Shlomo Kotev-Emeth

A NEW METHOD FOR QUANTITATIVE ANALYSIS OF WHOLE BLOOD PLATELET INTERACTION WITH EXTRACELLULAR MATRIX UNDER FLOW CONDITIONS

A new method and device in which whole blood platelet deposition and aggregation on extracellular matrix (ECM) under defined shear conditions is quantitatively evaluated was developed. A 0.25 mL aliquot of citrated whole blood is placed on ECM and a defined shear rate is applied for 2 min using a cone and plate device. This is followed by staining and measuring the number of stained objects, the percentage of ECM surface covered with stained objects and the average size of the objects using an image analyzer. When normal blood is analyzed, platelet deposition is a shear and a time dependent process, reaching maximal levels within 2 min at high shear rate (1300 s-1) of about 20% surface coverage and average aggregate size of about 40-50 microns 2. These two parameters demonstrated positive correlation with the platelet count and the hematocrit. Studies using samples from patients with von Willebrand disease (vWD) and Glanzmanns Thrombasthenia (GT) were performed and demonstrated the ability of the new method to detect these pathological conditions. Blood samples of vWD patients showed a very low adhesion and aggregation at high shear rate as reflected by very low surface coverage (5.2%) and average particle size of single platelets (21.3 microns2). GT samples at a high shear rate demonstrated surface coverage similar to normal blood samples (21.7%) but with average particle size of single platelets (21.3 microns2). The new method is an alternative method to clinically evaluate platelet function under close to physiological conditions.

Shear-induced platelet adhesion and aggregation on subendothelium are increased in diabetic patients

Increased platelet aggregation has been suggested to play a role in the accelerated atherosclerosis of diabetics. However the physiological relevance of the aggregation tests has been questioned. The purpose of this study was to determine platelet activation in diabetic patients, using a novel device--the cone and plate(let) analyzer--to measure shear-induced platelet adhesion and aggregation on extracellular matrix (ECM). Whole blood platelet adhesion and aggregation in patients with noninsulin-dependent diabetes mellitus (n=82) and in nondiabetic controls (n=71) were compared. Clinical and laboratory characteristics of the diabetic patients were analyzed for possible correlation with parameters of platelet activity. Patients with diabetes had a significantly increased platelet activation compared to nondiabetic subjects, demonstrated by an increased adhesion to the ECM (surface coverage, 23% [95% confidence interval, 22-25%] vs. 19% [95% confidence interval, 18-20%], respectively) and an increased average size of the ECM-bound aggregates (54 microm2 [95% confidence interval, 51-57 microm2] vs. 47 microm2 [95% confidence interval, 43-51 microm2], respectively). Platelet adhesion in the diabetic group was found to correlate with triglyceride levels (r=0.36) and hematocrit values (r=0.31) and inversely with high-density lipoprotein cholesterol levels (r=0.30). There were no correlation, however, between parameters of platelet reactivity and duration of diabetes, vascular complications and low-density lipoprotein levels. Our data demonstrate an increased platelet adhesion and aggregation in diabetic patients and suggest a modulatory role of diabetic dyslipidemia.

Estrogen receptor binding material in blood of patients after clomiphene citrate administration: determination by a radioreceptor assay.

The aim of the current study was to assess whether clomiphene citrate (CC) and/or active metabolites are present at presumed time of ovulation, nidation, or steroid-sensitive organogenesis, in serum of patients receiving CC for induction of ovulation. A radioreceptor assay, based on competitive replacement of 3H-estradiol on the rat uterus estrogen receptor, by ligands present in serum of patients after CC administration, was developed. Ligands reached maximal concentration 4 to 5 hours after a single dose of CC was administered, and declined with a half-life of 4.5 to 10 hours. In patients receiving CC on day 5 to day 9 in the cycle, ligands are still present on day 14 in the cycle and in some patients on day 22 of the cycle, but no ligands were detected 60 days after CC treatment.

Effect of maturation on the osteogenic response of cultured stromal Bone marrow cells to basic fibroblast growth factor

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Visible light induces no formation in sperm and endothelial cells

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.

