CSF-1 and Notch signaling cooperate in macrophage instruction and tissue repair during peripheral limb ischemia

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling via Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear. Using a mouse model of hind limb ischemia with CSF-1 inhibitor studies and Dll1 heterozygous mice we show that CSF-1 is induced in the ischemic niche by a subpopulation of stromal cells expressing podoplanin, which was paralleled by the development of ischemic macrophages. Inhibition of CSF-1 signaling with small molecules or blocking antibodies impaired macrophage differentiation but prolonged the inflammatory response, resulting in impaired perfusion recovery and tissue regeneration. Yet, despite high levels of CSF-1, macrophage maturation and perfusion recovery were impaired in mice with Dll1 haploinsufficiency, while inflammation was exaggerated. In vitro, CSF-1 was not sufficient to induce full MF differentiation from donor monocytes in the absence of recombinant DLL1, while the presence of DLL1 in a dose-dependent manner stimulated MF differentiation in combination with CSF-1. Thus, CSF-1 is an ischemic niche factor that cooperates with Notch signaling in a non-redundant fashion to instruct macrophage cell fate and maturation, which is required for ischemic perfusion recovery and tissue repair

A new fasciocutaneous flap model identifies a critical role for endothelial Notch signaling in wound healing and flap survival.

Flap surgery is a common treatment for severe wounds and a major determinant of surgical outcome. Flap survival and healing depends on adaptation of the local flap vasculature. Using a novel and defined model of fasciocutaneous flap surgery, we demonstrate that the Notch ligand Delta-like 1 (Dll1), expressed in vascular endothelial cells, regulates flap arteriogenesis, inflammation and flap survival. Utilizing the stereotyped anatomy of dorsal skin arteries, ligation of the major vascular pedicle induced strong collateral vessel development by end-to-end anastomosis in wildtype mice, which supported flap perfusion recovery over time. In mice with heterozygous deletion of Dll1, collateral vessel formation was strongly impaired, resulting in aberrant vascularization and subsequent necrosis of the tissue. Furthermore, Dll1 deficient mice showed severe inflammation in the flap dominated by monocytes and macrophages. This process is controlled by endothelial Dll1 in vivo, since the results were recapitulated in mice with endothelial-specific deletion of Dll1. Thus, our model provides a platform to study vascular adaptation to flap surgery and molecular and cellular regulators influencing flap healing and survival

Blood flow controls bone vascular function and osteogenesis

While blood vessels play important roles in bone homeostasis and repair, fundamental aspects of vascular function in the skeletal system remain poorly understood. Here we show that the long bone vasculature generates a peculiar flow pattern, which is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomotic fusion of endothelial buds. Impaired blood flow leads to defective angiogenesis and osteogenesis, and downregulation of Notch signalling in endothelial cells. In aged mice, skeletal blood flow and endothelial Notch activity are also reduced leading to decreased angiogenesis and osteogenesis, which is reverted by genetic reactivation of Notch. Blood flow and angiogenesis in aged mice are also enhanced on administration of bisphosphonate, a class of drugs frequently used for the treatment of osteoporosis. We propose that blood flow and endothelial Notch signalling are key factors controlling ageing processes in the skeletal system

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation

Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1

During angiogenesis, single endothelial cells (EC) specialize into tip cells that guide vessel sprouting towards growth factor gradients and instruct the adjacent vessel stalk. The balance between tip and stalk cells is regulated by endothelial Notch signalling through the expression of Notch ligand Delta-like 4 (Dll4) in tip cells, which suppresses a tip cell fate in adjacent stalk cells. Here we show, using genetic reporter and conditional deletion strategies, that myeloid cells regulate tip cell numbers and Dll4 expression via the Notch ligand Dll1 during vascular development in the retina. Dll1 is selectively expressed by a subpopulation of retinal myeloid cells, which progressively localizes to the sprouting vascular network. Conditional, myeloid-specific deletion of Dll1 impairs endothelial Dll4 tip-stalk gradient resulting in an increase of endothelial tip cells and EC filopodia, accompanied by an increase in vascular density and branching. In vitro, co-culture of human EC with monocyte-derived macrophages induced Dll1 upregulation in macrophages and Dll4 upregulation and an endothelial tip cell signature in EC. Furthermore, culturing human EC on recombinant DLL1 induced endothelial Dll4 expression and a tip cell program, indicating that changes are Dll1-dependent. Thus, myeloid cells regulate tip cell fate and angiogenesis through expression of Notch ligand Dll1

The chemokine receptor CX3CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury

Abstract Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX3CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6Clo monocytes expressing high levels of the cognate CX3CR1 chemokine receptor. In Cx3cr1‐deficient mice recruitment of Ly6Clo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6Clo monocytes. Furthermore, human non‐classical monocytes induce proliferation of endothelial cells in co‐culture experiments in a VEGFA‐dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo. Thus, CX3CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration

Peptidases released by necrotic cells control CD8+ T cell cross-priming.

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells