Collagens are the most abundant proteins in vertebrates, constituting approximately 30% of proteins in the body. Collagens play a prominent role in maintaining tissue structure and have additional functions in cell growth, differentiation, tissue repair, bone, cartilage, skin, and aging.
Collagens are a superfamily of extracellular matrix (ECM) proteins. In vertebrates, there are at least 27 types of collagens with 42 distinct polypeptide chains. Collagens type I, II, III, V, XI, XXIV, XXVII contain triple-helical structures which bundle into fibrils. Groups of fibrils bundle together to form collagen fibers. Hydroxyproline residues are amino acids that are nearly exclusive to collagen and function to stabilize the collagen triple helix.
While collagen types I, II and III are the most abundant throughout the body, some collagens have restricted tissue distribution. Collagen types II, IX and XI are found almost exclusively in cartilage and collagen type IV is the main component of basement membranes.
A delicate balance between collagen production and collagen degradation must be maintained to create homeostasis within the body. Key regulators of collagen degradation are matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and macrophages. Normally, important regulatory networks carefully manage collagen quantities within the body. However, disruptions to collagen synthesis, function, or degradation can lead to disease, including bone diseases, organ fibrosis, cancers, and more.
Collagens in disease
Collagen-related diseases can be caused by either increased collagen synthesis or excessive collagen degradation, resulting in disruptions to the intricate balance of matrix proteins. Reduced or defective collagen underlies aging, osteoporosis, osteoarthritis, and Ehlers-Danlos syndrome. Excessive deposition and accumulation of ECM proteins, including collagen, leads to tissue scarring and fibrosis that can affect the liver, lung, kidney, gut, and skin.
Chronic liver diseases
Chronic insults to the liver result in fibrosis that can ultimately lead to cirrhosis. Various types of collagen (type I, III, IV, V and VI) are increased in the liver during the progression of fibrosis. Liver fibrosis that involves collagen accumulation can occur in alcohol-associated liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Liver cirrhosis is also characterized by abundant and excessive collagen deposition. Research has suggested that collagens likewise promote the progression of hepatocellular carcinoma (HCC).
The rapid and accurate quantification of collagen in the liver is important while investigating liver diseases status. Liver tissue-derived collagens can be challenging to analyze due to low collagen concentrations and matrix effects of the liver. A sensitive method of collagen detection is required for liver tissue samples, especially during early stages of liver fibrosis.
Lungs maintain healthy tissue structure and integrity through the delicate management of continuous collagen production and collagen degradation. Following lung injury, beneficial collagen production and collagen degradation are both increased to repair damaged tissues and prevent scar tissue formation, respectively. Pulmonary fibrosis occurs when collagen production outperforms collagen degradation, resulting in collagen-rich matrix formation.
Pulmonary fibrosis can occur following acute lung injury, lung infections, damage caused by exposure to cigarette smoke, chronic obstructive pulmonary disease (COPD), and interstitial lung diseases (ILD) including idiopathic pulmonary fibrosis (IPF). COPD is characterized by lung inflammation that leads to impeded airflow and small airway fibrosis. ILD is a classification of chronic lung disorders that is characterized by inflammation and pulmonary fibrosis that develops in response to a variety of causes, including exposure to environmental or occupational toxins, auto-immune diseases, drug-induced diseases, and idiopathic reactions.
Recent research attention has been brought to the impact that impaired collagen degradation pathways have on pulmonary fibrosis, in addition to the influence of increased matrix production within damaged lungs. Analyzing collagens during pulmonary fibrosis is a key step towards developing antifibrotic therapies and understanding the mechanisms that drive pulmonary fibrosis.
Chronic kidney diseases
Chronic kidney disease is characterized by renal fibrosis that is caused by imbalanced ECM protein turnover. Injury to the kidney and glomeruli can drive the accumulation of collagen and renal fibrosis. This can occur in glomerulonephritis, when the filtering component of the kidney becomes damaged, and in diabetic nephropathy, which is characterized by high blood glucose levels that lead to nephron injury. There are additional causes of kidney disease such as Alport-Syndrome and glomerulopathies that predominantly involve ECM proteins. Renal fibrosis and increased collagen levels drive changes to renal cells and vascular smooth muscle cells, which further intensifies disease progression. Collagen biomarkers provide a deeper insight into ECM remodeling, fibrosis, and renal function during the progression of chronic kidney diseases.
