Role of heterotrimeric G proteins in platelet activation and clot formation in platelets treated with integrin αIIbß3 inhibitor.

Mechanisms of platelet activation are triggered by thrombin, adenosine diphosphate (ADP), epinephrine, thromboxane A2, and other soluble agonists which induce signaling via heterotrimeric Gαq, Gαi, and Gα12/13 proteins. We have undertaken a study addressing the contribution of these G proteins to platelet activation and clot formation in the presence of eptifibatide, thus excluding outside-in signaling provided by integrin αIIbß3-fibrinogen engagement. Selective and combined activation of the G proteins was achieved by using combinations of platelet agonists and inhibitors. Platelet activation in platelet-rich plasma was evaluated by P-selectin expression using flow cytometry. Contribution of platelets to whole blood clotting was assessed by rotation thromboelastometry (ROTEM). Selective signaling of Gαq or Gαi but not Gα12/13 promoted P-selectin expression. Further enhancement of P-selectin expression was achieved by ADP-induced combined signaling of Gαq and Gαi, and to more extent by U46619 at high concentration (1.5 µM) induced combined signaling of Gαq and Gα12/13 while maximal P-selectin expression was achieved by thrombin receptor-activating peptide (TRAP)-induced combined signaling of Gαq, Gαi, and Gα12/13. In ROTEM, selective activation of Gαq, Gαi, or Gα12/13 failed to affect blood clotting. Combined signaling of Gαq and Gαi or Gαq and Gα12/13 or all three G proteins shortened the clotting time and stimulated clot strength. Pretreatment of platelets with acetylsalicylic acid did not change the effect of ADP but inhibited the effect of TRAP. Signaling of Gαq and Gα12/13 triggered by U46619 also stimulated clot formation. Combined signaling of either Gαq and Gαi or Gαq and Gα12/13 is sufficient to stimulate maximal platelet activation and enhanced clot formation in platelets treated with inhibitor of integrin αIIbß3. It could be suggested that outside-in signaling is not necessarily required to fulfill these platelet functions.

Cetuximab intensifies cisplatin-induced testicular toxicity.

Epidermal growth factor receptor (EGFR) has proliferative properties in the testis. Cetuximab, an anti-EGFR, is administered together with chemotherapy to patients with various types of cancer. This studies aim was to investigate the effect of cetuximab on testicular function. Adult male mice were injected with cetuximab (10 mg/kg), cisplatin (8 mg/kg) or a combination of both, and killed one week or one month later. The doses were chosen by human equivalent dose calculation. Testicular function was evaluated by epididymal-spermatozoa total motile count and sperm motility, weights of testes and epididymides, and the level of anti-Müllerian hormone (AMH) in the serum. Immunohistochemistry was performed to examine germ cell proliferation (Ki-67), apoptosis (Terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labelling), reserve (DAZL-Deleted in azoospermia-like, Promyelocytic leukaemia zinc-finger), blood vessels (CD34) and Sertoli cells (GATA-4). Administration of cetuximab alone increased testicular apoptosis and decreased epididymal-spermatozoa total motile count over time. When added to cisplatin, cetuximab exacerbated most of the recorded testicular parameters, compared with the effect of cisplatin alone, including testis and epididymis weights, epididymal-spermatozoa total motile count, AMH concentration, meiosis and apoptosis. In conclusion, cetuximab has only a mild effect on testicular reserve, but when added to cisplatin, it exacerbates cisplatin-induced testicular toxicity.

Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbß3-actin cytoskeleton complex in platelets.

Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbß3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbß3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbß3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbß3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbß3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbß3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbß3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbß3-cytoskeleton association and the strongest αIIbß3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbß3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz.

Dexrazoxane exacerbates doxorubicin-induced testicular toxicity.

