Pathological neovascularization is intimately associated with the progression of several retinal diseases, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration. Neovascularization occurs in response to hypoxia, tissue ischemia, and the consequent production of angiogenic agonists, such as vascular endothelial growth factor A (VEGFA), a potent inducer of vessel formation and vascular leakage (Campochiaro, 2015; Semenza, 2012). The new vessels formed during retinal ischemia are often dysfunctional and fail to stabilize (Fruttiger, 2007; Krock et al., 2011), leading to vessel leakage or hemorrhaging and to retinal detachment, visual impairment, and even blindness. Therefore, suppression of neoangiogenesis, and thereby, retinal edema, is a therapeutic goal in the treatment of ischemic eye diseases (Daruich et al., 2018).

A number of therapeutic options designed to neutralize VEGFA by preventing binding to its receptor, VEGF receptor 2 (VEGFR2), such as bevacizumab, ranibizumab, and aflibercept, decrease neovascular formation as well as edema (Mintz-Hittner et al., 2011). However, they do not correct the underlying hypoxia; in fact, vessel regression may instead further aggravate hypoxia. Moreover, in many cases, anti-VEGF therapies can induce an elevation in intraocular pressure and hemorrhaging (Diabetic Retinopathy Clinical Research Network et al., 2015). The repeated intravitreal injections of anti-VEGF-therapy present a potential for infections and scarring (Patel et al., 2013); in addition, side effects including disrupted neural development in infants have been reported (Canadian Neonatal Network and the Canadian Neonatal Follow-Up Network Investigators et al., 2016). Thus, even though the current therapy improves vision for many patients in the early phases of disease, there is a clear need for developing new treatments to suppress proangiogenic stimuli while also achieving a long-lasting effect with safe administration.

Angiogenesis and vascular permeability in the retina are initiated by the VEGFA/VEGFR2 signaling pathway. VEGFR2 is found not only on blood vascular endothelial cells but also on neuronal cells in the retina, likely contributing to the side effect of VEGFA/VEGFR2 suppression on neural development in premature infants (Nishijima et al., 2007). VEGFR2 activity is initiated through VEGFA-induced receptor dimerization, kinase activation, phosphorylation of tyrosine residues in the receptor intracellular domain, and activation of signaling pathways. The VEGFR2 phosphotyrosine-initiated signaling pathways are now being unraveled. Phosphorylation of Y1212 in VEGFR2 is required for activation of phosphatidyl inositol three kinase and AKT (Testini et al., 2019), while phosphorylation of Y949 is required for activation of the c-Src pathway and regulation of vascular permeability through vascular endothelial (VE)-cadherin (Li et al., 2016). VE-cadherin is the main component of endothelial adherens junctions, strongly implicated in regulation of vascular permeability, leakage, and associated edema (Giannotta et al., 2013). In particular, phosphorylation of the Y685 residue in VE-cadherin correlates with VEGFA-induced vascular hyperpermeability (Smith et al., 2020; Wessel et al., 2014).

Endothelial nitric oxide synthase (eNOS), activated downstream of VEGFA through phosphorylation on S1177 (S1176 in mice) by AKT (Fulton et al., 1999), produces nitric oxide (NO) and regulates vascular permeability. Both mice with a constitutive eNOS gene (Nos3) inactivation (Fukumura et al., 2001) and mice expressing an eNOS serine to alanine point mutation (S1176A) (Nos3^{S1176A/S1176A}) (Di Lorenzo et al., 2013) show reduced extravasation of bovine serum albumin in the healthy skin in response to VEGFA challenge. An important mediator of the effect of eNOS-generated NO is the relaxation of peri-vascular smooth muscle cells (vSMCs), leading to increased vessel diameter and enhanced blood flow and thereby flow-driven vascular sieving (Sessa, 2004). In addition, eNOS activity correlates with tyrosine phosphorylation of VE-cadherin in cultured endothelial cells (Di Lorenzo et al., 2013), providing a mechanism for how eNOS activity may directly affect vascular permeability, distinct from vasodilation.

In contrast to the skin vasculature, the healthy retinal vasculature is protected by a stringent blood-retinal barrier. In retinal diseases, the barrier is disrupted, leading to increased vascular permeability (Zhao et al., 2015). In accordance, NO and related reactive oxygen species (ROS) are important pathogenic agents in retinopathy (Opatrilova et al., 2018). However, the molecular mechanisms whereby eNOS/NO interferes with the retinal vascular barrier and contributes to pathological vascular permeability in eye disease have remained unexplored.

Here, we show that suppressed NO formation via the use of the competitive NOS inhibitor, Nω-Methyl-l-arginine (L-NMMA), which inhibits NO formation from all three NOS variants (eNOS, inducible NOS, and neuronal NOS), or an eNOS mutant, S1176A, negates neovascular tuft formation and vascular leakage during retinal disease. Mechanistically, NO promotes c-Src Y418 phosphorylation at endothelial junctions and phosphorylation of VE-cadherin at Y685, required for dismantling of adherens junctions. Inhibition of NO formation by L-NMMA treatment suppresses vascular leakage also from established neovascular tufts, separating regulation of leakage from the angiogenic process as such. Mice expressing a VE-cadherin tyrosine to phenylalanine mutation (VEC-Y685F) are resistant to eNOS inhibition, supporting the model that NO regulates adherens junctions through direct effects on c-Src and VE-cadherin. These data suggest that eNOS/NO promote vascular permeability not only through the established effect on vSMC relaxation and increased flow-driven permeability to solute and small molecules in the precapillary arterial bed, but also through disruption of adherens junctions allowing leakage of larger molecules from the postcapillary venular bed.

