Transcriptional variations at perilesional site in vitiligo patients: probably a fight for cell-survival.

Exploration of gene expression variations is a potential source to unravel biological pathways involved in pathological changes in body and understand the mechanism underneath. Vitiligo patients were explored for gene expression changes transcriptionally at perilesional site in comparison to normal site of same patients for melanogenesis pathway (TYR, DCT & TYRP1) cell adhesion (MMPs & TIMP1), cell survival (BCL2 & BAX1) as well as proliferation, migration & development (SOX9, SOX10 & MITF) regulatory system, using skin biopsy samples. Results were also compared with changes in gene expression for melanocytes under stress after hydrogen peroxide treatment in-vitro. Gene amplification was carried out via real time PCR. We found increased expression of proliferation, migration & development regulatory genes as well as melanogenesis pathway genes at perilesional site of patients. In-vitro study also supports induced MITF expression and disturbed melanogenesis in melanocytes under stress. Expression level ratio of cell survival regulatory genes' (BCL2/BAX1) as well as cell adhesion regulatory genes (MMPs/TIMP1) was observed upregulated at patient's perilesional site however downregulated in hydrogen peroxide treated melanocytes in-vitro. Observed upregulated gene expression at perilesional site of patients may be via positive feedback loop in response to stress to increase cell tolerance power to survive against adverse conditions. Gene expression analysis suggests better cell survival and proliferation potential at perilesional site in vitiligo patients. It seems in-vivo conditions/growth factors supports cells to fight for survival to accommodate stressed conditions.

Catecholamines' accumulation and their disturbed metabolism at perilesional site: a possible cause of vitiligo progression.

Catecholamines (epinephrine, norepinephrine and dopamine) are considered toxic to the melanocytes and may play an important role in the development of depigmented patches on the skin. This study was done to evaluate the levels of catecholamines in skin and plasma samples of active vitiligo patients' and gene expression changes in catecholamines' metabolism regulatory genes (COMT and GTPCH1), immunoregulatory genes (CTLA4 and PTPN22), and Catalase in active vitiligo patients. In this single-centre, prospective, case-control study, 30 patients with active vitiligo were recruited and skin biopsies from the perilesional site and plasma samples were collected. Skin biopsies from the normal site in vitiligo patients and healthy controls (n = 15) and plasma samples from controls were also obtained. Catecholamines' estimation was done via high-performance liquid chromatography. Gene expression variations were investigated via reverse transcription-polymerase chain reaction (PCR) and real-time PCR. Epinephrine, norepinephrine and dopamine levels were significantly higher in perilesional skin biopsies as compared to controls (P = 0.035, 0.024, and 0.006, respectively). However, epinephrine, norepinephrine and dopamine levels observed in patients' plasma samples were comparable to controls. The mRNA expression level of the Catalase gene was found to be upregulated at the perilesional site of patients as compared to the non-affected site of same patients (P < 0.001) and healthy controls (P = 0.037). Transcriptional expression of GTPCH1 and COMT were observed to be increased significantly at the perilesional site of patients in comparison to controls (P = 0.004 and P = 0.046, respectively). Our results support the presence of oxidative stress, inflammation and induced immune response in vitiligo patients at the perilesional sites. The increased inflammatory response may lead to catecholamines upregulation resulting in oxidative stress and melanocyte damage.

Dopamine toxicity contributes to melanocyte loss via melanocytorrhagy: an in vitro study.