Fibrillar collagens are fundamentally important for healthy heart function. Collagens provide scaffolding to support heart architecture and are necessary for contractile force transmission so that the heart can pump and function properly. In particular, type I collagen forms thick fibers that provide tensile strength and type III collagen forms thin fibers that function to maintain elasticity.
Cardiac fibrosis is characterized by the excessive accumulation of ECM components, including collagens, within the heart. Fibrosis occurs in the heart when there is an imbalance between collagen production and degradation within the ECM. Cardiac fibrosis is associated with all forms of heart disease, hypertensive, diabetic, valvular, and ischemic heart disease. Cardiac fibrosis and collagen deposition can occur in response to damaged myocardium or can be reactive without underlying cardiac injury. Many cell types in the heart secrete collagen, yet activated fibroblasts are considered to be the leading collagen-producing cells in fibrotic areas of the heart. More research is needed to understand how to slow down the rate of cardiac fibrosis in order to to prevent the accumulation of collagen that drives irreversible fibrosis.
Basement membranes consist of dense ECM components that function as scaffolds and barriers to tumor invasion. In healthy tissue, the ECM of basement membranes is dynamic and undergoes remodeling through tightly regulated enzymatic pathways. However, during cancer and tumor growth, these dynamics are altered such that basement membranes can be abnormally remodeled or degraded. This results in the production of tumor-specific ECM that is collagen-rich and exhibits increased stiffness. Cancer cells stimulate the synthesis of atypical collagen to create a protective ECM environment that promotes cancer cell proliferation and immunosuppression.
Tumor cell invasiveness is also associated with atypical collagen turnover and degradation. In a mechanism that can be induced by collagen, cancer cells undergo an epithelial to mesenchymal transition (EMT) that leads to the invasion of adjacent tissues. Collagens are cross-linked in solid tumors, which increases matrix stiffness, promotes EMT, and drives cancer cell metastasis. Hence, high collagen density and collagen linearization are linked to poor prognoses of several cancers including breast cancer, pancreatic cancer, gastric cancer, and squamous cell carcinomas.
In addition to having direct effects on cancer cells, atypical collagens in tumor-specific microenvironments influence the function of tumor-infiltrating immune cells. Collagens within tumor microenvironments impede T cell migration and repel the infiltration of T cells into the tumor. Collagen-dense environments block the interactions of T cells with antigen-presenting cells, thereby reducing T cell activation. Collagen-dense environments also impact macrophage migration speed, morphology, proliferation, and activity. Identifying the many roles that collagens have on cancer cells and in mediating immune function could aid in developing effective cancer treatments and novel targets.
Collagens are localized to the endothelial basement membrane of the vascular wall to maintain vessel integrity and ECM stability. Pathologic fibrosis may coincide with imbalanced collagen turnover in vasculature. Accumulated collagens may lead to thrombosis, while decreased collagen levels can lead to vessel rupture and bleeding.
Mature collagen is crosslinked, making collagen insoluble and difficult to purify and extract from tissues. The QuickZyme suite of collagen assays circumvent many of these challenges by leveraging chromogenic hydroxyproline quantification to measure total collagen levels. The assay principle detects hydroxyproline, an amino acid that is nearly unique to collagen, making the total collagen assays capable of detecting all types and forms of collagen in a manner that is species independent.
Read more on “How to choose your collagen assay” for your research study.
The QuickZyme Total Collagen assay provides a quantitative colorimetric read-out to measure total collagen levels independent of extraction method.
The QuickZyme Sensitive Tissue Collagen assay is specialized to analyze total collagen levels in samples with low collagen content or high matrix effects, which is typical of liver tissue samples. This assay has been enhanced to neutralize the matrix effect in a manner that is especially suited for liver and formalin-fixed, paraffin-embedded (FFPE) samples without sample dilution.
The QuickZyme Soluble Collagen assay quantitively measures soluble fibrillar collagen levels through the precipitation of collagen with Sirius-Red followed by colorimetric detection. This assay should be applied in cell-based experiments that use cell culture media or cellular extract samples.
The QuickZyme Total Protein assay provides a quantitative measurement of amino acids within fresh tissue, frozen tissue, and FFPE hydrolyzed samples. This can be used in combination with the QuickZyme Total Collagen assay or QuickZyme Hydroxyproline assay kits to determine the quantity of collagen or hydroxyproline per total protein within a sample.