The results presented here show that eNOS/NO plays a role in the postcapillary venous system by directly affecting the endothelial barrier to exacerbate vascular hyperpermeability in retinopathy (Figure 6). According to the consensus model, eNOS-generated NO regulates vascular permeability by inducing the relaxation of perivascular smooth muscle cells. NO produced in endothelial cells diffuses across the vascular wall and activates soluble guanylate cyclase leading to protein kinase G activation in vSMC, lowering cellular Ca²⁺, and promoting vascular relaxation, increased blood flow, and reduced blood pressure (Surks et al., 1999). Indeed, vessel dilation is a part of the tissue deterioration seen in diabetic retinopathy (Bek, 2013; Grimm and Willmann, 2012). However, there is also evidence for a direct role for eNOS and NO in endothelial cells, as constitutive eNOS deficiency inhibits inflammatory hyperpermeability in the mouse cremaster muscle treated with platelet-activating factor (Hatakeyama et al., 2006). Moreover, NO can regulate phosphorylation of VE-cadherin in adheren junctions in vitro in microvascular endothelial cell cultures (Di Lorenzo et al., 2013). It is likely that these multifaceted effects of eNOS/NO are differently established in different vessel types. eNOS/NO-dependent vessel dilation is dependent on the vSMC coverage in arterioles and arteries; in contrast, adherens junction stability affects mainly the postcapillary venular bed (Orsenigo et al., 2012). In the skin, prevenular capillaries and postcapillary venules, with sparse vSMC coverage, respond to VEGFA with increased paracellular permeability, while arterioles/arteries do not (Honkura et al., 2018). These distinctions are important as exaggerated and chronic VEGFA-driven paracellular permeability in disease leads to edema and eventually to tissue destruction (Nagy et al., 2012). Therefore, we explored the consequences of NO deficiency using a mutant eNOS mouse model, with a serine to alanine exchange at 1176, as well as treatment with the NO inhibitor L-NMMA, to examine how attenuating NO production affects VEGFA-dependent pathological angiogenesis in ocular disease.

Figure 6

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Our data shows that while the attenuation of eNOS S1176 phosphorylation was dispensable for vascular development in the retina and for endothelial survival during hyperoxia, growth of pathological vessel tufts in the subsequent phase of relative hypoxia was suppressed, while pericyte coverage and inflammation remained unaffected. Also, treatment with L-NMMA during the hypoxic phase reduced growth of vascular tufts in the retina. In agreement with earlier literature, we conclude that eNOS activity and NO formation influence pathological angiogenesis in the eye (Ando et al., 2002; Brooks et al., 2001; Edgar et al., 2012). Of note, tufts that were established in the Nos3^{S1176A/S1176A} mice leaked less, in spite of similar levels of VEGFA being produced as in the wild-type retina. In an attempt to mimic the clinical situation, we treated P16 mice with established vascular tufts with a single dose of L-NMMA. At the examination 24 hr later, tuft area remained unaffected, while the leakage of 25 nm microspheres was reduced by 50–60%. Whether leakage regulation is separable from the regulation of growth of new vessels has been a matter of debate. Pathological angiogenesis in the retina is intimately associated with leakage and edema (Smith et al., 2020). Exactly how junction stability plays a role in the neoangiogenic process is unclear. However, leakage and the production of a provisional matrix is postulated to be a prerequisite for the growth of angiogenic sprouts (reviewed in Nagy et al., 2012). With the effects of the L-NMMA intervention treatment shown here, we can conclude that these responses indeed can be separated, at least in the short term.

L-NMMA and its analog, L-NAME, induce vasoconstriction by halting the NO/soluble guanylyl cyclase signaling pathway and consequent vasodilation (reviewed in Ahmad et al., 2018; Thoonen et al., 2013). Thus, a daily intake of L-NAME in rats (40–75 μg/g/day for 4–8 weeks) leads to vasoconstriction (Ribeiro et al., 1992; Simko et al., 2018; Vrankova et al., 2019; Zanfolin et al., 2006). NOS inhibitors have also been used clinically, for example, by administration of L-NMMA to increase the mean arterial pressure in cardiogenic shock. In a typical regimen, 1 mg.kg⁻¹.hr⁻¹ of L-NMMA is administered over 5 hr (Cotter et al., 2000). While we cannot exclude an effect on vasoconstriction with the single IP injection of L-NMMA used here (60 μg/g), blood flow appeared unaffected as equal amounts of microspheres arrived in the retinal vasculature, while leakage into the extravascular space was substantially reduced in the L-NMMA treated mice compared to the controls. The leakage suppression did not involve changes in pericyte coverage of vessels or in the degree of inflammation. While perfusion was enhanced in tufts that still formed in the Nos3^{S1176A/S1176A} retinas after OIR-challenge, there was no effect on perfusion in tufts remaining after multiple- or single-dose L-NMMA treatment, possibly reflecting differences in tuft morphology and the degree of vessel patency.

