Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment.

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

A plasma cell-based pericardial effusion leading to tamponade in a patient with multiple myeloma - a case report and review of the literature.

A rare case of extramedullary multiple myeloma causing cardiac tamponade secondary to a plasma cell-based pericardial effusion is described. A systematic search using PubMed (National Library of Medicine) was used to identify a further 27 cases dating back to 1970. Case characteristics, treatment strategies, and survival time following tamponade are discussed. Linear regression demonstrated a weak but statistically significant correlation between survival time following tamponade and treatment with systemic chemotherapy and steroids (ß=16.8 weeks, P=.009). However, this manifestation of extramedullary multiple myeloma still conveys a dismal prognosis with a median survival following tamponade of only 6 weeks based on our review.

Infiltrating Cardiac Synovial Sarcoma Presenting as Acute Cerebrovascular Accident.

Primary cardiac sarcoma is a rare malignant myocardial neoplasm that does not exhibit gender predominance or age predilection. The classification of these tumors includes several subtypes, of which synovial sarcoma is a rare manifestation. When present, these tumors portend a poor prognosis with high morbidity and mortality that is attributable to their inherent infiltrative capacity, especially in the absence of treatment. The general consensus for treatment is surgical excision and neoadjuvant chemotherapy and radiotherapy. In this report, a case of synovial sarcoma involving the left ventricular outflow tract and aortic valve is presented.

Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants.

Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPO(M)) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPO(P) from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPO(P). Using this process, up to 31% of the asialo-rhuEPO(P) could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO(P) expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.

C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants.

KEY MESSAGE: C -terminally fused Strep -tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at the C -terminus of rhuEPO should be avoided. Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low-cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPO(P) was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep -tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPO(P) expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPO(P) revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPO(P). However, Strep-tag II was detected on asialo-rhuEPO(P) that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.

ST-elevation myocardial infarction and myelodysplastic syndrome with acute myeloid leukemia transformation.

Acute myocardial infarction and acute myeloid leukemia are rarely reported as concomitant conditions. The management of ST-elevation myocardial infarction (STEMI) in patients who have acute myeloid leukemia is challenging: the leukemia-related thrombocytopenia, platelet dysfunction, and systemic coagulopathy increase the risk of bleeding, and the administration of thrombolytic agents can be fatal. We report the case of a 76-year-old man who presented emergently with STEMI, myelodysplastic syndrome, and newly recognized acute myeloid leukemia transformation. Standard antiplatelet and anticoagulation therapy were contraindicated by the patient's thrombocytopenia and by his reported ecchymosis and gingival bleeding upon admission. He declined cardiac catheterization, was provided palliative care, and died 2 hours after hospital admission. We searched the English-language medical literature, found 8 relevant reports, and determined that the prognosis for patients with concomitant STEMI and acute myeloid leukemia is clearly worse than that for either individual condition. No guidelines exist to direct the management of STEMI and concomitant acute myeloid leukemia. In 2 reports, dual antiplatelet therapy, anticoagulation, and drug-eluting stent implantation were used without an increased risk of bleeding in the short term, even in the presence of thrombocytopenia. However, we think that a more conservative approach--balloon angioplasty with the provisional use of bare-metal stents--might be safer. Simultaneous chemotherapy for the acute myeloid leukemia is crucial. Older age seems to be a major risk factor: patients too frail for emergent treatment can die within hours or days.

Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and ß1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P) bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P) (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M) (21%). The cytoprotective effect of the asialo-rhuEPO(P) was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

Weekly doxorubicin increases coronary arteriolar wall and adventitial thickness.

BACKGROUND: Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening. METHODS: Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, n = 7), or low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of DOX. The animals were treated for 2-12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness). RESULTS: Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm; p = 0.22). In comparison to animals receiving weekly saline, animals receiving weekly injections of 2.5 mg/kg of DOX experienced an increase in medial (23±2 µm vs. 13±3 µm; p = 0.005), and total wall thickness (51±4 µm vs. 36±5 µm; p = 0.022), respectively. These increases, as well as adventitial thickening became more prominent after normalizing for lumen diameter (p<0.05 to p<0.001) and after adjusting for age, weight, and total cumulative DOX dose (p = 0.02 to p = 0.01). Animals receiving low dose DOX trended toward increases in adventitial and total wall thickness after normalization to lumen diameter and accounting for age, weight, and total cumulative DOX dose (p = 0.06 and 0.09, respectively). CONCLUSION: In conclusion, these data demonstrate that weekly treatment of rats with higher doses of DOX increases coronary arteriolar medial, adventitial, and total wall thickness. Future studies are warranted to determine if DOX related coronary arteriolar effects are reversible or preventable, exacerbate the known cardiomyopathic effects of DOX, influence altered resting or stress-induced myocardial perfusion, or contribute to the occurrence of myocardial infarction.

