RESUMO
Background: The RECcording COurses of vasculaR Diseases (RECCORD) registry established by the German Society of Angiology - Society for Vascular Medicine aimed to address the lack in contemporary real-world data regarding current practice of medical and interventional care in vascular patients. We herein report the demographic and procedural characteristics of the first 1000 patients undergoing endovascular revascularization (EVR) for symptomatic peripheral artery disease (PAD). Patients and methods: RECCORD is an observational, prospective, multicenter, all-comers registry. Only patients undergoing EVR for symptomatic PAD are included and followed up for at least 1 year. Demographic characteristics, comorbidities, previous peripheral vascular interventions, medication, clinical stage of lower extremity artery disease (Rutherford category), hemodynamic parameters, and procedural data including complications are recorded via an entirely web-based platform. Results: Of the first 1000 patients (mean age 70 ± 10 years, 35% female) with 1096 EVR at 1477 vascular segments of the lower extremities, 25.0% were at the stage of chronic limb threatening ischemia (CLTI) and 75.0% at non-CLTI. The femoropopliteal segment was the dominant target lesion site (61.0%), followed by iliac (26.4%) and below-the-knee EVR (10.3%). Only angioplasty was performed in 130 EVR (11.9%), adjunctive drug coated balloons (DCB) in 498 (45.4%), additional stenting in 633 (57.8%). Debulking devices were used in 106 (9.7%) EVR. Clinical (Rutherford categories) and hemodynamic parameters (ankle-brachial-index) as well as secondary preventive medication were significantly improved post EVR. Periprocedural complications occurred in 63 (5.7%) EVR with pseudoaneurysm as the leading complication type in 26 (2.4%) EVR. Conclusions: The baseline data of the first 1000 patients from the RECCORD registry representing the real-world setting illustrate that the majority of EVR are performed in patients with claudication. Adjunctive use of DCB and stenting are the dominant types of EVR, while periprocedural complications are at an acceptable low rate.
Assuntos
Sistema de Registros , Idoso , Idoso de 80 Anos ou mais , Angioplastia com Balão , Demografia , Feminino , Artéria Femoral , Humanos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica , Artéria Poplítea , Estudos Prospectivos , Resultado do Tratamento , Grau de Desobstrução VascularRESUMO
BACKGROUND: The prevalence of peripheral artery disease (PAD) is increasing worldwide. Revascularization procedures constitute a cornerstone of the therapy in PAD, not only in critical limb ischaemia but increasingly also in patients with intermittent claudication. The German Society of Angiology - Society for Vascular Medicine is establishing a nationwide, prospective, multicentre registry to address the lack of contemporary real life data regarding current practice of medical and interventional care in vascular patients and its subsequent long-term outcome. PATIENTS AND METHODS: The RECording COurses of vasculaR Diseases registry (RECCORD registry) is an observational, prospective, multicentre, all-comers registry platform. In the initial phase, patients referred for endovascular revascularization of PAD of the lower limbs will be prospectively included and followed up for at least one year. At baseline, data on patients' demographic characteristics, comorbidities, previous peripheral interventions, medication, and clinical stage of PAD (Rutherford category), haemodynamic parameters, and procedural data including complications will be assessed. Major adverse cardiac and limb events will be recorded at planned (at six and 12 months) and at any unplanned visits. The therapeutic management will be exclusively left to the discretion of the vascular specialists. RESULTS AND CONCLUSIONS: The RECCORD registry will provide a comprehensive dataset depicting the current real life practice and outcome of vascular care. The seven predefined quality indicators will be used for benchmarking the participating centres. Moreover, identifying factors promoting a favourable outcome might pave the way for an evidence-based therapeutic strategy and a dedicated therapeutic pathway for patients with PAD including patient-oriented best interventional approaches. In the future, the RECCORD registry may provide a general platform to study the courses of various defined vascular diseases in order to get detailed insights into the real life current practice of health care provided to vascular patients.
