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1.
Stem Cells ; 42(3): 230-250, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38183264

RESUMO

Chronic inflammation and dysregulated repair mechanisms after epithelial damage have been implicated in chronic obstructive pulmonary disease (COPD). However, the lack of ex vivo-models that accurately reflect multicellular lung tissue hinders our understanding of epithelial-mesenchymal interactions in COPD. Through a combination of transcriptomic and proteomic approaches applied to a sophisticated in vitro iPSC-alveolosphere with fibroblasts model, epithelial-mesenchymal crosstalk was explored in COPD and following SARS-CoV-2 infection. These experiments profiled dynamic changes at single-cell level of the SARS-CoV-2-infected alveolar niche that unveiled the complexity of aberrant inflammatory responses, mitochondrial dysfunction, and cell death in COPD, which provides deeper insights into the accentuated tissue damage/inflammation/remodeling observed in patients with SARS-CoV-2 infection. Importantly, this 3D system allowed for the evaluation of ACE2-neutralizing antibodies and confirmed the potency of this therapy to prevent SARS-CoV-2 infection in the alveolar niche. Thus, iPSC-alveolosphere cultured with fibroblasts provides a promising model to investigate disease-specific mechanisms and to develop novel therapeutics.


Assuntos
COVID-19 , Células-Tronco Pluripotentes Induzidas , Doença Pulmonar Obstrutiva Crônica , Humanos , SARS-CoV-2 , Proteômica , Imunoterapia , Inflamação
2.
Am J Respir Cell Mol Biol ; 62(4): 524-534, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922883

RESUMO

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.


Assuntos
Colágeno Tipo VIII/metabolismo , Endostatinas/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Pulmão/metabolismo , Apoptose/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno Tipo XVIII/metabolismo , Genética Humana/métodos , Humanos , Transdução de Sinais/fisiologia
3.
Physiol Rep ; 6(11): e13721, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29894584

RESUMO

The importance of myeloid cells in promoting neovascularization has been shown in a number of pathological settings in several organs. However, the specific role of macrophages in promoting systemic angiogenesis during pulmonary ischemia is not fully determined. Our past work suggested that cells of monocytic lineage contributed to systemic angiogenesis in the lung since clodronate-induced depletion of all macrophages resulted in attenuated neovascularization. Our current goals were to define the population of macrophages important for systemic vessel growth into the lung after the onset of pulmonary ischemia in mice. Interstitial macrophages (CD64+ MerTK+ CD11b+ ) increased significantly as did the percent of CD45+ Ly6G+ neutrophils 1 day after the induction of left lung ischemia, despite the fact there was limited cell recruitment due to complete obstruction of the left pulmonary artery in this ischemia model. Since both interstitial macrophages and neutrophils express CD11b, we used CD11b+ DTR mice and showed the critical role for these cells since CD11b+ depleted mice showed no systemic angiogenesis 7 days after the onset of ischemia when compared to control mice. Coculture of mouse aortic endothelial cells with macrophages showed increased proliferation relative to endothelial cells in culture without inflammatory cells, or pulmonary artery endothelial cells. We conclude that CD11b+ leukocytes, trapped within the lung at the onset of ischemia, contribute to growth factor release, and are critical for new blood vessel proliferation.


Assuntos
Isquemia/complicações , Pulmão/irrigação sanguínea , Pulmão/imunologia , Macrófagos/imunologia , Neovascularização Patológica , Animais , Antígeno CD11b/imunologia , Células Cultivadas , Citocinas/imunologia , Células Endoteliais/imunologia , Inflamação/imunologia , Leucócitos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Artéria Pulmonar/imunologia
4.
Theranostics ; 7(2): 377-389, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28042341

RESUMO

Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvß3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvß3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvß3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvß3-No-Drug micelles while αvß3-Dxtl-PD or αvß3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvß3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvß3-Dxtl-PD micelles. Additionally, αvß3-Dxtl-PD decreased BAL eosinophil and αvß3+ CD45+ leukocytes relative to αvß3-No-Drug micelles, whereas αvß3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.