Infertility induced by anti-cancer treatments pose a major concern for cancer survivors. Doxorubicin (DXR) has been previously shown to exert toxic effects on the testicular germinal epithelium. Based upon the cardioprotective traits of dexrazoxane (DEX), we studied its potential effect in reducing DXR-induced testicular toxicity. Male mice were injected with 5  mg/kg DXR, 100  mg/kg DEX, combination of both or saline (control) and sacrificed either 1, 3 or 6 months later. Testes were excised and further processed. Glutathione and apoptosis assays were performed to determine oxidative stress. Immunohistochemistry and confocal microscopy were used to study the effects of the drugs on testicular histology and on spermatogonial reserve. DXR and the combined treatment induced a striking decline in testicular weight. DEX prevented DXR-induced oxidative stress, but enhanced DXR-induced apoptosis within the testes. Furthermore, the combined treatment depleted the spermatogonial reserve after 1 month, with impaired recovery at 3 and 6 months post-treatment. This resulted in compromised sperm parameters, testicular and epididymal weights as well as significantly reduced sperm motility, all of which were more severe than those observed in DXR-treated mice. The activity of DEX in the testis may differ from its activity in cardiomyocytes. Adding DEX to DXR exacerbates DXR-induced testicular toxicity.

Locally applied leptin induces regional aortic wall degeneration preceding aneurysm formation in apolipoprotein E-deficient mice.

OBJECTIVE: Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aortic aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process. METHODS AND RESULTS: Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor, as well as mRNA for matrix metalloproteinases (MMP)-9 and MMP-12. Leptin and leptin receptor antigen were evident in all AAAs, and leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched nondilated controls. To simulate in vivo local leptin synthesis, ApoE(-/-) mice were subjected to a paravisceral periaortic application of low-dose leptin. Leptin-treated aortas exhibited decreased transforming growth factor-ß and increased MMP-9 mRNA levels 5 days after surgery, and leptin receptor mRNA was upregulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12, and periadventitial macrophage clustering. Furthermore, the combination of local periaortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size. CONCLUSIONS: Leptin is locally synthesized in human AAA wall. Paravisceral aortic leptin in ApoE(-/-) mice induces local medial degeneration and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation.

S-Allylmercapto-N-Acetylcysteine (ASSNAC) Attenuates Osteoporosis in Ovariectomized (OVX) Mice.

Osteoporosis is a bone-debilitating disease, demonstrating a higher prevalence in post-menopausal women due to estrogen deprivation. One of the main mechanisms underlying menopause-related bone loss is oxidative stress. S-allylmercapto-N-acetylcysteine (ASSNAC) is a nuclear factor erythroid 2-related factor 2 (Nrf2) activator and cysteine supplier, previously shown to have anti-oxidation protective effects in cultured cells and animal models. Here, we studied the therapeutic potential of ASSNAC with and without Alendronate in ovariectomized (OVX) female mice. The experimental outcome included (i) femur and L3 lumbar vertebra morphometry via Micro-Computed Tomography (µCT); (ii) bone remodeling (formation vs. resorption); and (iii) oxidative stress markers in bone marrow (BM) cells. Four weeks after OVX, there was a significant bone loss that remained evident after 8 weeks, as demonstrated via µCT in the femur (cortical and trabecular bone compartments) and vertebra (trabecular bone). ASSNAC at a dose of 50 mg/Kg/day prevented bone loss after the four-week treatment but had no significant effect after 8 weeks, while ASSNAC at a dose of 20 mg/Kg/day significantly protected against bone loss after 8 weeks of treatment. Alendronate prevented ovariectomy-induced bone loss, and combining it with ASSNAC further augmented this effect. OVX mice demonstrated high serum levels of both C-terminal cross-linked telopeptides of type I collagen (CTX) (bone resorption) and procollagen I N-terminal propeptide (P1NP) (bone formation) after 2 weeks, and these returned to control levels after 8 weeks. Alendronate, ASSNAC and their combination decreased CTX and increased P1NP. Alendronate induced oxidative stress as reflected by decreased glutathione and increased malondialdehyde (MDA) levels, and combining it with ASSNAC partially attenuated these changes. These results portray the therapeutic potential of ASSNAC for the management of post-menopausal osteoporosis. Furthermore, ASSNAC ameliorates the Alendronate-associated oxidative stress, suggesting its potential to prevent Alendronate side effects as well as improve its bone-protective effect.

Brachial artery endothelial function predicts platelet function in control subjects and in patients with acute myocardial infarction.