Assuntos
Pesquisa Biomédica/métodos , Atenção à Saúde , Procedimentos Endovasculares , Extremidade Inferior/irrigação sanguínea , Doenças Vasculares Periféricas/terapia , Sistema de Registros , Benchmarking , Atenção à Saúde/normas , Procedimentos Endovasculares/efeitos adversos , Procedimentos Endovasculares/normas , Alemanha/epidemiologia , Humanos , Doenças Vasculares Periféricas/diagnóstico , Doenças Vasculares Periféricas/epidemiologia , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Diabetes mellitus type II and arterial hypertension are major risk factors for peripheral arterial disease and have been considered to reduce collateral growth (arteriogenesis). Collateral growth proceeds through different stages. Vascular proliferation and macrophage accumulation are hallmarks of early collateral growth. MATERIAL AND METHODS: We here compare the impact of arterial hypertension and diabetes mellitus type II on collateral proliferation (Brdu incorporation) and macrophage accumulation (ED 2 staining) as well as collateral vessel function (collateral conductance) in a rat model of peripheral vascular disease (femoral artery occlusion), diabetes mellitus type II (Zucker fatty diabetic rats and Zucker lean rat controls) and arterial hypertension (induced via clip placement around the right renal arteriy). We furthermore tested the impact of monocyte chemoattractant protein-1 (MCP1) on collateral proliferation and macrophage accumulation in these models RESULTS: Diabetic animals showed reduced vascular proliferation and macrophage accumulation, which however did not translate into a change of collateral conductance. Hypertensive animals on the contrary had reduced collateral conductances without altered macrophage accumulation and only a marginal reduction in collateral proliferation. Infusion of MCP1 only enhanced vascular proliferation in diabetic animals. CONCLUSIONS: These findings illustrate that impaired monocyte/macrophage recruitment is responsible for reduced collateral growth under diabetic conditions but not in arterial hypertension suggesting that diabetes mellitus in particular affects early stages of collateral growth whereas hypertension has its impact on later remodeling stages. Successful pro-arteriogenic treatment strategies in a patient population that presents with diabetes mellitus and arterial hypertension need to address different stages of collateral growth and thus different molecular and cellular targets simultaneously.
Assuntos
Circulação Colateral/fisiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Hipertensão/fisiopatologia , Macrófagos/citologia , Animais , Artérias/crescimento & desenvolvimento , Artérias/fisiopatologia , Diabetes Mellitus Tipo 2/imunologia , Angiopatias Diabéticas/imunologia , Modelos Animais de Doenças , Hipertensão/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Obesidade/fisiopatologia , Ratos ZuckerRESUMO
Several catheter-based systems have been developed for interventional recanalization of pulmonary embolism. These include local ultrasound assisted thrombolysis (EKOS), in-toto-thrombectomy via retriever and aspiration system (FlowTriever) and the Indigo mechanical aspiration system. Safety and efficacy in the removal of thrombus have been demonstrated for all systems. Interventional recanalization strategies for high- and intermediate-high risk pulmonary embolism are potentially more effective in the removal of thrombus and restoration of right heart function than systemic thrombolysis with a lower risk of major bleeding complications. Preliminary data from registries and observational studies are very promising whereas the evidence for systemic thrombolysis treatment in high and intermediate-high risk pulmonary embolism is low. Randomized controlled clinical trials are currently performed comparing catheter based interventional therapies to systemic thrombolysis for the treatment of intermediate-high risk pulmonary embolisms. Primary outcome measurements include mortality, hemodynamic collapse, and major bleedings. Results are expected in 2025. The introduction of interventional therapies for pulmonary embolism was accompanied by an increased awareness of the complexity of pulmonary embolism management. The need for specialized interdisciplinary pulmonary embolism response teams (PERT-teams) and a well-structured approach including a PDCA cycle was recognized.
