ROLE OF novel PKCs IN PLATELET PRODUCTION AND FUNCTION

Protein kinase C (PKC) is a family of serine-threonine kinase involved in many cellular function, including cell death, proliferation and differentiation (Corbalán-García S et al. 2006). In particular PKCε and PKCδ could be considered the yin and yang of novel PKCs, because of their antithetical role in many cellular mechanisms such as proliferation, apoptosis, tumor growth and cardioprotection. PKCε is largely considered as an oncogene because of its anti-apoptotic and pro-proliferation functions (Newton PM and Messing RO 2011), conversely PKCδ generally slows proliferation, induces cell cycle arrest and apoptosis (Steinberg SF 2004). In the heart, they are among the most widley expressed PKC isozymes and they play an antithetical role in the ischemic-reperfusion preconditioning process (i.e.: enhancing role forPKCε and inhibiting role for PKCδ (Chen L et al. 2001). Moreover, the importance of these two PKC isoforms in pathological conditions has been proven by clinical trials based on specific PKCε and PKCδ inhibitor peptides (Mochly-Rosen D et al. 2011). Platelets are the smallest blood cells with a primary physiological role in hemostasis. They are produced by megakaryocytes as anucleated cells that contain proteins and mRNA derived from megakaryocyte. Platelets retain also the protein synthesis machinery, therefore, platelet mRNAs can be efficiently translated during platelet life, during around 10 days. PKCs have been established as important regulators of several platelet functions. The physiological expression of the different PKC isoforms varies significantly in mature platelets, with relevant differences in humans and mice. Concerning novel PKC isoforms, it has been demonstrated that PKCδ is expressed in human platelets and regulates the activation response to GPVI agonists and adhesion to collagen (Hamm CW et al. 1987). This behavior of PKCδ in humans is similar to the one described for PKCε in mice, where it plays a relevant role in the activatory signaling cascade emanating from the GPVI receptor (Pears CJ et al. 2008). On the other hand, PKCε expression and function in human platelets is still very controversial (Crosby D and Poole AW. 2003; Muruggapan S et al. 2004; Beunsuceso S et al. 2005; Pears CJ et al. 2008). On these basis, during the first period of my doctoral fellowship, I focused my research on PKCε expression in human platelets, exploring whether its levels could be associated to platelet hyper-reactivity and related diseases (i.e. acute myocardial infarction), in order to clarify PKCε role in platelet function. The results of my research showed that the majority of HD-derived platelets did not express PKCε. This is in agreement with previous data (Gobbi G et al. 2007) describing the down-modulation of PKCε expression in in vitro human megakaryocytopoiesis from day 6 onward of TPO-driven MK differentiation of CD34 precursors. Platelets play a central role in the genesis and propagation of atherothrombosisis and the development of platelet thrombus is a critical, final phase in myocardial infarction. I demonstrated that in human platelets, PKCε is selectively de novo expressed in myocardial infarction, but not in stable coronary artery disease patients, and its expression returns negative after 15 days of follow-up after the acute event. Additionally, by functional experiments, I demonstrated that PKCε-transfected normal human platelets enhance their adhesion properties to collagen-coated surfaces under physiologically high shear forces. Myocardial infarction patients express PKCε mRNA at significantly higher frequency than healthy donors and stable coronary artery disease. Considering the dimensions of the first intronic sequence of the PKCε gene, that would virtually preclude the persistence of a potential PKCε pre-mRNA in the platelet, my findings suggest that platelet generations produced before the acute event of myocardial infarction might retain PKCε-mRNA that is not down-regulated during terminal MK differentiation. An alternative explanation would be an anticipated release of platelets, before physiological PKCε down-modulation. This possibility is however unlikely, as PKCε down-modulation takes place around day 6 of in vitro MK differentiation, that would be too early. Besides, the analysis conducted on the reticulated platelets of some myocardial infarction patients, randomly selected from the recruited cohort, did not show any difference in terms of PKCε expression as compared to mature platelets, excluding the possibility that the appearance of PKCε positive platelets in myocardial infarction patients could be selectively