Platelet and shear rate promote tumor cell adhesion to human endothelial extracellular matrix--absence of a role for platelet cyclooxygenase

Blood platelets are thought to be involved in certain aspects of malignant dissemination. To study the role of platelets in tumor cell adherence to vascular endothelium we performed studies under static and flow conditions, measuring tumor cell adhesion in the absence or presence of platelets. We used highly metastatic human adenocarcinoma cells of the lung, cultured human umbilical vein endothelial cells (ECs) and extracellular matrices (ECM) prepared from confluent EC monolayers. Our results indicated that under static conditions platelets do not significantly increase tumor cell adhesion to either intact ECs or to exposed ECM. Conversely, the studies performed under flow conditions using the flat chamber perfusion system indicated that the presence of 2 x 10(5) pl/microliters in the perfusate significantly increased the number of tumor cells adhered to ECM, and that this effect was shear rate dependent. The maximal values of tumor cell adhesion were obtained, in presence of platelets, at a shear rate of 1,300 sec-1. Furthermore, our results with ASA-treated platelets suggest that the role of platelets in enhancing tumor cell adhesion to ECM is independent of the activation of the platelet cyclooxygenase pathway.     

Role of red cells in preventing the growth of platelet aggregation

Using high-resolution real-time two-dimensional ultrasound, we have investigated the role of red cells in the growth of already established platelet aggregates under controlled flow conditions. Platelet rich plasma (PRP) was circulated in vitro in horizontally and vertically arranged tubing at mean shear rate ranging from 60 to 0 sec-1, and adenosine diphosphate (ADP) was used to induce platelet aggregation. ADP-induced platelet aggregates grew in size and tended to sediment as shear rate decreased, in particular, below 10 sec-1. At 0 sec-1 (stasis), large clusters of platelet aggregates formed. The addition of washed red cells to produce a hematocrit of only 2% significantly interfered with the growth and sedimentation of platelet aggregates as shear rate was reduced. Formaldehyde-hardened erythrocytes had a similar effect in preventing the growth of platelet aggregates, suggesting that mechanical collision of red cells with platelet aggregates may be the cause of growth inhibition. Therefore, the thrombotic process may be enhanced in red cell poor zones in circulation resulting from flow disturbances associated with vascular stenosis or within artificial organs and extracorporeal systems. The present study also suggested that red cell free PRP should be carefully administered therapeutically.     

The effect of vascular cell seeding on platelet deposition in an in vitro capillary perfusion model

In view of the reported beneficial effects of cell seeding on the performance of synthetic vascular grafts, we determined the deposition of 111Indium-labelled human platelets in polyethylene capillary tubes covered with human endothelial cells (HEC) or smooth muscle cells (SMC) using an in vitro perfusion model. Platelet deposition decreased with increasing vascular cell coverage and was virtually absent when the number of adherent vascular cells of both types exceeded 50,000/cm2. Platelet deposition increased with increasing shear rate (300-900 s-1) only when surface coverage was less than 50,000 vascular cells/cm2. Deposition of Ca2+-ionophore A 23187-activated platelets in capillary tubes completely covered with SMC was significantly higher compared to capillaries covered with a similar number of HEC. When HEC-lined capillaries were treated with aspirin, only the deposition of activated platelets increased slightly, but significantly. This platelet reactivity was more evident when the endothelial lining was not confluent. These results demonstrate that, although seeding of HEC and SMC may both prevent the deposition of non-activated platelets to surfaces, non-aspirin-treated confluent HEC linings offer the best protection against deposition of activated platelets.     

The subendothelium of the HMEC-1 cell line supports thrombus formation in the absence of von Willebrand factor and collagen types I, III and VI

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1's ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1's ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2,600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC's ECM. Interestingly, large thrombi were formed at 2,600/s when HMEC-1's ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1's ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.     

Glial extracellular matrix modulates γ-glutamyl transpeptidase activity in cultured bovine brain capillary and bovine aortic endothelial cells

Glial extracellular matrix (ECM) elevated γ-glutamyl transpeptidase (γ-GTP) activity in cultured bovine brain capillary and aortic endothelial cells (BBCEC, BAEC). In particular, the ECM of glial cells cultured with the conditioned medium of BAEC (BAEC CM) dramatically elevated γ-GTP activity in BBCEC and BAEC. The ECM of glial cells cultured with BBCEC CM also had a marked effect. The ECM of 3T3 cells cultured with BAEC CM, and the ECM of glial cells cultured with 3T3 CM had no effect. Glial CM had no effect on γ-GTP activity in BBCEC and BAEC. These findings indicate that γ-GTP activity in endothelial cells (EC) is modulated by glial ECM, and that the factor of ECM that affects γ-GTP activity in EC arises from the interaction between glial cells and EC.     

