Neuraminidase-3 Inhibition Mitigates Vascular Endothelial Dysfunction and Reduces Arterial Stiffness in Type 2 Diabetic Mice

Endothelial dysfunction and arterial stiffening are hallmarks of type 2 diabetes (T2D) and major contributors to cardiovascular disease. A common feature of these vascular abnormalities is endothelial glycocalyx degradation. The endothelial glycocalyx is a mechanosensitive structure that modulates endothelial cell responses to blood flow-dependent shear stress and maintains vascular wall homeostasis. We and others have shown that plasma neuraminidase (Neu) is increased in diabetes and that its activity degrades the glycocalyx and impairs arterial flow-mediated dilation (FMD). We have further demonstrated that inhalation of the Neu inhibitor, Zanamivir (10 mg every 12 hrs for 5 days, as FDA-approved), improves endothelial function in diabetic (db/db) mice without reducing arterial stiffness. However, these effects of Zanamivir did not translate to diabetic humans. The Neu family of enzymes degrades the glycocalyx by removing sialic acid moieties from glycoproteins and glycolipids. In mammals, it consists of four members, of which Neu3 is the most prominently present in plasma. As Zanamivir has only a moderate IC 50 of 6.8±3.1 µM against Neu3, we hypothesized that changing the route of administration and increasing the duration of treatment would further improve endothelial function and reduce arterial stiffness in diabetic mice, providing an impetus for a new clinical trial. We further hypothesized that the novel Neu3-specific inhibitor (C22601, Neu3i) would also ameliorate endothelial dysfunction and arterial stiffening in diabetic mice via endothelial glycocalyx restoration. We tested our hypotheses using human umbilical vein endothelial cells (HUVECs), isolated mesenteric arteries from 12-14 week-old male C57BL/6 mice, and 13-15 week-old male db/db mice. All differences reported herein are significant at P < 0.05. We found that, compared to vehicle (163 mM Cyclodextrin for 28 days), minipump subcutaneous infusion of Zanamivir (671 µg/kg/day for 28 days) in db/db mice decreased pulse wave velocity (PWV, 2.78±0.08 vs 3.96±0.14 m/s), increased atomic force microscopy measurements of aortic endothelium glycocalyx length (379±10 vs 276±28 nm), reduced arterial incremental modulus of elasticity (E inc , 10.39±0.70 vs 16.61±1.64 x10 6 dyn/cm 2 ), and improved FMD (98.2±4.1 vs 56.2±10.2 %) of isolated mesenteric arteries without affecting sodium nitroprusside (SNP)-induced vasodilation. In HUVECs, exposure to Neu3i (100 μM for 24 hrs) vs vehicle control improved wheat germ agglutinin (WGA) staining of the glycocalyx (1.23±0.08 fold), while in isolated mesenteric arteries, exposure to Neu3i for 18 hours improved FMD (49.8±4.9 vs 32.7±4.6 %). In db/db mice, Neu3i subcutaneous infusion (692 µg/kg/day for 28 days) reduced PWV (2.51±0.13 vs 3.96±0.14 m/s), increased endothelial glycocalyx length (419±41 vs 276±28 nm), reduced arterial E inc (12.50±1.30 vs 16.61±1.64 x10 6 dyn/cm 2 ), and improved isolated artery FMD (88.5±6.9 vs 56.2±10.2 %) without affecting SNP responses. These results indicated that Neu inhibition with Zanamivir or the novel Neu3i, C22601, improves glycocalyx structures and ameliorates endothelial dysfunction and arterial stiffening in diabetic mice, providing a potential therapeutic strategy for mitigating cardiovascular disease in individuals with T2D. LM-L and JP are supported by the National Institutes of Health grant: R01HL153264. FIR-P and LF-S are supported by the Research Excellence Program at the University of Missouri. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.