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1.
Am J Physiol Lung Cell Mol Physiol ; 327(3): L319-L326, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38860847

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-ß (TGF-ß) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFß-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.


Assuntos
Hipertensão Pulmonar , Músculo Liso Vascular , Inibidor 1 de Ativador de Plasminogênio , Remodelação Vascular , Animais , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Camundongos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Transdução de Sinais , Masculino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proliferação de Células , Camundongos Knockout , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Apoptose , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética
2.
Health Expect ; 27(4): e14155, 2024 08.
Artigo em Inglês | MEDLINE | ID: mdl-39044675

RESUMO

BACKGROUND: An estimated 2.2 million people from Central and Eastern Europe (CEE) live in the United Kingdom. It has been documented that CEE migrants underutilise health services in the United Kingdom and, as an alternative, seek healthcare in their home country. However, reasons for seeking healthcare abroad are not always clear. This review aims to identify the reasons for the uptake of transnational healthcare among CEE migrants resident in the United Kingdom. METHODS: Informed by discussions with community members, medical stakeholders and academics, a systematic scoping review was undertaken following the nine-stage Joanna Briggs Institute framework for scoping reviews. A search strategy with MeSH terms, where relevant, was used and adapted in five academic databases, two grey literature databases and Google Scholar. Included records encompassed four concepts: migration, CEE nationalities, UK nations and healthcare utilisation, which were written in English and published between May 2004 and 2022. Data from the literature were coded, grouped and organised into themes. RESULTS: A total of 16 publications fulfilled the inclusion criteria. There is evidence that some CEE migrants exclusively use healthcare services in the United Kingdom. However, many CEE migrants utilise healthcare both in the United Kingdom and their country of origin. Four themes were identified from the literature as to why migrants travelled to their country of origin for healthcare: cultural expectations of medical services, distrust in the UK NHS, barriers and transnational ties. CONCLUSION: Push factors led CEE migrants to seek healthcare in their country of origin, facilitated by ongoing transnational ties. CEE migrants frequently combine visits to their country of origin with medical appointments. Utilising healthcare in their country of origin as opposed to the United Kingdom can result in fragmented and incomplete records of medications, medical tests and surgeries and risk of unnecessary treatments and complications. This review highlights the need for more targeted health outreach with CEE groups within the United Kingdom, as well as the need for further research on the impact of national events, for example, COVID-19 and Brexit, on transnational healthcare-seeking behaviours. PATIENT OR PUBLIC CONTRIBUTION: The concept for this scoping review was informed by discussions with community members, medical professionals and academics, who identified it as a current issue. The results of this scoping review were discussed with healthcare stakeholders.


Assuntos
Aceitação pelo Paciente de Cuidados de Saúde , Migrantes , Humanos , Reino Unido , Migrantes/estatística & dados numéricos , Aceitação pelo Paciente de Cuidados de Saúde/etnologia , Europa Oriental/etnologia , Acessibilidade aos Serviços de Saúde
3.
Blood ; 135(23): 2085-2093, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32187355

RESUMO

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Heparina/toxicidade , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Trombocitopenia/imunologia , Tromboplastina/metabolismo , Animais , Anticoagulantes/toxicidade , Complexo Antígeno-Anticorpo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Receptores Fc/genética , Receptores Fc/imunologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/metabolismo , Trombocitopenia/patologia
4.
Blood ; 133(5): 481-493, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30442678

RESUMO

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.


Assuntos
Fibrina/imunologia , Ativação de Neutrófilo , Trombose/imunologia , alfa-Defensinas/imunologia , Animais , Coagulação Sanguínea , Fibrina/análise , Fibrinólise , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Calicreínas/sangue , Calicreínas/imunologia , Masculino , Camundongos , Conformação Proteica , Estabilidade Proteica , Trombose/sangue , Trombose/patologia , alfa-Defensinas/sangue
5.
J Biol Chem ; 292(50): 20528-20543, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-28972182

RESUMO

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin-positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.


Assuntos
Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Linfangioleiomiomatose/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Angiomiolipoma/tratamento farmacológico , Angiomiolipoma/genética , Angiomiolipoma/metabolismo , Angiomiolipoma/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/tratamento farmacológico , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Interferência de RNA , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genética
6.
J Biol Chem ; 291(29): 15029-45, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27151212

RESUMO

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(-/-)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(-/-) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(-/-) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis.


Assuntos
Proteínas de Homeodomínio/metabolismo , Neovascularização Fisiológica , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Células K562 , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
7.
Blood Adv ; 8(14): 3798-3809, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38805575

RESUMO

ABSTRACT: Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.


Assuntos
Megacariócitos , Ativador de Plasminogênio Tipo Uroquinase , Megacariócitos/metabolismo , Megacariócitos/citologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Humanos , Animais , Camundongos , Fibrinólise/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Plaquetas/metabolismo , Trombose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Grânulos Citoplasmáticos/metabolismo , Antígenos CD34/metabolismo
8.
Biochem J ; 443(2): 491-503, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22280367

RESUMO

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ß1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ß1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ß1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.


Assuntos
Movimento Celular , Proteínas da Matriz Extracelular/metabolismo , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Células Cultivadas , Proteínas da Matriz Extracelular/deficiência , Humanos , Camundongos , Camundongos Knockout , Ligação Proteica , Ativador de Plasminogênio Tipo Uroquinase/genética
9.
bioRxiv ; 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38106191

RESUMO

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbß3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points: Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbß3 receptors and is functionally available from activated platelets.

10.
bioRxiv ; 2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37790328

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

11.
J Biol Chem ; 286(26): 23044-53, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21540184

RESUMO

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of ß-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.


Assuntos
Barreira Alveolocapilar/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Mucosa Respiratória/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Barreira Alveolocapilar/patologia , Permeabilidade Capilar/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Edema Pulmonar/genética , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Mucosa Respiratória/patologia , Serpina E2/genética , Serpina E2/metabolismo , Serpina E2/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
12.
Blood ; 115(25): 5241-8, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20410503

RESUMO

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.


Assuntos
Sistemas de Liberação de Medicamentos , Precursores Enzimáticos/farmacologia , Eritrócitos , Fibrinolíticos/farmacologia , Pró-Fármacos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Trombose/prevenção & controle , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Humanos , Camundongos , Proteínas Recombinantes/farmacologia , Fatores de Tempo
13.
Biochim Biophys Acta Mol Cell Res ; 1869(1): 119157, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34619163

RESUMO

Endothelial cells (ECs) degrade the extracellular matrix of vessel walls and contact surrounding cells to facilitate migration during angiogenesis, leading to formation of an EC-tubular network (ETN). Mesenchymal stromal cells (MSC) support ETN formation when co-cultured with ECs, but the mechanism is incompletely understood. We examined the role of the urokinase-type plasminogen activator (uPA) system, i.e. the serine protease uPA, its inhibitor PAI-1, receptor uPAR/CD87, clearance by the low-density lipoprotein receptor-related protein (LRP1) and their molecular partners, in the formation of ETNs supported by adipose tissue-derived MSC. Co-culture of human umbilical vein ECs (HUVEC) with MSC increased mRNA expression levels of uPAR, MMP14, VEGFR2, TGFß1, integrin ß3 and Notch pathway components (Notch1 receptor and ligands: Dll1, Dll4, Jag1) in HUVECs and uPA, uPAR, TGFß1, integrin ß3, Jag1, Notch3 receptor in MSC. Inhibition at several steps in the activation process indicates that uPA, uPAR and LRP1 cross-talk with αv-integrins, VEGFR2 and Notch receptors/ligands to mediate ETN formation in HUVEC-MSC co-culture. The urokinase system mediates ETN formation through the coordinated action of uPAR, uPA's catalytic activity, its binding to uPAR and its nuclear translocation. These studies identify potential targets to help control aberrant angiogenesis with minimal impact on healthy vasculature.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Antígenos CD18/metabolismo , Células Cultivadas , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Receptores Notch/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
15.
Sci Rep ; 11(1): 8205, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859248

RESUMO

N-methyl-D-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A-D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.


Assuntos
Músculo Liso Vascular/metabolismo , Artéria Pulmonar/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animais , Células Cultivadas , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Vasoconstrição/genética
16.
J Pharmacol Exp Ther ; 332(3): 1022-31, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19952305

RESUMO

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.


Assuntos
Eritrócitos/efeitos dos fármacos , Fibrinolíticos/farmacologia , Ativadores de Plasminogênio/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Animais , Agregação Eritrocítica , Eritrócitos/fisiologia , Fibrinolíticos/farmacocinética , Glicoforinas/imunologia , Hemólise , Humanos , Técnicas In Vitro , Veias Jugulares , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/farmacocinética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/genética , Trombose Venosa/sangue , Trombose Venosa/prevenção & controle
17.
Blood ; 112(1): 100-10, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18337556

RESUMO

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle alpha-actin (alpha-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.


Assuntos
Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Actinas/genética , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica , Células HeLa , Humanos , Cinética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transdução de Sinais , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Nucleolina
18.
J Clin Invest ; 126(2): 483-94, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26690701

RESUMO

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbß3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbß3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.


Assuntos
Plaquetas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Pró-Fármacos/farmacologia , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pró-Fármacos/farmacocinética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Trombose/sangue , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/farmacocinética
19.
Blood Adv ; 1(1): 62-74, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-29296696

RESUMO

Heparin-induced thrombocytopenia (HIT) is a thrombotic disorder initiated by antibodies to complexes between platelet factor 4 (PF4) and heparin. The risk of recurrent thromboembolism persists after heparin is cleared and platelet activation leading to release of PF4 has dissipated. We asked whether antigenic complexes between polyphosphates and PF4 released from activated platelets might intensify or sustain the prothrombotic phenotype of HIT. PF4 forms stable, ultralarge complexes with polyphosphates of various sizes, including those released from platelets, which are recognized by the HIT-like monoclonal KKO, an immunoglobulin G2bκ monoclonal heparin/PF4 binding antibody, and by human HIT antibodies. KKO helps to protect PF4/polyphosphate complexes from degradation by phosphatases. Complement is activated when HIT antibodies bind to PF4/polyphosphate complexes and PF4 reverses the inhibition of complement by polyphosphates. Polyphosphates and PF4 are stored primarily in separate granules in resting platelets, but they colocalize when the cells are activated. Platelets activated by subaggregating doses of thrombin receptor activating peptide release polyphosphates and PF4, which form antigenic complexes that allow KKO to further activate platelets in the absence of heparin and exogenous PF4. These studies suggest that thrombin- or immune complex-mediated release of endogenous antigenic PF4/polyphosphate complexes from platelets may augment the prothrombotic risk of HIT and perpetuate the risk of thrombosis after heparin has been discontinued.

20.
Cell Rep ; 7(2): 412-423, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24726356

RESUMO

Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome, which is caused by mutations of the tumor suppressor gene folliculin (FLCN). The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcn deletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcn(f/f):SP-C-Cre mice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.


Assuntos
Apoptose , Síndrome de Birt-Hogg-Dubé/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Síndrome de Birt-Hogg-Dubé/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Deleção de Genes , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Alvéolos Pulmonares/patologia , Ratos , Mucosa Respiratória/patologia , Proteínas Supressoras de Tumor/genética
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