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Published in final edited form as: J Invest Dermatol. 2018 Nov 30;139(5):1150–1160. doi: 10.1016/j.jid.2018.11.016

Cyclophosphamide Pulse Therapy Normalizes Vascular Abnormalities in a Mouse Model of Systemic Sclerosis Vasculopathy

Takashi Yamashita 1, Yoshihide Asano 1, Ryosuke Saigusa 1, Takashi Taniguchi 1, Megumi Hirabayashi 1, Takuya Miyagawa 1, Kouki Nakamura 1, Shunsuke Miura 1, Ayumi Yoshizaki 1, Maria Trojanowska 2, Shinichi Sato 1
PMCID: PMC6604062  NIHMSID: NIHMS1030468  PMID: 30508546

Abstract

Intravenous cyclophosphamide pulse, a standard treatment for systemic sclerosis (SSc)-related interstitial lung disease, elicits a disease-modifying effect on SSc vasculopathy, such as fostering microvascular de-remodeling. To investigate the molecular mechanism by which cyclophosphamide mitigates SSc vasculopathy, we employed endothelial cell–specific Fli1 knockout mice that mimic the functional and structural vascular abnormalities characteristic of SSc. Biweekly cyclophosphamide injection improved vascular permeability and structural abnormalities of endothelial cell–specific Fli1 knockout mice in 2 weeks and in 3 months, respectively. In endothelial cell–specific Fli1 knockout mice, a single dose of cyclophosphamide was sufficient to normalize the decreased expression of α–smooth muscle actin in dermal blood vessels and improve the impaired neovascularization in skin-embedded Matrigel plug. Under the same condition, the decreased expression of vascular endothelial cadherin, platelet-derived growth factor B, S1P1, and CCN1 (molecules associated with angiogenesis and/or vasculogenesis) was reversed along with the reversal of endothelial Fli1 expression. In SSc patients, serum CCN1 levels were significantly increased after intravenous cyclophosphamide pulse. Taken together, these results indicate that cyclophosphamide improves Fli1 deficiency-dependent vascular changes by normalizing the expression of angiogenesis- and vasculogenesis-related molecules and endothelial Fli1, which may help to explain the beneficial effect of cyclophosphamide on SSc vasculopathy.

INTRODUCTION

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterized by extensive tissue fibrosis following aberrant autoimmune inflammation and vasculopathy. So far, SSc pathogenesis remains elusive and no curative treatment has been established (Asano and Sato, 2015; Denton, 2015). Currently, interstitial lung disease (ILD) is a leading cause of death, as well as pulmonary arterial hypertension (Steen and Medsger, 2007), and the combination of intravenous cyclophosphamide pulse (IVCY) with systemic corticosteroid is a standard treatment for SSc-ILD (Kowal-Bielecka et al., 2009). Although its efficacy is limited and variable in individual cases, cyclophosphamide exerts a variety of disease-modifying effects on SSc pathology (Borghini et al., 2015; Caramaschi et al., 2009; Furuya et al., 2010; Masui et al., 2013; Takahashi et al., 2012, 2013). Therefore, it would be helpful for the better understanding of SSc pathogenesis to elucidate the mechanism by which cyclophosphamide elicits a disease-modifying effect on SSc.

It is widely accepted that vascular injury is an early pathological event in SSc-ILD preceding the activation of interstitial lung fibroblasts (Castillo-Tandazo et al., 2013). Indeed, the proliferation of capillary endothelial cells is an initial pathologic change (Beon et al., 2004), reflecting aberrant vascular activation in response to injury caused by immune attacks of anti–endothelial cell antibodies, natural killer cells, and γδT cells (Asano and Sato, 2015; Castillo-Tandazo et al., 2013). During this process, endothelial-to-mesenchymal transition occurs (Mendoza et al., 2016), providing interstitial lung fibroblasts producing excessive amount of extracellular matrix. Also, endothelial cell apoptosis becomes evident, leading to the decrease in microvessels and the subsequent interstitial fibrosis due to tissue hypoxia and apoptotic cell–related responses (Beon et al., 2004). Correspondingly, the presence of anti–endothelial cell antibodies is closely associated with the development of SSc-ILD (Mihai and Tervaert, 2010). Cyclophosphamide, a cytotoxic DNA alkylating agent widely used as an immunosuppressant and anti-tumor drug, is basically applied to SSc-ILD to suppress autoimmune inflammation, but also likely exerts its effect on SSc-ILD by promoting vascular repair through the induction of circulating endothelial progenitor cells from bone marrow and the reduction of circulating anti-angiogenic factors (Borghini et al., 2015; Furuya et al., 2010). Consistent with this idea, cyclophosphamide improves nailfold capillary changes from active patterns to early patterns in SSc (Caramaschi et al., 2009). Thus, cyclophosphamide potentially has pleiotropic effects on SSc vasculopathy, but the underlying molecular mechanism has not been fully investigated.