Experimental isovolemic haemodilution-induced augmentation of carotid blood flow and oxygen transport through graded carotid stenoses.

The effect of haemodilution without volume expansion (isovolemic haemodilution) was assessed with respect to blood flow and oxygen transport across stenotic lesions of progressive severity in the dog carotid artery. As the mean haematocrit (Hct) was reduced from 40 +/- 1% (+/- SEM) to 32 +/- 0% (p less than 0.001), reductions in vascular resistance were significant across the 90% (p less than 0.001) and 95% (p less than 0.0003) relative carotid stenoses. Isovolemic haemodilution reduced fresh blood viscosity significantly by 27 +/- 3% (p less than 0.001) and 42 +/- 4% (p less than 0.001) at the low shear rates of 10 sec-1 and 1 sec-1 which are typical of low-flow states. Following a 20% reduction in Hct 30 to 35% increase (p less 0.001) in carotid blood flow occurred at non-critical degrees of stenosis while a mean 83% increase (p less than 0.001) occurred at the highly critical 95% relative stenosis. Oxygen transport after a 22% decrease in blood haemoglobin was significantly increased by 28% (p less than 0.01) at the 95% relative stenosis level. These data provide a physiologic rationale for the beneficial effects of haemodilution in acute cerebral ischaemia, cerebral vasospasm and cerebral revascularization or carotid endarterectomy.     

Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase

The degradation of extracellular matrix (ECM) adhesive glycoproteins, fibronectin (FN), thrombospondin (TSP) and von Willebrand factor (vWF), by human leukocyte cathepsin G and elastase, and by plasmin or thrombin, was analysed by immunoblotting after incubation of physiologic doses of the proteases with confluent human umbilical vein endothelial cells. Elastase induced an almost complete disappearance of intact FN, TSP, and vWF from the ECM at 0.02 units/ml within 5 minutes of incubation at 37 degrees C. Plasmin (0.2 units/ml) was also active on all three substrates, whereas cathepsin G (0.2 units/ml) had a preferential effect on TSP. Most remarkably, these degradations occurred with no apparent change in endothelial cell morphology, as shown by phase-contrast microscopy. In contrast, thrombin (0.2 units/ml) had no apparent proteolytic action on ECM glycoproteins, where it induced cell retraction and rounding. The release of adhesive glycoproteins from the ECM was accompanied by the detection of proteolytic fragments in the conditioned medium. Kinetic studies indicated that proteolysis started within minutes and proceeded for at least 1 hour. TSP was extremely sensitive to degradation by all enzymes except thrombin, whereas vWF released from the ECM was more resistant to proteolysis than constitutively secreted vWF, and FN was poorly degraded by plasmin. Our results indicate that serine proteinases, locally produced during inflammation and/or thrombolysis, can release extracellular matrix components and generate proteolytic fragments with potential biological activities.     

Antiphospholipid antibody positive sera enhance endothelial cell procoagulant activity--studies in a thrombosis model.

The effect of sera and IgG from 12 patients with systemic lupus erythematosus (SLE) on the endothelial cell (EC) procoagulant activity (PCA) was investigated in an in vitro thrombosis model. Six of the 12 SLE sera contained antiphospholipid antibodies (aPL). EC were stimulated for 8 h at 37 degrees C with or without 50 pM tumor necrosis factor (TNF) in culture medium containing 20% patient or control serum. Then the endothelial cell matrix (ECM) was isolated and subsequently exposed in a perfusion chamber to circulating normal whole blood, anticoagulated with low molecular weight heparin (LMWH). The PCA of the ECM was determined as the amount of generated fibrinopeptide A (FPA) in samples taken before and after perfusion. Furthermore, cross sections were made of the perfused matrix and analyzed for platelet adhesion and aggregate formation. All six aPL containing sera induced a small, but significant increase of ECM procoagulant activity. When added in combination with a low dose of TNF (50 pM), a synergistic enhancement of ECM procoagulant activity was found. The FPA generation was increased to 150-614% from the values obtained after stimulation with TNF and control serum. Also a shift towards the formation of larger platelet thrombi was observed. After stimulation with TNF and patient serum the surface of ECM covered with large aggregates (greater than 5 microns) was increased by 124-329% compared to the results obtained after stimulation with control serum and TNF. When patient sera were depleted from IgG the effects were strongly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

