Megakaryocyte- and erythroblast-specific cell-free DNA patterns in plasma and platelets reflect thrombopoiesis and erythropoiesis levels.

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.

Integrin α2ß1-Targeted Self-Assembled Nanocarriers for Tumor Bioimaging.

Recent developments in near-infrared (NIR) dyes and imaging modalities enable tumor fluorescent images in preclinical and clinical settings. However, NIR dyes have several drawbacks, and therefore, there is an unmet diagnostic need for NIR dye encapsulation in appropriate pharmaceutical nanocarriers with targeting abilities for the purpose of achieving effective diagnosis and image-guided surgeries. Because integrin receptors are established diagnostic targets, the cyclic Arg-Gly-Asp (RGD) peptides, recognizing the αVß3 integrin, have been extensively investigated for radiology and bioimaging of tumors. However, the Lys(Arg)-Thr-Ser [K(R)TS] cyclic peptides, selective for collagen receptors α1ß1/α2ß1 integrins, which are overexpressed in many tumors, were not yet investigated and therefore used here for tumor bioimaging with a unique α2ß1-integrin-targeted nanocarrier, encapsulating the indocyanine green NIR dye. We synthesized three kinds of peptides: two cyclic RTS peptides functional only in the cyclic conformation and a linear peptide lacking the cyclic cysteine constrained RTS loop. We used them for the preparation of integrin-targeted self-assembled nanocarriers (ITNCs), referred to as OF5 and OF27, and a nontargeted control nanocarrier, referred to as OF70. Their selective association was demonstrated with α2ß1 integrin expressing cell cultures and three-dimensional tumor spheroids and by competition with a α2ß1 selective disintegrin. Cytotoxicity experiments in vitro demonstrated the safety of the ITNCs. The targeting potential and the biodistribution of the ITNCs, applied intravenously in A431 tumor-bearing nude mice, were evaluated in vivo using NIR bioimaging. Time-dependent biodistributions indicated that the ITNC OF27 showed higher fluorescent signals in main tissues, with no cytotoxic effects to major organs, and presented higher accumulation in tumors. Cumulatively, these results highlight the potential of the ITNC OF27 as an optical and innovative pharmaceutical bioimaging system, suitable for integrin α2ß1 receptor in vivo tumor targeting and visualization in the NIR region.

Bleeding Risk Assessment and the Role of Primary Hemostasis Screening in Patients Undergoing Kidney Biopsy.

BACKGROUND: Risk factors for bleeding complications after percutaneous kidney biopsy (PKB) and the role of primary hemostasis screening are not well established. OBJECTIVES: To determine the role of primary hemostasis screening and complication outcomes among individuals who underwent PKB. METHODS: We reviewed data of 456 patients who underwent PKB from 2010 to 2016 in a large university hospital. In 2015, bleeding time (BT) testing was replaced by light transmission aggregometry (LTA) as a pre-PKB screening test. RESULTS: Of the 370 patients who underwent pre-PKB hemostasis screening by BT testing, prolonged BT was observed in 42 (11.3%). Of the 86 who underwent LTA, an abnormal response was observed in 14 (16.3%). Overall, 155 (34.0%) patients experienced bleeding: 145 (31.8%) had minor events (hemoglobin fall of 1-2 g/dl, macroscopic hematuria, perinephric hematoma without the need for transfusion or intervention) and 17 (3.7%) had major events (hemoglobin fall > 2 g/dl, blood transfusion or further intervention). Abnormal LTA response did not correlate with bleeding (P = 0.80). In multivariate analysis, only prolonged BT (P = 0.0001) and larger needle size (P = 0.005) were identified as independent predictors of bleeding. CONCLUSIONS: Bleeding complications following PKB were common and mostly minor, and the risk of major bleeding was low. Larger needle size and prolonged BT were associated with a higher bleeding risk. Due to the relatively low risk of major bleeding and lack of benefit of prophylactic intervention, the use of pre-PKB hemostasis screening remains unestablished.

Assessment of procoagulant potential in patients with reactive thrombocytosis and its association with platelet count.

OBJECTIVE: We aimed to determine hemostatic changes and characterize the procoagulant potential among patients with reactive thrombocytosis (RT). METHODS: Sixty patients with RT (median platelet count 718 × 109 /L) and 20 healthy persons were tested for complete blood count, C-reactive protein, von Willebrand factor (VWF), factor VIII and fibrinogen, and thrombin generation. Platelet studies, including light transmission aggregometry and Cone and Plate(let) Analyzer, were also conducted. Reticulated platelets and platelet P-selectin expression were measured using flow cytometry. RESULTS: Compared to patients with mild thrombocytosis (platelet count 500-700 × 109 /L; n = 27), those with moderate-to-severe thrombocytosis (platelet count >700 × 109 /L; n = 33) had significantly higher fibrinogen, factor VIII, and VWF antigen and activity levels; higher endogenous thrombin potential, peak thrombin generation and velocity index levels, and shorter time-to-peak thrombin level. VWF antigen and activity, fibrinogen, and factor VIII were positively associated with platelet count, whereas VWF activity/antigen ratio was inversely correlated. In a multivariate analysis of RT and control participants, only platelet count predicted endogenous thrombin potential with a positive-linear correlation. No patients developed acquired von Willebrand syndrome. CONCLUSIONS: As determined by thrombin generation, RT was associated with in vitro prothrombotic tendency, which correlated with platelet count. This may explain the increased thromboembolic risk previously reported in patients with RT.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

Differential impact of selective serotonin reuptake inhibitors on platelet response to clopidogrel: a randomized, double-blind, crossover trial.

STUDY OBJECTIVE: To assess the effect of two selective serotonin reuptake inhibitors (SSRIs), fluvoxamine and citalopram, that markedly differ in their level of cytochrome P450 (CYP) 2C19 inhibition, on the laboratory response to clopidogrel, a prodrug requiring metabolism by the CYP system, and especially CYP2C19, to produce its active form. DESIGN: Randomized, double-blind, crossover trial. SETTING: Clinical research unit of an academic medical center. SUBJECTS: Fifteen healthy male volunteers. INTERVENTION: All subjects received clopidogrel as a 300-mg loading dose on day 1, followed by 75 mg/day on days 2 and 3. Platelet function was tested at baseline and then after clopidogrel treatment on day 3. After a washout period of 2 weeks, subjects were randomly assigned in a double-blind manner to receive either citalopram 20 mg/day or fluvoxamine 100 mg/day for 7 days. On day 5, platelet function was tested while receiving the SSRI treatment alone; then, a 300-mg clopidogrel loading dose was administered, followed by clopidogrel 75 mg/day on days 6 and 7. Platelet function was then reassessed on day 7 while receiving the combination of the SSRI and clopidogrel. The treatment protocol was then repeated after a washout period of 2 weeks in all subjects with the other SSRI. MEASUREMENTS AND MAIN RESULTS: The antiplatelet effects of fluvoxamine and citalopram and their interactions with clopidogrel were assessed. The response to these three drugs was assessed by light transmittance aggregometry and vasodilator-stimulated phosphoprotein phosphorylation, reporting P2Y12 receptor reactivity. Both fluvoxamine and citalopram tended to reduce adenosine diphosphate-induced aggregation: 80.8 ± 3.4% at baseline, 67.3 ± 6.3% while receiving citalopram, and 65.8 ± 6.4% while receiving fluvoxamine. All subjects had a good laboratory response to clopidogrel, with a mean aggregation of 23.5 ± 3.2% and a mean platelet reactivity index of 47.7 ± 3.9% (p<0.001 compared with baseline for both methods). Laboratory response to clopidogrel was significantly attenuated in the presence of fluvoxamine compared with the response in the presence of citalopram as tested both by aggregometry (32.3 ± 4.2% vs 23.4 ± 3%, p=0.04) and by vasodilator-stimulated phosphoprotein phosphorylation (52.7 ± 5.1% vs 35.9 ± 4.2%, p=0.02). CONCLUSION: Fluvoxamine attenuated the laboratory response to clopidogrel, possibly through inhibition of CYP2C19, whereas citalopram did not affect this response. These potential drug interactions should be taken into consideration in the selection of the appropriate antidepressant agent for patients who are treated with clopidogrel.

Vipegitide: a folded peptidomimetic partial antagonist of α2ß1 integrin with antiplatelet aggregation activity.

Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2ß1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10(-10) M and 1.5×10(-10) M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2ß1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2ß1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.

A mouse model to study thrombotic complications of thalassemia.

Patients with ß-thalassemia major and mainly intermedia have an increased risk for developing venous and arterial thrombosis which may be related to circulating pathological red blood cells (RBC) and continuous platelet activation. In the present study we used a modified thalassemic mice model in conjunction with a "real-time" carotid thrombus formation procedure to investigate thrombotic complications of thalassemia. Heterozygous Th3/+ mice, which lack one copy of their ß-major and ß-minor globin genes, exhibit anomalies in RBC size and shape, chronic anemia and splenomegaly which recapitulate the phenotype of human ß-thalassemia intermedia. Flow cytometry measurements showed higher reactive oxygen species generation, indicating oxidative stress, in platelets and RBC of the thalassemic mice compared with wild type mice concomitant with an increase in reduced glutathione content which may represent a compensatory response to oxidative stress, and exposed phosphatidylserine which indicates platelet activation. To elucidate the effect of thalassemia on the development of arterial thrombosis, we studied photochemical-induced real-time thrombus formation in the carotid artery of these mice. The results indicated a significantly shorter "time to occlusion" in the thalassemic mice compared to wild type mice, which was prolonged following in vivo aspirin treatment. We suggest that this mouse model may contribute to our understanding of platelet activation and the hypercoagulable state in thalassemia and lay foundations to screening of anti-platelet drugs as well as anti-oxidants as possible therapeutics for prevention of thrombosis in thalassemia patients.

Platelet lysates stimulate angiogenesis, neurogenesis and neuroprotection after stroke.

Platelets contain chemo-attractants and mitogens that have a major role in tissue repair. Therefore we hypothesised that tissue regeneration secondary to activation of endogenous neural stem cells (eNSC) can be enhanced by delivering platelets to the ischaemic brain. To examine these potential therapeutic effects we injected platelet-poor plasma (PPP), fibroblast growth factor (FGF2) and platelet lysate (PLT) to the lateral ventricles after permanent middle cerebral artery occlusion (PMCAO) in rats. The animals were tested with the neurological severity score, and infarct volumes were measured at 90 days post-PMCAO. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischaemic brain. Platelets significantly increased eNSC proliferation and angiogenesis in the subventricular zone (SVZ) and in the peri-lesion cortex. Functional outcome was significantly improved and injury size was significantly reduced in rats treated with PLT suggesting additional neuroprotective effects. In conclusion, local delivery of PLT to the lateral ventricles induces angiogenesis, neurogenesis and neuroprotection and reduces behavioural deficits after brain ischaemia.

Identification of α2ß1 integrin inhibitor VP-i with anti-platelet properties in the venom of Vipera palaestinae.

Integrins are receptors of the extracellular matrix (ECM), playing a vital role in pathophysiological processes. They bind to ECM ligands like collagens and can mediate wound healing as well as tumor metastasis and thrombosis, thus being a part of cell adhesion and migration as well as platelet aggregation. For this reason, identifying α2ß1 integrin-specific antagonists can assist in the development of drugs to treat tumor progression, angiogenesis, and cardiovascular diseases. Snake venoms have been shown to contain antagonists which target collagen-binding integrins. EMS16, rhodocetin, and VP12 are three toxins belonging to the C-type lectin-related protein family and have been proven to inhibit the α2ß1 integrin, specifically the α2 integrin A domain. To specifically isolate antagonists targeting the α2ß1 integrin A domain, we developed a protocol based on affinity chromatography. Using this novel approach, the toxin VP-i was isolated from Vipera palaestinae venom. We show that VP-i binds to the α2 integrin A domain and that it successfully inhibits adhesion of various cells to type I collagen as well as cell migration. Moreover, our results indicate that VP-i differs structurally from the previously purified VP12, although not functionally, and therefore is a further venom compound which can be utilized for drug development.

The role of platelets and their microparticles in rehabilitation of ischemic brain tissue.

Stroke is a leading cause of mortality and chronic disability. Therapies aimed at reducing stroke related morbidity are currently limited. Therefore it is very important to develop effective treatments that will maximize rehabilitation after stroke. Current efforts in the field of cellular therapy focus on stem cell transplantations. This approach involves biological and ethical complications and therefore, the use of endogenous neural stem cells (eNSC) for repairing damage in various neurological disorders has been suggested. eNSCs reside in specialized vascular niches in defined regions, such as the subventricular zone (SVZ) of the lateral ventricle. These cells have an unlimited potential to create newborn cells. Interrelations between newborn neural and endothelial cells have an important role in eNSC survival, maturation, migration and differentiation and neurogenesis occurs in close spatio-temporal association with vessel growth in these niches. Previous studies have shown that application of external factors can boost long-term endogenous repair mechanisms in the cerebral cortex. Activated platelets and their microparticles contain a variety of growth and trophic factors essential to angiogenesis and neurogenesis and may therefore serve as novel therapeutic agents for brain injury. Specifically, factors from platelets and their microparticles may promote neurogenesis by stimulating eNSC proliferation, migration and differentiation, and by stimulating niche angiogenesis and the release of neurogenic signals from endothelial cells and astrocytes. In this review we will show that combined augmentation of angiogenesis, neurogenesis and neuroprotection using platelets and their microparticles is feasible and results in improved functional gain after stroke.

