Combination of Platelet expression of PKCepsilon and cardiac troponin-I for early diagnosis of chest pain patients in the emergency department.

A rapid differential diagnosis of the clinical conditions underlying chest pain is a relevant clinical issue. Specifically, a fast rule-in or -out of acute myocardial infarction (AMI) is mandatory to improve diagnostic outcome and cost-effectiveness of patient management. We demonstrated that Protein Kinase C (PKC) epsilon is selectively expressed by platelets from AMI patients, accounting for increased platelet activation. Thus, we hypothesized that PKCepsilon-expressing platelets may represent a pathophysiological marker of AMI that could be utilized in combination with troponin-I, the conventional marker of cardiac injury, to add diagnostic information in chest pain workup. In 94 chest pain patients consecutively admitted to Parma University Hospital, we tested the diagnostic performance of flow-cytometric detection of PKCepsilon expressing platelets in discriminating AMI vs. non-AMI conditions. We demonstrated that PKCepsilon-expressing platelets were significantly higher in patients with AMI. Flow cytometry detection of PKCepsilon-expressing platelets showed high sensitivity and specificity (87.5% and 84.4%, respectively) and good diagnostic accuracy (AUC: 0.875). The combination of PKCepsilon expressing platelets and cardiac troponin clearly discriminates patients with 100% and 0% of probability to be affected by AMI. Overall, we highlighted a dual marker strategy potentially useful for a rapid rule-in or -out of myocardial infarction in chest pain patients.

Protein kinase C ε expression in platelets from patients with acute myocardial infarction.

OBJECTIVE: Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease. METHODS AND RESULTS: We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen. CONCLUSIONS: Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.

High-fat diet activates pro-inflammatory response in the prostate through association of Stat-3 and NF-κB.

BACKGROUND: Signal transducer and activator of transcription (Stat)-3 and nuclear factor-kappa B (NF-κB) are important signaling pathways constitutively activated during inflammation. We previously reported that high-fat diet (HFD) intake induces oxidative stress in the prostate through elevated expression of NADPH oxidase subunits causing NF-κB activation. We sought to determine whether Stat-3 is involved in the activation of NF-κB in the prostate as a result of HFD feeding, leading to inflammation. METHODS: C57BL/6 mice were either fed with regular diet (RD) or HFD for 4 and 8 weeks. Plasma cytokine levels were determined by multiplex analysis. Western blotting was performed to determine the expression of NF-κB, Stat-3, Akt, PDK1, PKCε, and their phosphorylated forms along with pathologic evaluation of the prostate. Immunoprecipitation and electrophoretic mobility shift assay (EMSA) were conducted to study the association between Stat-3 and NF-κB. RESULTS: C57BL/6 mice fed with HFD showed a significant increase in the plasma levels of IL-1ß, IL-6, IL-17, and TNFα after 4 and 8 weeks of feeding, compared with RD controls. HFD feeding elevated the intraprostatic expression of IL-6 and caused activation of PKCε and Akt, the upstream kinase regulating Stat-3 and NF-κB. Nuclear extracts from the prostates of mice fed with HFD exhibited constitutively activated levels of Stat-3 and NF-κB/p65. Increased association between the activated forms of Stat-3 and NF-κB/p65 was observed in the nucleus as a result of HFD feeding, a finding that was accompanied by morphologic evidence of increased intraprostatic inflammation. CONCLUSIONS: Our findings suggest that HFD activates Stat-3 and NF-κB/p65 in the prostate, and their interaction is associated with increased inflammation in the prostate.

[Apoptosis induction and phosphorylated protein kinase C epsilon expression in 32D cells by sera from patients with aplastic anemia].

OBJECTIVE: To investigate the effect of phosphorylated protein kinase C epsilon (pPKC epsilon) on apoptosis of 32D cells induced by sera from patients with aplastic anemia (AA). METHODS: The expression of pPKC epsilon and apoptosis in 32D cells were measured by Western blotting and flow cytometry after incubation with sera from healthy individuals (controls, n = 8), patients with severe AA ( SAA, n = 8)and non severe AA (NSAA, n = 6). RESULTS: After incubation for 0, 12, 24, 36 and 48 hours in the presence of serum and for another 4 hours in medium deprived of serum, the levels of pPKC epsilon in cells in SAA and NSAA group increased gradually, peaked at 24 hours, and then declined (P < 0.05). Compared with that in control group (0.54 +/- 0.08), pPKC epsilon was overexpressed in both SAA group (0.90 +/- 0.10) and NSAA group (0.64 +/- 0.08) (P < 0.05) after 24 hours incubation with serum and subsequent 4 hours without serum. pPKC epsilon level was higher in SAA group than in NSAA group (P < 0.05). A greater proportion of 32D cells showed apoptosis after 24 hours incubation with sera from SAA patients [(4.05 +/- 1.05)%] and subsequent 4 hours incubation without serum than that in controls [(2.45 +/- 0.51)%, P < 0.05], which was correlated with the same serum-induced expression of pPKC epsilon (r = 0.869, P < 0.05). Although the mean level of pPKC epsilon expression was higher in NSAA group than in control group, no significant difference of apoptosis was found between the two groups [(2.45 +/- 0.51)% vs (3.24 +/- 0.56)%, P > 0.05]. CONCLUSION: Sera from both SAA and NSAA patients could upregulate the expression of pPKC epsilon in 32D cells. The SAA sera induce apoptosis in 32D cells significantly, but the latter do not.