RESUMEN
Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) ß2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFß2-neutralizing antibody. The inhibitory effect of silibinin on TGFß2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM.
Asunto(s)
Anticarcinógenos/farmacología , Fibroblastos/efectos de los fármacos , Próstata/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/prevención & control , Silimarina/farmacología , Factor de Crecimiento Transformador beta2/metabolismo , Anticarcinógenos/química , Antioxidantes/química , Antioxidantes/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Silybum marianum/química , Próstata/citología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Silibina , Silimarina/químicaRESUMEN
Although changes in the intracellular levels of calcium (Ca(2+)) are a central step in platelet activation, the underlying mechanism of Ca(2+) entry is still unclear. Previous studies have demonstrated that TRPC6, a member of the canonical transient receptor potential channel (TRPC) family is expressed in platelets in a significant amount, and is predominantly found on the plasma membrane. Based on these considerations, we hypothesized that TRPC6 plays a critical role in platelet function. To characterize the role of TRPC6 in platelet function in vivo, we employed a genetic approach, subjecting TRPC6 knockout mice to the tail bleeding time test and a carotid artery injury thrombosis model. We found that TRPC6-deficient animals displayed a prolonged bleeding time, and an increased time for occlusion of the injured carotid artery, compared to their wild-type littermates. Taken together, our data demonstrate for the first time, that TRPC6 deletion in mice results in defects in hemostasis and protection against thrombogenesis, suggesting a vital role in platelet function. Furthermore, TRPC6 may define a new therapeutic target for managing multiple thrombosis-based disorders.
Asunto(s)
Plaquetas/fisiología , Hemostasis/genética , Canales Catiónicos TRPC/fisiología , Trombosis/sangre , Trombosis/genética , Animales , Tiempo de Sangría , Plaquetas/metabolismo , Eliminación de Gen , Ratones , Ratones Noqueados , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6RESUMEN
In efforts to define new targets for antithrombotic purposes, there is interest in utilizing antibodies targeting ligand binding domains of platelet receptors. To this end, we have recently shown that an antibody (designated C-EL2Ab), which targets the C-terminus of the 2nd extracellular loop (C-EL2) of the thromboxane A(2) receptor (TPR), selectively blocks TPR-mediated platelet aggregation, under both in vitro and ex vivo experimental conditions. In the current studies we sought to determine whether C-EL2Ab exhibits in vivo antithrombotic activity, by employing a carotid artery injury thrombosis model. It was found that mice treated with C-EL2Ab, exhibited a significant increase in time for occlusion, when compared to controls such as normal rabbit IgG, or an antibody which targets a region separate from the ligand binding site (i.e., EL1). We next examined the effect of C-EL2Ab on hemostasis, and found no increase in tail bleeding times in C-EL2Ab treated mice, compared to the aforementioned controls. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, and is devoid of increased bleeding risk. Moreover, the identification of a functionally active TPR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Fibrinolíticos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Modelos Animales de Enfermedad , Fibrinolíticos/uso terapéutico , Ligandos , Ratones , Ratones Endogámicos C57BL , Inhibidores de Agregación Plaquetaria/uso terapéutico , Estructura Terciaria de Proteína , Receptores de Tromboxano A2 y Prostaglandina H2/química , Receptores de Tromboxano A2 y Prostaglandina H2/inmunologíaRESUMEN
While there have been many reports investigating the biological activity and signaling mechanisms of isoprostanes, their role in biology, particularly in platelets, appears to still be underestimated. Moreover, whether these lipids have their own receptors is still debated, despite multiple reports that discrete receptors for isoprostane do exist on platelets, vascular tissues, amongst others. This paper provides a review of the important literature of isoprostanes and provides reasoning that isoprostanes should be classified as orphan ligands until their receptor(s) is/are identified.
