Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids

Abstract Purpose Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids. Methods In this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1. Results Inhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network. Conclusion Combined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.


Introduction
Diabetic retinopathy (DR) is the major vision-threatening complication of diabetes that dramatically decreases the quality of life of the affected patients. 1 As the visual cycle is an active source of oxidation in the retina, a balance change between oxidation and anti-oxidation mechanisms leads to DR onset and progression. 2 The high glucose environment and insulin deficiency in the retina leads to increased oxidative stress, tissue hypoxia, chronic inflammation, microglia activation, and loss of retinal ganglion cells (RGC). 3,4 Microglia cell activation leads to constant retinal inflammation by producing proinflammatory cytokines such as Tumor necrosis factor-alpha (TNF-a), Interleukin-6 (IL-6), (C-C motif) ligand 2 (CCL-2; previously known as MCP-1). 5,6 Early stages of non-proliferative DR (NPDR) involve various degrees of vascular abnormalities, including loss of pericytes, reduced blood-retinal barrier (BRB) function through the decrease of ZO-1 and Occludin-1, acellular capillaries, microaneurysms, retinal hemorrhages. 7,8 With the DR progression, these changes lead to reduced perfusion, retinal ischemia, and further loss of BRB. Clinically these changes in the retinal vasculature become noticeable in patients' fluorescent angiography (FA). 9 While the loss of ganglion cells remains largely unnoticed by the patient, diabetic macular edema (DME) resulting from increased proinflammatory signaling and serum leakage into the retina dramatically reduces the patient's vision. The advancement of DR to the proliferative stage (PDR) is driven by increased production of proangiogenic factors such as hypoxia-inducible factor-1 alpha (HIF-1a), vascular endothelial growth factor (VEGF), which drive microvascular endothelial cell proliferation, migration, angiogenic growth. Pathological neo-vasculatures grow across the inner limiting membrane into the retina and vitreous. These neo-vessels are fragile, leading to retinal and vitreal hemorrhages, eventually resulting in retinal detachment and loss of vision. 1 PlGF is the second member of the VEGF family of ligands cloned in 1991 (VEGF-A was discovered in 1989 10 ). 11 PlGF is involved in pathological angiogenesis in a wide range of disease conditions, such as ischemia, inflammation, and cancer, through the interaction with VEGF-A and its VEGFR1 (NRP1/NRP2) signaling. [12][13][14][15] The downstream molecules of the PlGF signaling pathway have been elucidated in detail. 16,17 PlGF's role in eye diseases has been extensively characterized. [18][19][20][21][22] Especially, PlGF was found to promote choroidal and retinal angiogenesis (retinal neovascularization) and is a potential therapeutic target to treat vasculopathies, such as wet AMD, PDR, and DME. 7,[23][24][25] PlGF expression is increased in the vitreous in the DR/DME patients and the increase correlated with the disease severity. 22,26,27 PlGF, in addition to VEGF-A and VEGF-B, is directly targeted by the FDA-approved drug EYLEAV R (Aflibercept), leading to a more superior therapeutic effect than treatments that target anti-VEGF (e.g. Ranibizumab, Bevacizumab) in the treatment of DME patients with worse baseline. 28 Angiopoietins are a class of secreted glycosylated peptides (i.e. Ang-1, Ang-2, Ang-, and Ang-4) that significantly regulate vascular integrity and quiescence in numerous inflammatory-related conditions. Ang-3 signaling is not well documented, while angiopoietins 1, 2, and 4 are ligands for the Tie-2 receptor, 29 predominantly expressed by endothelial cells. It has been shown that Ang 1 signaling via Tie-2 maintains quiescence in the adult vascular endothelium. 30 Interestingly, Ang-2, a member of the angiopoietin family of glycosylated peptides, is widely believed to antagonize Ang-1 signaling. Despite the weak expression of Ang-2 by quiescent vascular endothelium, Ang-2 directly stimulates the phosphorylation of Tie-2. Ang-2 also de-stabilizes quiescent endothelium priming the vasculature to respond rapidly to exogenous stimuli such as inflammatory and angiogenic mediators. The central role of Ang-2 in regulating the rapid vascular response to stimuli is partly attributed to the readymade, stored Ang-2 in Weibel-Palade bodies within endothelial cells, 31 from where Ang-2 molecules are released quickly following stimulation to exert their effect. Functional studies indicated that Ang-2 affects pericyte coverage of the vasculature leading to BRB leakage and, thus, a potential therapeutic target for treating PDR and DME. [32][33][34] This study investigated a novel PlGF's/Ang-1/-2 and Tie-2 signaling axis in vitro, using human cell co-cultures, i.e. human retinal pericytes (HRP) and HREC, and blood vessel or vascular organoids (VO) derived from human induced pluripotent stem cells (iPSC). In line with this model system, we recently developed a three-dimensional (3D) human VO as a model for diabetic vasculopathy. This model showed similarities to human vasculatures as detailed elsewhere. 35 Pericyte coverage was examined using this model system in this study, and the relative expression and colocalization of PDGFRb relative to the endothelium were investigated. The goal was to provide insight into PlGF/VEGFR1 and Ang-1/ Tie-2 signaling, with the knowledge generated serving as a basis for future therapeutic interventions for conditions affecting the back of the eye, such as DR, among others.