Fli1 is a member of the Ets transcription factor family, which is downregulated in a variety of cells, such as dermal fibroblasts, endothelial cells, epithelial cells, and perivascular inflammatory cells, in lesional and non-lesional skin of SSc patients (Kubo et al., 2003; Takahashi et al., 2017a). Notably, Fli1 is epigenetically suppressed in SSc skin specimens and SSc dermal fibroblasts (Wang et al., 2006), suggesting that Fli1 is a predisposing factor of this disease. Supporting a critical role of Fli1 deficiency in SSc pathogenesis, endothelial cell–specific Fli1 knockout (Fli1 ECKO) mice recapitulate the vascular disintegrity characteristic of SSc vasculopathy, resulting in the development of arteriolar stenosis, capillary dilation, and increased vascular permeability (Asano et al., 2010). Importantly, Fli1 ECKO mice lack tissue fibrosis, autoimmunity, and inflammation, allowing us to investigate the vascular aspect of SSc without the influence of its fibrotic and autoimmune/inflammatory aspects. Indeed, we previously demonstrated that bosentan elicits its disease-modifying effect on SSc vasculopathy by reversing the expression and DNA binding ability of Fli1 in a series of studies with Fli1 ECKO mice and SSc skin samples (Akamata et al., 2015; Saigusa et al., 2016). Thus, this animal model is quite useful to elucidate a molecular mechanism of drugs acting on SSc vasculopathy. Therefore, we investigated the effect of cyclophosphamide on vascular features of Fli1 ECKO mice.

RESULTS

Cyclophosphamide improves vascular permeability of Fli1 ECKO mice

Initially, we evaluated vascular permeability by Evan Blue dye injection in mice receiving cyclophosphamide. Because vascular permeability is regulated primarily by endothelial cell–cell interaction and endothelial cell–pericyte/vascular smooth muscle cell interaction (Asano et al., 2010), it was expected that cyclophosphamide would modulate vascular permeability in a short time period. Therefore, we examined vascular leakage 2 weeks after cyclophosphamide treatment. In control groups treated with phosphate buffered saline (PBS), consistent with previous reports (Akamata et al., 2015; Asano et al., 2010), vascular leakage was prominently increased in Fli1 ECKO mice compared with control littermates (upper panels of Figure 1a). Upon administration of cyclophosphamide, vascular leakage was markedly decreased in Fli1 ECKO mice, while not altered in control littermates (lower panels of Figure 1a). These findings were confirmed by formamide-dependent extraction of Evans blue dye from macroscopically normal skin (Figure 1b). We also assessed vascular structure of the back skin by FITC-dextran injection. As reported previously (Akamata et al., 2015; Asano et al., 2010; Noda et al., 2014), Fli1 ECKO mice exhibited vascular structural abnormalities, such as arteriolar stenosis, capillary dilation, and mild distortion 2 weeks after PBS or cyclophosphamide injection (data not shown). However, six courses of biweekly cyclophosphamide injection improved vascular structural abnormalities in Fli1 ECKO mice (middle and right panels of Figure 1c) without affecting the organized dermal vascular structure in control littermates (left panels of Figure 1c). Taken together, these results indicate that cyclophosphamide rapidly improves vascular leakiness and gradually improves disorganized vascular structure in Fli1 ECKO mice.

Figure 1. The effect of CY on vascular permeability and structural abnormalities of Fli1 ECKO mice.

Figure 1.

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(a) Fli1 ECKO mice (right panels) and control litter mates (left panels) were administered a single dose of CY or PBS. Two weeks later, vascular permeability was evaluated by Evans blue dye injection (n = 6–7 per group). (b) Evans blue dye was extracted from the macroscopically normal skin by formamide. Extracted Evans blue dye was assessed by spectrophotometer. The results are summarized as a graph (n = 6–7 per group). (c) Fli1 ECKO mice (middle panels) and control litter mates (left panels) were administered six courses of CY biweekly. Two weeks after the final administration, vascular structure was visualized by FITC-dextran injection (n = 5 per group). A right upper panel depicted arteriolar stenosis (shown with arrows), which corresponds to the area surrounded with a dotted square. AU, arbitrary unit; CY, cyclophosphamide; Fli1 ECKO, endothelial cell-specific Fli1 knockout; PBS, phosphate buffered saline. Scale bars = 1 cm for (a) and 100 μm for (c).*P < 0.01 versus control-PBS group. #P < 0.01 versus Fli1 ECKO-PBS group.

Cyclophosphamide increases the expression levels of key molecules regulating vascular integrity in Fli1 ECKO mice

To elucidate the molecular mechanism underlying the efficacy of cyclophosphamide in improving dermal vasculature of Fli1 ECKO mice, we assessed the expression of key molecules regulating vascular integrity, such as vascular endothelial cadherin, platelet endothelial cell adhesion molecule-1, S1P1, and platelet-derived growth factor-B, which are downregulated in endothelial cells of Fli1 ECKO mice (Asano et al., 2010). Consistent with our previous findings, Cdh5, S1p1, and Pdgfb mRNAs were significantly decreased, while Pecam1 was moderately decreased in the skin of Fli1 ECKO mice compared with that of control littermates. Notably, cyclophosphamide enhanced mRNAs of these molecules in Fli1 ECKO mice to levels comparable with those in control littermates with or without cyclophosphamide treatment (Figure 2a), with the most pronounced increase in Pdgfb mRNA expression compared with baseline. These results indicate that cyclophosphamide broadly reverses the expression of vascular integrity regulating molecules in Fli1 ECKO mice, leading to the normalization of vascular permeability and structure. This notion was further confirmed by the normalization of vascular α–smooth muscle actin expression, a marker of pericytes with a contractile phenotype (Beamish et al., 2010), by cyclophosphamide injection in Fli1 ECKO mice (Figure 2b).

Figure 2. The effect of CY on vascular disintegrity of Fli1 ECKO mice.

Figure 2.

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(a) Fli1 ECKO mice and control littermates were injected with a single dose of CYor PBS. Two weeks later, skin samples were obtained from back skin. mRNA levels of vascular stability-related molecules were determined by quantitative reverse transcriptase PCR (n = 4–6 per group). (b) Under the same condition, the expression of α–smooth muscle actin in blood vessels was assessed by immunohistochemistry (n = 5 per group). Left upper insets of images depicted representative dermal small vessels shown with dotted squares. (c, d) Fli1 expression was determined at mRNA levels by quantitative reverse transcriptase PCR (n = 4–6 per group), (c) and at protein levels by immunohistochemistry (n = 5 per group), (d) in the same samples used for the above experiments. Strongly stained nuclei were indicated by solid line arrows and weakly stained nuclei were indicated by dotted line arrows. Representative photos are shown for each experiment. Each graph indicates mean ± standard error of the mean of the indicated parameters. AU, arbitrary unit; CY, cyclophosphamide; Fli1 ECKO, endothelial cell-specific Fli1 knockout; PBS, phosphate buffered saline. Scale bars = 50 μm for (b) and 10 μm for (d). *P < 0.05 versus control-PBS group. #P < 0.05 versus Fli1 ECKO-PBS group.

We also assessed the impact of cyclophosphamide on endothelial Fli1 expression in the skin of Fli1 ECKO mice. Of note, cyclophosphamide increased Fli1 mRNA expression in Fli1 ECKO mice to levels comparable with those in control littermates (Figure 2c). Furthermore, Fli1 protein expression was also elevated in endothelial cells of Fli1 ECKO mice treated with cyclophosphamide (Figure 2d). These results indicate that the normalization of endothelial Fli1 deficiency may account for the improvement of vasculopathy by cyclophosphamide in Fli1 ECKO mice.

