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. 2006 Jan;116(1):49-58.
doi: 10.1172/JCI24787. Epub 2005 Dec 8.

Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2

Kush M Parmar  1 , H Benjamin Larman, Guohao Dai, Yuzhi Zhang, Eric T Wang, Sripriya N Moorthy, Johannes R Kratz, Zhiyong Lin, Mukesh K Jain, Michael A Gimbrone Jr, Guillermo García-Cardeña
Affiliations

Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2

Kush M Parmar et al. J Clin Invest. 2006 Jan.

Abstract

In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway. Overexpression and silencing of KLF2 in the context of flow, combined with findings from genome-wide analyses of gene expression, demonstrate that the induction of KLF2 results in the orchestrated regulation of endothelial transcriptional programs controlling inflammation, thrombosis/hemostasis, vascular tone, and blood vessel development. Our data also indicate that KLF2 expression globally modulates IL-1beta-mediated endothelial activation. KLF2 therefore serves as a mechano-activated transcription factor important in the integration of multiple endothelial functions associated with regions of the arterial vasculature that are relatively resistant to atherogenesis.

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Figures

Figure 1
Figure 1
Flow-dependent expression of KLF2 and its regulation by a MEK5/ERK5/MEF2 pathway. (A) Archetypal atheroprotective and atheroprone shear stress waveforms derived from a human carotid artery, as previously described (4). These 2 shear stress waveforms were recreated using a dynamic flow system and applied to cultured HUVECs. (B) HUVECs were cultured under static (no flow), atheroprone, or atheroprotective flow conditions for 24 hours, and KLF2 mRNA expression was measured by RT-PCR (n = 3; mean ± SEM). (C) Whole-mount in situ hybridization of WT or sih mutant embryos at 48 hours, probed for Flk or KLF2a. Inserts show close-ups of the trunk vasculature. Anal sphincter staining is indicated by arrowheads. (D) ChIP of MEF2A and MEF2C with the KLF2 promoter under static conditions and flow. (E) KLF2 mRNA in HUVECs infected with control (GFP) or dominant negative MEF2 (MEF2ASA) adenovirus 24 hours before exposure to the static (no flow) or atheroprotective waveform. (F) Western blot of total immunoprecipitated ERK5 and p-ERK5 from HUVECs under static or flow 24 hours after infection with control (GFP) or MEK5-DN adenovirus. (G) KLF2 mRNA levels from experimental samples represented in F. (H) Western blot of ERK5 immunoprecipitates under conditions described in F from HUVECs infected with GFP or MEK5-CA adenovirus. (I) KLF2 mRNA from samples represented in H. *P < 0.05, **P < 0.01 vs. control; Student’s t test. ctrl, control.
Figure 2
Figure 2
Elucidation of KLF2-regulated global gene expression programs. (A) Visualization of whole-genome expression patterns controlled by KLF2 and/or IL-1β in HUVECs. HUVECs were infected with either Ad-GFP or Ad-KLF2 and incubated for 24 hours before RNA was isolated and analyzed using whole genome microarrays. Self-organizing map software was used to cluster similarly regulated genes and colorize them based on intensity of expression relative to GFP-expressing (control) cells. (B) Selected KLF2-responsive genes grouped by clusters shown in A, with major associated biological functions listed. Dev, development.
Figure 3
Figure 3
KLF2 regulates genetic programs controlling blood vessel development, vascular tone, and thrombosis/hemostasis. (A) HUVECs under the same conditions as in Figure 2A were analyzed by quantitative TaqMan RT-PCR (n = 3) of the indicated genes; Ad-GFP was performed as the control. (B) Ang-2 protein levels in supernatants were measured by ELISA under the same conditions (n = 3) as in A, but with an 8-hour incubation with IL-1β. Ang-2 levels shown are after subtraction of the amount in the media before conditioning. (C) Western blot of Tie2 and GAPDH (loading control) on whole-cell lysate of HUVECs infected with Ad-GFP or Ad-KLF2 for 24 hours. (D) Quantitative TaqMan RT-PCR was performed to assess the levels of expression of eNOS, ASS, CNP, and ET-1. (E) CNP protein levels in supernatant measured by ELISA. Supernatant was collected from HUVECs infected with the indicated adenoviruses for 24 hours with or without IL-1β. nd, not detectable. (F) Effect of KLF2 and/or IL-1β on the expression of major genes involved in thrombosis. Quantitative TaqMan RT-PCR of indicated genes was performed as described in A. EDG-1, endothelial differentiation gene 1; PAI-1, plasminogen activatory inhibitor 1. (G) FACS analysis of surface TM (TM/CD141) expression on HUVECs infected with Ad-GFP or Ad-KLF2 for 24 hours. (H) FACS analysis of cell-surface TF expression on HUVECs infected with the indicated virus followed by incubation with media or IL-1β (10 U/ml) for 6 hours. Bar graphs represent mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