Assuntos
Remodelação das Vias Aéreas , Inibidores da Angiogênese/administração & dosagem , Asma/tratamento farmacológico , Asma/patologia , Nanoestruturas/administração & dosagem , Animais , Asma/diagnóstico por imagem , Cicloexanos/administração & dosagem , Modelos Animais de Doenças , Docetaxel , Portadores de Fármacos/administração & dosagem , Ácidos Graxos Insaturados/administração & dosagem , Imageamento por Ressonância Magnética , Microscopia de Fluorescência , Pró-Fármacos/administração & dosagem , Pyroglyphidae/patogenicidade , Ratos , Sesquiterpenos/administração & dosagem , Taxoides/administração & dosagem , Resultado do Tratamento
5.
Angiogenesis ; 20(1): 73-84, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27787629

RESUMO

Although bronchial angiogenesis has been well documented in allergic asthma, lymphangiogenesis has not been widely studied. Therefore, we evaluated changes in lung lymphatics in a rat model of allergen-induced asthma using house dust mite (Der p 1; 100 µg/challenge). Additionally, properties of isolated lung lymphatic endothelial cells (CD45-, CD141+, LYVE-1+, Prox-1+) were studied in vitro. Three weeks after the onset of intranasal allergen exposure (twice-weekly), an increase in the number of lung lymphatic vessels was measured (34% increase) by lung morphometry. New lymphatic structures were seen predominantly in the peribronchial and periarterial interstitial space but also surrounding large airways. Isolated lymphatic endothelial cells from sensitized lungs showed enhanced proliferation (% Ki67+), chemotaxis, and tube formation (number and length) compared to lymphatic endothelial cells isolated from naive rat lungs. This hyper-proliferative lymphangiogenic phenotype was preserved through multiple cell passages (2-8). Lymphatic endothelial cells isolated from naive and HDM-sensitized rats produced similar in vitro levels of VEGF-C, VEGF-D, and VEGFR3 protein, each recognized as critical lymphangiogenic factors. Inhibition with anti-VEGFR (axitinib, 0.1 µM) blocked proliferation and chemotaxis. Results suggest that in vivo sensitization causes fundamental changes to lymphatic endothelium, which are retained in vitro, and may relate to VEGFR downstream signaling.


Assuntos
Asma/fisiopatologia , Linfangiogênese , Animais , Asma/parasitologia , Asma/patologia , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Secções Congeladas , Pulmão/patologia , Masculino , Pyroglyphidae/fisiologia , Ratos Endogâmicos BN , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo
6.
Cancer Res ; 76(20): 5962-5969, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27569207

RESUMO

Angiogenesis is vital for tumor growth but in well-vascularized organs such as the lung its importance is unclear. This situation is complicated by the fact that the lung has two separate circulations, the pulmonary and the systemic bronchial circulation. There are few relevant animal models of non-small cell lung cancer, which can be used to study the lung's complex circulations, and mice, lacking a systemic bronchial circulation cannot be used. We report here a novel orthotopic model of non-small cell lung cancer in rats, where we have studied the separate contributions of each of the two circulations for lung tumor growth. Results show that bronchial artery perfusion, quantified by fluorescent microspheres (206% increase in large tumors) or high-resolution computed tomography scans (276% increase in large tumors), parallels the growth in tumor volume, whereas pulmonary artery perfusion remained unchanged. Ablation of the bronchial artery after the initiation of tumor growth resulted in a decrease in tumor volume over a subsequent course of 4 weeks. These results demonstrate that although the existing pulmonary circulation can supply the metabolic needs for tumor initiation, further growth of the tumor requires angiogenesis from the highly proliferative bronchial circulation. This model may be useful to investigate new therapeutic approaches that target specifically the bronchial circulation. Cancer Res; 76(20); 5962-9. ©2016 AACR.


