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1 Departments of Cancer Biology and 2 Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1ß and tumor necrosis factor- caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1ß down-regulation of Ang-1. Furthermore, treatment of cells with IL-1ß indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1ß regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1ß would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1ß decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1ß on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1ß produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1ß indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1ß may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor that is frequently overexpressed in a variety of human cancers (8) . VEGF expression in tumor cells can be up-regulated by specific cytokines such as interleukin-1ß (IL-1ß), which has been implicated in the malignant progression and angiogenesis of numerous malignancies (9 , 10) . VEGF and the Angs seem to play complementary and coordinated roles in the development of new blood vessels (3) . IL-1ß is a potent immunoregulatory and proinflammatory cytokine secreted by a variety of activated immune cells that can infiltrate solid and tumors (11) ; it has been shown to be a proangiogenic factor in solid tumors (12) . In the present study, we examined the effects of IL-1ß on the regulation of Ang-1 and VEGF in ECs. In addition, we investigated the effect of IL-1ß on Ang-1 expression in human vascular smooth muscle cells (hVSMCs) as a surrogate for pericytes. IL-1ß decreased Ang-1 expression in ECs, and this decrease in expression was mediated via the P38 mitogen-activated protein kinase (MAPK) pathway. In addition, IL-1ß down-regulated Ang-1 in hVSMCs, an important observation considering the role the pericytes play in regulating EC function (13) . Similar to results observed in previous studies of colon cancer cells and hVSMCs (9 , 14) , IL-1ß led to an increase in VEGF expression. The fact that IL-1ß can up-regulate VEGF and down-regulate Ang-1 suggests that IL-1ß may be an important indirect regulator of tumor angiogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Isolation and Northern Blot Analysis.
For Northern blot analyses, total RNA was extracted from cells by TRIzol (Life Technologies, Inc.) according to the manufacturer’s instructions. Northern blot analysis was performed as described previously (6)
. Briefly, probes for Ang-1 (a 535-bp fragment of Ang-1 was a gift from Tona Gilmer, GlaxoSmithKline Inc., Research Triangle Park, NC), VEGF (a 204-bp cDNA probe was a gift from Dr. Brygida Berse, Harvard Medical School, Boston, MA), or for glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Collection) were radiolabeled by the random primer technique with a commercially available kit (Rediprime II; Amersham Biosciences, Piscataway, NJ), and nylon membranes (Hybond-N+; Amersham Biosciences) were subsequently hybridized overnight at 65°C (Rapid-hyb Solution; Amersham Biosciences). Autoradiography was performed thereafter in the linear range of the film (Hyperfilm MP; Amersham Biosciences). For in vitro experiments investigating cytokine-dependent Ang-1 mRNA expression, cells were treated with insulin-like growth factor-1 (100 ng/ml), platelet-derived growth factor-BB (10 ng/ml), IL-1ß (10 ng/ml), tumor necrosis factor- (TNF-; 10 ng/ml), or VEGF (10 ng/ml; all cytokines were purchased from R&D Systems Inc., Minneapolis, MN). Studies to identify relevant signaling pathways for IL-1ß-regulated Ang-1 expression in HUVECs were performed with specific inhibitors to extracellular signal-regulated kinase 1/2 (Erk-1/2; 50 mM UO126; New England Biolabs Inc., Beverly, MA), P38 MAPK (10 µM SB203580; Calbiochem, San Diego, CA), or phosphatidylinositol 3'-kinase/Akt (200 nM Wortmannin; Sigma, St. Louis, MO). HUVECs were treated with individual inhibitors for 1 h in 1% FBS-MEM before the addition of IL-1ß (10 ng/ml). Doses for each signaling inhibitor were previously tested for their toxicity to HUVECs to ensure cell viability throughout the experiments.
Western Blot Analysis.
