Deletion of FHL2 gene impaired ischemia-induced blood flow recovery by modulating circulating proangiogenic cells.

OBJECTIVE: The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in inhibiting inflammatory angiogenesis. Here, we tested the hypothesis that impaired ischemia-induced neovascularization in mice lacking FHL2 is related to a defect in proangiogenic cell mobilization and functions in vasculogenesis. APPROACH AND RESULTS: Unilateral hindlimb ischemia surgery was conducted in FHL2(-/-) mice and wild-type (FHL2(+/+)) mice. After hindlimb ischemia surgery, expression of FHL2 protein was noted in ischemic tissues of wild-type mice. All FHL2-null mice (100%) suffered from spontaneous foot amputation, but only 20% of wild-type mice had ischemia-induced foot amputation after ischemic surgery. Blood flow recovery was significantly impaired in FHL2(-/-) mice when compared with that in wild-type mice as determined by laser Doppler imaging. Histological analysis revealed that the capillary density in the ischemic limb was increased in wild-type mice, whereas no such increase was noted in FHL2(-/-) mice. Flow cytometry demonstrated that the number of CD34(+) or CD34(+)/Sca-1(+)/Flk-1(+) in peripheral blood after ischemic surgery significantly decreased in FHL2-null mice than those in wild-type mice after hindlimb ischemia surgery. FHL2 deficiency impaired ex vivo angiogenesis in mouse aortic-ring culture assay, which revealed that the mean density of the microvessels was significantly higher in the wild-type aorta than in the FHL2(-/-) aorta. Western blot analysis showed that vascular endothelial growth factor (VEGF), interleukin-6, matrix metalloproteinase-2, matrix metalloproteinase-9, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 levels were significantly downregulated in ischemic muscles in FHL2-null mice compared with wild-type mice. Deletion of FHL2 protein by FHL2 small interfering RNA impaired VEGF production under hypoxia conditions, and also suppressed endothelial progenitor cell angiogenic functions, but these effects could be recovered by administration of VEGF. CONCLUSIONS: Deficiency of FHL2 impairs ischemia-induced neovascularization, and these suppressive effects may occur through a reduction in proangiogenic cell mobilization, migration, and vasculogenesis functions.

Matrix metalloproteinase-9 is essential for ischemia-induced neovascularization by modulating bone marrow-derived endothelial progenitor cells.

OBJECTIVE: Both matrix metalloproteinases (MMPs) and endothelial progenitor cells (EPCs) have been implicated in the process of neovascularization. Here we show that the impaired neovascularization in mice lacking MMP-9 is related to a defect in EPC functions in vasculogenesis. METHODS AND RESULTS: Hindlimb ischemia surgery was conducted in MMP-9(-/-) mice and wild-type (MMP-9(+/+)) mice. Blood flow recovery was markedly impaired in MMP-9(-/-) mice when compared with that in wild-type mice as determined by laser Doppler imaging. Flow cytometry demonstrated that the number of EPC-like cells (Sca-1(+)/Flk-1(+)) in peripheral blood increased in wild-type mice after hindlimb ischemia surgery and exogenous vascular endothelial growth factor stimulation, but not in MMP-9(-/-) mice. Plasma levels and bone marrow concentrations of soluble Kit-ligand (sKitL) were significantly elevated in wild-type mice in response to tissue ischemia, but not in MMP-9(-/-) mice. C-kit positive bone marrow cells of MMP-9(-/-) mice have attenuated adhesion and migration than those isolated from wild-type mice. In in vitro studies, incubation with selective MMP-9 inhibitor suppressed the colony formation, migration, and tube formation capacities of EPC. Transplantation of bone marrow cells from wild-type mice restored collateral flow formation in MMP-9(-/-) mice. CONCLUSIONS: These findings suggest that MMP-9 deficiency impairs ischemia-induced neovascularization, and these effects may occur through a reduction in releasing the stem cell-active cytokine, and EPC mobilization, migration, and vasculogenesis functions.

Hedgehog is involved in prostate basal cell hyperplasia formation and its progressing towards tumorigenesis.

The role of Hedgehog signaling in human basal cell hyperplasia formation and its progressing towards tumorigenesis was investigated. Hedgehog signaling members including PTCH1, GLI1, GLI2, and GLI3 were found co-localized with p63 expression in most hyperplastic basal cells, but rarely in normal basal cells, suggesting Hedgehog involvement in basal cell hyperplasia formation. Both CK-14 and CK-8 markers were found co-localized in the majority of hyperplastic basal cells, but relatively few in the normal basal cells, indicating a Hedgehog-promoted transitory differentiation. Furthermore, CK-14 and PTCH1 were found co-localized with CD44 in the hyerplastic basal cells, in a way similar to the CD44 co-localization with PTCH1 and GLI1 in the cancer cells. Together, the present study indicated Hedgehog involvement in forming basal cell hyperplasia and its progressing towards cancer, presumably by transforming the normal basal stem cells into the cancer stem cells where persistent Hedgehog activation might be mandatory for tumorigenesis.