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); pulmonary hypertension and renal crisis are potentially lethal and silent at an early stage. Thus, better characterizing patients at risk is a crucial matter. In this context, we focused on pigmentary disorders in SSc, which affect about half of the patients and can impact their quality of life (
). Here, we reveal microscopic distribution of melanin in SSc skin among patients presenting hyperpigmentation or not.
In this observational cross-sectional study, we characterized arm skin melanin distribution in 26 patients with SSc patients in a blinded manner. Written informed consent was obtained from all participants and the study was approved by the institutional ethical committee (CPP, 2012 A00081-42, Aquitaine). Patients with SSc were recruited in a case-control manner to compare melanin distribution between patients with hyperpigmented or non-hyperpigmented SSc. Demographic and disease characteristics of patients with SSc are presented in Table 1. Diffuse hyperpigmentation was present in 14 (54%) patients with SSc (Figure 1a), accompanied with hypopigmented macules in seven patients. The hyperpigmented group included a significantly higher proportion of diffuse cutaneous SSc forms as previously reported (
). The proportion of patients with clinical vascular involvement was not statistically different between the two groups, which allows for the avoidance of a potential bias related to the previously reported association between hyperpigmentation and vascular involvement (
In brightfield images, we observed a striking pattern of dark brown pigment deposit in the dermis of eight patients with SSc, particularly lining dermal vessels. We could not easily evidence any pigmented deposit in the superficial dermis as its thickness was greatly reduced (
). CD31 staining allowing the identification of endothelial cells confirmed that pigment was specifically located near the outside layer of dermal vessels (Figure 1b). We did not find any evidence of perivascular pigment around lymphatic vessels (Supplementary Figure S1a). Fontana-Masson stain showing black pigment pointed out that the dermal pigment was at least partly constituted by melanin (Figure 1c), as supported by high-power view showing melanin granules (Figure 1c and Supplementary Figure S1b). Perls and Sudan black B stains were negative, ruling out hemosiderin and lipofuscin presence, respectively, in the perivascular pigmented deposit (Supplementary Figure S1c and d). Finally, bleaching with hydrogen peroxide completely washed out the perivascular pigment, confirming the melanin content (Supplementary Figure S1e). CD68 immunostaining identifying macrophages showed that, in some cases, the pigment was inside the cytoplasm of dermal melanophages, but not always (Figure 1d).
Among patients with SSc, those that exhibited such a pattern of perivascular pigmentation all belonged to a subgroup associating hyperpigmentation and vascular involvement (defined as the presence of digital ulcers and/or pulmonary hypertension and/or renal crisis) (Figure 1e). Among patients with hyperpigmented SSc, the presence of this histological pattern was significantly associated with vascular involvement (P = 0.0063, chi-square test). Overall, the specificity of this histological pattern to indicate vascular involvement was 100% in our cohort; moreover, among patients with SSc with clinical hyperpigmentation, the sensitivity of the pattern to indicate vascular involvement was 80%. Demographic and disease characteristics of the eight patients with SSc that presented this pattern are presented in Supplementary Table S1.
This finding raises several points. First, such dermal pigmentation could account for the persistent tanning that some patients report, maybe owing to the absence of pigment clearance once in the dermis (dermal tattoo); it could also explain the peculiar skin color (copper-like) observed in some patients. In this case, clinical hyperpigmentation would be explained by dermal pigmentation rather than increased epidermal pigmentation, and some samples even exhibited a marked decrease in epidermal pigmentation (Supplementary Figure S1f).
Second, this histological pattern probably is not restricted to SSc skin, and dermal pigment is a common phenomenon among dark phototypes (
), including in the case of post-inflammatory hyperpigmentation, although the perivascular location has not been commonly described. However, in our case, dermal pigmentation was also observed in fair phototypes; moreover, such pigment deposit was only incidentally seen in a cohort of 26 healthy control skin samples matched on sample location and phototype. Overall, intensity of pigment deposit around dermal vessels was significantly higher in patients with SSc compared with healthy controls (Supplementary Figure S1g; P = 0.0337).
Third, the specific perivascular localization of pigment deposit suggests, in an original way, a pathophysiological relationship between vasculature and pigmentation, as seen in other diseases such as melasma (
). This also raises the question of the origin of the pigment deposit. It is unlikely that the observed deposits are the result of drugs inducing dermal pigmentation, such as hydroxychloroquine; indeed, pigmentary changes usually differ clinically (bluish-green macules) and microscopically (pigmented deposit Perls-positive and in the superficial dermis) (
). However, and most importantly, of the eight patients that presented this pattern, three had no history of current or past drug uptake known to modify skin pigmentation, including hydroxychloroquine. In any case, the similar distribution suggests a shared mechanism, possibly triggered by a circulating factor.
Thus, one could imagine an enhanced secretion of melanogenic factors by SSc endothelial cells, such as endothelin-1 (
). Dermal melanin would then be phagocyted by macrophages (Figure 1d) migrating toward the nearest vessel. However, this hypothesis does not fully explain the presence of melanin inside the cytoplasm of CD68-negative perivascular cells. The nature of these cells remains to be elucidated; notably, one should consider the possibility of a local melanin production by transdifferentiation of perivascular cells.
Altogether, perivascular melanin could help to detect vascular involvement, specifically among patients with SSc presenting clinical hyperpigmentation. However, this finding needs to be assessed in larger cohorts of patients with hyperpigmented SSc to validate the association with clinical vascular involvement. Moreover, the cross-sectional design of the study does not allow determining if the perivascular melanin deposit occurred before or after the development of the vascular injury. Prospective studies are warranted to address the possibility of using this histological pattern as a predictive marker. Despite various durations of pigmentary changes in patients with SSc, we did not see any correlation between the intensity of the pigmented deposit and disease duration (data not shown). Nevertheless, this does not refute the hypothesis that the pigmented deposit occurs early in the development of the disease. Future research should also focus on the mechanism of melanin deposit in the dermis, particularly this peculiar localization around dermal vessels, to further establish the link between hyperpigmentation and vasculopathy.
Data availability statement
No datasets were generated or analyzed during this study.
This work was funded by Association des Sclérodermiques de France and Société Française de Dermatologie . The authors wish to thank Dr Hugues Begueret, from the Pathology Department of Bordeaux University Hospital, for providing the Perls stain, as well as Catherine Pain for technical assistance, and all the clinicians and patients who took part in the SSc-VISS cohort.