Figure 4
Figure 4
KLF2 mediates a global antiinflammatory program in endothelial cells. (A) Protein levels of cytokines in supernatants of HUVECs infected with the indicated virus and then incubated with either normal media or IL-1β (10 U/ml) for 24 hours. Samples were analyzed by multiplex ELISA or cytokine chip. IP-10, IFN-γ–inducible protein 10. (B) Regulation of PTGDS gene expression by KLF2 and/or IL-1β. (C) ELISA of 15d-PGJ2 after extraction from supernatants of cells infected with the indicated virus for 24 hours. (D) ELAFIN secretion measured by ELISA from supernatants under conditions described in A. All data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
Figure 5
Figure 5
Endothelial transcriptional programs evoked by atheroprotective flow require KLF2 expression. (A) Effects of suppressing flow-dependent KLF2 upregulation on gene expression. Cells were treated with either scrambled siRNA or siRNA targeting KLF2 for 24 hours and then placed under static or atheroprotective conditions for an additional 24 hours. Shown are RT-PCR data for the indicated genes. hPGT, human prostaglandin transporter; NOV, nephroblastoma overexpressed gene. (B) Monitoring of the interferon response in HUVECs treated with control or KLF2 siRNAs. Gene expression of 2′-5′-oligoadenylate synthetase (OAS2) or interferon-inducible transmembrane protein 1 (IFITM1) was assessed by TaqMan RT-PCR. As a positive control for the interferon response, HUVECs were incubated with long double-stranded RNA (dsRNA) poly I:C for 24 hours (n = 3). (C) Western blot for proteins in the same samples as represented in A. (D) Surface TM FACS on HUVECs under the indicated conditions. (E) ELISA of CNP from supernatants under the conditions described in A. (F) 15-d-PGJ2 levels in supernatants under the indicated conditions. (G) Histogram (blue bars) of genes binned according to fold regulation under flow. Overlaid on the histogram is a graph showing the percentage of genes within each bin that are KLF2 dependent (red diamonds and trend line). Gray shading indicates the portion of the histogram representing the 74 most highly regulated genes under flow (see text). All data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
Figure 6
Figure 6
KLF2 expression is essential for endothelial cellular phenotypes conferred by flow. (A) HL-60 cell adhesion to HUVEC monolayers under the indicated conditions. After preconditioning (static or atheroprotective flow), the cells were treated with 1 U/ml of IL-1β for 6 hours and then incubated with fluorescently labeled HL-60 cells. Shown are representative fields of HUVEC monolayers (blue) and attached monocytes (yellow). The bar graph on the right displays quantification of bound HL-60 cells. (B) Role of KLF2 in flow-mediated resistance to oxidant stress. Cells under static conditions (top row) were infected with either Ad-GFP or Ad-KLF2 for 24 hours, and cells preconditioned with atheroprotective flow (bottom row) were treated with control or KLF2 siRNA. Cells were then incubated with or without 200 μM tert-butyl H2O2 for an additional 4 hours. (C) Total protein from GFP- or KLF2-expressing HUVECs blotted for catalase, manganese superoxide dismutase (MnSOD), or α-tubulin as a loading control. All data are expressed as mean ± SEM (n = 3). *P < 0.05 versus control; Student’s t test.
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References

    1. Gimbrone MA, Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann. N. Y. Acad. Sci. 2000;902:230–239; discussion 239–240. - PubMed
    1. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler. Thromb. Vasc. Biol. 1998;18:677–685. - PubMed
    1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol. Rev. 1995;75:519–560. - PMC - PubMed
    1. Dai G, et al. Distinct endothelial phenotypes evoked by arterial wave forms derived from atherosclerosis-susceptible and -resistant regions of human vasculature. Proc. Natl. Acad. Sci. U. S. A. 2004;101:14871–14876. - PMC - PubMed
    1. Buckley AF, Kuo CT, Leiden JM. Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc–dependent pathway. Nat. Immunol. 2001;2:698–704. - PubMed

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