Attenuated hyaluronan synthesis impairs glycocalyx integrity and leads to arterial dysfunction

Abstract

The glycocalyx (GC) is a semi-solid layer lining the endothelium that plays a key role in maintaining barrier function and mechanosensing shear forces. With advanced age and pathological conditions, the GC deteriorates, and this is accompanied by arterial dysfunction characterized by impaired endothelial-dependent dilation (EDD) and large elastic artery stiffening. Alterations in hyaluronan (HA), a primary component of the GC, may contribute to the development of a dysfunctional artery phenotype. Here, we sought to determine the role of HA in GC integrity and arterial function. We have generated a tamoxifen-inducible, endothelial cell-specific, knockout mouse model of the hyaluronan producing enzyme, hyaluronan synthase 2 (HAS2, HAS2 f/f- Vecad-CreERT2). Endothelial HAS2 knockout (HAS2 f/f- Vecad-creERT2+: ecHAS2 KO, n=10) and wildtype (HAS2 +/+- Vecad-creERT2+: ecHAS2 WT, n=10) young mice were injected intraperitoneally with 4 mg of tamoxifen for 5 consecutive days. HAS2 mRNA expression was reduced by ~47% in carotid endothelial cells of ecHAS2 KO mice (P=0.004). GC barrier function (perfused boundary region [PBR]) was evaluated in vivo in the mesenteric microcirculation using an intravital microscope equipped with an automated capture and analysis system. PBR was 8.9% higher in ecHAS2 KO compared to ecHAS2 WT (P<0.0001). Subsequently, video recordings acquired were used to manually determine GC thickness from individual microvascular segments. GC thickness was significantly reduced in ecHAS2 KO vs ecHAS2 WT (7.9μm±0.02 vs 0.37μm±0.02, P<0.0001). Together, these results indicated that GC integrity was deteriorated in response to a reduction in endothelial HAS2. This GC deterioration impairs EDD, as evidenced by a 43.4% reduction in flow-induced dilation in isolated carotid arteries from ecHAS2 KO compared to ecHAS2 WT (P=0.013). To directly assess the role of GC plays in the differences in flow-induced EDD observed, the lumen of the arteries was infused with hyaluronidase, a HA degrading enzyme, and this abolished the differences in EDD between groups (P>0.05). Endothelial-independent dilation in response to sodium nitroprusside was not different between groups (P>0.05). Furthermore, aortic pulse wave velocity, a measure of large elastic artery stiffness, as well as systolic, diastolic, and mean arterial blood pressure were all greater in ecHAS2 KO compared to ecHAS2 WT (all P≤0.033). These results demonstrate that endothelial cell-specific impairment in HA synthesis compromises GC integrity and leads to arterial dysfunction and elevated blood pressure. Funded in part by awards from National Institutes of Health Awards R01 AG060395, R01 AG048366, T32 HL007576, T32 HL139451 and Veteran's Affairs Merit Review Award I01 BX004492. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.