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 ...

Ludwig-Maximilians-Universität München

Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.

Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning.

 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.

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.

Manuel Lasch

Ludwig-Maximilians-Universität München

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