Recently, Su Jin’s research team from the lab made major discoveries in the field of pulmonary fibrosis. Related research titled “Intracellular Hydroxyproline Imprinting Following Resolution of Bleomycin-Induced Pulmonary Fibrosis” was published in Eur Respir J (IF=16.67). With a variety of new technologies and methods, the research revealed “intracellular hydroxyproline imprinting” in classic animal models of pulmonary fibrosis, which not only settled the 20-year controversy about animal models in this field, but also provided multi-dimensional technologies for evaluating the severity of pulmonary fibrosis, which can be said to “move the field forward”.

Research Background

Idiopathic pulmonary fibrosis (IPF) is called “a disease second to cancer”, and the five-year survival rate of patients is only 25%. In the past ten years, a total of 145 clinical trials for IPF have been carried out around the world, but only two relatively effective drugs which cannot improve the overall survival rate of patients were approved in 2014 under the context that no anti-fibrotic drugs are available in the world.

The high failure rate of IPF drug development is considered to be related to animal models. Mouse with bleomycin-induced pulmonary fibrosis is the most important animal model for preclinical evaluation of drugs for pulmonary fibrosis because of its pathological characteristics closest to human IPF. However, the biggest controversy regarding this model is whether fibrosis will eventually go away by itself.

Excessive deposition of collagen is the main characteristic of fibrosis. Because hydroxyproline (HYP) is a unique amino acid of collagen, accounting for about 13% of the total content of amino acids of collagen, using biochemical methods (such as colorimetry, high performance liquid chromatography) to measure the total content of hydroxyproline in tissues becomes the most commonly used method for evaluating the severity of fibrosis. This method was further established as the “gold standard” for preclinical evaluation of pulmonary fibrosis by the guidelines of the American Thoracic Society in 2017.

Major Research Content and Significance

1. Preliminary discovery of contradictory results in experiments

Professor Su Jin’s team spent three years combining a variety of the latest technical methods with traditional evaluation methods, to carry out multiple rounds of 16-week tracking and comprehensive and detailed analysis of mice with bleomycin-induced pulmonary fibrosis. Eventually, they came to reproducible positive and negative conclusions: ① With the traditional biochemical method, it was found that the total content of hydroxyproline in the lung remained constant during 4-16 weeks after modeling (see Figure 1), indicating that fibrosis cannot go away by itself. ② The pathological full scan combined with technical methods like AI analysis, Picro Sirius Red solution, and collagen hybrid peptide (CHP) showed that pulmonary fibrosis in mice caused by a single blow gradually got away by itself 10 weeks after the injury; ③ CT images of small animals and pulmonary function tests also showed that the damage of lungs tends to heal in the same body.

Figure 1 Changing trend of the content of hydroxyproline in mice with pulmonary fibrosis

2. Hypotheses for contradictory results in experiments

The phenomenon that the total content of hydroxyproline in models with pulmonary fibrosis remains constant after the fibrosis peak is also supported by the article published in Eur Respir J in 2013. However, previous researches simply combined this result with image to support the conclusion that mice with fibrosis cannot heal by itself. Based on the previous researches on collagen degradation, the research team boldly speculated that if the degraded collagen fragments are not completely removed after the fibrosis goes away, the content of hydroxyproline may be constant (see Figure 2).

Figure 2 Possible mechanism of the constant content of hydroxyproline in mice with self-healing lung fibrosis

3. Perfectly confirm the hypothesis with basic theories in biochemistry

The hydroxylation of proline is a post-translational chemical modification of collagen peptides, and such specific modified amino acids can be immune-detected with the help of specific antibodies. Based on this idea, the research team applied an “anti-hydroxyproline antibody” from Cell Signaling Technology to the IHC detection of fibrotic lung tissue at different stages for the first time, and observed the phenomenon that perfectly confirmed the hypothesis of Figure 2: During the self-healing period of fibrosis, local and intracellular aggregation appeared in collagen fragments rich in hydroxyproline content, forming a unique “intracellular hydroxyproline imprinting” (see Figure 3a). Accurate analysis of pathology software revealed that the total intensity of the positive signal of hydroxyproline during the self-healing period of fibrosis showed no difference from the fibrosis peak (see Figure 3b).

Figure 3 “Intracellular hydroxyproline imprinting” during the self-healing period of pulmonary fibrosis

4. Research highlights and its significance to the field of organ fibrosis

The above-mentioned innovative research results were highly praised by the three reviewers of Eur Respir J. They believed that the discovery of “intracellular hydroxyproline imprinting” prompted the industry that traditional biochemical methods to detect the content of hydroxyproline cannot truly reflect the presence of collagen. In the evaluation of anti-fibrosis pharmacodynamics, this method should be carefully applied to evaluate the severity of fibrosis.

Collagen deposition is a common characteristic of all organ fibrosis. According to authoritative statistics, 45% of deaths caused by human disease are related to organ fibrosis. Therefore, the findings of this research and the establishment of related methodology will also have a broader influence on the study of other organ fibrosis.

Establishment of a multi-dimensional assessment methodology for pulmonary fibrosis

In the face of abnormal experimental results, the research team was able to make bold hypotheses based on the establishment and application of multi-dimensional evaluation techniques for pulmonary fibrosis, which mainly include the following three aspects:

1. Analysis of pulmonary function based on micro-CT images: According to the HU (Hounsfield Unit) value of micro-CT images, areas with different pulmonary functions are carefully cut out in the lungs, and a new methodology for the non-invasive dynamic analysis of the pulmonary status of fibrotic lung tissue is established.

Figure 4 Analysis of pulmonary function based on CT images

2. Methodology for exploring collagen degradation in the process of fibrosis: This research used a CHP probe that specifically recognizes single strand of collagen to detect collagen degradation in fibrotic lung tissue. This probe was developed and created by Professor Li Yang from the Key Laboratory of Molecular Imaging of the Fifth Affiliated Hospital of Sun Yat-Sen University. This research applied the probe to the recovery model of pulmonary fibrosis for the first time, which provides a reference for subsequent applications.

Figure 5 CHP probe explores the principle of collagen degradation

3. Anti-Hydroxyproline antibody probes the location of collagen molecules: Anti-Hydroxyproline antibody was mainly used in the detection of hydroxylation modification of a small number of intracellular signal transduction molecules in combination with immunoprecipitation. This research applied this antibody to the analysis of histocytological localization of collagen and its degraded fragments in fibrotic tissue for the first time.

Original paper：https://erj.ersjournals.com/content/early/2021/09/16/13993003.00864-2021