Platelet dysfunction in diabetes mellitus : Impact of hyperglycemia and glycoprotein GPIIb/IIIa inhibition

Diabetes mellitus (DM) is associated with increased cardiovascular morbidity and mortality, due to diabetic angiopathy. Hyperglycemia is one of the factors that may cause platelet dysfunction in diabetic patients. This work investigated mechanisms underlying hyperglycemia-induced platelet dysfunction, and its impact in DM patients. Antiplatelet treatment by glycoprotein (GP) IIb/IIIa blockade provides more efficient thrombotic protection in diabetic than in non-diabetic subjects. We therefore also examined the effect(s) of GPIIb/IIIa blockade on platelet procoagulant activity in vitro, and their possible impact in type 2 DM (T2DM) patients. High glucose dose-dependently enhanced agonist-induced platelet activation in vitro through increased osmolality, as shown by experiments with different sugars. High glucose enhanced platelet P-selectin expression but not fibrinogen binding in ADP-stimulated samples, and enhanced both platelet P-selectin expression and fibrinogen binding in thrombin receptor activating peptide (TRAP)-stimulated whole blood. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression by high glucose, but abolished the enhancement of TRAP-induced platelet fibrinogen binding. Superoxide anion scavenging by superoxide dismutase attenuated the enhancement by high glucose of ADP- and TRAPinduced platelet P-selectin expression, but did not influence platelet fibrinogen binding. The influence of a standardized carbohydrate-rich meal on platelet reactivity was assessed in T2DM patients and matched healthy controls; the patients also received oral antidiabetic treatment with repaglinide and glibenclamide in a cross-over study. In T2DM patients food intake caused postprandial hyperglycemia and markedly augmented platelet P-selectin expression and platelet-leukocyte aggregation induced by the thromboxane A2 (TxA2) analogue U46619, and slightly enhanced ADP-induced platelet P-selectin expression. The meal caused no postprandial hyperglycemia or enhancement of platelet reactivity in the healthy controls. Both repaglinide and glibenclamide treatment only mildly reduced the postprandial hyperglycemia, and did not attenuate the meal-induced enhancement of platelet reactivity in the T2DM patients. Platelet procoagulant activity was assessed by annexin V binding, reflecting phosphatidylserine appearance on the platelet surface membrane (PS exposure), and effects of GPIIb/IIIa blockade were examined in washed platelets from healthy volunteers. Thrombin-induced PS exposure was cell-cell contact dependent. GPIIb/IIIa blockade inhibited this by enhancing translocase activity and inhibiting scramblase activity. Thrombin-induced platelet derived microparticle (PDMP) formation was not influenced by GPIIb/IIIa blockade. Platelet procoagulant activity, i.e, PS and factor Va (FVa) expression, and PDMP generation, was compared in T2DM patients and healthy subjects. The unstimulated platelet procoagulant activity was only slightly higher in patients compared to controls. TRAP enhanced the PS exposure and FVa expression of platelets, and PDMP generation more markedly among T2DM patients. These hyperprocoagulant alterations in T2DM patients were associated with elevated thrombin generation and a shortened plasma clotting time. GPIIb/IIIa blockade with c7E3 or SR121566 reduced the platelet PS exposure and FVa expression, and also reduced the procoagulant activity seen in T2DM patients. In conclusion, high glucose levels enhance the platelet reactivity to agonist stimulation through elevated osmolality. This occurs via superoxide anion production that enhances platelet P-selectin expression (secretion), and PKC signalling that enhances TRAP-induced fibrinogen binding (aggregablity). Food intake markedly enhances TxA2-induced platelet activation in type 2 diabetic patients but not in healthy subjects, presumably due to postprandial hyperglycemia. The procoagulant PS exposure is cell-cell contact dependent and it is inhibited by GPIIb/IIIa blockade. The inhibition of PS exposure by GPIIb/IIIa blockade occurs via increased translocase and reduced scramblase activity. Moreover, GPIIb/IIIa blockade attenuates the platelet hyperprocoagulant activity which is seen in T2DM patients. This thesis provides additional information on mechanisms that probably contribute to the clinical benefits of glycemic control, and of GPIIb/IIIa blocker treatment, which might be useful in shaping more efficient antithrombotic therapy for T2DM patients

