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The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre– lox -based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2 + and Vav1 + compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury. The lymphatic system is thought to be derived by transdifferentiation of venous endothelium; this study shows that the origin of cardiac lymphatics is in fact more heterogeneous, including both venous and non-venous origins and that lymphangiogenesis occurs in the adult heart following myocardial infarction and can be enhanced to improve heart function. Embryonic origins of lymphatic vessels The lymphatic endothelium wass thought to arise entirely from transdifferentiation of the venous endothelium. Two studies published in this issue of Nature demonstrate that the origins of this vasculature are more varied than anticipated. Karina Yaniv and colleagues use live imaging of transgenic zebrafish embryos to trace the lineage of individual lymphatic endothelial cells and their progeny. They find that lymphatic progenitors emerge from a previously uncharacterized pool of multipotent angioblasts located in the floor of the cardinal vein, which bears the potential to generate not only lymphatic, but also arterial and venous fates. Paul Riley and colleagues used genetic fate-mapping techniques in mice to demonstrate a dual origin of cardiac lymphatic endothelial cells, involving both venous endothelium and a separate non-venous progenitor source arising from yolk sac cells. They also show that the lymphangiogenesis in the heart can be influenced by myocardial infarction, and they suggest that promoting lymphangiogenesis with VEGF-C improves cardiac function after infarction

Emerging evidence indicates that angiopoietin-2 (Angpt2), a well-recognized vascular destabilizing factor, is a biomarker of poor outcome in ischemic heart disease. However, its precise role in postischemic cardiovascular remodeling is poorly understood. Here, we show that Angpt2 plays multifaceted roles in the exacerbation of cardiac hypoxia and inflammation after myocardial ischemia. Angpt2 was highly expressed in endothelial cells at the infarct border zone after myocardial infarction (MI) or ischemia/reperfusion injury in mice. In the acute phase of MI, endothelial-derived Angpt2 antagonized Angpt1/Tie2 signaling, which was greatly involved in pericyte detachment, vascular leakage, increased adhesion molecular expression, degradation of the glycocalyx and extracellular matrix, and enhanced neutrophil infiltration and hypoxia in the infarct border area. In the chronic remodeling phase after MI, endothelial- and macrophage-derived Angpt2 continuously promoted abnormal vascular remodeling and proinflammatory macrophage polarization through integrin α5β1 signaling, worsening cardiac hypoxia and inflammation. Accordingly, inhibition of Angpt2 either by gene deletion or using an anti-Angpt2 blocking antibody substantially alleviated these pathological findings and ameliorated postischemic cardiovascular remodeling. Blockade of Angpt2 thus has potential as a therapeutic option for ischemic heart failure.

Caspase-8 is the initiator caspase of extrinsic apoptosis 1 , 2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality 3 , which can be rescued by deletion of either Ripk3 or Mlkl 4 – 6 . Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8 −/− mice 3 , 7 , Casp8 C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8 C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice 8 . Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1β. Both embryonic lethality and premature death were completely rescued in Casp8 C362S/C362S Mlkl −/− Asc −/− or Casp8 C362S/C362S Mlkl −/− Casp1 −/− mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood. The enzymatic activity of caspase-8 controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood in mice.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application. Angiopoietin (Angpt)-Tie receptor 2 (Tie2) regulates vascular stability and is thus a potential therapeutic target in vascular diseases. Here, the authors report a Tie2-agonistic antibody which targets a site distinct from the Angpt 1-binding site and which influences Tie2 clustering and activation in an Angpt2 inhibition-resistant manner.

The blood and lymphatic vascular systems are regulated by angiopoietin (ANGPT) growth factors, which signal via endothelial TIE receptor tyrosine kinases and integrins. However, mechanistic understanding of how these receptors crosstalk is limited. Here, we show how β1-integrin inactivation regulates endothelial ANGPT/TIE2 signaling. By integrating biophysical analyses, X-ray crystallography, size-exclusion chromatography-small-angle X-ray scattering and atomistic molecular dynamics simulations, we show that ANGPT2 binds through its asymmetrically positioned C-terminal fibrinogen-like domains to both TIE2 and α5β1-integrin, forming a trimeric complex compatible with the inactive α5β1-integrin conformation. Inactive β1-integrin colocalizes with ANGPT-induced TIE2 in cell-cell junctions and stabilizing β1-integrin in its inactive state enhances junctional TIE2 accumulation and promotes nuclear exclusion of the TIE2 transcriptional effector FOXO1 in cultured endothelial cells. Endothelial-specific β1-integrin deletion in adult mice reduces venous TIE2 phosphorylation, whereas endotoxemia diminishes junctional β1-integrin along with decreased phosphorylated TIE2. In contrast, without TIE2, ANGPT2 uniquely engages active β1-integrin, via its N-terminal superclustering domain. Altogether, our results provide structural and mechanistic evidence of ANGPT signaling via α5β1-integrin and support a model in which inactive α5β1-integrin acts as a junctional scaffold for ANGPT/TIE2/FOXO1 signaling, explaining how integrin conformational switching spatially organizes growth factor signaling in the endothelium.

Elevated angiopoietin-2 is associated with diverse inflammatory conditions, including sepsis, a leading global cause of mortality. During inflammation, angiopoietin-2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, angiopoietin-2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here, we show that inflammation-induced proteolytic cleavage of angiopoietin-2 converts this ligand from Tie2 agonist to antagonist. Conditioned media from stimulated macrophages induced endothelial angiopoietin-2 secretion. Unexpectedly, this was associated with reduction of the 75 kDa full-length protein and appearance of new 25 and 50 kDa C-terminal fragments. Peptide sequencing proposed cathepsin K as a candidate protease. Cathepsin K was necessary and sufficient to cleave angiopoietin-2. Recombinant 25 and 50 kDa angiopoietin-2 fragments (cANGPT2 25 and cANGPT2 50 ) bound and antagonized Tie2. Cathepsin K inhibition with the phase 3 small-molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Full-length angiopoietin-2 enhanced survival in endotoxemic mice administered odanacatib and, conversely, increased mortality in the drug’s absence. Odanacatib’s benefit was reversed by heterologous cANGPT2 25 . Septic humans accumulated circulating angiopoietin-2 fragments, which were associated with adverse outcomes. These results identify cathepsin K as a candidate marker of sepsis and a proteolytic mechanism for the conversion of angiopoietin-2 from Tie2 agonist to antagonist, with therapeutic implications for inflammatory conditions associated with angiopoietin-2 induction.

The role of endothelial dysfunction in tubulointerstitial fibrosis associated with chronic kidney disease (CKD) is not well understood. In this study, we demonstrate that the activation of the endothelial tyrosine kinase TIE2 alleviates renal pathology in experimental CKD in mice. TIE2 activation was achieved using a human angiopoietin-2–binding and TIE2-activating antibody (ABTAA) or through adult-induced endothelium-specific knockout of the vascular endothelial protein tyrosine phosphatase gene ( Veptp ). Both methods markedly protected CKD mice from endothelial dysfunction, peritubular capillary loss, tubular epithelial injury, and tubulointerstitial fibrosis. Conversely, silencing TIE2 through adult-induced endothelium-specific knockout of the Tie2 gene exacerbated CKD pathology. Additionally, we found that endothelial dysfunction promoted renal fibrosis not through endothelial-to-mesenchymal transition, as previously expected, but by inducing the expression of profibrotic PDGFB in tubular epithelial cells, a process that is inhibited by TIE2 activation. Our findings suggest that TIE2 activation via ABTAA warrants investigation as a therapy in human CKD, where there is a substantial unmet medical need.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro . These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have opposing effects on vascular stability through their receptor Tie2, but there is evidence for Tie2-independent functions of Ang2. Here, Hakanpaa et al. show that Ang2 directly activates β1-integrin, leading to rearrangement of the actin cytoskeleton and decreased VE-cadherin in cell–cell junctions.

The pathophysiology of acute kidney injury (AKI) after cardiac surgery is not completely understood. Recent evidence suggests a pivotal role for the endothelium in AKI. In experimental models of AKI, the endothelial specific receptor Tie2 with its ligands Angiopoietin (Ang) 1 and Ang2 are deranged. This study investigates their status after cardiac surgery, and a possible relation between angiopoietins and AKI. From a cohort of 541 patients that underwent cardiac surgery, blood and urine was collected at 5 predefined time points. From this cohort we identified 21 patients who had at least 50% post-operative serum creatinine increase (AKI). We constructed a control group (n = 21) using propensity matching. Systemic levels of Ang1, Ang2, and sTie2 were measured in plasma and the AKI markers albumin, kidney injury molecule-1 (KIM-1) and N-acetyl-beta-D-glucosaminidase (NAG) were measured in the urine. Ang2 plasma levels increased over time in AKI (from 4.2 to 11.6 ng/ml) and control patients (from 3.0 to 6.7 ng/ml). Ang2 levels increased 1.7-fold more in patients who developed AKI after cardiac surgery compared to matched control patients. Plasma levels of sTie2 decreased 1.6-fold and Ang1 decreased 3-fold over time in both groups, but were not different between AKI and controls (Ang1 P = 0.583 and sTie2 P = 0.679). Moreover, we found a positive correlation between plasma levels of Ang2 and urinary levels of NAG. The endothelial Ang/Tie2 system is in dysbalance in patients that develop AKI after cardiac surgery compared to matched control patients.

This review summarizes the latest findings in the literature of Angiopoietin-2 (Ang-2), Tyrosine-protein kinase receptor (Tie-2) complex, and faricimab along with their involvement for the treatment of retinal vascular diseases in various clinical trials. In ischemic diseases, such as diabetic retinopathy, Ang-2 is upregulated, deactivating Tie-2, resulting in vascular leakage, pericyte loss, and inflammation. Recombinant Angiopeotin-1 (Ang-1), Ang-2-blocking molecules, and inhibitors of vascular endothelial protein tyrosine phosphatase (VE-PTP) decrease inflammation-associated vascular leakage, showing therapeutic effects in diabetes, atherosclerosis, and ocular neovascular diseases. In addition, novel studies show that angiopoietin-like proteins may play an important role in cellular metabolism leading to retinal vascular diseases. Current therapeutic focus combines Ang-Tie targeted drugs with other anti-angiogenic or immune therapies. Clinical studies have identified faricimab, a novel bispecific antibody designed for intravitreal use, to simultaneously bind and neutralize Ang-2 and VEGF-A for treatment of diabetic eye disease. By targeting both Ang-2 and vascular endothelial growth factor-A (VEGF-A), faricimab displays an improved and sustained efficacy over longer treatment intervals, delivering superior vision outcomes for patients with diabetic macular edema and reducing the treatment burden for patients with neovascular age-related macular degeneration and diabetic macular edema. Phase 2 results have produced promising outcomes with regard to efficacy and durability. Faricimab is currently being evaluated in global Phase 3 studies.