SummaryLipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS-and cytokine-related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA-7, on the LPS-mediated release of tumour necrosis factor-a, interleukin-1b and interleukin-6 from THP-1 macrophages and the pathology of dextran sulphate sodium (DSS)-induced colitis in mice. SMA-7 suppressed the LPS-induced cytokine release and nuclear factorjB activation. LPS stimulation caused a four-fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 lM SMA-7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT-29 cells had significantly decreased cell viability when incubated with media from LPS-stimulated THP-1 macrophages. However, incubating the colon cells in media from both SMA-7 and LPStreated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS-stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA-7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS-induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease.

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Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor-2 (VEGFR-2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR-2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of K K,K K-di£uoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their e¡ects on VEGF-induced angiogenesis. DFPMPA-3, the most e¡ective inhibitor of human PTP-1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more e¡ectively than any other DFPMPAs. The inhibitor promoted the VEGF-induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR-2. Its e¡ectiveness was proven through neo-vascularization in mice. The present ¢ndings suggest that targeting PTP to promote therapeutic neo-vascularization may be a potential strategy. ß