Effects of high glucose on platelet activation

Diabetes mellitus (DM) is associated with increased cardiovascular mortality and morbidity, due to diabetic angiopathy. Hyperglycaemia is one of the factors that may cause platelet dysfunction in diabetic patients. This work investigated the mechanisms underlying hyperglycaemia-induced platelet hyperreactivity and its impact in diabetic patients. The studies involved healthy subjects and type 2 DM patients. Platelet and leukocyte activation and platelet- leukocyte aggregate formation were measured by whole blood flow cytometry. High glucose dose-dependently enhanced Agonist-induced platelet activation in vitro. The enhancement was agonist-specific. High glucose elevated platelet P-selectin expression (reflecting platelet secretion) but not fibrinogen binding (platelet aggregability) in adenosine diphosphate (ADP)stimulated samples, whilst it enhanced both platelet P-selectin expression and fibrinogen binding in thrombin receptor activating peptide (TRAP)-stimulated blood. High glucose also elevated TRAP-, but not ADP-induced platelet-leukocyte aggregate formation. Similar effects were seen with 30 mM L-glucose, sucrose, and galactose. Protein kinase C (PKC) blockade by bisindolylinaleimide I (BIM-I) did not counteract the enhancement of platelet P-selectin expression by high glucose, but abolished the enhancement by high glucose of TRAP-induced platelet fibrinogen binding. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the enhancement by high glucose of ADP- and TRAP-induced platelet P-selectin expression, but did not influence platelet fibrinogen binding. The influence of a carbohydrate-rich meal on platelet reactivity was investigated in type 2 DM patients and matched healthy subjects. Platelet activation was measured before and after a standardized meal in an open, randomized, cross-over study of oral antidiabetic treatment with repaglinide and glibenclamide. ADP and the thromboxane A2 (TxA2) analogue U46619 were used as platelet agonists. Measurements were performed on three occasions: at baseline (on diet only), and after six weeks of treatment with either drug. The results revealed that food intake markedly augmented platelet P-selectin expression and platelet-leukocyte aggregation induced by U46619. The meal also slightly enhanced ADP-induced platelet P-selectin expression, but did not influenced platelet activation in unstimulated samples. Similar enhancements of platelet reactivity were, however, not seen in healthy subjects who did not have postprandial hyperglycemia. Both repaglinide and glibenclamide treatment mildly reduced the postprandial hyperglycemia in the patients, but did not attenuate the meal-induced enhancement of platelet reactivity. In conclusion, high glucose levels enhance platelet reactivity to agonist stimulation through elevated osmolality. This occurs via superoxide anion production that enhances platelet P-selectin expression (secretion), and PKC signalling that enhances TRAP-induced fibrinogen binding (aggregablity). Food intake enhances TxA2- and ADP-induced platelet activation in type 2 diabetic patients but not in healthy subjects, presumably due to postprandial hyperglycemia

Whole-exome sequencing identifies R1279X of MYH6 gene to be associated with congenital heart disease

Abstract Background Myosin VI, encoded by MYH6, is expressed dominantly in human cardiac atria and plays consequential roles in cardiac muscle contraction and comprising the cardiac muscle thick filament. It has been reported that the mutations in the MYH6 gene associated with sinus venosus atrial septal defect (ASD type III), hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Methods Two patients in an Iranian family have been identified who affected to Congenital Heart Disease (CHD). The male patient, besides CHD, shows that the thyroglossal sinus, refractive errors of the eye and mitral stenosis. The first symptoms emerged at the birth and diagnosis based on clinical features was made at about 5 years. The family had a history of ASD. For recognizing mutated gene (s), whole exome sequencing (WES) was performed for the male patient and variants were analyzed by autosomal dominant inheritance mode. Results Eventually, by several filtering processes, a mutation in MYH6 gene (NM_002471.3), c.3835C > T; R1279X, was identified as the most likely disease-susceptibility variant and then confirmed by Sanger sequencing in the family. The mutation frequency was checked out in the local databases. This mutation results in the elimination of the 660 amino acids in the C-terminal of Myosin VI protein, including the vital parts of the coiled-coil structure of the tail domain. Conclusions Our study represents the first case of Sinus venosus defect caused directly by MYH6 stop codon mutation. Our data indicate that by increase haploinsufficiency of myosin VI, c.3835C > T mutation with reduced penetrance could be associated with CHD

Reduced menin expression impairs rapamycin effects as evidenced by an increase in mTORC2 signaling and cell migration

BACKGROUND: Mammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin. METHODS: In addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells. RESULTS: PDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin-/- MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin-/- cells as compared to menin+/+ MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin-/- MEFs than menin+/+ MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin-/- MEFs, as well as in menin-silenced BON1 cells. CONCLUSIONS: Menin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin

Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D

<p>Abstract</p> <p>Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca²⁺/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.</p

The first case of NSHL by direct impression on EYA1 gene and identification of one novel mutation in MYO7A in the Iranian families

Objective(s): Targeted next-generation sequencing (NGS) provides a consequential opportunity to elucidate genetic factors in known diseases, particularly in profoundly heterogeneous disorders such as non-syndromic hearing loss (NSHL). Hearing impairments could be classified into syndromic and non-syndromic types. This study intended to assess the significance of mutations in these genes to the autosomal recessive/dominant non-syndromic genetic load among Iranian families. Materials and Methods: Two families were involved in this research and two patients were examined by targeted next-generation sequencing. Here we report two novel mutations in the MYO7A and EYA1 genes in two patients detected by targeted NGS. They were confirmed by Sanger sequencing and quantitative real-time PCR techniques. Results: In this investigation, we identified a novel mutation in MYO7A, c.3751G>C, p.A1251P, along with another previously identified mutation (c.1708C>T) in one of the cases. This mutation is located in the MYTH4 protein domain which is a pivotal domain for the myosin function. Another finding in this research was a novel de-novo deletion which deletes the entire EYA1 coding region (EX1-18 DEL). Mutations in EYA1 gene have been found in branchiootorenal (BOR) syndrome. Interestingly the patient with EYA1 deletion did not show any other additional clinical implications apart from HL. This finding might argue for the sole involvement of EYA1 function in the mechanism of hearing. Conclusion: This investigation exhibited that the novel mutations in MYO7A, c.3751G>C, p.A1251P, and EYA1, EX1-18 DEL, were associated with NSHL. Our research increased the mutation spectrum of hearing loss in the Iranian population