Mechanistically, our results place c-Src downstream of eNOS activity. c-Src has also been placed upstream of eNOS by c-Src’s regulation of the PI3K-AKT pathway, of importance for eNOS activation (Haynes et al., 2003). VEGFR2-induced activation of c-Src has however been mapped to the pY949 residue in VEGFR2 (Li et al., 2016), while activation of Akt is dependent on Y1212 (Testini et al., 2019). In VEGFA-stimulated endothelial cells, c-Src is implicated in phosphorylation of VE-cadherin at Y685 and potentially other tyrosine residues, to induce vascular permeability (Orsenigo et al., 2012; Wallez et al., 2007). The fact that the mutant VEC-Y685F mice in which the Y685 residue has been replaced with a non-phosphorylatable phenylalanine were resistant to L-NMMA inhibition, indicates that the VEGFR2 Y1212/eNOS/NO pathway acts on adherens junction through c-Src (Li et al., 2016). Combined, our data support a model in which eNOS phosphorylation at S1176 is required for the activation of c-Src, which in turn phosphorylates VE-cadherin at Y685, inducing transient disintegration of adherens junctions and increased paracellular permeability (Figure 6). These in vivo results potentially provide meaningful mechanistic and therapeutic insights with regard to retinopathy of prematurity (Cunha-Vaz et al., 2011).

A considerable challenge in the analysis of c-Src activity is the close structural relatedness between the kinase domains of c-Src and the related SFKs Yes and Fyn (Sato et al., 2009), which are also expressed in endothelial cells. The amino acid sequence covering the activating tyrosine is entirely conserved between these three cytoplasmic tyrosine kinases such that the antibodies against pY418 c-Src in fact reacts with all three SFKs. In agreement, immunoblotting using antibodies against pY418 c-Src resulted in biphasic peaks of activity possibly reflecting activation of different SFKs. Moreover, pY418 immunostaining of wild-type and Nos3^{S1176A/S1176A} retinas after OIR failed to reveal a dependence on eNOS catalytic activity for activation of c-Src, in agreement with the findings of Di Lorenzo et al., 2013. However, combining oligonucleotide-linked secondary antibodies reacting with c-Src protein and pY418 Src in a PLA on isolated endothelial cells demonstrated a critical role for eNOS activity on accumulation of active, pY418-positive c-Src at endothelial junctions. The question remains how eNOS/NO influences c-Src activity? NO can couple with cysteine thiols to form S-nitroso-thiols, which may affect the folding and function of the target protein. In accordance, Rahman et al. demonstrated S-nitrosylation of c-Src at the kinase domain cysteine 498, correlating with increased c-Src activity (Rahman et al., 2010).

Although not experimentally addressed in this study, retinal diseases, such as diabetic retinopathy and adult macular degeneration, are accompanied by excessive permeability (Antonetti et al., 2012; Campochiaro, 2015). Of note, patients with diabetic retinopathy have elevated levels of NO in the aqueous humor, and particular eNOS polymorphisms are associated with protection or increased risk for diabetic retinopathy (for a review, see Opatrilova et al., 2018) and macular edema (Awata et al., 2004). Our results show that pharmacological inhibition of NO production after OIR challenge in mice can prevent vascular leakage. Thus, NO inhibitors applied in combination with anti-VEGF therapy, delivered in low but still efficient doses, could possibly be of clinical benefit. Local delivery of NOS inhibitors may be needed to avoid drawbacks with systemic delivery, such as hypertension, or any other vasoconstriction-associated adverse events. A limitation of the findings reported here was that neither the multiple-dose nor single-dose L-NMMA treatment improved perfusion of remaining tufts after OIR challenge. However, as tuft formation and leakage were suppressed with the multiple-dose treatment, a relative larger fraction of vessels remained healthy, with normal perfusion.

In conclusion, the data presented here establish a critical role for eNOS in endothelial cells, regulating c-Src activation downstream of VEGFA/VEGFR2, and thereby, in VE-cadherin-regulated endothelial junction stability and vascular leakage in retinal pathology.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–5. Source data files have also been deposited with Dryad: https://doi.org/10.5061/dryad.x69p8czhv.

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Author details

1. Takeshi Ninchoji

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

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Writing - review and editing

   Contributed equally with

   Dominic T Love

   For correspondence

   nincho830@gmail.com

   Competing interests

   No competing interests declared

2. Dominic T Love

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

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Contributed equally with

   Takeshi Ninchoji

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8530-1352

3. Ross O Smith

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

   Contribution

   Supervision, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

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

4. Marie Hedlund

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

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. 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

6. William C Sessa

   Yale University School of Medicine, Department of Pharmacology and Vascular Biology and Therapeutics Program, New Haven, United States

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5759-1938

7. Lena Claesson-Welsh

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

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, 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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