Asunto(s)
Plaquetas/fisiología , Hemostasis/fisiología , Isoprostanos/metabolismo , Activación Plaquetaria/fisiología , Transducción de Señal/fisiología , Ácido Araquidónico/química , Plaquetas/metabolismo , Humanos , Isoprostanos/química , Modelos Moleculares , Estructura MolecularRESUMEN
AIM: To investigate the potential antagonistic activity of the antidiabetic agent glybenclamide for the human platelet thromboxane A(2) receptor (abbreviated as TPR). METHODS: Platelets were obtained from healthy donors. Aggregation studies were performed in a model 700 aggregometry system. Radioactivity was counted in a Beckman LS 6000 liquid scintillation counter and calcium imaging was performed using an LS50B PerkinElmer Fluorescence Spectrometer. RESULTS: It was found that glybenclamide: 1) inhibited aggregation induced by the TPR agonist U46619 (IC(50)=2.3+/-0.31 micromol/L) and by the thromboxane A(2) precursor arachidonic acid (IC(50)=2.6+/-0.24 micromol/L); 2) displaced SQ29,548 from its binding sites on platelets; 3) lacked any detectable effects on aggregation stimulated by ADP, or the thrombin receptor activating-peptide 4; 4) blocked calcium mobilization induced by U46619, but not by ADP; and 5) failed to raise cAMP levels. CONCLUSION: The findings indicate that glybenclamide exerts inhibitory effects on platelets by interacting with TPR. Thus, glybenclamide or a rationally designed derivative has the potential to serve as an antithrombotic agent.
Asunto(s)
Gliburida/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina Difosfato/farmacología , Sitios de Unión , Plaquetas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Calcio/metabolismo , AMP Cíclico/sangre , Ácidos Grasos Insaturados , Humanos , Hidrazinas/farmacología , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Conteo por Cintilación , Espectrometría de FluorescenciaRESUMEN
High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.
Asunto(s)
Enfermedades de la Aorta/etiología , Arteriosclerosis/etiología , Arteritis/etiología , Grasas de la Dieta/efectos adversos , Células Endoteliales/efectos de los fármacos , Hipertrigliceridemia/complicaciones , Proteínas Relacionadas con Receptor de LDL/metabolismo , Lipoproteínas HDL/toxicidad , Lipoproteínas LDL/toxicidad , Lipoproteínas VLDL/toxicidad , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Receptores de LDL/metabolismo , Triglicéridos/toxicidad , Factor de Necrosis Tumoral alfa/farmacología , Aorta , Apolipoproteína C-III/metabolismo , Apolipoproteína C-III/farmacología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Quilomicrones/sangre , Grasas de la Dieta/administración & dosificación , Selectina E/biosíntesis , Selectina E/genética , Endocitosis , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Emulsiones Grasas Intravenosas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipertrigliceridemia/sangre , Hipoglucemia , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/farmacología , Proteínas Relacionadas con Receptor de LDL/efectos de los fármacos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Lipoproteínas VLDL/sangre , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/efectos de los fármacos , Proteínas de Transporte de Membrana/efectos de los fármacos , Modelos Cardiovasculares , Monocitos/citología , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , Estrés Oxidativo , Receptores de LDL/efectos de los fármacos , Reología , Transducción de Señal/efectos de los fármacos , Triglicéridos/sangre , Factor de Necrosis Tumoral alfa/fisiología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CD11c, a member of the leukointegrin family, is expressed prominently on tissue macrophages and dendritic cells and binds to complement fragment (iC3b), provisional matrix molecules (fibrinogen), and the Ig superfamily cell adhesion molecule, ICAM-1. CD11c has been proposed to function in phagocytosis, cell migration, and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells. Using assays to quantify CD11c-mediated cell adhesion, we demonstrate that CD11c recognizes ICAM-2 and VCAM-1. The CD11c-binding site on VCAM-1 appears to be different from that used by the integrin alpha4. CD11c and alpha4beta1 contributed to monocyte capture and transmigration on inflamed human aortic endothelial cells. We discovered that the anti-mouse CD11c mAb N418 blocks CD11c binding to iC3b, ICAM-1, and VCAM-1. Treatment of mice with N418 reduced SRBC-induced delayed-type hypersensitivity significantly. CD11c appeared to contribute predominantly to the sensitization phase and somewhat less to the response to SRBC challenge. This suggests a novel role for CD11c during leukocyte recruitment, antigen uptake, and the survival of APC.
