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Inhibition of hypoxia-induced angiogenesis by cigarette smoke exposure: impairment of the HIF-1/VEGF pathway¹

Department of Cardiovascular Research, Centre Hospitalier de l'Université de Montréal, Montréal, Québec, Canada

²Correspondence: Centre Hospitalier de l'Université de Montréal, 1560 Sherbrooke Est, Suite Y-3605, Montreal, Quebec, H2L 4M1, Canada. E-mail: rivardal{at}total.net

SPECIFIC AIMS

Cigarette smoking is the leading modifiable risk factor associated with premature death. This increased mortality is mainly attributed to the effect of cigarette smoke exposure on promoting the incidence of ischemic vascular diseases. Although the association between smoking and atherosclerosis is well established, the precise mechanisms involved are not completely understood. Moreover, the effects of smoking on compensatory mechanisms such as angiogenesis in the setting of severe vascular obstructions and tissue ischemia are currently unknown. The aim of this study was to investigate the effect of cigarette smoking on the development of angiogenesis in response to vascular insufficiency and hypoxia. Because of the importance of HIF-1 and VEGF on the development of angiogenesis in response to hypoxia, we also studied the effect of cigarette smoke exposure on the expression of these factors.

PRINCIPAL FINDINGS

1. Cigarette smoke exposure impairs hypoxia-induced angiogenesis
We investigated the effect of cigarette smoke exposure on angiogenesis in vitro and in vivo. As determined using the Matrigel assay, 10% cigarette smoke extracts (CSE) exposure was associated with an important inhibition of hypoxia-induced cellular migration and capillary-like tubules formation in HUVECs (Fig. 1 ). In vivo, blood flow perfusion in surgically induced ischemic hind limbs was significantly reduced in mice exposed to cigarette smoke (MES) compared with control mice (P<0.05) (Fig. 1C ). The capillary density in ischemic muscles was also significantly reduced in MES compared with controls (P<0.01) (Fig. 1D ).

View larger version (52K): [in this window] [in a new window]   Figure 1. A) Inhibition of hypoxia-induced angiogenesis by smoke exposure in vitro. HUVECs formed capillary-like structures when cultured under normoxia for 24 h on growth factor-reduced Matrigel in the absence of serum or exogenous growth factors. Formation of capillary structures was further increased by hypoxia but inhibited by 10% cigarette smoke extract (CSE). One representative experiment of 3 is shown. B) Inhibition of hypoxia-induced migration of HUVECs by CSE (modified Boyden chamber). Cells that had migrated were counted in 3 representative fields. Assays were conducted in triplicate and repeated 3 times. Data are presented as the mean ± SE, **P < 0.001. C, D) Inhibition of ischemia-induced angiogenesis by smoking in vivo. Angiogenesis was evaluated in a murine ischemic hind limb model. C) Representative results of laser Doppler measurements recorded at serial times after surgery in control mice (n=15) and MES (n=16, left panel). A color scale illustrates blood flow variations from minimal (dark blue) to maximal (red) values. NI indicates nonischemic (right) limb; T, tail; and I, ischemic (left) limb. The right panel represents laser Doppler perfusion ratio over time after surgery in control mice and MES. Data are presented as the mean ± SE, *P < 0.05 vs. control mice. D) CD31 immunostaining of ischemic muscles from control mice and MES at time of death (28 days) showed a significant reduction in the capillary density in MES. Data are presented as the mean ± SE, *P < 0.05 vs. control mice.

2. The inhibitory action of cigarette smoke exposure on hypoxia-induced angiogenesis is associated with a reduced expression of VEGF and HIF-1
In HUVECs, VEGF and HIF-1 are significantly induced after 6 h of hypoxia; in the presence of CSE, expression of VEGF and HIF-1 under hypoxic conditions is significantly reduced (Fig. 2 A, P<0.05). In ischemic hind limbs, VEGF and HIF-1 are markedly induced from day 3 to day 14 after surgery. However, the expression of these proteins in ischemic muscles is significantly reduced in MES vs. controls (P<0.01) (Fig. 2B ). Pulse-chase experiments showed that CSE reduces hypoxia-induced HIF-1 protein half-life in HUVECs (Fig. 2C ).

View larger version (46K): [in this window] [in a new window]   Figure 2. A–C) Smoke exposure inhibits hypoxia-induced protein expression of VEGF and HIF-1 in vitro and in vivo. A) Western blot analysis of VEGF and HIF-1 (top panel) in HUVECs after 6 h under various conditions. VEGF indicates VEGF-specific band (46 kDa). VEGF and HIF-1 density values were normalized to ß-actin (middle panel). Values are mean ± SE, *P < 0.05, **P < 0.001. EMSA of HIF-1 in HUVECs after 6 h under various conditions (bottom panel). One representative experiment of 4 is shown. B) Western blot analysis of VEGF and HIF-1 (top panel) in ischemic muscles harvested at different times after surgery in MES and control mice. VEGF and HIF-1 density values normalized to ß-actin (middle panel). Values are mean ± SE, *P < 0.05, **P < 0.001. EMSA of HIF-1 in ischemic muscles harvested at different times after surgery in MES and control mice (bottom panel). One representative experiment of 4 is shown. C) Pulse-chase metabolic labeling with [S35]methionine in HUVECs shows that CSE decreases HIF-1 half-life under hypoxic conditions. D–G) Ad.HIF-1/VP16 restores VEGF expression and reverses the cigarette smoke inhibition of angiogenesis. D) Western blot analysis of HIF-1/VP16 and VEGF in HUVECs infected with Ad.HIF-1/VP16 or Ad5.Null (control) after 6 h. E) Western blot analysis of HIF-1/VP16 and VEGF in ischemic muscles harvested at different times in mice locally infected with Ad.HIF-1/VP16 or Ad5.Null. F) Laser-Doppler perfusion ratios over time and capillary density analysis on day 28 after surgery (G) in mice locally infected with Ad.HIF-1/VP16 (n=6) or Ad5.Null (n=6). Data are presented as the mean ± SE, *P < 0.05 vs. Ad5.Null mice.

3. HIF-1/VP16 gene transfer completely reverses the cigarette smoke inhibition of VEGF expression and angiogenesis in hypoxic conditions
We used an adenoviral vector encoding for HIF-1/VP16, a hybrid transcription factor that is stable in normoxic and hypoxic conditions, to rescue cigarette smoke-related impairment of VEGF expression and angiogenesis in vitro and in vivo. HUVECs infected with this vector show high levels of HIF-1/VP16 and VEGF protein expression that are not influenced by oxygen concentration or CSE exposure (Fig. 2D ). VEGF protein up-regulation by Ad. HIF-1/VP16 in HUVECs increases capillary-like tubules formation under normoxic conditions and rescues the CSE inhibition of in vitro angiogenesis under hypoxic conditions. In vivo, MES injected intramuscularly with Ad.HIF-1/VP16 at the time of surgery demonstrate increased VEGF expression in ischemic muscles compared with MES injected with Ad5.Null (Fig. 2E ). This leads to a significant increase in blood flow perfusion in MES treated with Ad. HIF-1/VP16 vs. Ad5.Null (P<0.05) (Fig. 2F ). A significant increase in capillary density in the ischemic muscles of MES injected with Ad. HIF-1/VP16 was also documented (P<0.01) (Fig. 2G ).

CONCLUSIONS

Hypoxia is a major factor involved in tissue injury following vascular insufficiency and ischemia. Hypoxia also triggers compensatory mechanisms that are important for tissue protection, such as angiogenesis. In the setting of severe vascular obstructions with reduced blood flow, the angiogenic response is seen as an attempt by the organism to improve perfusion and preserve tissue integrity. The possibility of increasing the "natural" angiogenic response in order to improve tissue perfusion has been tested in animal models and in recent clinical trials.

Here we show that HUVECs exposed to CSE lose their ability to organize in a capillary-like tubule network in response to hypoxia. In vivo, we used a model of hind-limb ischemia to investigate the effect of cigarette smoke exposure on angiogenesis. We found that smoking significantly impairs blood flow recuperation (laser-Doppler imaging) after surgically induced ischemia. At the microvascular level, smoking was associated with an important reduction of capillary density in ischemic muscles. Therefore, our study is the first to document the negative effect of smoking on postnatal angiogenesis in the context of ischemic vascular diseases.

The mechanisms by which smoking impairs angiogenesis are potentially diverse. Angiogenesis is a complex process that includes activation, migration and proliferation of endothelial cells. VEGF has been shown to be a critical factor for the induction of angiogenesis. In vascular ischemia, the angiogenic response is dependent on the stabilization of HIF-1 by hypoxia and the subsequent transcriptional activation of VEGF by HIF-1. Here we showed that cigarette smoke exposure significantly reduces the expression of VEGF in HUVECs under hypoxic conditions and in ischemic muscles. Cigarette smoke exposure was also associated with a reduction in HIF-1 expression, DNA binding activity, and protein half-life under hypoxic conditions. To compensate for the defect in HIF-1 expression with cigarette smoke exposure, we used an adenoviral vector encoding for HIF-1/VP16, a hybrid transcription factor that is stable in normoxic and hypoxic conditions. We found that this vector could completely rescue the cigarette smoke-related impairment of VEGF expression under hypoxic conditions in vitro and in vivo. Moreover, smoking-induced defect in angiogenesis after hypoxia was reversed in HUVECs and in ischemic muscles treated with the HIF-1/VP16 vector.

The exact components of cigarette smoke responsible for the impairment of angiogenesis are unknown. Cigarette smoke is composed of nearly 4000 different chemicals, many of which (carbon monoxide, cadmium, hydrocarbons, acetaldehyde, etc.) are toxic to endothelial cells and detrimental to health. Carbon monoxide has been shown to destabilize HIF-1 and suppress the activation of target genes. This could help explain the findings of the present study. Nicotine, on the other hand, was shown to promote angiogenesis in different models. Nonetheless, our experiments suggest that the cumulative effect of the different components of cigarette smoke has a negative effect on angiogenesis.

In summary, our study demonstrates for the first time that smoking has a detrimental effect on the protective angiogenic response in the setting of vascular insufficiency and hypoxia. We propose that cigarette smoke exposure 1) impairs HIF-1 stabilization and accumulation under hypoxic conditions, leading to 2) down-regulation of hypoxia-induced VEGF expression, which in turn results in 3) reduced angiogenesis in response to hypoxia (Fig. 3 ). We also show that the cigarette smoke inhibition of angiogenesis can be reversed by the administration of an adenoviral vector encoding for a stable form of HIF-1. These findings could have important clinical implications for patients with severe ischemic vascular diseases who are exposed to cigarette smoke. The elucidation of specific mechanisms that negatively modulate angiogenesis could lead to the development of novel therapeutic strategies to protect against hypoxia and maintain tissue integrity.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic representation of the antiangiogenic actions of cigarette smoke. Under hypoxia, HIF-1 accumulates and triggers the transcription and protein expression of VEGF, which promotes angiogenesis. In the presence of cigarette smoke, HIF-1 accumulation is compromised, causing reduced VEGF expression and inhibition of hypoxia-induced angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0172fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0172fje

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