Platelet activation occurs in an endothelium-dependent flow-mediated dilation (FMD) impairment environment. The aim of this study was to explore the association between platelet reactivity and brachial artery FMD in individuals without established cardiovascular disease (controls) and acute myocardial infarction (AMI) patients. We prospectively assessed brachial artery FMD in 151 consecutive subjects, 104 (69%) controls, and 47 (31%) AMI patients; 115 (76%) men, mean age 53 ± 11 years. Following overnight fasting and discontinuation of all medications for ≥ 12 h, percent change in brachial artery FMD (%FMD) and endothelium-independent, nitroglycerin-mediated vasodilation (%NTG) were assessed. Platelet aggregation was assessed by conventional aggregometry, and platelet adhesion and aggregation under flow conditions by cone-and-plate(let) technology (Impact-R). Smoking, diabetes, and hypertension were more common in AMI compared to control subjects (p < 0.01 for all). Furthermore, aspirin, clopidogrel, beta-blockers, angiotensin-converting enzyme inhibitors, and statin administration were more common in AMI compared to controls (p < 0.01 for all). %FMD but not %NTG was significantly lower in AMI patients compared to controls (10.2 ± 4.2% vs. 15.4 ± 4.4%; p < 0.001 and 17.2 ± 3.9% vs. 18.0 ± 3.7%, p = 0.803, respectively). %FMD was significantly and inversely associated with all platelet functions tests (p < 0.001) in all study participants. In a multivariate logistic regression (unadjusted and adjusted for age, gender, smoking status, diabetes mellitus, hypertension, hypercholesterolemia, overweight, family history, and concomitant medications), %FMD remained the best predictor of platelet function, irrespective of group allocation (AMI patients or controls). In conclusion, FMD is inversely correlated to platelet reactivity in both controls and AMI patients.

S-allylmercapto-N-acetylcysteine protects bone cells from oxidation and improves femur microarchitecture in healthy and diabetic mice.

Oxidative stress is involved in the deterioration of bone quality and mechanical strength in both diabetic and aging adults. Therefore, we studied the ability of the antioxidant compound, S-allylmercapto-N-acetylcysteine (ASSNAC) to protect bone marrow stromal cells (BMSCs) from advanced glycation end-products (AGEs) cytotoxicity and improve bone microarchitecture of adult healthy and obese/diabetic (db/db) female mice. ASSNAC effect on AGEs-treated cultured rat BMSCs was evaluated by Neutral Red and XTT cell survival and reactive oxygen species (ROS) level assays. Its effect on healthy (C57BL/6) and obese/diabetic (C57BLKS/J Leprdb+/+; db/db) female mice femur parameters, such as (1) number of adherent BMSCs, (2) percentage of CD73+/CD45- cells in bone marrow (BM), (3) glutathione level in BM cells, and (4) femur microarchitecture parameters by microcomputed tomography, was studied. ASSNAC treatment protected BMSCs by significantly decreasing AGEs-induced ROS production and increasing their cellular resistance to the cytotoxic effect of AGEs. ASSNAC treatment of healthy female mice (50 mg/kg/day; i.p.; age 12-20 weeks) significantly increased the number of BMSCs (+60%), CD73+/CD45- cells (+134%), and glutathione level (+110%) in the femur bone marrow. Furthermore, it increased the femur length (+3%), cortical diameter (+3%), and cortical areal moment of inertia (Ct.MOI; +10%) a surrogate for biomechanical strength. In db/db mice that demonstrated a compromised trabecular bone and growth plate microarchitecture, ASSNAC treatment restored the trabecular number (Tb.N, +29%), bone volume fraction (Tb.BV/TV, +130%), and growth plate primary spongiosa volumetric bone mineral density (PS-vBMD, +7%) and thickness (PS-Th, +18%). In conclusion, this study demonstrates that ASSNAC protects bone marrow cells from oxidative stress and may improve bone microarchitecture in adult healthy and diabetic female mice.

Statins have an early antiplatelet effect in patients with acute myocardial infarction.

