Collagenolytic activity is suppressed in organ-cultured human skin exposed to a gadolinium-based MRI contrast agent

Abstract

Objective: Human skin produces increased amounts of matrix metalloproteinase-1 (MMP-1) when exposed in organ culture to Omniscan, one of the gadolinium-based MRI contrast agents (GBCA). MMP-1, by virtue of its ability to degrade structural collagen, contributes to collagen turnover in the skin. The objective of the present study was to determine whether collagenolytic activity was concomitantly up-regulated with increased enzyme.

Materials and methods: Skin biopsies from normal volunteers were exposed in organ culture to Omniscan. Organ culture fluids obtained from control and treated skin were examined for ability to degrade type I collagen. The same culture fluids were examined for levels of MMP-1, tissue inhibitor of metalloproteinases-1 (TIMP-1), and complexes of MMP-1 and TIMP-1.

Results: Although MMP-1 was increased in culture fluid from Omniscan-treated skin, there was no increase in collagenolytic activity. In fact, collagenolytic activity declined. Increased production of TIMP-1 was also observed in Omniscan-treated skin, and the absolute amount of TIMP-1 was greater than the amount of MMP-1. Virtually all of the MMP-1 was present in MMP-1-TIMP-1 complexes, but the majority of TIMP-1 was not associated with MMP-1. When human dermal fibroblasts were exposed to TIMP-1 (up to 250 ng/mL), no increase in proliferation was observed, but an increase in collagen deposition into the cell layer was seen.

Conclusion: Gadolinium-based MRI contrast agent exposure has recently been linked to a fibrotic skin condition in patients with impaired kidney function. The mechanism is unknown. The increase in TIMP-1 production and concomitant reduction in collagenolytic activity demonstrated here could result in decreased collagen turnover and increased deposition of collagen in lesional skin.

Figure 1. Collagen fragmentation by organ culture fluid from control and Omniscan-treated skin

Polymerized type I collagen was exposed to organ culture fluid from untreated and Omniscan-treated (50 µM) organ-cultured skin. SDS-PAGE analysis was used to resolve collagen fragments, which were then digitized and quantified. Values obtained with untreated (control) skin cultures were arbitrarily set to 1.0 and the Omniscan value compared to this. Values are means and standard errors based on tissue from 6 different subjects. Statistical significance of the data was determined using the Student t-test. *indicates statistically significant decrease compared to control at p<0.05 level. Inset: The collagen alone lane shows a doublet corresponding to the α1 and α2 chains of intact type I collagen, but bands corresponding to the ¼- and ¾-sized fragments are not seen. Collagen exposed to culture fluid from a basal cell carcinoma (BCC) demonstrates bands corresponding to the ¼- and ¾-sized fragments of type I collagen. Multiple additional fragments are seen as the ¼- and ¾-sized fragments are susceptible to further degradation. Collagen fragments are seen in the lane corresponding to collagen plus control skin culture fluid but are not seen in the lane corresponding to collagen plus culture fluid from Omniscan-treated skin.

Figure 2. MMP-1, TIMP-1, and MMP-1–TIMP-1 complexes in organ culture fluid from control and Omniscan-treated skin

Tissue was incubated for three days under control conditions or in the presence of Omniscan (50 µM). At the end of the incubation period, culture fluid was collected and assayed as follows: Upper panel. MMP-1 assessed by western blotting (n=6). Inset: western blot from one subject. Middle panel. TIMP-1 assessed by ELISA (n=6). Lower panel. MMP-1 - TIMP-1 complex assessed by ELISA (n=5). Values shown are means and standard errors. Statistical significance of the data was determined using the Student t-test. *indicates statistically significant increase compared to control at p<0.05 level.

Figure 3. Inhibition of MMP-1-mediated collagen fragmentation by Omniscan

Polymerized type I collagen was exposed to purified, activated MMP-1 from synovial fibroblasts in the absence or presence of Omniscan (5–500 µM). SDS-PAGE analysis was used to resolve collagen fragments, which were then digitized and quantified. Values obtained with MMP-1 alone were arbitrarily set to 1.0 and the other values compared to this. Values are means and standard deviations based on three separate experiments. Statistical significance of the data was determined by ANOVA, followed by paired-group comparisons. *indicates statistically significant decrease compared to control at p<0.05 level. Inset: Collagen fragmentation pattern observed in one experiment. Lane 1 demonstrates intact collagen; lane 2 demonstrates the fragmentation pattern observed in the presence of activated MMP-1 alone; lanes 3–7 demonstrate fragmentation observed in the presence of activated MMP-1 and increasing concentrations (5–500 µM) of Omniscan.

Figure 4. Inhibition of MMP-1-mediated collagen fragmentation by organ culture fluid from control and gadolinium chloride-treated skin

Polymerized type I collagen was exposed to purified, activated MMP-1 from synovial fibroblasts in the absence or presence of organ culture fluid from control and gadolinium chloride-treated (10–50 µM) skin. SDS-PAGE analysis was used to resolve collagen fragments, which were then digitized and quantified. Values obtained with MMP-1 alone were arbitrarily set to 1.0 and the other values compared to this. Values are based on organ-cultured skin from one subjects. The experiment was repeated with skin from two subjects with similar results. Inset: Collagen fragmentation pattern observed with one subject. Lane 1 demonstrates intact collagen; lane 2 demonstrates the fragmentation pattern observed in the presence of activated MMP-1 alone; lanes 3–6 demonstrate fragmentation observed in the presence of activated MMP-1 and culture fluids from tissue expose to 0, 10, 20 and 50 µM gadolinium chloride.

Figure 5. Proliferation and collagen deposition in human dermal fibroblasts exposed to exogenous TIMP-1

Proliferation: Proliferation was assessed under serum-free conditions in a growth factor-enriched medium as described in the Materials and Methods section. Values shown are means and standard deviations based on n=4 separate experiments. Deposition of type I collagen. Cells were incubated under control conditions or in the presence of exogenous TIMP-1 (150 ng/ml) for three days. At the end of the incubation period, lysates were prepared from the cell layer and assayed for type I collagen by western blotting. Bands were digitized and quantified. The value from the control was set to 1.0 and the value from TIMP-1 treatment compared to this. Values shown are means and standard deviations based on three separate experiments. Statistical significance of the data was assessed using the Student t-test. *indicates statistically significant increase compared to control at p<0.05 level. Inset: Intact α1 and α2 chains of type I collagen from one of the replicate experiments with β-tubulin (no change; not shown) used as control.