BACKGROUND: Catecholamines (epinephrine, norepinephrine, and dopamine) have been proposed as a possible cause of melanocyte loss. Dopamine has been observed to cause apoptosis in melanocytes via reactive oxygen species development, but the effect on melanocyte adhesion and proliferation still remains to be elucidated. Thus, we explored the dose- and time-dependent toxicity of catecholamines and the effect of dopamine on the proliferation and adhesion potential of melanocytes. MATERIALS AND METHODS: Primary culture of melanocytes was investigated in vitro for toxic effects of epinephrine, norepinephrine, and dopamine on metabolic activity via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay. Cells were observed microscopically for any phenotypic changes. Further cell proliferation, cell adhesion, and cell death pathway were explored under dopamine toxicity in dose- and time-dependent manner with RNase/PI method of cell cycle analysis, cell adhesion assay, and Annexin V-FITC/PI assay, respectively. Altered gene expressions were confirmed with a real-time polymerase chain reaction. RESULTS: Metabolic activity of cells varied with time and different doses of epinephrine, norepinephrine, and dopamine. Dopamine was observed to be more toxic than epinephrine and norepinephrine. Melanocytes were observed to follow different cell death pathways at comparatively lower and higher concentrations of dopamine. Persistent exposure to dopamine resulted in decreased cell proliferation and adhesion potential with apoptotic changes. Gene expression changes also confirmed the weak adhesion and survival potential of cells under the toxic effects of dopamine. CONCLUSION: Dopamine can alter melanocytes' adhesion and survival potential, leading to apoptotic cell death or melanocytorrhagic loss.

Combination of Follicular and Epidermal Cell Suspension as a Novel Surgical Approach in Difficult-to-Treat Vitiligo: A Randomized Clinical Trial.

Importance: Epidermal cell suspension (ECS) and follicular cell suspension (FCS) are successful surgical modalities for the treatment of stable vitiligo. However, repigmentation in generalized and acrofacial vitiligo and over acral or bony sites (eg, elbows, knees, iliac crests, and malleoli), which are difficult to treat, is challenging. Objective: To study the efficacy of transplanting a combination of autologous, noncultured ECS and FCS (ECS + FCS) compared with ECS alone in stable vitiligo. Design, Setting, and Participants: A prospective, observer-blinded, active-controlled, randomized clinical trial was conducted at a tertiary care hospital, with treatment administered as an outpatient procedure. Thirty participants who had stable vitiligo with symmetrical lesions were recruited between October 18, 2013, and October 28, 2016. All of the lesions were resistant to medical modalities with minimum lesional stability of 1 year. Intent-to-treat analysis was used. Interventions: ECS + FCS was prepared by mixing equal amounts (in cell number) of FCS with ECS. After manual dermabrasion, ECS was applied to 1 lesion and ECS + FCS was applied to the anatomically based paired lesion of the same patient. No adjuvant treatment was given. Main Outcomes and Measures: Patients were followed up at 4, 8, and 16 weeks by a blinded observer and extent of repigmentation, color match, pattern of repigmentation, patient satisfaction and complications were noted. Both the visual and the computerized image analysis methods were used for outcome assessment. Cell suspensions were assessed post hoc for OCT4+ stem cell counts using flow cytometry; expression of stem cell factor and basic fibroblast growth factor was evaluated using quantitative relative messenger RNA expression. Results: Of the 30 patients included in the study, 18 (60%) were women; mean (SD) age was 23.4 (6.4) years. Seventy-four percent of the lesions (62 of 84) were difficult-to-treat vitiligo. ECS + FCS showed superior repigmentation outcomes compared with ECS: extent (76% vs 57%, P < .001), rapidity (48% vs 31%, P = .001), color match (73% vs 61%, P < .001), and patient satisfaction (mean [SD] patient global assessment score, 23.30 [6.89] vs 20.81 [6.61], P = .047). Melanocyte stem cell counts (2% in ECS + FCS vs 0.5% in ECS) as well as expression of basic fibroblast growth factor (11.8-fold) and stem cell factor (6.0-fold) were higher in ECS + FCS suspension (P<.05 for both). Conclusions and Relevance: The findings from this study establish ECS + FCS as a novel approach in vitiligo surgery for attaining good to excellent repigmentation in a short period with good color match, even in difficult-to-treat vitiligo. Trial Registration: ctri.nic.in Identifier: CTRI/2017/05/008692.