A Molecular Cascade Showing Nitric Oxide-Heme Oxygenase-1-Vascular Endothelial Growth Factor-Interleukin-8 Sequence in Human Endothelial Cel
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内分泌学杂志 2005年第5期
Department of Microbiology and Immunology (H.-O.P., G.-S.O., B.-M.C., H.-T.C.), Wonkwang University School of Medicine, Iksan-Shi, Chonbug 570-749, Republic of Korea; and Vascular System Research Center and Department of Molecular and Cellular Biochemistry (Y.-M.K.), Kangwon National University School of Medicine, Chunchon, Kangwon-Do 200-701, Republic of Korea
Address all correspondence and requests for reprints to: Hun-Taeg Chung, M.D., Ph.D., Department of Microbiology and Immunology, Wonkwang University School of Medicine, 344-2 Shinyoung-Dong, Iksan-Shi, Chonbug 570-749, Republic of Korea. E-mail: htchung@wonkwang.ac.kr.
Abstract
Heme oxygenase (HO)-1 has been shown to be an important biological target of nitric oxide (NO). NO can induce HO-1 expression and IL-8 production, particularly, in endothelial cells. Interestingly, HO-1 tends to induce the production of vascular endothelial growth factor (VEGF) that is involved in endothelial IL-8 syntheses. Whether HO-1 expression by NO may provide a link with IL-8 or VEGF synthesis was investigated in human umbilical vein endothelial cells (HUVECs). The NO donor S-nitroso-N-acetyl-penicillamine (SNAP) dose-dependently increased IL-8 and VEGF productions and HO-1 expression in HUVECs. Transfection with either HO-1 small interfering RNA or HO-1 antisense oligodeoxynucleotide abrogated the ability of SNAP to induce HO-1 expression and IL-8 and VEGF productions. Both pharmacological induction and gene transfer of HO-1 directly induced IL-8 and VEGF productions. Anti-VEGF neutralizing antibody blocked SNAP-mediated IL-8 production and VEGF itself induced IL-8 production, whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs. Neither anti-VEGF nor anti-IL-8 antibodies influenced SNAP-induced HO-1 expression. Moreover, neither VEGF nor IL-8 showed an additive effect on SNAP-induced HO-1 expression. HO-1 transfection had no significant effect on productions of other CXC chemokines, such as growth-related oncogen- and epithelial neutrophil activation peptide-78. Taken together, these results provide a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
Introduction
CHEMOKINES ARE CHARACTERIZED by their ability to induce chemotaxis in leukocytes. Secretion of chemokines is regarded as a pivotal step in cell recruitment necessary during initiation and maintenance of inflammatory responses (1). The CXC chemokine IL-8 is a regulator of leukocyte survival (2, 3) and is supposed to be a mediator of angiogenesis (4). IL-8 is produced from a variety of cells including monocytes and endothelial cells when the cells are exposed to oxidative stress, proinflammatory cytokines including TNF-, or some growth factors including vascular endothelial growth factor (VEGF) (5). In addition, the potential of nitric oxide (NO) to induce IL-8 synthesis has been demonstrated (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
NO plays a central role in a variety of physiological and pathological processes (21). NO synthases (NOSs), enzymes that produce NO, are composed of three isoforms, endothelial and neuronal enzymes and inducible NOS (22). NO produced by the actions of constitutively expressed NOS serves as a messenger/modulator agent vital for the functions of immune and other cells. On the other hand, induction of inducible NOS results in the production of NO at higher levels (23). Elevated NO is believed to contribute to tissue damage through its action to induce an inflammatory response. Recently, it has been evidenced that NO-producing cells possess regulatory pathways where protective mechanisms can operate to control proinflammatory responses via its induction of cytoprotective enzymes and thus limit the destructive potential (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Among the cytoprotective enzymes, inducible heme oxygenase (HO)-1 plays regulatory roles in the modulation of inflammatory responses (26, 27) and NO-induced cytotoxicity (32, 33).
HO-1 is a rate-limiting enzyme in heme catabolism, leading to the formation of biliverdin, which is reduced to bilirubin, carbon monoxide, and free iron. There is a large body of evidence suggesting that HO-1 is antiinflammatory and cytoprotective (34, 35, 36, 37, 38, 39), although the precise mechanisms by which HO-1 can be induced by specific stimuli remain unclear. Among the possible biological actions of HO-1 in the vessel wall is the regulation of VEGF synthesis (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), which has been proven to be an important growth factor critical for blood vessel formation. It has been demonstrated that HO-1 overexpression in human endothelial cells induced VEGF synthesis and consequently angiogenesis (40, 45, 48, 54).
VEGF has been shown to have at least five human isoforms (55, 56) that develop from alternate splicing of a single gene. Based on the affinity for binding to heparan sulfate, the isoforms have been shown to have soluble (VEGF121), cell-associated (VEGF189), or both (VEGF165) properties. There are a growing number of reports investigating the isoforms and their different roles in disease, and it is now clear that there are isoform-specific differences. VEGF165 has been associated with pathologic intravitreous neovascularization (57, 58) and has been found to be potently more proinflammatory than VEGF121 (56). VEGF165 has been detected in human proliferative diabetic retinopathy and was associated with a poor prognosis in certain tumors (56). The cell-associated isoform, VEGF189, is important in endothelial cell migration and adhesion, whereas the other isoforms were not (56). In endothelial cells, VEGF165 has been reported to induce HO-1 expression (46) and IL-8 production (57, 58).
Considering that NO can induce HO-1 expression (32, 33) as well as IL-8 synthesis (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), and HO-1 tends to induce VEGF synthesis (45, 54) associated with endothelial IL-8 production (57, 58), we have investigated whether HO-1 expression by NO may provide a link with IL-8 or VEGF syntheses in human endothelial cells. The results presented in this study provide, for the first time, a molecular cascade showing the NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
Materials and Methods
Reagents, cytokine, growth factor, antibodies, and HO-1 gene
Human recombinant IL-8, TNF-, VEGF121, VEGF165, and anti-IL-8 and anti-VEGF neutralizing antibodies were purchased from R&D Systems, Inc. (Minneapolis, MN). S-Nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) were obtained from Sigma-Aldrich Chemical (St. Louis, MO). Tin protoporphyrin (SnPP), cobalt protoporphyrin (CoPP), and hemin were from Porphyrin Products (Logan, UT). Human anti-HO-1 and anti--actin antibodies were from StressGen Biotech (Victoria, British Columbia, Canada). HO-1 cDNA was a kind gift from Dr. A.M.K. Choi (University of Pittsburgh, Pittsburgh, PA). HO-1 small interfering RNA (siRNA) and its transfection kit were from Santa Cruz Biotechnology (Santa Cruz, CA). Sense and antisense oligodeoxynucleotides (ODNs) were supplied by Invitrogen (San Diego, CA) and transfected with the Superfect transfection reagent (Qiagen, Hilden, Germany).
Cell culture
Human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMECs) were obtained from the American Type Culture Collection (Manassas, VA). These cell lines were maintained in culture as an adherent monolayer cell medium supplemented with 10% fetal bovine serum and endothelial cell growth supplement (EGM-2; Cambrex Biosciences, Walkersville, MD). HUVECs and HMECs were grown up to 80% confluence and then transferred to serum and EGM-2-free medium overnight before being used for transfection experiments.
HO-1 and HO-1 siRNA transfection
HO-1 was cloned into pcDNA3 (Invitrogen). HUVECs (5 x 106) or HMECs (5 x 106) were transfected with 10 μg constructs by electroporation at 270 V, 950 μF in serum-free RPMI 1640 using a Gene Pulser (Bio-Rad, Richmond, CA) followed by culture in EGM-2 supplemented with 10% fetal bovine serum for 48 h and plating on 96-well plates at 5 x 105 cells/well in the presence of 1.25 mg/ml G418. Single stable clones of transfectant were isolated and expanded.
Inhibition of HO-1 expression was assessed by Western blotting analysis after transfection of HUVECs with HO-1 siRNA. Briefly, HUVECs were grown in 24- or six-well plates and transiently transfected with 1.5 and 3 μg HO-1 siRNA (Santa Cruz Biotechnology) mixed with siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer’s protocol. After incubation at 37 C and 5% CO2 for 36 h, the cells were treated with NO donors. Samples were then prepared and analyzed for cytokines or immunoblotting.
HO-1 sense and antisense ODNs were directed against the flanking translation initiation codon in the human HO-1 cDNA (27). The antisense sequence was 5'-CGC CTT CAT GGT GCC-3', whereas the sense sequence was 5'-GGC ACC ATG AAG GCG-3'. ODNs were phosphorothioated on the first three bases on the 3' end. HUVECs were transfected using the Superfectr transfection reagent following the manufacturer’s instructions. Briefly, the cells were seeded in culture plates before transfection. The proportions used were 1 μg DNA/5 μl transfection reagent/well. HUVECs were incubated for 4 h with the ODNs.
