Distribution of Translation Efficiency Score (TES) across novel lncRNAs identified in this study and RefSeq genes. Box limits indicate the 25^th and 75^th percentiles as determined by R software; whiskers extend till 5^th and 95^th percentiles.

Differential expression analysis revealed 156 endothelial‐enriched lncRNAs with a fold change of at least 10 (closed circles) and 685 lncRNAs at 2‐fold (open circles).

UCSC browser snapshot of the zebrafish vascular endothelial‐associated lncRNA 2 (veal2) transcript. 5’ RACE and 3’ RACE data confirmed ends of the veal2 transcript.

Ribosomal pulldown shows lack of occupancy of ribosomes on veal2. fli1a and actb were used as positive controls.

e‐GFP fusion assay confirms lack of peptide formation from veal2 sequence. (F–G) mitfa‐eGFP fused transcript. (H–I) veal2‐eGFP fused transcript. Arrowheads indicating e‐GFP expression in mitfa‐eGFP‐injected embryos. Scale bar‐100μm.

Relative abundance analysis from different subcellular fractions revealed veal2 is a cytoplasmic lncRNA. Bar graph represents the relative abundance of veal2 and actb transcripts across different fractions of the cell. Data from three different experiments plotted as mean percentage values ± standard deviation.

Relative expression of veal2 across fluorescence‐activated cell sorted (FACS) GFP(+) endothelial cells (EC) and GFP(‐) non‐endothelial cells (NEC). fli1a and actb were taken as positive control and normalization control, respectively. Data from three different experiments represented as fold change relative to EC values ± standard deviation.

Whole‐mount in situ expression analysis of the veal2 transcript across different stages of zebrafish embryos. (LI,II) 1K cell stage, (LIII,IV) 10 hpf stage, and (LV,IV) 28 hpf stage. LI,III,V‐Anti‐sense‐veal2 probe. (LII,IV,VI) Sense‐veal2 probe. Magnification‐2.5X and scale bars‐100μm.

Expression of veal2 transcript (FPKM scores) across (M) 11 developmental stages and (N) all publically available RNA‐seq data of zebrafish’s different tissues or cell types compiled by ZFLNC database (Hu et al, 2018).

List of sequences showing indels at the veal2 locus at somatic level in F₀ zebrafish embryos injected with TALEN arms.

Schematic representation of outcross of veal2 heterozygous mutant veal2^gib005Δ8/+ and the chromatograms representing the two different types of alleles identified on genotyping of 20 embryos randomly.

Bar graph representing the number of animals which displayed the hemorrhage phenotype across the progeny derived from breeding of two control gib004Tg(fli1a:EGFP;gata1a:DsRed) zebrafish and progeny derived from an outcross of veal2^gib005Δ8/+ zebrafish. Data from three different experiments plotted as mean percentage values ± standard deviation.

Bar graph representing the number of animals which displayed the hemorrhage phenotype across the progeny derived from an outcross of veal2^gib005Δ8/+ zebrafish injected with veal2 RNA and vehicle control separately. Data from three different experiments plotted as mean percentage values ± standard deviation.

Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2^gib005Δ8/+ zebrafish upon complementing with the wild‐type (WT) veal2 RNA. (F–J) Control zebrafish embryos. (K–O) veal2^gib005Δ8/+ embryos injected with vehicle control. (P–T) veal2^gib005Δ8/+ embryos complemented with veal2 RNA. (F, K, P) Bright field. (G, L, Q) mRFP. (H, M, R) Animals stained with O‐dianisidine stain. (I, N, S) eGFP. (J, O, T) Merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F–H, K–M, P–R) Magnification‐5× and scale bar‐100 μm. (I–J, N–O, S–T) Magnification‐20× and scale bar‐50 μm.

RAP‐MS followed by Western blotting validated Prkcbb and Ephb3 as interacting protein partners with veal2.

RNA immunoprecipitation of veal2 was performed by pulldown of Prkcbb and Ephb3, followed by qRT–PCR. IgG pulldown was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.

qRT–PCR‐based quantification of veal2 transcript across Prkcbb, Ephb3, and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation.

Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various concentrations of the WT veal2 RNA, veal2‐Δ8 RNA, and veal2‐AS RNA. 52 nM of the PRKCB2 protein was used per reaction. Data from three different experiments plotted as mean fold change values ± standard deviation.

Enzastaurin treatment rescues hemorrhage phenotype in veal2^gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2^gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates, and a total no. of embryos scored are mentioned in figure. (F–I) Magnification‐5× and scale bar‐100 μm. (J–K) Magnification‐20× and scale bars‐50μm.