Statins confer an antiplatelet effect in hypercholesterolemic subjects and in stable coronary artery disease patients. We explored the antiplatelet effects of statins in ST-elevation myocardial infarction (STEMI) patients undergoing primary angioplasty. Of 120 STEMI patients, 80 (67%) received statins while 40 (33%) did not. Ex vivo platelet reactivity was studied on admission and 72 hours later by conventional aggregometry and under flow conditions (Impact R). Measures of platelet reactivity under flow conditions included aggregate size and surface coverage, signifying platelet aggregation and adhesion respectively. The effect of statins on platelet function under flow conditions and platelet aggregation was studied in?vitro in platelets from 10 STEMI patients. Platelets from each patient were incubated in?vitro with lovastatin or PBS as a control. The effect of lovastatin in the presence of a nitric oxide synthase inhibitor (L-NMMA) was also studied. Patients treated with statins were compared with those who did not have significantly lower ADP-induced platelet aggregation on the 4th day (56 ± 18% vs. 64 ± 17%, p=0.02). Platelet deposition under flow conditions as measured by surface coverage was reduced from admission to 72 hours later among statin-treated patients (19 ± 28% reduction, p<0.01), but was unchanged in non-treated patients (for comparison p<0.01). The extent of platelet inhibition was unrelated to patient characteristics, including lipid profile and type of statin administered (lipophylic vs. hydrophilic). In the in vitro study platelet incubation with statin compared with PBS resulted in a lower aggregate-size (29 ± 9 µm(2) vs. 39 ± 15 µm(2), p<0.01), and lower surface coverage (8.5 ± 4% vs. 12 ± 4%, p<0.01). The effect of the statin on both parameters was significantly blunted by L-NMMA. Incubation with statin also resulted in a reduction in collagen-induced platelet aggregation (31 ± 20% vs. 54 ± 25%, p<0.01). We concluded that in acute myocardial infarction patients, statins have an early antiplatelet effect, in addition to that afforded by standard antiplatelet therapy.

Visible light induces nitric oxide (NO) formation in sperm and endothelial cells.

BACKGROUND: Visible light-based stimulation using low-intensity lasers, LEDs, and broadband visible light devices has been recently introduced for therapy of human tissues in the absence of exogenous photosensitizers. Nitric oxide (NO) formation might be a potential mechanism for photobiomodulation because it is synthesized in cells by nitric oxide synthase (NOS), which contains both flavin and heme groups that absorb visible light. NO synthesis may also result from increased reactive oxygen species (ROS), which are found in various cell cultures following visible light illumination. NO is mainly known for inducing blood vessel dilation by endothelial cells, and in sperm cells NO is considered as an important agent in acrosome reaction and capacitation process, which are essential for successful fertilization. PURPOSE: To study NO formation in endothelial and sperm cells following visible light irradiation. MATERIALS AND METHODS: Sperm and endothelial cells were illuminated with broadband visible light, 400-800 nm, 130 mW/cm(2), for 5 minutes. During illumination, the endothelial cells were incubated in PBS free of Ca(+2) and Mg(+2), and the sperm cells were incubated in NKM buffer, to induce "stress conditions." NO production was quantified by using the Griess reagent which reacts with nitrite in the medium to yield an Azo compound which has an absorption band at 540 nm. RESULTS: Visible light illumination increased NO concentration both in sperm and endothelial cells. Blue light was more effective than red. Light-induced NO occurred only when endothelial cells were incubated in PBS free of Ca(+2) and Mg(+2), and in sperm cells, only when incubated in NKM. CONCLUSION: Light induces NO formation in endothelial and sperm cells. In endothelial cells, NO formation may explain previous results demonstrating enhanced wound healing and pain relief following illumination. In illuminated sperm cells, NO formation may account for the enhanced fertilization rate.

Allicin up-regulates cellular glutathione level in vascular endothelial cells.

