Endothelial Progenitor Cells: Relevant Players in the Vasculopathy and Lung Fibrosis Associated with the Presence of Interstitial Lung Disease in Systemic Sclerosis Patients

Endothelial progenitor cells (EPC), which are key effectors in the physiologic vascular network, have been described as relevant players in autoimmune diseases. We previously showed that EPC frequency may help to identify the presence of interstitial lung disease (ILD) in rheumatoid arthritis patients. Given that ILD constitutes the main cause of mortality in systemic sclerosis (SSc) patients, we aimed to determine the EPC contribution to the pathogenic processes of vasculopathy and lung fibrosis in SSc-ILD+. EPC quantification was performed by flow cytometry on blood from 83 individuals: 21 SSc-ILD+ patients and subjects from comparative groups (20 SSc-ILD− and 21 idiopathic pulmonary fibrosis (IPF) patients and 21 healthy controls (HC)). EPC were considered as CD34+, CD45low, CD309+, and CD133+. A significant increase in EPC frequency was found in SSc-ILD+ patients when compared to HC (p < 0.001). SSc-ILD+ patients exhibited a higher EPC frequency than SSc-ILD− patients (p = 0.012), whereas it was markedly reduced compared to IPF patients (p < 0.001). EPC frequency was higher in males (p = 0.04) and negatively correlated to SSc duration (p = 0.04) in SSc-ILD+ patients. Our results indicate a role of EPC in the processes of vasculopathy and lung fibrosis in SSc-ILD+. EPC frequency may be considered as a biomarker of ILD in SSc patients.


Introduction
Endothelial progenitor cells (EPC) are known to be key cellular effectors in the homeostasis of the physiologic vascular network, being implicated in vascular regeneration, both in new vessel formation and in the repair mechanisms of existing vessels [1][2][3]. The ability of these cells to differentiate towards mature endothelial cells and to incorporate into the injured vasculature, promoting neovascularization, has raised great interest over the last several decades [2,4]. Accordingly, growing evidence has shown the relevant contribution of EPC to the pathogenesis of different vascular diseases [2,[4][5][6]. In fact, we recently proposed that the degree of EPC frequency may be useful as a biomarker to identify the presence of interstitial lung disease (ILD) in patients with rheumatoid arthritis (RA) [7].
Taking all this into account and given the essential role of EPC in endothelial repair, the objective of this study was to determine the contribution of EPC to the pathogenic processes of vasculopathy and lung fibrosis in SSc-ILD + .

Study Population
A total of 83 individuals constituted by 21 SSc-ILD + patients and subjects from three comparative groups (20 SSc-ILD − patients, 21 idiopathic pulmonary fibrosis (IPF) patients, and 21 healthy controls (HC)) were recruited from the Pneumology and Rheumatology departments of Hospital Universitario Marqués de Valdecilla (Santander, Spain).
Patients with SSc fulfilled the 2013 American College of Rheumatology/European League Against Rheumatism criteria for the classification of SSc [31]. Pulmonary fibrosis was assessed in all the patients by high-resolution computed tomography (HRCT) images of the chest and pulmonary function tests (PFTs). Additionally, pulmonary hypertension (PH) was diagnosed by transthoracic echocardiogram in all the patients. SSc patients who lacked lung involvement (absence of pulmonary fibrosis and PH) were considered as SSc-ILD − patients, whereas those who fulfilled the American Thoracic Society/European Respiratory Society criteria for ILD were classified as SSc-ILD + [32]. IPF patients met the criteria proposed by the American Thoracic Society/European Respiratory Society [32]. HC did not present any history of autoimmune or lung diseases.
For further clinical characterization, demographic and clinical features of patients including sex, age, smoking history, duration of SSc disease (early: ≤ 5 years; late: >5 years), antibodies status, C-reactive protein, erythrocyte sedimentation rate, PFTs, PH, pulmonary fibrosis on HRCT, HRCT patterns, and other SSc clinical manifestations at the time of the study were collected (Table 1). HRCT patterns of ILD patients were stratified according to the Fleischner society's criteria for usual interstitial pneumonia pattern [33].
All the experiments involving humans and human blood samples were carried out in accordance with the approved guidelines and regulations, according to the Declaration of Helsinki. All experimental protocols were approved by the Ethics Committee of Clinical Research of Cantabria, Spain (2016.092). All subjects gave written informed consent to participate in this study prior to their inclusion.  HRCT: high resolution computed tomography; UIP: usual interstitial pneumonia; NSIP: non-specific interstitial pneumonia.

Statistical Analyses
Data were expressed as mean ± standard deviation (SD) for continuous variables and as number of individuals (n) and percentage (%) for categorical variables. Comparisons of EPC frequency between two study groups were performed by Student's t-test. The relationships of EPC frequency with continuous variables and categorical variables related to demographic and disease features were established via estimation of Pearson's correlation coefficient (r) and one-way ANOVA, respectively. p-values < 0.05 were considered as statistically significant. Statistical analysis was performed using STATA 12/SE statistical software (Stata Corp., College Station, TX, USA).

