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von Hippel Lindau tumor suppressor and HIF-1: new targets of NSAIDs inhibition of hypoxia-induced angiogenesis ¹

MICHAEL K. JONES^*, IMRE L. SZABÓ^*, HIROFUMI KAWANAKA, SYEDA S. HUSAIN^* and ANDRZEJ S. TARNAWSKI^*2

Departments of
^* Medicine and
Surgery, Veterans Affairs Medical Center, Long Beach, California 90822, and University of California, Irvine, California 92697, USA

²Correspondence: DVA Medical Center, 5901 East Seventh St., Long Beach, CA 90822, USA. E-mail: atarnawski{at}yahoo.com

SPECIFIC AIMS

Nonsteroidal anti-inflammatory drugs (NSAIDs, inhibitors of cyclooxygenase enzymes that synthesize prostaglandins) inhibit angiogenesis essential for tissue repair and colon cancer growth. The mechanisms of NSAIDs inhibition of angiogenesis remain unexplained. Since hypoxia is a major stimulus for angiogenesis, the aim of this study was to determine the molecular mechanism(s) of NSAIDs inhibition of hypoxia-induced angiogenesis in gastric microvascular endothelial cells (RGMEC). We studied whether NSAIDs affect expression levels of the hypoxia-inducible transcription factor-1 (HIF-1) and/or the von Hippel Lindau tumor suppressor (VHL). The rationale for this study is that HIF-1 and VHL control hypoxia-induced expression of VEGF (the most potent angiogenic factor) and its specific receptor, Flt-1, major mediators of hypoxia-induced angiogenesis.

PRINCIPAL FINDINGS

1. NSAIDs impair hypoxia-induced angiogenesis by inhibiting hypoxia-induced expression of VEGF and its specific receptor, Flt-1
We investigated whether NSAIDs inhibit hypoxia-induced angiogenesis and/or hypoxia-induced VEGF/Flt-1 expression in RGMEC cells. Hypoxia strongly induced in vitro angiogenesis in RGMEC by 271%, and this induction was completely inhibited by both a nonselective NSAID (indomethacin) and a COX-2-selective inhibitor (NS-398) (Table 1 ). Indomethacin and NS-398 also significantly inhibited hypoxia-induced VEGF and Flt-1 expression at both the transcriptional and translational levels (Fig. 1 C–F).

View larger version (42K): [in this window] [in a new window]   Figure 1. A, B) NSAIDs inhibit hypoxia-induced expression of HIF-1 but do not alter expression of HIF-1ß. A) HIF-1 protein. expression after 6 h under hypoxia. *P < 0.004 vs. normoxia control; #P < 0.0001 vs. hypoxia control. B) HIF-1ß protein expression after 6 h. C–F) NSAIDs inhibit hypoxia-induced expression of VEGF and Flt-1 mRNA and protein. C) Upper panel: VEGF mRNA after 6 h under hypoxia by RT-PCR. Middle panel: ß-actin used as an internal control. Left lane shows 100 bp ladder as size marker. Lower panel: Quantitative data of the relative expression levels of VEGF mRNA. *P < 0.00001 vs. normoxia control; #P < 0.006 vs. hypoxia control. D) Upper panel: Flt-1 mRNA after 6 h under hypoxia. Middle panel: ß-actin used as an internal control. Left lane shows 100 bp ladder as size marker. Lower panel: Quantitative data of the relative expression levels of Flt-1 mRNA. *P < 0.001 vs. normoxia control; #P < 0.002 vs. hypoxia control. Data are expressed as the ratio of the VEGF or Flt-1 product to ß-actin product. E) Upper panel: VEGF165 protein levels by immunoblot analysis after 6 h under hypoxia. Lower panel: Quantitative data of the relative expression levels of VEGF165 protein from RGMEC cells cultured under the various conditions. *P < 0.04 vs. normoxia control #P < 0.03 vs. hypoxia control. F) Upper panel: Flt-1 protein expression after 6 h under hypoxia. Lower panel: Quantitative data of the relative expression levels of Flt-1 protein from RGMEC cells cultured under the various conditions. *P < 0.03 vs. normoxia control; #P < 0.03 vs. hypoxia control. G–H) NSAIDs induce increased expression of VHL and increase protein ubiquitination under the condition of hypoxia. G) Upper panel: VHL protein expression after 6 h under hypoxia. Lower panel: Quantitative data of the relative expression levels of VHL protein from RGMEC cells cultured under the various conditions. *P < 0.01 vs. normoxia control; #P < 0.02 vs. hypoxia control. H) Left panel: Total ubiquitinated protein after 6 h under hypoxia. Whole cell lysates of cells cultured under normoxia (lane 1); hypoxia (lane 2); hypoxia + indomethacin (lane 3); hypoxia + NS-398 (lane 4). Right panel: Quantitative data of the relative amounts of ubiquitinated protein from RGMEC cells cultured under the various conditions. *P < 0.002 vs. normoxia control; #P < 0.002 vs. hypoxia control. Data represent the mean ± SD of 5 independent determinations, each performed in triplicate.

