Circulating levels of the CXCL4 are raised in the patients with SSc and the patients with a very early diagnosis of SSc (VEDOSS)
We first examined serum CXCL4 in SSc and VEDOSS patients in the identification cohort, and evaluated the possible correlation with specific clinical features of the disease. A total of 58 patients with SSc, 10 patients not fulfilling the 2013 ACR/EULAR classification criteria for SSc (total score < 9) and identified as VEDOSS (presented with Raynaud’s phenomenon, puffy figures, and positivity of antinuclear antibodies, together with SSc specific antibodies and / or pathognomonic microvascular alteration at capillaroscopy), and 80 healthy controls (HC) were recruited and stratified as described in Methods. In this identification cohort, serum CXCL4 levels were 60.59% higher in patients with SSc and 132.95 % higher in patients with VEDOSS than matched HC (HC: 1751 ± 917.1; SSc: 2812 ± 1445; VEDOSS: 4079 ± 1978 ng / ml; p < 0.0001 compared with HC respectively; Fig. 1A). Notably, significantly higher serum CXCL4 levels were detected in the patients with VEDOSS than the patients with SSc (p = 0.0089, Fig. 1A). These results were subsequently validated in the replication cohort, where circulating CXCL4 resulted significantly increased in SSc (n=50) and in VEDOSS (n=12) serum compared with control serum (n=80) (HC: 1288 ± 839.2; SSc: 3423 ± 1617; VEDOSS: 5003 ± 1814 ng / ml; p < 0.0001 compared with HC respectively; Fig. 1B). Similarly, further elevation of CXCL4 was detected in the patients with VEDOSS compared to the patients with SSc (p = 0.0004, Fig. 1B).
We performed the pooled analysis by combining patients and controls from the identification and replication cohorts. Circulating CXCL4 levels were 103.62 % higher in patients with SSc and 201.51 % higher in the patients with VEDOSS than in healthy controls, also significantly higher in SSc patients relative to VEDOSS patients (HC: 1520 ± 906.4; SSc: 3095 ± 1551; VEDOSS: 4583 ± 1903 ng / ml; p < 0.0001 compared with each other; Fig. 1C).
Moreover, we found that CXCL4 levels gradually increased per group in the following order: patients with SSc sine scleroderma (ssSSc) (n=4), overlapping syndrome of SSc (n=13), limited cutaneous SSc (lcSSc) (n=39), diffuse cutaneous SSc (dcSSc) (n=52), and those with very early systemic sclerosis (VEDOSS) (n=22) (Fig. 1D). Additionally, the level of CXCL4 was remarkably increased in VEDOSS patients rather than those in the subsets of ssSSc, overlapping, lcSSc, or dcSSc (Fig. 1D).
Increased serum CXCL4 levels positively correlate to the much severer skin fibrosis and peripheral vasculopathy in SSc patients
We next assessed the association between CXCL4 levels and the clinical phenotype in the combined cohort, and the patients with SSc were stratified as described in Methods. The levels of CXCL4 in SSc were positively correlated with their mRSS score (Fig. 2A), demonstrating that the increased CXCL4 level may implicate much severer skin fibrosis. Notably, significantly higher serum CXCL4 levels were detected in the SSc patients with digital ulcer (DU) (n=41) than those without DU (n=67) (3686±1769 vs 2734±1285 ng/ml, p = 0.0016; Fig. 2B), and the levels of CXCL4 in SSc were positively correlated with the number of digital ulcers (Fig. 2C), indicating the close association of CXCL4 with peripheral microvascular involvement severity in SSc.
We also investigated whether CXCL4 could serve as a biomarker, which performed on the combined group using ROC analysis to select a threshold (Fig. 2D). Patients who had a higher baseline level of CXCL4 (over 2797 ng/ml) had a significantly increased prevalence of newly-onset of digital ulcer in 6 months (34.78% vs 65.22%, odds ratio 6.133, P = 0.0335; Fig. 2E), with unbiased background treatments (data not shown).
Next, we stratified SSc patients according to their nailfold videocapillaroscopy (NVC) pattern and compared the CXCL4 levels between subsets, we also evaluated the possible correlation of CXCL4 levels with the mean number of nailfold capillary in the patients with SSc (n = 58). SSc patients with early NVC pattern demonstrated elevated serum CXCL4 levels rather than those with active or late NVC pattern (p=0.0159 and p=0.0063 for each comparison) (Fig. 2F). The levels of CXCL4 were negatively correlated with the mean number of nailfold capillary in patients with SSc (Fig. 2G).
SSc derived CXCL4 disturbed angiogenesis
It is widely reported that the stimulation with SSc sera disturbed angiogenic performance of HUVECs[16-18], and, as we showed above, circulatory CXCL4 levels were raised in the patients with SSc and correlated with their peripheral vasculopathy. Thereby, we treated HUVECs using recombinant human CXCL4 or SSc sera, with or without CXCL4 neutralized antibody, in order to testify the contribution of SSc sera derived CXCL4 to the angiogenesis of endothelial cell line HUVECs, including viability, migration and tube formation.
