Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.
ABSTRACT: We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of ?2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted ?2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the ?2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.
Project description:Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus-1) as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/- mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.
Project description:OBJECTIVE:To elucidate the role of interferon regulatory factor (IRF)3 and IRF7 in neovascularization. METHODS:Unilateral hind limb ischaemia was induced in Irf3-/- , Irf7-/- and C57BL/6 mice by ligation of the left common femoral artery. Post-ischaemic blood flow recovery in the paw was measured with laser Doppler perfusion imaging. Soleus, adductor and gastrocnemius muscles were harvested to investigate angiogenesis and arteriogenesis and inflammation. RESULTS:Post-ischaemic blood flow recovery was decreased in Irf3-/- and Irf7-/- mice compared to C57BL/6 mice at all time points up to and including sacrifice, 28 days after surgery (t28). This was supported by a decrease in angiogenesis and arteriogenesis in soleus and adductor muscles of Irf3-/- and Irf7-/- mice at t28. Furthermore, the number of macrophages around arterioles in adductor muscles was decreased in Irf3-/- and Irf7-/- mice at t28. In addition, mRNA expression levels of pro-inflammatory cytokines (tnfα, il6, ccl2) and growth factor receptor (vegfr2), were decreased in gastrocnemius muscles of Irf3-/- and Irf7-/- mice compared to C57BL/6 mice. CONCLUSION:Deficiency of IRF3 and IRF7 results in impaired post-ischaemic blood flow recovery caused by attenuated angiogenesis and arteriogenesis linked to a lack of inflammatory components in ischaemic tissue. Therefore, IRF3 and IRF7 are essential regulators of neovascularization.
Project description:BACKGROUND:Angiogenesis improves reperfusion to the ischaemic tissue after vascular obstruction. The underlying molecular mechanisms of post-ischaemic angiogenesis are not clear. FAM3A belongs to the family with sequence similarity 3 (FAM3) genes, but its biological function in endothelial cells in regards to vascular diseases is not well understood. METHODS:Gain- and loss-of-function methods by adenovirus or associated-adenovirus (AAV) in different models were applied to investigate the effects of FAM3A on endothelial angiogenesis. Endothelial angiogenesis was analysed by tube formation, migration and proliferation in vitro, and the blood flow and capillary density in a hind limb ischaemic model in vivo. FINDINGS:Endothelial FAM3A expression is downregulated under hypoxic conditions. Overexpression of FAM3A promotes, but depletion of FAM3A suppresses, endothelial tube formation, proliferation and migration. Utilizing the mouse hind limb ischaemia model, we also observe that FAM3A overexpression can improve blood perfusion and increase capillary density, whereas FAM3A knockdown has the opposite effects. Mechanistically, mitochondrial FAM3A increases adenosine triphosphate (ATP) production and secretion; ATP binds to P2 receptors and then upregulates cytosolic free Ca2+ levels. Increased intracellular Ca2+ levels enhance phosphorylation of the transcriptional factor cAMP response element binding protein (CREB) and its recruitment to the VEGFA promoter, thus activating VEGFA transcription and the final endothelial angiogenesis. INTERPRETATION:In summary, our data demonstrate that FAM3A positively regulates angiogenesis through activation of VEGFA transcription, suggesting that FAM3A may constitute a novel molecular therapeutic target for ischaemic vascular disease.
Project description:Diabetes mellitus (DM) is associated with poor clinical outcomes in humans with peripheral arterial disease (PAD) and in pre-clinical models of PAD, but the effects of glycaemic control are poorly understood. We investigated the effect of glycaemic control on experimental PAD in mice with Type 1 DM and explored the effects of hyperglycaemia on vascular endothelial growth factor receptor 2 (VEGFR2) expression in ischaemia.Hind limb ischaemia was induced in non-diabetic, untreated Type 1 DM, and treated Type 1 DM mice. We assessed perfusion recovery, capillary density, VEGFR2 levels, and VEGFR2 ubiquitination in ischaemic hind limbs. We found that untreated Type 1 DM mice showed impaired perfusion recovery, lower hind limb capillary density 5 weeks post-ischaemia, and lower VEGFR2 protein in Day 3 post-ischaemic hind limbs when compared with non-DM controls. Treated Type 1 DM mice had perfusion recovery, capillary density, and VEGFR2 protein levels comparable with that of non-diabetic mice at the same time points. Treatment with anti-VEGFR2 antibody negated that the improved perfusion recovery displayed by treated Type 1 DM mice. In ischaemic Type 1 DM hind limbs and endothelial cells exposed to simulated ischaemia, high glucose impaired VEGFR2 expression and was associated with increased VEGFR2 ubiquitination. Inhibition of the ubiquitin-proteasome complex restored normal endothelial VEGFR2 expression in simulated ischaemia.Hyperglycaemia in Type 1 DM impairs VEGFR2 protein expression in ischaemic hind limbs, likely due to increased ubiquitination and degradation by the proteasome complex. Glycaemic control allows normal levels of VEGFR2 in ischaemia and improved perfusion recovery.
