SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations
Kuheli Banerjee, … , Martin A. Schwartz, Roxana Ola
Kuheli Banerjee, … , Martin A. Schwartz, Roxana Ola
Published July 25, 2023
Citation Information: J Clin Invest. 2023;133(18):e168352. https://doi.org/10.1172/JCI168352.
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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 2

KLF4 mediates the flow-induced hyperresponsiveness of SMAD4-depleted cells.

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KLF4 mediates the flow-induced hyperresponsiveness of SMAD4-depleted cel...
(A and B) Representative PECAM staining (negative images) of CTRL, SMAD4, and KLF4 and SMAD4;KLF4 siRNAs HUVECs subject to 12 DYNES/cm2 (A) and 1 DYNE/cm2 (B) for 24 hours. (C and D) Quantification of the length-to-width ratio and of EC alignment parallel to the flow direction (%) from experiments in A and B (average of n = 6 images (70–140 cells/image) per 3 independent experiments/group). (E) Quantification of PECAM signal intensity from experiments in A (n = 6 images per 3 independent experiments/group). (F) Colabeling for PECAM (white), GM130 (red), and DAPI (blue) of SMAD4 and KLF4 and SMAD4;KLF4 siRNAs HUVECs subject to 24 hours 1 DYNE/cm2. Black arrow indicates flow direction from right to left. (G) Quantification of cell polarization: against flow direction, with flow, or neutral (nonoriented) in pictures, as shown in F (n = 6 images (50–100 cells/image) per 3 independent experiments/group). (H and I) Representative VE-Cadherin staining (negative images) of HUVECs transfected with an empty lentiviral construct (CTRL OE) and an overexpression lentivirus for KLF4 (KLF4 OE) grown in static (H) or subject to 1 DYNE/cm2 for 48 hours (I). (J) KLF4 expression by qPCR (fold change) in CTRL-OE and KLF4-OE HUVECs (n = 4/group). (K) Quantification of the length-to-width ratio and of EC alignment parallel to the flow direction (%) in images as shown in H and I (n = 6 images (70–140 cells/image) per 3 independent experiments/group). (L) S-phase ratio ((EdU+) per total number of DAPI+ nuclei (%)) of CTRL-OE and KLF4-OE HUVECs grown in static and 12 DYNES/cm2 for 24 hours (n = 6 images (140–250 cells/image) per 3 independent experiments/group). Scale Bars: 100μm in A, B, H, and I, and 50μm in F. Data are represented as mean ± SEM. Mann-Whitney test (J and K-right), 1-way Anova (C, D, E, K-left, L), and Kruskal-Wallis tests (G) were used to determine statistical significance.*P < 0.05, **P < 0.01, ***P < 0.001.