BACKGROUND: Allicin in garlic is the primary active compound known to rapidly interact with free thiols. AIMS OF THE STUDY: To examine the effect of allicin on gene expression and glutathione cellular level in vascular endothelial cells. METHODS: Cultured endothelial cells were exposed to allicin; mRNA was prepared and subjected to Micro-array and Real-Time PCR. Glutathione cellular level was determined on cell lysates. RESULTS: Micro-array analysis demonstrated allicin-induced up- and down-regulation of 116 and 100 genes, respectively. Up-regulated genes included the phase II detoxifying enzymes thioredoxin reductase 1 and 2, heme oxygenase-1 and glutamate cysteine lygaze modifier subunit, the rate limiting enzyme in glutathione biosynthesis. Endothelial cells exposed to allicin and its derivatives containing glutathione or cysteine residues increased cellular glutathione. Allicin increased the glutathione level in a concentration and time-dependent manner up to 8-fold at a concentration of 10-20 microM after 28 h exposure. Furthermore, allicin derivative-treated cultures demonstrated a 50% decrease in tBuOOH cytotoxicity. CONCLUSIONS: These results may suggest a putative role for allicin and its derivatives in preventing reactive oxygen species damage by up-regulating the phase II detoxifying enzymes and increasing the cellular glutathione level.

S-Allylmercapro-N-Acetylcysteine Attenuates the Oxidation-Induced Lens Opacification and Retinal Pigment Epithelial Cell Death In Vitro.

The capacity of S-Allylmercapto-N-acetylcysteine (ASSNAC) to protect human retinal pigment epithelial (RPE) cells (line ARPE-19) and porcine lenses from oxidative stress was studied. Confluent ARPE-19 cultures were incubated with ASSNAC or N-acetyl-cysteine (NAC) followed by exposure to oxidants and glutathione level and cell survival were determined. Porcine lenses were incubated with ASSNAC and then exposed to H2O2 followed by lens opacity measurement and determination of glutathione (reduced (GSH) and oxidized (GSSG)) in isolated lens adhering epithelial cells (lens capsule) and fiber cells consisting the lens cortex and nucleus (lens core). In ARPE-19 cultures, ASSNAC (0.2 mM; 24 h) increased glutathione level by 2⁻2.5-fold with significantly higher increase in GSH compared to NAC treated cultures. Similarly, ex-vivo exposure of lenses to ASSNAC (1 mM) significantly reduced the GSSG level and prevented H2O2 (0.5 mM)-induced lens opacification. These results demonstrate that ASSNAC up-regulates glutathione level in RPE cells and protects them from oxidative stress-induced cell death as well as protects lenses from oxidative stress-induced opacity. Further validation of these results in animal models may suggest a potential use for ASSNAC as a protective therapy in retinal degenerative diseases as well as in attenuation of oxidative stress-induced lens opacity.

Effectiveness of reloading to overcome clopidogrel nonresponsiveness in patients with acute myocardial infarction.

Whether increasing doses of clopidogrel can overcome nonresponsiveness was evaluated. Clopidogrel nonresponsiveness was found in up to 25% of treated patients and was associated with worse prognosis in patients with acute coronary syndrome and patients undergoing coronary intervention. Adenosine diphosphate (ADP)-induced platelet aggregation was prospectively determined on day 4 of acute myocardial infarction in 200 consecutive patients, who received clopidogrel 300 mg as a loading dose and 75 mg/day thereafter. Thirty patients (15%) had ADP-induced platelet aggregation >or=80% using light transmittance aggregometry and were considered clopidogrel nonresponders. Nonresponders were reloaded with clopidogrel 600 mg, followed by 150 mg/day for 4 weeks. A 75-mg/day dose was resumed thereafter. ADP-induced platelet aggregation was reassessed 4 hours after reloading and biweekly for 10 weeks. Flow cytometry was used to determine platelet P-selectin expression and fibrinogen binding before and 4 hours after reloading. ADP-induced platelet aggregation significantly decreased 4 hours after reloading (from 83 +/- 6% to 56 +/- 14%; p <0.01). The decrease in platelet aggregation was maintained throughout the 4-week doubled maintenance dose. After resuming a maintenance dose of 75 mg/day, ADP-induced platelet aggregation returned to 66 +/- 12% (p <0.001), and 5 patients (17%) had ADP-induced platelet aggregation >or=80%. Flow cytometry showed a significant decrease in P-selectin expression (from 37 +/- 16% to 26 +/- 13%; p <0.01) and fibrinogen binding (from 84 +/- 7% to 70 +/- 13%; p <0.01) in ADP-stimulated platelets 4 hours after reloading. In conclusion, clopidogrel reloading and increased maintenance dose may overcome clopidogrel nonresponsiveness in patients with acute myocardial infarction.