Total and phosphorylated protein levels were determined by Western blot analyses as described previously (14)
. Briefly, protein was extracted from cell lysates by use of RIPA buffer, and 40-µg protein samples were subjected to Western blot analysis on denaturing 6% or 10% SDS-PAGE gels (15)
. Activated signaling pathways were identified by use of the following antibodies (all from Cell Signaling Technology, Beverly, MA): anti-Erk-1/2, anti-phosphospecific Erk-1/2 [Phospho-p44/42 MAPK (Thr202/Tyr204)], anti-Akt, anti-phosphospecific Akt (Phospho-Akt Ser 473), anti-phosphospecific c-jun amino-terminal kinase/stress-activated protein kinase [Phospho-SAPK/JNK (Thr183/Try 185)], anti-P38, and anti-phosphospecific P38 [Phospho-p38 MAPK (Thr180/Tyr 182)].
Quantification of VEGF Protein in Conditioned Medium (CM) from HUVECs Treated with IL-1ß.
CM from HUVECs was prepared as follows. Cells were grown to 80–90% confluence and were incubated for 48 h in 1% FBS with IL-1ß (10 ng/ml; "activated" CM) or without IL-1ß (control CM). The CM was collected and centrifuged, followed by filtration through Amicon Centriprep filters (PM10) according to the manufacturer’s protocol (Millipore, Bedford, MA). The CM was quantitated spectrophotometrically by the BCA (Pierce) assay. VEGF ELISA was performed according to the manufacturer’s protocol (R&D Systems).
Immunoprecipitation of Ang-1 or Phosphorylated Tie-2.
HUVECs were incubated for 24 or 48 h in the presence or absence of IL-1ß (10 ng/ml) in 1% FBS-MEM. Protein was extracted as described above. Aliquots (500 µg) of protein samples were immunoprecipitated with goat antihuman Ang-1 antibody or rabbit antihuman Tie-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) by overnight rotation at 4°C in RIPA buffer containing agarose beads (A/G Plus Agarose; Santa Cruz Biotechnology). The beads were washed three times with cold RIPA buffer and resolved on denaturing 10% (for Ang-1) or 6% (for Tie-2) SDS-PAGE gels as described above. Western blot membranes were probed with mouse antihuman Ang-1 antibody (R&D Systems) or mouse antiphosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). For Tie-2, equal loading was verified by reprobing the membrane with Tie-2 antibody. For immunoprecipitation studies, a nonspecific IgG antibody was used to confirm the absence of nonspecific binding.
Gelfoam in Vivo Angiogenesis Assay.
Effects of IL-1ß on angiogenesis were investigated in a Gelfoam in vivo angiogenesis assay using male BALB/c mice. Sterile absorbable sponges (Pharmacia, Peapack, NJ) were cut into 5 x 5 x 7-mm pieces and hydrated overnight at 4°C in sterile PBS. Excess PBS was then drained by blotting on sterile filter paper. The sponges were then soaked in 0.4% agarose (100 µl) containing either PBS (control) or IL-1ß (1 µg/ml). The agarose-Gelfoam plugs were then allowed to harden for 1 h at room temperature before being implanted s.c. into BALB/c mice (five mice/group). Mice were anesthetized with Nembutal (50 mg/kg i.p.), and the plugs were implanted s.c. via a midline incision of the abdominal skin; one plug was placed s.c. at least 2 cm away from the incision site on either side. The wound was closed with surgical metal clips. Fourteen days later, mice were sacrificed. The Gelfoam plugs were removed and washed once in PBS and then frozen in OCT compound. Specimens were snap-frozen in liquid nitrogen for subsequent immunohistochemical analysis.
Immunohistochemical Analyses of Vessel Density.
Rat antimouse CD31/PECAM-1 antibody was obtained from PharMingen (San Diego, CA) and peroxidase-conjugated goat antirat IgG from Jackson Research Laboratories (West Grove, PA). Gelfoam plugs that had been frozen in OCT were sectioned in 20-µm slices, mounted on positively charged slides, and air-dried for 30 min. Tissue sections were then fixed in cold acetone followed by 1:1 acetone–chloroform and acetone, each for 5 min, and then washed with PBS. Specimens were then incubated with 3% H2O2 in methanol for 12 min at room temperature to block endogenous peroxidase, washed three times with PBS (pH 7.5), and incubated for 20 min at room temperature in a protein-blocking solution consisting of PBS supplemented with 1% normal goat serum and 5% normal horse serum. The primary antibody directed against CD31 was diluted 1:800 in protein-blocking solution and applied to the sections, which were then incubated overnight at 4°C. Sections were then rinsed in PBS and incubated for 10 min in protein-blocking solution before the addition of peroxidase-conjugated secondary antibody. The secondary antibody used for CD31 (peroxidase-conjugated goat antirat IgG) staining was diluted 1:200 in protein-blocking solution. After incubation with the secondary antibody for 1 h at room temperature, the samples were washed and incubated with stable diaminobenzidine (Research Genetics, Huntsville, AL) substrate. Staining was monitored under a bright-field microscope, and the reaction was stopped by washing with distilled water. Sections were mounted with Universal Mount (Research Genetics). CD31-stained vessels were counted (at x100 magnification) in five different quadrants of each Gelfoam plug (2 mm inside the edge), and averages were calculated. For all immunohistochemical studies, the primary antibody was omitted as a negative control.
