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OBJECTIVE: Stem cell-based regenerative therapies have been intensively studied with the aim to define an ideal cell type for the treatment of myocardial infarction. We tested systemically delivered, platelet-targeted induced vascular progenitor cells (iVPCs) to study their potential to salvage damaged myocardium after ischemia-reperfusion injury. METHODS: Using a mouse model of ischemia-reperfusion injury, we tested the potential of platelet-targeted iVPCs (1 × 106 targ-iVPCs) compared to non-targ-iVPCs and a saline control. Bioluminescence imaging, echocardiography, and histological analyses were performed. RESULTS: Four weeks after ischemia-reperfusion injury, systemic delivery of targ-iVPCs led to reduced fibrosis and infarct size (PBS: 25.7 ± 3.9 vs targ-iVPC: 18.4 ± 6.6 vs non-targ-iVPC: 25.1 ± 3.7%I/LV, P < 0.05), increased neovascularization, and restored cardiac function (PBS: 44.0 ± 4.2 vs targ-iVPC: 54.3 ± 4.5 vs non-targ-iVPC: 46.4 ± 3.8%EF, P < 0.01). Cell tracking experiments revealed entrapment of intravenously injected iVPCs in the pulmonary microvasculature in both cell-treated groups. CONCLUSIONS: Systemic delivery of iVPCs after cardiac ischemia-reperfusion injury is limited by pulmonary entrapment of the cells. Nevertheless, targ-iVPCs reduced infarct size, fibrosis, increased neovascularization, and most importantly retained cardiac function. These findings contribute to the mechanistic discussion of cell-based therapy and ultimately identify activated platelet-targeted iVPCs as candidates for cell therapy and also describe cell therapy benefits without the necessity of engrafting.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Microvasos/citologia , Traumatismo por Reperfusão Miocárdica/terapia , Animais , Plaquetas/citologia , Comunicação Celular , Rastreamento de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Pulmão/irrigação sanguínea , Camundongos , Comunicação Parácrina , Resultado do TratamentoRESUMO
Aims: CD39 is a cell membrane NTPase with anti-inflammatory and anti-platelet effects. However, its clinical use is limited by its bleeding side effect. With the goal of harnessing its therapeutic potential while avoiding haemostatic problems, we designed a fusion protein consisting of the extracellular domain of CD39 and a single-chain antibody (Targ-CD39) that specifically binds to activated glycoprotein (GP)IIb/IIIa and thus to activated platelets. Through this enrichment at activated platelets, the required systemic dose is below the dose impairing haemostasis. Methods and results: Using an ischaemia/reperfusion mouse model (left anterior descending artery ligated for 1 h) we achieved remarkable protection of the reperfused tissue with Targ-CD39 compared with Non-targ-CD39 (mutated, non-binding version of Targ-CD39) and PBS control. Targ-CD39 restored ejection fraction and fractional shortening to a level indistinguishable from pre-injury status, while controls showed functional deterioration. Employing advanced clinically relevant methods of ultrasound analysis, we observed that both radial and longitudinal strain and strain rate showed infarct-typical changes of myocardial deformation in controls, but not in Targ-CD39 treated mice. Histological assessment confirmed strong reduction of infarct size and increase in neovascularization. Furthermore, attenuation of post-ischaemic inflammation was seen in cytokine profiling. Conclusion: Overall, we demonstrate that Targ-CD39 holds promise for treatment of myocardial infarction.
Assuntos
Adenosina Trifosfatases/uso terapêutico , Anticorpos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Proteínas Recombinantes de Fusão/uso terapêutico , Adenosina Trifosfatases/farmacologia , Animais , Anticorpos/farmacologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/patologia , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.
