The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit(+)/CXCR-4(+) cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.

Key Points Shear stress–induced release of RNA from endothelial cells is crucial for initiation of arteriogenesis by controlling mechanotransduction. Extracellular RNA is essential for VWF release from endothelial cells initiating the inflammatory process driving arteriogenesis.

Midkine is a pleiotropic factor, which is involved in angiogenesis. However, its mode of action in this process is still ill defined. The function of midkine in arteriogenesis, the growth of natural bypasses from pre-existing collateral arteries, compensating for the loss of an occluded artery has never been investigated. Arteriogenesis is an inflammatory process, which relies on the proliferation of endothelial cells and smooth muscle cells. We show that midkine deficiency strikingly interferes with the proliferation of endothelial cells in arteriogenesis, thereby interfering with the process of collateral artery growth. We identified midkine to be responsible for increased plasma levels of vascular endothelial growth factor A (VEGFA), necessary and sufficient to promote endothelial cell proliferation in growing collaterals. Mechanistically, we demonstrate that leukocyte domiciled midkine mediates increased plasma levels of VEGFA relevant for upregulation of endothelial nitric oxide synthase 1 and 3, necessary for proper endothelial cell proliferation, and that non-leukocyte domiciled midkine additionally improves vasodilation.The data provided on the role of midkine in endothelial proliferation are likely to be relevant for both, the process of arteriogenesis and angiogenesis. Moreover, our data might help to estimate the therapeutic effect of clinically applied VEGFA in patients with vascular occlusive diseases.

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.

Tissue‐resident mast cells (MCs) are well known for their role in inflammatory responses and allergic and anaphylactic reactions, but they also contribute to processes of arterial remodeling. Although ribosomes and cytosolic RNAs are located around secretory granules in mature MCs, their functional role in MC responses remains unexplored. Previous studies by our group characterized extracellular RNA (eRNA) as an inflammatory and pathogenetic factor in vitro and in vivo. In the present study, RNA‐containing MCs and eRNA were located in close proximity to growing collateral arteries in vivo. In vitro, various agonists were found to induce the degranulation of MCs and the concomitant release of eRNA in association with microvesicles (MVs). The liberation of eRNA from MCs was abolished by MC stabilizers or by preventing the increase of intracellular Ca2+ in MCs. eRNA was found to be mainly contained inside MVs, as demonstrated by electron microscopy and immunocytochemistry. The exposure to and the uptake of MC‐released MVs by cultured endothelial cells increased their expression of cytokines, such as monocyte chemo‐attractant protein or IL‐6, in a dose‐ and time‐dependent manner. These results indicate that RNA‐containing MC‐derived MVs are likely to be involved in inflammatory responses, relevant, for example, to processes of vascular remodeling.—Elsemüller, A.‐K., Tomalla, V., Gärtner, U., Troidl, K., Jeratsch, S., Graumann, J., Baal, N., Hackstein, H., Lasch, M., Deindl, E., Preissner, K. T., Fischer, S. Characterization of mast cell‐derived rRNA‐containing microvesicles and their inflammatory impact on endothelial cells. FASEB J. 33, 5457–5467 (2019). http://www.fasebj.org

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.

L-Arginine is the common substrate for nitric oxide synthases (NOS) and arginase. Whereas the contribution of NOS to collateral artery growth (arteriogenesis) has been demonstrated, the functional role of arginase remains to be elucidated and was topic of the present study. Arteriogenesis was induced in mice by ligation of the femoral artery. Laser Doppler perfusion measurements demonstrated a significant reduction in arteriogenesis in mice treated with the arginase inhibitor nor-NOHA (N ω -hydroxy-nor-arginine). Accompanying in vitro results on murine primary arterial endothelial cells and smooth muscle cells revealed that nor-NOHA treatment interfered with cell proliferation and resulted in increased nitrate/ nitrite levels, indicative for increased NO production. Immuno-histological analyses on tissue samples demonstrated that nor-NOHA administration caused a significant reduction in M2 macrophage accumulation around growing collateral arteries. Gene expression studies on isolated growing collaterals evidenced that nor-NOHA treatment abolished the differential expression of Icam1 (intercellular adhesion molecule 1). From our data we conclude that arginase activity is essential for arteriogenesis by promoting perivascular M2 macrophage accumulation as well as arterial cell proliferation.

Collateral artery growth (arteriogenesis) involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Whereas the proliferation of ECs is directly related to shear stress, the driving force for arteriogenesis, little is known about the mechanisms of SMC proliferation. Here we investigated the functional relevance of the potassium channels KV1.3 and KCa3.1 for SMC proliferation in arteriogenesis. Employing a murine hindlimb model of arteriogenesis, we found that blocking KV1.3 with PAP-1 or KCa3.1. with TRAM-34, both interfered with reperfusion recovery after femoral artery ligation as shown by Laser-Doppler Imaging. However, only treatment with PAP-1 resulted in a reduced SMC proliferation. qRT-PCR results revealed an impaired downregulation of α smooth muscle-actin (αSM-actin) and a repressed expression of fibroblast growth factor receptor 1 (Fgfr1) and platelet derived growth factor receptor b (Pdgfrb) in growing collaterals in vivo and in primary murine arterial SMCs in vitro under KV1.3. blockade, but not when KCa3.1 was blocked. Moreover, treatment with PAP-1 impaired the mRNA expression of the cell cycle regulator early growth response-1 (Egr1) in vivo and in vitro. Together, these data indicate that KV1.3 but not KCa3.1 contributes to SMC proliferation in arteriogenesis.