Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress–related AMPK/FoxO1 signaling
Robert P. Richter, Amit R. Ashtekar, Lei Zheng, Danielle Pretorius, Tripathi Kaushlendra, Ralph D. Sanderson, Amit Gaggar, Jillian R. Richter
Robert P. Richter, Amit R. Ashtekar, Lei Zheng, Danielle Pretorius, Tripathi Kaushlendra, Ralph D. Sanderson, Amit Gaggar, Jillian R. Richter
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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 6

Decreased AMPK activity following HS cleavage from flow-conditioned HLMVECs may increase Ang-2 expression through FoxO1 activation.

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Decreased AMPK activity following HS cleavage from flow-conditioned HLMV...
(A) Relative FOXO1 and KLF2 gene expression in HLMVECs following 48 hours of either static culture or 15 dyn/cm2 (n = 3–4 per group). **P < 0.01, ***P < 0.001. (B) Relative FOXO1 and KLF2 gene expression in flow-conditioned HLMVECs (15 dyn/cm2, 48 hours) exposed to an additional 24 hours of 15 dyn/cm2 in the absence or presence of HepIII 200 mU/mL (n = 3–4 per group). **P < 0.01. (C) Representative 100× original magnification epifluorescence images of HLMVECs stained for p-FoxO1 following 48 hours of static culture (top) or 15 dyn/cm2 (bottom) (with DAPI overlay). Scale bar: 10 μm. (D) Relative staining intensity for p-FoxO1 in HLMVEC cytoplasm following static culture or flow conditioning (from 8 representative 40× original magnification images, normalized to number of nuclei within field of view). ***P < 0.001. (E) Representative 100× original magnification epifluorescence images of p-FoxO1 staining in flow-conditioned HLMVECs exposed to an additional 24 hours of 15 dyn/cm2 in the absence (top) or presence (bottom) of HepIII 200 mU/mL (with DAPI overlay). Scale bar: 10 μm. (F) Relative staining intensity for p-FoxO1 in the cytoplasm of flow-conditioned HLMVECs exposed to an additional 24 hours of 15 dyn/cm2 in the absence or presence of HepIII 200 mU/mL (from 8 representative 40× original magnification images, normalized to number of nuclei within field of view). **P < 0.01. Representative 100× original magnification epifluorescence images of p-AMPK (G) and p-FoxO1 (H) staining in statically cultured HLMVECs treated with AICAR 1 mM or vehicle for 24 hours (with DAPI overlay). Scale bar: 10 μm. (I) Relative ANG2 gene expression in statically cultured HLMVECs treated with the FoxO1 inhibitor AS1842856 or vehicle for 24 hours (n = 4 per group). ***P < 0.001. All data are presented as mean ± SEM and analyzed by Student’s 2-sided t test (A, B, D, F, and I).