Variable responsiveness to clopidogrel and aspirin among patients with acute coronary syndrome as assessed by platelet function tests.

Unresponsiveness to clopidogrel or aspirin has been reported in patients with acute coronary syndrome (ACS). Platelet aggregometry (PA) and the Impact-R [Cone and Plate(let) Analyzer (CPA) technology, measuring whole blood platelet adhesion under flow conditions] were compared in detecting laboratory unresponsiveness to clopidogrel and aspirin among ACS patients. Platelet-rich plasma (PRP) samples were evaluated in 404 patients by PA using adenosine diphosphate (ADP) and arachidonic acid (AA) and whole blood samples by the Impact-R ADP- and AA-response tests. The first cohort (n=114) was assayed by PA on days 1 and 4 of the onset of ACS. A patient with relative decrease of /=70%. A patient with an absolute value of AA-induced maximal aggregation >/=60% was defined as laboratory NR patient to aspirin. The second cohort (n=290) was tested on day 4 by both systems and results analyzed by receiver operating characteristic curve. The following cut-off values of the Impact-R surface coverage were obtained:

Phacoemulsification-induced injury in corneal endothelial cells mediated by apoptosis: in vitro model.

PURPOSE: To characterize the mechanism of injury caused by phacoemulsification and examine the protective effect of ascorbic acid in cultured bovine corneal endothelial cells (CECs). SETTING: Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. METHODS: To minimize the direct mechanical effects of phacoemulsification on CECs, cells cultured on gas-permeable flexible membranes were exposed to phacoemulsification. Apoptosis was assayed by immunohistochemistry using anticaspase-3 antibody. RESULTS: Phacoemulsification caused a significantly higher rate of apoptosis (mean apoptotic nuclei per square millimeter, 334+/-29 [SD] versus 45+/-12) 48 hours after exposure (P<.001). Results were similar in CECs exposed to hydrogen peroxide for 48 hours as a positive control (mean 345+/-6 and 376+/-1 at hydrogen peroxide concentrations of 50 microM and 100 microM, respectively). Addition of ascorbic acid (1 or 10 mM) significantly decreased the 48-hour apoptotic nuclei count induced by phacoemulsification (mean 219+/-15 and 130+/-29, respectively) (P<.001). Application of shear forces up to 2000 seconds(-1) for 60 minutes did not increase the rate of apoptotic nuclei. CONCLUSIONS: Flexible membranes covered with cultured CECs, used as a new model that mimics in vivo conditions, minimized the mechanical damage caused by ultrasonic vibrations and turbulent currents, which destroy cells grown on hard surfaces. Phacoemulsification damage was not mediated by mechanical or shear forces but resulted from free-radical formation that apparently triggered cellular cascades, leading to apoptosis. Cell death was significantly reduced by the addition of ascorbic acid, probably via a free radical-scavenging mechanism.

S-allylmercapto-N-acetylcysteine protects against oxidative stress and extends lifespan in Caenorhabditis elegans.

S-allylmercapto-N-acetylcysteine (ASSNAC) was shown in our previous study to activate Nrf2-mediated processes and increase glutathione level and resistance to oxidative stress in cultured endothelial cells. In this study, we explored the antioxidant protective effect of ASSNAC in Caenorhabditis elegans (C. elegans). Treatment of gst-4 reporter strain (CL2166) with increasing concentrations of ASSNAC (0.2 to 20 mM) for 24 hours and with ASSNAC (10 mM) for various time periods demonstrated a significant concentration- and time-dependent increase in Glutathione S-transferase (GST) gene expression (up to 60-fold at 20 mM after 24 hours). In addition, ASSNAC (2 mM; 24 hours) treatment of C. elegans strains N2 (wild type strain), gst-4 reporter (CL2166) and temperature sensitive sterile strain (CF512) significantly increased GST enzyme activity by 1.9-, 1.5- and 1.8-fold, respectively. ASSNAC (2.0 mM; 24 hours) increased the reduced glutathione content in N2 and CF512 strains by 5.9- and 4.9-fold, respectively. Exposure of C. elegans (N2 strain) to a lethal concentration of H2O2 (3.5 mM; 120 min) resulted in death of 88% of the nematodes while pretreatment with ASSNAC (24 hours) reduced nematodes death in a concentration-dependent manner down to 8% at 2.0 mM. C. elegans nematodes (strain CF512) cultured on agar plates containing ASSNAC (0.5 to 5.0 mM) demonstrated a significant increase in lifespan compared to control (mean lifespan 26.45 ± 0.64 versus 22.90 ± 0.59 days; log-rank p ≤ 0.001 at 2.0 mM) with a maximal lifespan of 40 versus 36 days. In conclusion, ASSNAC up-regulates the GST gene expression and enzyme activity as well as the glutathione content in C. elegans nematodes and thereby increases their resistance to oxidative stress and extends their lifespan.

ADAMTS-13 regulates platelet adhesion under flow. A new method for differentiation between inherited and acquired thrombotic thrombocytopenic purpura.

ADAMTS-13 cleaves large and ultra-large von Willebrand factor multimers normally secreted by endothelial cells. Severe deficiency of this enzyme leads to thrombotic thrombocytopenic purpura (TTP). We applied the Impact-R system [Cone and plate(let) Analyzer, CPA] to determine optimal conditions for ADAMTS-13 function, to assess it's activity in TTP patients and to distinguish inherited TTP (inTTP) from acquired TTP (acTTP). The role of ADAMTS-13 in platelet adhesion under different conditions was investigated applying recombinant forms of VWF and ADAMTS-13. rVWF was first treated by rADAMTS-13 either in solution or when immobilized on the surface of the well, under static or flow conditions, in the presence or absence of BaCl2. The resulting cleaved VWF fragments were then immobilized and served to assess type 3 von Willebrand disease whole blood platelet adhesion under flow. Maximal effect of the rADAMTS-13 (decrease of platelet adhesion in the absence compared to the presence of BaCl2), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma ofTTP patients with normal blood (1:3) yielded a 1.6- to 2-fold increase of platelet adhesion under flow compared to mixing normal plasma with normal blood, at shear rate range of 1,800 - 2,500 s(-1) . Maximal increase of platelet adhesion was observed under 2,050 s(-1) . Under these conditions, the extent of adhesion was similarly higher in patients with inTTP and acTTP versus control [surface coverage (SC) 14.5 +/- 2.8% and 14.6 +/- 2.5% vs.7.4 +/- 1.7%, respectively]. ADAMTS-13 activity measured by collagen-binding test was similarly low (4.2 +/- 3.8% and 3.5 +/- 2.4% vs. 72.2 +/- 8.0%, respectively). An inverse correlation between SC and ADAMTS-13 activity was observed in a patient with inTTP assayed eight times during plasma infusion treatment. Addition of BaCl2 to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in inTTP (by 51%) but not in acTTP. The lack of reduction of platelet adhesion in acTTP could presumably be due to the presence of ADAMTS-13 inhibitor in these patients. These results suggest that VWF immobilization and high shear flow yielded optimal conditions for ADAMTS-13 activity, and that introduction of BaCl2 in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.

Platelets enhance CD4+ lymphocyte adhesion to extracellular matrix under flow conditions: role of platelet aggregation, integrins, and non-integrin receptors.

The purpose of this study was to examine the role of platelets in CD4+ T lymphocyte adhesion to subendothelial extracellular matrix (ECM). Herpesvirus saimiri (HVS)-infected CD4+ T cells were incubated on ECM. An image analysis was used to evaluate T cell adhesion. Under static condition, T cell activation with 4-alpha-Phorbol 12-myristate 13-acetate (PMA) resulted in a 2.6-fold increase in cell adhesion. However, adhesion was not affected by platelets. In contrast, under flow (200s(-1)), platelets markedly enhanced both resting and PMA-activated T cell adhesion (33- and 48-fold), forming lymphocyte-platelet co-aggregates that contain approximately 90% of the adherent T cells. Abrogation of platelet aggregation with tirofiban inhibited formation of platelet-T cell co-aggregates under flow and reduced T cell adhesion by 74%. Separate and combined blockade of CD40L and P-selectin glycoprotein-1 (PSGL-1) on PMA-activated lymphocytes reduced adhesion under flow in the presence of platelets by 28%, 33%, and 55%, respectively. Blockade of beta1-integrins decreased adhesion under both static and flow conditions (by 35% and 44%, respectively), while blockade of beta2-integrin reduced adhesion only under static condition (by 23%). A similar adhesion pattern was observed using CD4+ T cells isolated from normal donor peripheral blood. In conclusion, platelets support CD4+ lymphocyte adhesion to ECM under flow by formation of heterotypic platelet-lymphocyte coaggregates involving alphaIIbbeta3 integrin and beta1-related integrins, as well as CD40L and PSGL-1.