Assuntos
Embolia Pulmonar , Trombectomia , Terapia Trombolítica , Embolia Pulmonar/terapia , HumanosRESUMO
OBJECTIVE: Previously, we demonstrated the relevance for endothelial carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression in collateral formation. However, a proarteriogenic role for CEACAM1(+) myeloid cells is unknown. Here, we investigated the contribution of CEACAM1(+) myeloid cells on collateral formation. METHODS AND RESULTS: Collateral growth and vascular remodeling were analyzed in CEACAM1-competent and CEACAM1 null mice after femoral artery ligation in hindlimb ischemia. Reperfusion of the adductor muscles was evaluated by Laser Doppler measurements and microcomputed tomography imaging. In CEACAM1 null mice, poor reperfusion and reduced collateral formation were observed, accompanied by reduction in arterial diameters. Using flow cytometry, we identified an increase of the muscle-resident CD11b(+)/granulocyte receptor-1+ (Gr-1+) population in CEACAM1 null mice only, pointing toward a CEACAM1-dependent functional deviation. Direct and reciprocal bone marrow transplantations between CEACAM1-competent and CEACAM1 null mice, and antibody-mediated depletion of the CD11b(+)/Gr-1(+) population, confirmed the requirement of CEACAM1 expression on the CD11b(+)/Gr-1(+) population for reestablishment of perfusion after arterial occlusion. CONCLUSIONS: CEACAM1 expression on CD11b(+)/Gr-1(+) myeloid cells is a prerequisite for adequate collateral formation.
Assuntos
Antígeno CD11b/metabolismo , Antígeno Carcinoembrionário/metabolismo , Circulação Colateral , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Células Mieloides/metabolismo , Neovascularização Fisiológica , Receptores de Quimiocinas/metabolismo , Animais , Transplante de Medula Óssea , Antígeno Carcinoembrionário/genética , Modelos Animais de Doenças , Citometria de Fluxo , Membro Posterior , Isquemia/diagnóstico por imagem , Isquemia/genética , Isquemia/fisiopatologia , Fluxometria por Laser-Doppler , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/transplante , Fluxo Sanguíneo Regional , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.
Assuntos
Antígeno Carcinoembrionário/análise , Inflamação/fisiopatologia , Leishmaniose Cutânea/fisiopatologia , Células Mieloides/fisiologia , Neovascularização Patológica/fisiopatologia , Animais , Anticorpos Antiprotozoários/biossíntese , Transplante de Medula Óssea , Antígeno CD11b/análise , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Colágeno , Combinação de Medicamentos , Edema/etiologia , Edema/patologia , Glicoproteínas/biossíntese , Imunidade Celular , Implantes Experimentais , Inflamação/etiologia , Inflamação/imunologia , Interferon gama/biossíntese , Laminina , Leishmania major/imunologia , Leishmaniose Cutânea/complicações , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Vasos Linfáticos/metabolismo , Macrófagos/parasitologia , Macrófagos/fisiologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/química , Células Mieloides/classificação , Neovascularização Patológica/patologia , Proteoglicanas , Quimera por Radiação , Células Th1/imunologiaRESUMO
The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p(Ser239)-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Endoteliais/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Vimentina/biossíntese , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Ratos , Serina/metabolismo , Vimentina/genéticaRESUMO
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Endoteliais , Neovascularização Fisiológica , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Laminina/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microesferas , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteoglicanas/metabolismoRESUMO
BACKGROUND: Neutrophils and monocytes are centrally linked to vascular inflammatory disease, and leukocyte-derived myeloperoxidase (MPO) has emerged as an important mechanistic participant in impaired vasomotor function. MPO binds to and transcytoses endothelial cells in a glycosaminoglycan-dependent manner, and MPO binding to the vessel wall is a prerequisite for MPO-dependent oxidation of endothelium-derived nitric oxide (NO) and impairment of endothelial function in animal models. In the present study, we investigated whether heparin mobilizes MPO from vascular compartments in humans and defined whether this translates into increased vascular NO bioavailability and function. METHODS AND RESULTS: Plasma MPO levels before and after heparin administration were assessed by ELISA in 109 patients undergoing coronary angiography. Whereas baseline plasma MPO levels did not differ between patients with or without angiographically detectable coronary artery disease (CAD), the increase in MPO plasma content on bolus heparin administration was higher in patients with CAD (P=0.01). Heparin treatment also improved endothelial NO bioavailability, as evidenced by flow-mediated dilation (P<0.01) and by acetylcholine-induced changes in forearm blood flow (P<0.01). The extent of heparin-induced MPO release was correlated with improvement in endothelial function (r=0.69, P<0.01). Moreover, and consistent with this tenet, ex vivo heparin treatment of extracellular matrix proteins, cultured endothelial cells, and saphenous vein graft specimens from CAD patients decreased MPO burden. CONCLUSIONS: Mobilization of vessel-associated MPO may represent an important mechanism by which heparins exert antiinflammatory effects and increase vascular NO bioavailability. These data add to the growing body of evidence for a causal role of MPO in compromised vascular NO signaling in humans.
Assuntos
Anti-Inflamatórios/farmacologia , Endotélio Vascular/metabolismo , Heparina/farmacologia , Óxido Nítrico/metabolismo , Peroxidase/metabolismo , Idoso , Disponibilidade Biológica , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Estudos de Casos e Controles , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/sangue , Peroxidase/sangueRESUMO
Collateral growth is characterized by macrophage accumulation, suggesting an important role of circulating cells. To study origin and function of macrophages during arteriogenesis, we related the extent of macrophage accumulation to vascular proliferation and investigated the fate of fluorescently (CMFDA) labeled blood cells that were injected at the time of femoral artery occlusion. The effect of bone marrow depletion via cyclophosphamide before femoral artery occlusion on collateral proliferation and macrophage accumulation was studied, and we looked for the presence of bone marrow-derived stem cells in the vicinity of growing collateral vessels. Finally, we investigated the arteriogenic effect of macrophage activation via MCP-1 in bone marrow-depleted animals. Maximal macrophage accumulation occurred during the first 3 days after femoral artery occlusion and paralleled the extent of vascular proliferation. Fluorescently labeled leukocytes homed to spleen and wound but they were absent in proliferating collateral arteries during maximal macrophage accumulation. Depletion of circulating cells did neither affect macrophage accumulation nor collateral growth. Staining of monocyte-depleted animals for BrdUrd and ED2, alphaSMA, or VE-Cadherin demonstrated local proliferation of macrophages and vascular cells, whereas C-Kit, SSEA1, or Thy1-positive bone marrow-derived stem cells were not detectable. Enhancement of macrophage accumulation via MCP-1 was independent of circulating monocytes and promoted arteriogenesis in the absence of direct effects on vascular cells. We propose that the initial phase of vascular growth is characterized by local proliferation of tissue resident precursors rather than by migration of blood born cells. The full text of this article is available online at http://circres.ahajournals.org.
Assuntos
Quimiocina CCL2/fisiologia , Circulação Colateral/fisiologia , Linfócitos/fisiologia , Macrófagos/fisiologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/fisiologia , Actinas/análise , Animais , Antígenos CD , Biomarcadores , Células da Medula Óssea/fisiologia , Caderinas/análise , Movimento Celular , Células Cultivadas/citologia , Quimiocina CCL2/farmacologia , Vasos Coronários/citologia , Ciclofosfamida/toxicidade , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Artéria Femoral , Humanos , Antígenos CD15/análise , Ligadura , Macrófagos/classificação , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Pancitopenia/induzido quimicamente , Pancitopenia/fisiopatologia , Proteínas Proto-Oncogênicas c-kit/análise , Ratos , Baço/citologia , Antígenos Thy-1/análise , Cicatrização/fisiologia , Fator de von Willebrand/análiseRESUMO
Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa ß3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.
