Limits...
Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Mutations of the endothelial BMP9/10 receptors Alk1 and endoglin are associated with vascular malformations in hereditary hemorrhagic telangiectasia (HHT). Baeyens et al. report that fluid flow potentiates BMP activation of Alk1 signaling to stabilize blood vessels. HHT lesions thus result from a defect in a synergistic mechanical/biochemical signaling pathway.

No MeSH data available.


Alk1 signaling in response to FSS. (A) Nuclear translocation of Smad1, Smad5, and Smad8 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min (n = 3–8, ANOVA two-way; *, P < 0.05; ****, P < 0.0001). (B) Representative Western blots of phosphorylated Smad1,5,8, Smad1, Alk1 and endoglin in response to 1 ng/ml BMP9 or 12 dynes/cm2 FSS for the indicated times. Actin was used as a loading control. (C) Representative staining of Smad1 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min in HUVECs transfected with the indicated siRNAs (siRNA 1: QIAGEN; siRNA 2: GE Healthcare; n = 3–11, two-way ANOVA; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bar, 10 µm. (D) Representative Western blot of Smad1/5/8 phosphorylation in response to 15 min of 12 dynes/cm2 FSS in cells transfected with the indicated siRNAs (n = 3, ANOVA; *, P < 0.05; ***, P < 0.001). NS, not significant.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5037412&req=5

fig2: Alk1 signaling in response to FSS. (A) Nuclear translocation of Smad1, Smad5, and Smad8 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min (n = 3–8, ANOVA two-way; *, P < 0.05; ****, P < 0.0001). (B) Representative Western blots of phosphorylated Smad1,5,8, Smad1, Alk1 and endoglin in response to 1 ng/ml BMP9 or 12 dynes/cm2 FSS for the indicated times. Actin was used as a loading control. (C) Representative staining of Smad1 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min in HUVECs transfected with the indicated siRNAs (siRNA 1: QIAGEN; siRNA 2: GE Healthcare; n = 3–11, two-way ANOVA; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bar, 10 µm. (D) Representative Western blot of Smad1/5/8 phosphorylation in response to 15 min of 12 dynes/cm2 FSS in cells transfected with the indicated siRNAs (n = 3, ANOVA; *, P < 0.05; ***, P < 0.001). NS, not significant.

Mentions: These results led us to consider whether fluid shear stress (FSS) from blood flow might directly modulate Alk1 signaling. To test this idea, human umbilical vein endothelial cells (HUVECs) in low serum medium (0.2% FBS) were treated with 1 ng/ml BMP9 or subjected to FSS in the physiological range (12 dynes/cm2), and Smad activation was assessed. FSS induced both Smad phosphorylation and nuclear translocation, with a time course that was slightly slower than soluble BMP9, though of similar magnitude (Fig. 2, A and B; and Fig. S1 A). Smad1 was most strongly affected, followed by Smad5, with little change in Smad8. We therefore focused on Smad1. To test whether these effects were mediated by the known BMP9 receptors, Alk1 and endoglin levels were reduced using siRNA. Depletion of Alk1 blocked Smad1 activation by both BMP9 and FSS, whereas depletion of endoglin only affected the response to flow (Fig. 2, B and C). To validate this observation, we analyzed the response to BMP9 over a wide range of concentrations. Depletion of endoglin had no effect at any dose (Fig. S1 B). Reexpression of siRNA-resistant proteins rescued Smad activation, indicating specificity (Fig. S1 C). Our previous work identified a junctional mechanosensory complex, comprising PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs), whose activation controls a number of flow responses, including PI3K activation and alignment in the direction of flow (Tzima et al., 2005; Coon et al., 2015). However, depletion of PECAM-1 or VEGFR2 only slightly altered Smad1/5/8 phosphorylation in response to flow. Thus, flow signaling through Alk1–endoglin appears to be independent of the PECAM–VE-cadherin–VEGFR complex.


Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia
Alk1 signaling in response to FSS. (A) Nuclear translocation of Smad1, Smad5, and Smad8 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min (n = 3–8, ANOVA two-way; *, P < 0.05; ****, P < 0.0001). (B) Representative Western blots of phosphorylated Smad1,5,8, Smad1, Alk1 and endoglin in response to 1 ng/ml BMP9 or 12 dynes/cm2 FSS for the indicated times. Actin was used as a loading control. (C) Representative staining of Smad1 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min in HUVECs transfected with the indicated siRNAs (siRNA 1: QIAGEN; siRNA 2: GE Healthcare; n = 3–11, two-way ANOVA; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bar, 10 µm. (D) Representative Western blot of Smad1/5/8 phosphorylation in response to 15 min of 12 dynes/cm2 FSS in cells transfected with the indicated siRNAs (n = 3, ANOVA; *, P < 0.05; ***, P < 0.001). NS, not significant.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037412&req=5

fig2: Alk1 signaling in response to FSS. (A) Nuclear translocation of Smad1, Smad5, and Smad8 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min (n = 3–8, ANOVA two-way; *, P < 0.05; ****, P < 0.0001). (B) Representative Western blots of phosphorylated Smad1,5,8, Smad1, Alk1 and endoglin in response to 1 ng/ml BMP9 or 12 dynes/cm2 FSS for the indicated times. Actin was used as a loading control. (C) Representative staining of Smad1 in response to 1 ng/ml BMP9 or 12 dynes/cm2 for 45 min in HUVECs transfected with the indicated siRNAs (siRNA 1: QIAGEN; siRNA 2: GE Healthcare; n = 3–11, two-way ANOVA; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bar, 10 µm. (D) Representative Western blot of Smad1/5/8 phosphorylation in response to 15 min of 12 dynes/cm2 FSS in cells transfected with the indicated siRNAs (n = 3, ANOVA; *, P < 0.05; ***, P < 0.001). NS, not significant.
Mentions: These results led us to consider whether fluid shear stress (FSS) from blood flow might directly modulate Alk1 signaling. To test this idea, human umbilical vein endothelial cells (HUVECs) in low serum medium (0.2% FBS) were treated with 1 ng/ml BMP9 or subjected to FSS in the physiological range (12 dynes/cm2), and Smad activation was assessed. FSS induced both Smad phosphorylation and nuclear translocation, with a time course that was slightly slower than soluble BMP9, though of similar magnitude (Fig. 2, A and B; and Fig. S1 A). Smad1 was most strongly affected, followed by Smad5, with little change in Smad8. We therefore focused on Smad1. To test whether these effects were mediated by the known BMP9 receptors, Alk1 and endoglin levels were reduced using siRNA. Depletion of Alk1 blocked Smad1 activation by both BMP9 and FSS, whereas depletion of endoglin only affected the response to flow (Fig. 2, B and C). To validate this observation, we analyzed the response to BMP9 over a wide range of concentrations. Depletion of endoglin had no effect at any dose (Fig. S1 B). Reexpression of siRNA-resistant proteins rescued Smad activation, indicating specificity (Fig. S1 C). Our previous work identified a junctional mechanosensory complex, comprising PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs), whose activation controls a number of flow responses, including PI3K activation and alignment in the direction of flow (Tzima et al., 2005; Coon et al., 2015). However, depletion of PECAM-1 or VEGFR2 only slightly altered Smad1/5/8 phosphorylation in response to flow. Thus, flow signaling through Alk1–endoglin appears to be independent of the PECAM–VE-cadherin–VEGFR complex.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Mutations of the endothelial BMP9/10 receptors Alk1 and endoglin are associated with vascular malformations in hereditary hemorrhagic telangiectasia (HHT). Baeyens et al. report that fluid flow potentiates BMP activation of Alk1 signaling to stabilize blood vessels. HHT lesions thus result from a defect in a synergistic mechanical/biochemical signaling pathway.

No MeSH data available.