Exploring the relationship between mineralization and digestion of elastinYen-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:The formation of mineral deposits in the vasculature, known as vascular calcification, is a characteristic 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 degradation2. Determine whether calcification of the extracellular matrix affects digestion of elastin by Cathepsin

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

Figure 3. CatK Activity on Mineralized Congo Red Elastin. Back scatter electron micrographs (2500x) showing native (A) and mineralized (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 37oC 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 mineralizationCatK-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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