Involvement of platelet derived microparticles in tumor metastasis and tissue regeneration.

Platelets play a major role in hemostasis, but are also involved in vascular biology processes such as angiogenesis and tumor metastasis. Activated platelets release many proteins favoring wound healing and promoting angiogenesis. Microparticles (MP) are small plasma membrane vesicles shed from cells upon their activation or apoptosis. Platelet-derived microparticles (PMP) constitute the majority of the pool of MP circulating in the blood. PMP express and may transfer functional receptors, stimulate the release of cytokines, activate intracellular signaling pathways, promote angiogenesis, and are involved in tissue regeneration and cancer metastasis. We investigated the effect of PMP on cancer cells metastasis and their potential beneficial effect in an ischemic stroke model.

Identification of a promising drug candidate for the treatment of type 2 diabetes based on a P2Y(1) receptor agonist.

The activation by extracellular nucleotides of pancreatic P2Y receptors, particularly, the P2Y(1)R subtype, increases insulin secretion. Therefore, we developed analogues of the P2Y(1)R receptor agonist 2-MeS-ADP, as potential antidiabetic drugs. Analogue 3A was found to be a potent P2Y(1)R agonist (EC(50) = 0.038 µM vs 0.0025 µM for 2-MeS-ADP) showing no activity at P2Y(2/4/6)Rs. Analogue 3A was stable at pH 1.4 (t(1/2) = 7.3 h) and resistant to hydrolysis vs 2-MeS-ADP by alkaline phosphatase (t(1/2) = 6 vs 4.5 h), human e-NPP1 (4% vs 16% hydrolysis after 20 min), and human blood serum (30% vs 50% hydrolysis after 24 h). Intravenous administration of 3A in naive rats decreased blood glucose from 155 mg/dL to normal values, ca. 87 mg/dL, unlike glibenclamide, leading to subnormal values (i.e., 63 mg/dL). Similar observations were made for streptozotocin (STZ)-treated and db(+)/db(-) mouse models. Furthermore, 3A inhibits platelet aggregation in vitro and elongates bleeding time in mice (iv administration of 30 mg of 3A/kg), increasing bleeding time to 16 vs 9 min for Prasugrel. Oral administration of 30 mg/kg 3A to rats increased tail bleeding volume, similar to aspirin. These findings suggest that 3A may be an effective treatment for type 2 diabetes by reducing both blood glucose levels and platelet aggregation.

Platelet microparticles induce angiogenesis and neurogenesis after cerebral ischemia.

Activated platelets shed microparticles, which contain a variety of growth factors central to angiogenesis and neurogenesis. The aim of this study was to explore whether platelet derived microparticles (PMP) can boost endogenous neural stem cells dependent repair mechanisms following stroke in a rat model. To examine the effects of PMP therapy in-vivo, we delivered PMP or vehicle via a biodegradable polymer to the brain surface after permanent middle cerebral artery occlusion (PMCAO) in rats. Rats were tested with the neurological severity score and infarct volumes were measured at 90 days post-ischemia. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischemic brain. The results show that PMP led to a dose dependent increase in cell proliferation, neurogenesis and angiogenesis at the infarct boundary zone and significantly improved behavioral deficits.

Comparative analysis of platelet-derived microparticles reveals differences in their amount and proteome depending on the platelet stimulus.

Platelet-derived microparticles (PMP) are elevated in a number of disorders (e.g. cardiovascular disease and cancer). In the present study, we have carried out a high-resolution 2-DE-based proteomic analysis of PMP originated following platelet activation with different stimulus. Flow cytometric analysis revealed a higher average number of microparticles when platelets are activated with shear (1800 s(-1)) compared to 0.5 U/mL thrombin. Regarding the proteomic analysis, 30 protein features were found to be differentially regulated between shear and thrombin groups in the pI 4-7 range, from which 28 were successfully identified by MS, corresponding to 26 open-reading frames. Signaling proteins constituted the major functional group, including membrane receptors, and adapters. Ingenuity Pathways Analysis (IPA) software revealed that 21 of the 26 differentially regulated unique proteins identified are part of a common network related to cell assembly and organization and cell morphology. Western blotting analyses confirmed that Dok-2 and the integrin α6 are differentially regulated in PMP depending on the platelet stimulus. Interestingly, both proteins participate in mechanisms regulating angiogenesis so could be part of PMP regulation of endothelial cells function. In conclusion, this study shows that platelets shed microparticles in different amounts and with a different proteome depending on the stimulus.This article is part of a Special Issue entitled: Integrated omics.

Platelet microparticles promote neural stem cell proliferation, survival and differentiation.

