The number of people with impaired vision and blindness is increasing in Western society due to the aging population and the increased prevalence of diabetes. This has led to eye diseases, such as age-related macular degeneration and diabetic retinopathy becoming more common. In both these eye diseases, new blood vessels grow in the retina – the light-sensitive part of the eye – to bring oxygen and nutrients to the tissue. However, these new blood vessels are leaky and allow molecules to leave the bloodstream and enter the retinal tissue. This causes the retina to swell and impair a person’s vision. The leaky blood supply also reduces the amount of oxygen that gets to the tissue, resulting in further damage to the retina.

When tissues experience low levels of oxygen, cells start making a protein called vascular endothelial growth factor (or VEGF for short). Whilst this protein is important for helping form new blood vessels, it also makes these vessels leaky. Current treatments for age-related macular degeneration and diabetic retinopathy decrease swelling in the eye by blocking the action of VEGF. However, these treatments also cause existing blood vessels and nerve cells to die, leading to irreversible damage. Now, Smith et al. have set out to find whether the effects of VEGF can be blocked without causing further damage to existing cells.

To investigate this possibility, the eyes and retinas of mice were treated with a laser or exposed to changing oxygen levels to create injuries that resembled human age-related macular degeneration and diabetic retinopathy. Each of the tested mice had specific mutations in proteins known to interact with VEGF. Fluorescent particles were injected into the bloodstream of the mice to assess how these different mutations affected blood vessel leakage: if fluorescent particles could no longer be detected outside the blood vessels, this suggested that the mutation had stopped the vessels from leaking. Further experiments showed these specific mutations affected leakage and did not prevent new blood vessels from forming.

In the future it will be important to see if drugs, rather than mutations, can also decrease the leakiness of blood vessels in the retina. Such chemical compounds could then be tested in mouse experiments. If successful, these drugs might be used to treat patients with age-related macular degeneration and diabetic retinopathy.

Blood vessel dysfunction in the retina is a critical component of several blinding eye diseases. In particular, the retina is sensitive to the fluid accumulation that can result from excessively leaking blood vessels in the eye and reduction of the ensuing edema is a therapeutic goal in the clinic (Daruich et al., 2018). Vascular endothelial growth factor A (VEGFA) is one of the key factors controlling endothelial cell biology in the eye (Daruich et al., 2018). The importance of VEGFA signaling is underscored by the clinical success of antibodies (bevacizumab, ranibizumab) neutralizing VEGFA, or soluble receptor fusion proteins (aflibercept), neutralizing several VEGF family members. These drugs preserve vision in many patients, but over time disease progression may resume, and for others there is no benefit even from the initial anti-VEGF treatment (Bressler et al., 2017). The role of VEGFA for survival of VEGFR2-expressing retinal neurons presents an additional challenge (Nishijima et al., 2007). Moreover, anti-VEGF treatment has been linked to capillary loss and rarefaction of normal adult vasculature (Kamba and McDonald, 2007). While a focus in the development of new therapies to treat retinopathies has been on increasing the potency of anti-VEGFA therapy, the detrimental effects of long-lasting VEGFA suppression are now being recognized (Usui-Ouchi and Friedlander, 2019). Taken together, the development of alternative treatments to block excessive vascular leakage in the eye while sparing other VEGF functions is very important.

The retina is protected by a blood retinal barrier (BRB) which, under normal conditions, limits the extravasation of blood components into the tissue. The BRB consists of an inner barrier at the level of retinal vessels and an outer barrier at the cell junctions of the retinal pigment epithelium and it serves to stringently protect the tissue from pathogens, edema and inflammation (Zhao et al., 2015). The BRB is established in conjunction with vessel entry in the central nervous system during early development (Chow and Gu, 2017). In eye diseases such as diabetic retinopathy, hypoxia and the ensuing increased production of VEGFA is accompanied by breakdown of the BRB (Campochiaro, 2015). The molecular mechanisms downstream of VEGFA leading to this disruption have not been defined.