Asunto(s)
Antígeno CD11c/inmunología , Antígenos CD18/inmunología , Hipersensibilidad Tardía/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Aorta/citología , Sitios de Unión , Antígeno CD11c/metabolismo , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Complemento C3b/inmunología , Células Endoteliales/inmunología , Células Endoteliales/fisiología , Eritrocitos/inmunología , Humanos , Integrina alfa4/metabolismo , Ligandos , Ratones , Monocitos/inmunología , Monocitos/fisiología , Unión Proteica , Ovinos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
While blood platelets express several G-protein-coupled receptors (GPCRs) that play pivotal roles in their activation, several diseases, for example thrombotic disorders, may develop if these receptors are inappropriately activated. Thus, these receptors have been the subject of investigations to design therapeutic interventions for managing multiple thrombosis-based disease states. One such GPCR, the thromboxane A(2) receptor (TPR), remains resistant to such interventions. The present review provides a critical examination of the binding, structural biology, and signaling of TPRs. The review also provides a rationale for using principles of "drug rediscovery" as an alternative/viable approach for the therapeutic targeting of TPRs. To this end, it is noteworthy that many US Food and Drug Administration (FDA)-approved drugs have been found to selectively (and nonselectively) block TPR-mediated functional responses, for example platelet aggregation, as described in this review. Therefore, while none of the antagonists, thus far developed for targeting TPRs, have made it into clinical use, this peculiar receptor can be antagonized by a large number of drugs used for indications unrelated to thrombosis.
Asunto(s)
Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Anticoagulantes/farmacología , Plaquetas/metabolismo , Diseño de Fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Trombosis/tratamiento farmacológico , Trombosis/prevención & controlRESUMEN
There is considerable interest in discovering novel antiplatelet approaches with an enhanced safety profile. To this end, in our efforts to define new targets for antithrombotic activity, we investigated the utility of antibodies which recognize the ligand binding domains of the platelet thromboxane A(2) receptor (TPR). We hypothesized that an antibody (abbreviated as C-EL2Ab), which interacts with the C-terminus of the second extracellular loop (C-EL2; i.e., ligand binding domain) of TPR exhibits antagonistic activity. Our findings demonstrate that C-EL2Ab did indeed inhibit TPR-mediated platelet aggregation. However, it was devoid of any apparent effects on aggregation triggered by ADP or the thrombin receptor activating peptides 1 or 4. Furthermore, results from radiolabeled ligand binding studies indicate that C-EL2Ab competitively displaced the classical TPR antagonist [(3)H]SQ29,548 from its binding sites. On the other hand, control experiments indicated that normal rabbit IgG and an antibody which targets a TPR domain separate from those involved in ligand recognition, failed to inhibit aggregation in response to TPR activation. Collectively, these findings demonstrate that C-EL2 of TPR plays a critical role in platelet activation, and establish C-EL2Ab as a function blocking antibody. Furthermore, our data suggest a potential for the therapeutic application of C-EL2Ab, which may serve either as an alternative to, or a complement for current treatments. Finally, the identification of a functionally active TPR sequence should aid molecular modeling study predictions for organic derivatives which possess in vivo activity.
