The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbß3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

An autologous platelet-rich plasma stimulates periodontal ligament regeneration.

BACKGROUND: Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts. METHODS: The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western blot. RESULTS: This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor-treated cells compared to non-stimulated cells, although no statistically significant differences were observed. CONCLUSION: This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

Cigarette smoke extract induces select matrix metalloproteinases and integrin expression in periodontal ligament fibroblasts.

BACKGROUND: The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. METHODS: PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. RESULTS: Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). CONCLUSIONS: This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

Effect of 17ß-estradiol on adhesion of Mytilus galloprovincialis hemocytes to selected substrates. Role of alpha2 integrin subunit.

The process of hemocyte adhesion to extracellular matrix (ECM) proteins plays a crucial role in cell immunity. In most of these interactions between ECM proteins and cells, integrins are involved. The results of the present study showed that incubation of Mytilus galloprovincialis hemocytes with 17ß-estradiol caused significant increased adhesion of hemocytes to ECM proteins and specifically to laminin-1, collagen IV and oxidized collagen IV, in relation to control cells. The adhesion of hemocytes to oxidized collagen was significantly higher than to either collagen IV or to laminin-1. In accordance with this, inhibition of either NADPH oxidase or nitric oxide (NO) synthase attenuated 17ß-estradiol effect on hemocyte adhesion, suggesting that the high levels of free radicals, produced after 17ß-estradiol effect, could contribute to the high adhesion of hemocytes to laminin-1 and collagen IV. The implication of ROS was further confirmed by the use of the oxidant rotenone, which caused elevation of cell adhesion in relation to control and by the antioxidant NAC which attenuated 17ß-estradiol effect. The mechanism of 17ß-estradiol induced adhesion to laminin-1, collagen IV and oxidized collagen IV involves a large number of intracellular components, as Na+/H+ exchanger (NHE), all isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK) as well as alpha2 integrin subunit. Maintenance of high cyclic adenosine-3'-5'-monophosphate (cAMP) levels caused non significant higher adhesion of hemocytes to ECM proteins in relation to control cells. Our results showed that 17ß-estradiol caused a significant increase in α2 integrin subunit levels, which was reduced after inhibition of NHE, PI3K, PKC, NO synthase, NADPH oxidase and JNK. In addition, our results showed that apart from 17ß-estradiol, high cAMP and high ROS levels caused significantly higher induction of α2 integrin subunit levels in relation to control. Our results imply a potential involvement of cAMP in immune responses of Mytilus hemocytes, which needs further investigation.

Evaluation of α2-integrin expression as a biomarker for tumor growth inhibition for the investigational integrin inhibitor E7820 in preclinical and clinical studies.

E7820 is an orally active inhibitor of α(2)-integrin mRNA expression, currently tested in phases I and II. We aimed to evaluate what levels of inhibition of integrin expression are needed to achieve tumor stasis in mice, and to compare this to the level of inhibition achieved in humans. Tumor growth inhibition was measured in mice bearing a pancreatic KP-1 tumor, dosed at 12.5-200 mg/kg over 21 days. In the phase I study, E7820 was administered daily for 28 days over a range of 0-200 mg, followed by a 7-day washout period. PK-PD models were developed in NONMEM. α(2)-Integrin expression measured on platelets, corresponding to tumor stasis at t = 21 in 50% and 90% of the mice (I(int,50), I(int,90)) were calculated. It was evaluated if these levels of inhibition could be achieved in patients at tolerable doses. One hundred nineteen α(2)-Integrin measurements and 210 tumor size measurements were available from mice. The relationship between PK and α(2)-integrin expression was modeled using an indirect-effect model, subsequently linked to an exponential tumor growth model. I(inh,50) and I(inh,90) were 14.7% (RSE 7%) and 17.9% (RSE 8%). Four hundred sixty two α(2)-integrin measurements were available from 29 patients. Using the schedule of 100 mg qd (MTD), α(2)-integrin expression was inhibited more strongly than the I(int,50) and I(int,90) in greater than 95% and greater than 50% of patients, respectively. Moderate inhibition of α(2)-integrin expression corresponded to tumor stasis in mice, and similar levels could be reached in patients with the dose level of 100 mg qd.

Role of glycoprotein Ia gene polymorphisms in determining platelet function in myocardial infarction patients undergoing percutaneous coronary intervention on dual antiplatelet treatment.

Response variability to antiplatelet treatment has been described and the widespread use of acetylsalicylic acid (ASA) and clopidogrel requires clarification of the residual platelet reactivity (RPR). Various glycoprotein Ia (GpIa) polymorphisms have been investigated, but their influence on platelet reactivity in myocardial infarction (MI) patients undergoing percutaneous coronary intervention (PCI) on dual antiplatelet treatment is not still elucidated. Aim of this study was to evaluate the effect of C807T, G873A and T837C polymorphisms of GpIa on modulating platelet function in MI patients on dual antiplatelet treatment undergoing PCI. We measured platelet function by both a point-of-care assay (PFA100) and platelet-rich-plasma aggregation in 289 MI patients undergoing PCI and receiving dual antiplatelet treatment. Our data show that C807T/G873A polymorphisms, but not T837C, are associated with higher platelet reactivity. Carriers of the 807T/873A allele had significantly higher platelet aggregation values after arachidonic acid (AA) and collagen stimuli and, even if they did not reach the statistical significance, after 2 and 10 microM ADP stimuli; 807T/873A allele carriers had also significantly shorter closure times on PFA100/epinephrine membranes. At the multiple analyses, C807T/G873A polymorphisms resulted an independent risk factor for RPR defined by both AA induced platelet aggregation (OR=3.0, 95%CI 1.17-7.89, p=0.022) or by PFA100/epinephrine (OR=4.1, 95%CI 1.53-10.89, p=0.005). In conclusion, this study shows the 807T/873A allele of the GpIa gene is an independent risk factor for the RPR on dual antiplatelet treatment, and extends, in a larger acute coronary syndrome population, the observation that the 807T/873A allele is associated with higher platelet reactivity.

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice.

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.

Variability in platelet aggregation following sustained aspirin and clopidogrel treatment in patients with coronary heart disease and influence of the 807 C/T polymorphism of the glycoprotein Ia gene.

A broad variability in patient response to dual antiplatelet treatment has been described during the first month of treatment. Data on platelet function profiles in patients on dual antiplatelet therapy for a more sustained period are limited. Whether gene sequence variations of the glycoprotein Ia/IIa receptor influence platelet aggregation in these patients is also unknown. The aim of this study was to characterize platelet aggregation profiles in patients on dual antiplatelet treatment (aspirin plus clopidogrel) for >1 month and to assess whether these may be influenced by the C807T polymorphism of the glycoprotein Ia gene. We included 82 patients, who were divided into 2 groups: carriers (CT + TT genotypes; n = 51) and noncarriers (CC genotype; n = 31) of the mutant T allele. Platelet aggregation was assessed using light transmittance aggregometry after stimuli with adenosine diphosphate (20 micromol/L), collagen (6 microg/ml), and epinephrine (20 micromol/L). A significant variability in the distribution of platelet aggregation was observed in the overall study population, as well as in carriers and noncarriers of the T allele. T allele carriers had increased platelet aggregation compared with noncarriers after stimuli with adenosine diphosphate, collagen, and epinephrine (p <0.05 for all platelet aggregation assays). Thus, platelet aggregation varied significantly in patients on long-term dual antiplatelet treatment and was increased in T allele carriers of the 807C/T polymorphism of the glycoprotein Ia gene. These findings may contribute to the increased ischemic risk observed in these patients.