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RASA1-dependent cellular export of collagen IV controls blood and lymphatic vascular development
Di Chen, … , Philip E. Lapinski, Philip D. King
Di Chen, … , Philip E. Lapinski, Philip D. King
Published June 11, 2019
Citation Information: J Clin Invest. 2019;129(9):3545-3561. https://doi.org/10.1172/JCI124917.
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Research Article Angiogenesis Vascular biology

RASA1-dependent cellular export of collagen IV controls blood and lymphatic vascular development

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Abstract

Combined germline and somatic second-hit inactivating mutations of the RASA1 gene, which encodes a negative regulator of the Ras signaling pathway, cause blood and lymphatic vascular lesions in the human autosomal-dominant vascular disorder capillary malformation–arteriovenous malformation (CM-AVM). How RASA1 mutations in endothelial cells (ECs) result in vascular lesions in CM-AVM is unknown. Here, using different murine models of RASA1 deficiency, we found that RASA1 was essential for the survival of ECs during developmental angiogenesis, in which primitive vascular plexuses are remodeled into hierarchical vascular networks. RASA1 was required for EC survival during developmental angiogenesis, because it was necessary for export of collagen IV from ECs and deposition in vascular basement membranes. In the absence of RASA1, dysregulated Ras/MAPK signal transduction in ECs resulted in impaired folding of collagen IV and its retention in the endoplasmic reticulum (ER), leading to EC death. Remarkably, the chemical chaperone 4-phenylbutyric acid and small-molecule inhibitors of MAPK and 2-oxoglutarate–dependent collagen IV–modifying enzymes rescued ER retention of collagen IV and EC apoptosis and resulted in normal developmental angiogenesis. These findings have important implications for a better understanding of the molecular pathogenesis of CM-AVM and possible means of treatment.

Authors

Di Chen, Joyce M. Teng, Paula E. North, Philip E. Lapinski, Philip D. King

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Figure 7

RASA1 knockdown in HUVECs results in collagen IV accumulation in the ER that can be rescued by 4PBA.

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RASA1 knockdown in HUVECs results in collagen IV accumulation in the ER ...
HUVECs were transfected with control or RASA1 siRNA and cultured for 24 hours in the presence or absence of 4PBA. (A) Cells were stained with Hoechst and antibodies against collagen IV and calnexin. Representative images are shown. Note the intracellular accumulation of collagen IV in RASA1 siRNA–treated cells and its rescue by 4PBA treatment. Scale bars: 10 μm. (B) Higher-magnification images of RASA1 siRNA–treated cells in A show large intracellular accumulations of collagen IV surrounded by calnexin. Scale bar: 5 μm. (C) Knockdown of RASA1 was confirmed by reverse transcriptase qPCR (RT-qPCR). Data represent the mean ± 1 SEM of the amount of RASA1 mRNA normalized to RASA1 mRNA levels in control siRNA–treated HUVECs in the same experiment (n = 2). (D) Data show the mean ± 1 SEM of the percentage of HUVECs per field with evidence of intracellular collagen IV accumulation (n = 12). *P < 0.05 and ****P < 0.0001, by 1-way ANOVA with Dunnett’s multiple comparisons post hoc test.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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