Differences in EPC Frequency between SSc-ILD + Patients and the Comparative Groups
Patients with SSc-ILD + showed a significantly higher EPC frequency than HC (p < 0.001) ( Figure 1 and Table S1). Likewise, EPC frequencies were increased in IPF and SSc-ILD − patients when compared to HC, but in the latter case it was marginally statistically significant (p < 0.001 and p = 0.057, respectively) ( Figure 1). Moreover, EPC frequency was increased in SSc-ILD + patients compared to patients with SSc-ILD − (p = 0.012), whereas it was markedly reduced compared to IPF patients (p < 0.001) (Figure 1 and Table S1). Furthermore, patients with SSc-ILD − exhibited a lower frequency of EPC than IPF patients (p < 0.001) (Figure 1).
In a further step, SSc-ILD + and SSc-ILD − patients were stratified by disease duration. In this sense, EPC frequency was greater both in early and late SSc-ILD + patients when compared to HC (p < 0.001 in both cases) (Figure 2a). No significant differences were observed between early and late SSc-ILD + patients (Figure 2a). Regarding SSc-ILD − , early SSc-ILD − patients showed EPC frequencies significantly greater than HC (p = 0.030), while no differences were found between late SSc-ILD − patients and HC (p = 0.332) (Figure 2b). No significant differences were observed between early and late SSc-ILD − patients (Figure 2b).

Relationship of EPC Frequency with Demographic and Clinical Features
We found a negative correlation between the frequency of EPC and the SSc disease duration in patients with SSc-ILD + (r = −0.45; p = 0.04) ( Table 2). Moreover, EPC frequency was higher in male SSc-ILD + patients when compared to female patients (p = 0.04) ( Table 3). No significant relationship between EPC frequency and the other demographic and clinical features assessed was found in SSc-ILD + patients (Tables 2 and 3).
Regarding patients with SSc-ILD − and IPF, no significant results were obtained (Tables 2 and 3).

Discussion
EPC have been described as important players in the pathogenesis of vascular diseases [2,[4][5][6]. Given that we recently identified EPC as a potential biomarker of endothelial damage in RA-ILD + [7], and based on the relevance of ILD as a main cause of mortality in patients with SSc [8][9][10][11][12], we wondered if EPC may also play a crucial role in SSc-ILD + . Accordingly, we assessed the contribution of EPC to the pathogenic processes of vasculopathy and lung fibrosis in SSc-ILD + .
Our study found a relevant role of EPC in the vasculopathy process of SSc-ILD + . In particular, we found an increase in EPC production and mobilization into the systemic circulation in SSc-ILD + patients, since a higher EPC frequency was found in these patients when compared to HC. This difference remained significant when SSc-ILD + patients were stratified into early and late disease duration. Given that endothelial injury and insufficient endothelial repair are strong contributors to the vasculopathy underlying SSc-ILD + , we hypothesize that EPC are increasing in peripheral blood to be recruited at the sites of vascular damage and to exert their reparative function as a compensatory mechanism. This finding is consistent with a previous result of our group from a study performed in RA-ILD + patients [7]. Likewise, other groups demonstrated an increase in circulating EPC in SSc patients in relation to HC [14][15][16]20,[26][27][28]30], mainly in the early stage of the disease [14,20,[26][27][28]. In keeping with this, our study showed significantly greater EPC frequencies in early SSc-ILD − patients than HC, unlike late SSc-ILD − patients. The latter may explain the lack of statistically significant difference between HC and SSc-ILD − patients, regardless of SSc duration. We also observed an increase in EPC frequency in IPF patients in relation to HC, as described in a previous work [6], further supporting the compensatory mechanism of these cells.
Interestingly, an increase in EPC linked to the presence and severity of ILD was found in our study. According to this, patients with SSc-ILD + exhibited a higher EPC frequency than those patients with SSc-ILD − who did not present lung disease. Notably, the greatest EPC frequencies were found in IPF patients, who experience the most aggressive form of ILD. This behavior of EPC is similar to that observed in our previous work, in which we showed that the degree of EPC frequency may help to identify the presence of ILD in RA patients [7]. Therefore, although the exact mechanism responsible for these findings is not clear, it can be speculated that it may be associated with an enhancement of EPC production in response to the lung fibrotic process and, consequently, to the presence of ILD in patients with autoimmune diseases. In favor of this suggestion, previous reports in SSc indicated an association of EPC with lung involvement [15,16]. Our results further support the idea that EPC frequency may be considered as a useful complementary tool to identify the presence of ILD in SSc patients, mirroring the severity of SSc disease.
It is known that several factors influence the development of SSc-ILD + , including shorter disease duration and male sex [9,11]. Notably, our results revealed an inverse correlation of EPC frequency with SSc disease duration in SSc-ILD + patients, as reported in SSc by different authors [16,20,25,34]. It is conceivable that the decrease in EPC in patients with long-standing disease is related to the recruitment of such cells into damaged tissues, leading to a decrease in circulating EPC. Furthermore, we found a higher frequency of EPC in men, which seems to be expected considering that the male sex is a known SSc-ILD + risk factor. Nevertheless, no further association was found between EPC and demographic or disease features in SSc-ILD + patients or in SSc-ILD − and IPF patients. In agreement with this, previous reports showed a lack of association of EPC with PFTs and smoking status in IPF patients [37], as well as with age, inflammation markers, antibodies status, and PH presence in SSc patients [14,19,24,25,27].
In conclusion, our findings provide evidence for a potential role of EPC in the pathogenic processes of vasculopathy and lung fibrosis in SSc-ILD + . Interestingly, EPC frequency may be considered a promising marker for vascular damage and disease progression, particularly regarding the presence of ILD in patients with SSc.

Supplementary Materials:
The following is available online at https://www.mdpi.com/article/10 .3390/biomedicines9070847/s1, Table S1: Differences in EPC frequency between SSc-ILD + patients and the three comparative groups (healthy controls, SSc-ILD − and IPF patients). Institutional Review Board Statement: All subjects gave their informed consent to be included in the study. The procedures followed were in accordance with the ethical standards of the approved guidelines and regulations, in accordance with the Declaration of Helsinki. All experimental protocols were approved by the Ethics Committee of Clinical Research of Cantabria, Spain (2016.092).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All data generated or analyzed during this study are included in this published article.