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Endotoxins induce and interferon suppresses vascular endothelial growth factor (VEGF) production in human peripheral blood mononuclear cells¹

PETRI SALVEN², KRISTA ANTTONEN^*,, HEIKKI REPO^,¶, HEIKKI JOENSUU and ARTO ORPANA^*,

Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA; Departments of
^* Clinical Chemistry,
Medical Genetics,
Department of Medicine, Infectious Disease Unit,
Oncology, Helsinki University Central Hospital, 00290 Helsinki, Finland; and
^¶ Department of Bacteriology and Immunology, Haartman Institute, 00290 Helsinki, Finland

²Correspondence: Division of Hematology-Oncology, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. USA. E-mail: pjs2004{at}med.cornell.edu; petri.salven{at}helsinki.fi

SPECIFIC AIM

Both circulating and resident inflammatory cells release diverse factors known to modify vascular permeability, enhance angiogenic phenotypes, and contribute to many vascular events, including atherosclerotic plaque development and rupture, aortic aneurysm formation, ischemia/reperfusion damage and repair, and tumor angiogenesis. The aim of the present study was to explore the role of human peripheral blood mononuclear cells (PBMNCs) as producers of vascular endothelial growth factor (VEGF), and to identify factors that regulate the VEGF biosynthesis and release by PBMNCs.

PRINCIPAL FINDINGS

1. Unstimulated PBMNCs from healthy donors are able to release VEGF protein continuously into culture media
Freshly isolated PBMNCs from healthy volunteers cultured for 24 h in serum-free medium in the absence of any stimulus released VEGF continuously into the surrounding medium in a time-dependent manner as determined by ELISA.

2. Physiological endotoxin concentrations cause a dose-dependent increase in VEGF secretion by PBMNCs
PBMNCs were incubated for 24 h in serum-free medium with various concentrations of endotoxin. At a concentration range of Salmonella typhimurium endotoxin as low as 20–200 pg/ml a discernible increase in VEGF secretion was detectable. Higher concentrations of endotoxin increased VEGF secretion until the process started to saturate at concentrations higher than 2 ng/ml (Fig. 1 ). Stimulation of PBMNCs with endotoxin (20 ng/ml) caused a significant increase in VEGF secretion that was already detectable after 3 h. After 24 h stimulation with endotoxin at 20 ng/ml, the mean VEGF production of endotoxin stimulated cells (157 pg/10⁶ cells) was fourfold higher than that of nonstimulated cells (39 pg/10⁶ cells).

View larger version (14K): [in this window] [in a new window]   Figure 1. Dose-dependent secretion of VEGF (pg/106 cells) by PBMNCs stimulated with Salmonella typhimurium endotoxin. The PBMNCs were incubated for 24 h with various concentrations of endotoxin. The endotoxin concentration (ng/ml) is shown on the X-axis (log scale). The values are given as means ± 1 SE of seven independent experiments. *P < 0.05 (the Wilcoxon signed rank test).

3. The increase in VEGF secretion of PBMNCs by endotoxin represents induction of de novo VEGF production
The VEGF secretion induced by endotoxin represents de novo VEGF production, as an induction of the expression of the major 3.7 kb VEGF mRNA transcripts was observed and the release of VEGF was blocked by cycloheximide. A 14 h incubation of PBMNCs with endotoxin at 2 or 20 ng/ml enhanced VEGF mRNA levels 1.7-fold and 1.9-fold, respectively, over those in unstimulated cells.

4. Unstimulated and endotoxin-stimulated PBMNCs express the mRNAs encoding for VEGF121, VEGF165, VEGF189, and VEGF206
Amplification of cDNA from unstimulated PBMNCs and cells stimulated for 24 h with endotoxin gave rise to four bands corresponding to the mRNA sizes predicted for VEGF121, VEGF165, VEGF189, and VEGF206. All four transcripts were also expressed in unstimulated cells. The expression pattern of the VEGF mRNAs in endotoxin-stimulated cells was comparable to that in nonstimulated cells. The mRNAs encoding for the smaller, freely diffusible VEGF isoforms VEGF121 and VEGF165 gave the major signals both in unstimulated and endotoxin-stimulated cells.

5. Interferon (IFN-), a modulator of immune system and inhibitor of angiogenesis, inhibits VEGF release from PBMNCs dose dependently
Treatment of cells with IFN- (2 ng/ml) caused a significant decrease in the VEGF secretion by PBMNCs that became detectable after 6 h. The inhibitory effect of 2 ng/ml IFN- on the VEGF secretion was strongest at 12 h and started to diminish toward 24 h. At concentrations of IFN- as low as 2–20 pg/ml discernible decreases in VEGF secretion were detectable. Higher concentrations of IFN- further decreased VEGF secretion in a dose-dependent manner (Fig. 2 ). At the maximum dose tested (20 ng/ml), VEGF production of IFN--treated cells after a 24 h incubation was half of that of nontreated cells (18 vs. 35 pg/10⁶ cells, respectively; Fig. 2 ).

View larger version (16K): [in this window] [in a new window]   Figure 2. Dose-dependent inhibition of secretion of VEGF (pg/106 cells) by PBMN cells treated with IFN-. The PBMNCs were incubated for 24 h with various concentrations of IFN-. The IFN- concentration (ng/ml) is shown on the X-axis (log scale). The values are given as means ± 1 SE of seven independent experiments. *P < 0.05 (the Wilcoxon signed rank test).

6. Treatment of BPMNCs with IFN- suppresses VEGF mRNA levels
After a 14 h incubation with IFN- at 2 or 20 ng/ml, VEGF mRNA levels were decreased to 0.8-fold and 0.5-fold, respectively, compared with those of PBMNCs incubated in the absence of IFN-.