Assuntos
Vasos Sanguíneos/imunologia , Circulação Colateral/imunologia , Inflamação/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Vasos Sanguíneos/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/imunologia , Células Progenitoras Endoteliais/imunologia , Inflamação/patologia , RatosRESUMO
It has been demonstrated in several studies that collateral growth is associated with accumulation of macrophages around proliferating vessel. Macrophages are known to secrete vascular growth factors and metalloproteinases. Both are necessary for the development of a proper vasculature. Recent studies suggest that certain subpopulations of macrophages are also capable of transdifferentiating into vascular cells. There are good reasons to assume that shear force rises dramatically in preexisting arteriolar shunts after occlusion of the main supplying vessel. Based upon these two findings it was hypothesized that high shear forces lead to homing of circulating monocytes to the growing collateral artery. The majority of studies, however, indicate that monocytes home under low shear force conditions. Our own observations in monocyte depleted animals suggest that proliferation and transdifferentiation of tissue macrophages occurs locally in growing collateral vessels and is independent of circulating cells. We thus propose that local proliferation and transdifferentiation of tissue macrophages rather than homing of circulating monocytes play a major role in arteriogenesis.
Assuntos
Artérias/citologia , Artérias/crescimento & desenvolvimento , Macrófagos/fisiologia , Diferenciação Celular , Circulação Colateral , Células Endoteliais/metabolismo , Substâncias de Crescimento/metabolismo , Humanos , Modelos Biológicos , Monócitos/fisiologiaRESUMO
"Therapeutic angiogenesis" requires targeted delivery of growth factors for maximal benefit and limitation of potential hazards such as enhancement of tumor or plaque angiogenesis. Physiological distinctions between angiogenesis and collateral growth suggest the possibility of targeting selectively collateral endothelium. This article describes the generation of collateral-targeting single-chain antibodies (scFv). Membrane preparations of growing collateral arteries from rats were used to produce collateral-targeting antibodies (CTAs) via immunization of mice. ScFv were generated from CTA-producing hybridoma and cloned into the pV gene of M13 phages. Phages expressing collateral-targeting scFv (CT scFv) were selected via repeated exposure to activated collateral arteries followed by reamplification. CT scFv could specifically be amplified, selected, and sequenced. Phages expressing CT scFv bound selectively to proliferating collateral vessels as identified by positive Polycyclic Nuclear Antigen (PCNA) staining and homed specifically to collateral endothelium after in vivo injection but bound neither to control vessels nor to tumor vessels. This study reveals major differences between angiogenic and collateral endothelium and delivers a tool that will allow the stimulation of collateral growth without promoting tumor or plaque angiogenesis.
Assuntos
Anticorpos Monoclonais/genética , Artérias/crescimento & desenvolvimento , Neovascularização Fisiológica , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Artérias/metabolismo , Bacteriófago M13/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Circulação Colateral , Células Endoteliais/metabolismo , Artéria Femoral/crescimento & desenvolvimento , Regulação da Expressão Gênica , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Biblioteca de Peptídeos , Ratos , Ratos Sprague-DawleyRESUMO
Migration of endothelial cells plays an important role during angiogenesis and the late remodeling phase of arteriogenesis. To investigate mechanisms responsible for cell migration, the authors subcloned a rat heart endothelial cell line (RHE) into a migrating and a nonmigrating cell line (RHE-A and RHE-neg, respectively). Both cell lines form cobblestone patterns in confluent cultures similar to the originating cell line, but RHE-neg cells grow in dense cell islets of several layers whereas RHE-A cells grow in a less dense monolayer. Both cell lines show the same expression pattern of known endothelial cell surface antigens (e.g., FIK-1). The authors used two-dimensional gel electrophoresis technique to look for differentially regulated proteins with possible functional importance for cell migration. The analysis of the cytosolic fraction as well as the membrane fraction revealed differences in the protein expression patterns of RHE-neg and RHE-A cells. Regulated spots were isolated and analyzed by mass spectrometry (MS/MS technique), leading to the identification of proteins potentially responsible for endothelial cell migration, e.g., the intermediate filament vimentin that was exclusively expressed in RHE-A cells. The authors thus have generated a reproducible model that allows the analysis of the proteome responsible for endothelial cell migration.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica/fisiologia , Proteoma/metabolismo , Animais , Antígenos de Superfície/metabolismo , Biomarcadores , Divisão Celular/fisiologia , Linhagem Celular , Eletroforese em Gel Bidimensional , Células Endoteliais/imunologia , Queratinas/metabolismo , Espectrometria de Massas , Especificidade de Órgãos , Proteoma/química , Proteoma/imunologia , Ratos , Vimentina/metabolismoRESUMO
Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.
Assuntos
Moléculas de Adesão Celular/deficiência , Osteoclastos/citologia , Animais , Antígenos CD/genética , Medula Óssea/metabolismo , Remodelação Óssea , Moléculas de Adesão Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , FenótipoAssuntos
Angioplastia com Balão/instrumentação , Arteriopatias Oclusivas/terapia , Stents Farmacológicos , Artéria Femoral/cirurgia , Doença Arterial Periférica/terapia , Artéria Poplítea/cirurgia , Idoso , Stents Farmacológicos/efeitos adversos , Stents Farmacológicos/estatística & dados numéricos , Terapia por Exercício , Alemanha , Humanos , Resultado do Tratamento , CaminhadaRESUMO
Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.
Assuntos
Derme/transplante , Células Endoteliais/citologia , Células Endoteliais/transplante , Regeneração Tecidual Guiada , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular , Ensaios de Migração Celular , Derme/patologia , Camundongos , Camundongos Nus , Modelos Animais , Miocárdio/citologia , Neovascularização Fisiológica , Ratos , Transplante de Células-Tronco , Engenharia Tecidual , Alicerces TeciduaisRESUMO
BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.
Assuntos
Fibroblastos/transplante , Membro Posterior/patologia , Isquemia/terapia , Animais , Células Cultivadas , Circulação Colateral/genética , Circulação Colateral/fisiologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Masculino , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Plasmídeos/genética , Ratos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: The involvement of collateral blood flow/fluid shear stress, nitric oxide (NO), and macrophages during collateral growth (arteriogenesis) is established, but their interplay remains paradoxical. METHODS: In order to further elucidate the "fluid shear stress/NO/macrophage" paradox, we investigated the time course of collateral blood flow (using a Doppler flow probe) and NOS expression (immunohistochemistry, Western blot) in growing rat collateral vessels after femoral artery occlusion and their impact on macrophage recruitment and collateral proliferation (immunohistochemistry, angiographies). RESULTS: (values are given as mean ± standard error of mean) Early after occlusion, collateral blood flow was significantly reduced (pre- 90.0 ± 4.5 vs. post-occlusion 62.5 ± 5.9 µl/min; p < 0.01), and local inducible NOS (iNOS) and endothelial NOS (eNOS) expression were down-regulated (expression in % of non-occluded: eNOS 49.4 ± 11.8% and iNOS 54.5 ± 7.9% vs. non-occluded at 12 h after occlusion; p < 0.03). An artificial rise (induced by a peripheral vasodilatation) of the initially decreased collateral blood flow back to pre-occlusion levels reduced collateral macrophage recruitment (macrophages per collateral section: post- 42.5 ± 4.4 vs. artificial pre-occlusion 27.8 ± 2.0; p < 0.05) and diminished collateral proliferation (proliferative index: post- 0.54 ± 0.02 vs. artificial pre-occlusion 0.19 ± 0.04; p < 0.001) significantly 72 h after femoral artery occlusion. CONCLUSIONS: We propose the following resolution of the "fluid shear stress/NO/macrophage" paradox: Collateral blood flow and NOS expression are initially reduced during arteriogenesis allowing macrophages to accumulate and therewith enhancing collateral proliferation. After homing of macrophages (24 h after occlusion), collateral blood flow and NOS expression recover in order to join the effects of macrophages for restoring blood flow.