Assuntos
Adenocarcinoma/patologia , Artérias Brônquicas/fisiologia , Neoplasias Pulmonares/patologia , Neovascularização Fisiológica , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Artéria Pulmonar/fisiologia , Circulação Pulmonar , Ratos , Tomografia Computadorizada por Raios X , Carga Tumoral
7.
Am J Respir Cell Mol Biol ; 54(3): 394-401, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26244419

RESUMO

Lymphocytes have been shown to modulate angiogenesis. Our previous work showed that T regulatory (Treg) cell depletion prevented angiogenesis. In the present study, we sought to examine T-cell populations during lung angiogenesis and subsequent angiostasis. In a mouse model of ischemia-induced systemic angiogenesis in the lung, we examined the time course (0-35 d) of neovascularization and T-cell phenotypes within the lung after left pulmonary artery ligation (LPAL). T cells increased and reached a maximum by 10 days after LPAL and then progressively decreased, suggestive of a modulatory role during the early phase of new vessel growth. Because others have shown IFN-γ to be angiostatic in tumor models, we focused on this effector T-cell cytokine to control the magnitude of angiogenesis. Results showed that IFN-γ protein is secreted at low levels after LPAL and that mice required Treg depletion to see the full effect of effector T cells. Using Foxp3(DTR) and diphtheria toxin to deplete T regulatory cells, increased numbers of effector T cells (CD8(+)) and/or increased capacity to secrete the prominent angiostatic cytokine IFN-γ (CD4(+)) were seen. In vitro culture of mouse systemic and pulmonary microvascular endothelial cells with IFN-γ showed increased endothelial cell apoptosis. CD8(-/-) mice and IFN-γR(-/-) mice showed enhanced angiogenesis compared with wild-type mice, confirming that, in this model, IFN-γ limits the extent of systemic neovascularization in the lung.


Assuntos
Células Endoteliais/imunologia , Isquemia/imunologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Neovascularização Fisiológica , Linfócitos T Reguladores/imunologia , Animais , Apoptose , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Isquemia/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Ativação Linfocitária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interferon/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Receptor de Interferon gama
8.
Am J Respir Cell Mol Biol ; 52(5): 603-10, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25275926

RESUMO

Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4(+) lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4(+) CD25(+) forkhead homeobox protein-3 (Foxp3)(+) cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3(+) Treg cells can be depleted with diphtheria toxin (DT; Foxp3(DTR)), we unexpectedly found that Foxp3(+) Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3(gfp) controls). Adoptive transfer studies using CD4(+) CD25(+) splenocytes from congenic CD45.1 mice into Foxp3(+) Treg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3(gfp) controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-ß, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII](int), CD11C(+)) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Isquemia/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Fisiológica , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Isquemia/genética , Isquemia/imunologia , Isquemia/fisiopatologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Fatores de Tempo
9.
Angiogenesis ; 18(1): 1-11, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25149641

RESUMO

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 µg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvß3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.


Assuntos
Asma/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Modelos Animais de Doenças , Neovascularização Patológica/parasitologia , Pyroglyphidae/patogenicidade , Resistência das Vias Respiratórias/fisiologia , Análise de Variância , Animais , Artérias Brônquicas/patologia , Hiper-Reatividade Brônquica/parasitologia , Primers do DNA/genética , Fluorocarbonos , Pulmão/patologia , Imageamento por Ressonância Magnética , Cloreto de Metacolina , Nanopartículas , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Elastômeros de Silicone , Fatores de Tempo
10.
PLoS One ; 9(9): e106092, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25181540

RESUMO

The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules. Yet, endothelial nitric oxide has been shown to be largely anti-inflammatory in other systemic venules. Using intravital microscopy to visualize post-capillary tracheal venules in anesthetized, ventilated mice, the number of adherent leukocytes was significantly decreased in eNOS-/- mice under baseline conditions (2±1 cell/60 min observation) vs wild type (WT) C57BL/6 mice (7±2 cells). After exposure to PEEP (8 cmH2O for 1 min; 5 times), adherent cells increased significantly (29±5 cells) in WT mice while eNOS-/- mice demonstrated a significantly decreased number of adherent cells (11±4 cells) after PEEP. A similar response was seen when thrombin was used as the pro-inflammatory stimulus. In addition, mouse tracheal venular endothelial cells studied in vitro after exposure to cyclic stretch (18% elongation) or thrombin both demonstrated increased p-selectin expression that was significantly attenuated by NG-nitro-L-arginine methyl ester, N-acetylcysteine amide (NACA) and excess BH4. In vivo treatment with the ROS inhibitor NACA or co-factor BH4 abolished completely the PEEP-induced leukocyte adherence. These results suggest that pro-inflammatory stimuli cause leukocyte recruitment to tracheal endothelium in part due to eNOS uncoupling.


Assuntos
Leucócitos/enzimologia , Leucócitos/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Mecânico , Traqueia/patologia , Vênulas/patologia , Animais , Adesão Celular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/deficiência , Selectina-P/metabolismo , Fosforilação/efeitos dos fármacos , Respiração com Pressão Positiva , Espécies Reativas de Oxigênio/metabolismo , Trombina/farmacologia
11.
Pulm Circ ; 4(2): 260-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25006445

RESUMO

Abnormal lung microvascular endothelial vascular barrier function may contribute to pulmonary inflammation, such as that occurring during inhalation of cigarette smoke (CS). Cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in both epithelial and endothelial cells, regulates the organization of tight junctions between epithelial cells and has also been implicated in the transport of sphingosine-1 phosphate (S1P), a vascular barrier-enhancing sphingolipid. Because CS has been shown to affect CFTR function, we hypothesized that CFTR function contributes to lung endothelial cell barrier and that CFTR dysfunction worsens CS-induced injury. CFTR inhibitors GlyH-101 or CFTRinh172 caused a dose-dependent increase in pulmonary or bronchial endothelial monolayer permeability, which peaked after 4 hours. CFTR inhibition was associated with both intercellular gaps and actin stress fiber formation compared with vehicle-treated cells. Increasing endothelial S1P, either by exogenous treatment or by inhibition of its degradation, significantly improved the barrier function in CFTR-inhibited monolayers. Both cultured lung endothelia and the lung microcirculation visualized in vivo with intravital two-photon imaging of transgenic mice deficient in CFTR showed that CFTR dysfunction increased susceptibility to CS-induced permeability. These results suggested that CFTR function might be required for lung endothelial barrier, including adherence junction stability. Loss of CFTR function, especially concomitant to CS exposure, might promote lung inflammation by increasing endothelial cell permeability, which could be ameliorated by S1P.

12.
PLoS One ; 8(6): e66432, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23776670

RESUMO

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0-24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.


Assuntos
Brônquios/metabolismo , Brônquios/patologia , Quimiocinas/metabolismo , Animais , Brônquios/efeitos dos fármacos , Broncopatias/metabolismo , Proliferação de Células , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Dexametasona/uso terapêutico , Células Endoteliais/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Neovascularização Patológica/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Respir Res ; 13: 93, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23061826

RESUMO

BACKGROUND: Systemic neovascularization of the lung during chronic ischemia has been observed in all mammals studied. However, the proteins that orchestrate the complex interaction of new vessel growth and tunneling through lung tissue matrix have not been described. Although previous work has demonstrated the CXC chemokines are essential growth factors in the process of angiogenesis in mice and rats, key matrix proteins have not been identified. METHODS: Since the degradation of chemokines has been shown to be dependent on metalloproteinases (MMP), we first surveyed gene expression patterns (real time RT-PCR) of several lung matrix proteins in DBA/J (D2) mice and C57Bl/6 (B6) mice, strains known to have divergent parenchymal responses in other lung disease models. We studied changes in the time course of MMP-12 activity in D2 and B6 mice. Functional angiogenesis was determined 14 days after the onset of complete left lung ischemia induced by left pulmonary artery ligation (LPAL), using fluorescent microspheres. RESULTS: Our results confirmed higher levels of MMP-12 gene expression in D2 mice relative to B6, which corresponded to a phenotype of minimal systemic angiogenesis in D2 mice and more robust angiogenesis in B6 mice (p < 0.01). MMP-12 activity decreased over the course of 14 days in B6 mice whereas it increased in D2 mice (p < 0.05). MMP-12 was associated largely with cells expressing the macrophage marker F4/80. Genetic deficiency of MMP-12 resulted in significantly enhanced neovascularization (p < 0.01 from B6). CONCLUSION: Taken together, our results suggest macrophage-derived MMP-12 contributes to angiostasis in the ischemic lung.