VEGFA, originally identified as vascular permeability factor (VPF) (Senger et al., 1986), binds to VEGFR2 inducing receptor phosphorylation and the propagation of signaling cascades regulating endothelial survival, proliferation and motility (Simons et al., 2016). Acute responses to VEGFR2 activity include destabilization of endothelial junctions leading to increased permeability. Notably, phosphorylation of VEGFR2 at Y949 (951 in human) enhances permeability via the disruption of adherens junctions in the dermal vasculature, but also in pathologies such as neuroendocrine cancer, melanoma and glioblastoma (Li et al., 2016a). Phosphorylation of Y949 creates a binding site for the adaptor molecule T cell specific adaptor (TSAd), which in turn binds c-Src at endothelial junctions (Li et al., 2016a; Matsumoto et al., 2005; Sun et al., 2012). c-Src is implicated in phosphorylation of vascular endothelial (VE)-cadherin (Li et al., 2016a; Adam et al., 2010; Vestweber, 2008; Wallez et al., 2007). VE-cadherin is the main component of endothelial adherens junctions, forming homophilic interactions between endothelial cells, and is critical in regulation of permeability in response to VEGFA as well as inflammatory cytokines (Li et al., 2016a; Akla et al., 2018; Lampugnani et al., 2018). Tyrosine phosphorylation of VE-cadherin is associated with different biological functions. Thus, phosphorylation of Y658 and Y685 is associated with the control of vascular permeability (Orsenigo et al., 2012), and angiogenic sprouting (Gordon et al., 2016), and Y658 with junction stability (Garrett et al., 2017; Schulte et al., 2011), whereas phosphorylation of Y731 in VE-cadherin is linked to leukocyte extravasation (Wessel et al., 2014). Serine phosphorylation of VE-cadherin at S665 is also implicated in regulation of endothelial junctions (Gavard and Gutkind, 2006).

Here, the molecular mechanisms underlying vessel leakage in eye diseases were studied using mouse models deficient in the VEGFR2-TSAd-VE-cadherin pathway. Interruption of the VEGFR2 pY949F pathway resulted in suppressed vascular leakage at the choroid or at the superficial retinal vasculature in mouse models of age-related macular degeneration and diabetic retinopathy, respectively. Furthermore, disrupting this pathway correlated with reduced VE-cadherin pY685 levels and reduced pathological neoangiogenesis of the superficial retinal vasculature. These data identify VEGFR2 pY949 signaling as an important contributor to edema in retinopathies which may serve as a basis for development of new therapies selectively suppressing VEGFA-dependent disruption of the vascular barrier.

Here we demonstrate that blocking VEGFA-induced VEGFR2 pY949 signaling at any of several steps in the downstream pathway, leads to reduced vascular leakage in two independent retinopathy models, OIR and CNV. While exposure to high oxygen in the OIR procedure causes pathological vascularization in the superficial retinal vessels, the CNV laser injury targets the choroidal vasculature. Thus, we implicate VEGFR2 pY949 signaling in exaggerated vascular leakage in two very different vascular beds in the retina.

Importantly, we identify components of a signaling axis that can be targeted in order to control excessive vascular leakage and pathological neoangiogenesis without interfering with physiological functions of VEGFA. Although pathological vascular tuft formation was diminished in the Kdr^Y949F/Y949F strain, careful characterization of the vasculature in different organs of this strain has not revealed deficiencies in vessel density or morphology during development (Li et al., 2016a). Of note, the improved barrier properties in the Kdr^Y949F/Y949F vasculature do not exclude infiltration of CD68/CD45+ inflammatory cells (Li et al., 2016a and Figure 2—figure supplement 2). The effect of bacterial infections and wound healing properties remain to be tested. Nevertheless, based on the currently available information, we favor the notion that suppressed macromolecular leakage in response to VEGFA plays an important role in pathologies while it is dispensable in physiology. We do not exclude that leakage suppression to some extent interferes with neoangiogenesis due to the lack of a fibrinogen mesh for the new vessel to grow on Dvorak et al. (1995). However, as leakage was suppressed in the Kdr^Y949F/Y949F retinas in both the CNV and OIR (normalized to neovascular area) models, our data support the concept that leakage is regulated separately from angiogenesis per se. We conclude that in a range of tissues, either in the CNS or in peripheral organs, the VEGFR2 – TSAd – VE-cadherin pathway regulates vascular leakage in the context of disease, independent of angiogenesis.

