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Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress–related AMPK/FoxO1 signaling
Robert P. Richter, … , Amit Gaggar, Jillian R. Richter
Robert P. Richter, … , Amit Gaggar, Jillian R. Richter
Published June 28, 2022
Citation Information: JCI Insight. 2022;7(15):e155010. https://doi.org/10.1172/jci.insight.155010.
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Research Article Vascular biology

Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress–related AMPK/FoxO1 signaling

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Abstract

Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress–mediated 5′-adenosine monophosphate–activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

Authors

Robert P. Richter, Amit R. Ashtekar, Lei Zheng, Danielle Pretorius, Tripathi Kaushlendra, Ralph D. Sanderson, Amit Gaggar, Jillian R. Richter

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Figure 3

Flow conditioning HLMVECs alters HS distribution and suppresses ANG2 mRNA expression.

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Flow conditioning HLMVECs alters HS distribution and suppresses ANG2 mRN...
(A) Stacked 20× original magnification confocal micrographs demonstrating staining for HS (10E4 epitope), F-actin, β-catenin, and cell nuclei (DAPI) on or in HLMVECs after 48 hours of static culture (top row) or 15 dyn/cm2 (bottom row). Scale bar: 100 μm. (B) Stacked 60× original magnification confocal micrographs demonstrating HS distribution relative to cell edges (demarcated by β-catenin) on HLMVECs after 48 hours of static culture (left panel) or 15 dyn/cm2 (right panel) (with DAPI overlay). Scale bar: 20 μm. (C) Supernatant Ang-2 levels (ELISA) from a confluent monolayer of HLMVECs over the course of 24 hours of static culture following media change (n = 4–8 per group). ***P < 0.001, versus 5 minutes. (D) Relative HLMVEC ANG2 gene expression following 48 hours of either static culture (n = 9) or 15 dyn/cm2 (n = 10). ***P < 0.001. All data are presented as mean ± SEM and analyzed by 1-way ANOVA corrected by Tukey’s multiple comparisons test (C) or by Student’s 2-sided t test (D).

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