Dane MJ, van den Berg BM, Lee DH, Boels MG, Tiemeier GL, Avramut MC, van Zonneveld AJ, van der Vlag J, Vink H, Rabelink TJ. A microscopic view on the renal endothelial glycocalyx.
Objective:
Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown.
Approach and Results:
Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with
13
C-labeled glucose.
Conclusions:
These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.
Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality.
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