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Objective— Angiogenesis is the formation of new blood vessels through endothelial cell sprouting. This process requires the mitogen-activated protein kinases, signaling molecules that are negatively regulated by the mitogen-activated protein kinase phosphatase-1 (MKP-1). The purpose of this study was to evaluate the role of MKP-1 in neovascularization in vivo and identify associated mechanisms in endothelial cells. Approach and Results— We used murine hindlimb ischemia as a model system to evaluate the role of MKP-1 in angiogenic growth, remodeling, and arteriogenesis in vivo. Genomic deletion of MKP-1 blunted angiogenesis in the distal hindlimb and microvascular arteriogenesis in the proximal hindlimb. In vitro, endothelial MKP-1 depletion/deletion abrogated vascular endothelial growth factor–induced migration and tube formation, and reduced proliferation. These observations establish MKP-1 as a positive mediator of angiogenesis and contrast with the canonical function of MKP-1 as a mitogen-activated protein kinase phosphatase, implying an alternative mechanism for MKP-1–mediated angiogenesis. Cloning and sequencing of MKP-1–bound chromatin identified localization of MKP-1 to exonic DNA of the angiogenic chemokine fractalkine, and MKP-1 depletion reduced histone H3 serine 10 dephosphorylation on this DNA locus and blocked fractalkine expression. In vivo, MKP-1 deletion abrogated ischemia-induced fractalkine expression and macrophage and T-lymphocyte infiltration in distal hindlimbs, whereas fractalkine delivery to ischemic hindlimbs rescued the effect of MKP-1 deletion on neovascular hindlimb recovery. Conclusions— MKP-1 promoted angiogenic and arteriogenic neovascular growth, potentially through dephosphorylation of histone H3 serine 10 on coding-region DNA to control transcription of angiogenic genes, such as fractalkine. These observations reveal a novel function for MKP-1 and identify MKP-1 as a potential therapeutic target.

Physical cues such as the mechanical properties of the extracellular matrix are known to modulate blood vessel formation, but the underlying mechanisms remain poorly understood. Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) were recently identified as key mediators of cellular mechanotransduction [1], but the roles of YAP and TAZ in neovascular mechanobiology have not been investigated. We hypothesized that YAP and TAZ mediate neovascular mechanotransduction of extracellular matrix rigidity by vasculogenic endothelial colony forming cells (ECFCs) cultured on/in matrices of controllable rigidity. YAP and TAZ depletion abrogated ECFC migration (Fig. 1A), and YAP/TAZ expression and nuclear localization (i.e., activation) was significantly upregulated in migrating cells (Fig. 1B). Combinatorial depletion also abrogated tubular network formation and 3D vasculogenesis in collagen matrices of variable rigidity but constant matrix density (data not shown). In 2D, PDMS rigidity regulated YAP/TAZ subcellular localization (data not shown). In 3D, vasculogenesis was enhanced in stiff (G’= 1049 Pa) collagen oligomer matrices compared to soft (G’ = 305 Pa) monomer matrices formulated at constant collagen matrix density. YAP and TAZ were nuclear localized (activated) in the stiff oligomer matrices, but cytosolic in soft monomer matrices (Fig. 1C). Mechanistically, YAP and TAZ depletion reduced cytoskeletal stress fiber formation (data not shown), implicating YAP and TAZ in a feedback mechanism to control cytoskeletal remodeling. Delivered in oligomer matrices in vivo , ECFCs formed a functional human neovascular plexus and exhibited strong expression and nuclear localization of YAP and TAZ and expression of downstream genes, Cyr61 and CTGF (data not shown). In summary, YAP and TAZ are essential for mechanical control of ECFC migration and vasculogenesis. [1] Dupont et al. 2011 Nature. 474:179-185.