A considerable strength of our study is in the use of intravenously injected microspheres to assess leakage, as we are able to address both pathological vessel growth and leakage simultaneously. This is superior to traditional methodologies using Evans blue injection, to determine the area of dye leakage over the retina surface, or measure the relative amount of dye extracted from retinas using formamide (Xu et al., 2001). Unlike the methodology described here, neither of these methods allow for the precise visualization of the leakage or how it relates to vessel morphology.

Studies in rodent models have suggested that a complete loss of VEGFA-induced signaling in the eye can lead to atrophy of the neural layers, which mechanistically can be explained by the presence of VEGFR2 on retinal neurons (Okabe et al., 2014; Saint-Geniez et al., 2008). In rats, anti-VEGFA treatment does not lead to damage of neural cells or loss of photoreceptor function (Long et al., 2018), indicating that VEGFA might indeed be dispensable for the neural retina in this species. However, retrospective and long term follow-up studies in human patients treated with anti-VEGFA therapies do provide examples of rare negative side effects such as hemorrhages, potentially linked to a systemic reduction of VEGFA (Avery, 2014; Falavarjani and Nguyen, 2013; Keir et al., 2017). Furthermore, anti-VEGFA treatment may contribute to the progression of geographic atrophy due to insufficient vascularization or to the fact that VEGFA may serve as a vital survival factor for the retinal pigment epithelium (RPE) (Byeon et al., 2010). Increased treatment frequency in patients receiving ranibizumab or bevacizumab is associated with greater loss of RPE, indicating geographic atrophy (Enslow et al., 2016; Grunwald et al., 2014; Rofagha et al., 2013). The necessity of repeated intraocular injections (Patel et al., 2013) for the administration of current anti-VEGFA therapy reduces patient compliance (Polat et al., 2017) and results in rare but serious instances of eye damage caused by the injection procedure, such as endophthalmitis or retinal detachment (Falavarjani and Nguyen, 2013; Li et al., 2016b). Ultimately, the interaction between VEGFR2 pY949/pY951 and TSAd would be an ideal target to specifically block leakage but save other aspects of VEGFR2 biology.

The literature strongly implicates Src family kinases (SFK) in the phosphorylation of tyrosine residues on VE-cadherin, thereby playing an essential role in controlling vessel permeability (Adam et al., 2010; Orsenigo et al., 2012; Wessel et al., 2014; Trani and Dejana, 2015). In the context of OIR, direct inhibition of c-Src activation by kinase inhibitors (Seo and Suh, 2017; Werdich and Penn, 2006), indirect inhibition of c-Src kinase (Toutounchian et al., 2017), or suppressed c-Src expression using siRNA (Zhang et al., 2010), all result in decreased tuft formation. Here, we found that neovascular tufts displayed abundant pY418 c-Src at endothelial junctions. Although we may have expected decreased p418 c-Src levels in the Kdr^Y949F/Y949F tufts, the levels were similar in the mutant and Kdr^+/+ littermates. These data indicate that while activation of c-Src alone may be required for induction of permeability and tuft formation, it may not be sufficient to induce these events. In agreement, a triggering event, in addition to c-Src activity, is needed for opening of paracellular junctions (Adam et al., 2010; Adam et al., 2016). Also, Orsenigo and coworkers detected constitutive, flow-dependent pY418 c-Src in leakage-permissive venules, but described an additional triggering event required for leakage to become established (Orsenigo et al., 2012). Our data suggests the scenario that VEGFR2 pY949 signaling leads to close proximity and complex formation between VEGFR2 and VE-cadherin potentially involving c-Src (Li et al., 2016a), which may be activated in a VEGFR2-independent manner. A caveat in the interpretation of these data is that the pY418 antibody used to detect activated c-Src may cross-react with the related Yes and Fyn antibodies. Currently, reagents specifically recognizing only c-Src are missing.

