In the present study, the potential correlation between hypoxia and MIF expression was investigated in cultured HUASMCs. The major findings of this study are: (1) hypoxia at 3% O2, but not 5 or 10% oxygen, up-regulates MIF expression in VSMCs; (2) transcription factor HIF-1α is necessary and sufficient in the signaling pathway of MIF induction under hypoxia; (3) enhanced generation of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) might be important in mediating the hypoxia-induced expression of HIF-1α and MIF; (4) blockade of MIF results in the inhibition of hypoxia-induced proliferation and migration in the HUASMC cells. Our data clearly indicate that moderate hypoxia plays a crucial role in the modulation of MIF expression in VSMCs. These observations also establish a role of MIF in mediating hypoxia-induced proliferation and migration in vascular smooth muscle cells. In addition, our results suggest that inhibition of MIF might be useful for preventing abnormal VSMC proliferation and migration evoked by hypoxia.
Both MIF and hypoxia play critical roles in inflammation and atherosclerosis. MIF has been identified as a hypoxia-induced gene in cancer cells. Recent evidences show that MIF is up-regulated by hypoxia (1% O2) in human cancer cells, such as colon tumor cells and breast carcinoma cells . Moreover, MIF mRNA expression is induced in a HIF-dependent manner in HeLa cells . However, by using siRNA approach, Larsen et al reported that MIF regulation in MCF-7 breast cancer cells is due to other hypoxia-induced regulatory mechanisms, independent of HIF-1 and HIF-2 activation. Consistent with previous report , we observed that HIF-1α mRNA and protein expression and DNA binding activity could be induced in HUASMCs exposed to hypoxia stimulation (3% O2). In our experimental model, over-expression of HIF-1α, the key transcription factor activated during hypoxia, was able to up-regulate MIF gene and protein expression under normoxia, whereas knockdown of HIF-1α expression in HUASMCs inhibited hypoxia-induced MIF expression. Our data demonstrate that, in cultured HUASMCs, hypoxia (3% O2) increased MIF expression and synthesis via the HIF-1α pathway, suggesting that the mechanism involved in up-regulation of MIF expression is dependent on cell type.
HIF-1 regulation by non-hypoxic stimuli has gained considerable interest [8, 9]. In addition to angiotensin II and thrombin , studies have showed that HIF-1α stabilization and activation is also induced by several pro-inflammatory cytokines, such as IL-1 and TNFα [25, 26]. As a regulator of MIF expression, HIF-1α might be of potential importance during inflammatory diseases. It is reported that MIF regulates HIF-1 activity in a p53-dependent manner in human cancer cells. Therefore, whether MIF could activate HIF-1α transcriptional activity in VSMCs needs further studies.
Activation of several signaling pathways such as ERK might mediate HIF-1α activation  and MIF expression . By using a specific inhibitor, the present observations demonstrate that hypoxia upregulates MIF via activation of ERK in HUVSMCs. Another pathway implicated in the hypoxia induced response is the generation of intercellular ROS [9, 20]. Our results showed that the antioxidant Tiron, a cell permeable scavenger of ROS, partially blocks MIF production elicited by hypoxia, suggesting that ROS acts as intermediates of hypoxia-induced MIF expression.
VSMCs proliferation and migration that respond to vascular injury contribute to vessel narrowing and play an important role in the atherosclerotic process. It has been recognized that hypoxia is a stimulus to VSMCs proliferation and migration, a process known as the vascular remodeling [29, 30]. Hypoxia plays an important role in vascular remodeling and directly affects VSMCs functions. Some growth factors, such as PDGF  and VEGF , are involved in the hypoxia-induced proliferation of VSMCs. Consistent with previous reports [23, 31], we observed that HIF-1α is essential to VSMC proliferation exposed to hypoxia. Moreover, our results show that the specific MIF-siRNA partially blocks HUASMCs proliferation elicited by hypoxia, suggesting that MIF acts as one of the mediators of hypoxia-induced VSMC proliferation.
In addition to proliferation of VSMCs within the vessel wall, the migration of VSMCs from the media into the neointima is another important feature in the pathogenesis of atherosclerosis [5, 32, 33]. This process is regulated by multiple factors, and MIF is one of the multiple factors that could increase VSMCs migration. MIF is an important mediator of vessel wall remodeling and acts on the migration of VSMCs in an autocrine and paracrine manner. Previous report shows that hypoxia induces the migration of human coronary artery smooth muscle cells, and the migration is elicited by thrombospondin-1. In the present study, treatment of HUASMCs with the specific MIF-siRNA abolished hypoxia-induced migration, suggesting a role for MIF in the migration of VSMCs in response to hypoxia. Altogether, our data demonstrate that HIF-1α and MIF are both important mediators of vascular cell proliferation and migration.