Firstly, the addition of CXCL4 or the SSc sera to the medium of HUVECs inhibited endothelial cell proliferation determined using CCK-8 assay, which could be significantly ameliorated by antibody-mediated neutralization (***P < 0.001, Fig. 3A and 3B).
Furthermore, the stimulation with CXCL4 significantly decreased the ability of the tube formation and migration of HUVECs, which were dramatically improved by treating with anti-CXCL4 antibody (*P < 0.05, ***P < 0.001, Fig. 3C and 3D). Similarly, both of tube formation and migration was significantly impaired after challenging with 10% SSc sera, and this inhibitory effects on HUVECs were significantly reversed by pretreatment with an anti-CXCL4 antibody (Fig. 3E and 3F). Therefore, these data revealed the anti-angiogenic effects of SSc derived CXCL4.
As CXCR3 mediated Ca2+ mobilization and chemotaxis in response to C-X-C chemokines, including CXCL4, we also confirmed the expression of CXCR3 in HUVECs (Supplemental Fig. 1A), we thereby wondered if CXCL4 inhibited endothelial cell proliferation via its receptor CXCR3. However, the addition of AMG487, a specific antagonist of CXCR3, did not show to reverse the inhibition of cell viability, tube formation and migration induced by CXCL4 or SSc sera (P > 0.05 against the CXCL4 group or SSc sera group; Supplemental Fig. 1B-G). Therefore, the data showed that CXCL4 exerted its anti-angiogenic effect on HUVECs not through CXCR3.
CXCL4 regulated ET-1, c-Abl/Fli-1 pathway in HUVECs
As ET-1 increased and Fli-1 deficiency participate in SSc peripheral vasculopathy[19-21], we examine whether CXCL4 exerted its anti-angiogenic effect through these mediators. We found that CXCL4 induced the upregulation of ET-1 and downregulation of Fli-1 in a dose-dependent manner at mRNA and protein levels in HUVECs, and these changes were reversed by the pre-treatment of anti-CXCL4 antibody (*P < 0.05, **P < 0.01, ***P < 0.001; Fig. 4 A-D), but not CXCR3 pharmacological inhibition (P > 0.05; Supplemental Fig.1H and 1I).
Since the activation of c-Abl pathway is a negative regulating Fli-1 deficiency[22, 23], we initially looked at the effect of CXCL4 on the c-Abl signaling. As shown in Fig.5A, CXCL4 increased the expression of c-Abl in a dose-dependent manner, and CXCL4 neutralizing antibody inhibited the overexpression of c-Abl induced by CXCL4 (P < 0.001 for each comparison). Next, we showed the time course of c-Abl induction by CXCL4 treatment in HUVECs (P < 0.001 for each comparison; Fig. 5B). As ponatinib[24] could inhibit c-Abl pathway (Fig. 5C), we also investigated that ponatinib normalized the reduced levels of Fli-1(*P < 0.05, **P < 0.01, ***P < 0.001; Fig. 5D and 5E). Collectively, these data suggest that CXCL4 regulates Fli-1 via c-Abl pathway in SSc vasculopathy.
CXCL4 blocked the pro-angiogenic effect of TGF-β and PDGF in HUVECs
Numerous pro-angiogenic mediators like transforming growth factor (TGF)-β and platelet-derived growth factor (PDGF) activated in SSc[25, 26]. Since CXCL4 could bind to growth factors directly to exert its effect, we examine whether TGF-β and PDGF are involved in the progressive vasculopathy of CXCL4 induced. As shown in Fig.6 A and 6B, TGF-β and PDGF significantly induced the proliferation of HUVECs as previously reported[27-30] and the inhibitors against TGF-β or PDGF blocked their pro-angiogenic effect respectively (P < 0.0001 for each comparison). Interestingly, the addition of CXCL4 reduced the cell proliferation of HUVECs induced by TGF-β and PDGF, which were completely reversed by the pre-treatment of the CXCL4 neutralizing antibody. These data suggested that CXCL4 exerted its anti-angiogenic effect by antagonizing TGF-β and PDGF signaling.
CXCL4 was recently recognized to induce macrophage into a specific phenotype as “M4”, which typically express interleukin (IL) -6, S100A8, matrix metalloproteinase (MMP) -7 and tumor necrosis factor (TNF) -α, activate endothelial cells in atherosclerosis[7]. We herein studied whether CXCL4 exerted its anti-angiogenic effect by shifting the phenotype of macrophage to “M4”. Similar to the previous study[11], we induced THP-1 into “M4” macrophage after CXCL4 stimulation (*P < 0.05, **P < 0.01, ***P < 0.001; Supplemental Fig. 2). The Addition of the conditional medium (CM) from “M4” induced by CXCL4 to the cultures of HUVECs did not change the expressions of ET-1 or Fli-1 (P > 0.05; Supplement Fig. 3). Moreover, when co-culturing with this “M4” macrophage (CXCL4-induced), the HUVECs did not show the changed expressions of these genes in HUVECs (P > 0.05; Supplement Fig. 4). Hence, these results suggested that CXCL4 contributed to anti-angiogenesis independent of triggering macrophage to the “M4” phenotype.