Angiogenesis is a multistep process that requires coordinated migration, proliferation, and junction formation of vascular endothelial cells (ECs) to form new vessel branches in response to growth stimuli. Major intracellular signaling pathways that regulate angiogenesis have been well elucidated, but key transcriptional regulators that mediate these signaling pathways and control EC behaviors are only beginning to be understood. Here, we show that YAP/TAZ, a transcriptional coactivator that acts as an end effector of Hippo signaling, is critical for sprouting angiogenesis and vascular barrier formation and maturation. In mice, endothelial-specific deletion of Yap/Taz led to blunted-end, aneurysm-like tip ECs with fewer and dysmorphic filopodia at the vascular front, a hyper-pruned vascular network, reduced and disarranged distributions of tight and adherens junction proteins, disrupted barrier integrity, subsequent hemorrhage in growing retina and brain vessels, and reduced pathological choroidal neovascularization. Mechanistically, YAP/TAZ activates actin cytoskeleton remodeling, an important component of filopodia formation and junction assembly. Moreover, YAP/TAZ coordinates EC proliferation and metabolic activity by upregulating MYC signaling. Overall, these results show that YAP/TAZ plays multifaceted roles for EC behaviors, proliferation, junction assembly, and metabolism in sprouting angiogenesis and barrier formation and maturation and could be a potential therapeutic target for treating neovascular diseases.

The blood–retinal barrier (BRB) consists of tightly interconnected capillary endothelial cells covered with pericytes and glia, but the role of the pericytes in BRB regulation is not fully understood. Here, we show that platelet-derived growth factor (PDGF)-B/PDGF receptor beta (PDGFRβ) signalling is critical in formation and maturation of BRB through active recruitment of pericytes onto growing retinal vessels. Impaired pericyte recruitment to the vessels shows multiple vascular hallmarks of diabetic retinopathy (DR) due to BRB disruption. However, PDGF-B/PDGFRβ signalling is expendable for maintaining BRB integrity in adult mice. Although selective pericyte loss in stable adult retinal vessels surprisingly does not cause BRB disintegration, it sensitizes retinal vascular endothelial cells (ECs) to VEGF-A, leading to upregulation of angiopoietin-2 (Ang2) in ECs through FOXO1 activation and triggering a positive feedback that resembles the pathogenesis of DR. Accordingly, either blocking Ang2 or activating Tie2 greatly attenuates BRB breakdown, suggesting potential therapeutic approaches to reduce retinal damages upon DR progression.

Hypoxia-inducible factor-1α (HIF-1α) mediates hypoxic responses and regulates gene expression involved in angiogenesis, invasion and metabolism. Among the various HIF-1α posttranslational modifications, HIF-1α methylation and its physiological role have not yet been elucidated. Here we show that HIF-1α is methylated by SET7/9 methyltransferase, and that lysine-specific demethylase 1 reverses its methylation. The functional consequence of HIF-1α methylation is the modulation of HIF-1α stability primarily in the nucleus, independent of its proline hydroxylation, during long-term hypoxic and normoxic conditions. Knock-in mice bearing a methylation-defective Hif1a^KA/KA allele exhibit enhanced retinal angiogenesis and tumour vascularization via HIF-1α stabilization. Importantly, S28Y and R30Q mutations of HIF-1α, found in human cancers, are involved in the altered HIF-1α stability. Together, these results demonstrate a role for HIF-1α methylation in regulating protein stability, thereby modulating biological output including retinal and tumour angiogenesis, with therapeutic implications in human cancer.

Primary open-angle glaucoma (POAG) is often caused by elevated intraocular pressure (IOP), which arises due to increased resistance to aqueous humor outflow (AHO). Aqueous humor flows through Schlemm’s canal (SC), a lymphatic-like vessel encircling the cornea, and via intercellular spaces of ciliary muscle cells. However, the mechanisms underlying increased AHO resistance are poorly understood. Here, we demonstrate that signaling between angiopoietin (Angpt) and the Angpt receptor Tie2, which is critical for SC formation, is also indispensable for maintaining SC integrity during adulthood. Deletion of Angpt1/Angpt2 or Tie2 in adult mice severely impaired SC integrity and transcytosis, leading to elevated IOP, retinal neuron damage, and impairment of retinal ganglion cell function, all hallmarks of POAG in humans. We found that SC integrity is maintained by interconnected and coordinated functions of Angpt-Tie2 signaling, AHO, and Prox1 activity. These functions diminish in the SC during aging, leading to impaired integrity and transcytosis. Intriguingly, Tie2 reactivation using a Tie2 agonistic antibody rescued the POAG phenotype in Angpt1/Angpt2-deficient mice and rejuvenated the SC in aged mice. These results indicate that the Angpt-Tie2 system is essential for SC integrity. The impairment of this system underlies POAG-associated pathogenesis, supporting the possibility that Tie2 agonists could be a therapeutic option for glaucoma.

Blood vessels are essential for blood circulation but also control organ growth, homeostasis, and regeneration, which has been attributed to the release of paracrine signals by endothelial cells. Endothelial tubules are associated with specialised mesenchymal cells, termed pericytes, which help to maintain vessel wall integrity. Here we identify pericytes as regulators of epithelial and endothelial morphogenesis in postnatal lung. Mice lacking expression of the Hippo pathway components YAP and TAZ in pericytes show defective alveologenesis. Mutant pericytes are present in normal numbers but display strongly reduced expression of hepatocyte growth factor leading to impaired activation of the c-Met receptor, which is expressed by alveolar epithelial cells. YAP and TAZ are also required for expression of angiopoietin-1 by pulmonary pericytes, which also controls hepatocyte growth factor expression and thereby alveologenesis in an autocrine fashion. These findings establish that pericytes have important, organ-specific signalling properties and coordinate the behavior of epithelial and vascular cells during lung morphogenesis.

Purpose. To investigate the role of angiopoietin-1 (Ang1) in choroidal neovascularization (CNV) and vascular leakage. Methods. We generated laser-induced CNV in mice and measured the size of CNV and vascular leakage after intravitreal administration of Ang1. The expressions and distributions of endothelial junctional proteins were analyzed using immunohistochemistry and Western blot. Moreover, we compared the sizes of CNV and vascular leakage in Ang1-overexpressing, Ang1-deficient, and their littermate control mice. In addition, following the transplantation of GFP⁺ bone marrow cells into these Ang1-genetically modified mice, we evaluated the recruitment of VEGF-A producing macrophages from the bone marrow after CNV induction. Results. Intravitreal administration of Ang1 was as effective as VEGF-Trap in inhibiting CNV formation. Furthermore, Ang1 suppressed vascular leakage by increasing endothelial junctional proteins, which was more effective than VEGF-Trap. Genetic deletion of Ang1 exacerbated, while overexpression of Ang1 suppressed CNV formation and vascular leakage. We attribute these Ang1-induced, anti-angiogenic, and anti-leakage effects to its inhibitory actions against the recruitment and infiltration of VEGF-A–producing macrophages from bone marrow into the inflammatory lesions. Conclusions. Ang1 supplementation can be established as a therapeutic strategy to suppress the CNV formation and vascular leakage by inhibiting the recruitment of angiogenic macrophages and tightening the endothelial junctions.