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

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 3

Flow-induced KLF4 is a key determinant in AVM formation.

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Flow-induced KLF4 is a key determinant in AVM formation. (A) Schematic o...
(A) Schematic of the experimental strategy. Arrowheads indicate intragastric injection of 100μg tamoxifen (Tx) at P0–P2 in Klf4fl/fl and Klf4iΔEC, i.p. injection of IgG and blocking antibodies for BMP9 and BMP10 (BMP9/10blAb) at P4 and P5 and analysis at P6. (B–E) Representative IB4 labeling (negative images) of P6 Klf4fl/fl (B and C) and Klf4iΔEC (D and E) retinas treated with IgG (B and D) and BMP9/10blAb (C and E). Higher magnification of the small insets (red squares) from B and C labelled for KLF4 (green) and IB4 (white). Small red/blue arrowheads in B indicate the branch points in arteries and veins. Yellow/white arrowheads in C indicate increased KLF4 intensity in AVM capillary ECs and lower KLF4 in vessels outside of AVMs, respectively. (F) Quantification of KLF4 pixel intensity per EC in arteries, capillaries, and veins in Klf4fl/fl retinas treated with IgG and BMP9/10blAb (n = 8 images from 4 retinas/group). (G and H) Quantification of P6 AVM numbers (G) (n = 6 retinas/group) and of vascular density at the retinal front (%) (H) (n = 8 retinas/group) in the indicated genotypes. (I) Schematic of the experimental strategy used to delete Smad4 and Klf4 in neonates (P0–P6). (J–L) IB4 labeling (negative images) of P6 Smad4;Klf4fl/fl (J), Smad4iΔEC (K), and Smad4;Klf4iΔEC (L) retinas. (M) Smad4 and Klf4 mRNA expression in mouse lung ECs (mLECs) from P6 mice (n = 6 /group). (N and O) Quantification of AVM numbers (N) (n = 6 retinas/group) and of vascular density at the retinal front (%) (O) (n = 10 (2 images per retina)/group) of the indicated genotypes. Red arrows in C, K, and L mark AVMs. Blue squares in B–E and J–L indicate the vascular front. Scale Bars: 500 μm in B–E and J–L and 100 μm in higher magnification images from B and C. a, artery; v, vein. Data are represented as mean ± SEM. Statistical significance was determined by Mann-Whitney test (M) and 1-way Anova (F, G, H, N, and O).*P < 0.05, **P < 0.01, ***P < 0.001.