8% to 92.6% and 96.7% with treatment with IP-10 1 and 10 µg/mL, respectively, for 24 hours. In contrast, the percentage of apoptotic A375 cells that were gated increased minimally from 2.5% to 2.6% and 7.5% with 24 hours of treatment with IP-10 1 and 10 µg/mL, respectively. Exposure of fibroblasts to IP-10 at either dose did not increase apoptosis at 24 hours over the baseline level of 2% to 3%. Similar data were observed for all cell types at 48 hours (results not shown).


Figure 3
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  		FIG. 3. Deoxyuride-5'-triphosphate biotin nick end labeling assays for apoptosis in various cell lines with and without exposure to interferon {gamma} –inducible protein 10 (IP-10). Gating determined by positive and negative controls as described in Materials and Methods and cells above the gating threshold were considered apoptotic. The first column represents fluorescence-activated cell-sorting (FACS) analysis of apoptosis in HUVECs with 24 hours of exposure to media alone (top row), IP-10 1 µg/mL (middle row), and IP-10 10 µg/mL (bottom row). The second column demonstrates FACS analysis of apoptosis in A375 cells with 24 hours of exposure to media alone (top row), IP-10 1 µg/mL (middle row), and IP-10 10 µg/mL (bottom row). The third column shows FACS analysis of apoptosis in fibroblasts with 24 hours of exposure to media alone (top row), IP-10 1 µg/mL (middle row), and IP-10 10 µg/mL (bottom row). The results for 48 hours are similar and are not shown.

 
Quantitative Reverse Transcriptase-Polymerase Chain Reaction for CXCR3A/B
To investigate the selective antiproliferative and proapoptotic effects of IP-10 on HUVECs, CXCR3 messenger RNA (mRNA) expression was evaluated by using quantitative reverse transcriptase-polymerase chain reaction. CXCR3A (activating) and CXCR3B (inhibitory) receptor expression varied for different cell types (Table 1Go). As predicted, T lymphocytes stimulated with IL-2 had a preponderance of the high-affinity CXCR3A receptor, consistent with the literature that IP-10 is a potent chemoattractant for T cells.15 Confluent HUVECs (plated at 800,000 cells per well for 48 hours), A375 cells, WIDR cells, and fibroblasts expressed mRNA for both variants of CXCR3. The ratio of CXCR3B to CXCR3A mRNA expression was highest in HU-VECs, at 41.5, as compared with fibroblasts and WIDR and A375 cell lines, which were all <10. Receptor mRNA expression of either variant in fibroblasts was generally low. Both tumor cell lines had greater expression of CXCR3A mRNA as compared with either HUVECs or fibroblasts.


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  		TABLE 1. Gene copy number standardized to 1 x 105 copies of ß-actin for IP-10 receptors, CXCR3A (activating) and CXCR3B (inhibitory), in various cell types
 
We then investigated whether the degree of confluence altered levels of CXCR3B mRNA expression. HUVECs were plated at progressively increasing cell densities, incubated for 48 hours, and then observed with a light microscope. Those plated at 50,000 cells per well displayed sparse confluence, 200,000 to 400,000 cells per well had sparse to intermediate confluence, and 600,000 to 800,000 cells per well demonstrated 80% to 100% confluence at 48 hours. It is interesting to note that reverse transcriptase-polymerase chain reaction analysis revealed that actively proliferating HUVECs had the highest levels of CXCR3B expression when compared with confluent, nonproliferating cells (P < .05, Student’s t-test; Fig. 4Go). CXCR3A expression was minimal at all levels of endothelial cell confluence.


Figure 4
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  		FIG. 4. Quantitative reverse transcriptase-polymerase chain reaction for CXCR3 variants in human umbilical vein endothelial cells plated at increasing cell densities. Gene copy number is normalized to 1 x 105 copies of ß-actin. There was a statistically significant decrease in CXCR3B messenger RNA expression between the initial cell inocula of 50,000 to 200,000 cells per well as compared with 800,000 cells per well (*P < .05; Student’s t-test). CXCR3A did not vary with cell density (solid bar, CXCR3A; open bar, CXCR3B).

 
In Vivo Studies and Immunohistochemistry
Subcutaneously implanted A375 tumors were measured at regular intervals, and their areas were calculated by using the product of the greatest perpendicular diameters once the tumors became palpable after cell inoculation. IP-10 or carrier (n = 10/group) was injected once daily for 5 days and then every other day beginning at the time of A375 cell inoculation. All tumor measurements were conducted before IP-10 or carrier injection so as not to interfere with the accuracy of measurement. None of the mice in either group displayed any toxicity from the protein or its administration. Subcutaneously implanted flank tumors had significantly slower growth rates in nude athymic mice treated with IP-10 compared with control (P = .005; analysis of variance; Fig. 5AGo).


Figure 5
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  		FIG. 5. (A) In vivo tumor growth curves of interferon {gamma}-inducible protein 10 (IP-10)--treated and control mice. The tumor area of mice injected with IP-10 or carrier was assessed. Each point represents the mean volume of 10 mice. There was a significantly larger tumor area in control mice as compared with IP-10-treated mice (P = .005; analysis of variance). (B) Histopathologic findings in subcutaneous tumors are shown. (I) Microvessel staining from a representative section of a tumor of a control mouse (anti-CD31 immunostaining; original magnification, x 200). (II) Microvessel staining from a representative section of a tumor of a mouse treated with IP-10 (anti-CD31 immunostaining; original magnification, x 200).

