Astrocytes propel neurovascular dysfunction during cerebral cavernous malformation lesion formation
Miguel Alejandro Lopez-Ramirez, … , Issam A. Awad, Mark H. Ginsberg
Miguel Alejandro Lopez-Ramirez, … , Issam A. Awad, Mark H. Ginsberg
Published May 27, 2021
Citation Information: J Clin Invest. 2021;131(13):e139570. https://doi.org/10.1172/JCI139570.
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Astrocytes propel neurovascular dysfunction during cerebral cavernous malformation lesion formation

Abstract

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2- and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1α in astrocytes, resulting in increased VEGF production and expression of a “hypoxic” program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1α target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non–cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

Authors

Miguel Alejandro Lopez-Ramirez, Catherine Chinhchu Lai, Shady Ibrahim Soliman, Preston Hale, Angela Pham, Esau J. Estrada, Sara McCurdy, Romuald Girard, Riya Verma, Thomas Moore, Rhonda Lightle, Nicholas Hobson, Robert Shenkar, Orit Poulsen, Gabriel G. Haddad, Richard Daneman, Brendan Gongol, Hao Sun, Frederic Lagarrigue, Issam A. Awad, Mark H. Ginsberg

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Figure 7

KLF2 and KLF4 regulate increased eNOS expression during CCM.

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KLF2 and KLF4 regulate increased eNOS expression during CCM. (A) Express...
(A) Expression levels of NOS3 mRNA as assessed by RT-qPCR from human CCM lesions and compared with nonneurological disease controls (SEM, n = 4 or 6 in each group). (B) Immunofluorescence staining of eNOS (green) and collagen IV (Col IV; red) of human CCM lesion matched to CCM lesion-free brain tissue (n = 3). Asterisks denotate vascular lumen of CCM lesion. Nuclei were counterstained with DAPI (white). (C) Human umbilical vein endothelial cells (HUVECs) were transduced with lentivirus encoding KLF2 or KLF4, as previously reported (6), and analysis of NOS3 mRNA levels by RT-qPCR was determined in cells overexpressing KLF2 or KLF4 and compared with lentivirus encoding GFP as a control (n = 3 or 4). (D) Analysis of eNOS protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4, as determined by Western blot analysis (40); lentivirus encoding GFP was used as a control (SEM, n = 3). (E) Analysis of NOS3 mRNA levels by RT-qPCR in hCMEC/D3 cells transduced with lentivirus encoding shKRIT1 or scrambled control, followed by transfection with KLF2- and KLF4-specific small interfering RNAs (siRNA; siK2/K4) or small interfering RNA control (siCtrl) (SEM, n = 4). (F) Schematic model. KLF2- and KLF4-mediated elevation of eNOS in CCM endothelium by a cell-autonomous mechanism. Data are mean ± SEM. **P < 0.01, ***P < 0.001, as determined by Student’s t test and 1-way ANOVA, followed by the Tukey post hoc test. Scale bar: 100 μm (B).