1. Mineralization of elastin reduces its degradation by cathepsin K, as mineral deposits prevent the enzyme's access. 2. Cathepsin K binds instantly to elastin, then releases over time as it digests the substrate and becomes denatured. 3. Peptide fragments from cathepsin K digestion of elastin increase calcium mineral deposition on elastin at high concentrations.

1. Exploring the relationship between mineralization and digestion of elastin
Yen-Wen Chen, Neil Mackenzie, Dieter Brömme
Department of Oral and Biological Sciences, Faculty of Dentistry; Department of Biochemistry and Molecular Biology, Faculty of Medicine;
Center for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, V6T1Z3, BC, Canada.
Introduction:
Theformationofmineraldepositsinthevasculature,knownasvascularcalcification,isacharacteristic
of aging and can be linked to many diseases such as atherosclerosis, diabetes and renal failure.
Calcification occurs mainly in the extracellular matrix of blood vessels, making these vessel walls
brittle and restricting blood flow. This is predictive of some of the main causes of death in Canada,
including heart attacks and strokes.
Elastin is a highly stable protein found in the extracellular matrix (ECM), and is particularly important
in the blood vessels, lungs and skin. One of the roles of elastin is to provide the vasculature with
elasticity and resilience, allowing blood pressure to be maintained. When various stressors act upon
the vasculature, including mechanical stress or infection, the ECM undergoes structural changes,
including degradation of elastin, which has been shown to be primarily mediated by cathepsins.
It has been reported that peptide fragments derived from the proteolysis of elastin affect cellular
signaling leading to mineralization, but the biochemical effects are not fully understood.
Elastin is produced only during early development and childhood and undergoes little turnover
throughout its lifespan, thus any proteolytic damage is essentially irreparable.We hypothesized that
the calcification may occur as a protective mechanism that loses control during diseases, leading to
significant pathology.
Figure 1. Schematic of the role of cathepsins and elastin digestion in the development of an atherosclerotic plaque.
Aims:
1. Optimize protocols for testing the effects of mineralization on elastin degradation
2. Determine whethercalcificationoftheextracellularmatrixaffectsdigestionofelastinbyCathepsin
K (catK)
3. Determine whether peptide fragments derived from catK digestion of elastin have a biochemical
effect on mineralization
Methods:
• Z-FR-MCA Assay: Cathepsin K enzyme mixed with Cathepsin K Buffer with 0.25% dithiothreitol
(DTT) is added to 1 M substrate Z-FR-MCA and read in spectrophotometer.
• Elastin Mineralization: Elastin incubated for 14 days using Eagle’s Minimum Essential Medium
(MEM), 2 mM Sodium Phosphate (NaPi; pH = 7.4), 10% Fetal Calf Serum (FCS), 0.02% sodium azide
(NaN3
). Medium was changed every 48 hours.
• CatK Digestion: Elastin incubated with Cathepsin K enzyme at 37o
C, 200 rpm with Cathepsin K
Buffer (Sodium Acetate buffer; pH = 5.5) with 0.25% dithiothreitol (DTT).
• Congo Red Assay: Congo Red Elastin digested overnight with Cathepsin K enzyme. Supernatant
removed and released Congo Red dye read at 450 nm.
• Electron Microscopy: Samples fixed in 2.5% Glutaraldehyde in PBS for 5 min.; dehydrated with
increasing concentrations of ethanol. Samples coated with carbon viewed on a FEI Helios NanoLab
650 dual beam scanning electron microscope. The vCD back scatter electron detector was used to
determine density changes in mineralized samples. This imaging was carried out using a voltage
of 5kV and a probe current of 0.2nA at a working distance of 4mm.
• Calcium Assay: Calcium extracted from elastin samples by incubation in 0.6 M hydrochloric acid
over 24 hours. Ca2+
ions detected through addition of O-Cresolphthalein complexone forming a
violet complex which can be read at 562 nm. Concentrations of calcium normalized to weight of
sample
+ CatK
+ Cat K + T12
***
Results:
Effects of mineralization on cathepsin degradation
It has been hypothesized that deposition of mineral crystals on the matrix will prevent cathepsin-
catalyzed degradation. 7-day and 14-day mineralization periods were tested to determine whether
there is a relation between the degree of mineralization and the amount of degradation.