Assays of chemokines and VEGF
Supernatants were analyzed for IL-8, growth-related oncogen (Gro)-, epithelial neutrophil activation peptide (ENA-78), monocyte chemotactic peptide (MCP)-1, and VEGF using ELISA obtained commercially (R&D Systems, Inc.). The assays were performed according to the manufacturer’s protocol. Chemokine and VEGF concentrations are expressed in nanograms per milliliter. A standard curve was prepared from a group of serially diluted standard samples of either chemokine or VEGF. Absorbance was read against a blank at 450 nm using a microtiter ELISA reader.
Cell extracts and Western blotting analysis
Cells were harvested via first rinsing in ice-cold PBS and then immediately resuspended in cell lysis buffer containing a complete protease inhibitor mixture (New England Biolabs, Beverly, MA). Cellular protein extracts were electrophoresed under denaturing conditions (10- 12.5% polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad). HO-1 expression was detected using anti-HO-1 monoclonal antibody. Actin expression was detected by using anti--actin monoclonal antibody. Primary antibodies were detected by using horseradish peroxidase-conjugated antirabbit or antimouse IgG secondary antibodies (Santa Cruz Biotechnology). Peroxidase was visualized using the enhanced chemiluminescence assay (New England Biolabs) according to the manufacturer’s instructions.
RT-PCR
Total RNA was isolated by the guanidine isothiocyanate method using Tri Reagent (Sigma-Aldrich Chemical). First strand cDNAs were synthesized from RNA using the ImProm-II RT system (Promega, Madison, WI). Then, PCR with Taq DNA polymerase (Promega) was performed for 27 cycles using the following protocol: 95 C for 40 sec, 58 C for 40 sec, and 72 C for 50 sec. PCR products were analyzed by electrophoresis in 2% agarose gels. PCRs were carried out using primers for VEGF121 and VEGF165 (5'-CAC CGC CTC GGC TTG TCA CAT and 5'-CTG CTG TCT TGG GTG CAT TGG), for IL-8 (5'-GGA CAA GAG CCA GGA AGA AAC C and 5'-CTT CAA CTT CTC CAC AAC CC), or for the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (5'-CGT ATT GGG CGC CTG GTC ACC and 5'-GGG ATG ATG TTC TGG AGA GCC C). The product length was 431 bp for the VEGF121, 563 bp for VEGF165, and 335 bp for IL-8 and glyceraldehyde-3-phosphate dehydrogenase.
Statistical analysis
Data in this study present the means ± SD where applicable. Statistical evaluation was determined with Student’s t test or with ANOVA followed by the Tukey test. P < 0.01 was taken to indicate statistical significance.
Results
Effects of the NO on IL-8 production and HO-1 expression
Previous studies have demonstrated that a variety of NO donors induce IL-8 production (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and HO-1 expression in monocytic, epithelial, and endothelial cells (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). In the current study, treatment of HUVECs with the NO donor SNAP at noncytotoxic doses induced IL-8 mRNA and protein in a dose-dependent manner as measured 24 h after exposure (Fig. 1A). Another NO donor GSNO also induced IL-8 mRNA and protein (Fig. 1A). To examine the role of the gaseous molecule NO in IL-8 production by SNAP treatment, the endothelial cells were incubated with the NO scavenger PTIO before SNAP treatment. PTIO almost completely reduced SNAP-induced IL-8 mRNA and protein levels (Fig. 1A). Both SNAP and GSNO also induced HO-1 expression and increased HO activity (Fig. 1B); these effects were paralleled by increased IL-8 mRNA and protein (Fig. 1A). Similar to its effects on SNAP-induced IL-8 mRNA and protein, PTIO abrogated the SNAP-induced increases in HO-1 expression and HO activity (Fig. 1B). SNAP, GSNO, or PTIO did not have significant effect on the viability of the endothelial cells (data not shown).
FIG. 1. Effects of NO donors on IL-8 production and HO-1 expression in HUVECs. A, Cells were incubated for 8 h (for IL-8 mRNA; upper panel) or 24 h (for IL-8 protein; lower panel) with SNAP at indicated concentrations or GSNO in the absence or presence of PTIO (500 μM). RT-PCR for IL-8 mRNA analysis and ELISA for the quantification of IL-8 protein were performed as described in Materials and Methods. NO donors significantly increased IL-8 mRNA levels and IL-8 production (* and #, P < 0.01), and PTIO significantly reduced IL-8 mRNA expression and IL-8 production by NO donors (** and ##, P < 0.01). B, Cells were incubated for 6 h with SNAP at indicated concentrations or GSNO in the absence or presence of PTIO (500 μM). Western blotting for HO-1 expression (upper panel) and bilirubin quantification for HO activity (lower panel) were performed as described in Materials and Methods. NO donors significantly increased HO-1 expression and HO activity (* and #, P < 0.01), and PTIO significantly reduced HO-1 expression and HO activity by NO donors (** and ##, P < 0.01). RT-PCR and Western blotting were performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of HO-1 siRNA and HO-1 antisense ODN on NO-induced IL-8 production and HO-1 expression
We transfected HUVECs with HO-1 siRNA to knock down HO-1 expression and examined the effect of this on the ability of SNAP to induce IL-8 production. Transfection of HUVECs with HO-1 siRNA abolished the ability of SNAP to induce HO-1 expression and IL-8 production (Fig. 2A). HO-1 siRNA also blocked GSNO-induced IL-8 production (Fig. 2A). Whether NO donors could also induce HO-1 expression and IL-8 production in HMECs, another type of endothelial cells, was determined. As in HUVECs, treatment with SNAP or GSNO significantly induced HO-1 expression and increased IL-8 production in HMECs (Fig. 2B). Again, HO-1 siRNA reversed the ability of SNAP to induce HO-1 expression and IL-8 production (Fig. 2B). In addition, transfection with HO-1 antisense ODN, but not with HO-1 sense ODN, also abolished HO-1 expression and IL-8 production in SNAP-treated HUVECs (Fig. 2C). SNAP or GSNO had no significant effect on the viability of HMECs (data not shown).
FIG. 2. Effects of HO-1 siRNA and HO-1 antisense ODN on HO-1 expression and IL-8 production in HUVECs and HMECs. HUVECs (A) or HMECs (B) were transfected with HO-1 siRNA, HO-1 sense ODN (C; HO-1 S) or HO-1 antisense ODN (C; HO-1 AS), and transfected and untransfected cells were incubated for 6 h with 100 μM SNAP or GSNO. Western blotting for HO-1 expression (upper panel) and ELISA for IL-8 protein (lower panel) were performed as described in Materials and Methods. NO donors significantly increased HO-1 expression and IL-8 production in HUVECs and HMECs (* and #, P < 0.01). Transfection with HO-1 siRNA or HO-1 antisense ODN into HUVECs or HMECs significantly reduced HO-1 expression and IL-8 production (** and ##, P < 0.01). Western blotting was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of pharmacological induction and gene transfer of HO-1 on IL-8 production
We addressed whether CoPP and hemin, inducers of HO-1, could mimic the ability of NO to induce IL-8 production in HUVECs. Like NO donors, two HO-1 inducers increased HO-1 expression and IL-8 production (Fig. 3A). The effects of HO-1 inducers on IL-8 production were significantly inhibited when the cells were transfected with HO-1 siRNA (Fig. 3A). We next transfected HUVECs with the human HO-1 gene. HO-1 gene transfer resulted in significant increases in HO-1 expression and IL-8 production (Fig. 3B). Blockage of HO activity with the HO inhibitor SnPP reversed the effect of HO-1 gene transfer on the IL-8 production (Fig. 3B). SnPP had no significant effect on the viability of HUVECs (data not shown).