ADAM 15 is an adhesion receptor for platelet GPIIb-IIIa and induces platelet activation

Cell adhesion and proteolytic matrix degradation are central processes in atherosclerosis. Being a member of the family of ADAMs ("a disintegrin and metalloproteinase"), metargidin (ADAM15) combines a metalloproteinase domain and an RGD aminoacid sequence. We studied the potential role of ADAM15 as an adhesion receptor on endothelial cells and interactions between platelets and ADAM15 with respect to platelet adhesion, activation and thrombus formation. ADAM15 was found to be expressed on cultured endothelial cells (HUVEC). Platelet adhesion to immobilized recombinant ADAM15 was effectively enhanced under both static and high shear rate conditions reaching the maximum level of adhesion to fibrinogen. Consistently, platelet adhesion onto ADAM15 overexpressing endothelial cells was significantly increased. Adhesion to ADAM15 was reduced by blockade of GPIIb-IIIa using neutralizing anti-alpha(IIb)beta3 mAbs (7E3, 2G12), but not by anti-alpha(v)beta3 (LM609). Soluble ADAM15 binds to activated but not to resting GPIIb-IIIa. Moreover, platelets adherent to ADAM15 additionally attracted platelets under high shear rates indicating an initial role of platelet-ADAM15 interactions for thrombus formation. Furthermore, incubation of platelets with soluble ADAM15 showed a dose-dependent increase in secretion of CD62P and CD40L. ADAM15 is expressed on endothelial cells and can serve as an adhesion receptor for platelets via GPIIb-IIIa binding. Platelet adhesion to ADAM15 leads to platelet activation, secretion and promotes thrombus formation. Thus, ADAM15 may represent a novel target for antithrombotic strategies in cardiovascular pathologies.     

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.     

Platelet adhesion to subendothelium--effect of shear rate, hematocrit and platelet count on the dynamic equilibrium between platelets adhering to and detaching from the surface

The adherence of human 3H-adenine-labeled platelets to rat subendothelium was quantitated using a rotating probe device. Platelet adhesion increased in relation to the rotation time, reaching a plateau value in about 4-6 min without any further increase. A non-linear fitting analysis of experimental data allowed calculations of initial rate and plateau value of platelet adhesion. Increasing the shear rates (from 35 to 150 sec-1) or the hematocrit (from 10% to 40%), both the adhesion rate and the plateau value were increased. When different platelet concentrations were used the adhesion rate and the plateau calculated increased with platelet concentration. Different plateau values were obtained in the experimental conditions considered. This suggests that the plateau was not reached for the complete occupation of the subendothelial surface by the adherent platelets. Experiments using two different vessels rotated in the same platelet suspension or, viceversa, the same vessel rotated successively in two fresh platelet suspensions, showed that the plateau was not determined by reduced platelet reactivity. Rotating the same vessel first in radiolabeled platelets, until the plateau was reached, and secondly in non labeled platelets, or viceversa, showed that the plateau was indeed a dynamic condition where the number of platelets adhering and detaching reached equilibrium. These observations suggest that the platelet adhesion to subendothelium is the final equilibrium of two platelet fluxes, one adhering to the surface and another detaching from the surface.     

Platelet interaction with the extracellular matrix produced by cultured endothelial cells: A model to study the thrombogenicity of isolated subendothelial basal lamina

Cultured bovine endothelial cells produce an extensive underlying extracellular matrix (ECM) which closely resemble the vascular subendothelial basal lamina in its organization and chemical composition. This naturally produced ECM was used to study the interaction of platelets with the subendothelium when exposed or covered with vascular endothelial cells. Incubation of platelet rich plasma with the ECM induced a rapid and massive platelet adherence, aggregation, thromboxane formation and release reaction. These were demonstrated using phase and scanning electron microscopy, Indium-111 or (¹⁴C)-serotonin labelled platelets, and a radioimmunoassay for thromboxane B₂. In contrast to the ECM no platelet activation was induced either by uncoated plastic dishes or ECM covered with a confluent endothelial cell monolayer. Aspirinized platelets failed to undergo aggregation and degranulation, when incubated with the ECM. Culture dishes coated with characteristic constituents of the basal lamina such as collagen type IV and type V or fibronectin induced a much lower platelet reactivity as compared with ECM coated dishes. Digestion of ECM components (collagen, fibronectin, hyaluronic acid, and chondroitin sulphate) by specific enzymes was not associated with a substantial decrease in its platelet reactivity. Furthermore, exposure of ECM to sodium dodecyl sulphate or sodium periodate, or freezing and thawing did not decrease its biological activity. In contrast, platelet activation was completely abolished following heat denaturation or glutaraldehyde fixation of the ECM. The availability of a naturally produced ECM provides an appropriate model to study the interaction of platelets with the subendothelium in a controlled system which is isolated from other components of the vessel wall.     