 
Tumors of mice treated with IP-10 had a lower level of microvessel density as compared with tumors of control mice (Fig. 5BGo). CD31 staining demonstrated larger vessels and individual endothelial cells. The scores for the representative sections of the 2 tumors of treated mice were 4 and 2.8 microvessels per high-power field, compared with 2 control mice that had scores of 10.6 and 11.7.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The CXC chemokines include both angiogenic and angiostatic compounds based on distinct amino acid motifs. IP-10 was first described as an {alpha} chemokine induced by interferon {gamma} in U937 lymphoma cells.17 It was subsequently demonstrated to be a powerful chemoattractant for monocytes and T lymphocytes,15 and its antitumor properties were attributed to this mechanism.14 In athymic mice, IP-10 has been shown to inhibit angiogenesis;12,13 however, the mechanism of angiostatic activity has not been defined.

The receptor for IP-10 is CXCR3, which was recently demonstrated to have two splice variants: CXCR3A and CXCR3B.16 The classic CXCR3 receptor, renamed CXCR3A, mediated the proliferation of human mesangial cells in response to CXCL9, CXCL10 (IP-10), and CXCL11 and was responsible for both the increased survival and the angiogenic properties of HMVECs transfected with CXCR3A.16 In contrast, the same study demonstrated that HMVECs transfected with and overexpressing CXCR3B died within fewer passages than those that did not overexpress the receptor, had lower proliferation rates, and had higher levels of apoptosis. In addition, primary cultures of human mesangial cells expressed only CXCR3A, whereas those of HMVECs had only CXCR3B present.16

We demonstrated that IP-10 seems to have selective inhibitory effects on the proliferation of HUVECs and does not affect the proliferation of fibroblasts, A375, or WIDR cells in vitro. In addition, IP-10 counteracts the augmented proliferative effects of VEGF on HUVECs. Similarly, it selectively induces apoptosis in HUVECs without changing the levels of apoptosis in IP-10–exposed fibroblasts or A375 cells. It is interesting to note that HUVECs have a predominance of CXCR3B, the inhibitory receptor, and this may explain the selective effects of IP-10 on proliferation and apoptosis in endothelial cells. In contrast, both tumor cells lines exhibit CXCR3A mRNA, the activating variant, as well as CXCR3B mRNA expression, with a notably lower ratio of CXCR3B to CXCR3A. A375 and WIDR cell lines do not respond to treatment with IP-10 with regard to proliferation or apoptosis. The balance that is conferred by the ratio of both variants and the increased affinity of CXCR3A over CXCR3B may explain such observations. IP-10 induces chemotaxis of T lymphocytes, and the predominance of CXCR3A expression is a plausible explanation for this effect. In contrast, IP-10 does not affect the proliferation or apoptosis of fibroblasts; this may be attributed to the generally low levels of CXCR3A and CXCR3B expression in fibroblasts.

It is particularly worth noting that CXCR3B mRNA expression is inversely related to the degree of cell confluence in HUVECs. As cells approach confluence, they become quiescent and decrease expression of the inhibitory and angiostatic variant of the receptor, CXCR3B. Tumor angiogenesis involves the proliferation of endothelial cells; thus, subconfluent endothelial cell mRNA expression may be more representative of the response of activated tumor neovasculature to treatment with IP-10 in vivo.

The inhibition of tumor growth of A375 melanoma subcutaneous xenografts in nude athymic mice supports the role of non–T cell–dependent antitumor properties. This is evident in both our systemic model of administration of the recombinant protein and that of Feldman et al.,12 in which IP-10–transduced cell lines were used. The more pronounced effects on tumor growth inhibition with the transduced cell lines may in fact be due to the difference in pharmacokinetics between systemic administration and local production, as well as to the unknown quantities of the IP-10 produced by the altered cell lines in vivo. Immunostaining for CD31 again reinforces the antiangiogenic properties of IP-10: decreased microvessel density and dispersion of vessel architecture are exhibited in tumors of mice treated with IP-10.

In conclusion, IP-10 is an antiangiogenic protein that selectively affects endothelial cells with respect to apoptosis and proliferation. The addition of other antiangiogenics that have complementary mechanisms of action or chemotherapeutic agents may enhance its usefulness in the clinical setting. Further work is warranted to clarify the intermediaries responsible for the observed effects of endothelial cell treatment with IP-10.


   ACKNOWLEDGMENTS
 
The authors thank Romi Sawhney for her assistance with the preparation of this manuscript, Dr. Dominique Lorang for her invaluable assistance with the immunohistochemical staining, and Shawn Farid for his expertise in the use of fluorescence-activated cell-sorting analysis.

Received for publication March 4, 2005. Accepted for publication August 2, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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R. J. Bodnar, C. C. Yates, M. E. Rodgers, X. Du, and A. Wells
IP-10 induces dissociation of newly formed blood vessels
J. Cell Sci., June 15, 2009; 122(12): 2064 - 2077.
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M. Funk, K. Kriechbaum, F. Prager, T. Benesch, M. Georgopoulos, G. J. Zlabinger, and U. Schmidt-Erfurth
Intraocular Concentrations of Growth Factors and Cytokines in Retinal Vein Occlusion and the Effect of Therapy with Bevacizumab
Invest. Ophthalmol. Vis. Sci., March 1, 2009; 50(3): 1025 - 1032.
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