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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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Research Article Vascular biology Article has an altmetric score of 1

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

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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 1

SMAD4 maintains the FSS set point to restrict flow-mediated EC responses.

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SMAD4 maintains the FSS set point to restrict flow-mediated EC responses...
(A) Volcano plot highlighting the 10 most significantly upregulated genes upon 24 hours 12 DYNES/cm2 FSS in CTRL and SMAD4 siRNA HUVECs (n = 3/group). Color key shows log2 change after SMAD4 depletion. (B and C) qPCR for SMAD4 (B) and KLF4 (C) in CTRL and SMAD4 siRNAs HUVECs grown in static versus 1, 5, and 12 DYNES/cm2 FSS for 24 hours (n = 5/group). (D–I) VE-Cadherin staining (negative images) of CTRL (D–F) and SMAD4 (G–I) siRNAs HUVECs grown in static (D and G), subject to 12 DYNES/cm2 (E and H) or 1 DYNE/cm2 (F and I) for 24 and 48 hours. Flow direction: right to left. (J and K) Quantification of length-to-width ratio and EC alignment parallel to flow direction (%) in CTRL and SMAD4 siRNA HUVECs in static and 12 DYNES/cm2 (J) or 1 DYNE/cm2 (K) (n = 6 average of 3 images (70–140 cells/image) per 3 independent experiments/group). (L and M) VE-Cadherin (white), GM130 (red), and DAPI (blue) staining of CTRL and SMAD4 siRNA HUVECs upon 24 hours 1 DYNE/cm2. Arrow indicates flow direction. (N) Quantification of EC polarization (%) against, with the flow’s direction, or nonoriented (neutral) in experiments from L and M (n = 12 images [50–100 cells/image] per 3 independent experiments/group). (O) S-phase ratio (EdU+) per total number of DAPI+ nuclei (%) in CTRL and SMAD4 siRNA HUVECs grown in static, 1, and 12 DYNES/cm2 for 24 hours (n = 12 images [200–250 cells/image] per 3 independent experiments/group). (P) FACS analysis in Smad4fl/fl and Smad4iΔEC P6 retinas (n = 5 retinas/group). (Q) S-phase ratio (EdU+/ERG+) per total ECs (ERG+) in capillaries, arteries, and veins of fl/fl and Smad4iΔEC retinas engaged or not in AVMs (n = 15 images from 4 retinas/group). Scale Bars: 100μm in panels D–I, L, and M. Data are represented as mean ± SEM. Mann-Whitney test (B, C, N, and P) and 1-way Anova (J, K, O, and Q) were used to determine statistical significance. *P < 0.05,**P < 0.01,***P < 0.001.

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