In Vitro Sirius Red Collagen Assay Measures the Pattern Shift from Soluble to Deposited Collagen
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In this study, we compared two in vitro collagen production assays ([3H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-β), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [3H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agent’s capacity for collagen production and (b) Sirius Red is more useful than [3H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.
Ionizing radiation (IR) often leads to fibrosis, which impairs the given organ’s functions, reduces the host’s quality of life, and can even lead to death [1, 2]. The underlying mechanism for IR-induced fibrosis is still largely unknown and the effort to reduce IR-induced fibrosis has progressed slowly with unsatisfactory results. In part, this lack of progress is due to the difficulties associated with using an in vitro system to mimic the long-term in vivo pathological process. Fibrosis characteristically deposits a large amount collagen in the organs; collagen production is a transition from a single alpha-chain procollagen secreted from cells to 3 alpha chains, which is the final mature and deposited form [3, 4, 5, 6]. We believe that blocking procollagen production from cells early in the process is the key to reducing fibrosis. In this study, the two most frequently used in vitro collagen production assays, [3H]-proline incorporation  and the Sirius Red method , were compared. In addition, the simultaneous measurement of collagen in both media and cell lysates revealed the transition from procollagen to collagen. Finally, the effect of the antifibrotic agent, triptolide (TPL), was used to determine the ability of Sirius Red to screen effective agents.
For the Sirius Red assay, 12-well plates were seeded with 20 × 104 fibroblast cells in 1 ml Iscove’s minimal essential medium (IMEM) with 10 % newborn calf serum (NCS); thereafter, the cells were grown to 70–80 % confluence. To stimulate the production of collagen, we used 0, 2.5, or 5 ng/ml doses of transforming growth factor-beta (TGF-β). Both culture media and cell lysate were collected on days 1, 2, 4, and 6 after TGF-β treatment. To prove that collagen was stimulated by TGF-β, interleukin-1 alpha (IL-1α), which did not strongly stimulate collagen production, was used as negative cytokine control.
For the [3H]-proline incorporation assay, 0.5 μCi/well [3H]-proline cells were added to cells cultured similar to those above 24 h after being seeded. On days 1, 2, 4, and 6 after TGF-β treatment, the media and cell lysates were collected with 0.1 mg/ml pepsin in 0.5 M acetic acid. Since [3H]-proline was incorporated into many proteins during biosynthesis in order to distinguish the collagen from other proteins, the samples were divided into two groups in which: (1) total incorporated [3H]-proline was counted and (2) collagenase was used to digest the collagen. After incubation at 37 °C for 3 h, the incorporated [3H]-proline was precipitated with 10 % trichloric acid. The pellets were washed three times with phosphate buffer and dissolved in 0.2 N NaOH; a scintillation counter read the proteins labeled with [3H]-proline. The following calculation was used: collagen level = total [3H]-proline protein − noncollagenous protein.
We also compared the Sirius Red and [3H]-proline incorporation assays side-by-side to determine not only which assay more sensitively distinguished the alterations caused by TGF-β but also which assay was easier to perform. Since collagen production closely correlated with the cell number, a thiazolyl blue tetrazolium bromide (MTT) assay was used according to standard protocol  to determine if the cell number was similar in all groups. MTT determined the cell number when cells were seeded and during the experiment when cells were treated with different factors. The transition of procollagen in media to deposited collagen was examined. In all experiments, the culture media and cell lysates were collected on days 1, 2, 4, and 6 after treatment and analyzed for the procollagen in media and deposited collagen in cell lysates as a function of time.
Finally, two experiments were performed to determine the effect of TPL on the reduction of collagen production. (1) In vivo study: the whole chest of C57BL/6 mice (female, 8 weeks old) received 15 Gy at a dose rate of 1.8 Gy/min with a cesium-137 source. Mice were then divided into two groups for treatment with vehicle alone (saline) or TPL (0.25 mg/kg, intravenous, twice a week, for 3 months). Nonirradiated mice were also treated with saline as an aging control or TPL as agent toxicity control. Eight months after lung IR, mice were euthanized, and lung hydroxyproline levels, representing the deposited collagen, were measured with a biochemistry method  and compared among the four groups. The protocol was approved by Institutional Animal Use Committee. (2) In vitro study: NIH 3T3 or HFL-1 cells (ATCC) were treated with 5 ng/ml of TPL 4 h before 5 ng/ml of mouse or human TGF-β (mTGF-β or hTGF-β) was added. The conditional media was collected on days 1 and 2 after treatment for procollagen production.
All data were statistically analyzed using either t-test or analysis of variance. P < 0.05 was considered significant.
Measurement of collagen in both cell media and cell lysates detected the transition of procollagen to collagen. Figures 7.1 and 7.2 show that both assays detected the same pattern of collagen production. (1) In media: on day 1 TGF-β stimulated increased procollagen, which continued to increase on day 2 in a dose-dependent manner. However, on days 4 and 6, the assays revealed no difference. (2) In cell lysates: the stimulatory effect of TGF-β on collagen production was not seen until day 4. This time-shift pattern from an early increase in the media to a later increase in the lysates is consistent with the collagen production progress from a soluble alpha single chain to a deposited insoluble triple helix.
Through this comparison of two collagen assays, we concluded that Sirius Red is superior to [3H]-proline incorporation because it is easier to use, more convenient, less time consuming, and does not require radioactive material. Unlike [3H]-proline, which incorporates all proteins that contain proline, Sirius Red has better specificity; it mainly stains collagen types I and III, the major forms found in the human body, and negatively stains other extracellular matrixes. The increase in collagen level by TGF-β further proves that the assay is specific for collagen level.
The measurement of collagen in media seems to allow us to measure the cell number with MTT in the same culture well, which is a more reliable control. In addition, the media is easier to collect and to assay. More importantly, the alteration magnitude is higher than the assessment of lysates, indicating that media can more sensitively reflect the alterations induced by different factors.
The level of collagen in media is an overall result of the balance between production and deposition. On days 1–2, the increased collagen reflected that production exceeded deposition; on day 4, the single-chain collagen increased by TGF-β became an insoluble triple helix and deposited as a matrix into the cell layer; on day 6, the effect of TGF-β was reduced, and the production and deposition tended to be flat and similar for all groups. Taken together, measuring media on day 2 with the Sirius Red assay is the best method to study the effect of collagen production.
TPL is a known inhibitor of fibrosis, as evidenced by the in vivo reduction of hydroxyproline levels in lungs treated with TPL. However, these data can only be obtained 8 months after irradiation. With the in vitro Sirius Red assay and 2 day media, we see the same tendency, which suggests that this assay is likely to be a fast, simple, and reliable tool for screening antifibrotic agents.
As compared to [3H]-proline incorporation, Sirius Red is the superior in vitro assay to study the effect of collagen production; it allows for faster and easier screening of antifibrotic agents.
This project is supported in part by U19 AI067733, RC1AI078519, RC2-AI-087580, RC1-AI081274 (NIAID/NIH), and Shands Cancer Center startup funds (University of Florida). We thank Kate Casey-Sawicki for editing this manuscript and Dr. Chihray Liu and his group for setting up the physical dosimetry for accurate radiation.
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