Endothelial Membrane Remodeling Is Obligate for Anti-Angiogenic Radiosensitization during Tumor Radiosurgery

Background

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either “normalize” dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.

Methodology/Principal Findings

Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C₁₆-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase^+/+ mice or asmase^−/− littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase^−/− mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.

Conclusions/Significance

These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials.     

Diabetes,physical activity and breast cancer among Hispanic women

Purpose: We assessed the association between diabetes and breast cancer and whether physical activity modified the effect of diabetes on breast cancer in Hispanic women. Methods: We used data from a case-control study of breast cancer among Hispanic women aged 30–79 conducted between 2003 and 2008 on the Texas–Mexico border. In-person interviews were completed with 190 incident breast cancer cases ascertained through surgeons and oncologists, and 979 controls who were designated as both high-risk (n = 511) and low-risk (N = 468) for breast cancer (with respective response rates of 97%, 83% and 74%). Results: After adjustment for menopausal status and mammography screening, there was no effect of diabetes on breast cancer risk (high-risk control group odds ratio [OR] 1.02, 95% confidence interval [CI] 0.71–1.48; low-risk control group OR 0.87, 0.58–1.30). Women who had a diabetes history and did not exercise were at no risk of breast cancer (OR 0.96, 95% CI 0.63–1.48) or a slightly reduced breast cancer risk (low-risk control group OR 0.72, 95% CI 0.46–1.15) depending on the control group used, while women with diabetes who did exercise had significantly reduced breast cancer risk (OR 0.41, 95% CI 0.21–0.83) regardless of the control group used (high-risk control group p-value for interaction = 0.013, low-risk control group p-value for interaction 0.183). Conclusions: Should other studies confirm our results, physical activity should be explored as a means of reducing breast cancer risk in diabetic women.     

The Lipid Accumulation Product and All‐cause Mortality in Patients at High Cardiovascular Risk: A PreCIS Database Study

The BMI is the most frequently used marker to evaluate obesity‐associated risks. An alternative continuous index of lipid over accumulation, the lipid accumulation product (LAP), has been proposed, which is computed from waist circumference (WC, cm) and fasting triglycerides (TGs) (mmol/l): (WC ? 65) × TG (men) and (WC ? 58) × TG (women). We evaluated LAP and BMI as predictors of mortality in a high‐risk cohort. Study population included 5,924 new consecutive patients seen between 1995 and 2006 at a preventive cardiology clinic. Fifty‐eight percent of patients were discordant for their LAP and BMI quartiles. Patients whose LAP quartile was greater than BMI quartile had higher mortality compared with those with LAP quartile was lower than BMI quartile (8.2 vs. 5.4% at 6 years, P = 0.007). After adjustment for age, gender, smoking, diabetes mellitus, blood pressure, low‐density lipoprotein‐cholesterol (LDL‐C) and high‐density lipoprotein‐cholesterol (HDL‐C), (ln)LAP was independently associated with mortality (hazard ratio (HR) = 1.46, P < 0.001). BMI was not associated with increased mortality (HR = 1.06, P = 0.39). Adding LAP to a model including traditional risk factors for atherosclerosis increased its predictive value (C statistic 0.762 vs. 0.750, P = 0.048). Adding BMI to the same model did not change its predictive value (0.749 vs. 0.750, P = 0.29). Subgroup analyses showed that LAP predicted mortality in the nondiabetic patients (adjusted HR for (ln)LAP 1.64, P < 0.001), but did not reach significance in the diabetic patients (HR = 1.21, P = 0.11). In conclusion, LAP and not BMI predicted mortality in nondiabetic patients at high risk for cardiovascular diseases. LAP may become a useful tool in clinical practice to stratify the risk of unfavorable outcome associated with obesity.     

Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia

The aim of this study was to investigate the epidemiological relationships of clinical and environmental isolates of the Cryptococcus neoformans species complex in Colombia. The current study reflects data from 1987 to 2004. In Colombia serotypes A and B are most frequently recovered from patients and the environment. Of the 178 clinical isolates studied, 91.1% were of serotype A, 8.4% serotype B and 0.5% serotype C. Of the 247 environmental isolates, 44.2% were of serotype A, 42.6% serotype B and 13.2% serotype C. No serotype D isolates were isolated. Serotype AD has not been recovered in Colombia. PCR fingerprinting with the primers M13, (GACA)4 and (GTG)5 and URA5 gene restriction fragment length polymorphism analysis grouped the majority of clinical serotype A and environmental serotype B isolates into the molecular types VNI (98.1%) and VGII (100%), respectively. Mating type alpha was determined in 99.3% of serotype A isolates, but 96.6% of serotype B isolates were of mating type a. Similar profiles between clinical and environmental isolates suggest that the patients may have acquired the infection from the environment. The data presented form part of the Colombian contribution to the ongoing global survey of the C. neoformans species complex.     

Heparin modulation of human plasma kallikrein on different substrates and inhibitors

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.     

TRPM channels, calcium and redox sensors during innate immune responses

Melastatin-related TRPM ion channels have emerged as novel therapeutic targets due to their potential ability to modulate the function and fate of immune cells during inflammation, innate, and adaptive immunity. Four family members, TRPM1, TRPM2, TRPM4 and TRPM7 have a strong presence in the immune system. TRPM channels regulate ion-homeostasis by sensing cellular redox status and cytoplasmic calcium levels. TRPM2 for example, is highly expressed in phagocytes. This channel is activated by intracellular ADP-ribose upon exposure to oxidative stress and induces cell death. Here we will review the functional links between TRPM-mediated ion conductance, chemotaxis, apoptosis, and innate immunity.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.     

Characterization of Vibrios Diversity in the Mucus of the Polychaete Myxicola infundibulum (Annellida,Polichaeta)

Vibrios are among the most abundant culturable microbes in aquatic environments. They can be either free-living in the water column or associated with several marine organisms as mutualists, saprophytes, or parasites. In the present study we analysed vibrios abundance and diversity in the mucus of the polychaete Myxicola infundibulum, complementing culture-based with molecular methods. Vibrios reached 4.6?×?10³ CFU mL^?1 thus representing a conspicuous component of the heterotrophic culturable bacteria. In addition, luminous vibrios accounted for about 60 % of the total culturable vibrios in the mucus. The isolates were assigned to: Vibrio gigantis, Vibrio fischeri, Vibrio jasicida, Vibrio crassostreae, Vibrio kanaloae, and Vibrio xuii. Two Vibrio isolates (MI-13 and MI-15) may belong to a new species. We also tested the ability of the Vibrio isolates to grow on M. infundibulum mucus as the sole carbon source. All strains showed appreciable growth in the presence of mucus, leading us to conclude that this matrix, which is abundant and covers the animal entirely, may represent a microcosm and a food source for some bacteria, playing a crucial role in the structuring of a mucus-associated beneficial microbial community. Moreover, the trophic relationship between vibrios and M. infundibulum mucus could be enhanced by the protection that mucus offers to vibrios. The results of this study represent a contribution to the growing evidence for complex and dynamic invertebrate-microbe associations present in nature and highlight the importance of exploring relationships that Vibrio species establish with marine invertebrates.