Cell cultures and antibody treatments
The primary human retinal endothelial cells (HREC; Cat#: ACBR1 181) and human retinal pericytes (HRP; Cat#: ACBR1 183) were purchased from Cell Systems (Kirkland, WA) and cultured based on the experimental procedures as described previously. 36,37 In brief, HREC were seeded on fibronectin-coated (1 mg/cm 2 , Cat#: 1030-FN, R&D Systems). Plastic vessels and cultured with EBM2-MV medium (Cat#: cc-4176, Lonza) supplemented with EC growth factors (Cat#: cc-4147, Lonza). HRP cells were cultured with a complete pericyte culture medium (Cat#: 4N0-500, Cell Systems) with supplementation of normal glucose, culture boost (Cat#: 4CB-500, Cell Systems), and attachment factor (Cat#: 4Z0-201, Cell Systems). HREC and HRP cells were co-cultured at the ratio of 2:1 with the combination of two culture media. After HREC monolayer formation and HREC-HRP co-culture stabilization, the culture media were replaced with fresh ones with the following desired treatment agents: D-glucose (25 mM), mannitol (25 mM), hydrocortisone (50 mM), anti-PlGF antibody (PL5D11D4, Oxurion, Leuven, Belgium), and anti-VEGFR1 antibody (MF1, ImClone Systems). The treatment duration was 2 days. It is worth noting that the mouse anti-PlGF antibody was validated to bind with human PlGF protein in our previous study. 36 Both anti-PlGF and anti-VEGFR1antibodies showed robust efficacy in in-vivo and in-vitro experiments. [23][24][25]38 Human iPSC-derived vascular organoids cultures Human iPSC cells reprogrammed from a non-disease human subject were obtained from Infinity BiologiX LLC, NJ (Subject: NDS00249; iPSC: ND50018; Passage 11). The iPSC cells were cultured and maintained with the mTeSR1 medium (Cat#: 85850, STEMCELL Technologies), which was formulated from basal medium (Cat#: 85851 STEMCELL) and supplement (Cat#: 85852, STEMCELL). Blood vessels or vascular organoids were generated according to the protocols described by Wimmer et al. 35,39 Briefly, human iPSC cells were first aggregated on a 6-well low attachment plate with the mTeSR1 medium containing 50 mM Y27632 (ROCK inhibitor, Cat#: 688001, Calbiochem, Millipore/Sigma) for 1 day. Then, the iPSC cell aggregates were committed to mesoderm linage in the presence of 12 mM CHIR99021 (GSK3b inhibitor, Cat#: 4423, Tocris) and 30 ng/mL BMP-4 (Cat#: 78211, Stemcell Tech) for 3Days. The aggregated progenitor cells were further differentiated into vascular cells with 100 ng/ml VEGF-A and 100 ng/ml FGF-2 for 2 days. The final step was that vascular cells continue to sprout, grow and form a network in the collagen I-Matrigel matrix under the induction of VEGF-A and FGF-2 from 5 to 14 days. The success of vascular organoids was confirmed by three-dimensional (3D) structures, vascular networks, and pericyte coverage.

PlGF enzyme-linked immunosorbent assay
The levels of PlGF in the culture supernatants were determined by the commercial PlGF enzyme-linked immunosorbent assay (ELISA) kit (DPG00, R&D Systems). The culture supernatants from HREC and HREC-HRP co-cultures were collected and filtered through Millex-GP Filter, 0.22 mm (LGPB5010, Sigma-Aldrich) to remove cells and cellular debris. The ELISA was performed according to manufacturer instructions using provided PlGF control standard curve. The resultant absorbances were read at 450 nm using an 800 TS Absorbance Reader (BioTek Instruments, Winooski, VT, USA).

