Loss of Smad4 drives vascular malformations via c-KIT–dependent high–fluid shear stress mimicry
Johannes Gahn, Fan Wu, Qing Zhang, Yuxi Di, Tanmaya Behera, Yonggang Ren, Zohrah Hashemi, Kuheli Banerjee, Julio Cordero, Claudia Gherman, Kornelia Andorfer, Caroline T. Seebauer, Fatemeh Mirzapour-Shafiyi, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
Johannes Gahn, Fan Wu, Qing Zhang, Yuxi Di, Tanmaya Behera, Yonggang Ren, Zohrah Hashemi, Kuheli Banerjee, Julio Cordero, Claudia Gherman, Kornelia Andorfer, Caroline T. Seebauer, Fatemeh Mirzapour-Shafiyi, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
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Research Article Cell biology Genetics Vascular biology

Loss of Smad4 drives vascular malformations via c-KIT–dependent high–fluid shear stress mimicry

Abstract

Vascular endothelial cells (ECs) encode a homeostatic fluid shear stress (FSS) set point that is essential for vascular stability. Deviations above or below this threshold trigger adaptive remodeling to restore physiological shear levels. Disruption of this control mechanism leads to enlarged arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia, a vascular disorder caused by heterozygous loss-of-function (LOF) mutation in ALK1, ENG, or SMAD4. Mechanistically, Smad4-deficient ECs are reset to a lower FSS set point value, resulting in AVMs that show characteristics of high-FSS remodeling with elevated KLF4 and high activation of the downstream Akt. Here, we investigated the KLF4/Akt upstream mechanisms by which SMAD4 sets the physiological FSS set point. We identified the receptor tyrosine kinase c-KIT as a component and regulator of the junctional mechanosensory receptor complex, which is highly upregulated in murine and human AVMs. SMAD4 restrains flow signaling by limiting c-KIT–dependent ERK5 activation and KLF4 induction. Thus, SMAD4 LOF leads to sustained c-KIT engagement in the sensory junctional apparatus, driving enhanced and prolonged activation of the ERK5/KLF4/Akt signaling axis. These results show that Smad4-LOF mutations induce malformations by disabling a key homeostatic mechanism and identify c-KIT as a potentially previously unrecognized therapeutic target.

Authors

Johannes Gahn, Fan Wu, Qing Zhang, Yuxi Di, Tanmaya Behera, Yonggang Ren, Zohrah Hashemi, Kuheli Banerjee, Julio Cordero, Claudia Gherman, Kornelia Andorfer, Caroline T. Seebauer, Fatemeh Mirzapour-Shafiyi, Gergana Dobreva, Martin A. Schwartz, Roxana Ola

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Figure 7

Ligand-independent c-KIT activation in SMAD4 KD sustains flow signaling.

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Ligand-independent c-KIT activation in SMAD4 KD sustains flow signaling....
(A) Relative KITL mRNA expression in RNA-seq groups (n = 3/group). (B) KITL qPCR in CTRL and SMAD4 siRNA HUVECs grown statically versus 2 h of 12 dynes/cm2 (n = 4/group). (C) WB for the indicated proteins in SMAD4 siRNA HUVECs grown statically or subjected to 12 dynes/cm2 for 5-30-120 minutes and treated with IgG or SCF blocking antibodies (SCF blAb). (D) Quantifications of SFK (Y418) and Akt (S473) levels normalized to total SRC and Akt, respectively, and of ERK5 gel shift (upper band, quantified by densitometry) (n = 3/group). (E, H, and K) KLF4 qPCR in CTRL and SMAD4 siRNA HUVECs treated with SCF blAb subjected to 12 dynes/cm2 FSS for 2 h (n = 4/group) (E) or treated with SCF for 2 h and subjected to 12 dynes/cm2 FSS (n = 4/group) (H) or subjected to 2 h of 12 dynes/cm2 treated with DMSO versus avapritinib (Ava) (n = 4/group) (K). (F) WB for the indicated proteins in CTRL and SMAD4 siRNA HUVECs subjected to 12 dynes/cm2 for 2 h in the presence of SCF (50 ng/mL) for the indicated time (0 minute — no SCF; 5 and 120 minutes of SCF). (G) Quantifications of SFK (Y416) and Akt (S473) levels normalized to total SRC and Akt, respectively, and of ERK5 gel shift (n = 3/group). (I) WB for the indicated proteins in SMAD4 siRNA HUVECs subjected to 12 dynes/cm2 for 2 h treated with DMSO or Ava. (J) Quantifications of SFK (Y416) and Akt (S473) levels normalized to total SRC and Akt, respectively, and of ERK5 gel shift (n = 3/group). Data are represented as mean ± SEM. Statistical significance for the interaction between siRNA groups and shear stress was determined by DESeq2 Likelihood Ratio Test (A). Two-way ANOVA (B, D, E, G, H, J, and K) was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.