Multicenter trial of desirudin for the prophylaxis of thrombosis: an alternative to heparin-based anticoagulation (DESIR-ABLE).

Desirudin, a subcutaneously (SC) administered direct thrombin inhibitor, is indicated for prevention of venous thromboembolic events (VTEs) after total joint replacement surgery. DESIR-ABLE (multicenter trial of desirudin for the prophylaxis of thrombosis: an alternative to heparin-based anticoagulation) was a multicenter, open-label, single-arm study of hospitalized patients requiring VTE protection designed to extend the safety profile for desirudin to include a broad population of perioperative/critically ill patients. The primary end point was major bleeding. A total of 516 patients undergoing major surgery (378, 73%) or who were medically ill with prolonged immobility (138, 27%) were enrolled at 19 centers and received desirudin 15 mg Q12H. Many patients had high-risk features for bleeding and thrombosis such as thrombocytopenia (<100 x 10(9)/mL, n = 50), severe obesity (body mass index >35, n = 145), and renal impairment (creatinine clearance <60 mL/min, n = 292). There were no major bleeds and no VTE-related deaths in this study. The DESIR-ABLE demonstrated the safety of desirudin in critically ill perioperative and medical patients. Trials in specific surgical or medically ill patients are needed to confirm these findings.

Interference of thrombin in immunological assays for hirudin specific antibodies.

Recombinant hirudins (desirudin, lepirudin) are direct thrombin inhibitors administered as anticoagulants for heparin-induced thrombocytopenia (HIT) and venous thromboembolism (VTE) prophylaxis. Although these small polypeptides are widely used, concern exists over reports of antigenicity. In the largest study of r-hirudin immunogenicity to-date, we evaluated the prevalence, quantity and specificity of IgG immune responses to desirudin (15 mg SC q12h for as long as clinically required) in 245 surgical and medically-ill subjects enrolled in DESIRABLE, a multicenter, open-label, clinical trial of hospitalized patients requiring VTE prophylaxis. Sera obtained before and 30 days after desirudin administration were analyzed for IgG anti-desirudin by immunoenzymetric assay using immobilized desirudin to bind desirudin-reactive antibody and peroxidase conjugated monoclonal-anti-human IgG Fc to detect bound IgG antibody. Of 245 study subjects, 19 (7.7%) were antibody "responders" (>2-fold increase in IgG antibody levels with >50% inhibition by desirudin 30 days post-treatment). There were no differences between responders and non-responders in incidence of clinical outcomes or bleeding-related adverse events. Forty-six patients had detectable desirudin-reactive IgG antibody prior to treatment, with no significant increase in antibody levels after exposure and no increase in clinical events. The origin of pre-existing hirudin-reactive IgG antibody requires further investigation involving suspected anti-thrombin-thrombin interactions. These results indicate a low potential for immunogenicity, with <8% of patients developing IgG antibodies after desirudin administration for VTE prophylaxis. In contrast to reports on lepirudin, production of anti-hirudin antibodies to desirudin has no apparent effect on clinical events.

N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin.

UNLABELLED: Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and ß1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human ß1,4-galactosyltransferase. The penultimate ß1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human ß1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal ß1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).

Calcification after myocardial infarction is independent of amniotic fluid stem cell injection.

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. (J Mol Cell Cardiol. 42 (2007) 746-759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

Chronic kidney disease and dipstick proteinuria are risk factors for stent thrombosis in patients with myocardial infarction.

BACKGROUND: Kidney failure (stage 5 chronic kidney disease [CKD]) is an independent risk factor for stent thrombosis (ST). Moderate (stage 3-4) CKD and proteinuria are both associated with adverse cardiovascular events, including worse outcomes after myocardial infarction (MI). Whether moderate CKD and proteinuria increase the risk of ST after MI is not known. This study evaluated the risk of ST associated with moderate CKD and dipstick proteinuria. METHODS: We retrospectively analyzed clinical and laboratory data from 956 non-stage 5 CKD patients who were admitted with MI and received intracoronary stenting. Clinical follow-up was collected at 1 year for definite or probable ST, as well as for all-cause mortality, nonfatal MI or death, and target vessel revascularization or coronary artery bypass graft surgery. RESULTS: After adjustment for multiple clinical and biochemical covariates, patients with both estimated glomerular filtration rate (GFR) of 15 to 59 mL min(-1) 1.73 m(-2) and > or =30 mg/dL dipstick proteinuria had increased cumulative incidence of ST (hazard rate [HR] 3.69, 95% CI 1.54-8.89), all-cause mortality (HR 2.68, 95% CI 1.34-5.37), and nonfatal MI or death (HR 3.20, 95% CI 1.77-5.81) at 1 year. In addition, estimated GFR of 15 to 59 mL min(-1) 1.73 m(-2) was a significant independent predictor of ST (HR 2.61, 95% CI 1.33-5.10). Dipstick proteinuria > or =30 mg/dL was associated with a trend toward increased risk for all outcomes. CONCLUSIONS: In an acute MI population, moderate CKD was identified as a novel prognostic marker for ST. In addition, patients with both decreased GFR and proteinuria had higher incidences of all-cause mortality and nonfatal MI or death than patients with either condition alone.

Derived fibrinogen compared with C-reactive protein and brain natriuretic peptide for predicting events after myocardial infarction and coronary stenting.

BACKGROUND: After myocardial infarction (MI), biomarkers can be helpful to identify patients who might benefit from more intensive therapies. The prothrombin time-derived fibrinogen (PTDF) assay is widely available and relatively inexpensive. We determined whether PTDF predicts events in patients with MI and compared this assay with brain natriuretic peptide (BNP) and C-reactive protein (CRP). METHODS: We retrospectively analyzed data from 915 patients admitted with MI. Follow-up was collected at 1 year for major adverse cardiac events (MACE) defined as death from any cause, nonfatal MI or death, target vessel revascularization, or coronary artery bypass grafting. RESULTS: Patients in the fourth quartile of PTDF were older and had more risk factors but fewer ST-elevation MI and lower peak troponin values. The fourth quartiles of PTDF, CRP, and BNP were associated with increased MACE compared with the first quartiles with hazard ratios of 2.08 (1.30-3.33), 1.94 (1.22-3.07), and 2.56 (1.57-4.18), respectively, findings that remained significant after adjustment. When outcomes by strata of PTDF were examined, CRP failed to add additional prognostic value. Higher BNP levels predicted MACE in the upper but not lower stratum of PTDF. CONCLUSION: In patients with MI, PTDF is a predictor of MACE at 1 year, with equivalent value compared to BNP and CRP. With low PTDF levels, neither BNP nor CRP adds prognostic value. At elevated PTDF values, higher BNP, but not CRP, identifies a higher-risk population. Therefore, PTDF can be substituted for CRP, with BNP being useful in the presence of elevated PTDF.

Transglutaminases: the missing link in nephrogenic systemic fibrosis.

Nephrogenic systemic fibrosis (NSF), also known as nephrogenic fibrosing dermopathy (NFD), occurs in renal failure patients after gadolinium contrast exposure. The fibrosis of the dermis and subcutaneous septae accompanies fibrosis of other organs, including the heart, liver, lungs, and muscle. The fibrotic skin demonstrates increased dermal collagen, fibroblasts, and mucin. The mechanism by which gadolinium is associated with fibrosis is not known. We tested the hypothesis that upregulation of transglutaminases contributes to the fibrosis seen in the organs, including skin, of renal failure patients exposed to gadolinium contrast. We performed immunohistochemical studies using antibodies to transglutaminase-2, factor XIIIa, transglutaminase isopeptide, and the histiocyte marker CD68 on five archived skin biopsies of NSF. The results indicate that the dermal fibroblasts and histiocytes of NSF express transglutaminase-2, CD68, factor XIIIa, and transglutaminase isopeptide, indicating increased expression and/or activation of transglutaminases in NSF. We recommend further research into the use of transglutaminase inhibitors in the treatment and prevention of NSF.

Multimer formation by FKBP-12: roles for cysteine 23 and phenylalanine 36.

FKBP-12 mediates the immunosuppressive actions of FK506 and rapamycin, and modulates the activities of the ryanodine, IP3 and type 1 TGF-ss receptors. Additionally, FKBP-12 possesses cis-trans peptidylprolyl isomerase (rotamase) activity. We have discovered that recombinant FKBP-12 readily forms a dimer and a small amount of trimer under nonreducing conditions. A mutant with substitution at the sole cysteine residue of FKBP-12 (C23S) did not form dimers or trimers. Using mutants with 5% or less rotamase activity, the formation of dimers was independent of enzymatic activity. The formation of trimers was abrogated by a F36Y substitution, even though dimer formation was preserved. Dimers were also observed with native FKBP-12 that was detached from rabbit skeletal muscle ryanodine receptors using FK590. The multimers of FKBP-12 could interact with molecular targets distinctly from the FKBP-12 monomer, for example, by facilitating the assembly of multimeric receptors or coordinating the activity of receptor subunits.

