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1.
Nat Commun ; 13(1): 6941, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396957

RESUMO

Chronic obstructive airway diseases are a global medical burden that is expected to increase in the near future. However, the underlying mechanistic processes are poorly understood so far. Herein, we show that the endocannabinoid anandamide (AEA) induces prominent airway relaxation in vitro and in vivo. In contrast to 2-arachidonlyglycerol-induced airway relaxation, this is mediated by fatty acid amide hydrolase (FAAH)-dependent metabolites. In particular, we identify mouse and also human epithelial and airway smooth muscle cells as source of AEA-induced prostaglandin E2 production and cAMP as direct mediator of AEA-dependent airway relaxation. Mass spectrometry experiments demonstrate reduced levels of endocannabinoid-like compounds in lungs of ovalbumin-sensitized mice indicating a pathophysiological relevance of endocannabinoid signalling in obstructive airway disease. Importantly, AEA inhalation protects against airway hyper-reactivity after ovalbumin sensitization. Thus, this work highlights the AEA/FAAH axis as a critical regulator of airway tone that could provide therapeutic targets for airway relaxation.


Assuntos
Ácidos Araquidônicos , Endocanabinoides , Animais , Camundongos , Humanos , Endocanabinoides/metabolismo , Ovalbumina , Ácidos Araquidônicos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
3.
Sci Transl Med ; 14(638): eabl6328, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-35353541

RESUMO

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fosfatidilinositol 3-Quinase , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Inflamação , Camundongos , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
4.
Pharmaceutics ; 15(1)2022 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-36678686

RESUMO

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

5.
ACS Pharmacol Transl Sci ; 4(2): 888-897, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33860209

RESUMO

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.

6.
Arterioscler Thromb Vasc Biol ; 41(12): 2974-2989, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34615374

RESUMO

OBJECTIVE: Pathological angiogenesis is a hallmark of various diseases characterized by local hypoxia and inflammation. These disorders can be treated with inhibitors of angiogenesis, but current compounds display a variety of side effects and lose efficacy over time. This makes the identification of novel signaling pathways and pharmacological targets involved in angiogenesis a top priority. Approach and Results: Here, we show that inactivation of FAAH (fatty acid amide hydrolase), the enzyme responsible for degradation of the endocannabinoid anandamide, strongly impairs angiogenesis in vitro and in vivo. Both, the pharmacological FAAH inhibitor URB597 and anandamide induce downregulation of gene sets for cell cycle progression and DNA replication in endothelial cells. This is underscored by cell biological experiments, in which both compounds inhibit proliferation and migration and evoke cell cycle exit of endothelial cells. This prominent antiangiogenic effect is also of pathophysiological relevance in vivo, as laser-induced choroidal neovascularization in the eye of FAAH-/- mice is strongly reduced. CONCLUSIONS: Thus, elevation of endogenous anandamide levels by FAAH inhibition represents a novel antiangiogenic mechanism.


Assuntos
Amidoidrolases/farmacocinética , Ácidos Araquidônicos/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Endocanabinoides/farmacologia , Endotélio Vascular/crescimento & desenvolvimento , Músculo Liso Vascular/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/patologia , Agonistas de Receptores de Canabinoides/farmacologia , Bovinos , Linhagem Celular , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Neovascularização Patológica
7.
Front Chem ; 8: 833, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33173765

RESUMO

The cyclic depsipeptide FR900359 (FR) isolated from the plant Ardisia crenata and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53-0.55 nM and limits of quantification (LOQ) of 1.6-1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.

8.
Mol Cell ; 80(6): 940-954.e6, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33202251

RESUMO

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fosfolipase C beta/genética , Cálcio/metabolismo , Sinalização do Cálcio/genética , Citosol/metabolismo , Células HEK293 , Humanos , Ligação Proteica/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética
9.
Cell Metab ; 32(1): 56-70.e7, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32589947

RESUMO

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.


Assuntos
Envelhecimento/metabolismo , Obesidade/metabolismo , Receptor A2B de Adenosina/metabolismo , Adolescente , Adulto , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Receptor A2B de Adenosina/deficiência , Transdução de Sinais , Adulto Jovem
10.
Br J Pharmacol ; 177(8): 1898-1916, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31881095

RESUMO

BACKGROUND AND PURPOSE: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. EXPERIMENTAL APPROACH: We have now developed Gq -specific, cell-permeable 3 H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. KEY RESULTS: FR and YM displayed low nanomolar affinity for Gαq , Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq -knockout cells, but not for Gα15 . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. CONCLUSIONS AND IMPLICATIONS: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Transdução de Sinais , Animais , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Cinética , Camundongos
11.
Aging Cell ; 19(1): e13049, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31692231

RESUMO

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi-organ investigation of age-associated changes of ceramide species, a unique and distinctive change pattern of C16:0 and C18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C16:0 and C18:0 acyl chains, respectively, was down-regulated in skeletal muscle of aged mice. Similarly, an age-dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1Δ/Δ and CerS5Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1- and CerS5-deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age-related muscle wasting disorders in humans.


Assuntos
Ceramidas/metabolismo , Resistência à Insulina/genética , Adulto , Envelhecimento , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/metabolismo , Força Muscular , Adulto Jovem
12.
J Control Release ; 305: 155-164, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31121282

RESUMO

Anti-angiogenic therapies are promising options for diseases with enhanced vessel formation such as tumors or retinopathies. In most cases, a site-specific local effect on vessel growth is required, while the current focus on systemic distribution of angiogenesis inhibitors may cause severe unwanted side-effects. Therefore, in the current study we have developed an approach for the local inhibition of vascularization, using complexes of lentivirus and magnetic nanoparticles in combination with magnetic fields. Using this strategy in the murine embryonic stem cell (ESC) system, we were able to site-specifically downregulate the protein tyrosine phosphatase SHP2 by RNAi technology in areas with active vessel formation. This resulted in a reduction of vessel development, as shown by reduced vascular tube length, branching points and vascular loops. The anti-angiogenic effect could also be recapitulated in the dorsal skinfold chamber of mice in vivo. Here, site-specific downregulation of SHP2 reduced re-vascularization after wound induction. Thus, we have developed a magnet-assisted, RNAi-based strategy for the efficient local inhibition of angiogenesis in ESCs in vitro and also in vivo.


Assuntos
Regulação para Baixo , Vetores Genéticos/genética , Lentivirus/genética , Células-Tronco Embrionárias Murinas/metabolismo , Neovascularização Fisiológica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Animais , Linhagem Celular , Vetores Genéticos/administração & dosagem , Imãs/química , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Interferência de RNA , Transdução Genética/métodos
13.
Sci Rep ; 8(1): 17582, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30514882

RESUMO

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM+ endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45+ hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Modelos Animais , Células-Tronco Embrionárias Murinas/citologia , Neovascularização Patológica , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Animais , Medula Óssea/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Genes Reporter , Camundongos , Camundongos Transgênicos , Remodelação Vascular
14.
Life Sci ; 215: 96-105, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30403990

RESUMO

AIMS: Pulmonary hypertension (PH) leads to right ventricular (RV) adaptation and remodeling and has deleterious long-term effects on RV function. The endocannabinoid receptor CB2 has been associated with protective effects in adaptation and remodeling of the left ventricle after ischemia. Therefore, we investigated the role of CB2 receptor in RV adaptation after occlusion of the left pulmonary artery (LPA) in a murine model. MAIN METHODS: C57/Bl6 (WT)- and CB2 receptor-deficient (Cnr2-/-)-mice underwent paramedian sternotomy and LPA was occluded using a metal clip. Right heart hemodynamic study (Millar®) preceded organ harvesting for immunohistochemistry and mRNA analysis 7 and 21 days (d) post-occlusion. KEY FINDINGS: LPA occlusion led to higher RV systolic pressure in Cnr2-/--hearts, while hemodynamics were comparable with WT-hearts after 21d. Cnr2-/--hearts showed higher macrophage infiltration and lower interleukin-10 expression after 7 d, but otherwise a comparable inflammatory mediator expression profile. Cardiomyocyte-hypertrophy was stronger in Cnr2-/--mice, presenting with higher tenascin-C expression than WT-hearts. Planimetry revealed higher collagen area in Cnr2-/--hearts and small areas of cardiomyocyte-loss. Surrounding cardiomyocytes were cleaved caspase-3- and TUNEL positive in Cnr2-/--hearts. This was associated by maladaptation of myosin heavy-chain isoforms and lower reactive oxygen scavenger enzymes induction in Cnr2-/--hearts. We found comparable morphological changes in both lungs between the two genotypes. SIGNIFICANCE: LPA occlusion led to increased systolic pressure and adaptation of RV in CB2-deficient mice. CB2 receptor seems to modulate RV adaptation through expression of contractile elements, reactive oxygen scavenger enzymes, and inflammatory response in order to prevent cardiomyocyte apoptosis.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/fisiopatologia , Miócitos Cardíacos/patologia , Artéria Pulmonar/fisiopatologia , Receptor CB2 de Canabinoide/genética , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Hipertrofia Ventricular Direita/genética , Inflamação/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Direita/fisiologia
15.
Int J Cardiol ; 272S: 4-10, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30314839