Trans-endothelial electrical resistance measurement by an electrical cell-impedance sensing system
HRECs and HRP were seeded and co-cultured at a 2:1 ratio on the 8-well cultureware (PC, 8W10E). Trans-endothelial electrical resistance (TEER) changes were measured in realtime with the electrical cell-impedance sensing system (ECIS)-ZƟ system (Applied Biophysics, NY). The ECIS software-embedded mathematical model of impedance change was used to calculate the TEER (X/cm 2 ), measuring the cellcell barrier and cell-matrix resistance. 40 The single-frequency model (4000 Hz) measured resistance and impedance with a 300 s interval. After the resistance stabilized and reached a platform, indicating the formation of confluent monolayer and functional barrier, The various treatments: IgG control, PlGF antibody (50 mg/ml), and PlGF antibody (50 mg/ml) þ VEGFR1 antibody (50 mg/ml) was added to the medium and then continued to culture for two days. The normalized resistance values were expressed as a percentage relative to vehicle control.

Western blot and densitometric quantification
Western blot (WB) was performed according to the previously described methods with some modifications. 24,36,41 HREC and RRP were mono-and co-cultured to confluency in 6-well plates and used for WB analysis. In brief, the cells were washed with cold PBS three times, detached with a cell scraper, and collected by centrifugation. The harvested cell pellets were sonicated in cold RIPA buffer containing FAST protease inhibitors (Cat#: S8830, Sigma, St. Louis, MO). The protein concentration was determined with the DC TM Protein Assay kit (Bio-Rad) and Qubit 4 fluorometer.
Before the electrophoretic transfer to 0.45 lm pore-size nitrocellulose membranes, 30-50 lg total protein per lane were separated by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% non-fat milk (Bio-Rad) or 2% BSA at room temperature for 1 hr and then incubated overnight at 4 C with the following primary antibodies (Table 1). After being washed with PBST buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology) for 1 hr at room temperature. Signals were developed with enhanced chemiluminescence with a SuperSignal West Pico kit (Thermo-Fisher) and detected with an ImageQuant LAS 500 (GE Healthcare).

RNA isolation and quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was conducted accruing to the previously described protocol with some modifications. 42 Vascular organoids were washed with PBS, and total RNA was extracted using RNeasy Plus Mini Kit; (Qiagen) according to the kit protocol with the addition of RLT plus buffer supplemented with 20ul of 2 M Dithiothreitol (DTT). RNA was analyzed for quality and quantified using a NanoDrop One (Thermo Fisher Scientific) and reverse transcribed to cDNA using Maxima TM H Minus cDNA Synthesis Master Mix, with dsDNase (M1682; Thermo Fisher Scientific), according to the manufacturer's protocol in SimpliAmp Thermal Cycler; (Life Technology, MA, USA). Gene expression analysis was performed using Power SYBR Green Master Mix (Thermo Fisher Scientific) with the following mouse-specific primers: (Table 2) in a Quant Studio 3 RT-PCR system (Applied Biosystems, CA, USA). The relative expression values of target genes were normalized to Cyclophilin as the housekeeping gene, and the fold change was calculated using the relative quantification (2 ÀDDCT ) method. Four biological replicates per treatment group were run in three technical replicates.

Immunofluorescence staining analysis
HRP cells and vascular organoids (VO) were cultured as described above. At the experiments' endpoints, the cells and VO samples were cryo-sectioned or flat-mounted, fixed in 4% paraformaldehyde (VVR Life Science), permeabilized by incubation in 0.05% Triton X-100, and blocked with 10% normal goat serum (NGS). The fixed samples were then incubated with the primary antibodies (Table 1). After washing with PBST buffer, the staining signals were visualized by goat anti-rabbit IgG (H þ L) Cyanine5, goat anti-mouse IgG (H þ L) Alexa Fluor 488, and goat anti-rat IgG (H þ L) pacific blue. The non-specific staining and background tissue autofluorescence were established by incubating the sections of vascular organoids with secondary antibodies alone ( Figure S1). The cell nuclei were stained with DAPI (1:5,000). The slides were mounted with a ProLong Diamond antifade reagent (Thermo-Fisher) and imaged with an LSM 700 inverted laser confocal microscope (Carl Zeiss, Oberkochen, Germany).

TUNEL assay for detection of apoptotic nuclei
The terminal dUTP nick-end labeling (TUNEL) assay was performed according to previously described procedures with some modifications using the commercial kit (ApopTAG Red In Situ Apoptosis Detection Kit; Millipore, Temecula, CA). 6 In brief, the cells were fixed in 1% paraformaldehyde for 10 minutes at room temperature and then washed twice for 5 minutes in PBS (pH7.4). After the tailing of digoxigenin-dNTP catalyzed by the TdT enzyme, the sections were incubated with the anti-digoxigenin-rhodamine antibody for 30 minutes at room temperature. The processed specimen was mounted with the antifade mounting medium for fluorescence-containing DAPI (Vectashield; Vector, Burlingame, CA) and viewed fluorescence microscope.
Cytotoxicity assay of the HRP cell cultures HRP cells were cultured in 96 well plates with normal glucose (5 mM D-glucose þ 20 mM mannitol) and high glucose (25 mM D-glucose) with or without anti-VEGFR1 antibody (50 mg/ml) for 5 days. MTT assay was performed to quantitively evaluate the cell viability caused by high glucose and anti-VEGFR1 ab. The resultant absorbances were read at 570 nm using an 800 TS Absorbance Reader (BioTek Instruments, Winooski, VT, USA).

Colocalization analysis of the double-stained vascular organoids
The vascular organoids treated IgG control, and PlGF ab was immuno-stained with the primary antibodies against EC marker CD31 and pericyte marker PDGFRb and the secondary goat anti-rabbit IgG (H þ L) Cyanine5, goat antimouse IgG (H þ L) Alexa Fluor 488. Immunofluorescence images were taken with EVOS M7000 fluorescent microscopy. ImageJ software analyzed the colocalization correlation between green and red signals by the JACoP (Just Another Co-localization Plugin) plugin. Briefly, after being imported into ImageJ software, the two channels were converted to 8bit for analysis. Then, the JACoP plugin was used for colocalization analysis between two channels (green and red), followed by an online document  35 In summary, iPS cells were differentiated into vascular organoids and treated for 3 weeks with a diabetic media containing 75 mM Glucose, 1 ng/mL TNF-a, 1 ng/mL IL6 (DI) or were untreated in 17 mM Glucose (NG). The endothelial cells were sorted by FACS using CD31 as the marker. CD31 and sorted cells were stored at 80 C until use. The two NG (Normal Glucose) and 2 DI (Diabetes Induced) are pools of sorted endothelial cells from multiple vascular organoids (>100) from 2 independent differentiations/ treatments. Raw reads were mapped to the genome (hg19) using HISAT2 0.1.6. 43 Only reads with unique mapping were considered for further analysis. Gene expression levels were calculated using the HISAT2 software package (http:// daehwankimlab.github.io/hisat2/). Normalization and differential expression analysis were done using the DESeq2 R package (Bioconductor, https://bioconductor.org/packages/ release/bioc/html/DESeq2.html). Differentially expressed genes were selected using a twofold change cut-off between at least two populations, and p < 0.05 was adjusted for multiple gene testing. The Gene expression matrix was clustered using a k-means algorithm with correlation as the distance metric.

Statistical analysis
The values were expressed as the mean ± standard deviation (SD) for the respective groups. Statistical analyses were Table 2. A list of mouse-specific primers used in the study.

Gene
Forward/reverse Primer sequence Reference sequence performed with GraphPad Prism 8 software. Analysis of variance (ANOVA) or a linear mixed model was used for the statistical comparisons of multiple groups. 44 The p-values were adjusted for multiple comparisons with Dunnett's test. The non-parametric Mann-Whitney U test was performed to determine the significance level between the two groups. Statistical significance was set at p < 0.05.

Results
PlGF blockade regulates ang-1 and tie-2 in HREC-HRP co-cultures in an antibody concentrationdependent manner To investigate whether PlGF is involved in pericyte-endothelial cross-talks, we first examined the PlGF level in HREC culture and HREC-HRP co-culture media. The PlGF level was 32.6 ± 2.8 pg/ml in the HREC cultures and 46.5 ± 3.7 pg/ ml in the co-cultures. Next, we investigated the effect of PlGF blockade on the expression levels of Ang-1, Tie-2, VE-Cadherin, and N-Cadherin proteins in the HREC-HRP cocultures. The confluent co-cultures were treated with the neutralizing PlGF antibody (PL5D11D4) at three concentrations (25, 50, and 100 mg/ml) for two days. WB analysis was performed to examine the protein expression changes. WB barely detected Tie-2 protein expression in the vehicle control samples (Figure 1(A)). The PlGF antibody at the dose of 25 mg/ml slightly increased the Tie-2 protein level compared with vehicle control. 50 and 100 mg/ml antibody doses up-regulated the Tie-2 protein expression compared with vehicle control and 25 mg/ml antibody. WB detected N-Cadherin, VE-Cadherin, and Ang-1 protein expressions in all four groups (Figure 1(A,B)). 25 mg/ml PlGF antibody decreased the protein abundance of N-Cadherin (N-Cad) and Ang-1 but not VE-Cadherin (VE-Cad) compared with vehicle control. 50 and 100 mg/ml PlGF antibody concentrations up-regulated protein levels of Ang-1 but not N-Cad or VE-Cad compared with control and 25 mg/ml antibody. The protein blots were quantified to determine whether there were significant differences between treatment groups. The quantitative results (Figure 1(C,D)) revealed no significant differences for Tie-2 between vehicle control and 25 mg/ml antibody, between 50 and 100 mg/ml PlGF antibody. However, the two higher antibody doses (50 and 100 mg/ml) significantly up-regulated Tie-2 protein expression compared with the lower antibody dose (25 mg/ml) and control conditions. 25 mg/ml PlGF antibody downregulated N-cadherin compared with vehicle control. There were no significant differences for VE-cadherin between any groups. 25 mg/ml antibody downregulated, but the two higher doses up-regulated Ang-1 compared with vehicle control. These results indicate that PlGF regulates the protein expressions of Ang- 1 and Tie-2 but not N-Cad or VE-Cad depending on antibody concentration.