FIG. 3. Effects of HO-1 inducers and HO-1 gene transfer on HO-1 expression and IL-8 production in HUVECs. A, Cells untransfected or transfected with HO-1 siRNA were incubated for 6 h (for HO-1 expression; upper panel) or 24 h (for IL-8 protein; lower panel) with 20 μM CoPP or 20 μM hemin. Western blotting for HO-1 expression and ELISA for IL-8 protein were performed as described in Materials and Methods. HO-1 inducers significantly increased HO-1 expression and IL-8 production (* and #, P < 0.01). Transfection with HO-1 siRNA significantly reduced HO-1 expression and IL-8 production by HO-1 inducers (** and ##, P < 0.01). B, Cells were transfected with control vector (Vector) or human HO-1 gene (HO-1), and the cells were incubated for 24 h with medium or 20 μM SnPP. Western blotting for the confirmation of HO-1 overexpression (upper panel) and ELISA for the determination of IL-8 protein (lower panel) were performed as described in Materials and Methods. Transfection with HO-1 gene significantly increased HO-1 expression and IL-8 production (*, P < 0.01). SnPP significantly reduced IL-8 production in HO-1-transfected cells (**, P < 0.01). Western blotting was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of NO on TNF--mediated IL-8 production
NO has been also known to suppress cytokine-mediated IL-8 production in human endothelial cells (14). Thus, we examined the effect of NO on TNF--mediated IL-8 production. Exposure of HUVECs to the proinflammatory cytokine TNF- induced HO-1 expression and increased IL-8 production (Fig. 4). Treatment of HUVECs with SNAP partially, but not completely, suppressed TNF--mediated IL-8 production, and the blockade of NO-induced expression of HO-1 by HO-1 siRNA markedly suppressed IL-8 production (Fig. 4). These results suggest that NO-induced partial suppression of IL-8 in response to TNF- occurs through HO-1-independent pathways; NO may impair TNF- signaling pathway(s) leading to IL-8 production.
FIG. 4. Effects of NO on TNF--mediated IL-8 production in HUVECs. Cells untransfected or transfected with HO-1 siRNA were incubated with medium, 10 ng/ml TNF-, 50 μM SNAP, or SNAP plus TNF- for 6 (for HO-1 expression; upper panel) or 24 (for IL-8 protein; lower panel) h. Western blotting for HO-1 expression and ELISA for IL-8 protein were performed as described in Materials and Methods. Both SNAP and a combination of SNAP and HO-1 siRNA significantly reduced IL-8 production (* and ** P < 0.01). Each column with bar represents the means ± SD (n = 6).
Effects of HO-1 on TNF--mediated Gro-, ENA-78, and MCP-1 productions
We asked whether HO-1 could also affect other chemokine productions in endothelial cells. The CXC chemokine family (IL-8, Gro- and ENA-78) and the CC chemokine family (MCP-1) were chosen and examined in HO-1-transfected HUVECs stimulated with or without TNF-. No significant change in the productions of Gro-, ENA-78, and MCP-1 was observed in HUVECs transfected with HO-1 gene, as compared with nontransfected or vector-transfected HUVECs, whereas IL-8 production was significantly increased in HO-1-transfected HUVECs (Fig. 5). Interestingly, HO-1 overexpression significantly reduced TNF--mediated productions of Gro-, ENA-78 and MCP-1, but not of IL-8 (Fig. 5). These experiments indicate that HO-1 expression selectively increases IL-8 production in HUVECs.
FIG. 5. Effects of HO-1 on TNF--mediated productions of chemokines in HUVECs. Cells were transfected with control vector (Vector) or human HO-1 gene (HO-1), and the cells were incubated for 24 h with medium or 20 μM 10 ng/ml TNF-. Concentrations of IL-8 (A), Gro- (B), ENA-78 (C), and MCP-1 (D) were determined by using ELISA as described in Materials and Methods. *, P < 0.01 with respect to each TNF--untreated group. Each column with bar represents the means ± SD (n = 6).
Effects of NO treatment and HO-1 expression on VEGF production
A previous report suggests that HO-1 expression by NO positively correlates with enhanced VEGF production in rat vascular smooth muscle cells (50, 53). Thus, we investigated the role of NO-induced HO-1 in the modulation of VEGF synthesis in HUVECs. NO released by NO donors induced VEGF mRNA and protein in HUVECs (Fig. 6). Transfection with HO-1 siRNA abolished the ability of SNAP or GSNO to induce VEGF production (Fig. 7A). The ability of SNAP to induce VEGF production in HUVECs was also abolished by HO-1 antisense ODN (data not shown). Treatment with HO-1 inducers, CoPP and hemin, induced VEGF production; this effect was abrogated by blockade of HO-1 expression by HO-1 siRNA (Fig. 7B). Notably, the cells transfected with human HO-1 gene synthesized more VEGF protein than the cells transfected with control vector but did not in the presence of the HO inhibitor SnPP (Fig. 7C). Similarly, SNAP induced VEGF production via HO-1-dependent pathway in HMECs (data not shown).
FIG. 6. Effects of NO donors on VEGF production in HUVECs. Cells were incubated for 8 (for VEGF mRNA, upper panel) or 24 (for VEGF protein, lower panel) h, with indicated concentrations of SNAP or GSNO in the absence or presence of 500 μM PTIO. RT-PCR for VEGF mRNA analysis and ELISA for the quantification of VEGF protein were performed as described in Materials and Methods. NO donors significantly increased VEGF mRNA levels and VEGF production (* and #, P < 0.01), and PTIO significantly reduced VEGF mRNA expression and VEGF production by NO donors (** and ##, P < 0.01). RT-PCR was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
FIG. 7. Effects of NO donors, HO-1 inducers, and HO-1 gene transfer on VEGF production in HUVECs. A and B, Cells untransfected or transfected with HO-1 siRNA were incubated for 24 h with 100 μM SNAP, 100 μM GSNO, 20 μM CoPP, or 20 μM hemin. C, Cells transfected with control vector (Vector) or HO-1 gene (HO-1) were incubated for 24 h with medium or 20 μM SnPP. ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. NO donors, HO-1 inducers, and HO-1 gene transfer significantly increased VEGF production (* and #, P < 0.01). Transfection with HO-1 siRNA and incubation with SnPP significantly reduced VEGF production (** and ##, P < 0.01). ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. Each column with bar represents the means ± SD (n = 6).
Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on VEGF and IL-8 productions by NO
Anti-IL-8 and anti-VEGF neutralizing antibodies were employed to examine whether IL-8 and VEGF induced by the NO donor SNAP might affect each other in HUVECs. Anti-VEGF neutralizing antibody blocked IL-8 production (Fig. 8A), whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs (Fig. 8B). In addition, VEGF itself induced IL-8 production and further enhanced SNAP-induced IL-8 production (Fig. 8C). When comparing the effects of VEGF121 and VEGF165, it appeared that IL-8 production induced by VEGF165 was greatly higher than that induced by VEGF121 (Fig. 8C), suggesting that NO-mediated IL-8 production in HUVECs might be due to VEGF165. Exposure of IL-8 (20 ng/ml) to HUVECs did not induce VEGF production (data not shown). As previously reported (58), either LY294002 (10 μM), a phosphatidylinositol 3-kinase inhibitor, or pyrrolidine dithiocarbamate (15 μM), a nuclear factor-B inhibitor, significantly inhibited VEGF165-induced IL-8 production in HUVECs (data not shown).
FIG. 8. Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on VEGF and IL-8 productions in SNAP-treated HUVECs. A and B, Cells were incubated for 24 h with 100 μM SNAP in the absence or presence of either 5 μg/ml anti-VEGF (A) or 5 μg/ml anti-IL-8 (B) antibody. C, Cells were incubated for 24 h with 2 ng/ml VEGF121, 2 ng/ml VEGF165, or combination of VEGF121 and VEGF165 in the absence or presence of 100 μM SNAP. SNAP significantly increased IL-8 and VEGF productions (*, P < 0.01). ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. Each column with bar represents the means ± SD (n = 6). Anti-VEGF antibody significantly reduced SNAP-induced IL-8 production by SNAP (**, P < 0.01), whereas anti-VEGF antibody had no significant effect on SNAP-induced VEGF production. VEGF165 significantly increased IL-8 production (#, P < 0.01) and enhanced SNAP-induced IL-8 production (##, P < 0.01). No remarkable effects of VEGF121 on IL-8 production in SNAP-untreated or -treated HUVECs were observed.
Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on NO-induced HO-1 expression
Neither anti-IL-8 nor anti-VEGF neutralizing antibody had a significant effect on NO-induced HO-1 expression in HUVECs (Fig. 9A). As in HUVECs, neutralizing antibodies had no significant effect on SNAP-induced HO-1 expression in HMECs (data not shown). In another experimental set, we examined whether IL-8 or VEGF could directly induce HO-1 expression in HUVECs. As previously reported (46), VEGF itself induced HO-1 expression slightly at 24 h and significantly at 48 h (Fig. 9B), whereas IL-8 had no effect on HO-1 expression (data not shown). In addition, combination of VEGF and a low concentration of SNAP did not further enhance SNAP-induced HO-1 expression (Fig. 9B).
FIG. 9. Effects of IL-8 and VEGF on HO-1 expression in HUVECs. A, Cells were incubated for 12 h with 100 μM SNAP in the absence or presence of either 5 μg/ml anti-IL-8 antibody or 5 μg/ml anti-VEGF antibody. B, Cells were incubated for 24 or 48 h with medium, 5 ng/ml VEGF121, 5 ng/ml VEGF165, or combination of VEGF121 and VEGF165 in the absence or presence of 10 μM/ml SNAP. Western blotting was performed two to three times with essentially similar results as described in Materials and Methods. It appeared that neither neutralizing antibodies nor VEGF had a significant effect on SNAP-induced HO-1 expression.
