Exposure to cigarette smoke has a negative effect on endothelial function, serum lipid profile, and hemostatic factors [17
]. Smoking plays a central role in the initiation and progression of Buerger's disease, a nonatherosclerotic condition that most commonly affects small and medium-sized arteries and veins as well as nerves of the extremities [18
]. To examine the effects of cigarette smoke on angiogenesis, we treated mice with B[a]P, one of the polycyclic and halogenated aromatic hydrocarbons found in tobacco smoke. Oral exposure to B[a]P resulted in significant inhibition of the increase in blood flow induced by hypoxia in WT mice. Furthermore, we found that this inhibition of angiogenesis by B[a]P was associated with inhibition of hypoxia-induced up-regulation of VEGF expression. Cigarette smoke was previously shown to inhibit secretion of the soluble form of the VEGF receptor Flt-1 in a dose-dependent manner [19
]. Inhibition of the VEGF receptor Flk-1 was also shown to augment the vascular and endothelial dysfunction induced by cigarette smoke [20
]. However, we detected no significant effect of B[a]P on the abundance of Flt-1 or Flk-1 mRNAs in ischemic hindlimb of WT mice in the present study.
MTs are cysteine-rich metal-binding proteins with diverse physiological functions, including protection against metal toxicity and oxidants [21
]. Furthermore, MT expression has been shown to be increased at the transcriptional level by hypoxia acting through metal response elements in the proximal promoter region of MT genes [22
]. In the present study, we detected a marked increase in the expression of MT-1 and MT-2 in the ischemic hindlimb of both AHR-null and WT mice. Moreover, B[a]P induced a further marked increase in MT expression in the ischemic hindlimb of WT mice. This observation is consistent with previous studies showing that MT expression was induced in response to B[a]P exposure [23
]. Such an increase in MT expression may reflect a protective response to B[a]P-induced oxidative stress. We also found that B[a]P induced a decrease in the amount of IL-6 in the ischemic hindlimb of WT mice. IL-6 has been shown to induce MT expression by binding to IL-6-responsive elements in the gene promoter [24
]. On the other hand, the lipopolysaccharide-induced increases in the circulating concentration of IL-6 as well as in the expression of IL-6 in lung, kidney, and liver were markedly greater in mice deficient in MT-1 and MT-2 than in WT mice [25
]. Furthermore, overexpression of MT-1 inhibited up-regulation of ectopic IL-6 expression in astrocytes of transgenic mice [26
]. IL-6 has also been shown to support angiogenesis by up-regulating the expression of VEGF [27
]. The increase in MT expression in the ischemic hindlimb of WT mice induced by B[a]P in the present study may thus result in down-regulation of IL-6 expression, which in turn may lead to impairment of hypoxia-induced angiogenesis.
AHR-null mice are relatively unaffected by the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p
-dioxin (TCDD) at doses that induce severe toxic and pathological effects in WT mice [28
]. Moreover, genotoxic and carcinogenic responses to B[a]P or dibenzo[a,l
]pyrene are greatly diminished in AHR-null mice [29
]. In the present study, the increase in the amount of CYP1A1 mRNA induced by B[a]P in WT mice was not apparent in AHR-null mice, consistent with our finding that AHR deficiency attenuated the inhibition of ischemia-induced angiogenesis by B[a]P. The expression of MT induced by B[a]P in the ischemic hindlimb of WT mice was not detected in that of AHR-null mice. The amount of IL-6 in the ischemic hindlimb of AHR-null mice was not affected by exposure to B[a]P, consistent with the lack of an effect of B[a]P on MT expression and possibly contributing to the diminution of the impairment of ischemia-induced angiogenesis by B[a]P in these mice.
In conclusion, oral exposure to B[a]P resulted in a marked impairment of angiogenesis in response to surgically induced hindlimb ischemia. Furthermore, the impairment of ischemia-induced angiogenesis by B[a]P was greatly reduced in AHR-null mice. Our observations suggest that the loss of the inhibitory effect of B[a]P on ischemia-induced angiogenesis apparent in AHR-null mice may be attributable to maintenance of IL-6 expression and consequent promotion of angiogenesis through up-regulation of VEGF expression.