PF-4var/CXCL4L1 Predicts Outcome in Stable Coronary Artery Disease Patients with Preserved Left Ventricular Function

Background: Platelet-derived chemokines are implicated in several aspects of vascular biology. However, for the chemokine platelet factor 4 variant (PF-4var/CXCL4L1), released by platelets during thrombosis and with different properties as compared to PF-4/CXCL4, its role in heart disease is not yet studied. We evaluated the determinants and prognostic value of the platelet-derived chemokines PF-4var, PF-4 and RANTES/CCL5 in patients with stable coronary artery disease (CAD). Methodology/Principal Findings: From 205 consecutive patients with stable CAD and preserved left ventricular (LV) function, blood samples were taken at inclusion and were analyzed for PF-4var, RANTES, platelet factor-4 and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Patients were followed (median follow-up 2.5 years) for the combined endpoint of cardiac death, non-fatal acute myocardial infarction, stroke or hospitalization for heart failure. Independent determinants of PF-4var levels (median 10 ng/ml; interquartile range 8-16 ng/ml) were age, gender and circulating platelet number. Patients who experienced cardiac events (n = 20) during follow-up showed lower levels of PF-4var (8.5 [5.3-10] ng/ml versus 12 [8-16] ng/ml, p = 0.033). ROC analysis for events showed an area under the curve (AUC) of 0.82 (95% CI 0.73-0.90, p<0.001) for higher NT-proBNP levels and an AUC of 0.32 (95% CI 0.19-0.45, p = 0.009) for lower PF-4var levels. Cox proportional hazard analysis showed that PF-4var has an independent prognostic value on top of NT-proBNP. Conclusions: We conclude that low PF-4var/CXCL4L1 levels are associated with a poor outcome in patients with stable CAD and preserved LV function. This prognostic value is independent of NT-proBNP levels, suggesting that both neurohormonal and platelet-related factors determine outcome in these patients

Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions

Staphylococcus aureus enterotoxins induce IL-8 secretion by human nasal epithelial cells

BACKGROUND: Staphylococcus aureus produces a set of proteins which act both as superantigens and toxins. Although their mode of action as superantigens is well understood, little is known about their effects on airway epithelial cells. METHODS: To investigate this problem, primary nasal epithelial cells derived from normal and asthmatic subjects were stimulated with staphylococcal enterotoxin A and B (SEA and SEB) and secreted (supernatants) and cell-associated (cell lysates) IL-8, TNF-α, RANTES and eotaxin were determined by specific ELISAs. RESULTS: Non-toxic concentrations of SEA and SEB (0.01 μg/ml and 1.0 μg/ml) induced IL-8 secretion after 24 h of culture. Pre-treatment of the cells with IFN-γ (50 IU/ml) resulted in a further increase of IL-8 secretion. In cells from healthy donors pretreated with IFN-γ, SEA at 1.0 μg/ml induced release of 1009 pg/ml IL-8 (733.0–1216 pg/ml, median (range)) while in cells from asthmatic donors the same treatment induced significantly higher IL-8 secretion – 1550 pg/ml (1168.0–2000.0 pg/ml p = 0.04). Normal cells pre-treated with IFN-γ and then cultured with SEB at 1.0 μg/ml released 904.6 pg/ml IL-8 (666.5–1169.0 pg/ml). Cells from asthmatics treated in the same way produced significantly higher amounts of IL-8 – 1665.0 pg/ml (1168.0–2000.0 pg/ml, p = 0.01). Blocking antibodies to MHC class II molecules added to cultures stimulated with SEA and SEB, reduced IL-8 secretion by about 40% in IFN-γ unstimulated cultures and 75% in IFN-γ stimulated cultures. No secretion of TNF-α, RANTES and eotaxin was noted. CONCLUSION: Staphylococcal enterotoxins may have a role in the pathogenesis of asthma

Thiocyanate-Dependent Inhibition of Spontaneous and Agonist-Induced Eosinophil Apoptosis by Eosinophil Peroxidase (EPO): A Potential Physiologic Role for Endogenously Generated HOSCN in Maintaining Eosinophil Viability.

Abstract Apoptotic eosinophils (EOs), atypically, release cytotoxic specific granule proteins that promote tissue damage in eosinophilic inflammatory states such as asthma. PMN myleloperoxidase strongly promotes PMN apoptosis by generating HOCl. We tested whether EPO plays a similarly pivotal role in EO apoptosis. In vivo, bromide (Br−), nitrite (NO2−), and thiocyanate (SCN−) compete for oxidation by EPO and H2O2 yielding, respectively, HOBr, NO2°, and HOSCN. We have shown that SCN- is the strongly preferred substrate for EPO in vivo and that HOSCN, in striking contrast to HOBr, NO2° and HOCl, is a weak, sulfhydryl-specific oxidant. Because we recently showed that HOSCN is a uniquely potent oxidant activator of endothelial cell NF-kB (Blood 107:558 2006), a powerful antagonist of apoptosis, we hypothesized that endogenously generated HOSCN would inhibit EO apoptosis. Blood EOs were isolated from mildly atopic donors by immunomagnetic separation to &gt; 95% purity. EOs were cultured in RPMI + 10% FCS without added IL-5 and assayed for cell viability by Annexin V and Propidium Iodide (PI) staining and flow cytometry. Apoptosis was confirmed using an immunoassay of cytoplasmic histone-associated DNA fragments, caspase 3 activation and morphology. In every time course examined, EOs were first annexin+/PI-, then later annexin+/PI+. Therefore data are here reported as viability normalized to a control of EOS with 1 ng/ml IL-5, the remainder being comprised of early and late apoptotic cells. EOs cultured 2 days with 1 mM SCN− were 69% viable, a 77% relative increase (n=9, p &lt; 0.0001) over those cultured with nothing (i.e., Cl- only), 1 mM Br-, or 1 mM NO2-, all of which had the same viability (~39%). When 0.5 nM PMA was added to activate the respiratory burst, viability with SCN− after 2 days was 63%, Cl- 5%, Br- 2%, and NO2- 14%. Surprisingly, viability with PMA and SCN− was 20% higher than that with Cl- without PMA (p &lt; 0.05), suggesting that HOSCN not only fails to promote apoptosis but instead engenders an anti-apoptotic tone. Addition of the EPO inhibitor azide (1 mM) abrogated the protective effect of SCN− with PMA. Moreover, SCN- failed to protect EPO-devoid monocytes and lymphocytes from both spontaneous and PMA-induced apoptosis. EOs activated with the physiologic agonists C5a (33 nM) and PAF (5 μM) exhibited the same protective effect of SCN− and increased viability in activated vs. unactivated EOs. EOs treated 2 hours with and without 1mM H2O2 before adding an agonist anti-Fas antibody (1 μg/ml) had viabilities ~30% higher with SCN− than with the other halides at day 1 and at day 2, late-apoptotic cells were 42% that in Cl-. BAY 11-7085 (10μM), an inhibitor of NF-kB activation, caused rapid EO apoptosis (70% at day 1) but in this setting SCN− was not protective with or without PMA activation. Unlike PMN, Western blots for IkB-alpha showed no degradation with PMA irrespective of halide. We conclude that HOSCN generated endogenously in EOs by the EPO/H2O2/ SCN− system plays a previously unsuspected role to maintain both constitutive and agonist-stimulated EO survival. HOSCN antagonizes EO apoptosis through a mechanism that may require constitutive, but not inducible, activation of NF-kB. Because serum SCN− levels vary widely (10–300 μM) and are dietarily determined, oral supplementation with this inexpensive and innocuous pseudohalide may mitigate tissue injury in eosinophilic inflammatory states by inhibiting EO apoptosis in infiltrated organs.</jats:p

Rapid transcriptional down-regulation of c-myc expression during cyclic adenosine monophosphate-promoted differentiation of leukemic cells.