Low plasma levels of FGF-2 and PDGF-BB are associated with cardiovascular events in type II diabetes mellitus (diabetes heart study).

OBJECTIVE: We tested associations of the growth factors VEGF, FGF-2, HGF and PDGF-BB with coronary artery calcium scores and cardiovascular events (CVD) in type 2 diabetes mellitus (T2DM). METHODS: A cross-sectional study selected 40 frequency matched (by age, gender and race) subjects with T2DM from the first (0-111) and the third (> 1400) coronary artery calcium (CAC) score tertiles in the Diabetes Heart Study (DHS), in which 36 were with and 41 were without history of CVD events. Plasma levels of VEGF, FGF-2, HGF and PDGF-BB were measured in all subjects. RESULTS: None of the growth factors was significantly different between the first and third CAC score tertiles. Mean plasma FGF-2 and PDGF-BB levels were significantly higher in the group without prior CVD events compared with the group with prior CVD events [mean(95%CI): 219.20 (194.42-247.15) vs. 152.93 (135.64-172.43) pg/ml, p=0.03] and [mean(95%CI): 106.70 (89.12-127.74) vs. 61.56 (50.91-74.44) pg/ml, p=0.03], respectively. Subgroup analysis in the first CAC tertile showed a significantly higher PDGF-BB levels in those without compared with those with CVD events [mean (95%CI): 208.36 (190.57-228.15) vs. 102.93 (80.64-125.21) pg/ml, p=0.004]. CONCLUSION: Plasma growth factor levels were not significantly different between the extremes of CAC scores in T2DM. However, low plasma levels of PDGF-BB and FGF-2 are associated with prior cardiovascular events in T2DM. Studies are needed to confirm our results and also to establish temporality of this association.

Effect of genetic variations in platelet glycoproteins Ibalpha and VI on the risk for coronary heart disease events in postmenopausal women taking hormone therapy.

Millions of women still use postmenopausal hormone therapy (HT). We genotyped 2090 women in Heart and Estrogen/progestin Replacement Study for functional polymorphisms in GP1BA and GP6 and assessed the coronary heart disease (CHD) event rate over 5.8 years of follow-up. In patients receiving placebo, there was an increased CHD death/myocardial infarction (MI)/unstable angina (UA) event rate in carriers of the GP1BA -5C allele (adjusted [adj] P = .006). HT increased the hazard ratio (HR) of CHD events in patients with the GP1BA -5TT genotype by 16% and reduced the HR in patients with the TC+CC genotypes by 46% (adj interaction P < .001). HT reduced the HR in patients with the GP6 13254TT genotype by 17% but increased the HR in patients with the TC+CC genotypes by 35% (adj interaction P < .001). Furthermore, HT increased the HR of CHD events in patients with the GP1BA -5TT plus GP6 13254TC+CC genotypes by 57% and reduced the HR in patients with the GP1BA -5TC+CC plus GP6 13254TT genotypes by 55% (adj interaction P < .001). In postmenopausal women with established CHD, these polymorphisms of platelet genes were predictors of CHD events and significantly modified the effects of HT on CHD risk. It will be important to replicate these findings in other studies.

Roles of transglutaminases in cardiac and vascular diseases.

All transglutaminases share the common enzymatic activity of transamidation, or the cross-linking of glutamine and lysine residues to form N epsilon (gamma-glutamyl) lysyl isopeptide bonds. The plasma proenzyme factor XIII is responsible for stabilizing the fibrin clot against physical and fibrinolytic disruption. Another member of the transglutaminase family, tissue transglutaminase or TG2 is abundantly expressed in cardiomyocytes, vascular cells and macrophages. The transglutaminases have a variety of functions independent of their transamidating activity. For example, TG2 binds and hydrolyzes GTP, thereby fostering signal transduction by several G protein coupled receptors. Accumulating evidence points to novel roles for factor XIII and TG2 in cardiovascular biology including: (a) modulating platelet activity, (b) regulating glucose control, (c) contributing to the development of hypertension, (d) influencing the progression of atherosclerosis, (e) regulating vascular permeability and angiogenesis (f) and contributing to myocardial signaling, contractile activity and ischemia/reperfusion injury. In this review, we summarize the cardiovascular biology of two members of the family of transglutaminases, Factor XIII and TG2.

Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts.

BACKGROUND: P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. METHODS: Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. RESULTS: Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. CONCLUSION: Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. CONDENSED ABSTRACT: The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.