DiaPharma and QuickZyme Know Matrix Matters!
Matrix Metalloproteases (MMPs) degrade collagen and elastin in the extracellular matrix (ECM). As indicators of inflammation and disease, MMPs are key biomarkers and targets for drug development and research studies.
Alcohol-Associated Liver Disease (ALD)
MMP-2, MMP-8, and MMP-9 may be markers of alcohol-associated liver cirrhosis. Pre-clinical rat models show that MMP-9 levels are elevated following chronic alcohol consumption.
Biliary Atresia
MMP-7 has garnished research support for its role in mediating biliary injury in the disease.
Cancers
MMP-2, MMP-7, MMP-8, and MMP-9 can be used as biomarkers in a variety of cancers including colorectal, bladder, breast, and gastric cancers. Studies of squamous cell carcinoma suggest that MMP-9 activates epithelial-mesenchymal transitions (EMT), which can lead to tumor cell metastasis.
Cardiac Fibrosis
MMP-2, MMP-8, and MMP-9 have been identified to be involved with myocardial remodeling, which can lead to fibrosis. Elevated plasma MMP-14 levels correlate with cardiac fibrosis.
Chronic Obstructive Pulmonary Disease (COPD)
MMP-2 levels have been explored as a biomarker during MMP-inhibitor therapy.
Idiopathic Pulmonary Fibrosis (IPF)
MMP-7 levels can indicate IPF disease progression.
Kidney Fibrosis and Function
MMP-9 has been suggested to be a profibrotic factor, ultimately driving kidney fibrosis when secreted. Urinary MMP-7 levels have been analyzed as a biomarker during renal function research.
NAFLD and NASH
Serum MMP-7 is an indicator of fibrosis in non-alcoholic fatty liver disease (NAFLD). Serum MMP-9 is useful when researching NAFLD and is suggested to be a predictor of non-alcoholic steatohepatitis (NASH).
QuickZyme specializes in collagen-related assays and MMP activity assays (MMP-2, MMP-7, MMP-8, MMP-9, MMP-14). These assays were developed by scientists to fill an unmet need in their research studies. MMP activity assays provide a more granular insight to the role that fabrication and degradation of the ECM can play in fibrosis, inflammation, and metastatic tumors.
Finally, tests are available to quantify MMP activity accurately and specifically in relevant biological samples (blood, sputum, urine, cell cultures).
Available QuickZyme MMP activity assay kits
- QuickZyme Human MMP-2 Activity Assay Kit
- QuickZyme Human MMP-7 Activity Assay Kit
- QuickZyme Human MMP-8 Activity Assay Kit
- QuickZyme Human MMP-9 Activity Assay Kit
- QuickZyme Mouse MMP-9 Activity Assay Kit
- QuickZyme Human MMP-14 Activity Assay Kit
MMP Function and Regulation
MMPs secreted into the ECM undergo cleavage to actively degrade ECM proteins. MMPs are the only enzymes that degrade collagen, and thus are involved in many physiologic processes including wound healing and embryonic development. Due to differences in protein domains, MMPs are classified as archetypal MMPs (MMP-1,-3,-8,-10,-12,-13,-19,-20, -27), gelatinases (MMP-2,-9), matrilysins (MMP-7,-26), and convertase-activatable MMPs (MMP-11, -14, -15, -16, -17, -21, -23B, -24, -25, -28).
Under normal conditions, MMPs are regulated at the levels of transcription, proenzyme activation, localization, and enzymatic activity. MMP activity is inhibited by endogenous inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). MMPs and TIMPs normally exist in a delicate balance, but when the activity of MMPs outweighs the inhibitory functionality of TIMPs within a tissue, ECM breakdown occurs. Thus, TIMPs are also the focus of many research studies and are used as biomarkers for many of the same disease states as MMPs. (Side note: TIMPs are now clinically used in fibrosis tests, like the Enhanced Liver Fibrosis (ELF™) test for the identification of NASH subjects with advanced fibrosis.)
MMP Research Applications
The dysregulated expression, aberrant activity, or altered degradation of MMPs and ECM components underlie many disease states including cancer, inflammatory diseases of the gut and joints, vascular diseases, CNS diseases, bacterial and viral infections, sepsis, and fibrosis. Hence, MMP activity is analyzed as a biomarker in many diverse research applications investigating these disease states:
MMP-2 activity has been examined in the serum of subjects with COPD, bladder cancer, colorectal cancer, and pre-eclampsia.
MMP-7 activity has proven to be a useful biomarker in breast cancer, colorectal cancer, IPF, kidney fibrosis, and lung fibrosis studies.
MMP-8 has been analyzed in periodontitis, gastric cancer, and colorectal cancer research.
MMP-9 activity is used as a biomarker in studies of cardiac remodeling, colorectal cancer, squamous cell carcinoma, bladder cancer, early kidney damage, ocular inflammation (tear fluid), and pre-eclampsia.
MMP-14 has been investigated as a biomarker and potential target in familial amyloidotic polyneuropathy (FAP) studies. Increased levels of plasma MMP-14 correlate with cardiac fibrosis.
Analysis of MMP Enzymatic Activity
The enzymatic activities of MMPs are important to understand but have been difficult to study due to complex mechanisms and the lack of sensitive, specific tools to analyze MMPs in biological samples. While collagen degradation assays provide a direct readout on general MMP activity, they do not indicate the activity of specific MMPs. Quenched fluorescent (FRET) peptides are effective at evaluating protease kinetics but require purified enzymes and fail to indicate MMP specificity in biological samples. Zymography is not high throughput and can activate inactive proteases, skewing the limited semi-quantitative outputs.
Immunocapture activity methods have proven to be the best assays for assessing specific enzymatic activity in biological samples. Immunocapture activity assays are leading to a better understanding of how MMP activities participate in normal biological processes, disease states, and how they can be further leveraged as fluid-based biomarkers in therapeutic and drug development scenarios.
Quickzyme immunocapture activity assays have been developed to measure MMP-specific activity and improve technical specificity. This technology utilizes a monoclonal antibody to capture a specific MMP, much like the capture of a standard ELISA. This allows specific MMPs to be isolated from biological samples. Next, the proteolytic activity is measured with chromogenic or fluorogenic peptide substrates. MMPs require amino acid bonds on flanking sides of target cleavage sites, which makes classic chromogenic peptide substrate set-ups not possible. The Quickzyme chromogenic activity assays overcome this by utilizing a modified pro-urokinase that is activated following cleavage by MMPs. The modified urokinase design increases assay sensitivity due to the amplification of the detection enzyme. The chromogenic readout can be measured over time and offers a wide dynamic range by kinetic read-out, unlike standard antigen ELISAs.
QuickZyme Chromogenic Activity Assay Strategy
In addition to the existing activity measurement, there is an optional enzyme activation step of the QuickZyme kits that makes it possible to detect both active enzymes and pro-enzymes, after urokinase activation. This is an advantage over standard total antigen ELISAs, as the enzymatic specificity of what is captured (the pro-form, active form, inhibitor complex, or a combination) in antigen ELISAs is generally not known.