Project description:Arterial bypass grafts remain the gold standard for the treatment of end-stage ischaemic disease. Yet patients unable to tolerate the cardiovascular stress of arterial surgery or those with unreconstructable disease would benefit from grafts that are able to induce therapeutic angiogenesis. Here, we introduce an approach whereby implantation of 3D-printed grafts containing endothelial-cell-lined lumens induces spontaneous, geometrically guided generation of collateral circulation in ischaemic settings. In rodent models of hind-limb ischaemia and myocardial infarction, we demonstrate that the vascular patches rescue perfusion of distal tissues, preventing capillary loss, muscle atrophy and loss of function. Inhibiting anastomoses between the construct and the host's local capillary beds, or implanting constructs with unpatterned endothelial cells, abrogates reperfusion. Our 3D-printed grafts constitute an efficient and scalable approach to engineer vascular patches able to guide rapid therapeutic angiogenesis and perfusion for the treatment of ischaemic diseases.
Project description:The effects on angiogenesis of a novel CXC chemokine receptor 4 (CXCR4) antagonist, SDF-1betaP2G, derived from human stromal cell-derived factor-1beta (SDF-1beta), were examined in a model of hind limb ischaemia in mice.The antagonistic activities of SDF-1betaP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1betaP2G were evaluated by inflammatory and apoptotic markers. SDF-1betaP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1beta but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1betaP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1betaP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1betaP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1betaP2G administration.Our findings indicate that the novel CXCR4 antagonist, SDF-1betaP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.
Project description:Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.
Project description:The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase-9 (MMP-9) on the angiogenic function of EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP-9/knockout (MMP-9/KO) and wild-type (WT) mice. EPCs were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel(™) assays and time-lapse imaging were conducted to monitor angiogenic function of WT and MMP9-deficient EPCs or after treatment with MMP-9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP-9 deficiency decreased their number in non-ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP-9 deficiency reduced the angiogenic abilities of OECs to form vascular networks, in vitro. Treatment with the MMP inhibitor GM6001 and the specific MMP-9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OECs, while exogenous MMP-9 could not revert the impaired angiogenic function in MMP-9/KO OECs. Finally, time-lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP-9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPCs, while MMP-9 plays a key role in the formation of vascular networks by EPCs.
Project description:Restoration of cerebral blood flow (CBF) and upregulation of angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Here, we investigated for the first time the role of PTX3 in long-term CBF, angiogenesis, and neuronal viability after ischaemic stroke induced by transient middle cerebral artery occlusion (MCAo). Lack of PTX3 had no effect on early brain damage, but significantly impaired restoration of CBF, 14 and 28 days after MCAo, compared to wild-type (WT) mice. Immunohistochemical analysis revealed that PTX3 KO mice have significantly greater neuronal loss, significantly decreased vessel diameter, vessel proliferation, vascular density, and reactive astrocytes and decreased expression of vascular endothelial growth factor receptor 2 (VEGR2), vascular extracellular matrix (ECM)-proteins (collagen IV, laminin), and integrin-?, in the ipsilateral (stroke) hemisphere compared to WT mice, 28 days after MCAo. Therefore, PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Collectively, these findings demonstrate the potential and clinical relevance of PTX3 as a promising therapeutic target, providing sustained long-term post-stroke neurovascular repair and reducing the loss of neurons. KEY MESSAGES: Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Restoration of cerebral blood flow (CBF) and angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia.
Project description:AIMS:Cardiac remodelling in the ischaemic heart determines prognosis in patients with ischaemic heart disease (IHD), while enhancement of angiogenesis and cell survival has shown great potential for IHD despite translational challenges. Phosphoinositide 3-kinase (PI3K)/Akt signalling pathways play a critical role in promoting angiogenesis and cell survival. However, the effect of PI3K? in the ischaemic heart is poorly understood. This study investigates the role of endothelial and cardiomyocyte (CM) PI3K? in post-infarct cardiac remodelling. METHODS AND RESULTS:PI3K? catalytic subunit-p110? level was increased in infarcted murine and human hearts. Using cell type-specific loss-of-function approaches, we reported novel and distinct actions of p110? in endothelial cells (ECs) vs. CMs in response to myocardial ischaemic injury. Inactivation of endothelial p110? resulted in marked resistance to infarction and adverse cardiac remodelling with decreased mortality, improved systolic function, preserved microvasculature, and enhanced Akt activation. Cultured ECs with p110? knockout or inhibition displayed preferential PI3K?/Akt/endothelial nitric oxide synthase signalling that consequently promoted protective signalling and angiogenesis. In contrast, mice with CM p110?-deficiency exhibited adverse post-infarct ventricular remodelling with larger infarct size and deteriorated cardiac function, which was due to enhanced susceptibility of CMs to ischaemia-mediated cell death. Disruption of CM p110? signalling compromised nuclear p110? and phospho-Akt levels leading to perturbed gene expression and elevated pro-cell death protein levels, increasing the susceptibility to CM death. A similar divergent response of PI3K? endothelial and CM mutant mice was seen using a model of myocardial ischaemia-reperfusion injury. CONCLUSION:These data demonstrate novel, differential, and cell-specific functions of PI3K? in the ischaemic heart. While the loss of endothelial PI3K? activity produces cardioprotective effects, CM PI3K? is protective against myocardial ischaemic injury.