Assuntos
Apolipoproteínas E , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/biossíntese , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell-based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair. METHODS AND RESULTS: We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function. CONCLUSIONS: These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Quimiocina CXCL12/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Ataque Isquêmico Transitório/terapia , Infarto do Miocárdio/terapia , Glicoproteínas da Membrana de Plaquetas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/uso terapêutico , Colágeno/metabolismo , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/uso terapêutico , Ligação Proteica , Receptores CXCR4/metabolismo , Resultado do TratamentoRESUMO
Platelets play a critical role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases. In a multiple step process, they adhere to activated endothelium and release proinflammatory cytokines thereby promoting the inflammatory process. Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and triggers platelet activation and primary hemostasis. Activation of GPVI leads to stable platelet adhesion and degranulation of platelet granules. However, GPVI is critically involved in platelet adhesion to activated endothelium without exposure of subendothelial matrix. Earlier studies show that the soluble GPVI-Fc binds to collagen and protects mice from atherosclerosis and decreases neointima proliferation after arterial injury. Here, we show for the first time that recombinant GPVI-Fc binds to activated endothelium mainly via vitronectin and prevents platelet/endothelial interaction. Administration of GPVI-Fc reduced infarct size and preserved cardiac function in a mouse model of myocardial infarction. This process was associated with reduced GPVI-induced platelet degranulation and release of proinflammatory cytokines in vitro and in vivo. Taken together, administration of GPVI-Fc offers a novel strategy to control platelet-mediated inflammation and to preserve myocardial function following myocardial infarction.
Assuntos
Endotélio Vascular/metabolismo , Coração/fisiologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Ataque Isquêmico Transitório/metabolismo , Adesividade Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Ataque Isquêmico Transitório/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Multimerização Proteica/fisiologiaRESUMO
Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.
Assuntos
Traumatismo por Reperfusão , Trombose , Animais , Plaquetas/metabolismo , Humanos , Camundongos , Ativação Plaquetária , Reperfusão , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Trombose/metabolismoRESUMO
Thrombosis contributes to one in four deaths worldwide and is the cause of a large proportion of mortality and morbidity. A reliable and rapid diagnosis of thrombosis will allow for immediate therapy, thereby providing significant benefits to patients. Molecular imaging is a fast-growing and captivating area of research, in both preclinical and clinical applications. Major advances have been achieved by improvements in three central areas of molecular imaging: - (1) better markers for diseases, with increased sensitivity and selectivity, (2) optimised contrast agents with improved signal to noise ratio and (3), progress in scanner technologies with higher sensitivity and resolution. Clinically available imaging modalities used for molecular imaging include magnetic resonance imaging (MRI), X-ray computed tomography (CT), ultrasound, as well as nuclear imaging, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT). In the preclinical imaging field, optical (fluorescence and bioluminescent) molecular imaging has provided new mechanistic insights in the pathology of thromboembolic diseases. Overall, the advances in molecular imaging, driven by the collaboration of various scientific disciplines, have substantially contributed to an improved understanding of thrombotic disease and raise the exciting prospect of earlier diagnosis and individualised therapy for cardiovascular diseases. As such, these advances hold significant promise to be translated to clinical practice and ultimately to reduce mortality and morbidity in patients with thromboembolic diseases. LINKED ARTICLES: This article is part of a themed issue on Molecular imaging - visual themed issue. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.21/issuetoc.
Assuntos
Trombose , Trombose Venosa , Artérias , Biomarcadores , Humanos , Imagem Molecular , Trombose/diagnóstico por imagem , Trombose Venosa/diagnóstico por imagemRESUMO
New disease-modifying therapeutic options for immune diseases in general and immune-related cardiovascular diseases are urgently needed. Various innovative therapies are currently developed ranging from small molecules and natural compounds to biopharmaceuticals. This article summarizes the overview of possible points of intervention for an ongoing immune response and lists examples of currently developed therapeutic exploitations of these targets. Research and development for the treatment of autoimmune and inflammatory diseases in general will most likely be fruitful also for therapy of immune-related cardiovascular diseases such as myocarditis and atherosclerosis and should be closely followed by research groups focused on cardiovascular immunology.
Assuntos
Fenômenos do Sistema Imunitário/efeitos dos fármacos , Imunoterapia/métodos , Inflamação/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/terapia , Humanos , Imunomodulação , MiocarditeRESUMO
Cardiovascular diseases (CVD) cause about 1/3 of global deaths. Therefore, new strategies for the prevention and treatment of cardiovascular events are highly sought-after. Vitamin E is known for significant antioxidative and anti-inflammatory properties, and has been studied in the prevention of CVD, supported by findings that vitamin E deficiency is associated with increased risk of cardiovascular events. However, randomized controlled trials in humans reveal conflicting and ultimately disappointing results regarding the reduction of cardiovascular events with vitamin E supplementation. As we discuss in detail, this outcome is strongly affected by study design, cohort selection, co-morbidities, genetic variations, age, and gender. For effective chronic primary and secondary prevention by vitamin E, oxidative and inflammatory status might not have been sufficiently antagonized. In contrast, acute administration of vitamin E may be more translatable into positive clinical outcomes. In patients with myocardial infarction (MI), which is associated with severe oxidative and inflammatory reactions, decreased plasma levels of vitamin E have been found. The offsetting of this acute vitamin E deficiency via short-term treatment in MI has shown promising results, and, thus, acute medication, rather than chronic supplementation, with vitamin E might revitalize vitamin E therapy and even provide positive clinical outcomes.
RESUMO
Acute myocardial infarction (AMI) is the single leading cause of mortality and morbidity worldwide. A key component of AMI therapy is the timely reopening of occluded vessels to prevent further ischaemic damage to the myocardium. However, reperfusion of the ischaemic myocardium can itself trigger reperfusion injury causing up to 50% of the overall infarct size. In recent years, considerable research has been devoted to understanding the pathogenesis of ischaemia/reperfusion (I/R) injury and platelets have emerged as a major contributing factor. This review summarizes the role of platelets in the pathogenesis of I/R injury and highlights the potential of platelet-directed therapeutics to minimize cardiac I/R injury. Activated platelets infiltrate specifically into the ischaemic/reperfused myocardium and contribute to I/R injury by the formation of microthrombi, enhanced platelet-leucocyte aggregation, and the release of potent vasoconstrictor and pro-inflammatory molecules. This review demonstrates the benefits of platelet inhibition beyond their well-described anti-thrombotic effect and highlights the direct cardioprotective role of anti-platelet drugs. In particular, the inhibition of COX, the P2Y12 receptor and the GPIIb/IIIa receptor has demonstrated the potential to attenuate I/R injury. Moreover, targeting of drug candidates or regenerative cells to the activated platelets accumulated within the ischaemic/reperfused myocardium shows remarkable potential to protect the myocardium from I/R injury. Overall, activated platelets play a key role in the pathogenesis of I/R injury. Their direct inhibition as well as their use as epitopes for site-directed therapy is a unique and promising therapeutic approach for the prevention of I/R injury and ultimately the preservation of cardiac function.
Assuntos
Plaquetas/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/uso terapêutico , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Animais , Plaquetas/metabolismo , Inibidores de Ciclo-Oxigenase/efeitos adversos , Humanos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Inibidores da Agregação Plaquetária/efeitos adversos , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Transdução de Sinais , Resultado do TratamentoRESUMO
OBJECTIVE: Myocardial infarction (MI) is a leading cause of mortality and morbidity worldwide and new treatment strategies are highly sought-after. Paradoxically, reperfusion of the ischemic myocardium, as achieved with early percutaneous intervention, results in substantial damage to the heart (ischemia/reperfusion injury) caused by cell death due to aggravated inflammatory and oxidative stress responses. Chronic therapy with vitamin E is not effective in reducing the cardiovascular event rate, presumably through failing to reduce atherosclerotic plaque instability. Notably, acute treatment with vitamin E in patients suffering a MI has not been systematically investigated. METHODS AND RESULTS: We applied alpha-tocopherol (α-TOH), the strongest anti-oxidant form of vitamin E, in murine cardiac ischemia/reperfusion injury induced by ligation of the left anterior descending coronary artery for 60â¯min. α-TOH significantly reduced infarct size, restored cardiac function as measured by ejection fraction, fractional shortening, cardiac output, and stroke volume, and prevented pathological changes as assessed by state-of-the-art strain and strain-rate analysis. Cardioprotective mechanisms identified, include a decreased infiltration of neutrophils into cardiac tissue and a systemic anti-inflammatory shift from Ly6Chigh to Ly6Clow monocytes. Furthermore, we found a reduction in myeloperoxidase expression and activity, as well as a decrease in reactive oxygen species and the lipid peroxidation markers phosphatidylcholine (PC) (16:0)-9-hydroxyoctadecadienoic acid (HODE) and PC(16:0)-13-HODE) within the infarcted tissue. CONCLUSION: Overall, α-TOH inhibits ischemia/reperfusion injury-induced oxidative and inflammatory responses, and ultimately preserves cardiac function. Therefore, our study provides a strong incentive to test vitamin E as an acute therapy in patients suffering a MI.
Assuntos
Cardiotônicos/metabolismo , Inflamação/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , alfa-Tocoferol/metabolismo , Animais , Biomarcadores/metabolismo , Cardiotônicos/farmacologia , Citocinas/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Inflamação/tratamento farmacológico , Inflamação/etiologia , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/etiologia , Oxirredução/efeitos dos fármacos , Transcriptoma , alfa-Tocoferol/farmacologiaRESUMO
Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.
Assuntos
Antineoplásicos/administração & dosagem , Plaquetas/metabolismo , Imunoconjugados/administração & dosagem , Terapia de Alvo Molecular/métodos , Oligopeptídeos/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Microambiente Tumoral , Animais , Antineoplásicos/metabolismo , Modelos Animais de Doenças , Imunoconjugados/metabolismo , Camundongos , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Oligopeptídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Anticorpos de Cadeia Única/imunologia , Transplante Heterólogo , Resultado do TratamentoRESUMO
The plant Garcinia kola is used in African ethno-medicine to treat various oxidation- and inflammation-related diseases but its bioactive compounds are not well characterized. Garcinoic acid (GA) is one of the few phytochemicals that have been isolated from Garcinia kola. We investigated the anti-inflammatory potential of the methanol extract of Garcinia kola seeds (NE) and purified GA, as a major phytochemical in these seeds, in lipopolysaccharide (LPS)-activated mouse RAW264.7 macrophages and its anti-atherosclerotic potential in high fat diet fed ApoE-/- mice. This study outlines an optimized procedure for the extraction and purification of GA from Garcinia kola seeds with an increased yield and a purity of >99%. We found that LPS-induced upregulation of iNos and Cox2 expression, and the formation of the respective signaling molecules nitric oxide and prostanoids, were significantly diminished by both the NE and GA. In addition, GA treatment in mice decreased intra-plaque inflammation by attenuating nitrotyrosinylation. Further, modulation of lymphocyte sub-populations in blood and spleen have been detected, showing immune regulative properties of GA. Our study provides molecular insights into the anti-inflammatory activities of Garcinia kola and reveals GA as promising natural lead for the development of multi-target drugs to treat inflammation-driven diseases.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Garcinia kola/química , Nozes/química , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Animais , Biomarcadores , Cromatografia Líquida , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células RAW 264.7 , Sementes , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.
Assuntos
Antígenos CD/farmacologia , Apirase/farmacologia , Lesões das Artérias Carótidas/tratamento farmacológico , Fibrinolíticos/farmacologia , Glicoproteínas/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/farmacologia , Trombose/prevenção & controle , Animais , Antígenos CD/toxicidade , Apirase/farmacocinética , Apirase/toxicidade , Doenças das Artérias Carótidas/sangue , Doenças das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/induzido quimicamente , Lesões das Artérias Carótidas/patologia , Cloretos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Compostos Férricos , Fibrinolíticos/farmacocinética , Fibrinolíticos/toxicidade , Glicoproteínas/farmacocinética , Glicoproteínas/toxicidade , Hemorragia/induzido quimicamente , Humanos , Fragmentos Fc das Imunoglobulinas/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Placa Aterosclerótica , Inibidores da Agregação Plaquetária/farmacocinética , Inibidores da Agregação Plaquetária/toxicidade , Glicoproteínas da Membrana de Plaquetas/farmacocinética , Glicoproteínas da Membrana de Plaquetas/toxicidade , Proteínas Recombinantes de Fusão/farmacologia , Trombose/sangue , Trombose/induzido quimicamente , Trombose/patologiaRESUMO
Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer using three different imaging modalities. These findings warrant further development of this activated platelet specific scFvGPIIb/IIIa, potentially as a universal marker for cancer diagnosis and ultimately for drug delivery in an innovative theranostic approach.
