1. Proteoglycan Mimic of the Glycocalyx to Treat Endothelial Dysfunction Victoria L. Messerschmidt, James R. Wodicka, and Alyssa Panitch Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, 47907 Introduction Abstract Synthesis & Purification of Proteoglycans References Acknowledgements E-Selectin and ICAM Upregulation Methods Patients with kidney failure usually undergo hemodialysis, a process by which toxins produced by the body are filtered from the blood, in order to survive. These patients require a vascular assess site and the preferred form is called an arteriovenousfistula (AVF), a surgically created connection between an artery and vein that is utilized to undergo dialysis. However, AVFs have a failure rate of 50-60%. One of the contributions to AVF failure is endothelial cell dysfunction and loss of glycocalyx, which allows neutrophils and other native cells into the media of the vessel, which causes an additional inflammatory response and subsequent vessel narrowing. Our lab addresses endothelial dysfunction by mimicking the function of the glycocalyx to prevent transmigration of inflammatory cells and ultimately create a healthier vessel for hemodialysis. We have synthesized several glycocalyx mimics consisting of a dermatan sulfate backbone with multiple selectin and ICAM-binding peptides attached. Initial testing involved determining the ability of the variants to bind to inflamed endothelial cells. We also began cultures of human promyelocytic leukemia cells (HL60s) and determined conditions to differentiate them into neutrophils. Initial studies are underway to determine the ability of the glycocalyx mimics to prevent neutrophil binding and diapedesis. Thus far, we have observed that the glycocalyx mimics bind to endothelial cells and that this binding is dependent upon the type of selectin and/or ICAM-binding peptides as well as how many peptides are present per dermatan sulfate backbone. We have also shown that HL60 proliferation begins approximately 10 days after seeding, and that rentinoic acid (RA) differentiates HL60 cells into neutrophils, which is observed by their adherence to tissue culture flasks. The establishment of protocols for culturing HL60 cells and differentiating them into neutrophils will significantly aid in the ability to assess a promising set of glycocalyx mimics. Figure 6: Purification of proteoglycan mimics after BMPH linkage molecule. Culturing HL60 Cells Media: RPMI 1640 medium with 20% FBS, 1% pen-strep Conditions: 37°C with 5% CO 2. Media was changed every 2 to 3 days. Differentiation of HL60 to Neutrophils Media: RPMI 1640 with 20% FBS, 1% pen-strep and 5 μM Retinoic Acid. Conditions: 37°C with 5% CO2. Media was changed every 2 to 3 days. Diapedesis of Neutrophils Transwell plates (3.0 μm pore size) were seeded with a monolayer of human aortic endothelial cells, treated with selectin and ICAM treatments, and coated with neutrophils to determine the transmigration of neutrophils across the membrane. Synthesis of Proteoglycan Peptides [1] S. T. W. Morris et al, “Impaired endothelial function in isolated human uremic resistance arteries,” Kidney International, vol. 60, no. 3, pp. 1077-1082, Mar 2001. [2] B. M. van den Berg et al, “Glycocalyx and endothelial (dys) function: From mice to men,” Pharmocol Rep, vol. 58, 2006. [3] J. Wodika, A. Panitch, “Arteriovenous fistula maturation in hemodialysis patients,” Aug 2014, Manuscript in preparation. [4] E. O’Riordan et al, “Endothelial cell dysfunction: The syndrome in making,” Kidney International, vol. 67, no. 5, pp. 1654-1658, May 2005. NIH R01HL106792 Figure 5: ICAM receptors are upregulated when treated with an inflammatory response molecule, TNF-α for 24 hours. This graph shows that by making the endothelial cells inflamed, a treatment would be necessary to reduce the inflammation. Future Research on Glycocalyx Mimics Figure 7: Starting from a cell density of 4*10 4, it takes 10 days before proliferation has begun. Originally there is a loss in cell number, but with 20% FBS the culture can proliferate. When cells reach a certain density they may begin to spontaneously differentiate. This research has shown that there is promise in Glycocalyx mimics. In the future, the lab will create a way to optimize the concentration and dosage to find the more effective treatments. Later, animal models will be used to determine the in vivo effect of the therapeutics. If then it is shown that the neutrophils still do not migrate across and cause inflammation, the therapeutic can go into human trials.  End Stage Renal Disease (ESRD) patients are unable to filter toxins from their blood resulting in the need for dialysis [1,2].  Current methods [3]:  The best method, arteriovenous fistula, fails 50-60% of the time resulting in fistula failure [3].  Those that have ESRD have clogged arteries similar to those with heart disease. It has been shown that the glycocalyx has been a part in this [1].  E-Selectin and ICAM receptors bind and adhere to neutrophils, white blood cells.  When the glycocalyx is gone, the endothelial cells are permeable and allow native and foreign cells into the vessel walls, which further increases inflammation [4]. Figure 2:Tunneled Dialysis Cather Figure 1: Arteriovenous and graft Differentiation of HL60 to Neutrophils Figure 7: Neutrophils are the cells that migrate across the compromised endothelial layer. In order to make neutrophils, Human Leukemia cells (HL60) were cultured. Once proliferation was established, 5 μM of Retinoic Acid was added to the media. The neutrophils were differentiated from the HL60 cells by checking adherence. HL60 are cultured in suspension, while neutrophils are adherent. HL60 Growth Pattern End Stage Renal Disease Figure 4: Cycle of AVF failure. Our Methods Figure 3: E-Selectin and ICAM Function Dermatan Sulfate (DS) NaIO 4 Acetate Buffer Oxidized DS 1x PBS DS-BMPH X 1x PBS Peptide 1x PBS Semicarbazide Hydrochloride Peptide DS-BMPH X -Peptide X mAU 4000 3000 2000 1000 0 020406080100120 mL 280 nm254 nm215 nm HL60 Cells + Retinoic AcidNeutrophil =  This project focuses was inspired by the cycle shown in Figure 4.  Once the fistula fails to mature, then a cycle of damage and restenosis happens.  This project aims to stop this cycle by mimicking the body’s natural vessel protection system.