Cyclophosphamide accelerates vessel maturation in Fli1 ECKO mice

Previous reports demonstrated that an increase in circulating endothelial progenitors correlates with the clinical efficacy of IVCY for SSc-ILD, suggesting that IVCY may also modulate vasculogenesis in SSc-ILD patients responsive to this treatment (Furuya et al., 2010). Therefore, we next examined the impact of cyclophosphamide on neovascularization by in vivo Matrigel plug assay, a well-established method to evaluate angiogenesis and vasculogenesis (Tigges et al., 2008). When the Matrigel plugs were evaluated on day 7 after the pretreatment of PBS, there was no difference in the area of vascular lumens between Fli1 ECKO mice and control littermates. However, the pretreatment with cyclophosphamide significantly increased the area of vascular lumens in both strains to a similar extent (Figure 3a). In contrast, a clear difference between Fli1 ECKO mice and control littermates was seen in immunohistochemistry for α–smooth muscle actin. The signal intensity of α–smooth muscle actin in the Matrigel plugs was significantly decreased in Fli1 ECKO mice compared with control littermates when injected with PBS, while being comparable in these two strains after a single dose of cyclophosphamide (Figure 3b). Because an abundant expression of α–smooth muscle actin is a hallmark of mature blood vessels, the formation of functional blood vessels was confirmed by double immunofluorescence for CD31 and CD235a, markers for endothelial cells and erythrocytes respectively, demonstrating that the number of erythrocytecontaining blood vessels became much more evident in Fli1 ECKO mice treated with cyclophosphamide compared with those treated with PBS (Figure 4). These results indicate that cyclophosphamide improves impaired angiogenesis and vasculogenesis in Fli1 ECKO mice.

Figure 3. The effect of CY on neovascularization in skin-embedded Matrigel plug.

Figure 3.

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In vivo Matrigel plug assay was carried out with Fli1 ECKO mice and control littermates treated with a single dose of CY or PBS. Newly formed blood vessels were evaluated by hematoxylin and eosin staining (a) and by immunohistochemistry for α–smooth muscle actin (b). Representative images are shown (n = 5 per group). AU, arbitrary unit; CY, cyclophosphamide; Fli1 ECKO, endothelial cell-specific Fli1 knockout; PBS, phosphate buffered saline. Scale bars = 500 μm for (a) and 200 μm for (b). Ratio of vascular area and the signal intensity of α–smooth muscle actin were analyzed by ImageJ (National Institutes of Health, Bethesda, MD) and then summarized as graphs. Each graph indicates mean ± standard error of the mean of the indicated parameters. *P < 0.01 versus control-PBS group. #P < 0.01 versus Fli1 ECKO-PBS group.

Figure 4. The effect of CY on the formation of erythrocyte-containing functional vessels in skin-embedded Matrigel plug.

Figure 4.

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In vivo Matrigel plug assay was carried out with Fli1 ECKO mice and control littermates treated with a single dose of CY or PBS. Double immunofluorescence for CD31 and CD235a, markers for endothelial cells and erythrocytes respectively, was performed. Newly formed blood vessels containing erythrocytes were shown with arrows. Sequential sections were used for hematoxylin and eosin staining and immunofluorescence in each group. CY, cyclophosphamide; Fli1 ECKO, endothelial cell-specific Fli1 knockout; PBS, phosphate buffered saline. Scale bar = 100 μm.

Cyclophosphamide normalizes CCN1 expression in Fli1 ECKO mice

We next assessed the effect of cyclophosphamide on the expression levels of key molecules regulating angiogenesis and vasculogenesis. First, we assessed the expression of vascular endothelial growth factor–A and stromal cell derived factor–1 (also known as CXCL12), which promote the mobilization of endothelial progenitors from the bone marrow into circulation (Petit et al., 2007). As shown in Figure 5a, Vegfa and Sdf1 mRNA levels in the skin were comparable between PBS-treated Fli1 ECKO mice and control littermates. After cyclophosphamide injection, mRNA levels of Vegfa and Sdf1 did not show any statistically significant increase in any of the mouse strains. Therefore, endothelial Fli1 deficiency does not affect the expression of soluble factors related to the induction of endothelial progenitor cells.