Establishment of a Rat Long-Term Culture Expressing the Osteogenic Phenotype : Dependence on Dexamethasone and FGF-2

Rat stromal bone-marrow cells cultured in the presence of dexamethasone, ascorbic acid, g -glycerophosphate, and fibroblast growth factor-2 (FGF-2) express the osteogenic phenotype (Pitaru et al., J. Bone Miner. Res . 8:919-929, 1993). The purpose of this study was to establish a long-term homogeneous culture expressing the osteogenic phenotype. The cultures were routinely passaged every 5 days in the absence or presence of either or both dexamethasone and FGF-2, and the cumulative doubling number and the expression of the osteogenic phenotype were determined. Cultures treated with dexamethasone (10 m 7 M) ceased proliferation and only upon addition of FGF-2 (3 ng/ml) was a spontaneous immortalization achieved, as expressed by sustained proliferation for about 1 year, with a doubling time of 22 h and more than 300 doublings in 72 passages. Both FGF-2 and dexamethasone are required and act synergistically to maintain cell propagation, alkaline phosphatase expression, and osteocalcin secretion; however, protein content was FGF-2 dependent and the mineralization was dexamethasone dependent. Repetitive single-cell cloning tested the homogeneity and stability of the cells expressing the osteogenic phenotype in these long-term cultures. It was shown that 25% to 50% of subclones derived from clones with an osteogenic phenotype do not further express the osteogenic phenotype. In conclusion, we have established a spontaneously immortalized dexamethasone- and FGF-2-dependent rat stromal bone-marrow-derived long-term culture expressing the osteogenic phenotype. The cultures tend to lose the osteogenic phenotype, and dexamethasone supports the long-term preservation of the osteogenic phenotype.

Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli

Background Unstable carotid plaques cause cerebral emboli. Leptin promotes atherosclerosis and vessel wall remodeling. We hypothesized that carotid atherosclerotic lesion instability is associated with local leptin synthesis. Methods and Results Carotid endarterectomy plaques from symptomatic (n=40) and asymptomatic patients with progressive stenosis (n=38) were analyzed for local expression of leptin, tumor necrosis factor (TNF)-α, and plasminogen activator inhibitor type 1. All lesions exhibited advanced atherosclerosis inclusive of thick- and thin-cap fibroatheromas or lesion rupture. Symptomatic lesions exhibited more plaque ruptures and macrophage infiltration (P=0.001 and P=0.05, respectively). Symptomatic plaques showed preferential leptin, TNF-α, and plasminogen activator inhibitor type 1 transcript (P=0.03, P=0.04, and P=0.05, respectively). Leptin mRNA and antigen in macrophages and smooth muscle cells were confirmed by in situ hybridization and immunohistochemistry. Plasma leptin levels were not significantly different between groups (P=1.0), whereas TNF-α was significantly increased in symptomatic patients (P=0.006). Human aortic smooth muscle cell culture stimulated by TNF-α, lipopolysaccharide, or lipoteichoic acid revealed 6-, 6.7-, and 6-fold increased secreted leptin antigen, respectively, at 72 hours (P<0.05). Conclusions Neurologically symptomatic patients overexpress leptin mRNA and synthesize leptin protein in carotid plaque macrophages and smooth muscle cells. Local leptin induction, presumably by TNF-α, could exert paracrine or autocrine effects, thereby contributing to the pathogenesis of lesion instability. Clinical Trial Registration URL: www.Clinicaltrials.gov. Unique identifier: NCT00449306.

S-Allylmercapto-N-acetylcysteine (ASSNAC) protects cultured nerve cells from oxidative stress and attenuates experimental autoimmune encephalomyelitis.

Oxidative stress and/or low cellular glutathione are associated with development and progression of neurodegenerative diseases. We have shown that S-allylmercapto-N-acetylcysteine (ASSNAC) up-regulates the level of glutathione and phase II detoxifying enzymes in cultured vascular endothelial cells. The present study demonstrates that exposure of nerve cell lines to ASSNAC significantly increases the cellular level of glutathione probably via activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and protects the cells from tBuOOH-induced cytotoxicity. Furthermore, ASSNAC increases the level of mice spinal cord and brain glutathione (by 54% and 47%, respectively) and attenuates the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice. In conclusion, these data implicate ASSNAC to protect nerve cells, both in vitro and in vivo, from oxidative stress and thereby to attenuate the clinical symptoms of EAE, suggesting its potential use for the treatment of neurodegenerative diseases.

Involvement of cortactin and phosphotyrosine proteins in cell–cell contact formation in cultured bovine corneal endothelial cells

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell–cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including α-catenin, vinculin and cortactin, are localized at cell–cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell–cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y466, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y421 and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY421-cortactin was localized at the leading edge and pY466-cortactin at a perinuclear area. In confluent cultures both pY466- and pY421-cortactin isoforms were localized at the cell–cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell–cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y421 and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y466 and associated with the Triton-insoluble fraction.

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.