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

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 5

HS cleavage from flow-conditioned HLMVECs decreases shear stress–induced p-AMPK, contributing to Ang-2 secretion.

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HS cleavage from flow-conditioned HLMVECs decreases shear stress–induced...
(A) Representative 100× original magnification epifluorescence images of HLMVECs stained for p-AMPK following 48 hours of static culture (top) or 15 dyn/cm2 (bottom) (with DAPI overlay). Scale bar: 10 μm. (B) Relative staining intensity for p-AMPK within the cytoplasm of HLMVECs in static culture or following 15 dyn/cm2 (from 8 representative 40× original magnification images, normalized to number of nuclei within field of view). ***P < 0.001. (C) Representative 100× epifluorescence images of p-AMPK staining within flow-conditioned HLMVECs (15 dyn/cm2, 48 hours) exposed to 15 dyn/cm2 for an additional 24 hours in the absence (top) or presence (bottom) of HepIII 200 mU/mL (with DAPI overlay). Scale bar: 10 μm. (D) Relative staining intensity for p-AMPK within the cytoplasm of flow-conditioned HLMVECs (15 dyn/cm2, 48 hours) exposed to 15 dyn/cm2 for an additional 24 hours in the absence or presence of HepIII 200 mU/mL (from 8 representative 40× original magnification images, normalized to number of DAPI-stained nuclei within field of view). **P < 0.01. (E) Relative supernatant Ang-2 levels (ELISA) from flow-conditioned HLMVECs (15 dyn/cm2, 48 hours) exposed to 15 dyn/cm2 and HepIII 200 mU/mL for 24 hours in the presence or absence of AICAR 1 mM. **P < 0.01. All data are presented as mean ± SEM and analyzed by Student’s 2-sided t test (B, D, and E).