Local Application of Leptin Antagonist Attenuates Angiotensin II-Induced Ascending Aortic Aneurysm and Cardiac Remodeling.

BACKGROUND: Ascending thoracic aortic aneurysm (ATAA) is driven by angiotensin II (AngII) and contributes to the development of left ventricular (LV) remodeling through aortoventricular coupling. We previously showed that locally available leptin augments AngII-induced abdominal aortic aneurysms in apolipoprotein E-deficient mice. We hypothesized that locally synthesized leptin mediates AngII-induced ATAA. METHODS AND RESULTS: Following demonstration of leptin synthesis in samples of human ATAA associated with different etiologies, we modeled in situ leptin expression in apolipoprotein E-deficient mice by applying exogenous leptin on the surface of the ascending aorta. This treatment resulted in local aortic stiffening and dilation, LV hypertrophy, and thickening of aortic/mitral valve leaflets. Similar results were obtained in an AngII-infusion ATAA mouse model. To test the dependence of AngII-induced aortic and LV remodeling on leptin activity, a leptin antagonist was applied to the ascending aorta in AngII-infused mice. Locally applied single low-dose leptin antagonist moderated AngII-induced ascending aortic dilation and protected mice from ATAA rupture. Furthermore, LV hypertrophy was attenuated and thickening of aortic valve leaflets was moderated. Last, analysis of human aortic valve stenosis leaflets revealed de novo leptin synthesis, whereas exogenous leptin stimulated proliferation and promoted mineralization of human valve interstitial cells in culture. CONCLUSIONS: AngII-induced ATAA is mediated by locally synthesized leptin. Aortoventricular hemodynamic coupling drives LV hypertrophy and promotes early aortic valve lesions, possibly mediated by valvular in situ leptin synthesis. Clinical implementation of local leptin antagonist therapy may attenuate AngII-induced ATAA and moderate related LV hypertrophy and pre-aortic valve stenosis lesions. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/. Unique identifier: NCT00449306.

Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction.

BACKGROUND: Although clopidogrel reduces the risk of cardiovascular episodes after coronary events and stenting, a substantial number of incidents continue to occur. METHODS AND RESULTS: The antiplatelet effect of clopidogrel was studied prospectively in 60 consecutive patients who underwent primary angioplasty (percutaneous coronary intervention [PCI]) with stenting for acute ST-segment-elevation myocardial infarction (STEMI) to determine whether variability in response to clopidogrel affects clinical outcomes. Patients were stratified into 4 quartiles according to the percentage reduction of ADP-induced platelet aggregation. Although patients in the first quartile were resistant to the effects of clopidogrel (ADP-induced platelet aggregation at day 6, 103+/-8% of baseline), ADP-induced aggregation was reduced to 69+/-3%, 58+/-7%, and 33+/-12% of baseline, respectively, in patients in quartiles 2 through 4 (P<0.01 for all). In addition, epinephrine-induced platelet aggregation and platelet aggregation under flow conditions, assessed by the cone-and-plate(let) analyzer method, were reduced significantly less in the first quartile than in quartiles 2 through 4. Whereas 40% of patients in the first quartile sustained a recurrent cardiovascular event during a 6-month follow-up, only 1 patient (6.7%) in the second quartile and none in the third and fourth quartiles suffered a cardiovascular event (P=0.007). CONCLUSIONS: Up to 25% of STEMI patients undergoing primary PCI with stenting are resistant to clopidogrel and therefore may be at increased risk for recurrent cardiovascular events.