Platelet microparticles (PMP) are small subcellular fragments, shed upon platelet activation. PMP host a variety of cytokines and growth factor that were previously shown to affect angiogenesis and postischemic tissue regeneration. This study attempted to explore the effect of PMP on neural stem cell (NSC) proliferation, survival and differentiation. Cells were grown as neurospheres and treated with PMP, or relevant growth factors, sphere size and cell fates were evaluated. PMP treatment led to larger neurospheres with increased cell survival. PMP treatment was comparable with the effect of acceptable single growth factors such as fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). PMP treatment also increased the differentiation potential of NSC to glia and neurons. Specific growth factor inhibitors only partly blocked these effects, which were associated with increments in ERK and Akt phosphorylation. In this study, we show that various growth factors contained within the PMP promote neuronal cell proliferation, survival and differentiation. The results suggest a role for platelet microparticles in augmenting endogenous neural progenitor and stem cells angiogenesis and neurogenesis that might be utilized for treatment following brain injury.

Underdiagnosed menorrhagia in adolescents is associated with underdiagnosed anemia.

OBJECTIVE: To test the hypothesis that adolescent girls with menorrhagia rarely seek medical attention. STUDY DESIGN: A total of 705 adolescent girls attended a lecture on menorrhagia, completed an initial anonymous questionnaire, and were asked to participate in a more comprehensive study comprising a detailed bleeding questionnaire, a pictorial blood loss assessment chart, and blood tests. RESULTS: A total of 105 adolescents (15%) reported they had heavy periods on the initial questionnaire. Among the 94 girls who completed the full questionnaire, 34 reported menorrhagia (36%; 95% CI, 26.5%-46.7%). Almost one-third (11 of 34) of these girls did not perceive having menorrhagia according to their response to the initial questionnaire. Menorrhagia was not related to age, years since menarche, or family history of menorrhagia. Among the 62 girls who consented to blood testing, 6 had anemia (9.6%; 95% CI, 3.6%-19.6%), all of whom had bleeding symptoms. CONCLUSION: Using standardized questionnaires, we were able to identify adolescents with menorrhagia associated with anemia. Importantly, some of these adolescents were not aware of having menorrhagia and/or anemia. Screening programs for menorrhagia in schools could result in better detection of menorrhagia among adolescents and consequent appropriate referral for medical consultation.

Development, cell differentiation, angiogenesis--microparticles and their roles in angiogenesis.

Cells of various types release small membrane vesicles called microparticles (MP) on their activation, as well as during the process of apoptosis. The properties and roles of MP generated in different contexts are diverse and are determined by their parent cell and the pathway of their generation, which affects their content. MP are involved in multiple cellular functions, including immunomodulation, inflammation, coagulation, and intercellular communication. MP are able to deliver molecular signals in the form of lipids, proteins, nucleic acids, or functional transmembrane proteins from the parent cell to distantly located targets. In this review, we summarize some of the current knowledge regarding MP and their functional roles in transfer of proteins, nucleic acids, and signal transduction stimulators between cells of different origins in different settings. We will focus on the ability of MP to mediate angiogenesis-related signals and their effect on cell development. The investigation of MP could elucidate new cellular communication pathways and may lead to better understanding of pathophysiological processes. From a clinical point of view, MP may serve as biomarkers for disease status and may be found useful for developing novel therapeutic strategies targeting angiogenesis-related conditions.

Isolation and cultivation of integrin alpha(6)beta(1)-expressing salivary gland graft cells: a model for use with an artificial salivary gland.

Regeneration of the salivary glands' (SGs) normal function for patients with cancer of the head and neck treated with irradiation would be a major contribution to their quality of life. This could be accomplished by re-implantation of autologous SG cells into the residual irradiated tissue or by implantation of tissue-engineered artificial SGs. Both methods depend on the isolation of cells able to propagate and differentiate into SG epithelial cells. Recently, it has been shown that SG integrin alpha(6)beta(1)-expressing (SGIE) cells have stem cell capabilities, but these cells could be isolated only after duct ligation insult requiring surgical intervention. Because such an invasive procedure is not clinically acceptable for these patients, our aim in the present study was to explore the use of immuno-magnetic separation of untreated and short heat stress-conditioned rats as a less-insulting methodology for enhancement of these cells. Our results show that submandibular SGIE cells could be isolated and cultivated from untreated animals. However, short heat stress (HS) increased the number of isolated SGIE cells 4.7-fold and their proliferation and clonal capability 4.6-fold and 3 fold, respectively. We believe that SGIE graft cells may be suitable candidates for future tissue-engineered SGs that have been damaged by irradiation in patients with head and neck cancer.