The acute stimulation of blood vessels with bradykinin or histamine to induce leakage is linked to a strong and transient drop in VE-cadherin phosphorylation due to internalization and degradation (Orsenigo et al., 2012). We find that in the chronic disease models studied here, regions of greater VE-cadherin phosphorylation displayed increased leakage. The decrease in leakage observed in the Kdr^Y949F/Y949F mice challenged with OIR or CNV, correlated with a reduction in VE-cadherin pY685 immunostaining, identifying this phosphosite as a key mediator in leakage regulation. This finding is in line with the observation that sites of leakage in a cremaster model correlated with staining for VE-cadherin pY685 (Wessel et al., 2014). The mechanism underlying the important role of VE-cadherin pY685 compared to pY658 in vascular permeability is not understood. The pY685 phosphosite serves as the binding site for C-terminal Src Kinase (CSK), a negative regulator of c-Src activity. CSK phosphorylates c-Src at pY527, leading to its inactivation (Latour and Veillette, 2001). Reduced pY685 VE-cadherin levels, as seen in the Kdr^Y949F/Y949F mice, would predict less CSK at the junction and ultimately increased c-Src activity (Baumeister et al., 2005; Ha et al., 2008), which we did not observe. Moreover, CSK deletion in vitro does not alter barrier properties (Adam et al., 2010). Another important aspect of the role of VE-cadherin pY685 is its regulation of pathological angiogenesis. Notably, tuft area was decreased per se, in the VEC-Y685F mutant (Figure 3B,C). VE-cadherin has been implicated in regulation of VEGFR2 signaling by limiting receptor internalization. In the presence of VE-cadherin, VEGFR2 is dephosphorylated by density-enhanced phosphatase (DEP)1, thus decreasing the levels of active VEGFR2 and proliferative signaling (Lampugnani et al., 2006). We hypothesize that VE-cadherin pY685 downstream effectors are critical in VEGFR2 signaling at junctions.

The VE-cadherin Y658 site has also been identified as an integral regulator of junctional stability. Phosphorylation of the site acts to displace bound p120 catenin leading to destabilized adherens junctions (Garrett et al., 2017; Schulte et al., 2011). VE-cadherin Y658 is phosphorylated by Src family kinases, with low levels of shear stress leading to maximal pY658 immunostaining (Orsenigo et al., 2012; Conway et al., 2017). In the OIR model, tufts are known to have perturbed perfusion, which may drive the high pY658 intensity in tufts, to a similar extent in the WT and mutant models, as observed in this study.

At this point, VEGFA-targeting is the only pharmacological strategy exploited clinically and it is therefore of particular interest to understand how edema is established in response to VEGFA. Other molecular regulators of pathological leakage have been described that may operate in concert with or independently of the VEGFR2 Y949 signaling. Semaphorin 3A (Cerani et al., 2013), Neuropilin 1 (Fantin et al., 2017), activin-like kinase receptor type I (Akla et al., 2018), angiopoietin-like 4 (Babapoor-Farrokhran et al., 2015), VE-PTP and Tie2 (Frye et al., 2015; Shen et al., 2014) have all been implicated in the control of junctional integrity in different vascular beds. Here, we provide information on the specific VEGFR2 signaling pathway leading to edema that can be tested as a readout in other systems. We also present an effective methodology to quantify leakage in relation to pathological angiogenesis, which we foresee will aid in exploration of new targets to treat retinopathies.

All data generated or analysed during this study are included in the manuscript and supporting files. Text files containing the ImageJ macros used for automatic detection of microspheres in Figures 1 and 2 are provided.

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Author details

1. Ross O Smith

   Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory Uppsala University, Uppsala, Sweden

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4239-3204

2. Takeshi Ninchoji

   Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory Uppsala University, Uppsala, Sweden

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Emma Gordon

   Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory Uppsala University, Uppsala, Sweden

   Present address

   Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia

   Contribution

   Conceptualization, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

4. Helder André

   Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden

   Contribution

   Resources, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Elisabetta Dejana

   1. Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory Uppsala University, Uppsala, Sweden
   2. IFOM-IEO Campus Via Adamello, Milan, Italy

   Contribution

   Resources, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

6. Dietmar Vestweber

   Max Planck Institute for Molecular Biomedicine, Münster, Germany

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3517-732X

7. Anders Kvanta

   Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

8. Lena Claesson-Welsh

   Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Science for Life Laboratory Uppsala University, Uppsala, Sweden

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing

   For correspondence

   lena.welsh@igp.uu.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4275-2000

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