ascribed to newly formed platelets. These results suggest that the ectopic expression of PKCε in platelets could be used as a marker of probability to anticipate the acute event in patients at risk. Since many pathological conditions depend on an abnormal platelet function, the study of molecular mechanisms involved in platelet activation and thrombus formation, has always been a matter of great interest. On the other hand, many diseases are related to abnormal platelet production. Thrombocytopenia is a major clinical problem encountered across a number of conditions, including immune thrombocytopenic purpura, myelodysplastic syndromes, chemotherapy, aplastic anaemia, human immunodeficiency, virus infection, complications during pregnancy and delivery, and surgery. Patients with a low platelets number, are at increased risk of spontaneous bleeding or hemorrhage and, to prevent them, they are treated with platelets transfusion. However the use of apheresis-equivalent units derived from human donors, shows several limitations and challenges with platelet preparation and storage technologies , such as clinically significant immunogenicity, associated risk of sepsis and inventory shortages due to high and 5-days shelf life (Thon JN et al. 2014). On these basis, two strategy are possible: i) to potentiate an in vitro platelet producing system to obtain platelets for infusion and ii) to develop pharmacological treatment able to modulate in vivo megakaryocytopoiesis and platelet production. Therefore, a deeper understanding of molecular mechanisms that control megakaryocytopoiesis is a key element to regulate in vitro and in vivo platelet production for clinical applications. PKCε is involved in human and mouse megakaryocytopoiesis. In human CD34+-derived MKs, PKCε is down-modulated in the later phases of differentiation and its forced overexpression reduces cell polyploidization and platelet production via Bcl-xL up-regulation (Gobbi G et al 2007). Regarding to PKCδ although it is well established that PKCδ is present in human platelets regulating their function, little is known about its expression during megakaryocytopoiesis Given this background, during the second period of my doctoral fellowship I sought to demonstrate whether PKCδ expression in human platelet could be the expression of a specific modulation of this isoform during megakaryocyte differentiation, similarly to what described for PKCε. Using in vitro model of human megakaryocytopoiesis, I found that, conversely to PKCε levels, PKCδ is constantly expressed and its forced down-modulation results in reduced MKs differentiation and platelet release via Bcl-xL up-regulation and Bax down-modulation Finally, given the growing interest of the scientific community on ameliorating in vitro platelet production for transfusion purposes, I studied whether the modulation of PKCε/PKCδ balance could affect platelet production in vitro. The importance of PKCε and PKCδ balance in human thrombopoiesis is additionally proven by my findings in pathological conditions, ie. myeloproliferative disorders. Primary myelofibrosis is a chronic myeloproliferative neoplasm characterized by bone marrow hyperplastic MKs with an impaired capacity to generate proplatelets in vitro. Interestingly, I found that PMF-CD34+-derived megakaryocytes show an imbalance between PKCδ/ PKCε expression, with an increase in PKCε levels- in agreement with our more recently data (Masselli et al. In press) – and a decrease in PKCδ levels, than those from healthy subjects. Moreover, these expression levels of PKCs are associated with similar imbalance between their down-stream effectors Bcl-xL and Bax, respectively. Finally, using a pharmacological inhibitor and activator of PKCδ and PKCε function, I obtained a modulation of in vitro platelet production. The concomitant PKCδ inhibition and PKCε activation reduces platelet release; conversely, PKCδ activation associated with PKCε inhibition clearly demonstrates the possibility to potentiate platelet production. Collectively, these data suggest that novel PKCs i) should be adequately expressed in human circulating platelets and PKCε ectopic expression could be associated to cardiovascular deseases, suggesting its possible use as a risk marker; ii) have a crucial role during normal human megakaryocytopoiesis and platelet production, through a mechanism involving apoptotic pathway (specifically Bcl-xL and Bax); iii) might be modulated, as protein expression levels, during in vitro megakaryocytopoiesis and by modifying their rate is possible to revise platelet production, suggesting future strategy for platelet diseases theraphy and infusion-aimed platelet expansion