A C
B
Figure3.CatKActivityonMineralizedCongoRedElastin.Backscatterelectronmicrographs(2500x)showingnative(A)andmineralized
(B) Bovine Neck elastin following treatment with 2mM NaPi. (C) Congo Red Assay used to determine the relative digestion activity
following a 7-day and 14-day incubation of Congo Red Elastin in mineralization media.
In both the 7-day and 14-day incubations, the amount of digestion present in the mineralized
samples is significantly reduced compared to the unmineralized samples. A paired t-test revealed
no significant difference (P>0.005) in digestion activity of the mineralized elastin between the 7-day
and 14-day incubation time periods.
Cathepsin binding kinetics during digestion
A time course study was done to determine
the binding kinetics for Cathepsin K with an
elastin substrate.
Figure 2. Time dependent binding of cathepsin K to Elastin.
Aqueous phase of Bovine Neck Elastin digested with CatK
while incubating at 37o
C is tested for enzyme activity with
Z-FR-MCA every 30 min.
Initially, the catK shows immediate binding within the first 30 min. During digestion, the enzyme
will be released into the aqueous phase as substrate is digested, and enzyme becomes unbound.
Over time, activity of enzyme is reduced as it becomes denatured.
Effect of peptide fragments on elastin mineralization
CatK-digested elastin peptide fragments were tested to elucidate whether they had any biochemical
effects on the calcium mineral deposition on elastin.
A B
Figure 4. Calcium Assay of Mineralized Elastin Exposed to Increasing Peptide Fragment Concentrations. (A) Flowchart of peptide
fragment experiment (B) Bovine Neck Elastin mineralized for 14 days using MEM, 2mM NaPi, 10% FCS, 0.02% NaN3, and increasing
concentrations of Elastin fragments. Calcium assay performed to quantify calcium levels. n= 3; error bars represent 1 SEM. ***P < 0.005
Based on this preliminary study, high concentrations of peptide fragments (500 ng/mL) increase
the amount of calcium mineralization present. There is no significant difference in the lower
concentrations of peptide fragments.
Conclusions:
In this study, we have successfully optimized a protocol to test the effects of mineralization on elastin
degradation. From these initial experiments, we are able to draw 3 conclusions:
• Mineralized elastin has reduced Cathepsin K activity when compared to unmineralized elastin
• Cathepsin K binding occurs instantly in the first 30 minutes of activity
• Addition of peptides derived from catK digestion of elastin is correlated with increased calcium
concentration levels
Future Experiments:
Previous results in the lab have shown that an inhibitor, dihydrotanshinone-12 (T12) inhibits
mineralization.
A C
B
Figure 5. Elastase specific inhibitors reduce Cathepsin K initiated mineralization. (A) Back scatter electron micrographs (2000x)
showing CatK treated (A) and CatK + T12 inhibitor treated (B) elastin following treatment with 2mM NaPi. (C) Mineralization levels of
the elastin tissue compared between elastin digested with CatK and elastin digested with CatK under the effects of T12. ***P < 0.05
We would like to use methods developed in this project to determine the effects of various novel
catK inhibitors on:
• CatK enzyme activity by calculating IC50
curves for the various novel catK inhibitors
• Elastin mineralization following digestion with catK in the presence/absence of inhibitors
• Development of mineralization in vascular cells and ex vivo aortic ring cultures
Various novel catK inhibitors have been compiled from comprehensive reviews of the literature to
determine classes of compounds that have an inhibitory effect on elastase activity in cathepsins.
These inhibitors will have their inhibitory effect measured using the Congo Red assay to determine
their IC50
values.
Those that show specific inhibition of catK elastase activity will be tested using methods described
above to determine if they have an effect on mineralization.
We are hoping to eventually move the experiments into the in vitro environment by running these
experiments in a cellular model. Hopefully these novel inhibitors can be tested in animal models in
the future and one day be a potential therapeutic for diseases associated with vascular calcification.
Acknowledgments:
I would like to thank everyone at the Bromme Lab for all of the help they have provided, with Neil
Mackenzie in particular for his guidance. Our work was supported by the CIHR and the CBR (Summer
Studentship).
+2mM NaPi
Calcium Assay
+CatK
8 mg of BNE
3 mg of BNE
0.014 mg/µL
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*
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