Ang-1 upregulation by PlGF blockade in HREC-HRP cocultures, but not monocultures
It is well established that Ang-1 is secreted by the peri-endothelial cells, such as pericytes, and regulates Tie-2 signaling activity in endothelial cells in a paracrine manner. 45 Therefore, we examined whether the upregulation of Ang-1 protein expression by PlGF blockade requires HREC-HRP cross-talks. The confluent HREC, HRP mono-and co-cultures were treated with 50 mg/ml PlGF antibody for two days (the effective dose from above). The western blots detected Ang-1 protein expression in all three cultures: HRP monoculture, HREC monoculture, and HRP-HREC co-cultures ( Figure 2(A-C)). PlGF antibody up-regulated Ang-1 protein expression in the HRP-HREC co-cultures but not the two monocultures compared to vehicle control. Densitometry analysis of protein blots confirmed a significant difference between the PlGF antibody and IgG control in the co-cultures but not the monocultures (Figure 2(D-F)). We also examined the expression of the Ang-2 protein (a partial agonist/antagonist of Tie-2) and the effect of PlGF inhibition in the three culture systems. Ang-2 expression was detected in the HRP-HREC co-cultures and HRP monocultures. However, WB failed to detect Ang-2 expression in HREC monocultures. Because that ECs could express and store Ang-2, 46 we should be cautious about interpreting this result, possibly caused by the expression level of Ang-2 that is not enough for the immunoblotting to detect in the cultured HRECs. Compared with vehicle control, WB and densitometry analyses revealed that PlGF inhibition does not alter Ang-2 protein expression in all three culture systems.
These results indicate that Ang-1 upregulation by PlGF blockade is dependent on EC-pericytes cross-talk, and the relative ratio of Ang-1 to Ang-2 was increased due to the Ang-1 upregulation and Ang-2 non-alteration, which may contribute to the beneficiary effect of PlGF blockade on EC integrity and function, as we reported previously. 36

VEGFR1 is involved in PlGF's effect on ang-1 and tie-2 expression and EC barrier function
PlGF confers function (i) directly through VEGFR1, (ii) through heterodimerizing with VEGF-A and activation of VEGFR1 and R2, or (iii) through replacing VEGF-A from VEGFR1 to VEGFR2, then indirectly activating VEGF-A/ VEGFR2 signaling. We asked what mechanism through which PlGF regulates Tie-2 and Ang-1 gene expression and EC barrier function. First, we found that VEGFR1 inhibition reduced the increased Tie-2 and Ang-1 protein levels caused by PlGF blockade compared to the vehicle control, suggesting that VEGFR1 is involved in the expression regulation ( Figure 3(A,B)). VEGFR1 inhibition also reduced EC barrier function, as indicated by reduced resistance (Figure 3(C)). Since VEGFR1 inhibition antagonizes the effects of PlGF blockade, it is unlikely that PlGF exerts this function directly via VEGFR1 or heterodimerizing with VEGF-A and activating both VEGFR1 and R2 receptors. Therefore, it is

VEGFR1 inhibition reduces pericyte viability in highglucose conditions
We investigated whether VEGFR1 mediates pericytes viability in cell cultures in high glucose (HG, diabetes-like) conditions. HG treatment reduced viability, as shown by LDH and MTT results (Figure 4(A,B)). Apoptotic cells were significantly increased in HG þ VEGFR1 inhibition conditions than in normal glucose control and HG condition ( Figure  4(C)), indicating VEGFR1's involvement in pericyte survival. Additionally, by using an anti-phospho-(p)VEGFR1 antibody, we found that the phosphorylated or activated VEGFR1 form was co-localized with the apoptotic cells (either Caspase 3 þ or TUNEL þ ) induced by HG (Figure 4(D-F)).

iPSC-derived vascular organoids
Blood vessels or vascular organoids have recently been created from human ESC and iPSC as an appealing model for diabetic vasculopathy. 35,39 We have successfully generated 3dimensional (3D)-VO from human iPSC, which are structurally and functionally similar to human vasculatures and contain the key vascular components, including CD 31 (þ) EC-formed vessel lumen structures, the associated PDGFRb (þ) pericytes, and the Col IV (þ) basements deposits ( Figure 5(A-C)). 47 The results of whole mount stainings were further confirmed in immunostaining in cryosections ( Figure S1). The 3D reconstruction of CD31 staining deep z-stack images has revealed complex vascular meshwork within the organoid (Figure 5(D)).

