There is a critical need for predictive biomarkers in clinical research. Patients and payers demand greater efficacy and safety windows. Due to scarce research funding, drug developers are forced to select projects they are confident in early on in their development for investment and further investigation in expensive phase III studies. While the need is clear, the most frequently used methods of quantification within serum or plasma samples are more than two decades old. These methods quantify total proteins and overlook new developments and understanding of proteins as complex players that have multiple functions. Separately quantifying each part of the protein provides more information, by assessing the formation of the protein, the degradation of the proteins and potential signaling domains, rather than a crude measure of the total protein. In fact, many chronic diseases like osteoporosis, osteoarthritis, fibrosis of the liver, lung, kidney, skin, intestine, and heart result from an imbalance between the formation and degradation of tissue.  

Proteins have multiple domains, each with distinct functions. Some proteins have pro-peptides which may be used as biomarkers of protein formation. Other proteins have cryptic signals which are released after protease cleavage during tissue remodeling. Lastly, all proteins are degraded, which results in specific fragments that may be used as biomarkers for protein destruction. Our research group has focused on the extracellular matrix (ECM) and collagens for 20 years producing more than 500 publications. The collagens contain a variety of domains that can be quantified individually, providing an abundance of information. Some examples can be found below.

The N- and C-terminal end of fibrillar collagens contain pro-peptides, which are cleaved and separated from the molecule when the collagens are embedded into the matrix. The mature collagen structure will only be incorporated correctly into the ECM when the PRO-peptides are removed. The maturation and the correct processing of the collagens is essential for the quality of the ECM structure. The pro-peptides can be used as a surrogate measure of tissue formation. Biomarkers of type I collagen formation (PINP or PICP) have been used for the assessment of bone formation12 for decades. Only the neo-epitope Protein FingerprintTM technology allows for the assessment of the exact PRO-peptides generated during tissue formation, by measuring the cleavage site neo-epitope generated by proteases in this process. 


In order to sustain healthy tissues, the ECM is continuously being remodeled; as such, associated ECM proteins are degraded. This degradation results in the release of small protein fragments from larger complex proteins and the circulating fragments may be used as biomarkers. The smaller protein fragments contain information relating to specific tissue degradation. For example, bone is continuously remodeled by the bone degrading osteoclasts. The main destructive protease responsible is cathepsin K. The cleavage of type I collagen by cathepsin K yields the fragment referred to as CTX-I, which has been used as a biomarker of bone resorption for diseases in the osteology field3,4.

By assessing the formation and degradation of tissue separately, a greater understanding of tissue homeostasis can be obtained. Traditional protein measurement technologies do not permit the separate assessment of formation and degradation; rather, these methods quantify the total protein pool. In many diseases, the balance between degradation and formation of tissues is affected, leading to more non-functional tissue such as in skin, liver, lung, and kidney fibrosis or increased degradation leading to tissue loss in rheumatologic disorders. The figure below highlights the formation and degradation balance in a healthy individual, as compared to a diseased individual. This balance may be severely altered leading to the same amount of that protein in the blood, but with an altered composition. In COPD, 50% less formation and 50% more degradation is observed during excerbations5,6. This imbalance is not detected by traditional assays, but only by using the Protein FingerprintTM7,8 technology.


The extracellular matrix proteins are emerging as more than just passive structural proteins7,8. Collagens and other proteins contain sequences with potent signaling functions and these sequences become active when they are exposed and released by proteolytic cleavage. Assays have been developed for the bioactive fragments of these proteins. See the below examples.

Protein

Function

Collagen type

Tumstatin10

Signaling

Type IV

Endotrophin11

Hormone

Type VI

Vastatin12

Signaling

Type VIII

Restin10

Signaling

Type XV

Endostatin13

Signaling

Type XVIII

A protein is not just a protein, and a serological assessment of protein fragments - not a pool of the entire protein - may contain essential information for data interpretation as ECM signal peptides, tissue formation and tissue degradation fragments need to be quantified separately. By using more advanced protein assessment technologies, more accurate diagnostic and prognostic information may be obtained. 

1. Henriksen K, Tanko LB, Qvist P, et al: Assessment of osteoclast number and function: Application in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 18, 2007

2. Karsdal MA, Henriksen K, Leeming DJ, et al: Biochemical markers and the FDA Critical Path: how biomarkers may contribute to the understanding of pathophysiology and provide unique and necessary tools for drug development. [Internet]. Biomarkers 14:181–202, 2009Available from: http://www.ncbi.nlm.nih.gov/pubmed/19399662

3. Garnero P, Ferreras M, Karsdal MA, et al: The Type I Collagen Fragments ICTP and CTX Reveal Distinct Enzymatic Pathways of Bone Collagen Degradation. J Bone Miner Res 18, 2003

4. Henriksen K, Bohren KM, Bay-Jensen AC, et al: Should biochemical markers of bone turnover be considered standard practice for safety pharmacology? Biomarkers 15:195–204

5. Sand JMB, Leeming DJ, Byrjalsen I, et al: High levels of biomarkers of collagen remodeling are associated with increased mortality in COPD - results from the ECLIPSE study. Respir Res 17:125, 2016

6. Schumann DM, Leeming D, Papakonstantinou E, et al: Collagen degradation and formation are elevated in exacerbated COPD compared to stable disease. [Internet]. Chest , 2018Available from: http://www.ncbi.nlm.nih.gov/pubmed/29966667

7. Sand JMB, Knox AJ, Lange P, et al: Accelerated extracellular matrix turnover during exacerbations of COPD. Respir Res 16, 2015

8. Stolz D, Leeming DJ, Kristensen JHE, et al: Systemic Biomarkers of Collagen and Elastin Turnover Are Associated With Clinically Relevant Outcomes in COPD. Chest 151, 2017

9. Karsdal MA, Manon-Jensen T, Genovese F, et al: Novel insights into the function and dynamics of extracellular matrix in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 308:G807-30, 2015

10. Karsdal MA, Nielsen SH, Leeming DJ, et al: The good and the bad collagens of fibrosis - Their role in signaling and organ function. [Internet]. Adv Drug Deliv Rev , 2017Available from: http://www.ncbi.nlm.nih.gov/pubmed/28736303

11. Karsdal MA, Henriksen K, Genovese F, et al: Serum endotrophin identifies optimal responders to PPARγ agonists in type 2 diabetes. Diabetologia 60, 2017

12. Siebuhr AS, Karsdal MA: Type XIII Collagen. 2016

13. Schuppan D, Cramer T, Bauer M, et al: Hepatocytes as a source of collagen type XVIII endostatin. [Internet]. Lancet (London, England) 352:879–80, 1998Available from: http://www.ncbi.nlm.nih.gov/pubmed/9742985 

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