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OBJECTIVE: Mutations in Krüppel like factor-11 (KLF11), a gene also known as maturity-onset diabetes of the young type 7, contribute to the development of diabetes. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell (VSMC) biology and arterial thrombosis. APPROACH AND RESULTS: Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout (Klf11 KO) mice while bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that VSMC specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (TF, encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 KO mice, as determined by real-time quantitative PCR and immunofluorescence. Furthermore, VSMCs isolated from Klf11 KO mouse aortas showed increased TF expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, siRNA-mediated knockdown of KLF11 increased TF expression. Consistent results were observed upon adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. CONCLUSIONS: Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in VSMCs, constituting a potential molecular target for inhibition of arterial thrombosis.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Introduction: Recruitment of pericytes (PC), critical to microvascular maturation, requires deposition of basement membrane proteins such as von Willebrand factor (vWF). Loss of PCs ultimately leads to vessel regression and rarefaction. Hypothesis: Splice variants of tissue factor (TF) stabilize the vasculature by supporting EC-PC interactions. Results: We determined that alternatively spliced TF (asTF) resides in Weibel Palade bodies (WPB) of ECs. Overexpression of KLF-2 enhanced asTF protein levels in WPBs. In confluent EC monolayers, asTF was deposited into the extracellular matrix (ECM). In assays employing vascular plexus remodelling on 3D-basement membrane and purified recombinant asTF or full-length TF (flTF), only asTF improved EC-PC interactions. At sites of low KLF-2 expression (low or turbulent flow), total TF mRNA was diminished, leading to low asTF expression. However, upon EC activation by TNFα, total TF mRNA levels rapidly increased and TF protein expression shifted from the ECM-deposited asTF to luminally expressed flTF. Interestingly, flTF-driven EC conversion to a procoagulant state was associated with the release of vWF and asTF from WPBs, depleting the intracellular depots from these proteins. Immunohistochemical staining of kidney specimens of human living donors confirmed that asTF was present at high levels in WPBs of microvascular endothelium. Moreover, after 45 minutes of reperfusion of the transplanted kidney, asTF- and vWF-staining was markedly reduced, consistent with their release from WPB following an inflammatory insult. Conclusion: We show for the first time that quiescent, KLF-2 expressing ECs predominantly generate non-coagulant asTF, which is stored in WPBs and serves to stabilize the microvasculature by supporting EC-PC interactions. In contrast, in low-flow zones or sites with disturbed flow (e.g. at bifurcations), KLF-2 is downregulated, leading to decreased production of asTF and impaired stabilization of the vasculature. During inflammation, acute perturbation of ECs (e.g. exposure to TNFα) is associated with downregulation of KLF-2 and a post-transcriptionally regulated increase in the production of flTF, resulting in a pro-coagulant phenotype and loss of EC-PC interactions.