Rag1 Deficiency Impairs Arteriogenesis in Mice.

Increasing evidence suggests that lymphocytes play distinct roles in inflammation-induced tissue remodeling and tissue damage. Arteriogenesis describes the growth of natural bypasses from pre-existing collateral arteries. This process compensates for the loss of artery function in occlusive arterial diseases. The role of innate immune cells is widely understood in the process of arteriogenesis, whereas the role of lymphocytes remains unclear and is the subject of the present study. To analyze the role of lymphocytes, we induced arteriogenesis in recombination activating gene-1 (Rag1) knockout (KO) mice by unilateral ligation of the femoral artery. The lack of functional lymphocytes in Rag1 KO mice resulted in reduced perfusion recovery as shown by laser Doppler imaging. Additionally, immunofluorescence staining revealed a reduced vascular cell proliferation along with a smaller inner luminal diameter in Rag1 KO mice. The perivascular macrophage polarization around the growing collateral arteries was shifted to more pro-inflammatory M1-like polarized macrophages. Together, these data suggest that lymphocytes are crucial for arteriogenesis by modulating perivascular macrophage polarization.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Isolation of Decidual Macrophages and Hofbauer Cells from Term Placenta-Comparison of the Expression of CD163 and CD80.

(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163+ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80+ cells could be found in the decidual macrophages. Considering the percentage of CD80+CD163- and CD80-CD163+ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80+CD163+) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.

Depletion of γδ T Cells Leads to Reduced Angiogenesis and Increased Infiltration of Inflammatory M1-like Macrophages in Ischemic Muscle Tissue.

γδ T cells, a small subset of T cells in blood, play a substantial role in influencing immunoregulatory and inflammatory processes. The functional impact of γδ T cells on angiogenesis in ischemic muscle tissue has never been reported and is the topic of the present work. Femoral artery ligation (FAL) was used to induce angiogenesis in the lower leg of γδ T cell depleted mice and wildtype and isotype antibody-treated control groups. Gastrocnemius muscle tissue was harvested 3 and 7 days after FAL and assessed using (immuno-)histological analyses. Hematoxylin and Eosin staining showed an increased area of tissue damage in γδ T cell depleted mice 7 days after FAL. Impaired angiogenesis was demonstrated by lower capillary to muscle fiber ratio and decreased number of proliferating endothelial cells (CD31+/BrdU+). γδ T cell depleted mice showed an increased number of total leukocytes (CD45+), neutrophils (MPO+) and neutrophil extracellular traps (NETs) (MPO+/CitH3+), without changes in the neutrophils to NETs ratio. Moreover, the depletion resulted in a higher macrophage count (DAPI/CD68+) caused by an increase in inflammatory M1-like macrophages (CD68+/MRC1-). Altogether, we show that depletion of γδ T cells leads to increased accumulation of leukocytes and M1-like macrophages, along with impaired angiogenesis.

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 analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery. After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

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

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) (MPO+CitH3+), 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.

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

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.

Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ.

Peripheral nerve injury can cause debilitating disease and immune cell-mediated destruction of the affected nerve. While the focus has been on the nerve-regenerative response, the effect of loss of innervation on lymph node function is unclear. Here, we show that the popliteal lymph node (popLN) receives direct neural input from the sciatic nerve and that sciatic denervation causes lymph node expansion. Loss of sympathetic, adrenergic tone induces the expression of IFN-γ in LN CD8 T cells, which is responsible for LN expansion. Surgery-induced IFN-γ expression and expansion can be rescued by ß2 adrenergic receptor agonists but not sensory nerve agonists. These data demonstrate the mechanisms governing the pro-inflammatory effect of loss of direct adrenergic input on lymph node function.

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

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.

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

Background: 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. Methods and Results: 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+/CitH3 + [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. Conclusion: In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation.

A Simple and Effective Flow Cytometry-Based Method for Identification and Quantification of Tissue Infiltrated Leukocyte Subpopulations in a Mouse Model of Peripheral Arterial Disease.

Arteriogenesis, the growth of a natural bypass from pre-existing arteriolar collaterals, is an endogenous mechanism to compensate for the loss of an artery. Mechanistically, this process relies on a locally and temporally restricted perivascular infiltration of leukocyte subpopulations, which mediate arteriogenesis by supplying growth factors and cytokines. Currently, the state-of-the-art method to identify and quantify these leukocyte subpopulations in mouse models is immunohistology. However, this is a time consuming procedure. Here, we aimed to develop an optimized protocol to identify and quantify leukocyte subpopulations by means of flow cytometry in adductor muscles containing growing collateral arteries. For that purpose, adductor muscles of murine hindlimbs were isolated at day one and three after induction of arteriogenesis, enzymatically digested, and infiltrated leukocyte subpopulations were identified and quantified by flow cytometry, as exemplary shown for neutrophils and macrophages (defined as CD45+/CD11b+/Ly6G+ and CD45+/CD11b+/F4/80+ cells, respectively). In summary, we show that flow cytometry is a suitable method to identify and quantify leukocyte subpopulations in muscle tissue, and provide a detailed protocol. Flow cytometry constitutes a timesaving tool compared to histology, which might be used in addition for precise localization of leukocytes in tissue samples.

Contribution of the Potassium Channels KV1.3 and KCa3.1 to Smooth Muscle Cell Proliferation in Growing Collateral Arteries.

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.

Extracellular RNA released due to shear stress controls natural bypass growth by mediating mechanotransduction in mice.

Fluid shear stress in the vasculature is the driving force for natural bypass growth, a fundamental endogenous mechanism to counteract the detrimental consequences of vascular occlusive disease, such as stroke or myocardial infarction. This process, referred to as "arteriogenesis," relies on local recruitment of leukocytes, which supply growth factors to preexisting collateral arterioles enabling them to grow. Although several mechanosensing proteins have been identified, the series of mechanotransduction events resulting in local leukocyte recruitment is not understood. In a mouse model of arteriogenesis (femoral artery ligation), we found that endothelial cells release RNA in response to increased fluid shear stress and that administration of RNase inhibitor blocking plasma RNases improved perfusion recovery. In contrast, treatment with bovine pancreatic RNase A or human recombinant RNase1 interfered with leukocyte recruitment and collateral artery growth. Our results indicated that extracellular RNA (eRNA) regulated leukocyte recruitment by engaging vascular endothelial growth factor receptor 2 (VEGFR2), which was confirmed by intravital microscopic studies in a murine cremaster model of inflammation. Moreover, we found that release of von Willebrand factor (VWF) as a result of shear stress is dependent on VEGFR2. Blocking VEGFR2, RNase application, or VWF deficiency interfered with platelet-neutrophil aggregate formation, which is essential for initiating the inflammatory process in arteriogenesis. Taken together, the results show that eRNA is released from endothelial cells in response to shear stress. We demonstrate this extracellular nucleic acid as a critical mediator of mechanotransduction by inducing the liberation of VWF, thereby initiating the multistep inflammatory process responsible for arteriogenesis.

Insulin Treatment Forces Arteriogenesis in Diabetes Mellitus by Upregulation of the Early Growth Response-1 (Egr-1) Pathway in Mice.

The process of arteriogenesis is severely compromised in patients with diabetes mellitus (DM). Earlier studies have reported the importance of Egr-1 in promoting collateral outward remodeling. However, the role of Egr-1 in the presence of DM in outward vessel remodeling was not studied. We hypothesized that Egr-1 expression may be compromised in DM which may lead to impaired collateral vessel growth. Here, we investigated the relevance of the transcription factor Egr-1 for the process of collateral artery growth in diabetic mice. Induction of arteriogenesis by femoral artery ligation resulted in an increased expression of Egr-1 on mRNA and protein level but was severely compromised in streptozotocin-induced diabetic mice. Diabetes mellitus mice showed a significantly reduced expression of Egr-1 endothelial downstream genes Intercellular Adhesion Molecule-1 (ICAM-1) and urokinase Plasminogen Activator (uPA), relevant for extravasation of leukocytes which promote arteriogenesis. Fluorescent-activated cell sorting analyses confirmed reduced leukocyte recruitment. Diabetes mellitus mice showed a reduced expression of the proliferation marker Ki-67 in growing collaterals whose luminal diameters were also reduced. The Splicing Factor-1 (SF-1), which is critical for smooth muscle cell proliferation and phenotype switch, was found to be elevated in collaterals of DM mice. Treatment of DM mice with insulin normalized the expression of Egr-1 and its downstream targets and restored leukocyte recruitment. SF-1 expression and the diameter of growing collaterals were normalized by insulin treatment as well. In summary, our results showed that Egr-1 signaling was impaired in DM mice; however, it can be rescued by insulin treatment.

Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

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 chemoattractant 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.

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

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.

Midkine Controls Arteriogenesis by Regulating the Bioavailability of Vascular Endothelial Growth Factor A and the Expression of Nitric Oxide Synthase 1 and 3.

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.

Perivascular Mast Cells Govern Shear Stress-Induced Arteriogenesis by Orchestrating Leukocyte Function.

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.

Arginase inhibition attenuates arteriogenesis and interferes with M2 macrophage accumulation.

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.