Currently, the diagnosis of SSc relies heavily on clinical judgment. An improvement in diagnostic criteria through incorporation of objective measures is needed17,36
. Our electron microscopy findings corroborate previous descriptions of microvascular change in SSc, including swollen endothelial cells with a duplicated, lamellated appearance of the basement membrane26,27
. We found that regardless of SSc disease type, disease duration, or clinical features, all capillaries in SSc patients appeared to have these features of swollen endothelium and lamellation of basal lamina. These findings were also present in 2 patients with early SSc12
but not in a patient with RP without capillary nailbed changes. Thus, these features suggest oxidative injury and endothelial dysfunction, and establish SSc as a disease of the endothelium37
. Under standing the link between endothelium dysfunction and interstitial edema has diagnostic and therapeutic implications. It is particularly important to study additional patients with early, diffuse SSc and RP without SSc to understand this mechanism.
Whole-field digital microscopy offers a means of rapidly carrying out quantitative, reproducible measurements of microanatomical features of microvascular change in SSc. Endothelium in patients with SSc is morphologically abnormal and this is one of the earliest changes found in this disease38
. Previous histopathology descriptions of vasculopathy in SSc demonstrated that regardless of the extent of fibrosis, abnormalities of blood vessels are universal in patients with SSc26,27
. The dissociation between vasculopathy and fibrosis as part of the natural history of the disease might explain the finding that some patients have signs of vascular leak without fibrosis, the entity of SSc sine
scleroderma. Additionally, the mRSS decreases in a majority of patients over extended time, but the vascular complications of SSc seem to be progressive. Mast cells and CD34 cells have been proposed as important to the disease pathogenesis based on their presence in descriptive studies27,39,40
. Our study suggests that whole-field digital microscopy of skin biopsy specimens, particularly in regard to quantification of vessel number, cell types, and vascular leak, may be useful both for research and to personalize clinical care.
We found that interstitial edema, a marker of vascular leak, was a central feature in all SSc specimens. This early stage of disease without fibrosis may be more responsive to disease-targeted therapies, because successful antifibrotic therapies do not exist17
. It is possible that the interstitial edema is driven by another process, such as vascular injury, that does not improve over time. While total microvessel density (MVD) was not significantly different between patients with SSc and controls (likely because of differences in SSc disease duration), when CD34+ staining was quantified by MVD, SSc specimens had less CD34+ staining than controls. Further, there was significantly less CD34+ staining among SSc with ILD as compared to those without ILD. This finding is of interest because normal endothelial repair requires CD34 to be expressed on small-vessel endothelium41
to promote chemokine-mediated trafficking42
. In addition to leukocyte adhesion or “homing” during the inflammatory process, CD34 expression plays a role in stem/progenitor cell localization/adhesion in the basement membrane. Our findings highlight that the endothelium remains a principal cell type of interest in SSc pathogenesis, since endothelial activation and abnormalities in the vessel walls are thought to be essential for vascular leak27,43,44
. Although the initial trigger for endothelial injury in SSc remains unknown, it is thought that reperfusion injury, autoantibodies, infectious agents, and/or environmental factors conspire to perpetuate endothelial injury in genetically predisposed hosts45
. The endothelium is a critical regulator of subsequent immune dysfunction46
; thus understanding the process of vascular leak in SSc is of paramount importance.
Mast cells have been proposed as important in disease pathogenesis based on their presence in descriptive studies27,39,40,47
. In our study, all patients with SSc had mast cells in a perivascular and interstitial location. The numbers of mast cells were not different statistically from control samples and appeared to be within normal limits, in contrast to previous descriptions32
. This may be a sampling artifact or variation related to duration of SSc in our cases; nevertheless, the significance of mast cells as well as their function in SSc pathogenesis remains to be established.
Quantification of vasculopathy by whole-field digital microscopy may also be helpful for determining response to treatment. One patient who was rebiopsied 6 months after the initiation of immunosuppression and therapy for PAH showed no changes in clinical features, while the CD34 staining increased, suggesting that repeat biopsy may have a role in tracking therapeutic response.
Our study has some limitations. It was cross-sectional: a longitudinal study would be of greater value for determining the significance of our findings. While the pathologist was not blinded to the fact that the samples carried a diagnosis of SSc, she was blinded to the clinical severity, progression, and characteristics of the patients. In addition, we had only 2 participants with diffuse SSc; thus we were unable to do analysis by disease subtype. We have included these patients in the analysis because their findings are consistent with those of the limited subset, and suggest a common mechanism of disease. The importance of understanding the mechanism of vasculopathy in SSc is underscored by the fact that our findings did not display any correlation with a large number of other clinical variables. The population studied all had RP with capillary nailbed changes, but included only 1 patient with RP without SSc; thus we do not know if these findings are a consequence of RP, whether primary or secondary, or are related to SSc. Additionally, all patients with SSc were taking a CCB; thus the effects of this medication on the findings are also unknown. The value of repeat skin biopsies and the proper interval of time between biopsies remain to be determined. We restricted our controls to skin biopsy specimens from healthy participants requiring biopsy in the forearm location for nevus removal. We were able to find only 4 such patients during the study period. Given the small number of control specimens, our observations need to be confirmed with a larger number of samples. Nonetheless the study had adequate power to detect the differences in interstitial edema and MVD per post hoc analysis.
Our study demonstrates that whole-field digital microscopy can be used successfully to quantify microvascular change in SSc skin biopsies. It emphasizes that interstitial edema (vascular leak) was present in all SSc biopsies, and that the presence of finger/hand edema clinically was negatively associated to fibrosis. Endothelial cell changes, presence of perivascular mast cells, and CD34+ MVD may be helpful in establishing an earlier diagnosis of SSc than can be done by relying on the clinical recognition of fibrosis, and may be useful in gauging therapeutic response. Further research is needed to determine the usefulness of whole-field digital microscopy in defining disease mechanisms of pathogenesis and to determine whether SSc is indeed an intrinsic disease of the endothelium.