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Interleukin-1 promotes angiogenesis in vivo via VEGFR-2 pathway by inducing inflammatory cell VEGF synthesis and secretion¹

Division of Hematology-Oncology, Weill Medical College of Cornell University, New York, New York, USA

²Correspondence: Division of Hematology-Oncology, Weill Medical College of Cornell University, 1300 York Ave., Room C-606, New York, NY 10021, USA. E-mail: pjs2004@med.cornell.edu; petri.salven{at}helsinki.fi

SPECIFIC AIM

The immune system is thought to have an important role in the regulation of angiogenesis. Interleukin-1 (IL-1) is a prototypical proinflammatory cytokine that has been shown to be strongly angiogenic in in vivo assays for measuring angiogenesis, but the direct mechanism of this angiogenic effect has not been well defined. The inability of IL-1 to stimulate angiogenesis in vitro suggests that IL-1 requires the mediatory activity of accessory cells not present in the in vitro model. We have previously shown that unstimulated human peripheral blood mononuclear cells (PBMNCs) synthesize and release vascular endothelial growth factor (VEGF), a pivotal promoter of physiological and pathological angiogenesis. We now hypothesize that IL-1 may stimulate angiogenesis in vivo by modulating the expression of VEGF in inflammatory cells.

PRINCIPAL FINDINGS

1. IL-1 causes dose-dependent induction of VEGF synthesis and secretion by human PBMNCs
Freshly isolated PBMNCs from healthy donors cultured for 24 h in serum-free medium in the absence of any stimulus released VEGF continuously into the surrounding medium (Fig. 1 a). Stimulation of PBMNCs with IL-1 (4 ng/mL) caused a significant increase in VEGF secretion that was detectable at 12 h. After 24 h stimulation, the mean VEGF production of IL-1-treated cells (84 pg/10⁶ PBMNCs) was > twofold higher than that of unstimulated cells (37 pg/10⁶ PBMNCs; Fig. 1a ). The stimulated (Fig. 1a ) and unstimulated (not shown) release of VEGF was completely blocked by cycloheximide at 1 mM.

View larger version (28K): [in this window] [in a new window]   Figure 1. IL-1 induces the production and secretion of VEGF protein in human PBMNCs. a) Time course of VEGF protein secretion (pg/106 cells) by untreated PBMNCs incubated in serum-free medium (), by PBMNCs treated with IL-1 at 4 ng/mL (•), and PBMNCs treated with a combination of IL-1 (4 ng/mL) and cycloheximide at 1 mM (X). The values are given as means ± 1 SE of 7 independent experiments. b) Dose-dependent secretion of VEGF (pg/106 cells) by PBMNCs stimulated with IL-1. PBMNCs were incubated for 30 h with various concentrations of IL-1. The IL-1 concentration (ng/mL) is shown on the x axis (log scale). Values are given as means ± 1 SE of 5 independent experiments. *P < 0.05, paired t test.

The concentration dependence with which IL-1 increases the secretion of VEGF protein was next characterized. PBMNCs were incubated for 30 h with various concentrations of IL-1 (Fig. 1b ). At a concentration as low as 40 pg/mL, an increase in VEGF secretion could be observed. Higher concentrations of IL-1 further increased VEGF secretion in a dose-dependent manner (Fig 1b ).

2. IL-1 causes dose-dependent induction of VEGF mRNA in human PBMNCs
Northern analyses demonstrated that treatment of PBMNCs with IL-1 results in a significant induction of the major 3.7 kb transcripts of VEGF. A 14 h incubation with IL-1 at 0.4 or 4 ng/mL enhanced VEGF mRNA levels 1.8- and 2.3-fold, respectively, over those in unstimulated cells.

3. Four VEGF mRNA splice variants were detected by RT-PCR in unstimulated and IL-1-stimulated PBMNCs
Amplification of cDNA from unstimulated PBMNCs and cells stimulated for 14 h with IL-1 (4 ng/mL) gave rise to four bands corresponding to the sizes predicted for the transcripts encoding for VEGF121, VEGF165, VEGF189, and VEGF206. The expression pattern of the four VEGF transcripts in IL-1-stimulated cells was comparable to that in unstimulated cells. The mRNAs encoding for the freely diffusible isoforms VEGF121 and VEGF165 gave the major signals in both unstimulated and IL-1-stimulated PBMNCs.

4. IL-1 promotes angiogenesis in vivo in mice via VEGFR-2 pathway
We studied the in vivo effect of IL-1 on angiogenesis in mice injected subcutaneously (s.c.) with murine IL-1 or PBS every other day for 11 days (Fig. 2 ). Evaluation of the skin from the injection site by bright-field microscopy revealed few blood vessels in the control mice injected s.c. with PBS (Fig. 2a ). In contrast, a high number of blood vessels of varying sizes could be seen in the mice injected s.c. with IL-1 (Fig 2b ). The elevated number of s.c. blood vessels in the IL-1-treated mice was evident when the endothelial cells were stained for von Willebrand factor (vWF; Fig. 2b ). The blood vessel number was quantitated by counting vFW-positive vessel elements. It was estimated that mice injected with IL-1 had about fivefold more blood vessels than the control mice injected s.c. with PBS (59±18 vs. 12±4 vascular elements/field, respectively; P=0.01). High numbers of inflammatory cells expressing the leukocyte common antigen CD45 and VEGF were observed at the injection site in the mice treated with IL-1 (Fig. 2b ). In contrast, only a few cells positive for CD45 or VEGF could be seen in the control mice treated with PBS (Fig. 2a ). The IL-1-induced blood vessel growth was blocked when the mice were treated with VEGFR-2-blocking mAbs (Fig. 2c ). Mice injected with IL-1 and VEGFR-2-blocking mAbs had vascular densities comparable to those in the control mice treated with PBS (13±5 vs. 12±4, respectively). Treatment with blocking mAbs against VEGFR-1 had a marginal inhibiting effect on the angiogenic effect caused by the IL-1 injections; when compared to the mice treated with IL-1 alone, the difference failed to reach statistical significance (Fig. 2d ; 46±11 vs. 59±18, respectively; P >0.05). When used in combination with anti-VEGFR-2 mAbs, anti-VEGFR-1 mAbs were unable to add significant antiangiogenic effect to the effect of anti-VEGFR-2 mAbs used alone (Fig. 2e ; 12±5 vs. 13±5, respectively; P>0.05). In all mice injected s.c. with IL-1, an infiltrate of VEGF- and CD45-expressing cells was observed regardless of the treatment with VEGFR-blocking mAbs (not shown).