Relative number of animals that displayed the hemorrhage phenotype across the progeny of the outcross of veal2^gib005Δ8/+ zebrafish. Number of phenotypic animals upon treatment with Enzastaurin was normalized with the number of phenotypic animals when treated with DMSO. Data from three different experiments plotted as mean fold change values ± standard deviation.

Scatter plot showing the number of animals which displayed the hemorrhage phenotype across the progeny derived from an outcross of veal2^gib005Δ8/+ zebrafish injected with IVTs of different veal2 variants. Control indicates veal2^gib005Δ8/+ zebrafish without complementation. Data from three different experiments plotted as individual values; the middle bar represents mean percentage, and the error bar represents ± standard deviation.

The site of interaction of the motif‐3 of veal2 (CLM) with the Prkcbb protein indicates that it lies in a previously known DAG‐binding site. Both RNA (pink) and protein (purple) structures are shown as ribbon models. The distances between nucleotides of motif‐3 of veal2 and the 2 amino acids of Prkcbb are given in Å units.

Relative kinase activity of human PRKCB2 with various concentrations of DAG without or with different concentrations of veal2 WT RNA (0, 0.025, 0.25, 2.5, 25 nM). PRKCB2 activity without DAG was used for normalization. Data from three different experiments plotted as mean fold change values ± standard deviation.

Abundance of Prkcbb protein in total cell (T), cytoplasmic (C), and membrane (M) fractions of cells from 2 dpf gib004Tg(fli1a:EGFP;gata1a:DsRed) and veal2^gib005Δ8/+ zebrafish embryos. Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2^gib005Δ8/+ zebrafish embryos.

Relative quantification of Prkcbb localization in cytoplasmic and membrane fractions of control and veal2^gib005Δ8/+ embryos. Data from three different experiments plotted as mean percentage values ± standard deviation.

RIP‐seq of PRKCB in HUVECs identified a candidate lncRNA with significant q‐value (0.002) and high expression.

Representation of genomic location of AC008440.2‐human vascular endothelial‐associated lncRNA 2 (VEAL2) transcript. It is anti‐sense to 3’ UTR of known protein‐coding myeloid‐associated differentiation marker(MYADM) gene. 3’RACE analysis confirmed VEAL2 as an independent transcript. Reads from PRKCB‐RIP‐seq and IgG‐RIP‐seq mapping to VEAL2 loci are also given. Blue highlights reads mapping to +ve strand, and red highlights reads mapping to ‐ve strand.

Absolute quantification of VEAL2 in HUVECs revealed 0.024 copies per cell. 10² to 10⁸ copies of VEAL2 RNA were used to make the standard curve. Data from three different experiments plotted as mean values ± standard deviation.

e‐GFP fusion assay confirms lack of peptide formation from veal2 sequence. (E–F) mitfa‐eGFP fused transcript. (G–H) VEAL2‐eGFP fused transcript. Arrowheads indicating e‐GFP expression in mitfa‐eGFP‐injected embryos. Scale bar‐100 μm.

Complementation of VEAL2 in veal2^gib005Δ8/+ embryos significantly rescued hemorrhage phenotype. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I–L) Magnification‐5× and scale bar‐100 μm. (M–N) Magnification‐20× and scale bars‐50 μm.

Single molecule FISH (smFISH) of VEAL2 in HUVECs shows its cytoplasmic localization. (O) VEAL2 in CAL Fluor Red (610 nM). (P) DAPI. (Q) Merged image for VEAL2 and DAPI. Magnification‐100× and scale bar‐5 μm.

Co‐IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL Fluor Red (610 nM). (T) Merged image for PRKCB and VEAL2. Magnification‐100× and scale bar‐20 μm.

Bar graph represents the colocalization rate (%) of VEAL2 with PRKCB and CAMKIID proteins in HUVECs. Data from three different experiments plotted as individual values with mean percentage values ± standard deviation.

Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various concentrations of the WT VEAL2 RNA and VEAL2‐AS RNA. 52 nM of the PRKCB2 protein was used per reaction. Data from three different experiments plotted as mean fold change values ± standard deviation.

Bar graph representing relative fold change of VEAL2 expression in blood samples of patients with different diabetic stages with aggravating vascular dysfunctions from diabetic mellitus (DM) to non‐proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) compared to control patients. Data were collected from 50 different patients in each condition and represented as individual values with mean fold change ± standard deviation.

ROC curve shows sensitivity and specificity of VEAL2 as a diagnostic biomarker for proliferative diabetic retinopathy with endothelial dysfunction.

Complementation of VEAL2 and veal2 reverted increased permeability levels in the HUVEC monolayer model for hyperglycemia. Bar graph representing the effect of overexpression of VEAL2 and veal2 on permeability levels in hyperglycemia disease model, measured as efflux of dextran‐conjugated FITC. Data obtained from 3 different biological replicates and plotted as mean percentage fold change values ± standard deviation.

Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E–H, Q) CDH5 protein, (I–L, R) CTNNB1 protein, and (M–P, S) PRKCB protein. (E–P) Magnification‐60× and scale bar‐15 μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q–S) Data from cells of different fields of 3 technical replicates of 1 biological replicate are presented as representation. Data are shown as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation.

Representative images of morpholino‐injected zebrafish at 2 dpf under bright field and EGFP filter. (B, D, F) Embryos injected with scrambled morpholinos. (C, E, G) Embryos injected with the veal2 morpholino. veal2 knockdown induces sprouting defects (indicated by arrowheads). (B–E) 5× magnification. Scale bars represent 100 μm. (F–G) 20× magnification. Scale bars represent 50 μm.

Bar graph representing a number of animals displaying vascular sprouting defects in veal2 morpholino‐injected zebrafish at 2 dpf. Data from three different experiments plotted as mean percentage values ± standard deviation.

Representative images of morpholino‐injected 2 dpf zebrafish under bright field, mRFP filter and animals stained with O‐dianisidine. (I, K, M) Embryos injected with non‐targeting control (NTC) morpholino. (J, L, N) Embryos injected with the veal2 morpholino. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I–N) 5× magnification. Scale bars represent 100 μm.

Percentage of animals that showed vascular integrity defects at 2 dpf when injected with 3 nl of 500 μM scrambled morpholino, 500 μM veal2 morpholino and cocktail of 500 μM veal2 morpholino and 100 ng of veal2 RNA. Data from three different biological replicates represented as mean percentage ± standard deviation.

Gel represents the PCR‐amplified products using primers designed across the intron. The arrowhead indicates the product with retention of the intron due to the effect of morpholino.

Relative expression of veal2 across control and veal2 knockdown embryos. actb was taken as normalization control. Data from three different experiments represented as mean ΔΔC_T values normalized to EC values ± standard deviation.

The 3 bases of motif‐3 (CLM) in veal2 are highlighted in pink, and the base positions are mentioned. The 4 amino acids known to bind with DAG (Leonard et al, 2011) within C1 domain of Prkcbb have been highlighted.

Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various variants of veal2 lacking putative functional motifs and wt veal2 IVT RNA. Data from three different experiments plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation.

Dot plot representing wound closure rate at 0, 9, and 24 h post‐scratch in control cells and veal2‐overexpressed HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation.

Representative images showing wound closure rate in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer at 0, 9, and 24 h post‐scratch. Images taken at 10× magnification with scale bar representing 50 μm.

Dot plot representing wound closure rate at initial time, 9, and 24 h post‐scratch in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation.

smFISH of VEAL2 in HUVECs transfected with control siRNA and VEAL2 siRNA shows specificity of cytoplasmic signal of VEAL2. VEAL2 in CAL Fluor Red (610 nM). Merged image for VEAL2 and DAPI. Magnification‐100× and scale bar‐5 μm.

Dot plot representing endogenous kinase activity of human PRKCB2 in HUVECs under standard conditions and upon knockdown of VEAL2. Data from three different experiments are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation.

Retina scan of patients highlighting symptoms of retinopathy. (C) Fundus fluorescence angiography of patients showing vessel integrity defects. (D) Fundus photograph of a patient with symptoms of proliferative diabetic retinopathy and having subhyaloid hemorrhages. (E) Fundus photograph of a patient with symptoms of proliferative diabetic retinopathy with fibrovascular proliferation at disk and abnormal new blood vessels (NVE). (F) Optical coherence tomography of patients with diabetic macular edema.

Dot plot representing relative expression of VEAL2 (in fold change) in fibrous membrane isolated from control and PDR patients. Data obtained from 7 patients as biological replicates and represented as individual values with mean fold change values ± standard deviation.

Bar graph representing relative expression of VEAL2 in control of HUVECs and hyperglycemia stimulated HUVECs by growing under high glucose. Data are acquired from 3 different biological replicates and shown as individual values with mean fold change values ± standard deviation.