The Genetic Makeup of Myeloproliferative Neoplasms: Role of Germline Variants in Defining Disease Risk, Phenotypic Diversity and Outcome

Myeloproliferative neoplasms are hematologic malignancies typified by a substantial heritable component. Germline variants may affect the risk of developing a MPN, as documented by GWAS studies on large patient cohorts. In addition, once the MPN occurred, inherited host genetic factors can be responsible for tuning the disease phenotypic presentation, outcome, and response to therapy. This review covered the polymorphisms that have been variably associated to MPNs, discussing them in the functional perspective of the biological pathways involved. Finally, we reviewed host genetic determinants of clonal hematopoiesis, a pre-malignant state that may anticipate overt hematologic neoplasms including MPNs

Novel role of PKC epsilon in mitotic spindle stability

Mitosis is a highly regulated process characterized by dramatic and coordinated morphological changes to ensure the fidelity of chromosome segregation. Missegregation of mitotic chromosomes leads to a condition that underlies chromosomal instability(1), which is a hallmark of cancer. In order to assure symmetry and bipolarity of the cell division process, mitotic spindle microtubules properly segregate mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases, PKCε is one of the best understood for its role as a transforming oncogene, and it has been found overexpressed in different types of tumors. In 2008, Saurin and colleagues demonstrated the involvement of PKCε in the regulation of the late stage of mitosis (3). Through its association with 14-3-3 at the midbody, PKCε is essential for the successful completion of cytokinesis, and the inhibition of functional PKCε-14-3- 3 complex leads to abscission failure and multinucleated phenotype in cells. In this study, we found that PKCε is involved in mitotic spindle stability. Using fluorescence microscopy, we found that the active form of PKCε (phosphorylated at Ser-729), colocalizes to the centrosome in cells in metaphase, where the mitotic spindle nucleation occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of the tubulin superfamily localized to the microtubule organizing centers and is essential for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ expression affects spindle stability: PKCɛ downregulation by specific shRNA results in mitotic spindle disorganization with a reduction of the amount of centrosomal and mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using Nocodazole, known to interfere with the polymerization of microtubules, revealed that cells lacking PKCɛ were unable to regrow microtubules after depolymerization. These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely determinant for genome stability

PKC epsilon involvement in Th17 in vitro differentiation: implications in psoriasis pathogenesis

Psoriasis is a noncontagious, arytematous-squamose dermatitits affecting both sexes and all races. Although its exact etiology is largely unknown, it is now recognized as one of the most common immune-mediated disorders and several studies demonstrate an impairment of regulatory T-cells (Tregs) function and an up-regulation of IL-17 levels produced by T-helper 17 lymphocytes (Th17)(1,2). Protein kinase C epsilon (PKCε) is a serine/threonine kinase which plays a key role in the proliferation and differentiation of epidermal cells. We have previously demonstrated a role for PKCε in the pathogenesis of the autoimmune disease Hashimoto’s thyroiditis (3). PKCε is over-expressed in CD4+ T lymphocytes isolated from PBMC fraction in patients affected by this pathology and its forced down-modulation primed the TGF-mediated in vitro Treg polarization of human T CD4+ cells. Since it has been demonstrated that PKC-signalling is altered in psoriatic keratinocytes (4), we investigated the involvement of PKCε in Th17 in vitro differentiation and its potentially implication in immune response correlated to psoriasis. Using western blot and real time PCR, we have observed that PKCε protein levels and mRNA increase during Th17-lineage in vitro differentiation from naïve CD4+ T cells with a similar trend of Th17 markers of differentiation STAT3 and RoRyT. Moreover, PKCε overexpression significantly increases STAT3 and phosphorylated STAT3 levels, suggesting that PKCε boosts Th17 polarization. Thereafter, we sought to investigate PKCε expression in CD4+ lymphocytes obtained from peripheral blood of psoriatic patients and we observed that PKCε expression levels are significantly higher compared with healthy donors. Intriguingly, we observed a closely correlation of PKCε expression with PASI index, suggesting an involvement of the kinase with the severity of the disease. Collectively these data suggest that PKCε might be involved in Th17 differentiation, that it could be a key factor to regulate Th17 pathological expansion and therefore a potential psoriatic pharmacological target

Activation and nuclear translocation of PKCε promotes skeletal muscle cell differentiation via HMGA1 downregulation

The role of novel PKCs in skeletal muscle differentiation has recently emerged. PKCθ is the most expressed isoform of PKCs in muscle and it promotes the fusion of myoblasts [1]. Recently, we have demonstrated that PKCε is implicated in myocardiocyte differentiation of bone marrow mesenchymal stem cells [2] but the role of PKCε in skeletal muscle cell regeneration has only recently emerged [3]. We here demonstrate that both nuclear and cytoplasmic fractions of PKCε are up-regulated during in vitro C2C12 cell line and satellite cell differentiation. We also show that PKCε is able to modulate myogenic differentiation genes via a downmodulation of HMGA1 proteins that promotes myogenin accumulation and mature myoblast formation. To study the effects of PKCε on muscle regeneration, we have used the in vivo model of CTX-induced skeletal muscle injury. We show that the upregulation of PKCε also occurs in vivo, particularly in the centro-nucleated regenerating fibers that are derived from the fusion process of the resident satellite cells, suggesting a role for PKCε in human satellite cell-driven muscle repair and substitution, with clinically relevant implications in human muscle pathology

Correlation between Protein Kinase Cε expression and thrombotic risk in Primary Myelofibrosis (PMFs)

Myelofibrosis (MF) - either primary (PMF) or arising from a previous PV or ET - is a Philadelphia-negative MPNs characterized by aberrant platelet production and consequent variable platelet count with altered hemostatic function (1). It has already been demonstrated that the risk of thrombotic events is one of the most common co-morbidities associated with PV and ET (2-5). However, risk of thrombotic events in PMF has not been investigated yet. We previously demonstrated that PKCepsilon (PKCε) is over-expressed in platelets from patients with acute myocardial infarction and accounts for their increased reactivity (6). Additionally, we recently showed that PKCε overexpression plays a crucial role in PMF MK impaired differentiation and that its levels correlated with the disease severity (expressed by the IPSS/DIPPS risk category) (7,8). On these bases, we analyzed PKCε expression in platelets from PMF patients, investigating a potential correlation with thrombotic risk and the aggressiveness of the disease. For this study, peripheral blood samples from 6 PMF patients and 3 healthy donors (HD) were collected in Na-citrate tubes. PKCε mRNA and protein levels were determined in platelets purified as described by Carubbi C, 2012. Finally, patients are stratified according to the history of cardio-vascular events and the IPSS/DIPSS risk category. PMF platelets showed significantly higher mRNA levels of PKCε as compared to HD. Protein analysis confirm PKCε over-expression in PMF platelets, almost reaching statistical significance. We then found that platelet from PMF patients who suffered from cardiovascular events display significantly higher levels of PKCε as compared to the one with a negative history. Finally, similarly to what observed in PMF magakaryocytes, we showed a positive correlation between PKCε platelets levels and IPSS/DIPSS risk category, with the lowest levels in low-risk patients and higher levels in high-risk patients. Collectively, our preliminary results indicate that PMF platelets show an aberrant expression of PKCε which correlates with the disease burden and a history of cardiovascular events. This suggests that the over-expression of PKCε may account for PMF platelet altered reactivity and function