Figure 5. The effect of CY on the expression of vasculogenesis-related molecules in Fli1 ECKO mice.

Figure 5.

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(a) Fli1 ECKO mice and control littermates were injected with a single dose of CY or PBS. Two weeks later, mRNA levels of the Vegfa, Sdf1, and Ccn1 genes were determined in the back skin by quantitative reverse transcriptase PCR (n = 4– per group). (b) Under the same condition, CCN1 expression was evaluated by immunohistochemistry in the back skin of these mice (n = 5 per group). Representative photos are shown for each experiment. Left upper panels of images depicted representative dermal small vessels shown with dotted squares. AU, arbitrary unit; CY, cyclophosphamide; Fli1 ECKO, endothelial cell-specific Fli1 knockout; PBS, phosphate buffered saline. Scale bar = 50 μm.*P < 0.05 versus control-PBS group. #P < 0.05 versus Fli1 ECKO-PBS group.

We next focused on the expression levels of CCN1. CCN1 binds as a soluble factor to circulating endothelial progenitors through integrin αVβ3 and αMβ2, which aids in their attachment and transmigration into the area of neovascularization (Grote et al., 2007). Furthermore, CCN1 induces the release of various chemokines, cytokines, growth factors, and proteolytic enzymes from circulating endothelial progenitors, resulting in the promotion of vascular repair and angiogenesis (Grote et al., 2007). Because CCN1 is a direct target of Fli1 and is downregulated in endothelial cells of Fli1 ECKO mice (Saigusa et al., 2015), we examined the effect of cyclophosphamide on the expression levels of CCN1 in the skin of Fli1 ECKO mice. As shown in Figure 5a, cyclophosphamide injection reversed the decreased Ccn1 mRNA levels in Fli1 ECKO mice, whereas no effect was seen in control littermates. Furthermore, cyclophosphamide increased CCN1 protein expression in dermal small vessels of Fli1 ECKO mice (Figure 5b). These results suggest that CCN1 upregulation may contribute to the therapeutic effect of cyclophosphamide on vasculopathy of Fli1 ECKO mice by improving the recruitment of circulating endothelial progenitors to injured blood vessels (vasculogenesis) and promoting the subsequent vascular repair and angiogenesis.

IVCY significantly increases serum CCN1 levels in SSc-ILD patients

We previously reported that CCN1 expression is decreased in SSc dermal microvascular endothelial cells due to the Fli1 deficiency, and that serum CCN1 levels are decreased in SSc patients with severe vasculopathy (Saigusa et al., 2015). Therefore, we examined the effect of IVCY on serum CCN1 levels in SSc patients. As shown in Figure 6, IVCY treatment significantly increased serum CCN1 levels in SSc-ILD patients. These results suggest that the increased expression of endothelial CCN1 is involved in the molecular mechanism by which IVCY treatment improves vasculopathy in SSc patients.

Figure 6. Serum CCN1 levels in systemic sclerosis patients with interstitial lung disease treated with intravenous cyclophosphamide pulse.

Figure 6.

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In 12 patients treated with three to six courses of intravenous cyclophosphamide pulse, serum CCN1 levels were measured by ELISA before and after the treatment. Statistical analysis was carried out with Wilcoxon matched-pairs signed-rank test. IVCY, intravenous cyclophosphamide.

DISCUSSION

This study was undertaken to clarify a part of molecular mechanisms underlying a disease-modifying effect of cyclophosphamide on SSc vasculopathy through a series of experiments with Fli1 ECKO mice. In addition to loss of endothelial integrity similar to SSc vasculopathy (Asano et al., 2010), Fli1 ECKO mice exhibited impaired neovascularization in in vivo Matrigel plug assay, indicating the contribution of disturbed angiogenesis and vasculogenesis to the vascular phenotype of this animal model. Of note, a single dose of cyclophosphamide was sufficient to normalize vascular disintegrity and impaired vessel maturation in Fli1 ECKO mice, followed by reorganization of vascular structure after the six courses of biweekly cyclophosphamide injection. Importantly, Fli1 expression was reversed in Fli1 ECKO mice treated with cyclophosphamide along with the restoration of its targets, including vascular endothelial cadherin, platelet endothelial cell adhesion molecule–1, plateletderived growth factor–B, and CCN1 expression. Therefore, the increase in endothelial Fli1 expression at least partially accounts for the mechanism underlying the effect of cyclophosphamide on vasculopathy of Fli1 ECKO mice. Given that Fli1 deficiency reproduces many molecular features of SSc vasculature (Asano et al., 2010; Ichimura et al., 2014; Noda et al., 2012, 2013; Romano et al., 2016; Saigusa et al., 2016; Takahashi et al., 2015, 2016, 2017a, 2017b; Taniguchi et al., 2017a, 2017b; Toyama et al., 2017; van Bon et al., 2014; Yamashita et al., 2016, 2018), these findings in mice may be partly applicable to human SSc. This is supported by the finding that IVCY treatment increased serum CCN1 levels in SSc patients.

