Atherosclerosis, a cardiovascular disease (CVD) characterized by the accumulation and rupturing of plaque within the vessel walls, is responsible for approximately 75% of myocardial infarctions and remains the leading cause of death worldwide (1). Disruption and degradation of the endothelial glycocalyx (GCX), a protective sugar mesh on the surface of vascular endothelial cells, promotes atherosclerosis (2). Our lab has shown that a novel combination therapeutic consisting of heparin sodium and sphingosine-1-phosphate (HS-S1P) restores GCX structure and function in the disturbed flow (DF) conditions characteristic of early disease stages (3). However, this data is correlative, and we do not know how the therapeutic works. One possible mechanism of action for HS-S1P is through the S1P receptor 1 (S1PR1), an endothelial cell receptor that plays a role in maintaining vascular tone, barrier function, and inflammation (4). Another potential mechanism of action for HS-S1P is exotosin-like3 (EXTL3), a gene involved in heparan sulfate (HS) synthesis, a glycosaminoglycan that makes up over 70% of the GCX (5). Prior work has shown that exogenous HS-S1P restores enzymatically induced HS damage in rat fat pad endothelial cells (6), suggesting that the co-treatment could be triggering HS production. This work begins to evaluate S1PR1 and EXTL3 as mechanisms of action for our HS-S1P co-treatment through preliminary investigation into the effects of the co-treatment on S1PR1 and EXTL3 expression in DF conditions and through investigating the impact of S1PR1 KD on GCX structure and function.
Methods:
Two DF models were used to investigate the effects of the co-treatment on S1PR1 and EXTL3 expression. For in vivo, C57Bl/6 mice underwent a partial ligation of the left carotid artery to induce acute disturbed flow. The right carotid artery was left as a control for each mouse. Mice received a single dose of either HS-S1P or a vehicle control 5 days post-ligation and were sacrificed 30 minutes after to account for the co-treatment's in vivo half-life. The carotid arteries were collected, fixed, and stained for S1PR1 and EXTL3 expression as previously described (3). For in vitro, human coronary artery endothelial cells (HCAECs) were cultured to confluency on glass coverslips and subjected to a 12-hour flow experiment in media containing either the co-treatment or no co-treatment. The flow chamber simulated DF conditions upstream and uniform flow (UF) conditions downstream, providing an experimental and a control group for each coverslip. Cells were fixed and stained for S1PR1 expression as previously described (3).
S1PR1 KD mice were used to investigate the role of S1PR1 in GCX structure and function. The descending aorta serves as a uniform flow region, while the aortic arch serves as a chronic DF region. GCX components syndecan-1(SDC1), syndecan-2 (SDC2), and glypican-1 (GPC1) were stained.
All samples were imaged using confocal microscopy. Results are displayed as mean ± SEM, and significance was determined using one- or two-way ANOVA with Tukey’s multiple comparison tests.
Results:
Our analysis showed that HS-S1P co-treatment significantly increases S1PR1 and EXTL3 expression in vivo. In vivo, normalized S1PR1 expression in LCA vessels relative to RCA, was 0.48 ± 0.10 in untreated conditions, 0.38 ± 0.07 in vehicle-only conditions, and 1.00 ± 0.08 in co-treatment conditions. In vitro, normalized S1PR1 expression in no treatment DF regions was 0.74 ± 0.07, whereas normalized S1PR1 expression in treated DF regions increased to 1.35 ± 0.16. This increase in S1PR1 expression was significant in vivo, but not in vitro.
Investigation into KD models is ongoing, but our current analysis shows that S1PR1 KD impairs GCX structure, as is evident by decreases in SDC2 and GPC1 expression.
Discussion:
These preliminary studies provided insight into the effects of HS-S1P co-treatment on S1PR1 and EXTL3 expression. The significant increases in both following treatment with HS-S1P in vivo confirm their candidacy as potential mechanisms of action for the therapy. Increased S1PR1 expression validates prior work that has shown the roles of HS and S1P individually in reversing hyperpermeability7, a known consequence of GCX damage. HS and S1P are also anti-inflammatory agents, which is critical considering the role of S1PR1 in regulating inflammation. Increased EXTL3 expression could explain the restoration of GCX structure through HS synthesis in DF conditions. Further investigation into S1PR1 and EXTL3 is needed to determine their potentials as mechanisms of action.
These preliminary studies also provided insight into the effects of S1PR1 KD on GCX structure. This work provides baseline characteristic data that can be used to inform the design of a future experiment investigating the effects of HS-S1P co-treatment on S1PR1 KD models in DF conditions. This will be necessary to confirm the involvement of S1PR1 in HS-S1P co-treatment function, and in turn is crucial for the therapy’s continued commercial development.
Acknowledgements: We thank Northeastern’s Institute for Chemical Imaging of Living Systems (RRID:SCR_022681) for imaging support. We thank the Hla Lab at Boston Children’s Hospital for donating S1PR1 KD mice. Funding was provided by the Northeastern University Center for Research Innovation Spark Fund.
References:
(1): Cardiovascular diseases (CVDs). Accessed February 23, 2025. https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds)
(2): Mitra R, O’Neil GL, Harding IC, Cheng MJ, Mensah SA, Ebong EE. Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target. Curr Atheroscler Rep. 2017;19(12):63. doi:10.1007/s11883-017-0691-9
3: Mitra R, Pentland K, Kolev S, et al. Co-Therapy with S1P and Heparan Sulfate Derivatives to Restore Endothelial Glycocalyx and Combat Pro-Atherosclerotic Endothelial Dysfunction. Published online November 8, 2024:2024.11.06.622347. doi:10.1101/2024.11.06.622347
4: Cartier A, Leigh T, Liu CH, Hla T. Endothelial sphingosine 1-phosphate receptors promote vascular normalization and antitumor therapy. Proceedings of the National Academy of Sciences. 2020;117(6):3157-3166. doi:10.1073/pnas.1906246117
5: Reitsma S, Slaaf DW, Vink H, van Zandvoort MAMJ, oude Egbrink MGA. The endothelial glycocalyx: composition, functions, and visualization. Pflugers Arch. 2007;454(3):345-359. doi:10.1007/s00424-007-0212-8
6: Mensah SA, Cheng MJ, Homayoni H, Plouffe BD, Coury AJ, Ebong EE. Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication. PLOS ONE. 2017;12(10):e0186116. doi:10.1371/journal.pone.0186116
7: Wang L, Dudek SM. Regulation of vascular permeability by sphingosine 1-phosphate. Microvasc Res. 2009;77(1):39-45. doi:10.1016/j.mvr.2008.09.005