A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process

A model of in vitro mechanical injury of confluent human endothelial cells (EC) in culture was developed. Human EC were obtained from umbilical veins and grown to confluence. Application on the EC monolayer of a calibrated disk of cellulose polyacetate paper resulted in removal of the EC, leaving a continuous subendothelial extracellular matrix (ECM) on the culture dish. The regeneration time depended on the original size of the lesion. Regeneration was similar with EC grown on different substrates such as human fibronectin, human subendothelial ECM, bovine collagen type I or surfaces coated with Transglutine, a surgical glue containing adhesive proteins. A human brain extract containing growth factor activity accelerated significantly the repair of the lesion, especially at low serum concentration. This simple in vitro model of mechanical injury allows the quantitative study of the effects of matrices, growth factors and pharmacological agents on the repair process.     

Bone morphogenetic protein 4 inhibits TGF‐β2 stimulation of extracellular matrix proteins in optic nerve head cells: Role of gremlin in ECM modulation

The characteristic cupping of the optic nerve head (ONH) in glaucoma is associated with elevated TGF‐β2 and increased synthesis and deposition of extracellular matrix (ECM) proteins. In addition to TGF‐β2, the human ONH also expresses bone morphogenetic proteins (BMPs) and BMP receptors, which are members of the TGF‐β superfamily. We examined the potential effects of BMP4 and the BMP antagonist gremlin on TGF‐β2 induction of ECM proteins in ONH cells. BMP‐4 dose dependently inhibited TGF‐β2‐induced fibronectin (FN) and PAI‐1 expression in ONH astrocytes and lamina cribrosa (LC) cells and also reduced TGF‐β2 stimulation of collagen I, collagen VI, and elastin. Addition of gremlin blocked this BMP‐4 response, increasing cellular and secreted FN as well as PAI‐1 levels in both cell types. Gremlin was expressed in ONH tissues and ONH cells, and gremlin protein levels were significantly increased in the LC region of human glaucomatous ONH tissues. Interestingly, recombinant gremlin dose dependently increased ECM protein expression in cultured ONH astrocytes and LC cells. Gremlin stimulation of ECM required activation of TGF‐β receptor and R‐Smad3. TGF‐β2 increased gremlin mRNA expression and protein levels in ONH cells. Inhibition of either the type I TGF‐β receptor or Smad3 phosphorylation blocked TGF‐β2‐induced gremlin expression. In conclusion, BMP4 blocked the TGF‐β2 induction of ECM proteins in ONH cells. The BMP antagonist gremlin reversed this inhibition, allowing TGF‐β2 stimulation of ECM synthesis. Increased expression of gremlin in the glaucomatous ONH may further exacerbate TGF‐β2 effects on ONH ECM metabolism by inhibiting BMP‐4 antagonism of TGF‐β2 signaling. Modulation of the ECM via gremlin provides a novel therapeutic target for glaucoma. © 2008 Wiley‐Liss, Inc.     

Regulated complement deposition on the surface of human endothelial cells: effect of tobacco smoke and shear stress

Cigarette smoke and hemodynamic stress both contribute to vascular inflammation and associated atherosclerosis. We recently demonstrated direct activation of complement components C4 and C3 on human endothelial cells (EC). The present study was designed to explore complement activation on bone marrow microvascular endothelial cells (BMEC) and human umbilical vein endothelial cells (HUVEC) in response to endothelial cell injury by tobacco smoke extract, shear stress, or other known inflammatory and atherogenic mediators, lipopolysaccharide (LPS) and INF-gamma. Following treatment, confluent EC monolayers were exposed to plasma (60 min, 37 degrees C), and cell surface deposition of stable complement derivatives C4d, iC3b and SC5b-9 was measured in situ using an ELISA approach. Consistent with previous results, moderate levels of C4d, iC3b and SC5b-9 deposition were observed on native EC monolayers exposed to human plasma. Tobacco smoke and shear stress enhanced EC C4d deposition. In contrast, LPS and INF-gamma failed to affect EC mediated complement activation, despite evidence of EC activation illustrated by ICAM-1 expression. The combination of tobacco smoke and shear stress nearly doubled EC C4d expression. No increases in iC3b or SC5b-9 were noted, suggesting inhibition of classical and alternative pathway C3 convertase assembly or activity. Indeed, concomitantly increased surface expression of complement regulatory proteins CD35 (CR1) and CD55 was observed following EC exposure to tobacco smoke and shear stress. These results suggest that a balance between complement activation and regulation exists at the EC surface, and may impact vascular injury leading to thrombosis, arteriosclerosis, and atherogenesis.     