Assuntos
Isquemia/enzimologia , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Neovascularização Fisiológica , Animais , Antígenos de Diferenciação/metabolismo , Biomarcadores/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Isquemia/etiologia , Isquemia/genética , Isquemia/imunologia , Ligadura , Pulmão/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Metaloproteinase 12 da Matriz/deficiência , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Artéria Pulmonar/cirurgia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Fatores de Tempo
14.
PLoS One ; 6(11): e26716, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22110592

RESUMO

Angiogenesis after pulmonary ischemia is initiated by reactive O(2) species and is dependent on CXC chemokine growth factors, and its magnitude is correlated with the number of lavaged macrophages. After complete obstruction of the left pulmonary artery in mice, the left lung is isolated from the peripheral circulation until 5-7 days later, when a new systemic vasculature invades the lung parenchyma. Consequently, this model offers a unique opportunity to study the differentiation and/or proliferation of monocyte-derived cells within the lung. In this study, we questioned whether macrophage subpopulations were differentially expressed and which subset contributed to growth factor release. We characterized the change in number of all macrophages (MHCII(int), CD11C+), alveolar macrophages (MHCII(int), CD11C+, CD11B-) and mature lung macrophages (MHCII(int), CD11C+, CD11B+) in left lungs from mice immediately (0 h) or 24 h after left pulmonary artery ligation (LPAL). In left lung homogenates, only lung macrophages increased 24 h after LPAL (vs. 0 h; p<0.05). No changes in proliferation were seen in any subset by PCNA expression (0 h vs. 24 h lungs). When the number of monocytic cells was reduced with clodronate liposomes, systemic blood flow to the left lung 14 days after LPAL decreased by 42% (p<0.01) compared to vehicle controls. Furthermore, when alveolar macrophages and lung macrophages were sorted and studied in vitro, only lung macrophages secreted the chemokine MIP-2α (ELISA). These data suggest that ischemic stress within the lung contributes to the differentiation of immature monocytes to lung macrophages within the first 24 h after LPAL. Lung macrophages but not alveolar macrophages increase and secrete the proangiogenic chemokine MIP-2α. Overall, an increase in the number of lung macrophages appears to be critical for neovascularization in the lung, since clodronate treatment decreased their number and attenuated functional angiogenesis.


Assuntos
Isquemia , Pulmão/irrigação sanguínea , Macrófagos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Técnicas In Vitro , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Neovascularização Fisiológica , Fenótipo
15.
Am J Physiol Lung Cell Mol Physiol ; 301(5): L782-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21821727

RESUMO

Hyaluronan (HA), a glycosaminoglycan critical to the lung extracellular matrix, has been shown to dissociate into low-molecular-weight (LMW) HA fragments following exposure to injurious stimuli. In the present study we questioned whether lung HA changed during ischemia and whether changes had an effect on subsequent angiogenesis. After left pulmonary artery ligation (LPAL) in mice, we analyzed left lung homogenates immediately after the onset of ischemia (0 h) and intermittently for 14 days. The relative expression of HA synthase (HAS)1, HAS2, and HAS3 was determined by real-time RT-PCR, total HA in the lung was measured by an ELISA-like assay, gel electrophoresis was performed to determine changes in HA size distribution, and the activity of hyaluronidases was determined by zymography. A 50% increase in total HA was measured 16 h after the onset of ischemia and remained elevated for up to 7 days. Furthermore, a fourfold increase in LMW HA fragments (495-30 kDa) was observed by 4 h after LPAL. Both HAS1 and HAS2 showed increased expression 4-16 h after LPAL, yet no changes were seen in hyaluronidase activity. These results suggest that both HA fragmentation and activation of HA synthesis contribute to increased HA levels during lung ischemia. Delivery of LMW HA fragments in an in vitro tube formation assay or directly to the ischemic mouse lung in vivo both resulted in increased angiogenesis. We conclude that ischemic injury results in matrix fragmentation, which leads to stimulation of neovascularization.


