Clinical and laboratory characterization of patients with localized scleroderma and response to UVA-1 phototherapy: In vivo and in vitro skin models

Abstract

Background/purpose: Localized scleroderma (LS) is a rare disease leading to progressive hardening and induration of the skin and subcutaneous tissues. LS is responsive to UVA-1 phototherapy, though its exact mechanism of action dermal fibrosis is yet to be fully elucidated. We aimed to investigate the molecular changes induced by UVA-1 rays in human primary fibroblasts cultures.

Methods: A total of 16 LS patients were treated with medium-dose UVA-1 phototherapy. At baseline, during and after therapy, Localized Scleroderma Assessment Tool, Dermatology Life Quality Index and lesions' staging and mapping were performed along with high-frequency ultrasound (HFUS) examination for dermal thickness assessment. Gene expression analysis for 23 mRNA transcripts, in vitro UVA-1 irradiation and viability tests were realized on lesional fibroblasts' primary cultures, before and 3 months after therapy.

Results: The dermal thickness, the LoSCAT and the DLQI progressively decreased starting from the last phototherapy session up to the 6 and 9 month follow-ups (-57% and -60%, respectively). Molecular gene analysis (rt-PCR) revealed that UVA-1 phototherapy exerts multiple effects: the activation of specific anti-fibrotic pathways (e.g., overexpression of CTHRC1 and metalloproteases 1, 2, 7, 8, 9, 12, suppression of TIMP-1), the downregulation of peculiar pro-fibrotic pathways (e.g., downregulation of TGF-ß, TGF-ßrII, Grb2, SMAD 2/3, TNRSF12A, CTGF) through a significant overexpression of IL-1ß; the stabilization of collagen synthesis acting on genes COL1A1, COL3A1, COL8A1, COL10A1, COL12A1.

Conclusion: UVA-1 phototherapy adds significant benefits in local tissue remodeling, rebalancing the alteration between pro-fibrotic and anti-fibrotic pathways; these changes can be well monitored by HFUS.

Keywords: UVA-1 phototherapy; high-frequency ultrasound; in vitro UVA-1 irradiation; localized scleroderma; primary fibroblast cultures.

FIGURE 1

Example of the body mapping scheme that was filled for each patient during every evaluation time of the study (t0‐t7) (A). The position and the stage for each morphea lesion was indicated among 7 stages (i.e., erythematous (E), inflammatory (I), early sclerotic (ES), sclerotic (S), sclero‐atrophic (SC), atrophic (A) and dyspigmented (D); exemplificative images at different body sites took from 7 different patient enrolled in the study (B)

FIGURE 2

Ultrasound imaging 22 Mhz in a 65‐year‐old patient with plaque LS: examination at 5 points of an inflammatory patch of the abdomen (E = epidermis, D = dermis, H = hypodermis)

FIGURE 3

Clinical appearance of a sclerotic localized scleroderma (LS) patches of the lumbar‐gluteal area and of the posterior leg in a 57‐year‐old female, and corresponding high‐frequency ultrasound (HFUS) 22 MhZ estimation of dermal thickness in the center of the lesion (A,B); the same lesions examined at t5 (C,D). Clinical appearance of an inflammatory LS patch of the back in a 16‐year‐old male, and corresponding HFUS performed at t0 (E) and t5 (F), when the skin has retrieved its plicability as demonstrated by the skin pinching test, reaching an extension up to 2 cm (F)

FIGURE 4

The relative expression of mRNA levels of 12 out of the 21 genes analyzed in the study, involved in pro‐ and anti‐fibrotic pathway fibrotic pathways, according to qRT‐PCR analysis; average values derived from patients belonging to the three subgroups are compared: lesional fibroblasts from selected a localized scleroderma (LS) lesion in harvested before phototherapy in 16 patients (Pre); lesional fibroblasts from the same cleared lesions harvested after phototherapy (Post); control group of healthy fibroblasts of 16 healthy patients (Ctrl). The relative gene expressions normalized to Act‐β reference gene are shown. Bars represent mean ± Sd. Relative quantification of mRNA was measured by using the 2^−∆∆CT method. For each patient/control, tests were repeated at least for three clusters of fibroblasts cultures (having a total of 80 specimens and assuming the average results among the three values)