Blocking CD248 molecules in perivascular stromal cells of patients with systemic sclerosis strongly inhibits their differentiation toward myofibroblasts and proliferation: a new potential target for antifibrotic therapy

CD248 (also known as endosialin or tumor endothelial marker 1) is a transmembrane receptor whose known ligands are fibronectin and type I/IV collagen. It is widely expressed on mesenchymal cells during embryonic life and is required for proliferation and migration of pericytes and fibroblasts [1]. Although CD248 expression is dramatically reduced during adult life, it may be upregulated during specific conditions such as malignancy, inflammation, and fibrosis [2–4]. It is well known that CD248 is expressed on the surface of cells of mesenchymal origin, including tumor-associated pericytes and activated fibroblasts, which are thought to play a key role in the development of tumor neovascular networks and stromal interaction [1]. The interruption of endosialin function, with antibody blockade or genetic knockouts, negatively affects tumor growth and angiogenesis in numerous cancer types [5–7]. Furthermore, in the experimental model of kidney fibrosis after unilateral ureteral obstruction (UUO), CD248^−/− mice display downregulation of myofibroblast proliferation, thus decreasing the kidney fibrosis [8]. These biologic effects, in cancer and in reparative response, may be related to the ability of CD248 to modulate many signaling pathways involved in both cancer development and tissue repair, including platelet-derived growth factor BB (PDGF-BB), transforming growth factor-β (TGF-β), and Notch receptor protein [9]. Under normal conditions, pericytes that expressed high levels of CD248 were able to proliferate, responding to PDGF-BB stimulation [9], and higher expression of CD248 is required for imparting fibroblast sensitivity to the effects of TGF-β [10].

Owing to its multifunctional activities modulating innate immunity, cell proliferation, and vascular homeostasis [9, 11], CD248 may be considered a potential therapeutic target for several diseases [12], and currently, the results of a first-in-human, open-label, phase I study recruiting patients with extracranial solid tumors who failed standard chemotherapy and were treated with a biologic therapy targeting CD248 have been published, confirming the therapy’s safety and a positive impact on different cancers [13].

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology with multiorgan involvement and heterogeneous clinical manifestations. The hallmark of early SSc is endothelial involvement, whereas later stages are characterized by an excessive accumulation of extracellular matrix (ECM), resulting in extended fibrosis in skin and internal organs [14, 15]. In the last few years, it has been clarified that endothelial cells (ECs) and pericytes, after injury, may differentiate toward myofibroblasts, which are committed to producing increased amounts of collagen [16–18], and this process has been proposed as a key pathogenic mechanism in SSc.

Several polypeptide mediators are involved in fibrosis during SSc, such as TGF-β and PDGF-BB. The latter is a potent pro-proliferative signal for mesenchyme-derived cells, including myofibroblasts [19, 20], whereas TGF-β primarily promotes myofibroblast activation, α-smooth muscle actin (α-SMA) expression, and collagen deposition [16, 21–26]. Interestingly, CD248 modulates both these pathways because of CD248 is required for imparting fibroblast sensitivity to the effects of TGF-β [9] and is crucial for optimal migratory response of activated fibroblasts to PDGF-BB [19].

The goal of this work is to investigate the expression of CD248 in skin perivascular stromal cells from patients with SSc and its ability in mediating pericyte differentiation toward myofibroblasts. Although the role of CD248 in the pathogenesis of SSc has not yet been established, its potential role in controlling vessel regression and fibrosis makes this molecule a potential therapeutic target in a clinical setting, different from cancer, and in which an effective therapeutic approach to prevent fibrosis is still an important unmet need.

