multiphoton-intravital-imaging-for-monitoring-leukocyte-recruitment

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

whole-mount-immunofluorescence-staining-confocal-imaging-3d

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Ludwig Maximilian University Munich

Ludwig-Maximilians-Universität München

Ludwig Maximilians University (LMU) Munich

University Hospital Munich, Ludwig Maximilians University (LMU) Munich

 RNase A (the bovine equivalent to human RNase 1) and RNase 5 (angiogenin) are two closely related ribonucleases. RNase 5 is described as a powerful angiogenic factor. Whether RNase A shares the same angiogenic characteristic, or interferes with vessel growth as demonstrated for arteriogenesis, has never been investigated and is the topic of this present study.  To investigate whether RNase A shows a pro- or anti-angiogenic effect, we employed a murine hindlimb model, in which femoral artery ligation (FAL) results in arteriogenesis in the upper leg, and, due to provoked ischemia, in angiogenesis in the lower leg. C57BL/6J male mice underwent unilateral FAL, whereas the contralateral leg was sham operated. Two and seven days after the surgery and intravenous injection of RNase A (50 μg/kg dissolved in saline) or saline (control), the gastrocnemius muscles of mice were isolated from the lower legs for (immuno-) histological analyses. Hematoxylin and Eosin staining evidenced that RNase A treatment resulted in a higher degree of ischemic tissue damage. This was, however, associated with reduced angiogenesis, as evidenced by a reduced capillary/muscle fiber ratio. Moreover, RNase A treatment was associated with a significant reduction in leukocyte infiltration as shown by CD45 (pan-leukocyte marker), Ly6G or MPO (neutrophils), MPO/CitH  [neutrophil extracellular traps (NETs)], and CD68 (macrophages) staining. CD68/MRC1 double staining revealed that RNase A treated mice showed a reduced percentage of M1-like polarized (CD68/MRC1) macrophages whereas the percentage of M2-like polarized (CD68/MRC1) macrophages was increased.  In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation.

RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue.

Cold-inducible RNA-binding protein (CIRP) is an intracellular RNA-chaperone and extracellular promoter of inflammation, which is increasingly expressed and released under conditions of hypoxia and cold stress. The functional relevance of CIRP for angiogenesis and regeneration of ischemic muscle tissue has never been investigated and is the topic of the present study. We investigated the role of CIRP employing CIRP deficient mice along with a hindlimb model of ischemia-induced angiogenesis. 1 and 7 days after femoral artery ligation or sham operation, gastrocnemius muscles of CIRP-deficient and wildtype mice were isolated and processed for (immuno-) histological analyses. CIRP deficient mice showed decreased ischemic tissue damage as evidenced by Hematoxylin and Eosin staining, whereas angiogenesis was enhanced as demonstrated by increased capillary/muscle fiber ratio and number of proliferating endothelial (CD31/BrdU) cells on day 7 after surgery. Moreover, CIRP deficiency resulted in a reduction of total leukocyte count (CD45), neutrophils (myeloperoxidase, MPO), neutrophil extracellular traps (NETs) (MPO/CitH3), and inflammatory M1-like polarized macrophages (CD68/MRC1), whereas the number of tissue regenerating M2-like polarized macrophages (CD68/MRC1) was increased in ischemic tissue samples. In summary, we show that the absence of CIRP ameliorates angiogenesis and regeneration of ischemic muscle tissue, most likely by influencing macrophage polarization in direction to regenerative M2-like macrophages.

Absence of Cold-Inducible RNA-Binding Protein (CIRP) Promotes Angiogenesis and Regeneration of Ischemic Tissue by Inducing M2-Like Macrophage Polarization.

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Förster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research.

Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.

The use of fluorescent reporters and the development of new imaging technologies have revolutionized studies in cell biology. During recent years the number of fluorescent proteins offering the ability to visualize the distribution of proteins, organelles, and cells has increased tremendously. In parallel, the imaging tools available were refined rapidly enabling now the use of a huge spectrum of specialized methods to explore the cellular and subcellular localization and dynamics of fluorescently tagged markers. This chapter presents an overview of fluorescent reporters and methods available, and describes a selection of those that are routinely applicable in imaging studies using Dictyostelium discoideum.

Fluorescent reporters and methods to analyze fluorescent signals.

Coronin-1A (Coro1A) belongs to a family of highly conserved actin-binding proteins that regulate cytoskeletal re-arrangement. In mammalians, Coro1A expression is most abundant in the haematopoietic lineage, where it regulates various cellular processes. The role of Coro1A in platelets has been previously unknown. Here, we identified Coro1A in human and mouse platelets. Genetic absence of Coro1A in mouse platelets inhibited agonist-induced actin polymerization and altered cofilin phosphoregulation, leading to a reduction in spreading and low-dose collagen induced aggregation. Furthermore, Coro1A-deficient mice displayed a defect in ferric chloride-induced arterial thrombosis with prolonged thrombus formation and reduced thrombus size. Immunofluorescence analysis revealed a less compact thrombus structure with reduced density of platelets and fibrinogen. In summary, Coro1A has a role in platelet biology with impact on spreading, aggregation and thrombosis.

The Actin Regulator Coronin-1A Modulates Platelet Shape Change and Consolidates Arterial Thrombosis.

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3-/-) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3-/- mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&E staining. In addition, angiogenesis was increased in C3-/- mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31/BrdU). Moreover, our results showed that the total number of leukocytes (CD45) was increased in C3-/- mice, which was based on an increased number of neutrophils (MPO), neutrophil extracellular trap formation (MPO/CitH3), and macrophages (CD68) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68/MRC1). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.

C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue.

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31/CD45/BrdU). This was accompanied by a reduction of total leukocyte count (CD45), neutrophils (MPO), neutrophil extracellular traps (NETs) (MPOCitH3), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68/MRC1) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68/MRC1) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia.

ArticleTotal : 2	Year
Publication title Cited by 1	2021
Publication title Cited by 6	2020

Article	Year
RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue. Frontiers in physiology\| PubMed ID: 33240100	2020
Absence of Cold-Inducible RNA-Binding Protein (CIRP) Promotes Angiogenesis and Regeneration of Ischemic Tissue by Inducing M2-Like Macrophage Polarization. Biomedicines\| PubMed ID: 33916904	2021
Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM. Molecules (Basel, Switzerland)\| PubMed ID: 22469598	2012
Fluorescent reporters and methods to analyze fluorescent signals. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23494303	2013
The Actin Regulator Coronin-1A Modulates Platelet Shape Change and Consolidates Arterial Thrombosis. Thrombosis and haemostasis\| PubMed ID: 30453345	2018
C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue. International journal of molecular sciences\| PubMed ID: 34071589	2021
The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia. International journal of molecular sciences\| PubMed ID: 34502391	2021

Hellen Ishikawa-Ankerhold

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