Discussion
In human endothelial cells, NO has been shown to induce IL-8 production (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) as well as HO-1 expression (32, 33), and HO-1 expression is associated with VEGF production (45, 54) that is involved in endothelial IL-8 synthesis (57, 58). The molecular cascade that could explain their relationship in endothelial cells has not yet been identified. In the present study, we examined the mechanism(s) by which NO can induce IL-8 and VEGF productions in human endothelial cells. In particular, we explored, for the first time, the critical involvement of HO-1 in the production of IL-8 through VEGF synthesis. Our studies have provided a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
IL-8, originally discovered as a chemotactic factor for leukocytes, has recently been shown to contribute to tissue repair and cancer progression through its potential functions as a mitogenic, angiogenic, and motogenic factor (59). Several studies using NO donors have indicated that NO serves as an intracellular second messenger to induce IL-8 gene expression (15, 16, 17, 18, 19, 20). Conversely, NO was reported to be able to suppress IL-8 expression. Fowler et al. (14) found that exogenously supplied NO down-regulates cytokine-mediated secretion of IL-8. The apparent discrepancy may be due to the use of different cell lines and IL-8 stimulators. It is known that the concentration of NO is an important determinant of its impact on cell functions, including gene expression (60). The biological relevance of NO-mediated IL-8 regulation in either tissue-repairing or tumor-progressing angiogenesis remains to be determined.
NO has been reported to induce HO-1 expression in various cell types, including endothelial cells, probably via its induction of oxidative stress (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). However, whether HO-1 induced by NO could be associated with NO-mediated biological effects in the endothelial cells remains to be investigated. In HUVECs, NO donors induced IL-8 production as well as HO-1 expression in a dose-dependent manner (Fig. 1). To explore the involvement of HO-1 in IL-8 production, we transfected the endothelial cells with either HO-1 siRNA or HO-1 antisense ODN to knock down HO-1 expression. Transfection with either HO-1 siRNA or HO-1 antisense ODN abolished the ability of NO to induce IL-8 production (Fig. 2). Moreover, both pharmacological induction and gene transfer of human HO-1 (Fig. 3) directly induced IL-8 production, as clearly observed when the endothelial cells were treated with an NO donor alone (Fig. 1). These results demonstrate that IL-8 production is associated with increased expression of HO-1.
NO has been also known to partially inhibit cytokine-mediated IL-8 production in endothelial cells (14, 61). In our study, we observed that TNF--mediated IL-8 production was partially inhibited by an NO donor and almost completely by NO donor and HO-1 siRNA transfection (Fig. 4). This suggests that partial inhibition of TNF--mediated IL-8 production by NO occurs through HO-1-independent pathways; NO may impair TNF- signaling pathway(s) leading to IL-8 production. We also examined the effect of HO-1 on the productions of other CXC chemokines, such as Gro- and ENA-78, and the CC chemokine MCP-1 in HUVECs. By using the gene transfer of HO-1, we found that HO-1 expression had no significant effect on the productions of these chemokines (Fig. 5). These data indicate that HO-1 expression in endothelial cells may selectively induce IL-8 production. It is of interest that intercellular adhesion molecule-1 expression was induced in HUVECs transduced with an HO-1 recombinant adenovirus, whereas HO-1 overexpression had no significant effect on the expressions of endothelial-leukocyte adhesion molecule-1 (E-selectin) and vascular adhesion molecule-1 (62).
Because HO-1 expression up-regulates VEGF production in the endothelial cells (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), and VEGF treatment directly induces endothelial IL-8 production through activation of nuclear factor-B via calcium and phosphatidylinositol 3-kinase pathway (57, 58), we investigated the possible role of HO-1-induced VEGF in IL-8 production. According to our data, VEGF was produced by NO donors via the HO-1-dependent pathway (Fig. 6). In agreement with this, pharmacological induction and gene transfer of HO-1 also induced VEGF production (Fig. 7), which is in agreement with recent observations showing that HO-1 overexpression spontaneously induces VEGF production in human endothelial cells and rat vascular smooth muscle cells (45, 48, 53, 54). Moreover, anti-VEGF neutralizing antibody blocked NO-mediated IL-8 production, and VEGF itself induced directly IL-8 production (Fig. 8). These results indicate that IL-8 production is associated with VEGF synthesis resulting from HO-1 expression by NO.
Recent evidence indicates that HO-1 is both an upstream and a downstream mediator of VEGF (41). It is well demonstrated that HO-1 expression in endothelial cells induces VEGF secretion (48, 54). It is possible that such secreted VEGF can reach the underlying endothelial cells and, in this way, may stimulate HO-1 expression. Such a relationship was tested using anti-IL-8 and anti-VEGF neutralizing antibodies. IL-8 and VEGF secreted from NO-treated cells did not affect HO-1 expression because HO-1 was clearly expressed by an NO donor even in the presence of either anti-IL-8 or anti-VEGF neutralizing antibody (Fig. 9A). Moreover, VEGF did not enhance NO-induced HO-1 expression in the endothelial cells (Fig. 9B), although VEGF, but not IL-8, itself could induce HO-1 expression (Fig. 9B). HO-1 was more rapidly and strongly expressed by NO (Fig. 1B) than by VEGF (Fig. 9B), which might explain why secreted VEGF could not enhance NO-induced HO-1 expression in endothelial cells.
The key finding of this study is that HO-1 expression by NO up-regulated VEGF production in human endothelial cells (Figs. 6 and 7). This finding is further supported by recent reports demonstrating that knockout of either the inducible NOS gene or the HO-1 gene impairs VEGF production (63, 64). VEGF is produced by many cell types under both physiological and pathological conditions. Its production is higher at sites of inflammation in various organs, and it is necessary for cutaneous wound healing. Importantly, it is responsible for the formation of new blood vessels during tumor growth (65). In addition, VEGF acts as a proinflammatory cytokine by increasing endothelial cell permeability, by inducing the expression of endothelial cell adhesion molecules, and via its ability to act as a monocyte chemoattractant (65). Interestingly, it has been demonstrated that HO-1 plays an important role in VEGF-driven angiogenesis in vivo and acts as a key regulator in the control of the antiinflammatory vs. proinflammatory actions of VEGF (46).
The molecular basis of the antiinflammatory action of HO-1 in endothelial cells remains to be fully elucidated. It has been reported that HO-1 overexpression in human endothelial cells reduced TNF--mediated E-selectin and vascular adhesion molecule-1 but not intercellular adhesion molecule-1 expression (62) and inhibited monocyte chemotaxis (46). However, there was no available report showing the effect of HO-1 on cytokine-mediated productions of chemokines in endothelial cells. We now demonstrate that HO-1 overexpression in endothelial cells inhibited TNF--mediated productions of Gro-, ENA-78, and MCP-1 but not of IL-8 (Fig. 5). The published reports and the present data suggest that HO-1 may be a potentially important regulator of inflammatory cell trafficking within the vessel wall, most likely by inhibiting the expression of endothelial-leukocyte adhesion molecules and the production of certain proinflammatory chemokines. It is of interest that increased leukocyte adhesion to the vessel wall and spontaneous perivascular infiltration of leukocytes into the liver, lungs, and kidneys was observed in HO-1-deficient mice (66, 67).
Although the mechanism by which HO-1 expression induces VEGF production remains to be elucidated, we speculated that HO-1 might not contribute to endothelial inflammatory reaction because of its additional antiinflammatory actions. HO-1-induced VEGF and IL-8, which are known as proangiogenic factors, may enhance angiogenesis associated with the resolution of tissue injury. Enhancement of VEGF production is being tested as a way to facilitate blood vessel formation in ischemic tissues (68). Genetic augmentation of NOS or HO-1 may offer a possible therapeutic strategy for the treatment of impaired angiogenesis in cardiovascular diseases or for the enhancement of wound healing. In addition, the molecular cascade showing the NO-HO-1-VEGF-IL-8 sequence in endothelial cells may be important in maintaining the angiogenic phenotype of the endothelium and may provide opportunities to consider molecular targets to attenuate tumor angiogenesis.
In summary, our results show the existence of a novel pathway in the modulation of IL-8 and VEGF productions by an NO donor in human endothelial cells; NO induces HO-1 expression by which VEGF is secreted, thereby resulting in IL-8 production. The molecular mechanism(s) underlying the observed up-regulation of VEGF production by HO-1 expression afforded by exogenous NO remains to be clarified. It is possible that hypoxia-induced factor-1, the major factor involved in the regulation of VEGF synthesis under hypoxia, may be playing a role in this effect. Whether HO-1 expression could induce VEGF production via hypoxia-induced factor-1-dependent pathway is currently being investigated in our laboratory.