Although the specific mechanistic details of SSc vasculopathy are still lacking, it is generally accepted that impaired vascular remodeling following vascular injury plays a central role in the development of vascular abnormalities characteristic of SSc (Asano and Sato, 2015). Vascular remodeling consists of two distinct processes, such as angiogenesis and vasculogenesis. Accumulating evidence in clinical and animal studies suggests that both processes are impaired in SSc (Kuwana and Okazaki, 2014; Kuwana et al., 2004). In Fli1 ECKO mice, inherent dysregulation of angiogenesis-related gene program has been reported previously (Asano et al., 2010). In addition, our current study demonstrated impaired neovascularization in in vivo Matrigel plug assay accompanied by a decreased expression of endothelial CCN1, a key molecule regulating the recruitment of circulating endothelial progenitor cells (vasculogenesis) (Saigusa et al., 2015). Given the structural and molecular similarity of vascular changes in Fli1 ECKO mice and human SSc (Asano et al., 2010), this animal model is well suited to assess the efficacy of drugs in treating SSc vasculopathy.

Cyclophosphamide is widely used in clinical settings as immunosuppressant and anti-tumor agent against autoimmune diseases and lymphoproliferative disorders, respectively. The mechanism underlying its immune suppressive effect has been elucidated in animal models of systemic lupus erythematosus, Sjögren’s syndrome, and autoimmune uveitis (Archer et al., 1989; Caspi et al., 1990; Jonsson et al., 1988). On the other hand, its immunomodulatory effect has been examined in the context of cancer immune therapy and graft-versus-host disease. In murine models of cancer vaccination and adoptive transfer therapy, cyclophosphamide depletes and inhibits the suppressive function of regulatory T cells, increases the production of type I IFN, along with the induction of granulocyte-macrophage colony-stimulating factor, IL-1β, IL-2, IL-7, IL-15, IL-21, and IFN-γ, as well as activates cells of innate immune system, in particular dendritic cells (Bracci et al., 2007; Limpens et al., 1991; Lutsiak et al., 2005; Schiavoni et al., 2000). All or some of these effects potentially enhance the efficacy of cancer immune therapy. In a murine model of graft-versus-host disease, cyclophosphamide increases the number of regulatory T cells, resulting in the prevention of disease development or the amelioration of disease manifestation (Wang et al., 2017). Thus, the effect of cyclophosphamide has been well studied in immune-mediated disease conditions, while not at all so far in terms of non-immune aspects of disease pathology.

One of the indications of a potential disease-modifying effect of cyclophosphamide on SSc vasculopathy was our previous study reporting the normalization of elevated circulating angiopoietin-2 levels, a useful marker of SSc disease activity, in SSc-ILD patients successfully treated with IVCY (Takahashi et al., 2013). As endothelial cells are the main producers of angiopoietin-2, a plausible hypothesis is that the activation of pro-angiogenic gene program is suppressed by IVCY in SSc patients. This notion is further reinforced by our current and previous data revealing the normalization of elevated serum levels of LL-37 and CCN1, both of which are neovascularization-associated factors overexpressed in SSc endothelial cells, after IVCY infusions in SSc-ILD patients (Takahashi et al., 2017b). Also, in Fli1 ECKO mice, cyclophosphamide injection modified the expression of angiogenesis-related genes toward the stabilization of vasculature, resulting in the normalization of vascular permeability. Further evidence supporting the beneficial role of cyclophosphamide in SSc vasculopathy was the observation of improved morphology of nailfold capillaries in SSc patients treated with IVCY or oral cyclophosphamide. Of note, in the same study, patients treated with corticosteroids and other immunosuppressants, such as cyclosporin and azathioprine, did not show improvement of nailfold capillary changes (Caramaschi et al., 2009). This latter work suggests that cyclophosphamide exerts its disease-modifying effect on SSc vasculopathy through mechanisms other than immune suppression. Our current data strongly support this notion because cyclophosphamide improved vascular changes of a non-inflammatory model of SSc vasculopathy, Fli1 ECKO mice. Therefore, cyclophosphamide improves SSc vasculopathy directly acting on endothelial cells, as well as acting on immune cells and inducing endothelial progenitor cells. The present findings provide us with a potential molecular mechanism explaining the clinical effect of cyclophosphamide on SSc vasculopathy.