2. The inhibitory action of NSAIDs on hypoxia-induced angiogenesis and VEGF/Flt-1 expression is mediated by inhibition HIF-1 accumulation
We investigated the effect of NSAIDs on hypoxia-induced HIF-1 accumulation. Immunoblot analysis revealed that hypoxia strongly induced accumulation of HIF-1 by 450% within 6 h (Fig. 1A ). Treatment with indomethacin significantly inhibited the hypoxia-induced accumulation of HIF-1 by 66% (P<0.0001) whereas NS-398 completely abolished it (P<0.0001) (Fig. 1A ). This inhibition was not the result of a general decrease in protein stability under hypoxia since neither NSAID affected the expression of HIF-1ß, which is not regulated by hypoxia (Fig. 1B ).

3. NSAIDs up-regulate VHL expression and protein ubiquitination under conditions of hypoxia
Since VHL is known to target HIF-1 for ubiquitination and proteasomal degradation, we investigated the effects of NSAIDs on VHL expression and protein ubiquitination under hypoxia. Hypoxia itself strongly down-regulated VHL expression (Fig. 1G ). Indomethacin and NS-398 significantly increased VHL expression in RGMEC cells cultured under hypoxia as early 1 h, with maximal increases of 400% (P<0.002) and 320% (P<0.002), respectively, at 6 h (Fig. 1G ). The NSAIDs-induced expression of VHL under hypoxia was sustained for up to 16 h (data not shown). Under the condition of hypoxia, indomethacin and NS-398 both caused significant increases in total protein ubiquitination by 44% (P<0.002) and 57% (P<0.002), respectively, compared with hypoxia alone (Fig. 1H ). We were unable to demonstrate increased ubiquitination of HIF-1 specifically, because HIF-1 is degraded immediately upon ubiquitination.

CONCLUSIONS

In the present study, we have shown for the first time that both nonselective and COX-2-selective NSAIDs inhibit hypoxia-induced in vitro angiogenesis in gastric microvascular endothelial cells via increased expression of the VHL tumor suppressor protein, which in turn leads to increased ubiquitination of total protein. As a result, NSAIDs inhibit accumulation of HIF-1 (which is rapidly degraded on ubiquitination) under conditions of hypoxia resulting in inhibition of hypoxia-induced VEGF/Flt-1 expression. Since HIF-1 is the major trigger for hypoxia-induced activation of the VEGF and Flt-1 genes, this could explain how NSAIDs inhibit angiogenesis. Exogenous VEGF and, to a lesser extent, exogenous prostaglandins partly reversed the NSAIDs inhibition of hypoxia-induced angiogenesis. Inhibition of hypoxia-induced angiogenesis was also partly reversed by the antibiotic peptide PR39, known to prevent degradation of HIF-1, further indicating that NSAIDs inhibit hypoxia-induced angiogenesis by preventing HIF-1 accumulation under hypoxia.