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Address all correspondence and requests for reprints to: Hun-Taeg Chung, M.D., Ph.D., Department of Microbiology and Immunology, Wonkwang University School of Medicine, 344-2 Shinyoung-Dong, Iksan-Shi, Chonbug 570-749, Republic of Korea. E-mail: htchung@wonkwang.ac.kr.
Abstract
Heme oxygenase (HO)-1 has been shown to be an important biological target of nitric oxide (NO). NO can induce HO-1 expression and IL-8 production, particularly, in endothelial cells. Interestingly, HO-1 tends to induce the production of vascular endothelial growth factor (VEGF) that is involved in endothelial IL-8 syntheses. Whether HO-1 expression by NO may provide a link with IL-8 or VEGF synthesis was investigated in human umbilical vein endothelial cells (HUVECs). The NO donor S-nitroso-N-acetyl-penicillamine (SNAP) dose-dependently increased IL-8 and VEGF productions and HO-1 expression in HUVECs. Transfection with either HO-1 small interfering RNA or HO-1 antisense oligodeoxynucleotide abrogated the ability of SNAP to induce HO-1 expression and IL-8 and VEGF productions. Both pharmacological induction and gene transfer of HO-1 directly induced IL-8 and VEGF productions. Anti-VEGF neutralizing antibody blocked SNAP-mediated IL-8 production and VEGF itself induced IL-8 production, whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs. Neither anti-VEGF nor anti-IL-8 antibodies influenced SNAP-induced HO-1 expression. Moreover, neither VEGF nor IL-8 showed an additive effect on SNAP-induced HO-1 expression. HO-1 transfection had no significant effect on productions of other CXC chemokines, such as growth-related oncogen- and epithelial neutrophil activation peptide-78. Taken together, these results provide a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
Introduction
CHEMOKINES ARE CHARACTERIZED by their ability to induce chemotaxis in leukocytes. Secretion of chemokines is regarded as a pivotal step in cell recruitment necessary during initiation and maintenance of inflammatory responses (1). The CXC chemokine IL-8 is a regulator of leukocyte survival (2, 3) and is supposed to be a mediator of angiogenesis (4). IL-8 is produced from a variety of cells including monocytes and endothelial cells when the cells are exposed to oxidative stress, proinflammatory cytokines including TNF-, or some growth factors including vascular endothelial growth factor (VEGF) (5). In addition, the potential of nitric oxide (NO) to induce IL-8 synthesis has been demonstrated (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
NO plays a central role in a variety of physiological and pathological processes (21). NO synthases (NOSs), enzymes that produce NO, are composed of three isoforms, endothelial and neuronal enzymes and inducible NOS (22). NO produced by the actions of constitutively expressed NOS serves as a messenger/modulator agent vital for the functions of immune and other cells. On the other hand, induction of inducible NOS results in the production of NO at higher levels (23). Elevated NO is believed to contribute to tissue damage through its action to induce an inflammatory response. Recently, it has been evidenced that NO-producing cells possess regulatory pathways where protective mechanisms can operate to control proinflammatory responses via its induction of cytoprotective enzymes and thus limit the destructive potential (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Among the cytoprotective enzymes, inducible heme oxygenase (HO)-1 plays regulatory roles in the modulation of inflammatory responses (26, 27) and NO-induced cytotoxicity (32, 33).
HO-1 is a rate-limiting enzyme in heme catabolism, leading to the formation of biliverdin, which is reduced to bilirubin, carbon monoxide, and free iron. There is a large body of evidence suggesting that HO-1 is antiinflammatory and cytoprotective (34, 35, 36, 37, 38, 39), although the precise mechanisms by which HO-1 can be induced by specific stimuli remain unclear. Among the possible biological actions of HO-1 in the vessel wall is the regulation of VEGF synthesis (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), which has been proven to be an important growth factor critical for blood vessel formation. It has been demonstrated that HO-1 overexpression in human endothelial cells induced VEGF synthesis and consequently angiogenesis (40, 45, 48, 54).
VEGF has been shown to have at least five human isoforms (55, 56) that develop from alternate splicing of a single gene. Based on the affinity for binding to heparan sulfate, the isoforms have been shown to have soluble (VEGF121), cell-associated (VEGF189), or both (VEGF165) properties. There are a growing number of reports investigating the isoforms and their different roles in disease, and it is now clear that there are isoform-specific differences. VEGF165 has been associated with pathologic intravitreous neovascularization (57, 58) and has been found to be potently more proinflammatory than VEGF121 (56). VEGF165 has been detected in human proliferative diabetic retinopathy and was associated with a poor prognosis in certain tumors (56). The cell-associated isoform, VEGF189, is important in endothelial cell migration and adhesion, whereas the other isoforms were not (56). In endothelial cells, VEGF165 has been reported to induce HO-1 expression (46) and IL-8 production (57, 58).
Considering that NO can induce HO-1 expression (32, 33) as well as IL-8 synthesis (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), and HO-1 tends to induce VEGF synthesis (45, 54) associated with endothelial IL-8 production (57, 58), we have investigated whether HO-1 expression by NO may provide a link with IL-8 or VEGF syntheses in human endothelial cells. The results presented in this study provide, for the first time, a molecular cascade showing the NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
Materials and Methods
Reagents, cytokine, growth factor, antibodies, and HO-1 gene
Human recombinant IL-8, TNF-, VEGF121, VEGF165, and anti-IL-8 and anti-VEGF neutralizing antibodies were purchased from R&D Systems, Inc. (Minneapolis, MN). S-Nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) were obtained from Sigma-Aldrich Chemical (St. Louis, MO). Tin protoporphyrin (SnPP), cobalt protoporphyrin (CoPP), and hemin were from Porphyrin Products (Logan, UT). Human anti-HO-1 and anti--actin antibodies were from StressGen Biotech (Victoria, British Columbia, Canada). HO-1 cDNA was a kind gift from Dr. A.M.K. Choi (University of Pittsburgh, Pittsburgh, PA). HO-1 small interfering RNA (siRNA) and its transfection kit were from Santa Cruz Biotechnology (Santa Cruz, CA). Sense and antisense oligodeoxynucleotides (ODNs) were supplied by Invitrogen (San Diego, CA) and transfected with the Superfect transfection reagent (Qiagen, Hilden, Germany).
Cell culture
Human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMECs) were obtained from the American Type Culture Collection (Manassas, VA). These cell lines were maintained in culture as an adherent monolayer cell medium supplemented with 10% fetal bovine serum and endothelial cell growth supplement (EGM-2; Cambrex Biosciences, Walkersville, MD). HUVECs and HMECs were grown up to 80% confluence and then transferred to serum and EGM-2-free medium overnight before being used for transfection experiments.
HO-1 and HO-1 siRNA transfection
HO-1 was cloned into pcDNA3 (Invitrogen). HUVECs (5 x 106) or HMECs (5 x 106) were transfected with 10 μg constructs by electroporation at 270 V, 950 μF in serum-free RPMI 1640 using a Gene Pulser (Bio-Rad, Richmond, CA) followed by culture in EGM-2 supplemented with 10% fetal bovine serum for 48 h and plating on 96-well plates at 5 x 105 cells/well in the presence of 1.25 mg/ml G418. Single stable clones of transfectant were isolated and expanded.
Inhibition of HO-1 expression was assessed by Western blotting analysis after transfection of HUVECs with HO-1 siRNA. Briefly, HUVECs were grown in 24- or six-well plates and transiently transfected with 1.5 and 3 μg HO-1 siRNA (Santa Cruz Biotechnology) mixed with siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer’s protocol. After incubation at 37 C and 5% CO2 for 36 h, the cells were treated with NO donors. Samples were then prepared and analyzed for cytokines or immunoblotting.
HO-1 sense and antisense ODNs were directed against the flanking translation initiation codon in the human HO-1 cDNA (27). The antisense sequence was 5'-CGC CTT CAT GGT GCC-3', whereas the sense sequence was 5'-GGC ACC ATG AAG GCG-3'. ODNs were phosphorothioated on the first three bases on the 3' end. HUVECs were transfected using the Superfectr transfection reagent following the manufacturer’s instructions. Briefly, the cells were seeded in culture plates before transfection. The proportions used were 1 μg DNA/5 μl transfection reagent/well. HUVECs were incubated for 4 h with the ODNs.