Platelet gene expression profile in acute myocardial infarction

Acute myocardial infarction is a sudden event that is fatal in around one-third of patients. It is primarily due to coronary atherosclerotic plaque rupture with subsequent platelet (PLT) activation/aggregation and thrombus formation. PLTs have a key role in the genesis and progression of atherosclerosis and in thrombus formation1. PLTs are anucleated cells which retain mRNA from their megakaryocyte precursor, therefore PLT mRNA is unique in representing a nearly fixed transcriptome2. We tested the hypothesis that platelet transcriptome acts as a fingerprint indicating the development of a future myocardial infarction, with the final goal of identifying a specific STEMI gene-signature, able to discriminate patients with acute event from healthy donors (HD) and from patients affected by stable coronary artery disease (sCAD), the phenotypically closest clinical condition to STEMI. Peripheral blood samples (50mL) were collected in Na-citrate tubes from 20 myocardial infarction patients (MI), 20 sCAD and 20 HD. Highly purified platelets were obtained by leukocyte depletion as previously described3. Platelet RNA extraction were performed by TRIzolTM reagent according to the manufacturer’s protocol. Gene expression profile was analyzed using an Affymetrix GeneChip system (Cancer Genomics and Bioinformatics Laboratory Facility, Kimmel Cancer Center, Jefferson University. Philadelphia, US). The exploratory analysis of PLT transcriptome confirmed differences in gene expression between STEMI, sCAD and HD. Hence, the common differentially expressed genes (DEGs) derived from the STEMI vs sCAD and STEMI vs HD comparisons were obtained and tested by k-nearest neighbor classification and bootstrap. A set of 17 STEMI-related DEGs was identified, showing good sensitivity and specificity for the discrimination of STEMI patients. Overall, we described a STEMI-specific gene expression patterns, suggesting that PLT transcriptome allows to characterize a powerful fingerprint of STEMI theoretically able to predict a future acute event

Impact of sulphurous water Politzer inhalation on audiometric parameters in children with otitis media with effusion

Objectives: The positive effects of spa therapy on ear, nose, and throat pathology are known but robust literature in this field, is still lacking. The aim of this study was to assess through a retrospective analysis, the effects on otitis media with effusion of Politzer endotympanic inhalation of sulphurous waters in children aged 5-9 years. Methods: A cohort of 95 patients was treated with Politzer insufflations of sulphurous water: 58 patients did a cycle consisting of a treatment of 12 days per year for three consecutive years; 37 patients followed the same procedure for 5 years consecutively. The control population was represented by untreated, age-matched children. A standard audiometric test was used before and after each cycle of treatment. Results: One cycle of Politzer inhalation of sulphur-rich water improved the symptoms. Three cycles definitively stabilized the improvement of hearing function. Conclusion: Our results show that otitis media with effusion in children can be resolved by an appropriate non-pharmacological treatment of middle ear with sulphur-rich water

Physical Activity and Redox Balance in the Elderly: Signal Transduction Mechanisms

Reactive Oxygen Species (ROS) are molecules naturally produced by cells. If their levels are too high, the cellular antioxidant machinery intervenes to bring back their quantity to physiological conditions. Since aging often induces malfunctioning in this machinery, ROS are considered an effective cause of age-associated diseases. Exercise stimulates ROS production on one side, and the antioxidant systems on the other side. The effects of exercise on oxidative stress markers have been shown in blood, vascular tissue, brain, cardiac and skeletal muscle, both in young and aged people. However, the intensity and volume of exercise and the individual subject characteristics are important to envisage future strategies to adequately personalize the balance of the oxidant/antioxidant environment. Here, we reviewed the literature that deals with the effects of physical activity on redox balance in young and aged people, with insights into the molecular mechanisms involved. Although many molecular pathways are involved, we are still far from a comprehensive view of the mechanisms that stand behind the effects of physical activity during aging. Although we believe that future precision medicine will be able to transform exercise administration from wellness to targeted prevention, as yet we admit that the topic is still in its infancy