Upregulation of PlGF and ang-2 by the diabetesmimicking condition in vascular organoids
Using the RNA sequencing data of the VO treated with diabetes-like conditions versus normal medium available from the public database, 35 we performed bioinformatics data analysis. The results revealed that both PlGF and Ang-2 were significantly up-regulated in the diabetes-mimicking treatment group compared to the control group ( Figure  6(A-C)). Then, we examined their expression in the VO using double immunofluorescence staining. The results revealed that PlGF and Ang-2 were expressed in perivascular cells associated with CD31 þ ECs ( Figure 6(D-I)). The double stain of PlGF and Ang-2 showed the two proteins had similar but not identical expression patterns (Figure 6(J-L)), indicative of heterogenous peri-vascular cell types, such as pericytes, smooth muscle cells, and others.

PlGF blockade up-regulates ang-1 and tie-2 in human iPSC-derived vascular organoids
We further examined whether the PlGF blockade could upregulate Ang-1 and Tie-2 in human iPSC-derived vascular organoids; as PlGF antibody at 50 mg/ml for 2 days was effective in the human EC-pericyte co-cultures, we, therefore, doubled the antibody amount and treatment duration (100 mg/ml, 4 days) for the vascular organoids. The treated vascular organoids were cryosectioned for the double immunofluorescence staining analysis of Tie-2 and Ang-1. The results showed the increased staining signal intensity in the PlGF ab treatment group compared with the IgG control ( Figure 7(A,B)). The quantification of staining intensity revealed the increased Ang-1 and Tie-2 expression levels by PlGF ab treatment compared to the vehicle control (Figure 7(C)). qPCR further confirmed that the mRNA transcripts of Ang-2 and Tie-2 were up-regulated in the antibody treatment relative to the control (Figure 7(D)).

PlGF blockade promotes pericyte coverage and ECpericyte association in vascular organoids
Finally, we investigated whether PlGF blockade promotes pericyte coverage and pericyte-EC association in vascular organoids, which was evaluated by the expression levels and correlation coefficiency of the PDGFRb's staining signal Figure 3. VEGFR1 inhibition diminishes PlGF's effect on Ang-1 and Tie-2 expression in the HREC-HRP co-culture. HREC and HRP were co-cultured and treated with three groups: IgG control, PlGF antibody (ab) (50 mg/ml), and PlGF ab (50 mg/ml) þ VEGFR1 ab (50 mg/ml). The western blots (A) and densitometry quantification results (B). GAPDH was used as a protein loading control. (C) Resistance results were measured with the ECIS system. The results were expressed as a percentage relative to the control (mean ± SD, n ¼ 3 for WB, n ¼ 4 for resistance). Ã p < 0.05. ÃÃ p < 0.01.
relative to the CD31's. The two-channel images have been calculated for relative expression and colocalization: the green channel for pericytes and the red channel for ECs ( Figure 8(A-C), Figure S3). We used Image J with the JACop plugin to calculate Manders' overlap coefficient, which indicates the co-relations of the green (M1) and the . VEGFR1 inhibition reduces pericyte viability in high-glucose conditions. The pericytes were cultured with normal glucose (NG), high glucose (HG), and HG þ varying VEGFR1 antibody concentrations (10,20,50, and 100 mg/ml). The cell lysates were used for cell viability assay with MTT (A) and the supernatant for LDH assay (B). The results were expressed relative cell viability to NG (mean ± SD, n ¼ 6). (C and D) The TUNEL (þ) apoptotic cells were counted for NG, HG, and HG þ 50 mg/ml VEGFR1 antibody. # p < 0.05 (compared to normal glucose). Ã p < 0.05 (compared to high glucose). (E, F) Double immunofluorescent labeling was performed for: pVEGFR1 and TUNEL (E), activated Caspase 3 (a-Casp3), and pVEGFR1 (F). Scale bar: 50 mm. Note that pVEGFR1 staining signals were co-localized with the TUNEL(þ) and a-Casp3 (þ) apoptotic cells. The colocalization of TUNEL and DAPI was demonstrated with individual channels in Figure S4. red (M2). The M1 value for the green channel is 0.59 ± 0.5, and the M2 value for the red channel is 0.99 ± 0.0006 in control. The M1 is 0.89 ± 0.13, and the M2 is 0.99 ± 0.0017 in PlGF-ab treated group (Figure 8(D)). As indicated by the cytofluorogram (Figure 8(E,F)), PlGF ab treatment caused an increased Pearson's correlation coefficient of green signals versus red signals, which indicates that the PlGF blockade leads to increased coverage and colocalization of pericytes and ECs on organoid vasculatures.

Discussion
Primary findings described in this study include 1) PlGF blockade up-regulates Tie-2 and Ang-2 expression in both HREC-HRP co-culture and human iPSC-derived vascular organoids, 2) VEGFR1 is involved in PlGF's regulation of Tie-2 and Ang-2 expression, 3) VEGFR1 inhibition reduces pericyte cell viability in high glucose condition, 4) PlGF and Ang-2 are expressed in perivascular cells and up-regulated by diabetes-mimicking conditions in the VO, and 5) PlGF blockade promotes pericyte coverage and association with ECs in the VO. This study is the first to illustrate the direct regulation of the two signaling axis in human retinal cells and VO vasculatures. The data highlight PlGF's regulation of Ang-1 and Tie-2 in human cells and vascular organoids. The significance of the two signaling pathway regulations is relevant to stabilizing the endothelium facilitating vascular function by enhancing pericyte coverage and association with ECs.
Although angiopoietins are thought to directly affect cell adhesion by interacting with integrins, the expression of N-Cadherin in this study was not altered by the PlGF blockade. Alternative and compensatory regulatory mechanisms may be responsible for this observation which is in line with previous reports on the same. 48 PlGF inhibition by the varying antibody concentrations affects the regulation of Ang-1 and Tie-2 gene expression. Inhibition of PIGF at antibody doses of 50 and 100 mg/ml led to increased expression of Ang-1 and Tie-2, which signaling axis is important for ECpericyte cross-talk and vascular stability. Lower antibody inhibition (i.e. 25 mg/ml) of PIGF reduces N-Cadherin expression. N-Cadherin is highly expressed in mesenchymal cells, thus a marker of the EC to mesenchymal transition (EndMT) 49,50 and epithelial-to-mesenchymal (EMT). 51,52 Therefore, this result suggests that PlGF inhibition at a low level might be involved in reducing EndMT. [49][50][51][52] The difference between PlGF antibodies at low and high doses might reflect that the degree of PlGF inhibition has a different effect on endothelial cell function: EndMT reduction requires less N-Cadherin and Ang-1, while vascular stability needs more Ang-1 and Tie-2. The observation that upregulation of Ang-1 occurred only in HREC-HRP co-culture but not monoculture suggests that a positive feedback loop may exist between pericytes and ECs. For example, the secreted pericyte Ang-1 activates Tie-2 in the ECs; subsequently, the activated Tie-2 triggers the signaling cascades in the ECs that up-regulate Ang-1 expression in the pericytes. These findings agree with our previous in-vivo study showing that Pgf gene knockout prevents diabetes-caused pericyte loss and up-regulates Ang-1 expression in the retina using diabetic PlGF knockout (Akita.PlGF -/-) mice. 7 Despite Ang-2 (the antagonist of Ang-1) not being changed in our experimental settings (50 mg/ml PlGF dose and 1:1 ratio of EC/pericyte), we could not rule out that PlGF inhibition regulates Ang-2 expression under other experimental conditions.
To further confirm the regulatory mechanism observed in 2 D co-cultures of human retinal EC and pericytes, we exploited 3D vascular organoid cultures derived from human iPSC. As shown in Figure 5, vascular organoids contain critical vascular components similar to human vasculatures, including the lumen-forming ECs, the closely associated pericytes, and the deposited basements, similar to those described by Wimmer et al. 35,47 Vascular organoids are physiologically similar to the in-vivo vascular organs more than the traditional 2 D cell cultures, representing a new attractive model system to study vascular physiology and screen new drugs for vasculopathy. Moreover, iPSC can be derived from human patients, which renders the patientderived vascular organoids (PDVO) powerful tools to mimic Figure 7. PlGF blockade up-regulates Ang-1 and Tie-2 in human iPSC-derived vascular organoids. Vascular organoids were generated from human iPSC as described in the methods. 100 mg/ml PlGF antibody was supplemented into the culture medium and incubated for 4 days. IgG was used as a control. 10-micron cryopreserved sections were made for immunofluorescent staining with anti-Ang-1 and anti-Tie-2 antibodies. The secondary antibodies with Alex Fluor 647 (infrared) and 405 (blue) were used for visualization and microphotography with EVOS M7000 fluorescent microscope (A, B). Scale bar: 75 mm. (C) Quantification results of the fluorescent images measured with ImageJ software (n ¼ 6). ÃÃÃ p < 0.0001. The fluorescent images from each channel were made with the same exposure time for both control and antibody treatment conditions to minimize the variations caused by the fluorescent imaging process. The expression levels were measured based on the mean pixel intensity per image (6 images total). The two additional example images used for quantification were shown in Suppl. Figure 4. Note that Ang-1 had a peri-vascular (pericyte) expression pattern similar to PlGF and Ang2. (D) qRT-PCR results of Tie-2 and Ang-1 mRNA transcripts. The values represent the change folds relative to the control (n ¼ 6). ÃÃÃ p < 0.0001. vascular diseases, screen drugs, and design personalized or precision medicine to treat vascular diseases, such as cardiovascular diseases, diabetic complications, and ischemic stroke. We used human iPSC-derived vascular organoids in this study to evaluate the pericyte-endothelial cross-talk by PlGF and Ang signaling pathways. Furthermore, we performed transcriptome bioinformatic analysis for the RNA sequencing data from the blood vessel organoids treated with diabetic conditions (high glucose þ TNF-alpha þ IL-6) versus vehicle control. 35 The results revealed that both PlGF and Ang-2 are significantly up-regulated in the blood vessel organoids by diabetic conditions.
It is well documented that Ang mediates its effects via Tie-2, maintaining quiescence in the adult vascular endothelium 30 and that VEGF is the main angiogenic cue molecule. Given the well-known limitations of the current mono anti-VEGF treatments, alternative treatment targets and treatment approaches have been on the agenda of many preclinical and clinical research groups. An example is combinational treatments, which involve targeting at once two or more molecules involved in the pathophysiology of a condition. In line with this, ongoing combinational therapy trials modulate both Ang-2 and the VEGF signaling to generate healthy, properly formed neo-vessels rather than pathological leaky neovessels. 53,54 Also, modulation of PlGF and Ang (Ang-1 and Ang-2) is an attractive therapeutic target for treating DR and DME. 55,56 Given the importance of Ang-1 in the pathophysiology of DR, our results provide novel and important insight into the Ang-1 signaling axis involving PlGF-VEGFR1 signaling. Interestingly, In support of the combinational treatment strategy and in support of the idea of therapeutically targeting PIGF, two large-scale phases 3 clinical trials (NCT03622580 and NCT03622593) were carried out to evaluate the bispecific molecule (Faricimab), which targets VEGF-A and Ang-2, for the treatment of wet age-related macular degeneration (AMD) and DME, in comparison to Aflibercept which targets VEGF-A and PlGF. These trials lead to the recent approval of Faricimab for the treatment of nvAMD and DME in the US. 57 In line with the importance of PIGF signaling, two phase-2 clinical trials (NCT03071068 and NCT03499223) have completed the safety and efficacy of a humanized PlGF antibody (THR-317) in the treatment of DR and DME. 36,37 However, no significant improvement was observed in Best Corrected Visual Acuity (BCVA) with the combination therapy THR-317 and ranibizumab compared to ranibizumab monotherapy in the overall study population. The combination therapy showed improvement in patients with poor or no response to prior anti-VEGF agents and patients with baseline BCVA 6 letters. 58 However, the funding of further clinical trials of THR-317 was discontinued in 2019, as the company chose to focus resources on other DR and DME therapeutic agents.
This study investigated PlGF's regulation of the Ang-1 and Ang-2 expression in the HREC-HRP co-cultures and the vascular organoids derived from human iPSC. Herein, we provided evidence suggesting that PlGF blockade enhances EC-pericyte cross-talk by controlling the Ang-1/Tie-2 signaling axis. VEGFR1 acts as one downstream molecule of PlGF signaling to mediate cell viability and Ang-1 expression in the pericyte. 59 This study includes several limitations.
1. We characterized the levels of PlGF and PlGF-VEGF dimers in the supernatants of HREC cultures 36 but did not characterize their levels in HREC-HRP co-cultures and vascular organoids. 2. We evaluated the levels of Ang1 and Ang2 in cell lysates, but not the secreted proteins level is needed as low intracellular levels may be explained by increased secretion of the protein. 3. We only tested the effect of VEGFR1 inhibition, but not the effect of anti-PlGF Ab (PL5D11D4) or a combination of both Abs on the Ang2 and Tie2 levels. 4. We were unable to perform the immunoprecipitation or proximity ligation assay to confirm the physical interaction of PlGF and Ang-1.

Conclusion
This study provides new evidence for the association of PlGF and angiopoietin in human retinal EC-pericyte co-cultures and iPSC-derived vascular organoids. The outcomes are relevant to DR and other vascular pathophysiology. Further investigations are needed to elucidate the regulatory mechanisms further and promote translational potentials by targeting PlGF and Ang-1 signaling pathways.