Assays of chemokines and VEGF
Supernatants were analyzed for IL-8, growth-related oncogen (Gro)-, epithelial neutrophil activation peptide (ENA-78), monocyte chemotactic peptide (MCP)-1, and VEGF using ELISA obtained commercially (R&D Systems, Inc.). The assays were performed according to the manufacturer’s protocol. Chemokine and VEGF concentrations are expressed in nanograms per milliliter. A standard curve was prepared from a group of serially diluted standard samples of either chemokine or VEGF. Absorbance was read against a blank at 450 nm using a microtiter ELISA reader.
Cell extracts and Western blotting analysis
Cells were harvested via first rinsing in ice-cold PBS and then immediately resuspended in cell lysis buffer containing a complete protease inhibitor mixture (New England Biolabs, Beverly, MA). Cellular protein extracts were electrophoresed under denaturing conditions (10- 12.5% polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad). HO-1 expression was detected using anti-HO-1 monoclonal antibody. Actin expression was detected by using anti--actin monoclonal antibody. Primary antibodies were detected by using horseradish peroxidase-conjugated antirabbit or antimouse IgG secondary antibodies (Santa Cruz Biotechnology). Peroxidase was visualized using the enhanced chemiluminescence assay (New England Biolabs) according to the manufacturer’s instructions.
RT-PCR
Total RNA was isolated by the guanidine isothiocyanate method using Tri Reagent (Sigma-Aldrich Chemical). First strand cDNAs were synthesized from RNA using the ImProm-II RT system (Promega, Madison, WI). Then, PCR with Taq DNA polymerase (Promega) was performed for 27 cycles using the following protocol: 95 C for 40 sec, 58 C for 40 sec, and 72 C for 50 sec. PCR products were analyzed by electrophoresis in 2% agarose gels. PCRs were carried out using primers for VEGF121 and VEGF165 (5'-CAC CGC CTC GGC TTG TCA CAT and 5'-CTG CTG TCT TGG GTG CAT TGG), for IL-8 (5'-GGA CAA GAG CCA GGA AGA AAC C and 5'-CTT CAA CTT CTC CAC AAC CC), or for the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (5'-CGT ATT GGG CGC CTG GTC ACC and 5'-GGG ATG ATG TTC TGG AGA GCC C). The product length was 431 bp for the VEGF121, 563 bp for VEGF165, and 335 bp for IL-8 and glyceraldehyde-3-phosphate dehydrogenase.
Statistical analysis
Data in this study present the means ± SD where applicable. Statistical evaluation was determined with Student’s t test or with ANOVA followed by the Tukey test. P < 0.01 was taken to indicate statistical significance.
Results
Effects of the NO on IL-8 production and HO-1 expression
Previous studies have demonstrated that a variety of NO donors induce IL-8 production (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and HO-1 expression in monocytic, epithelial, and endothelial cells (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). In the current study, treatment of HUVECs with the NO donor SNAP at noncytotoxic doses induced IL-8 mRNA and protein in a dose-dependent manner as measured 24 h after exposure (Fig. 1A). Another NO donor GSNO also induced IL-8 mRNA and protein (Fig. 1A). To examine the role of the gaseous molecule NO in IL-8 production by SNAP treatment, the endothelial cells were incubated with the NO scavenger PTIO before SNAP treatment. PTIO almost completely reduced SNAP-induced IL-8 mRNA and protein levels (Fig. 1A). Both SNAP and GSNO also induced HO-1 expression and increased HO activity (Fig. 1B); these effects were paralleled by increased IL-8 mRNA and protein (Fig. 1A). Similar to its effects on SNAP-induced IL-8 mRNA and protein, PTIO abrogated the SNAP-induced increases in HO-1 expression and HO activity (Fig. 1B). SNAP, GSNO, or PTIO did not have significant effect on the viability of the endothelial cells (data not shown).
FIG. 1. Effects of NO donors on IL-8 production and HO-1 expression in HUVECs. A, Cells were incubated for 8 h (for IL-8 mRNA; upper panel) or 24 h (for IL-8 protein; lower panel) with SNAP at indicated concentrations or GSNO in the absence or presence of PTIO (500 μM). RT-PCR for IL-8 mRNA analysis and ELISA for the quantification of IL-8 protein were performed as described in Materials and Methods. NO donors significantly increased IL-8 mRNA levels and IL-8 production (* and #, P < 0.01), and PTIO significantly reduced IL-8 mRNA expression and IL-8 production by NO donors (** and ##, P < 0.01). B, Cells were incubated for 6 h with SNAP at indicated concentrations or GSNO in the absence or presence of PTIO (500 μM). Western blotting for HO-1 expression (upper panel) and bilirubin quantification for HO activity (lower panel) were performed as described in Materials and Methods. NO donors significantly increased HO-1 expression and HO activity (* and #, P < 0.01), and PTIO significantly reduced HO-1 expression and HO activity by NO donors (** and ##, P < 0.01). RT-PCR and Western blotting were performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of HO-1 siRNA and HO-1 antisense ODN on NO-induced IL-8 production and HO-1 expression
We transfected HUVECs with HO-1 siRNA to knock down HO-1 expression and examined the effect of this on the ability of SNAP to induce IL-8 production. Transfection of HUVECs with HO-1 siRNA abolished the ability of SNAP to induce HO-1 expression and IL-8 production (Fig. 2A). HO-1 siRNA also blocked GSNO-induced IL-8 production (Fig. 2A). Whether NO donors could also induce HO-1 expression and IL-8 production in HMECs, another type of endothelial cells, was determined. As in HUVECs, treatment with SNAP or GSNO significantly induced HO-1 expression and increased IL-8 production in HMECs (Fig. 2B). Again, HO-1 siRNA reversed the ability of SNAP to induce HO-1 expression and IL-8 production (Fig. 2B). In addition, transfection with HO-1 antisense ODN, but not with HO-1 sense ODN, also abolished HO-1 expression and IL-8 production in SNAP-treated HUVECs (Fig. 2C). SNAP or GSNO had no significant effect on the viability of HMECs (data not shown).
FIG. 2. Effects of HO-1 siRNA and HO-1 antisense ODN on HO-1 expression and IL-8 production in HUVECs and HMECs. HUVECs (A) or HMECs (B) were transfected with HO-1 siRNA, HO-1 sense ODN (C; HO-1 S) or HO-1 antisense ODN (C; HO-1 AS), and transfected and untransfected cells were incubated for 6 h with 100 μM SNAP or GSNO. Western blotting for HO-1 expression (upper panel) and ELISA for IL-8 protein (lower panel) were performed as described in Materials and Methods. NO donors significantly increased HO-1 expression and IL-8 production in HUVECs and HMECs (* and #, P < 0.01). Transfection with HO-1 siRNA or HO-1 antisense ODN into HUVECs or HMECs significantly reduced HO-1 expression and IL-8 production (** and ##, P < 0.01). Western blotting was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of pharmacological induction and gene transfer of HO-1 on IL-8 production
We addressed whether CoPP and hemin, inducers of HO-1, could mimic the ability of NO to induce IL-8 production in HUVECs. Like NO donors, two HO-1 inducers increased HO-1 expression and IL-8 production (Fig. 3A). The effects of HO-1 inducers on IL-8 production were significantly inhibited when the cells were transfected with HO-1 siRNA (Fig. 3A). We next transfected HUVECs with the human HO-1 gene. HO-1 gene transfer resulted in significant increases in HO-1 expression and IL-8 production (Fig. 3B). Blockage of HO activity with the HO inhibitor SnPP reversed the effect of HO-1 gene transfer on the IL-8 production (Fig. 3B). SnPP had no significant effect on the viability of HUVECs (data not shown).
FIG. 3. Effects of HO-1 inducers and HO-1 gene transfer on HO-1 expression and IL-8 production in HUVECs. A, Cells untransfected or transfected with HO-1 siRNA were incubated for 6 h (for HO-1 expression; upper panel) or 24 h (for IL-8 protein; lower panel) with 20 μM CoPP or 20 μM hemin. Western blotting for HO-1 expression and ELISA for IL-8 protein were performed as described in Materials and Methods. HO-1 inducers significantly increased HO-1 expression and IL-8 production (* and #, P < 0.01). Transfection with HO-1 siRNA significantly reduced HO-1 expression and IL-8 production by HO-1 inducers (** and ##, P < 0.01). B, Cells were transfected with control vector (Vector) or human HO-1 gene (HO-1), and the cells were incubated for 24 h with medium or 20 μM SnPP. Western blotting for the confirmation of HO-1 overexpression (upper panel) and ELISA for the determination of IL-8 protein (lower panel) were performed as described in Materials and Methods. Transfection with HO-1 gene significantly increased HO-1 expression and IL-8 production (*, P < 0.01). SnPP significantly reduced IL-8 production in HO-1-transfected cells (**, P < 0.01). Western blotting was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
Effects of NO on TNF--mediated IL-8 production
NO has been also known to suppress cytokine-mediated IL-8 production in human endothelial cells (14). Thus, we examined the effect of NO on TNF--mediated IL-8 production. Exposure of HUVECs to the proinflammatory cytokine TNF- induced HO-1 expression and increased IL-8 production (Fig. 4). Treatment of HUVECs with SNAP partially, but not completely, suppressed TNF--mediated IL-8 production, and the blockade of NO-induced expression of HO-1 by HO-1 siRNA markedly suppressed IL-8 production (Fig. 4). These results suggest that NO-induced partial suppression of IL-8 in response to TNF- occurs through HO-1-independent pathways; NO may impair TNF- signaling pathway(s) leading to IL-8 production.