This report is the first, to the best of our knowledge, indicating that CD248, first identified as a tumor vascular endothelial antigen [35] and considered a key molecule of myofibroblast generation, is deeply involved in TGF-β and PDGF-BB signaling transduction during the fibrosis associated with SSc and that its inhibition strongly interferes with the profibrotic pathways of these two cytokines. Recently, CD248 has been considered as a marker of stromal fibroblasts, pericyte subsets, and human MSC [36, 37], and in the experimental model of fibrosis, after UUO, a significant upregulation of CD248 was observed [3]. On the contrary, CD248^−/− mice were protected from renal fibrosis and capillary rarefaction, probably inhibiting pericyte differentiation toward α-SMA⁺ interstitial myofibroblasts and preventing vascular instability and collagen production [3]. Furthermore, it has been reported that CD248 expressed on pericytes can promote EC apoptosis, probably impairing the cross-talk between EC integrins and vascular endothelial growth factor (VEGF) receptor 2, leading to the attenuation of VEGF signaling [38]. On these bases, CD248 may be considered a key target in those pathologic processes in which vascular damage and fibrosis are strongly joined in SSc.

In our present study, we chose to include in the patient cohort only patients affected by the Scl70⁺ diffuse form of the disease, because this subset rapidly progresses from vascular damage to fibrosis and may be considered a good “human model” to evaluate the link between vascular damage and fibrosis. In fact, it is well known that anti-Scl70 antibody is one of the typical autoimmune markers in SSc, occurring in 60.8% of cases of diffuse SSc and 23.4% of cases of limited SSc [39]. The presence of anti-Scl70 antibody is associated with severity of the disease, decreased survival [40], and evidence of pulmonary fibrosis [41]. We observed that CD248 was constitutively overexpressed on SSc cells derived from mesenchymal lineage. In SSc skin, we observed that CD248 was expressed on stromal fibroblasts and perivascular cells located in close proximity to the vessel, when compared with healthy skin, and the number of CD248⁺ cells significantly increased over time. Furthermore, in LSS-SSc biopsy, the CD248 mRNA expression of whole biopsy was significantly increased when compared with EOS-SSc. Of note, IF showed that the CD248⁺ cells coexpressed CD90, a molecule highly expressed in all the undifferentiated MSC [42, 43]. Currently, although we do not have a single cell marker capable of defining MSC, available literature suggests that CD90, which is highly expressed in all MSC, regardless of the source, may be considered a good marker to identify undifferentiated MSC [34]. The increased CD248 expression in perivascular cells of patients with SSc highly coexpressing the stem marker CD90 may suggest that in these MSC, both the profibrotic machinery and cells’ differentiation toward myofibroblasts may be activated.

Furthermore, our results showed that increased CD248 expression may be observed also in EC of SSc skin biopsies. It has been reported that CD248 may be expressed by endothelial progenitor cells [44] and tumor EC, together with pericytes and tumor-associated fibroblasts, during active cancer angiogenesis [38, 45]. It has been proposed that CD248 expressed by EC may interact with ECM proteins as well as tumor stromal cells to promote vascular invasion and migration [46]; in fact, CD248 expression was induced in EC when cultured in Matrigel, suggesting that endosialin could be induced in EC exposed to a complex extracellular environment [46]. In our setting, we hypothesize that SSc-EC may express CD248 as a compensatory mechanism to support angiogenesis in the context of a disease characterized by progressive desertification of vascular tree.

We investigated the functional role of CD248 in vitro in the perivascular differentiation toward myofibroblasts by using MSC, a validated surrogate of perivascular cells [26, 47]. Although the possible role of the CD248 molecule in SSc pathogenesis is largely unknown, it has been reported that this molecule is involved in the fibroproliferative process by modulating the PDGF-BB pathway [21] and collaborating with the TGF-β pathway to induce α-SMA expression [48]. In HC cells, TGF-β induced significantly decreased of CD248 when compared with UT cells. On the contrary, SSc-MSC displayed significantly higher CD248 expression than HC cells in both TGF-β-stimulated and unstimulated cultures. Recently, it has been shown that TGF-β strongly suppresses CD248 expression in healthy murine fibroblast cell lines; on the contrary, during cancer, where significantly higher CD248 levels are reported, mirroring what we observed in our cells, TGF-β failed to downregulate CD248 expression [49]. Although TGF-β is not a promoter of CD248 expression, CD248 may collaborate with TGF-β to induce α-SMA, possibly via downregulation of Notch3 [9, 12] and upregulation of interleukin 6 (IL6), C-C motif chemokine ligand 2 (CCL2), TGF-β1, and TGF-βR1 [48]. In fact, it has been reported that Notch3 may prevent the TGF-β1 induction of α-SMA, working as a molecular brake on smooth muscle gene transcription [50]. Furthermore, IL6, CCL2, TGF-β1, and TGF-βR1, activated by CD248, may strongly stimulate α-SMA [51]. Thus, we may hypothesize that CD248 overexpression in SSc-MSC may play its profibrotic role, exacerbating the TGF-β effects [52], by removing the Notch3 control and promoting α-SMA expression by its own downstream mediators. In fact, after silencing CD248, cells were unable to induce α-SMA expression after TGF-β stimulus, thus confirming the key role of CD248 in increasing the TGF-β effects in SSc-MSC.

To date, conflicting results have been reported in available literature concerning the expression of different smooth muscle genes during fibroblast proliferation and activation. Transcript levels of Sm22 are elevated in fibroblasts derived from mice lacking the cytoplasmic domain of CD248 [12], and CD248 knockdown did not affect the in vitro expression of α-SMA in normal human lung fibroblast [31]. On the contrary, in in vitro cultured human pericytes, the ability of TGF-β to induce α-SMA expression was lost in the CD248-specific siRNA-transfected primary human brain vascular pericytes, suggesting that α-SMA induction is CD248-dependent [53]. Furthermore, in an experimental model of liver fibrosis, the total liver mRNA of collagen and α-SMA was reduced in CD248-deficient mice compared with wild-type mice [19], and in a model of renal fibrosis, no increase of α-SMA molecule was observed in CD248-deficient mice [8]. These discrepancies may be partially explained by the different experimental models, the differences in cell manipulation, the intensity and quality of stimuli, and the different levels of efficacy in CD248 silencing.

To assess the contribution of CD248 in modulating the proliferative ability of SSc-MSC, we evaluated the expression of Ki-67 gene expression, which strongly correlates with cellular proliferation [19]. PDGF-BB induced a significant increase of Ki-67 levels when compared with UT cells in both HC- and SSc-MSC.

The role of CD248 in PDGF-BB signaling has been studied in human pericytes and hepatic stellate cells (HSC), commonly considered the precursors of septal myofibroblasts in liver fibrosis. PDGF-BB currently is considered one of the most potent HSC mitogens known, and it plays a key regulatory role in HSC proliferation during hepatic fibrogenesis [19]. It has been proposed that CD248 regulates the PDGF-BB pathway, thus controlling cell proliferation [54].

After CD248 silencing, we observed a strong inhibition of PDGF-BB-mediated cell proliferation, suggesting that this process is under CD248 control, thus decreasing the myofibroblast accumulation and confirming previous reports [23] in which primary renal fibroblasts isolated from CD248-knockout mice displays both decreased cell proliferation and reduced collagen secretion when compared with wild-type mice. Currently, it is still unclear how CD248 may modulate this proliferative signal: via the matrix-binding properties of CD248 and/or by the well-known ability of this molecule to potentiate PDGF-BB signaling. Recent studies in hepatic fibrosis, however, have provided evidence that HSC CD248^−/− display normal levels of PDGF receptors, suggesting that the antiproliferative effect of CD248^−/− HSCs is not mediated through the modulation of PDGF receptor expression [19].

Our study shows that SSc perivascular cells overexpress CD248, which is involved in SSc pericyte transition toward myofibroblasts, and CD248 silencing may prevent pericyte-to-myofibroblast transition, proliferation, vascular instability, and tissue fibrosis. Taken together, our data suggest that targeting CD248 expression may be considered a potential target in order to block tissue fibrosis and vascular desertification during SSc.