7. IFN- suppresses the endotoxin-induced VEGF production by PBMNCs
IFN- inhibited also the endotoxin-induced VEGF production by PBMNCs when the cells were treated with endotoxin (20 ng/ml) alone or with a combination of endotoxin (20 ng/ml) and IFN- (2 ng/ml). After a 24 h incubation, the VEGF production of endotoxin-treated cells was nearly twofold higher than that of cells treated with the combination of endotoxin and IFN- (157 vs. 90 pg/10⁶ cells, respectively).

CONCLUSIONS AND SIGNIFICANCE

The results of the present study show for the first time that endotoxin promotes de novo VEGF production and release by PBMNCs in the absence of serum. Although our finding must be interpreted and extrapolated to in vivo conditions cautiously, VEGF released from endotoxin-activated leukocytes within the circulation and extravascular tissues may play an important role in the pathogenesis of a variety of acute and chronic infectious and inflammatory disorders. Endotoxins are ubiquitous and occur in healthy subjects at concentrations of up to 20 pg/ml of peripheral blood and 1 ng/ml of portal venous blood, i.e., at concentrations sufficient to promote VEGF production in PBMNCs, as shown in the present study. In patients with sepsis due to gram-negative bacteria, circulating endotoxin levels may increase up to 1 ng/ml of plasma. In patients with systemic inflammation triggered by gram-positive bacteria, which lack endotoxin, or by noninfectious insults such as major trauma or hemorrhagic pancreatitis, intestinal permeability increases, followed by the leakage of endotoxin molecules from the gut into the circulation.

Tissue edema attributable to increased vascular permeability and microvascular sequestration of inflammatory cells are all well-described features of indirect organ injury accompanied by systemic inflammation. Edema is typical of the failing organ in patients with systemic inflammation, but the link between tissue-sequestered leukocytes and edema formation per se is not fully understood. The results of the present study suggest that the mechanism of edema may involve the release of VEGF from circulating and tissue-migrating PBMNCs activated by endotoxins. As long-living cells, emigrating PBMNCs may provide a long-endurance source of VEGF in the end organ.

VEGF and its angiogenic function have previously been connected to the formation of atherosclerotic plaque. VEGF has been shown to be moderately to strongly expressed in atherosclerotic human arteries where smooth muscle cells and extracellular matrix contain extensive VEGF levels. Remarkably, in areas of inflammatory cell infiltration, double immunostaining has identified the prominent T cell infiltrate as responsible for VEGF production. Recently, the angiogenesis inhibitors endostatin or TNP-470 have been shown to be able to reduce intimal neovascularization and plaque growth in mouse model of atheroscerosis. Gram-negative bacteria can cause chronic infections, which maintain inflammation and may predispose an individual to atherosclerosis. In the body, endotoxins occur in tissues at foci of chronic infection. Further studies are now needed to disclose whether different types of bacterial endotoxins may in vivo enhance VEGF production of circulating PBMNCs and PBMNCs infiltrating atherosclerotic vessels, and thus support the development of atherosclerosis.

In the tumor microenvironment, the amount of VEGF may be crucial, as evidenced by the studies with heterozygous and homozygous VEGF-deficient transgenic mice indicating a tight dose-dependent regulation of embryonic vessel development by VEGF. The amount of VEGF in the tumor microenvironment may depend on the VEGF production by tumor cells and other cells at the site, including circulating and emi-grating PBMNCs. The results of the present study suggest that the critical balances governing tumor angiogenesis may be altered by endotoxins in the circulation and in the tumor milieu itself.

Interferons (IFNs) are widely studied modulators of the immune reaction. IFNs have established antitumor action; the mechanisms underlying this effect, however, are not clear. In mice inoculated with tumor cell lines IFN- inhibited immunologically induced angiogenesis, whether initiated by allogeneic lymphocytes or by the mouse’s own T cells in response to an exogenous antigen. In cancer patients, IFNs induce regression of various malignancies, including leukemias, lymphomas, and solid tumors, and increase survival. In patients with rheumatoid arthritis, IFN treatment decreased joint inflammation. In the present study, both basal and endotoxin-induced VEGF production were inhibited by half in the presence of 2 ng/ml IFN-. This ability to inhibit VEGF synthesis of PBMNCs is a novel mechanism that may explain, at least partly, the clinical efficacy of IFN-.

We conclude that tissue endotoxins may promote VEGF production by PBMNCs. Induction of VEGF expression in circulating and tissue emigrating PBMNCs by endotoxin may enhance the shift to angiogenic phenotype in cancer as well as in a variety of nonmalignant disorders designated by excessive angiogenesis. These include inflammatory disorders and vascular events such as atherosclerotic plaque development, ischemia/reperfusion damage, and repair. In addition, VEGF-producing PBMNCs may be a novel mechanism of tissue edema in systemic inflammation triggered by endotoxin. The ability to inhibit VEGF synthesis of PBMNCs may be a novel mechanism explaining in part the clinical efficacy of IFN-.FIGURE 3

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized involvement of endotoxins and IFN- in the angiogenic process and in the regulation of vascular permeability. Red arrows indicate inhibitory pathways; green arrows indicate stimulatory functions, leading to angiogenesis and increased vascular permeability. Induction of VEGF expression in circulating and emigrating PBMNCs by endotoxin may enhance the shift to angiogenic phenotype in a variety of disorders designated by excessive angiogenesis. These include inflammatory conditions and cancer, as well as vascular events such as atherosclerotic plaque development, ischemia/reperfusion damage, and repair. In addition, VEGF-producing PBMNCs may be a novel mechanism of tissue edema in systemic inflammation triggered by endotoxin. The ability to inhibit VEGF synthesis of PBMNCs may be a novel mechanism explaining the clinical efficacy of IFN-.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0627fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0627fje

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