FIG. 4. Effects of NO on TNF--mediated IL-8 production in HUVECs. Cells untransfected or transfected with HO-1 siRNA were incubated with medium, 10 ng/ml TNF-, 50 μM SNAP, or SNAP plus TNF- for 6 (for HO-1 expression; upper panel) or 24 (for IL-8 protein; lower panel) h. Western blotting for HO-1 expression and ELISA for IL-8 protein were performed as described in Materials and Methods. Both SNAP and a combination of SNAP and HO-1 siRNA significantly reduced IL-8 production (* and ** P < 0.01). Each column with bar represents the means ± SD (n = 6).
Effects of HO-1 on TNF--mediated Gro-, ENA-78, and MCP-1 productions
We asked whether HO-1 could also affect other chemokine productions in endothelial cells. The CXC chemokine family (IL-8, Gro- and ENA-78) and the CC chemokine family (MCP-1) were chosen and examined in HO-1-transfected HUVECs stimulated with or without TNF-. No significant change in the productions of Gro-, ENA-78, and MCP-1 was observed in HUVECs transfected with HO-1 gene, as compared with nontransfected or vector-transfected HUVECs, whereas IL-8 production was significantly increased in HO-1-transfected HUVECs (Fig. 5). Interestingly, HO-1 overexpression significantly reduced TNF--mediated productions of Gro-, ENA-78 and MCP-1, but not of IL-8 (Fig. 5). These experiments indicate that HO-1 expression selectively increases IL-8 production in HUVECs.
FIG. 5. Effects of HO-1 on TNF--mediated productions of chemokines in HUVECs. Cells were transfected with control vector (Vector) or human HO-1 gene (HO-1), and the cells were incubated for 24 h with medium or 20 μM 10 ng/ml TNF-. Concentrations of IL-8 (A), Gro- (B), ENA-78 (C), and MCP-1 (D) were determined by using ELISA as described in Materials and Methods. *, P < 0.01 with respect to each TNF--untreated group. Each column with bar represents the means ± SD (n = 6).
Effects of NO treatment and HO-1 expression on VEGF production
A previous report suggests that HO-1 expression by NO positively correlates with enhanced VEGF production in rat vascular smooth muscle cells (50, 53). Thus, we investigated the role of NO-induced HO-1 in the modulation of VEGF synthesis in HUVECs. NO released by NO donors induced VEGF mRNA and protein in HUVECs (Fig. 6). Transfection with HO-1 siRNA abolished the ability of SNAP or GSNO to induce VEGF production (Fig. 7A). The ability of SNAP to induce VEGF production in HUVECs was also abolished by HO-1 antisense ODN (data not shown). Treatment with HO-1 inducers, CoPP and hemin, induced VEGF production; this effect was abrogated by blockade of HO-1 expression by HO-1 siRNA (Fig. 7B). Notably, the cells transfected with human HO-1 gene synthesized more VEGF protein than the cells transfected with control vector but did not in the presence of the HO inhibitor SnPP (Fig. 7C). Similarly, SNAP induced VEGF production via HO-1-dependent pathway in HMECs (data not shown).
FIG. 6. Effects of NO donors on VEGF production in HUVECs. Cells were incubated for 8 (for VEGF mRNA, upper panel) or 24 (for VEGF protein, lower panel) h, with indicated concentrations of SNAP or GSNO in the absence or presence of 500 μM PTIO. RT-PCR for VEGF mRNA analysis and ELISA for the quantification of VEGF protein were performed as described in Materials and Methods. NO donors significantly increased VEGF mRNA levels and VEGF production (* and #, P < 0.01), and PTIO significantly reduced VEGF mRNA expression and VEGF production by NO donors (** and ##, P < 0.01). RT-PCR was performed two to three times with essentially similar results. Each column with bar represents the means ± SD (n = 6).
FIG. 7. Effects of NO donors, HO-1 inducers, and HO-1 gene transfer on VEGF production in HUVECs. A and B, Cells untransfected or transfected with HO-1 siRNA were incubated for 24 h with 100 μM SNAP, 100 μM GSNO, 20 μM CoPP, or 20 μM hemin. C, Cells transfected with control vector (Vector) or HO-1 gene (HO-1) were incubated for 24 h with medium or 20 μM SnPP. ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. NO donors, HO-1 inducers, and HO-1 gene transfer significantly increased VEGF production (* and #, P < 0.01). Transfection with HO-1 siRNA and incubation with SnPP significantly reduced VEGF production (** and ##, P < 0.01). ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. Each column with bar represents the means ± SD (n = 6).
Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on VEGF and IL-8 productions by NO
Anti-IL-8 and anti-VEGF neutralizing antibodies were employed to examine whether IL-8 and VEGF induced by the NO donor SNAP might affect each other in HUVECs. Anti-VEGF neutralizing antibody blocked IL-8 production (Fig. 8A), whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs (Fig. 8B). In addition, VEGF itself induced IL-8 production and further enhanced SNAP-induced IL-8 production (Fig. 8C). When comparing the effects of VEGF121 and VEGF165, it appeared that IL-8 production induced by VEGF165 was greatly higher than that induced by VEGF121 (Fig. 8C), suggesting that NO-mediated IL-8 production in HUVECs might be due to VEGF165. Exposure of IL-8 (20 ng/ml) to HUVECs did not induce VEGF production (data not shown). As previously reported (58), either LY294002 (10 μM), a phosphatidylinositol 3-kinase inhibitor, or pyrrolidine dithiocarbamate (15 μM), a nuclear factor-B inhibitor, significantly inhibited VEGF165-induced IL-8 production in HUVECs (data not shown).
FIG. 8. Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on VEGF and IL-8 productions in SNAP-treated HUVECs. A and B, Cells were incubated for 24 h with 100 μM SNAP in the absence or presence of either 5 μg/ml anti-VEGF (A) or 5 μg/ml anti-IL-8 (B) antibody. C, Cells were incubated for 24 h with 2 ng/ml VEGF121, 2 ng/ml VEGF165, or combination of VEGF121 and VEGF165 in the absence or presence of 100 μM SNAP. SNAP significantly increased IL-8 and VEGF productions (*, P < 0.01). ELISA for the quantification of VEGF protein was performed as described in Materials and Methods. Each column with bar represents the means ± SD (n = 6). Anti-VEGF antibody significantly reduced SNAP-induced IL-8 production by SNAP (**, P < 0.01), whereas anti-VEGF antibody had no significant effect on SNAP-induced VEGF production. VEGF165 significantly increased IL-8 production (#, P < 0.01) and enhanced SNAP-induced IL-8 production (##, P < 0.01). No remarkable effects of VEGF121 on IL-8 production in SNAP-untreated or -treated HUVECs were observed.
Effects of anti-VEGF and anti-IL-8 neutralizing antibodies on NO-induced HO-1 expression
Neither anti-IL-8 nor anti-VEGF neutralizing antibody had a significant effect on NO-induced HO-1 expression in HUVECs (Fig. 9A). As in HUVECs, neutralizing antibodies had no significant effect on SNAP-induced HO-1 expression in HMECs (data not shown). In another experimental set, we examined whether IL-8 or VEGF could directly induce HO-1 expression in HUVECs. As previously reported (46), VEGF itself induced HO-1 expression slightly at 24 h and significantly at 48 h (Fig. 9B), whereas IL-8 had no effect on HO-1 expression (data not shown). In addition, combination of VEGF and a low concentration of SNAP did not further enhance SNAP-induced HO-1 expression (Fig. 9B).
FIG. 9. Effects of IL-8 and VEGF on HO-1 expression in HUVECs. A, Cells were incubated for 12 h with 100 μM SNAP in the absence or presence of either 5 μg/ml anti-IL-8 antibody or 5 μg/ml anti-VEGF antibody. B, Cells were incubated for 24 or 48 h with medium, 5 ng/ml VEGF121, 5 ng/ml VEGF165, or combination of VEGF121 and VEGF165 in the absence or presence of 10 μM/ml SNAP. Western blotting was performed two to three times with essentially similar results as described in Materials and Methods. It appeared that neither neutralizing antibodies nor VEGF had a significant effect on SNAP-induced HO-1 expression.
Discussion
In human endothelial cells, NO has been shown to induce IL-8 production (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) as well as HO-1 expression (32, 33), and HO-1 expression is associated with VEGF production (45, 54) that is involved in endothelial IL-8 synthesis (57, 58). The molecular cascade that could explain their relationship in endothelial cells has not yet been identified. In the present study, we examined the mechanism(s) by which NO can induce IL-8 and VEGF productions in human endothelial cells. In particular, we explored, for the first time, the critical involvement of HO-1 in the production of IL-8 through VEGF synthesis. Our studies have provided a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.
IL-8, originally discovered as a chemotactic factor for leukocytes, has recently been shown to contribute to tissue repair and cancer progression through its potential functions as a mitogenic, angiogenic, and motogenic factor (59). Several studies using NO donors have indicated that NO serves as an intracellular second messenger to induce IL-8 gene expression (15, 16, 17, 18, 19, 20). Conversely, NO was reported to be able to suppress IL-8 expression. Fowler et al. (14) found that exogenously supplied NO down-regulates cytokine-mediated secretion of IL-8. The apparent discrepancy may be due to the use of different cell lines and IL-8 stimulators. It is known that the concentration of NO is an important determinant of its impact on cell functions, including gene expression (60). The biological relevance of NO-mediated IL-8 regulation in either tissue-repairing or tumor-progressing angiogenesis remains to be determined.
NO has been reported to induce HO-1 expression in various cell types, including endothelial cells, probably via its induction of oxidative stress (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). However, whether HO-1 induced by NO could be associated with NO-mediated biological effects in the endothelial cells remains to be investigated. In HUVECs, NO donors induced IL-8 production as well as HO-1 expression in a dose-dependent manner (Fig. 1). To explore the involvement of HO-1 in IL-8 production, we transfected the endothelial cells with either HO-1 siRNA or HO-1 antisense ODN to knock down HO-1 expression. Transfection with either HO-1 siRNA or HO-1 antisense ODN abolished the ability of NO to induce IL-8 production (Fig. 2). Moreover, both pharmacological induction and gene transfer of human HO-1 (Fig. 3) directly induced IL-8 production, as clearly observed when the endothelial cells were treated with an NO donor alone (Fig. 1). These results demonstrate that IL-8 production is associated with increased expression of HO-1.
NO has been also known to partially inhibit cytokine-mediated IL-8 production in endothelial cells (14, 61). In our study, we observed that TNF--mediated IL-8 production was partially inhibited by an NO donor and almost completely by NO donor and HO-1 siRNA transfection (Fig. 4). This suggests that partial inhibition of TNF--mediated IL-8 production by NO occurs through HO-1-independent pathways; NO may impair TNF- signaling pathway(s) leading to IL-8 production. We also examined the effect of HO-1 on the productions of other CXC chemokines, such as Gro- and ENA-78, and the CC chemokine MCP-1 in HUVECs. By using the gene transfer of HO-1, we found that HO-1 expression had no significant effect on the productions of these chemokines (Fig. 5). These data indicate that HO-1 expression in endothelial cells may selectively induce IL-8 production. It is of interest that intercellular adhesion molecule-1 expression was induced in HUVECs transduced with an HO-1 recombinant adenovirus, whereas HO-1 overexpression had no significant effect on the expressions of endothelial-leukocyte adhesion molecule-1 (E-selectin) and vascular adhesion molecule-1 (62).
Because HO-1 expression up-regulates VEGF production in the endothelial cells (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), and VEGF treatment directly induces endothelial IL-8 production through activation of nuclear factor-B via calcium and phosphatidylinositol 3-kinase pathway (57, 58), we investigated the possible role of HO-1-induced VEGF in IL-8 production. According to our data, VEGF was produced by NO donors via the HO-1-dependent pathway (Fig. 6). In agreement with this, pharmacological induction and gene transfer of HO-1 also induced VEGF production (Fig. 7), which is in agreement with recent observations showing that HO-1 overexpression spontaneously induces VEGF production in human endothelial cells and rat vascular smooth muscle cells (45, 48, 53, 54). Moreover, anti-VEGF neutralizing antibody blocked NO-mediated IL-8 production, and VEGF itself induced directly IL-8 production (Fig. 8). These results indicate that IL-8 production is associated with VEGF synthesis resulting from HO-1 expression by NO.
Recent evidence indicates that HO-1 is both an upstream and a downstream mediator of VEGF (41). It is well demonstrated that HO-1 expression in endothelial cells induces VEGF secretion (48, 54). It is possible that such secreted VEGF can reach the underlying endothelial cells and, in this way, may stimulate HO-1 expression. Such a relationship was tested using anti-IL-8 and anti-VEGF neutralizing antibodies. IL-8 and VEGF secreted from NO-treated cells did not affect HO-1 expression because HO-1 was clearly expressed by an NO donor even in the presence of either anti-IL-8 or anti-VEGF neutralizing antibody (Fig. 9A). Moreover, VEGF did not enhance NO-induced HO-1 expression in the endothelial cells (Fig. 9B), although VEGF, but not IL-8, itself could induce HO-1 expression (Fig. 9B). HO-1 was more rapidly and strongly expressed by NO (Fig. 1B) than by VEGF (Fig. 9B), which might explain why secreted VEGF could not enhance NO-induced HO-1 expression in endothelial cells.
The key finding of this study is that HO-1 expression by NO up-regulated VEGF production in human endothelial cells (Figs. 6 and 7). This finding is further supported by recent reports demonstrating that knockout of either the inducible NOS gene or the HO-1 gene impairs VEGF production (63, 64). VEGF is produced by many cell types under both physiological and pathological conditions. Its production is higher at sites of inflammation in various organs, and it is necessary for cutaneous wound healing. Importantly, it is responsible for the formation of new blood vessels during tumor growth (65). In addition, VEGF acts as a proinflammatory cytokine by increasing endothelial cell permeability, by inducing the expression of endothelial cell adhesion molecules, and via its ability to act as a monocyte chemoattractant (65). Interestingly, it has been demonstrated that HO-1 plays an important role in VEGF-driven angiogenesis in vivo and acts as a key regulator in the control of the antiinflammatory vs. proinflammatory actions of VEGF (46).
The molecular basis of the antiinflammatory action of HO-1 in endothelial cells remains to be fully elucidated. It has been reported that HO-1 overexpression in human endothelial cells reduced TNF--mediated E-selectin and vascular adhesion molecule-1 but not intercellular adhesion molecule-1 expression (62) and inhibited monocyte chemotaxis (46). However, there was no available report showing the effect of HO-1 on cytokine-mediated productions of chemokines in endothelial cells. We now demonstrate that HO-1 overexpression in endothelial cells inhibited TNF--mediated productions of Gro-, ENA-78, and MCP-1 but not of IL-8 (Fig. 5). The published reports and the present data suggest that HO-1 may be a potentially important regulator of inflammatory cell trafficking within the vessel wall, most likely by inhibiting the expression of endothelial-leukocyte adhesion molecules and the production of certain proinflammatory chemokines. It is of interest that increased leukocyte adhesion to the vessel wall and spontaneous perivascular infiltration of leukocytes into the liver, lungs, and kidneys was observed in HO-1-deficient mice (66, 67).
Although the mechanism by which HO-1 expression induces VEGF production remains to be elucidated, we speculated that HO-1 might not contribute to endothelial inflammatory reaction because of its additional antiinflammatory actions. HO-1-induced VEGF and IL-8, which are known as proangiogenic factors, may enhance angiogenesis associated with the resolution of tissue injury. Enhancement of VEGF production is being tested as a way to facilitate blood vessel formation in ischemic tissues (68). Genetic augmentation of NOS or HO-1 may offer a possible therapeutic strategy for the treatment of impaired angiogenesis in cardiovascular diseases or for the enhancement of wound healing. In addition, the molecular cascade showing the NO-HO-1-VEGF-IL-8 sequence in endothelial cells may be important in maintaining the angiogenic phenotype of the endothelium and may provide opportunities to consider molecular targets to attenuate tumor angiogenesis.
In summary, our results show the existence of a novel pathway in the modulation of IL-8 and VEGF productions by an NO donor in human endothelial cells; NO induces HO-1 expression by which VEGF is secreted, thereby resulting in IL-8 production. The molecular mechanism(s) underlying the observed up-regulation of VEGF production by HO-1 expression afforded by exogenous NO remains to be clarified. It is possible that hypoxia-induced factor-1, the major factor involved in the regulation of VEGF synthesis under hypoxia, may be playing a role in this effect. Whether HO-1 expression could induce VEGF production via hypoxia-induced factor-1-dependent pathway is currently being investigated in our laboratory.
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