SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations
Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
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Research Article Vascular biology

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations

Abstract

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression–mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

Authors

Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola

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Figure 6

KLF4-mediated TEK expression is required for flow-induced PI3K/Akt activation.

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KLF4-mediated TEK expression is required for flow-induced PI3K/Akt activ...
(A) Heatmap of potential mediators of PI3K signaling in CTRL siRNA HUVECs grown in static versus subject to 12 DYNES/cm2; n = 3/group. Color key shows log2 change upon FSS stimulation. (B) qPCR for TEK and PTEN (left panel) and FLT4 and CD31 (right panel) in CTRL versus KLF4 siRNAs HUVECs grown in static or subject to 12 DYNES/cm2 (n = 5/group). (C) TEK mRNA expression in CTRL-OE versus KLF4-OE HUVECs (n = 4/group). (D) Reanalysis of previous published CHIP-Seq data of KLF4 overexpression (caMEK5) in primary human pulmonary artery endothelial cells (PAEC) with the Integrative Genomics Viewer (IGV). Two distinct peaks within enhancer regions of the TEK gene were identified. (E) WB for indicated proteins of HUVECs transfected with CTRL and TEK siRNAs and CTRL-OE and KLF4-OE constructs grown in static or subject to 12 DYNES/cm2 for 4 hours. (F) Quantification of pAkt/Akt and TEK/GAPDH in indicated genotypes (n = 4/group). (G) Labeling of Tx-induced P6 fl/fl, Smad4iΔEC and Klf4iΔEC retinas with anti-TIE2 antibody (red) and IB4 (white). Green and yellow arrowheads indicate non-AVM versus AVM region, respectively. (H) Quantification of TIE2 expression per vascular area in the indicated genotypes (n = 6 [2 images/retina]/group). a, artery; v, vein. Scale Bars: 100μm in G. Mann-Whitney test (C) and 1-way Anova (B, F, and H) were used to determine statistical significance.Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.