Assuntos
Plaquetas/química , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Imagem Óptica/métodos , Ativação Plaquetária , Animais , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Anticorpos de Cadeia Única/metabolismoRESUMO
One of the major hurdles in intravenous regenerative cell therapy is the low homing efficiency to the area where these cells are needed. To increase cell homing toward areas of myocardial damage, we developed a bispecific tandem single-chain antibody (Tand-scFvSca-1+GPIIb/IIIa) that binds with high affinity to activated platelets via the activated glycoprotein (GP)IIb/IIIa receptor, and to a subset of peripheral blood mononuclear cells (PBMC) which express the stem cell antigen-1 (Sca-1) receptor. Methods: The Tand-scFvSca-1+GPIIb/IIIa was engineered, characterized and tested in a mouse model of ischemia-reperfusion (IR) injury applying left coronary artery occlusion for 60 min. Fluorescence cell tracking, cell infiltration studies, echocardiographic and histological analyses were performed. Results: Treatment of mice undergoing myocardial infarction with targeted-PBMCs led to successful cell delivery to the ischemic-reperfused myocardium, followed by a significant decrease in infiltration of inflammatory cells. Homing of targeted-PBMCs as shown by fluorescence cell tracking ultimately decreased fibrosis, increased capillary density, and restored cardiac function 4 weeks after ischemia-reperfusion injury. Conclusion: Tand-scFvSca-1+GPIIb/IIIa is a promising candidate to enhance therapeutic cell delivery in order to promote myocardial regeneration and thereby preventing heart failure.
Assuntos
Plaquetas/metabolismo , Testes de Função Cardíaca , Leucócitos Mononucleares/transplante , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/terapia , Animais , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Citocinas/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Inflamação/patologia , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Neovascularização Fisiológica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Anticorpos de Cadeia Única/metabolismo , Remodelação VentricularRESUMO
A reliable method for the diagnosis of minimal cardiac ischemia would meet a strong demand for the sensitive diagnosis of coronary artery disease in cardiac stress testing and risk stratification in patients with chest pain but unremarkable ECGs and biomarkers. We hypothesized that platelets accumulate early on in ischemic myocardium and a newly developed technology of non-invasive molecular PET imaging of activated platelets can thus detect minimal degrees of myocardial ischemia. To induce different degrees of minimal cardiac ischemia, the left anterior descending artery (LAD) was ligated for 10, 20 or 60 min. Mice were injected with a newly generated scFvanti-GPIIb/IIIa-64CuMeCOSar radiotracer, composed of a single-chain antibody that only binds to activated integrin GPIIb/IIIa (αIIbßIII) and thus to activated platelets, and a sarcophagine cage MeCOSar complexing the long half-life PET tracer copper-64. A single PET/CT scan was performed. Evans Blue/TTC staining to detect necrosis as well as classical serological biomarkers like Troponin I and heart-type fatty acid-binding protein (H-FABP) were negative, whereas PET imaging of activated platelets was able to detect small degrees of ischemia. Taken together, molecular PET imaging of activated platelets represents a unique and highly sensitive method to detect minimal cardiac ischemia.
Assuntos
Meios de Contraste/farmacologia , Angiografia Coronária/métodos , Sistemas de Liberação de Medicamentos , Isquemia Miocárdica/diagnóstico por imagem , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Tomografia por Emissão de Pósitrons/métodos , Anticorpos de Cadeia Única/farmacologia , Animais , Plaquetas , Humanos , CamundongosRESUMO
AIMS: Platelet adhesion and aggregation is essential for haemostasis and thrombosis. Cytoskeletal reorganization of activated platelets plays a crucial role in these processes and implies activation of Rho GTPases. Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signalling. In this study, we explored the regulation of Rho GTPases in platelets. METHODS AND RESULTS: Oligophrenin1 (OPHN1) is a RhoGAP-regulating cytoplasmic protein that has been investigated in patients with X-linked mental retardation. Here, we identified OPHN1 in mouse platelets where it co-localizes to actin-rich regions and Rho GTPases. OPHN1 exhibits strong GTPase-stimulating activity towards RhoA, Cdc42, and Rac1 and regulates cell adhesion and spreading. Furthermore, OPHN1 controls RhoA-mediated stress fibre and focal adhesion formation as well as filopodia and lamellipodia development. The analysis of different domains of OPHN1 revealed distinct functions in Rho hydrolysis. The C-terminus of OPHN1 displays an essential unit for Rho regulation, whereas the PH domain is a regulatory unit of OPHN1 controlling GAP function. The N-terminal BAR (Bin/amphiphysin/Rvs)-like domain is involved in GAP regulation but not in cytoskeleton rearrangements or Rho regulation and acts as a guidance domain to direct this GAP to its substrate. CONCLUSION: Our results suggest that OPHN1 is a powerful regulator of Rho GTPase activity in platelets that is critical for the reorganization of the cytoskeleton, which is a major process required for stable platelet adhesion and thrombus formation to occur.