We focused on analysing the expression and roles of Ang2 and Tie2, given that these are critical factors regulating vascular stabilization and integrity. Consistent with previous reports 25 , 26 , 27 , Ang2 was high in the pericyte-free tip ECs but low in ECs of the phalanx and plexus, while Tie2 expression showed the opposite pattern in the normally growing retinal vessels Fig.
This finding led us to hypothesize that pericyte coverage per se might regulate the expression of Ang2 or Tie2 in the adjacent ECs. Intriguingly, the inadequate pericyte attachment markedly increased Ang2 but reduced Tie2 in the ECs of retinal vessels Fig. Because Ang2 is upregulated together with ESM1 by the enhanced transcriptional activity of forkhead box protein O1 FOXO1 29 , 30 , 31 , we examined the changes of these molecules in vivo and in vitro. These data suggested that the ECs became unstable due to inadequate pericyte coverage, and that cleavage of Tie1 led to the suppression of Tie2 availability, which is required to inhibit Ang2 transcription by FOXO1 refs 33 , Unexpectedly, however, pericytes covering retinal vessels did not express Ang1, but those along choroidal vessels and neuronal cells in the ganglion cell and inner nuclear layers of the retina did Fig.
These results clearly indicate that retinal pericytes do not produce Ang1 to regulate vascular growth and BRB maturation. Note that most of GFP signals are detected in the retinal neurons yellow arrowheads located in ganglion cell layer and inner nuclear layer. Magnified images of boxed areas are shown below. GFP signals are not detected in pericytes or ECs, but detected in neurons that surround retinal vessels in superficial and intermediate vascular plexus layers.
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However, when Tie2 was specifically depleted in the ECs at adulthood, no noticeable changes including vascular leakage were detected in the retinal vessels compared with those of WT mice Supplementary Fig. These results indicate that Tie2 is indispensable for vascular growth and BRB maturation in the growing retinal vessels, but is dispensable for the maintenance of BRB integrity during adulthood. In fact, lung vessels continue to require Tie2 ref. Moreover, administration of Ang2-blocking antibody 42 also markedly attenuated micro-aneurysm formation, vascular enlargement, and RBC leakage upon APB5 administration Fig.
Thus, enhanced FOXO1 transcriptional activity upon inadequate pericyte attachment seems to be substantially responsible for deteriorating BRB maturation by upregulating several vascular destabilization factors including Ang2 refs 29 , 30 , However, the depletion did not cause pericyte detachment or loss from the stabilized retinal vessels and led to no significant changes in the retinal vessels compared with those in WT mice Supplementary Fig.
These findings indicate that the regulation of pericytes on BRB could be dependent on stability of the vessels, and a sudden pericyte loss from the stabilized retinal vessels is insufficient to induce a significant alteration in the integrity of retinal vessels and BRB, and additional insults may be required to mimic the hallmarks of DR.
Right lower panels, no visible hemorrhage is found in inner surface of retinal cups in both mice. NS, non-significant. Compared with Fc, ABTAA reduced vascular leakage and macrophage infiltration without restoration of pericyte coverage onto the vessels Fig. Boxed regions dotted-lines are magnified and presented in panels in the corner. In both cases with inadequate pericyte coverage around retinal vessels, increased Ang2 and Tie1 cleavage and reduced Tie2 further attenuated PI3 kinase-Akt signalling, leading to enhanced FOXO1-mediated transcription of vascular destabilizing factors in a positive feedback manner.
These exacerbating processes eventually trigger further disintegration of BRB, manifesting DR phenotypes and accelerating DR progression. Moreover, strikingly, a sudden pericyte loss did not significantly disturb the stabilized, quiescent adult retinal vessels and BRB for some time. Pericyte dropout is one of the major hallmarks of DR 15 , 16 , A long-standing question has been whether pericyte dropout is a primary causative factor, a secondary consequence of micro-vessel abnormality, or both in the pathogenesis of DR.
Our findings clearly demonstrate that pericyte dropout is not a direct causative factor for DR pathogenesis but rather is an essential factor that accelerate DR progression, further destabilizing retinal vascular ECs. In this regard, either preventing pericyte detachment or enhancing EC—pericyte interaction could be a potential therapeutic approach for ameliorating DR progression by maintaining BRB integrity and function.
In contrast to the prevailing concept 6 , 19 , 20 , 21 , we provide compelling evidence that pericytes that surround retinal vessels do not express Ang1. Despite the lack of Ang1 expression by pericytes, globally Ang1-depleted neonatal mice show reduced vascular growth and pericyte coverage in the retina In the context of Tie2, we showed that endothelial Tie2 plays an indispensable role in vascular development and BRB maturation.
It is well-known that Ang1-induced Tie2 clustering is critical for its activation 19 , 46 , However, the activation of Tie2 can also be strongly induced by shear stress 48 , which is an independent mode of action to Ang1-induced activation. These collectively imply that Ang1 may have derived from adjacent neurons or hematopoietic cells including platelets, which subsequently activate Tie2 or integrins for its roles in formation and maturation of the inner part of the BRB, or the abnormal vascular phenotypes after endothelial Tie2 deletion can be due to the absence of Tie2 activation induced by a blood flow-mediated shear stress.
The mechanism by which pericyte coverage regulates Tie2 can be explained in two ways. First, Tie2 shedding cleavage of Tie2 extracellular domain in ECs could have occurred immediately after pericyte loss, because ECs are directly exposed to a bunch of activated proteases and growth factors such as VEGF-A. In addition, recent elegant studies 33 , 34 , 50 have revealed that inflammation-induced cleavage of the Tie1 extracellular domain leads to reduced availability and signalling of Tie2, which in turn further degrades vascular stability through FOXO1-mediated upregulation of Ang2 in the ECs of tracheal vessels.
Thus, in agreement with these studies, inflammation induced by the BRB disruption after pericyte loss could also reduce functional Tie2 level and stimulate FOXO1 activation. Nevertheless, unlike FOXO1 and Ang2, the change in Tie2 upon pericyte depletion was different between growing retinal vessels and stable adult retinal vessels, implying that regulation of Tie2 by pericytes could also be dependent on vessel stability as well as its microenvironment, such as inflammation and hypoxia.
While PI3 kinases play key roles in the maintenance of vascular integrity and barrier function 51 , 52 , Akt also plays a vital role in maintaining vascular integrity against VEGF-induced impairments and increased permeability in blood vessels 53 , 54 , In fact, Akt acts as a negative regulator of FOXO1 by promoting its phosphorylation and degradation 19 , In addition, a previous study has shown that FOXO1 activity is induced together with Akt inactivation in primary cultured ECs of microvasculature under diabetic conditions Upregulation of Ang2 expression and enhanced secretion of Ang2 in ECs are highly reactive processes that occur in any unstable and stimulatory conditions, including acute inflammation, chronic hyperglycemia, cerebral cavernous malformation and excess of vascular stimulating factors and cytokines, which definitively contribute to further exacerbation of vascular destabilization in a positive feedback manner 57 , 58 , 59 , Ang2 is also detected in high levels in the vitreous fluid of patients with DR Moreover, a recent study 60 revealed that cerebral cavernous malformation 3 deficiency in brain ECs enhances the exocytosis and secretion of Ang2, which leads to destabilization of BBB and BRB together with pericyte dropout.
In the present study, our approaches with genetic deletion of Ang2 or FOXO1, or administration of Ang2 blocking antibody highlight the critical roles of both Ang2 and FOXO1 as negative regulators of EC stability in the absence of pericytes. However, neither genetic deletion nor antibody administration fully mitigated the vascular leakage caused by inadequate pericyte coverage. There could be several reasons for this partial rescue. First, there could be unidentified molecular actions or pathways that are responsible for the pericyte depletion-induced vascular leakage but independent of the action of FOXO1 or Ang2.
Animal care and experimental procedures were performed under the approval from the Institutional Animal Care and Use Committee No. Male and female mice were not distinguished in neonatal mice P5 to P15 , and male mice were used in adult after P28 experiments. The OIR mouse model was generated as previously reported Retinas were harvested at P Immunofluorescence IF staining of whole-mounted retinas and retinal sections were performed as previously described After extensive washes with PBS, for visualization of the retinal vessels, retinas were IF stained with a rabbit anti-type IV collagen polyclonal antibody Cosmo bio, LB, and a Cy3 conjugated anti-rabbit secondary antibody Jackson ImmunoResearch, , Vascular leakage was analysed by i.
EB concentration per weight of wet organs were calculated from a standard curve of EB in formamide, and normalized by control and presented as fold difference. Intracardiac perfusion with PBS was performed before mice sacrifice to remove circulating lectin. Radial length of the retinal vessels was measured as the shortest distance from the optic disc to the peripheral vascular front in each quadrant of the retina and averaged.
Branching point was measured manually in six 0.
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Number and length of filopodias were examined in four 0. RBC leakage and macrophage infiltration were measured in six random 0. NVT area and avascular areas in OIR model were measured using the Lasso tool of Adobe Photoshop software as previously described 37 and divided by total retinal area. Staining intensities were measured in four representative areas of retinal capillary vessels or, if indicated, other vascular regions in each retina and averaged.
For comparison, the values were normalized by the background signals in non-vascularized areas, and their ratio were normalized by control and presented as fold or percentage. We regarded the round and condensed signal of FOXO1 immunofluorescence staining in the retinal vessels as a nuclear-localized activated form of FOXO1, in accordance to a recently published report If needed, their values were normalized by control and presented as fold change.
The blood-retinal barriers.
GAPDH was used as a reference gene and the results were presented as relative expressions to control. Electroretinogram ERG was recorded to assess the retinal neuronal function at indicated time point. After pupil dilatation using tropicamide 0. To obtain scotopic dark-adapted condition a- and b-wave, a digital bandpass filter ranging from 0. Aortic ring assay was performed as previously described Briefly, aortas of the mice treated with tamoxifen as scheduled were harvested at the indicated time point.
To collect vitreous fluid in the eye, first, eyes were enucleated and washed with sterile ice cold PBS. Then, PBS around the eyes was removed using an absorbing paper. After making a small slit with a blade through the retina, vitreous fluid was collected using Nanoliter micro-injector World Precision Instruments fitted with a glass capillary pipette. After washing with PBS 3 times, the cells were lysed and sonicated using a Bioruptor sonicator Diagenode. Animals or samples were not randomized during experiments and not excluded from analyses. The investigators were not blinded to group allocation during experiments and outcome analyses.
The authors declare that all relevant data of this study are included within the article and its Supplementary Information files. These are also available from the authors upon request. How to cite this article: Park, D. Plastic roles of pericytes in the blood—retinal barrier. Cunha-Vaz, J. Blood-retinal barrier. Campochiaro, P. Molecular pathogenesis of retinal and choroidal vascular diseases.
The Blood–Retinal Barrier in Retinal Disease
Eye Res. Bergers, G. The role of pericytes in blood-vessel formation and maintenance. Neuro Oncol. Potente, M.
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Basic and therapeutic aspects of angiogenesis. Cell , — Hamilton, N.
Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease.