In the present study, we demonstrated increased levels of MPs derived from platelets, endothelial cells and monocytes in the blood of patients with SSc. In addition, a significant association with clinical features of SSc was found, including higher levels of PMPs in patients with positive anti-Scl-70 antibodies and longer disease duration, and lower EMPs levels in SSc patients with more severe cutaneous involvement, and more severe peripheral microangiopathy, as defined by NFC.

Our results are in accordance with previous studies, in which increased levels of different sources of MPs were found [12,13,14,15,16, 28]. As the elevated levels of various cytokines are a main feature of SSc, and platelets, endothelial cells and monocytes are activated in SSc [29, 30], the increased levels of MPs found in our study strengthen the hypothesis that MPs may be involved in SSc pathogenic mechanisms.

Our study is, thus far, the first to demonstrate increased plasma levels of PMPs in patients with SSc who were positive for anti-Scl-70 and had a longer disease duration (more than 3 years). The anti-Scl-70 autoantibody has a high specificity for the diagnosis of SSc [31, 32], and it is considered an important marker of disease progression, as it correlates with more severe phenotypes, such as diffuse cutaneous disease, pulmonary involvement, cardiac involvement and a higher risk of mortality [31,32,33]. Interestingly, in 2008, Nomura et al. [14] demonstrated an increase in serum levels of PMPs and MMPs in 42 patients with SSc compared to healthy controls, and this increase was significantly greater in patients with ILD. Recently, Leleu et al. [34] also found an increase in the serum level of PMPs in 96 patients with SSc compared to healthy controls, and this increase was more pronounced in patients with ILD and longer disease duration, similar to our study. Our findings, along with previous studies, therefore reinforce the possible role of these molecules as prognostic markers in SSc. It is important to highlight that, in both aforementioned studies, the prevalence of ILD was higher than in our sample (59.5% in the sample by Nomura et al. and 81% in the sample by Leleu et al.), which could explain the lack of association between ILD and MP levels in our study.

Endothelial cell-derived MPs have also been studied in SSc. Recently, Lammi et al. [35] demonstrated a significant increase in EMPs in 20 patients with SSc compared to healthy controls. Furthermore, our results are similar to those of Guiducci et al. [13], who showed a significant increase in PMPs, EMPs and MMPs in 37 patients with SSc compared to healthy controls, with lower levels of MPs in patients with an mRSS > 10. Significantly elevated levels of EMPs were also demonstrated in 47 scleroderma patients, with lower levels of EMPs in patients with higher mRSS [16]. This inverse correlation between EMP levels and the degree of skin thickening, measured by the mRSS, the best validated outcome measure for skin fibrosis in SSc [13], indicates that higher amounts of circulating MPs are linked to a lower degree of fibrosis in the dermis in patients with SSc [13, 35]. Considering that upregulation of matrix metalloproteinase expression by MPs can enhance the synthesis of fibroblasts involved in extracellular matrix (ECM) degradation and that MPs contain proteolytic enzymes involved in the degradation of ECM [9, 36], the potential antifibrotic properties of MPs deserve further investigation.

Interestingly, we demonstrated that EMP levels were lower in SSc patients with an avascular score higher than 1.5 in the NFC exam, indicating an association of lower EMPs with more severe peripheral vasculopathy. This is in accordance with the findings of Michalska-Jakubus et al. [16], where significantly lower levels of EMPs were correlated with late NVC patterns and higher NVC scores for capillary loss. NFC is a well-established method to evaluate morphological abnormalities in the microcirculation [37], allowing the observation of specific SSc capillaroscopic changes secondary to its microangiopathy. In patients with SSc, hemorrhages, enlarged capillaries, capillary loss and distortion of the capillary architecture are present in early stages of the disease. The late nailfold capillaroscopic pattern and more severe capillary loss are associated with severe internal organ involvement and mortality in SSc [37,38,39]. Thus, EMPs might be a useful biomarker of vascular damage in SSc. Further studies are necessary to evaluate the role of EMPs as a predictive marker of more severe vasculopathy in SSc. Nonetheless, we did not observe associations between MPs and vascular manifestations, such as DU and PAH. A lack of association between DU and MPs has already been shown [13, 16], but previous studies have shown higher levels of EMPs in SSc patients with PAH [35, 40], strengthening the potential role of EMPs as endothelial injury biomarkers. The different results observed in our report might have been due to the low prevalence of patients with PAH in our sample.

We also evaluated the level of MMPs in our patients. Remarkably, the greatest difference between patients and controls was found in this source of MPs. Monocytes play a pivotal role in SSc pathogenesis, as they present proinflammatory and profibrotic phenotypes and contribute to fibrogenesis owing to their high expression of TGF-β [20]. In addition, monocytes in SSc display low caveolin-1 function, collaborating with ILD, due to the increased monocyte maturation toward myofibroblasts and hyperaccumulation of fibrocytes in these patients, regardless of monocyte blood levels [14, 41].

The limitations of our study include its relatively small sample size and the use of flow cytometry. Although flow cytometry is the chosen method for evaluating MPs, the antibodies used across studies have varied, hindering direct comparisons. Since we did not use beads in the MPs acquisition, we were not able to express their quantification in absolute values. Furthermore, in vitro studies evaluating the behavior of MPs in SSc patients could confirm the hypothesis of the antifibrotic potential of these agents and explore their potential as new therapeutic targets, which are urgently needed. We were also not able to quantify ILD extension on chest HRCT, to better evaluate this abnormality in our patients.