RESUMO

The European guidelines, which focus on clinical aspects of pulmonary hypertension (PH), provide only minimal information about the pathophysiological concepts of PH. Here, we review this topic in greater detail, focusing on specific aspects in the pathobiology, pathology and genetics, which include mechanisms of vascular inflammation, the role of transcription factors, ion channels/ion channel diseases, hypoxic pulmonary vasoconstriction, genetics/epigenetics, metabolic dysfunction, and the potential future role of histopathology of PH in the modern era of PH therapy. In addition to new insights in the pathobiology of this disease, this working group of the Cologne Consensus Conference also highlights novel concepts and potential new therapeutic targets to further improve the treatment options in PAH.


Assuntos
Conferências de Consenso como Assunto , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Ensaios Clínicos como Assunto/métodos , Alemanha/epidemiologia , Humanos , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/metabolismo , Mediadores da Inflamação/metabolismo , Vasoconstrição/fisiologia
16.
Angiogenesis ; 21(2): 349-361, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29417260

RESUMO

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.


Assuntos
Ciclo Celular , Proteínas Contráteis/metabolismo , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteínas Contráteis/genética , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
17.
Cell Stem Cell ; 22(2): 262-276.e7, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29451855

RESUMO

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.


Assuntos
5'-Nucleotidase/metabolismo , Células da Medula Óssea/metabolismo , Células Endoteliais/metabolismo , Células-Tronco Multipotentes/metabolismo , Coloração e Rotulagem , Nicho de Células-Tronco , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Condrogênese , Células Endoteliais/citologia , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Especificidade de Órgãos , Células Estromais/citologia , Células Estromais/metabolismo
18.
FASEB J ; 32(5): 2519-2530, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29295855

RESUMO

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the ß2 adrenoceptor (AR) agonists terbutaline (TER) and salbutamol induced a dose-dependent vasorelaxation in large pulmonary arteries but not aortas of mouse. This effect was found to be independent of ß ARs and the endothelium but was determined by the type of the preconstrictor. Vasodilation by ß2 AR agonists occurred after pretreatment of pulmonary arteries with phenylephrine and serotonin, both agonists of α1 ARs, but was absent after preconstriction with the thromboxane analog U46619. These data indicated α-adrenolytic activity of ß2 AR agonists, which was confirmed by a right shift of the phenylephrine dose-response curve by TER. This effect was physiologically relevant because TER also relaxed small intrapulmonary arteries in lung slices and diminished pulmonary arterial pressure in an isolated perfused lung model under normoxia and hypoxia. Finally, TER applied as an aerosol also selectively decreased pulmonary arterial pressure without effects on systemic blood pressure and heart rate in mouse in vivo. Thus, ß2 AR agonists display α-adrenolytic activity in pulmonary arteries ex vivo and in vivo, and may provide a novel option to reduce pulmonary arterial pressure in pulmonary hypertension.-Neumann, V., Knies, R., Seidinger, A., Simon, A., Lorenz, K., Matthey, M., Breuer, J., Wenzel, D. The ß2 agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Pulmão , Artéria Pulmonar/fisiopatologia , Receptores Adrenérgicos alfa 1/metabolismo , Terbutalina/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Pulmão/irrigação sanguínea , Pulmão/fisiopatologia , Camundongos , Fenilefrina/farmacologia , Artéria Pulmonar/metabolismo
19.
Angew Chem Int Ed Engl ; 57(3): 836-840, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29194875

RESUMO

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.


Assuntos
Depsipeptídeos/biossíntese , Depsipeptídeos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Insetos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Bombyx/metabolismo , Cromossomos Artificiais Bacterianos , Biologia Computacional , Depsipeptídeos/metabolismo , Escherichia coli/genética , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Família Multigênica , Peptídeo Sintases/genética , Primulaceae/química , Células Sf9 , Espectrometria de Massas em Tandem
20.
Biomaterials ; 155: 176-190, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29179133

RESUMO

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.


Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Animais , Nanopartículas de Magnetita/efeitos adversos , Transplante de Células-Tronco
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