A bioengineered living cell construct activates metallothionein/zinc/MMP8 and inhibits TGFβ to stimulate remodeling of fibrotic venous leg ulcers

Abstract

Venous leg ulcers (VLU) represent a major clinical unmet need, impairing quality of life for millions worldwide. The bioengineered bilayered living cell construct (BLCC) is the only FDA-approved therapy demonstrating efficacy in healing chronic VLU, yet its in vivo mechanisms of action are not well understood. Previously, we reported a BLCC-mediated acute wounding response at the ulcer edge; in this study we elucidated the BLCC-specific effects on the epidermis-free ulcer bed. We conducted a randomized controlled clinical trial (ClinicalTrials.gov NCT01327937) enrolling 30 subjects with nonhealing VLUs, and performed genotyping, genomic profiling, and functional analysis on wound bed biopsies obtained at baseline and 1 week after treatment with BLCC plus compression or compression therapy (control). The VLU bed transcriptome featured processes of chronic inflammation and was strikingly enriched for fibrotic/fibrogenic pathways and gene networks. BLCC application decreased expression of profibrotic TGFß1 gene targets and increased levels of TGFß inhibitor decorin. Surprisingly, BLCC upregulated metallothioneins and fibroblast-derived MMP8 collagenase, and promoted endogenous release of MMP-activating zinc to stimulate antifibrotic remodeling, a novel mechanism of cutaneous wound healing. By activating a remodeling program in the quiescent VLU bed, BLCC application shifts nonhealing to healing phenotype. As VLU bed fibrosis correlates with poor clinical healing, findings from this study identify the chronic VLU as a fibrotic skin disease and are first to support the development and application of antifibrotic therapies as a successful treatment approach.

Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

© 2019 by the Wound Healing Society.

Figures

Figure 1.

CONSORT diagram. Patients included in VLU bed study as part of ClinicalTrials.gov NCT01327937.

Figure 2.

Inflammation and fibrosis in the chronic VLU bed transcriptome. (A) Gene networks of innate and adaptive immunity and(B) altered collagens and upregulated profibrotic factors present in nonhealing wound bed specimens from VLU patients (n = 6) in comparison with dermis from healthy donors immediately postwounding (n = 6). (C) Immunofluorescence staining of alpha-smooth muscle actin (red) with overlying DAPI staining of nuclei (blue) in nonhealing VLU bed sections. Image is representative of n = 4 study subjects. (D) Upregulated TGFB signaling pathway and a subset of target genes involved in fibrotic processes in the chronic VLU bed. Fold expression change over healthy donor dermis is indicated under each gene. (E) IPA Toxicity analysis. Percent overlap of VLU bed genes with IPA toxicity related lists. Overlap p-values were calculated using Fisher’s exact test.

Figure 3.

BLCC inhibits TGFB signaling in the chronic VLU bed. (A) IPA upstream regulator network identifying targets of TGFB1 that are up- or downregulated in BLCC-treated VLUs supporting a predicted inhibited state of TGFB1, as supported by IPAcalculated Z-score. (B) QPCR of TGFB2 expression in n = 7 pairs of chronic VLU bed specimens at baseline (Week 0, “WO”) and one week post-BLCC treatment (Week 1, “W1”). ** = P < 0.005 by ratio paired t test. (C) Box- and- whisker plots of tenascin C microarray probe expression intensity in the healthy donor dermis immediately postwounding (“Acute dermis,” n = 6) and in seven pairs of VLU before and 1 week after BLCC treatment (“W0” and “W1”). (D) QPCR of TIMP3 expression in n = 7 VLU before and after BLCC treatment. Mean ± SEM are shown; * = p < 0.05 by ratio paired t test. (E) Western blot and quantification of decorin expression in n = 7 VLC before and after BLCC treatment. Normalization was performed to ARPC2 expression. Men ± SEM are show; * = p < 0.05 by ratio paired t test.

Figure 4.

Extracellular matrix remodeling response to BLCC application. (A) Integrin-binding sialoprotein (IBSP) expression by microarray in chronic VLU patients (paired Week 1 vs. Week 0 biopsies) treated with standard-of-care compression (Control, n = 5) and BLCC (n = 7). ** = p < 0.005 by unpaired two-tailed t test with Welch’s correction. Dotted line denotes fold change threshold of 2. (B) Immunofluorescence of IBSP protein (red) in ulcer bed sections before and after BLCC treatment (Week 0 and Week 1, respectively). Images are representative of findings in n = 5 paired BLCC-treated VLUs. (C) Gene ontology analysis of enriched biological processes among genes displaying fold change >1.5 one week post- treatment with BLCC. Select overrepresented functions are listed in columns, and corresponding BLCC-regulated genes are shaded in gray.

Figure 5.

BLCC induces metallothionins, zinc release, and MMP8 expression in the VLU bed. (A) QPCR of MT1H, MT1X, and MT2A metallothioneins in n = 8 BLCC-treated VLU as compared with n = 8 control-treated VLU. * = p < 0.05 by Mann–Whitney U-test. (B) Western blot of metallothionein expression in chronic VLU before and after BLCC treatment. Band intensities of type 1 metallothionein proteins (6–20 kDa) were quantified relative to expression at Week 0. (C) Metallothionein expression by immunofluorescence (green) in VLU bed sections and DAPI staining of nuclei (blue). Images are representative of n = 5 BLCC-treated VLU patients. (D) Zinc quantification (absorbance at 560 nm) in n = 4 VLU before and after BLCC treatment. * = p < 0.05 by paired t test. (E) Western blotting of MMP8 expression (MW 70 kDa) post-BLCC treatment; images representative of n = 5 BLCC-treated and n = 3 control-treated VLU. * = p < 0.05 by unpaired t test with Welch’s correction. (F) Immunofluorescence staining of MMP8 (red) and CD45 (green) expression. Images representative of n = 5 BLCC-treated VLU.

Figure 6.

Model of antifibrotic BLCC effects on the nonhealing VLU bed to promote wound closure. The chronic VLU bed is characterized by unresolved inflammation which perpetuates fibrogenesis, as evidenced at the transcriptional level by activated TGFβ signaling and elevated markers of fibrosis including tenascin C. BLCC application reverses chronic inflammation and decreases fibrosis of the VLU bed by dampening TGFβ signaling and by stimulating matrix remodeling through activation of zinc-dependent collagenases and inhibition of TIMPs.