MiR-486-3p was downregulated at microRNA profiling of adrenals of multiple endocrine neoplasia type 1 mice, and inhibited human adrenocortical carcinoma cell lines

Adrenocortical carcinoma is a rare aggressive disease commonly recurring regardless of radical surgery. Although data on genomic alterations in malignant tumors are accumulating, knowledge of molecular events of importance for initiation of adrenocortical transformation is scarce. In an attempt to recognize early molecular alterations, we used adrenals from young multiple endocrine neoplasia type 1 conventional knock-out mice (Men1(+/-)) closely mimicking the human MEN1 trait (i.e. transformation of pituitary, parathyroid, endocrine pancreatic, and adrenocortical cells). MicroRNA array and hierarchical clustering showed a distinct pattern. Twenty miRNAs were significantly upregulated and eleven were downregulated in Men1(+/-) compared to wild type littermates. The latter included the known suppressor miRNA miR-486-3p, which was chosen for transfection in human adrenocortical carcinoma cell lines H295R and SW13. Cell growth decreased in miR-486-3p overexpressing clones and levels of the predicted target gene fatty acid synthase (FASN) and its downstream product, palmitic acid, were lowered. In conclusion, heterozygous inactivation of Men1 in adrenals results in distinct miRNA profile regulating expression of genes with impact on tumorigenesis, e.g. transcription, nucleic acid and lipid metabolism. Low levels of miR-486-3p in the early stages of transformation may contribute to proliferation by increasing FASN and thus fatty acid production. FASN as a potentially druggable target for treatment of the devastating disease adrenocortical carcinoma warrants further studies

Three Novel Variants identified in FBN1

Abstract Background Marfan syndrome (MFS) is a multi‐systemic autosomal dominant disease of the connective tissue characterized by the early development of thoracic aneurysms/dissections, along with various manifestations of the ocular and skeletal systems. Due to the genetic and clinical heterogeneity, the clinical diagnosis of this disorder is challenging. Loss‐of‐function mutations in FBN1 (encodes fibrillin‐1) lead to MFS type 1. Also, similar mutations in transforming growth factor β receptor 2 (TGFBR2) gene cause MFS type 2. Both proteins involve in TGF‐β signaling. Methods In this study, genetic screening using a panel involving 14 genes, especially FBN1 and TGFBR2, were performed on seven representatives affected members of seven unrelated Iranian families suspected with MFS. To confirm the variants, Sanger sequencing was applied to other affected/unaffected members of the families. Results A total of 13 patients showed MFS manifestations. Using genetic screening, two novel and three previously reported variants in FBN1 were identified. We also detected two variants (a novel and a previously reported variant) in the TGFBR2 gene. Conclusion In this study, we introduce three novel variants identified through gene screening in seven Iranian MFS families. This report is expected to considerably improve genetic counseling for Iranian MFS families. Early precise molecular diagnosis can be helpful for better management and improving the life expectancy of these patients

A homozygote variant in the tRNA splicing endonuclease subunit 54 causes pontocerebellar hypoplasia in a consanguineous Iranian family

Abstract Background Homozygous loss‐of‐function mutations in TSEN54 (tRNA splicing endonuclease subunit 54; OMIM: 608755) cause different types of pontocerebellar hypoplasias (PCH) including PCH2, PCH4, and PCH5. The study aimed to determine the possible genetic factors contributing to PCH phenotypes in two affected male infants in an Iranian family. Methods We subjected two affected individuals in a consanguineous Iranian family. To systematically investigate the susceptible gene(s), whole‐exome sequencing was performed on the proband and a novel identified variant was confirmed by Sanger sequencing. We also analyzed 26 relatives in three generations using PCR‐restriction fragment length polymorphism (PCR‐RFLP) followed and confirmed by Sanger sequencing. Results Physical and medical examinations confirmed PCH in the patients. Besides, the proband showed bilateral moderate sensorineural hearing loss and structural heart defects as the novel phenotypes. The molecular findings also verified that two affected individuals were homozygote for the novel synonymous variant, NM_207346.2: c.1170G>A; p.(Val390Val), in TSEN54. PCR‐RFLP and Sanger sequencing elucidated that the parents and 16 relatives were heterozygote for the novel variant. Conclusion We identified a novel synonymous variant, c.1170G>A, in TSEN54 associated with PCH in an Iranian family. Based on this study, we strongly suggest using “TSENopathies” to show the overlapped phenotypes among different types of PCH resulted from TSEN causative mutations