Analysis of Immunostained Tissue Sections.
The images were captured and analyzed by Optimas image analysis software (version 5.2; Bothell, WA). Positive cells were counted by NIH Image Analysis software (version 1.62) from the NIH (Bethesda, MD). The number of positive cells was expressed as the average of the number of positive staining cells per high-power field at x100. Five fields from one 20-µm-thick section/specimen were chosen randomly, and five mice/group underwent analysis.
Statistical Analyses.
All statistical analyses were done with InStat Statistical Software (version 2.03; GraphPad Software, San Diego, CA); with P < 0.05 considered statistically significant.
Densitometric Quantification.
Densitometric analysis of autoradiographs was performed with NIH Image Analysis software (version 1.62) from the NIH to quantify the results of Northern and Western blot analyses.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, or VEGF for 4 and 24 h. Northern blot analysis revealed that treatment with IL-1ß or TNF- led to marked decreases in Ang-1 mRNA expression at 4 h, with further decreases evident at 24 h. At 24 h, Ang-1 mRNA expression was decreased 70% by IL-1ß and 90% by TNF- (Fig. 1A)
. Similarly, Ang-1 protein expression was decreased at 24 h, as determined by immunoprecipitation (Fig. 1B)
. To confirm these observations in a different cell system, we treated hVSMCs with or without IL-1ß. Similar to our findings in HUVECs, Northern blot analysis demonstrated that IL-1ß decreased Ang-1 expression by 
50% at 4 h (Fig. 1C)
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3-fold increase in VEGF mRNA expression at 4 h and an 5-fold increase in VEGF mRNA expression at 24 h as determined by Northern blot (Fig. 2A)
. Similar to VEGF mRNA levels, we found that IL-1ß produced an 4-fold increase in VEGF protein expression at 48 h as determined by ELISA (Fig. 2B)
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70% compared with controls) at 24 h (Fig. 3A)
. This time point was selected for use in subsequent studies. We also investigated the effects of IL-1ß concentration on Ang-1 regulation by treating HUVECs with increasing doses of IL-1ß (0.01–10 ng/ml). Decreases in Ang-1 mRNA levels were detectable after IL-1ß treatment starting at a dose of 0.01 ng/ml, resulting in a 40% decrease in Ang-1 mRNA expression compared with control cells (Fig. 3B)
. Increasing the IL-1ß concentration to 10 ng/ml produced an additional down-regulation of Ang-1 expression (70%). Repeat studies verified the above results.
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. No changes were detected in JNK phosphorylation (data not shown). To identify the relevant signaling pathways involved in the IL-ß-mediated down-regulation of Ang-1 mRNA, we incubated HUVECs with inhibitors to phosphatidylinositol 3'-kinase/Akt (Wortmannin), Erk 1/2 (UO126), or P38 (SB203580), followed by a 24-h incubation with IL-1ß. Northern blot analysis revealed that inhibition of P38 activation blunted the IL-1ß-mediated down-regulation of Ang-1 mRNA (Fig. 4B)
. In contrast, the MAPK and phosphatidylinositol 3'-kinase inhibitors had minimal effects.
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. Repeat studies verified the above results.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decreased Ang-1 mRNA expression in human ECs and that the effects of IL-1ß were selectively mediated via the P38 MAPK signaling pathway. Considering the role of IL-1ß as a proangiogenic cytokine (9
, 16
, 17)
, we focused on elucidating its effects on the endothelial expression of Ang-1, which regulates EC survival and exerts antiangiogenic properties in vivo (2
, 6 , 18
, 19)
. Although it has previously been shown that IL-1ß and TNF- decrease Ang-1 expression in HUVECs (20)
, our study has expanded these observations to demonstrate the mechanism by which this occurs. We have also demonstrated that the reduction of Ang-1 in ECs leads to a decrease in the autocrine activation of Tie-2. IL-1ß has been implicated in colon cancer angiogenesis because of its ability to up-regulate VEGF expression in HT29 colon cancer cells in vitro (9) . We previously demonstrated that exposure of HT29 cells to IL-1ß caused a >5-fold increase in VEGF mRNA expression at 24 h that was associated with increases in VEGF promoter activity and protein expression (9) . Furthermore, IL-1ß may enhance tumor angiogenesis by promoting the induction of VEGF by other cell types within the tumor microenvironment. Small tumor vessels are composed of ECs and pericytes (or derivatives of VSMCs) that communicate with each other via cytokine signaling during the angiogenic process. We recently investigated the effects IL-1ß on VSMCs, showing that IL-1ß increased VEGF expression in VSMCs (14) . Data from this previous study suggested that IL-1ß may mediate EC survival and angiogenesis by induction of VEGF in a paracrine manner (14) . This regulation of VEGF expression in VSMCs was mediated by the P38 MAPK signaling pathway (14) . In the present study, we were able to demonstrate that the proangiogenic molecule IL-1ß can also down-regulate endogenous inhibitors of angiogenesis, such as Ang-1, in ECs and that this mechanism was mediated via the P38 MAPK pathway. Several cytokines have been shown to activate the P38 signaling pathway in ECs, and in general, P38 activation has been associated with decreased EC survival (21) . However, down-regulation of Ang-1, an important EC survival factor, by IL-1ß may play an additional role in the mechanism for P38-mediated decrease in cell survival.
Ang-1 is a secreted growth factor that activates the Tie-2 receptor tyrosine kinase, which enhances EC survival and capillary morphogenesis and also limits capillary permeability. Ang-1 is expressed by ECs, smooth muscle cells, pericytes, and their precursors and exerts paracrine effects on Tie-2-expressing ECs (1 , 4 , 5) . In the present study, we found that IL-1ß-mediated down-regulation of Ang-1 led to a significant reduction in Tie-2 phosphorylation in ECs. In contrast to the findings of others, where Tie-2 protein levels were slightly increased after cytokine treatment (22) , we did not observe any changes in total Tie-2 protein levels.
Another cytokine associated with angiogenesis and tumor growth is TNF-, which may also induce the expression of VEGF and other proangiogenic factors in cells (23)
. In the present study, treatment of HUVECs with TNF-, like IL-1ß, resulted in a marked reduction in endogenous Ang-1 mRNA expression. We chose to investigate IL-1ß rather than TNF- because IL-1ß inhibitors are already in clinical use for arthritis; the antiangiogenic effect of these agents may, in part, explain their efficacy (24)
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Regulation of cellular Ang-1 expression has important implications in angiogenesis given that Ang-1 exerts antiangiogenic activity in vivo and stabilizes ECs in vitro (4 , 6 , 7) . We and others have shown that stable overexpression of Ang-1 in various tumor systems inhibits tumor angiogenesis, tumor growth, and vascular permeability (6 , 7 , 18 , 19) .
In summary, our results demonstrate that IL-ß can down-regulate the expression of Ang-1 and that this occurs concomitant to an increase in VEGF expression in HUVECs. This mechanism results in a net gain of proangiogenic stimuli that overall may promote and potentiate neovascularization in tumors. Inhibition of IL-1ß-mediated effects may therefore be a valuable approach for antiangiogenic regimens to decrease VEGF expression in multiple cell types within tumors and maintain Ang-1 levels in ECs and pericytes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Lee M. Ellis, Department of Surgical Oncology, Box 444, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: (713) 792-6926, Fax: (713) 792-4689; E-mail: lellis{at}mdanderson.org
Received 2/24/03. Revised 1/15/04. Accepted 2/ 9/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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