Roger A. Sabbadini

Predicting obstructive coronary artery disease with serum sphingosine-1-phosphate

BACKGROUND Sphingolipids are emerging as important signaling molecules that may be produced by cardiac tissue during ischemic stress or as a consequence of inflammation. Because both inflammation and myocardial ischemia are associated with coronary artery disease (CAD), a study was designed to test the ability of serum sphingolipids to predict obstructive CAD. METHODS The study consisted of 308 consecutive patients undergoing coronary angiography for all indications. The primary data points were the assessment of coronary artery stenosis with angiography and the measurements of serum sphingolipids. RESULTS In this diverse population, serum sphingosine-1-phosphate (S1P) was a significant predictor of CAD (P <.001). Multivariate analysis with logistic regression demonstrated that serum S1P was more predictive of obstructive CAD (odds ratio = 7.61) than the traditional risk factors (age, sex, family history of CAD, diabetes mellitus, lipid profile, hypertension, etc.). A 3-variable S1PC composite score was derived by combining the power of the S1P marker with the 2 most important risk factors, age and sex. The relationship between the S1PC and CAD scores was continuous and progressive, such that patients with elevated S1PC scores had higher occurrences of obstructive CAD. S1PC was also predictive of disease severity; 53.2% of patients in the fourth S1PC quartile had 2 to 3 vessel CAD, whereas only 5.2% of patients in the first S1PC quartile had 2 to 3 vessel disease (RR = 10.2 for severity). CONCLUSIONS Serum S1P is a remarkably strong and robust predictor of both the occurrence and severity of coronary stenosis. An S1P-based composite score may be useful as a novel, non-invasive indicator of obstructive CAD.

TNFα receptor expression in rat cardiac myocytes: TNFα inhibition of L-type Ca2+ current and Ca2+ transients

Tumor necrosis factor‐α (TNFα) is a potentially powerful anti‐neoplastic agent; however, its therapeutic usefulness is limited by its cardiotoxic and negative inotropic effects. Accordingly, studies were undertaken to gain a better understanding of the mechanisms of TNFα‐mediated cardiodepression. Single cell RT‐PCR, [125I]TNFα ligand binding and Western immunoblotting experiments demonstrated that rat cardiac cells predominantly express type I TNFα receptors (TNFRI or p60). TNFα inhibited cardiac L‐type Ca2+ channel current (I Ca) and contractile Ca2+ transients. Thus, it is possible that the negative inotropic effects of TNFα are the result of TNFRI‐mediated blockade of cardiac excitation‐contraction coupling.

Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a “molecular sponge” to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.

Sphingosine-1-phosphate antibodies as potential agents in the treatment of cancer and age-related macular degeneration

Sphingosine‐1‐phosphate (S1P) is a pleiotropic bioactive lipid thought to be dysregulated in a variety of disease conditions. In this review, we discuss the roles of S1P in cancer and in wet age‐related macular degeneration. We also explore potential treatment strategies for these disorders, including the utility of anti‐S1P antibodies acting as molecular sponges to neutralize dysregulated S1P in relevant tissues.

Anti-sphingosine-1-phosphate monoclonal antibodies inhibit angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal neovascularization

The efficacy of novel monoclonal antibodies that neutralize the pro-angiogenic mediator, sphingosine-1-phosphate (S1P), were tested using in vitro and in vivo angiogenesis models, including choroidal neovascularization (CNV) induced by laser disruption of Bruchs membrane. S1P receptor levels in human brain choroid plexus endothelial cells (CPEC), human lung microvascular endothelial cells, human retinal vascular endothelial cells, and circulating endothelial progenitor cells were examined by semi-quantitative PCR. The ability of murine or humanized anti-S1P monoclonal antibodies (mAbs) to inhibit S1P-mediated microvessel tube formation by CPEC on Matrigel was evaluated and capillary density in subcutaneous growth factor-loaded Matrigel plugs was determined following anti-S1P treatment. S1P promoted in vitro capillary tube formation in CPEC consistent with the presence of cognate S1P(1-5) receptor expression by these cells and the S1P antibody induced a dose-dependent reduction in microvessel tube formation. In a murine model of laser-induced rupture of Bruchs membrane, S1P was detected in posterior cups of mice receiving laser injury, but not in uninjured controls. Intravitreous injection of anti-S1P mAbs dramatically inhibited CNV formation and sub-retinal collagen deposition in all treatment groups (p<0.05 compared to controls), thereby identifying S1P as a previously unrecognized mediator of angiogenesis and subretinal fibrosis in this model. These findings suggest that neutralizing S1P with anti-S1P mAbs may be a novel method of treating patients with exudative age-related macular degeneration by reducing angiogenesis and sub-retinal fibrosis, which are responsible for visual acuity loss in this disease.

The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 Å resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca2+. The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.

Lipid composition of transverse tubular membanes from normal and dystrophic skeletal muscle

Abstract Vesiculated fractions of transverse tubular (TT) membranes were isolated from normal and genetically dystrophic breast muscle of the chicken. The lipid composition of the TT was compared to the lipids of highly purified sarcoplasmic reticulum (SR) membranes and enriched fractions of sarcolemma (SL) membranes. Normal TT membranes contained a significantly higher molar ratio of cholesterol to phospholipid phosphorous (0.86) when compared to both SR (0.17) and SL (0.38) membranes. The phospholipid phosphorous-to-protein ratio was highest for SL (1.55 μmol phosphorous/mg), intermediate for TT (1.09), and lowest for the SR (0.79) membranes. TT membranes contained lower amounts of phosphatidylcholine and higher amounts of sphingomyelin when compared to SR while the TT phospholipid distribution was not significantly different from that of the SL. These data indicate that our putative TT fraction displays a lipid composition distinctly different from that of SR and SL and confirms other biochemical and morphological data which indicate that they are of TT origin. All three membrane classes isolated from dystrophic muscle displayed significant alterations in the composition of various lipids. Cholesterol levels were elevated most notably in SL (2.4-fold) followed by the SR (1.3-fold) but not TT membranes. Dystrophic TT and SL but not SR displayed elevated levels of phospholipid phosphorous. Few differences in the distribution of the individual phospholipid classes were found among the dystrophic membranes except for a 2.2-fold increase in the sphingomyelin content of the SR. The observed lipid abnormalities may be the underlying cause of reported alterations in excitation-contraction coupling and suggest that avian dystrophy may result from a generalized membrane defect.

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.

Background Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility. Methodology/Principal Findings Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility. Conclusions/Significance These results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.

The distribution of ATPase activities in purified transverse tubular membranes.

Abstract Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F 1 ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.

Sphingosine-1-phosphate (S1P) is a novel fibrotic mediator in the eye

Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P in the eye has not previously been investigated. In the current study, we evaluated cells from the anterior and posterior segments of the eye for the presence of S1P and their potential ability to produce and respond to S1P. In addition, we investigated the regulatory role of S1P as a mediator of proliferation, cellular transformation and pro-fibrotic protein expression in human retinal pigmented epithelial cells. Expression of S1P receptors and sphingosine kinases (the enzymes that produce S1P) was examined using RT-PCR, and intracellular localization of S1P was examined using immunoblotting, immunohistochemistry and ELISA in primary human retinal pigmented epithelial (RPE) cells, primary human conjunctival fibroblasts (ConF), and primary human corneal fibroblasts (CF). RPE cell proliferation was determined using an MTT-based cell proliferation assay, and RPE myofibroblast transformation, collagen type I production and profibrotic protein expression were assessed using immunofluorescence, ELISA and immunoblot. S1P(1-3, 5) receptors and sphingosine kinases 1 and 2 were expressed and intracellular pools of S1P were detected in RPE cells, ConF and CF. S1P stimulated RPE cell proliferation in a dose- and time-dependent manner. S1P induced myofibroblast transformation of RPE cells, as indicated by increased alpha-smooth muscle actin (alpha-SMA) expression and its incorporation into prominent stress fibers, and promoted collagen type I production. S1P stimulated the expression of plasminogen activator inhibitor-1 (PAI-1) and heat shock protein 47 (HSP47), two proteins that are linked to increased tissue fibrosis. Combined, these data demonstrate that RPE cells, ConF and CF from the human eye not only have the molecular ability to produce and respond to S1P, but also contain S1P. Furthermore, S1P promotes proliferation, myofibroblast transformation, collagen production and pro-fibrotic protein expression by human RPE cells. These data suggest that S1P is a previously unrecognized mediator of profibrotic cellular function and signaling in the eye.