Upstream thrombus growth impairs downstream thrombogenesis in non-anticoagulated blood: effect of procoagulant artery subendothelium and non-procoagulant collagen

In the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1. An axially-dependent decrease of platelet adhesion (34-57%, p less than 0.01-0.05) and thrombus volume (57-80%, p less than 0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29%; p less than 0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p less than 0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p less than 0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p less than 0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p less than 0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent ot the surface of platelets, leading to decreased platelet deposition further downstream.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple enzyme-immunoassay test for von Willebrand factor binding in human arterial subendothelium.

In order to determine the binding of vWF, subendothelium from everted human umbilical arteries was perfused with dialysed serum containing different concentrations of purified vWF using an annular perfusion chamber at a wall shear rate of 1100 sec-1 for 30 min. After perfusion, control (not perfused) and perfused vessel segments were washed and incubated with a diluted rabbit antibody against human vWF. Then the nonbound anti-vWF from both samples were used to determine indirectly vWF by EIA. Although in our experiments normal vWF serum concentrations were not enough to exert vWF binding, a substantial binding could be attained with vWF levels around 2.5 U/ml. To estimate the pre-existing subendothelial vWF amount, three different experiments were developed: a) diluted IgG from a nonimmunized rabbit, b) a diluted rabbit antibody to human vWF, c) PBS-BSA. After washing, vessel segments were incubated with rabbit antibody to human vWF. After incubation, the nonbound anti-vWF was used to determine indirectly vWF by EIA. The results obtained showed that the amount of pre-existing vWF was approximately 1.1x10(-3) U vWF/cm2 subendothelium.     

Neuropeptide degradation by large vessel and microvessel-derived endothelial cells in vitro: cell surface catabolism of thyrotropin releasing hormone (TRH)

Cell surface ectopeptidase activity of purified, cultured large vessel and microvessel-derived endothelial cells (EC) was studied. Degradation of thyrotropin releasing hormone (TRH), and production of cyclo-His-Pro was significantly increased (P less than 0.001) in large vessel EC compared with microcapillary EC. Since the rate of catabolism in the microvascular capillary bed is 5 times less than that in the large vessel wall, peptide concentrations are likely maintained longer in close proximity to their site of biosynthesis, where they are presumably most active.     

Different effects of aspirin, dipyridamole and UD-CG 115 on platelet activation in a model of vascular injury: studies with extracellular matrix covered with endothelial cells

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artificial intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 microM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.     

Impaired astrocytic extracellular matrix distribution under congenital hypothyroidism affects neuronal development in vitro

Astrocytes clearly play a role in neuronal development. An indirect mechanism of thyroid hormone (T3) in the regulation of neuronal development mediated by astrocytes has been proposed. T3 alters the production and organization of the extracellular matrix (ECM) proteins and proteoglycans, producing a high‐quality substrate for neuronal differentiation. The present study investigated the effect of hypothyroidism on the astrocyte production of fibronectin (FN) and laminin (LN) as well as their involvement in neuronal growth and neuritogenesis. Our results demonstrated that the amount of both FN and LN were significantly reduced in cultures of hypothyroid astrocytes from rat cerebellum compared with normal cells. This effect was accompanied by reduced numbers of neurons and neuritogenesis. Similarly, the proportions of neurons and neurons with neurites were reduced in cultures on ECM prepared from hypothyroid astrocytes in comparison with normal cells. The proportion of both normal and hypothyroid neurons is strongly reduced in astrocyte ECM compared with cocultures on astrocyte monolayers, suggesting that extracellular factors other than ECM proteins are involved in this process. Moreover, treatment of hypothyroid astrocytic cultures with T3 restored the area of both FN and LN immunostaining to normal levels and partially reestablished neuronal survival and neuritogenesis. Taken together, our results demonstrated that hypothyroidism involves impairment of the astrocytic microenvironment and affects the production of ECM proteins. Thus, hypothyroidism is implicated in impaired neuronal development. © 2010 Wiley‐Liss, Inc.