Repurposing diacerein for the treatment of chronic wounds in recessive-dystrophic epidermolysis bullosa patients by modulating matrix metalloproteinase-9 expression

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1, leading to loss or dysfunction of type-VII collagen (C7), a protein essential for skin stability. Clinically, patients suffer from severe skin blistering, chronic or recurrent wounds, and scarring, which predispose to early onset of aggressive squamous cell carcinoma. Previous studies showed that RDEB-keratinocytes (RDEB-KC) express high levels of matrix-metalloproteinase 9 (MMP-9), a molecule known to play a crucial role in wound chronification if dysregulated. We investigated the potential of diacerein, a small molecule that interferes with the MMP-9 regulatory pathway, to improve wound healing in a 5-year old RDEB patient presenting with chronic, generalized skin involvement unresponsive to previous treatment approaches. Upon 4 weeks of topical therapy applied to the patient's back, parents reported a nearly complete wound closure and a significant increase in quality of life. We also provide evidence that diacerein treatment of patient keratinocytes results in a downregulation of MMP-9 expression, accompanied by a reduction in their ability to degrade a fibrinogen matrix. These data characterize diacerein as a potential candidate for improving wound healing in RDEB through its impact on inflammatory as well as epithelial cells.

Keywords: RDEB; diacerein; matrix metalloproteinase; wound healing.

FIGURE 1

Treatment of a recessive dystrophic epidermolysis patient with diacerein. (a) Day 1 when starting the treatment with 1% diacerein, the patient presented with extensive wounding on the whole back. (b) After 28 days of treatment, all lesions but one (arrow) closed.

FIGURE 2

Baseline expression levels of matrix‐metalloproteinase 9 (MMP‐9) in healthy control keratinocytes (HC‐1090‐KC^{E6 / E7}) and recessive dystrophic epidermolysis bullosa keratinocytes (RDEB‐223‐KC^{E6 / E7}) on RNA and protein level. (a) Expression levels of MMP‐9 given relative to glyceraldehyde 3‐phosphate dehydrogenase are shown as 2^−ΔCq . (b) Representative Western blot showing MMP‐9 in whole cell lysates (WCL) of HC‐1090‐KC^{E6 /E7} and RDEB‐223‐KC^{E6 /E7} cells. The MMP‐9 upper band indicates the pro‐ (~92 kDa) and the lower band indicates the active form (~83 kDa) of MMP‐9. (c) Dosimetric quantification of MMP‐9 abundance in WCLs. Western blot data were normalized to ß‐tubulin, calculated as fold‐change RDEB‐ versus HC‐KC. Bar plots represent mean and standard error of mean (SEM) from 3 to 5 independent replicates. Differences between groups were analyzed using an unpaired, two‐sided t‐test. Non‐significant (ns): p > 0.05, *p ≤ 0.05.

FIGURE 3

Modulation of matrix‐metalloproteinase 9 (MMP‐9) expression upon diacerein treatment in recessive dystrophic epidermolysis keratinocytes (RDEB)‐223‐KC^{E6 /E7} . Representative Western blots show the levels of MMP‐9 expression in (a) whole cell lysates and (b) conditioned medium (supernatant: SN) of RDEB‐223‐KC^{E6 /E7} cells, either mock treated or treated for 24 h with 10 μg/mL diacerein. The upper band indicates the pro‐ (~92 kDa) and the lower band indicates the active form (~83 kDa) of MMP‐9. Data were normalized to ß‐tubulin and relative to levels in mock treated cells. Protein expression in supernatants was normalized to the total protein of harvested cells, approximating the number of cells where the conditioned medium derived from. Statistical analysis was performed using a paired t‐test, considering p < 0.05 as statistically significant, with non‐significant (ns): p > 0.05, *p ≤ 0.05. Bars represent mean and error bars standard error of mean (SEM) of (a) n = 5 and (b) n = 9 independently performed biological replicates. (c) Schematic showing matrix degradation assay. (d, e) RDEB‐223‐KC^{E6 /E7} were either pre‐treated for 24 h with vehicle control (mock) or 10 μg/mL diacerein, 6 μg/mL Lipopolysaccharides (LPS), or a combination thereof. Brightfield images of the whole well were acquired at 0 and 72 h post‐treatment and image analysis was performed with ImageJ. The degree of matrix degradation was measured as the percentage increase in area covered by cells 72 h post‐treatment (blue border) compared to area at 0 h (pink border). Bars show mean, error bars show standard error of mean (SEM) of increase in area (%) from 3 to 5 independent experiments. An unpaired t‐test was used for statistical analysis. Non‐significant (ns): p > 0.05, **p ≤ 0.01. Representative images are shown.

FIGURE 4

Expression profile of interleukin (IL‐)1β and matrix‐metalloproteinase 9 (MMP‐9) in recessive dystrophic epidermolysis (RDEB) skin in relation to the wound edge. (a) Hematoxylin and eosin staining of an RDEB patient skin biopsy shows the transition zone from (I) non‐lesional skin to (II) wound margin, and (III) chronic wound/denuded skin. Scale bar: 250 μM. (b) Immunofluorescence staining of the tissue sections for IL‐1β (green), and MMP‐9 (red) in combination with nuclear counterstain 4′,6‐diamidino‐2‐phenylindole, (DAPI) (blue). The dermo‐epidermal junction is indicated by white dashed lines. Scale bar: 50 μM. (c) Quantification of the mean fluorescence intensity of IL‐1β and MMP‐9 for keratinocytes of (non‐lesional) skin and keratinocytes at the wound margin, normalized by area. Differences between groups were analyzed using an unpaired, two‐sided t‐test. A p‐value of <0.05 was considered statistically significant with non‐significance (ns) at: p > 0.05, *p ≤ 0.05, **p ≤ 0.01.