Dataset Information

Comparative proteomic study of healthy retinal vascular tree from human donor eyes and fibrovascular membrane of PDR patients

ABSTRACT: In the retinal vascular unit, direct communication between pericytes (PCs) and endothelial cells (ECs) is important for vessel stability and maintenance of the properties of the blood-retinal barrier (BRB). ECs and PCs sharing a common basement membrane throughout vasculogenesis tend to lose vessel integrity in the retina during Diabetic Retinopathy (DR). The proteins required to maintain BRB integrity and EC-PCs homeostasis may be altered or lost during the late angiogenic stage of DR. The selective dropout of microvascular cells during mild DR, later recruitment of unstable cells, and thereby retinal detachment in proliferative DR are still speculative; therefore, a comprehensive profiling of healthy retinal vasculature and fibrovascular membrane from the retina of proliferative diabetic retinopathy patients may provide clues for the seriousness of retinal detachment due to angiogenesis. This study aimed to isolate the retinal vasculature (RV) from a healthy human retina, making it more suitable for comparison with the microvascular cells of the fibrovascular membrane (FVM) from PDR patients, and to perform proteome profiling to understand the switch in BRB homeostasis due to DR. We first isolated the VT-NRC by removing the non-vascular retinal components, further the presence of microvascular cells in VT-NRC and FVM using IHC studies confirmed that the vascular components expressing both SMA and CD-34, were differentially expressed for Desmin, PDGFR- and CD-31 in a hefty proportion of FVM. Additionally, to confirm angiogenesis, both samples were subjected to ki67 expression which is significantly expressed in the FVM. Proteins were extracted, quantified, and fractionated using 1D-SDS-PAGE to analyze the proteomes of VT-NRC and FVM. After in-gel tryptic digestion, the peptides were analyzed in duplicate using LC–MS/MS on a tandem mass spectrometer. A total of 60 and 49 highly confident unique proteins were identified in VT-NRC and FVM, respectively, with 40 shared proteins. Similarly, using two-dimensional (2D) analysis, VT-NRC and FVM protein profiles were analyzed using MALDI-TOF. Out of ten differentially expressed spots, 6 and 4 highly confident proteins were identified in VT-NRC and FVM, respectively. GO analysis of the FVM protein revealed that a large percentage of the identified proteins had negative and positive regulation of angiogenesis, cell death, apoptosis, and cellular adhesion, being the most enriched molecular functions. Interaction network analysis using the SHINYGO 0.80 tool showed the involvement of SFN, YWHAZ, YWHAG, CFL1, CALM3, HRNR and PRKDC in ECM-receptor interaction, regulation of the actin skeleton, negative regulation of Angiogenesis and PI3 kinase pathway. SFN and HRNR mRNA levels were increased significantly in PBMC isolated from whole blood of DR patients compared with Type II Diabetic and Healthy control patient. Similarly, CFL1 and CALM3 mRNA levels were decreased. Controlling the expression of the differentially regulated proteins may prevent the retinal angiogenesis thereby fibrovascular membrane formation

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Eye, Blood Vessel Endothelial Cell

DISEASE(S): Diabetic Retinopathy

SUBMITTER: Sharmila Rajendran

LAB HEAD: Sharmila Rajendran

PROVIDER: PXD052211 | Pride | 2026-06-15

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	230915_2309S08_KV-01.group	Other
	230915_2309S08_KV-01.wiff	Wiff
	230915_2309S08_KV-01.wiff.scan	Wiff
	230915_2309S08_KV-01__FDR.xlsx	Xlsx
	230915_2309S08_KV-02.group	Other

Items per page:

1 - 5 of 14

Publications

Retinal vasculature-derived proteins serve as potential systemic biomarkers for diabetic retinopathy.

Sharmila Rajendran R Angayarkanni Narayanasamy N Ratra Dhanashree D Shanmuganathan Seetharaman S Lawrence D'Cruze D Purushothaman Kathiresan K Lin Qingsong Q Radha Annamalai A Kaviarasan Kuppan K

BMJ open ophthalmology 20260601 2

<h4>Purpose</h4>Fibrovascular membrane (FVM), a pathological tissue causing retinal traction, represents an advanced stage of proliferative diabetic retinopathy (PDR). This study aimed to identify novel protein biomarkers associated with retinal microvascular damage leading to FVM formation and to evaluate the diagnostic potential of stratifin (SFN) and hornerin (HRNR) in patients with type 2 diabetes mellitus (T2DM) and diabetic retinopathy (DR).<h4>Methods</h4>Human retinal vasculature from no ...[more]

PMID: 42225355

Similar Datasets

Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. As a central element of the retinal neurovascular unit, Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression. Here, we aim to improve our understanding of Müller cell-specific signalling pathways that are altered during DR disease progression in order to develop novel gene therapy strategies that target Müller cells. We used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) most highly expressed in Müller cells. In cells from diabetic mice, GR mRNA and protein expression is significantly reduced and its target gene cluster downregulated in Müller cells. Importantly, GR was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. In an in vitro cortisol-stimulated retinal explant model cortisol not only increased GR phosphorylation, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and we found moderate improvements indicated by electroretinography. While synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insight into the role of GR signalling and glial alterations in the diabetic retina. This supports the therapeutic concept of locally enhancing the GR signaling axis. Our findings of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligand.

Project description:Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment. Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment.