In 1998, George Vande Woude's lab discovered that anthrax lethal factor (LF), the principal virulence component of anthrax toxin, was a zinc-metalloprotease that cleaved and inactivated mitogen-activated protein kinase kinases (MKK). It was perhaps not surprising, given the known roles of MKK1 and 2 in cell proliferation, that LF was subsequently found to dramatically inhibit tumor growth in vivo. What was not anticipated, however, was that the tumors treated with LF would have a substantially reduced vascular content. This intriguing result was one of the first indications that MKK signaling plays an important role in promoting tumor vascularization in vivo. In the following short review, we will compare in vitro and in vivo evidence that supports the hypothesis that MKK signaling pathways are essential for vascularization.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1-/- mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases.

Lower limb peripheral vascular disease (PVD) results from the occlusion of arteries leading to reduced blood flow and limb ischemia. Compensatory growth of blood vessels may be sufficient to overcome limb ischemia but for some patients therapeutic intervention, even limb amputation, is required. To gain insight into the mechanisms regulating compensatory growth of blood vessels we used a mouse model of hindlimb ischemia. The ischemic lesions were confirmed and compensatory arteriogenesis was evaluated by high frequency power Doppler ultrasound. In this model, compensatory growth occurs mainly through arteriogenesis. While signaling by mitogen activated protein kinase kinases 1 and 2 (MEK 1 and 2) is required for developmental and tumor angiogenesis, it is unknown whether MEK1/2 activity drives other physiologic vascular growth such as arteriogenesis. We hypothesized that MEK 1/2 activity is necessary for hindlimb re-vascularization following femoral artery ligation. To test this we ligated the femoral artery in mice and then treated them with PD0325901, an allosteric MEK1/2 inhibitor. Following femoral artery ligation active MEK signaling was detected in spindle-shaped cells located within regions of intense angiogenesis and myocyte proliferation. MEK inhibition with PD0325901 reversibly blocked neovascularization, muscle regeneration, and caused extensive coagulative necropathy. MEK inhibition not only prevented arteriogenesis in ischemic limbs but also caused a reduction in arterial diameter. Finally, MEK inhibition prevented accumulation of CD68+ cells in ischemic tissues and skewed systemic cytokine expression towards a persistent, pro-inflammatory phenotype. Our investigation provides pharmacologic evidence demonstrating an essential role for MEK signaling in angiogenesis and arteriogenesis in response to hind limb ischemia. Further, we observed activation of MEK signaling is required for accumulation of macrophages in ischemic tissues and prevents down-regulation of systemic pro-inflammatory cytokines. Lastly, since the effects of MEK inhibition on reperfusion are reversible, we can use MEK inhibitors to create a mouse model of chronic limb ischemia that mimics aspects of PVD.