Pharmacologic elevation of cyclic AMP (cAMP) promotes growth arrest and differentiation in a variety of transformed mammalian cells, including the HL-60 human promyelocytic leukemia cell line. However, mechanisms underlying this phenomenon are poorly understood. Because cellular oncogenes play a pivotal role in regulating proliferation and differentiation, we examined whether cAMP-promoted differentiation of HL-60 was preceded by a decrease in the expression of c-myc, a cellular oncogene both amplified and constitutively expressed in HL-60. We find that cyclic AMP elevation in HL-60 caused by three different pharmacologic regimens is followed by an abrupt, greater than 90% decrease in steady state c-myc mRNA levels within 3 h, well before detectable changes in proliferation and differentiation. This decrease, which occurs despite protein synthetic blockade, is attributable to transcriptional down-regulation of c-myc and is accompanied by changes in chromatin structure near c-myc promoter sites. Our findings establish that cAMP, a ubiquitous intracellular regulatory messenger previously known only to enhance gene transcriptional activity in higher eukaryotic cells, can also suppress transcription of a cellular oncogene, thereby suggesting a potential mechanism for cAMP-promoted differentiation

Endogenous Platelet PF4 Promotes In Vivo Activated Protein C (APC) Generation and Survival after Lethal Lipopolysaccharide Challenge in Mice: A Potential Physiologic Role for PF4.

Abstract Pharmacologic infusion of activated protein C (APC) improves survival in human sepsis and genetically induced deficiencies in APC generation increase mortality in a murine lipopolysaccharide (LPS) model of inflammation/sepsis. We have shown that the platelet α-granule-specific chemokine, platelet factor 4 (PF4), accelerates thrombin·thrombomodulin complex APC generation in vitro and in vivo when recombinant PF4 is pharmacologically co-infused in a primate thrombin infusion model system. Hypothesizing a potential physiologic role for PF4 in stimulating APC formation in vivo, we tested whether variations in the level of endogenous platelet PF4 content affect APC generation and also influence survival in the murine LPS challenge model. To stimulate APC formation and platelet PF4 release in mice, we injected murine thrombin (80U/kg IV). Ten min after thrombin injection into wildtype (WT) C57BL/6J mice, plasma APC levels, assessed by enzyme capture amidolytic assay, increased ~10-fold over basal levels. Injection of concurrent murine PF4 (7.5 mg/kg IV) further increased APC levels 2-2.6-fold. We then studied APC generation in mice that were either completely deficient in PF4 (PF4−/−) or transgenic mice that had 6-fold normal levels of human PF4 (hPF4+), both of which had been crossbred onto the same C57BL/6J background &amp;gt;10 times. Plasma APC generation by thrombin was 74 ± 6% in the PF4−/− mice (n = 7) compared to WT littermates (n = 15, p&amp;lt;0.1) and increased to 178 ± 49% in hPF4+ mice (n= 22, p&amp;lt; 0.003 compared to WT (n= 36)). Heterozygous protein C deficient (PC+/−) mice had 65 ± 21% of WT APC generation (n= 5, p= 0.05 compared to WT (n=7)), but APC generation was restored to 96 ± 6% level in hPF4+/PC+/− mice (n= 6, p= 0.88 compared to WT (n=12)). Only PF4−/− and PC+/− mice died during this thrombin injection trial with mortality rates of 30% and 58%, respectively. A lethal LPS challenge model (25 mg/kg IP) was used to test the susceptibility of mouse strains with varying PF4 levels. Significant numbers of WT and PF4−/− mice died before the end of the first day [(18 of 44 (40% mortality) and 6 of 21 (28% mortality), respectively], while none of the 19 hPF4+ animals died (p&amp;lt; 0.001). However, after the first day, mortality did not differ significantly between the groups and final mortality for the three groups was ~70%. In summary, these studies of mice with varying platelet PF4 suggest that endogenous PF4 plays a physiologic role to stimulate APC production. Moreover, mice with high platelet PF4 content appear to be protected from both thrombin- and LPS-provoked deaths. This suggests that PF4 plays a physiologic role to enhance APC generation, that patients with high PF4 levels may be intrinsically protected from sepsis and supports the possibility that PF4 infusions in patients with sepsis may be a viable therapeutic strategy.</jats:p