ABSTRACT
Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.
Subject(s)
CD18 Antigens/physiology , Inflammation/physiopathology , Intercellular Signaling Peptides and Proteins/physiology , Neutrophils/physiology , Animals , CD11 Antigens/physiology , CD18 Antigens/genetics , Cell Adhesion/immunology , Cell Adhesion/physiology , Cytokines/immunology , Cytokines/physiology , Humans , Inflammation/immunology , Inflammation/pathology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/immunology , Low Density Lipoprotein Receptor-Related Protein-1/immunology , Low Density Lipoprotein Receptor-Related Protein-1/physiology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Midkine , Nerve Growth Factors/genetics , Nerve Growth Factors/immunology , Nerve Growth Factors/physiology , Neutrophils/immunology , Neutrophils/pathology , Receptors, LDL/immunology , Receptors, LDL/physiology , Tumor Suppressor Proteins/immunology , Tumor Suppressor Proteins/physiologyABSTRACT
The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.
Subject(s)
Angiogenic Proteins/metabolism , Chorioallantoic Membrane/blood supply , Cytokines/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Ischemia/metabolism , Muscle, Skeletal/blood supply , Neovascularization, Physiologic , Angiogenic Proteins/administration & dosage , Angiogenic Proteins/deficiency , Angiogenic Proteins/genetics , Animals , Cell Hypoxia , Cell Proliferation , Cells, Cultured , Chick Embryo , Cytokines/administration & dosage , Cytokines/deficiency , Cytokines/genetics , Disease Models, Animal , Hindlimb , Human Umbilical Vein Endothelial Cells/ultrastructure , Humans , Ischemia/genetics , Ischemia/pathology , Ischemia/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Immunoelectron , Midkine , Monocytes/metabolism , Nerve Growth Factors/administration & dosage , Nerve Growth Factors/metabolism , Neutrophils/metabolism , Time Factors , Up-Regulation , Vascular Endothelial Growth Factor A/administration & dosageABSTRACT
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.