There are some limitations in this study. First of all, even though impaired vasculogenesis was suggested by in vivo Matrigel plug assay and endothelial CCN1 downregulation in Fli1 ECKO, the number of circulating endothelial progenitors and their vascular forming ability were not investigated due to the difficulty in obtaining endothelial progenitors enough to conduct these experiments. Second, it is unclear whether the property of endothelial progenitors is affected directly by cyclophosphamide or indirectly through its effect on hematopoietic stem cell niche. Lastly, the biological effect of cyclophosphamide is potentially different between mice and humans. To minimize the last issue, mice were injected with 5 mg of cyclophosphamide corresponding to the dose of 575 mg/m2 in human (average body weight of 8-week-old mice is 25 g; divided by 12.3 to obtain human equivalent dose; 170 cm and 60 kg [body surface area is 1.7 m2] for average SSc patients), which is within the rage of cyclophosphamide dose used for the treatment of SSc-ILD (400–1,000 mg/m2 per infusion), but further studies are required to clarify all of these issues in the future.

In summary, we demonstrated the effect of cyclophosphamide on vasculopathy of animal model mimicking SSc vasculopathy. A broad effect of cyclophosphamide on angiogenesis- and vasculogenesis-related molecules may explain a prominent disease-modifying effect of cyclophosphamide on SSc relative to corticosteroids and other immune suppressants. Although new therapies are emerging for SSc, the investigation of molecular mechanisms of conventional therapies provides insights to the pathological process of this complicated disorder.

MATERIALS AND METHODS

Methods

This study was performed according to the Declaration of Helsinki and approved by the ethical committee of The University of Tokyo Graduate School of Medicine. Written informed consent was obtained from SSc patients. Fli1 ECKO mice (8 weeks old) were intraperitoneally injected with cyclophosphamide (5 mg) or PBS biweekly. All animal protocols were approved by the Animal Care and Use Committee of University of Tokyo. Quantitative reverse transcription PCR, immunohistochemistry, immunofluorescence, vascular permeability assay, visualization of subcutaneous vascular network, and in vivo Matrigel plug assay are described in Supplementary Materials and Methods online.

Patients

Twelve SSc patients with ILD were included in this study. All patients underwent monthly IVCY therapy six to eight times together with oral intake of 15–25 mg/d prednisolone. Serum samples were obtained at the initial infusion and 1 month after the last infusion. The measurement of serum CCN1 were conducted as described previously (Saigusa et al., 2015).

Statistical analysis

Statistical analysis was carried out with one-way analysis of variance followed by Turkey post-hoc test for multiple comparison, with Mann–Whitney U test to compare the distributions of two unmatched groups and with Wilcoxon matched-pairs signed rank test for the comparison before and after treatment. Statistical significance was defined as a P < 0.05.

Supplementary Material

mmc1

ACKNOWLEDGMENTS

We thank T. Kaga, N. Toda, Y. Hasegawa, S. Itakura, and N. Watanabe for tissue processing and staining and technical assistance. This work was supported by a grant from Shimabara Science Promotion Foundation to Yoshihide Asano. Maria Trojwnowska was supported by the National Institutes of Health grant AR042334.

Abbreviations

Fli1 ECKO

endothelial cell-specific Fli1 knockout

ILD

interstitial lung disease

IVCY

intravenous cyclophosphamide pulse

PBS

phosphate buffered saline

SSc

systemic sclerosis

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2018.11.016.

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