Assuntos
Glucuronosiltransferase/metabolismo , Ácido Hialurônico/metabolismo , Isquemia/metabolismo , Pulmão/metabolismo , Neovascularização Patológica/metabolismo , Artéria Pulmonar/fisiopatologia , Animais , Colágeno , Combinação de Medicamentos , Eletroforese em Gel de Ágar , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Hialuronan Sintases , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Isquemia/fisiopatologia , Laminina , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Proteoglicanas , Artéria Pulmonar/cirurgia , Reação em Cadeia da Polimerase em Tempo Real
16.
Am J Physiol Lung Cell Mol Physiol ; 299(4): L535-41, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20693319

RESUMO

Pulmonary artery obstruction and subsequent lung ischemia have been shown to induce systemic angiogenesis despite preservation of normoxia. The underlying mechanisms, however, remain poorly understood. In a mouse model of lung ischemia induced by left pulmonary artery ligation (LPAL), we showed previously, the formation of a new systemic vasculature to the ischemic lung. We hypothesize that LPAL in the mouse increases reactive oxygen species (ROS) production, and these molecules play an initiating role in subsequent lung neovascularization. We used oxidant-sensitive dyes (DHE and H(2)DCF-DA) to quantify ROS and measured the antioxidant-reduced glutathione (GSH) and its oxidized form (GSSG) as indicators of ROS levels after LPAL. The magnitude of systemic neovascularization was determined by measuring systemic blood flow to the left lung with radiolabeled microspheres 14 days after LPAL. An increase in ROS was observed early (30 min: 55% increase in H(2)DCF-DA) after LPAL, with a return to baseline by 24 h. GSH/GSSG was decreased (∼50%) 4 h after LPAL, suggesting earlier ROS upregulation. Mice treated with the antioxidant N-acetylcysteine showed attenuated angiogenesis (62% of wild-type LPAL), and mice lacking Nrf2, a transcription factor important for antioxidant synthesis, resulted in increased neovascularization (207% of wild-type LPAL). Overall, GSH/GSSG was inversely associated with the magnitude of neovascularization. These results demonstrate that LPAL induces an early and transient ROS upregulation, and ROS appear to play a role in promoting ischemia-induced angiogenesis.


Assuntos
Modelos Animais de Doenças , Isquemia/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Patológica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/uso terapêutico , Acetilcisteína/toxicidade , Animais , Antioxidantes/uso terapêutico , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Isquemia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Neovascularização Patológica/tratamento farmacológico , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo
17.
Am J Physiol Lung Cell Mol Physiol ; 299(3): L323-33, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20453163

RESUMO

Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 microM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H(2)O(2)-challenged mouse lung microvascular (MLMVEC) monolayers. 8pCPT-cGMP also decreased perfusate LDH release in isolated mouse lungs exposed to H(2)O(2) or ischemia-reperfusion. The protective effect of increasing cGMP in MLMVECs was accompanied by enhanced endothelial H(2)O(2) scavenging (measured by H(2)O(2) electrode) and decreased intracellular ROS concentration (measured by 2',7'-dichlorofluorescin fluorescence) as well as decreased phosphorylation of p38 MAPK and Akt. The cGMP-mediated cytoprotection and increased H(2)O(2) scavenging required >2 h of 8pCPT-cGMP incubation in wild-type MLMVEC and were absent in MLMVEC from protein kinase G (PKG(I))-/- mice suggesting a PKG(I)-mediated effect on gene regulation. Catalase and glutathione peroxidase 1 (Gpx-1) protein were increased by cGMP in wild-type but not PKG(I)-/- MLMVEC monolayers. Both the cGMP-mediated increases in antioxidant proteins and H(2)O(2) scavenging were prevented by inhibition of translation with cycloheximide. 8pCPT-cGMP had minimal effects on catalase and Gpx-1 mRNA. We conclude that cGMP, through PKG(I), attenuated H(2)O(2)-induced cytotoxicity in MLMVEC by increasing catalase and Gpx-1 expression through an unknown posttranscriptional effect.


