RESUMEN
RATIONALE: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology. METHODS: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for in vivo labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent. RESULTS: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving in vivo injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM. CONCLUSION: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.
Asunto(s)
Vasos Sanguíneos/diagnóstico por imagen , Imagenología Tridimensional , Microscopía/métodos , Microtomografía por Rayos X , Animales , Colágeno/farmacología , Vasos Coronarios/diagnóstico por imagen , Combinación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Riñón/irrigación sanguínea , Laminina/farmacología , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/patología , Proteoglicanos/farmacologíaRESUMEN
In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP-/- mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow-derived cells was sufficient to mimic the increased blood flow recovery of global VASP-/- mice. In chemotaxis experiments, VASP-/- neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and ß-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP-/- leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Quimiotaxis de Leucocito , Isquemia/metabolismo , Leucocitos/metabolismo , Proteínas de Microfilamentos/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Peritonitis/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Miembro Posterior , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Macrófagos/metabolismo , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neuropéptidos/metabolismo , Peritonitis/genética , Peritonitis/patología , Peritonitis/fisiopatología , Fenotipo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transporte de Proteínas , Receptores CCR2/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Factores de Tiempo , Arrestina beta 2/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
AIMS: Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than [Formula: see text] . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. METHODS AND RESULTS: Tamoxifen-induced Nox4-knockout mice were crossed with ApoEâ»/â» mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. CONCLUSIONS: The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.