We have previously demonstrated that the mitogen-activated protein kinase ERK2 is a molecular target for the inhibition of in vitro angiogenesis by NSAIDs. It is clear that inhibition of hypoxia-induced angiogenesis by NSAIDs is a complex process and there now appear to be several targets for the inhibitory effects of NSAIDs on angiogenesis, including HIF-1 and VHL, as demonstrated here. Indeed, nonselective NSAIDs are known to elicit several cellular effects in addition to inhibition of COX activity. Even though exogenous prostaglandins (28 nM PGE₂ and 27 nM PGI₂) partly reversed the inhibition of hypoxia-induced angiogenesis, indicating a prostaglandin-dependent component, inhibition of hypoxia-induced angiogenesis caused by NSAIDs cannot be entirely accounted for by inhibition of COX activity alone. The COX-independent nature of the inhibitory action of NSAIDs on angiogenesis is uncertain. COX-2 is strongly induced by hypoxia, and at least PGE₂ may be involved in hypoxia-induced VEGF expression.

In this study, we have also shown that hypoxia induces apoptosis in gastric microvascular endothelial cells, which is further increased by NSAIDs. Although the NSAIDs-induced increase in apoptosis may contribute to the inhibition of hypoxia-induced angiogenesis, it is unlikely to be the primary factor since the magnitude of this increase (to a maximum of 3.2%±0.5% of the total cell population) was not sufficient to account for the dramatic inhibition of hypoxia-induced angiogenesis.

The rapid growth of solid tumors is limited by the amount of oxygen and nutrients the surrounding vasculature is able to provide and therefore is dependent on angiogenesis. VEGF expression is strongly induced by hypoxia in certain colon cancer cells such as the human colon adenocarcinoma HCT116. This action is mediated by increased HIF-1 levels. Down-regulation of VHL expression by histone deacetylases has been shown to induce angiogenesis by increasing HIF-1 levels, leading to increased VEGF expression. In the present report, we have shown that NSAIDs have the opposite action. They increase VHL expression, which leads to decreased HIF-1 accumulation, hypoxia-induced VEGF/Flt-1 expression, and inhibition of hypoxia-induced angiogenesis.

In summary, our study demonstrates that NSAIDs inhibit hypoxia-induced angiogenesis by 1) increasing VHL expression, leading to 2) increased ubiquitination and degradation of HIF-1, which in turn results in 3) down-regulation of hypoxia-induced VEGF/Flt-1 expression. The latter leads to reduced angiogenesis in response to hypoxia. These events are summarized in Fig. 2 . Our findings have potential implications for wound healing and cancer growth, which both depend critically on angiogenesis.

View larger version (29K): [in this window] [in a new window]   Figure 2. Diagrammatic representation of the anti-angiogenic actions of NSAIDs. Under hypoxia, VHL expression levels are suppressed leading to HIF-1 accumulation, VEGF/Flt-1 expression, and angiogenesis. In the presence of NSAIDs, VHL is up-regulated leading to increased ubiquitination and degradation of HIF-1, causing reduced VEGF/Flt-1 expression and inhibition of hypoxia-induced angiogenesis.

Our results further support the concept that 1) VHL targets HIF-1 for ubiquitination/degradation; 2) HIF-1 is an important mediator of hypoxia-induced VEGF/Flt-1 gene expression; and 3) this expression is required for hypoxia-induced angiogenesis. Our findings demonstrate that VHL and HIF-1 are novel targets of both nonselective and COX-2-selective NSAIDs that lead to inhibition of hypoxia-induced angiogenesis. The increase in VHL expression under hypoxia that results in decreased HIF-1 accumulation and VEGF/Flt-1 expression likely represents a primary molecular mechanism by which NSAIDs inhibit both the physiological angiogenesis crucial to gastrointestinal wound/ulcer healing and the pathological angiogenesis essential to the growth and metastasis of colon cancer.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0589fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0589fje

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