View larger version (47K): [in this window] [in a new window]   Figure 2. In vivo model of IL-1-induced angiogenesis in mice. Bright-field microscopy pictures of 11 x 5 mm sections of the skin are shown, with the injection sites at the center; x4. Immunohistochemical stainings for the endothelial cell marker von Willebrand factor (vWF) of the corresponding tissue samples are shown; x23. a) Only a low number of blood vessels are seen in mice injected s.c. with PBS. Likewise, only a few cells positive for the leukocyte common antigen CD45 or VEGF can be seen (inserts, x18). b) Numerous blood vessels can be seen in the mice injected s.c. with IL-1. The high number of blood vessels is evident when the endothelial cells are stained for vWF. Numerous inflammatory cells expressing CD45 and VEGF are seen (inserts, x18). c) IL-1-induced activation of angiogenesis was blocked when the mice were treated with VEGFR-2-blocking monoclonal antibodies. d) Treatment with blocking mAbs against VEGFR-1 had a marginal inhibiting effect on local angiogenesis caused by IL-1 injections. e) Treatment of mice with the combination of mAbs against VEGFR-2 and VEGFR-1. The anti-VEGFR-1 mAbs were unable to add a significant antiangiogenic effect to the effect of anti-VEGFR-2 mAbs used alone.

CONCLUSIONS AND SIGNIFICANCE

Excessive angiogenesis is integral to the pathology of conditions such as cancer, cardiovascular diseases (atherosclerosis), and chronic inflammation (rheumatoid arthritis, inflammatory bowel disease). The immune system is thought to have an important role in the regulation of angiogenesis. In the acute phase of inflammation, functional changes in the vasculature such as dilatation, increase in permeability, extensive endothelial cell mitotic activity, and remodeling of capillaries occur. Upon chronic stimulation, increases in capillary density and vascular dilatation can be observed. Recent studies have begun to reveal the nature of the link between inflammation and angiogenesis, which involves both augmentation of cellular infiltration and proliferation and overlapping roles of regulatory growth factors and cytokines.

The link between inflammation and angiogenesis may play an important role in atherosclerosis and cancer. An immune and inflammatory response accompanies the accumulation of lipids and fibrous materials in atheromatous arteries. This inflammatory response involves circulating leukocytes that participate in the disease process. Chronic inflammation is also a common feature of human neoplasia. The inflammatory microenvironment of tumors is characterized by the presence of host leukocytes in the supporting stroma and tumor areas, which may contribute to cancer growth and spread.

In the host, the angiogenic phenotype is determined by the balance between pro- and antiangiogenic molecules, which derive from various cells, including blood cells and stromal cells. The inability of IL-1 to stimulate angiogenesis in vitro suggests that the angiogenic activity of IL-1 observed in vivo requires the mediatory activity of accessory cells and/or molecules. IL-1 occurs in healthy humans at concentrations up to 5 ng/mL of peripheral blood, i.e., at concentrations sufficient to promote VEGF production in inflammatory cells, as shown in the present study. Other cell types might also contribute to IL-1-induced VEGF-mediated angiogenesis. However, in our animal model the inflammatory cells at the IL-1-injection site were the dominant cell type expressing VEGF. In vivo angiogenic activity of IL-1 could be completely inhibited using neutralizing antibodies against VEGFR-2, demonstrating that signaling via VEGFR-2 was required for the angiogenic effect of IL-1. VEGFR-1-blocking antibodies appeared to provide some reduction of angiogenesis and vascular branching at the IL-1 injection site, although the effect was not statistically significant in our series. This leaves open the possibility that signaling via VEGFR-1 on vascular endothelial cells, vascular smooth muscle cells, and/or inflammatory cells might contribute to angiogenesis in our model.

Taken together, our present data indicate that IL-1 promotes angiogenesis by activating the VEGF-VEGFR-2 signaling pathway between inflammatory cells and blood vessel endothelial cells. This novel mechanism of IL-1 action may play a role in several acute and chronic conditions with increased angiogenesis and/or vascular permeability. These include cancer, infections, inflammatory disorders including rheumatoid arthritis, and vascular events such as atherosclerotic plaque development.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic illustration of the involvement of IL-1 in angiogenesis. After stimuli produced by inflammation, infections, trauma, or ischemia, IL-1 is released into a local environment. IL-1 in turn activates the synthesis and secretion of VEGF by inflammatory cells, thereby promoting angiogenesis via VEGFR-2 on blood vessel endothelial cells. This mechanism of IL-1 action may play a role in several acute and chronic conditions with increased angiogenesis and/or vascular permeability. These include cancer, infections, inflammatory disorders (including rheumatoid arthritis), and vascular events such as atherosclerotic plaque development.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0134fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.02-0134fje

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