INTRODUCTION METHODS RESULTS RESULTS CONCLUSIONS
DESIGN OF AN IN VITRO PLATFORM TO STUDY LDL RETENTION BY GLYCOSAMINOGLYCANS Ana M. Porras, Taylor Weis & Kristyn Masters
Department of Biomedical Engineering, University of Wisconsin -‐ Madison INTRODUCTION
METHODS
RESULTS
Glycosaminoglycans in Healthy AorCc Valves Glycosaminoglycans (GAGs) are long polysaccharide chains usually found in the spongiosa layer of the aorFc valve, where they serve as lubricants1. The most abundant GAGs in aorFc valves are chondroiFn sulfate (CS) and hyaluronic acid (HA). While HA is not associated with a core protein, CS is found within the three most abundant valve proteoglycans: decorin, biglycan, and versican2.
Juvenile
1yo Adult 2yo Adult
LDL/oxLDL (100ug/ml) mixed prior to UV exposure. Amount of parFcles diffused out measured every 24 hrs.
Methacrylic Anhydride
LDL OxidaCon 5 nm CuSO4 37C, 24 hrs
Methacrylated GelaFn
GelaFn
I-‐2959 UV light
oxLDL
LDL
OR HA CS 1x = 1.6 mg/ml 1x = 8 mg/ml 1x is equivalent to physiologic concentraFons
GAGs and Calcific AorCc Valve Disease (CAVD) One of the earliest events in the development of CAVD is leaflet thickening due to proteoglycan (PG) enrichment of the spongiosa (Figure 1). AddiFonally, an accumulaFon of GAGs is evident in calcified aorFc valves, where they localize around areas of nodule formaFon3. These findings suggest GAGs may play a crucial role in the iniFaFon and progression of CAVD.
RetenCon Studies
Hydrogel Synthesis
Entrapment Studies
5% w/v gelaFn
Once synthesized, gels were incubated in 100 ug/ml LDL or oxLDL. Amount of parFcles diffused in measured every 24 hrs.
HA Contributes to LDL and oxLDL RetenCon 13 11 9 7 5 3 1 -‐1
Figure 1. AorFc valves from adult h y p e r c h o l e s t e r o l e m i c s w i n e exhibiFng hallmarks of early valve disease are enriched in GAGs3.
Low-‐Density Lipoprotein RetenCon by GAGs During atherosclerosis, GAGs in the vasculature are known to “trap” lipoproteins and increase their sensiFvity to oxidaFve modificaFon4. We have also observed an increase in LDL oxidaFon accompanying GAG enrichment in the aforemenFoned hypercholesterolemic swine model3. LDL retenFon by PGs is hypothesized to play an important role in the iniFaFon of aorFc valve disease5,6. However, this process remains largely uncharacterized and the roles of GAG enrichment and changes in GAG composiFon in facilitaFng the deposiFon of LDL and oxidized LDL (oxLDL) in the context of the aorFc valve have not previously been examined.
The aim of this study was to create an in vitro model to characterize how LDL and oxLDL deposiCon is impacted by changes in extracellular matrix composiCon that mimic early events in CAVD.
B)
oxLDL Retained (μg) Control GelaFn 1x HHA A 1x 2x HHA A 2x 4x HHA A 4x
0
24
* *
* * * 48
72
96
LDL Retained (μg) 13 11 9 7 5 3 1 -‐1
* *
120
*
*
*
0
24
Time (hours)
48
72
96
120
Time (hours)
Figure 2. QuanFficaFon of (A) oxLDL and (B) LDL retenFon in gelaFn-‐HA hydrogels over 6 days. Samples were normalized to a control containing nanoparFcles of the same diameter as LDL. *P
Department of Biomedical Engineering, University of Wisconsin -‐ Madison INTRODUCTION
METHODS
RESULTS
Glycosaminoglycans in Healthy AorCc Valves Glycosaminoglycans (GAGs) are long polysaccharide chains usually found in the spongiosa layer of the aorFc valve, where they serve as lubricants1. The most abundant GAGs in aorFc valves are chondroiFn sulfate (CS) and hyaluronic acid (HA). While HA is not associated with a core protein, CS is found within the three most abundant valve proteoglycans: decorin, biglycan, and versican2.
Juvenile
1yo Adult 2yo Adult
LDL/oxLDL (100ug/ml) mixed prior to UV exposure. Amount of parFcles diffused out measured every 24 hrs.
Methacrylic Anhydride
LDL OxidaCon 5 nm CuSO4 37C, 24 hrs
Methacrylated GelaFn
GelaFn
I-‐2959 UV light
oxLDL
LDL
OR HA CS 1x = 1.6 mg/ml 1x = 8 mg/ml 1x is equivalent to physiologic concentraFons
GAGs and Calcific AorCc Valve Disease (CAVD) One of the earliest events in the development of CAVD is leaflet thickening due to proteoglycan (PG) enrichment of the spongiosa (Figure 1). AddiFonally, an accumulaFon of GAGs is evident in calcified aorFc valves, where they localize around areas of nodule formaFon3. These findings suggest GAGs may play a crucial role in the iniFaFon and progression of CAVD.
RetenCon Studies
Hydrogel Synthesis
Entrapment Studies
5% w/v gelaFn
Once synthesized, gels were incubated in 100 ug/ml LDL or oxLDL. Amount of parFcles diffused in measured every 24 hrs.
HA Contributes to LDL and oxLDL RetenCon 13 11 9 7 5 3 1 -‐1
Figure 1. AorFc valves from adult h y p e r c h o l e s t e r o l e m i c s w i n e exhibiFng hallmarks of early valve disease are enriched in GAGs3.
Low-‐Density Lipoprotein RetenCon by GAGs During atherosclerosis, GAGs in the vasculature are known to “trap” lipoproteins and increase their sensiFvity to oxidaFve modificaFon4. We have also observed an increase in LDL oxidaFon accompanying GAG enrichment in the aforemenFoned hypercholesterolemic swine model3. LDL retenFon by PGs is hypothesized to play an important role in the iniFaFon of aorFc valve disease5,6. However, this process remains largely uncharacterized and the roles of GAG enrichment and changes in GAG composiFon in facilitaFng the deposiFon of LDL and oxidized LDL (oxLDL) in the context of the aorFc valve have not previously been examined.
The aim of this study was to create an in vitro model to characterize how LDL and oxLDL deposiCon is impacted by changes in extracellular matrix composiCon that mimic early events in CAVD.
B)
oxLDL Retained (μg) Control GelaFn 1x HHA A 1x 2x HHA A 2x 4x HHA A 4x
0
24
* *
* * * 48
72
96
LDL Retained (μg) 13 11 9 7 5 3 1 -‐1
* *
120
*
*
*
0
24
Time (hours)
48
72
96
120
Time (hours)
Figure 2. QuanFficaFon of (A) oxLDL and (B) LDL retenFon in gelaFn-‐HA hydrogels over 6 days. Samples were normalized to a control containing nanoparFcles of the same diameter as LDL. *P
