A Calcineurin-Independent Mechanism of Angiogenesis Inhibition by a Nonimmunosuppressive Cyclosporin A Analog

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.97). 05/2011; 338(2):466-75. DOI: 10.1124/jpet.111.180851
Source: PubMed


Cyclosporin A (CsA) is a widely used immunosuppressant drug. Its immunosuppressive activity occurs through the inhibition of the protein phosphatase calcineurin via formation of a ternary complex with cyclophilin A (CypA). CsA also inhibits endothelial cell proliferation and angiogenesis. This has been thought to occur through calcineurin inhibition as well. However, CsA is also a potent inhibitor of cyclophilins, a class of prolyl isomerases. Because calcineurin inhibition requires binding, and therefore inhibition of CypA, the relative contributions of calcineurin and cyclophilin inhibition in antiangiogenesis have not been addressed. We have taken a chemical biology approach to explore this question by dissociating the two activities of CsA at the molecular level. We have identified a nonimmunosuppressive analog of CsA that does not inhibit calcineurin but maintains inhibition of endothelial cell proliferation and in vivo angiogenesis. The same analog also maintains inhibition of all cyclophilin isoforms tested. We also show that a second, structurally distinct, cyclophilin inhibitor is sufficient to block endothelial cell proliferation. These results suggest that the inhibition of cyclophilins may play a larger role in the antiangiogenic activity of CsA than previously believed, and that cyclophilins may be potential antiangiogenic drug targets.

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Available from: Woong Sun, Jan 19, 2014
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    • "Whether combining cyclosporine A with cisplatinum can help to overcome PPIA induced chemoresistance in patients with malignant glioma is currently under investigation [31]. In addition, cyclosporin A plays a further potentially useful role as an inhibitor of angiogenesis [30] [31], and non-immunosuppressive analogues of might be safely applied as part of an anti-angiogenetic drug regimen [32]. As in our study, PPIC positive CTCs were more likely in follow-up samples of the platinum resistant than in platinum sensitive patients, we speculate that PPIC positive CTCs represent a subpopulation of circulating tumor cells which " survived " chemotherapy and have a more aggressive potential. "
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    ABSTRACT: Objective: The study aims at identifying novel markers for circulating tumor cells (CTCs) in patients with epithelial ovarian cancer (EOC), and at evaluating their impact on outcome. Methods: Microarray analysis comparing matched EOC tissues and peripheral blood leucocytes (N=35) was performed to identify novel CTC markers. Gene expression of these novel markers and of EpCAM was analyzed using RT-qPCR in blood samples taken from healthy females (N=39) and from EOC patients (N=216) before primary treatment and six months after adjuvant chemotherapy. All samples were enriched by density gradient centrifugation. CTC positivity was defined by over-expression of at least one gene as compared to the healthy control group. Results: CTC were detected in 24.5% of the baseline and 20.4% of the follow-up samples, of which two thirds were identified by overexpression of the cyclophilin C gene (PPIC), and just a few by EpCAM overexpression. The presence of CTCs at baseline correlated with the presence of ascites, sub-optimal debulking, and elevated CA-125 and HE-4 levels, whereas CTC during follow-up occurred more often in older and platinum resistant patients. PPIC positive CTCs during follow-up were significantly more often detected in the platinum resistant than in the platinum sensitive patient group, and indicated poor outcome independent from classical prognostic parameters. Conclusions: Molecular characterization of CTC is superior to a mere CTC enumeration or even be the rationale for CTC diagnostics at all. Ultimately CTC diagnostics may lead to more personalized treatment of EOC, especially in the recurrent situation.
    Gynecologic Oncology 09/2012; 128(1). DOI:10.1016/j.ygyno.2012.09.021 · 3.77 Impact Factor
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    • "The ability of CsA to inhibit endothelial cell proliferation and angiogenesis has also been known for some time, but CsA has not been used as a clinical angiogenesis inhibitor due to its immunosuppressive properties and nephrotoxic side effects at high doses [28], [29]. Interestingly, while the mechanism for immunosuppression by CsA is due to inhibition of the protein phosphatase calcineurin in T cells, the antiangiogenic properties of the drug are independent of calcineurin [30], [31]. "
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    ABSTRACT: Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50) dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.
    PLoS ONE 09/2011; 6(9):e24793. DOI:10.1371/journal.pone.0024793 · 3.23 Impact Factor
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    ABSTRACT: Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.
    PLoS ONE 03/2015; 10(3):e0119279. DOI:10.1371/journal.pone.0119279 · 3.23 Impact Factor
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