BME 545 Unit 2: ECM Remodeling and Aging/Disease

What are matrix metalloproteases (MMPs)?

MMPs are enzymes that degrade the ECM

What are the functions of degrading ECM?

1. Path clearing for cells through the ECM (allows cell movement)

2. ECM proteolysis generates signaling molecules (degrade ECM where GF are bound to release them)

3. Degradation of intercellular junctions and basement membrane (destroy barrier function)

4. Activation of a latent signal

5. Regulation of active signal

What are proteinases?

Enzymes that degrade the ECM

What is the characteristic structure of MMPs?

3 histidines with 4 conserved domains

What are the four characteristic domains of MMPs?

1. Pro-domain

2. Catalytic domain

3. Hinge domain

4. Hemopexin domain

What does the pro domain on an MMP do?

The pro-domain makes the MMP inactive, it must be removed for the MMP to become activated.

Zymogen

Inactive form of an enzyme until pro-peptide is cleaved

What does the catalytic domain of the MMP do?

Contains the 3 histidines that are linked to zinc

What does the hemopexin domain do on the MMP?

Binds to ECM or TIMPs, specific based on the substrate

How does a Pro-MMP remain inactive?

The cystidine on the pro-domain coordinates with Zn2+ to lock into inactive state

How do you activate the Pro-MMP?

Cleave the prodomain with the cystine so the Zn2+ can no longer lock the MMP in place.

What are some ways to disrupt the Cys-Zn2+ linkage in Pro-MMPs?

Proteases, chemicals, heat, pH

Collagenases

Cleave triple helix of fibrillar collagen (MMP 1, 8, 13)

Gelatinases

Degrade gelatin (2,9)

Stromelysins

Broad spectrum MMP but unable to cleave collagen triple helix (3,10)

Matrilysins

Activate MMPs into active form

What is the mode of action for MMPs?

Cleave the peptide before residue with hydrophobic side chain (ex: Leu, Met, Phe, Tyr)

What are ways to regulate MMPs?

1. Transcription (gene expression)

2. Zymogen activation (Pro-active)

3. Inhibited by endogenous inhibitors (TIMPs)

What is a TIMP?

Tissue inhibitor of MMPs; prevent MMPs from degrading ECM target in a 1:1 ratio; bind to hemopexin domain

What are serine proteases?

Broad spectrum proteases with 16 super families; can be secreted as zymogens

What are some examples of serine proteases?

Trypsin, plasmin, thrombin

What is the basic structure of serine proteases?

Catalytic triad with an acid/base/nucleophile

What does the catalytic triad do?

It is within the binding pocket and determines binding specificity; this is the part that does the cutting

What does chymotrypsin bind to?

Long hydrophobics (prefers aromatic side chains)

What does trypsin bind to?

Long positively charged (arginine and lysine); attached to negative charge

What does elastase bind to?

Small acids like Alanine, Serine and Glycine

What are the pathways for diseased ECM?

Increased ECM degradation

Increased ECM production (more of right or wrong ECM type)

Increase or change in X-Linking

What are AGEs?

Advanced glycation end products

How do AGEs work?

Non-enzymatic glycation of proteins and lipids by saccharide derivates

What are the results of Ages?

Excessive cross-linking and sped up oxidative damage to cells

Is glycation avoidable?

No it is not, as it is a feature of physiological metabolism

What are the affect of aging and injury to the ECM?

1. Increased glycation rate

2. Decreased ECM turnover

3. Increased expression for RAGEs

What are RAGEs?

Receptors for AGEs

What are the results of having more RAGEs for AGEs to bind to?

Increased reactive oxygen species and inflammatory factors

Describe the ECM changes involved in ligament and tendon aging.

1. Glycation causing collagen cross-linking

2. Reduction of GAGs causing less hydration

Both cause more stiffening

What is the impact of the ECM changes associated with ligament and tendon aging?

Reduced ability to withstand loading and recover from injury

What type of disease can affect cartilage in the ECM?

Osteoarthritis

What causes osteoarthritis?

Damage from mechanical stress with insufficient repair

What ECM changes occur with osteoarthritis?

• Increased degradation

• Increased MMPs

• Increased ADAMTS (degrade GAGs)

• increased collagen I replacing collagen II

What are the results of decreased GAGs/PGs and increased collagen I?

Cell phenotype changes from round to spread out (similar to fibroblast)

What is the impact of the ECM changes found in osteoarthritis?

Decreased lubrication and decreased compression modulus of cartilage

What are the steps of skin wound healing?

1. Initial fibrin clotting (to stop the bleeding)

2. Inflammation (drive vascularization and WBCs)

3. Proliferative phase (more fibroblasts to secrete temporary ECM)

4. Remodeling phase (ECM rearrangement and x-linking)

What are the ECM changes associated with scarring?

• Increased collagen I and III

• Decreased HA and decorin

• All leads to collagen disorganization

What impacts do the ECM changes related to scarring have?

Decreased tensile strength of skin

Describe the scarring feedback loop

Scar ECM changes cell phenotype causing increased fibrosis ECM production and more scarring

What type of disease affects the liver ECM and what is the end point of this disease?

Fibrosis which leads to liver cirrosis

What changes in the ECM occur during liver fibrosis?

3-5x increase in ECM where collagen I and III accumulate and replace basement membrane

What are the impacts of the ECM changes related to liver fibrosis?

Reduced diffusion/transport to hepatocytes

How are tumors imaged in ECMs?

TACs (tumor associated collagen signatures)

What are the types of TAC and their differences?

1. TAC1 locally dense collagen I

2. TAC2 straightened fibers around tumor

3. TAC3 radially aligned straight fibers 90 degrees from tumor

For tumors with increased TAC3, what does this mean?

Associated with poor prognosis and more metastasis

How to mimic aging in a scaffold?

Add AGEs/glycation to induce stiffening by adding sugars