Assuntos
Antioxidantes/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/farmacologia , Pulmão/irrigação sanguínea , Microvasos/fisiopatologia , Oxidantes/farmacologia , Animais , Fator Natriurético Atrial/farmacologia , Catalase/metabolismo , Morte Celular , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/deficiência , Células Endoteliais/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/metabolismo , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tionucleotídeos/farmacologia
18.
Microvasc Res ; 80(1): 18-22, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20144627

RESUMO

We showed previously in a mouse model of lung ischemia-induced angiogenesis, enhanced expression of the three ELR+ CXC chemokines (KC, LIX, and MIP-2) and that blockade of the ligand receptor CXCR(2) limited neovascularization. The present study was undertaken to determine the relative abundance and angiogenic potential of the three CXC chemokines and whether RhoA activation explained the measured differences in potencies. We found that LIX showed the greatest absolute amount in the in vivo model 4 h after left pulmonary artery obstruction (LIX>KC>MIP-2; p<0.05). In vitro, LIX induced the greatest degree of arterial endothelial cell chemotaxis and KC was without effect. A significant increase (approximately 40%) in active RhoA was observed with both LIX and MIP-2 compared with vehicle control (p<0.05). On average, LIX induced the greatest amount of tube formation within pleural tissue in culture. Thus, the results of the present study suggest that among the three ELR+ CXC chemokines, LIX predominates in eliciting a pro-angiogenic phenotype.


Assuntos
Proteínas Angiogênicas/farmacologia , Quimiocinas CXC/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Angiogênicas/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Aorta/citologia , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/farmacologia , Quimiocina CXCL2/metabolismo , Quimiocina CXCL2/farmacologia , Quimiocina CXCL5/metabolismo , Quimiocina CXCL5/farmacologia , Quimiocinas CXC/metabolismo , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Isquemia/metabolismo , Ligadura , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/metabolismo , Artéria Pulmonar/cirurgia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/imunologia , Técnicas de Cultura de Tecidos , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP
19.
Am J Physiol Lung Cell Mol Physiol ; 295(6): L1056-65, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18849438

RESUMO

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Guanilato Ciclase/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Barreira Alveolocapilar , AMP Cíclico/genética , AMP Cíclico/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Guanilato Ciclase/genética , Camundongos , Camundongos Knockout , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Lesão Pulmonar Induzida por Ventilação Mecânica/genética
20.
Am J Physiol Lung Cell Mol Physiol ; 295(5): L941-8, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18805956

RESUMO

We previously have shown in mice and rats, enhanced leukocyte recruitment to airway postcapillary venules after excessive distention imposed by the application of positive end-expiratory pressure. Because P-selectin was shown to be essential for this outcome, we sought to establish an in vitro endothelial cell model and determine the mechanisms whereby mechanical distension alters adhesion molecule expression. P-selectin surface expression on mouse jugular vein endothelial cells exposed to cyclic stretch (5 or 20% elongation for 5 min; Flexercell) were compared with static cells. The larger, pathophysiological regimen of cyclic stretch showed a 54% increase in P-selectin expression after stretch compared with static cells. This response was attenuated but confirmed in tracheal venular endothelium (29% increase). We questioned whether these changes were dependent on increases in intracellular Ca(2+) through voltage-gated Ca(2+) channels. The stretch-induced increase in P-selectin expression was substantially decreased by pretreatment with the T-type Ca(2+) channel inhibitor mibefradil (76% inhibition). Furthermore, when the Ca(v)3.1 T-type Ca(2+) channel expression was decreased in both endothelial cell subtypes with specific small-interfering RNA, the distension-induced expression of P-selectin decreased to levels less than that observed in static cells. We conclude that P-selectin expression on systemic venular endothelial cells contributes to a proinflammatory phenotype after mechanical stretch and can be selectively modulated by voltage-gated calcium channel inhibition.


Assuntos
Selectina-P/metabolismo , Pneumonia/metabolismo , Veias Pulmonares/metabolismo , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Mibefradil/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Respiração com Pressão Positiva , Veias Pulmonares/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Mecânico , Verapamil/farmacologia
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