Asunto(s)
Fragmentos de Péptidos/fisiología , Agregación Plaquetaria/fisiología , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Animales , Sitios de Unión/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
OBJECTIVE: The purpose of this study is to investigate the potential in vivo antiplatelet and thromboprotective properties of the antihypertensive drug losartan in mice. METHODS: Aggregometry studies were performed on platelets obtained from mice administered losartan for 5 days, via tail vein to examine the ex vivo effects (dose dependence) of this agent and to select an appropriate dose for the in vivo studies. Next, the tail bleeding time test and the time for occlusion in a carotid artery injury thrombosis model (ferric chloride) were also performed to assess the in vivo effects of losartan treatment. RESULTS: These data indicate that the antihypertensive agent losartan exerts dose-dependent inhibition of the thromboxane receptor-mediated (U46619/agonist)-induced platelet aggregation (ex vivo), whereas it produced no detectable effects on aggregation triggered by adenosine diphosphate or the thrombin receptor activating peptide 4. Findings from the in vivo analysis revealed that tail bleeding time of losartan-treated mice was not different from vehicle-treated mice. On the other hand, in the carotid artery injury thrombosis model, it was found that the losartan-treated mice had significantly longer time for occlusion in comparison with those treated with vehicle control. CONCLUSIONS: These findings provide evidence that administration of the antihypertensive drug losartan into live mice produces thromboxane A(2) receptor-specific antiplatelet effects. Furthermore, interestingly, this antiplatelet activity appears to translate into thromboprotective properties, without resulting in a bleeding phenotype. Consequently, aside from its potential use as an antithrombotic agent, losartan's chemistry may provide a "blueprint" for designing or repurposing novel derivatives which may have the potential to serve as an antiplatelet and thromboprotective agents but are deprived of the usually concomitant bleeding adverse effects.
Asunto(s)
Antihipertensivos/farmacología , Losartán/farmacología , Agregación Plaquetaria/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Adenosina Difosfato/farmacología , Animales , Ácido Araquidónico , Coagulación Sanguínea/efectos de los fármacos , Traumatismos de las Arterias Carótidas/patología , Ratones , Ratones Endogámicos C57BL , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Trombina/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/antagonistas & inhibidores , Trombosis/etiología , Trombosis/prevención & controlRESUMEN
While proper platelet function is a vital component of hemostasis, their inappropriate activation contributes to thrombotic disorders. One pathway for platelet activation involves their synthesis of the lipid mediator thromboxane A2 (TXA2). Although TXA2 acts by binding to a seven-transmembrane receptor (i.e., the prostanoid TP receptor) that participates in the genesis of thrombosis, currently, there are no antagonists available for clinical use. Since the only available drug targeting this pathway (aspirin) is associated with inherent limitations/serious side effects, developing prostanoid TP receptor antagonists is clearly warranted. To this end, we have previously employed the "repurposing old drugs for new uses" approach to identify prostanoid TP receptor antagonists and showed that the antidiabetic agent glybenclamide selectively inhibited human platelet prostanoid TP receptors (in vitro). On this basis, we hypothesized that glybenclamide exhibits in vivo antiplatelet potential, and therefore, may protect against thrombosis development. Using murine platelets, it was found that glybenclamide injections: 1) inhibited platelet aggregation induced by the prostanoid TP receptor agonist U46619 and the TXA2 precursor arachidonic acid, under ex vivo experimental settings, concentration-dependently; 2) lacked any detectable effects on aggregation stimulated by ADP, or the thrombin receptor activating-peptide 4; 3) impaired hemostasis by prolonging tail bleeding time; and 4) delayed the development of occlusive thrombi in a carotid artery injury model. Taken together, these findings indicate that glybenclamide does indeed exert, ex vivo and in vivo, prostanoid TP receptor-dependent inhibitory effects on platelet function. Thus, glybenclamide has the potential to be applied in the management of thromboembolic disorders.
Asunto(s)
Fibrinolíticos/farmacología , Gliburida/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inhibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Tiempo de Sangría , Trombosis de las Arterias Carótidas/prevención & control , Relación Dosis-Respuesta a Droga , Reposicionamiento de Medicamentos , Fibrinolíticos/administración & dosificación , Fibrinolíticos/uso terapéutico , Gliburida/administración & dosificación , Gliburida/uso terapéutico , Hemostasis/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Concentración Osmolar , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptores de Tromboxanos/agonistas , Receptores de Tromboxanos/antagonistas & inhibidores , Tromboembolia/prevención & control , Factores de TiempoRESUMEN
OBJECTIVE: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. METHODS: Human aortic endothelial cells were stimulated with TNF-alpha and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFkappa B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. RESULTS: VCAM-1 and E-selectin upregulation was greatest between 2-4 dyne/cm2 (6 and 4-fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM-1 expression and NFkappa B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E-selectin and VCAM-1 expression was greatest. CONCLUSIONS: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of approximately 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation.