Search

PurposePlacental 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.MethodsIn 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.ResultsInhibition 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.ConclusionCombined, these results suggest PlGFs 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.

Visual impairment from corneal stromal disease affects millions worldwide. We describe a cell-free engineered corneal tissue, bioengineered porcine construct, double crosslinked (BPCDX) and a minimally invasive surgical method for its implantation. In a pilot feasibility study in India and Iran (clinicaltrials.gov no.NCT04653922), we implanted BPCDX in 20 advanced kera- toconus subjects to reshape the native corneal stroma without removing existing tissue or using sutures. During 24 months of follow-up, no adverse event was observed. We document improvements in corneal thickness (mean increase of 209 +/- 18 mu m in India, 285 +/- 99 um in Iran), maximum keratometry (mean decrease of 13.9 +/- 7.9 D in India and 11.2 +/- 8.9 D in Iran) and visual acuity (to a mean contact-lens-corrected acuity of 20/26 in India and spectacle-corrected acuity of 20/58 in Iran). Fourteen of 14 initially blind subjects had a final mean best-corrected vision (spectacle or contact lens) of 20/36 and restored tolerance to contact lens wear. This work demonstrates restoration of vision using an approach that is potentially equally effective, safer, simpler and more broadly available than donor cornea transplantation., Funding Agencies|CareGroup India; European Union [667400]; LinkoCare Life Sciences AB; Linkoping University [LIO-609051]; Region Ostergotland in Sweden

Our previous studies found that the C-X-C motif chemokine receptor 5 (CXCR5) loss leads to retinal pigment epithelium (RPE) dysfunction and AMD pathogenesis. The current study aimed to characterize the G protein-coupled receptor (GPCR) structure of CXCR5 and analyze its interactions with AMD-related risk genes. The sequence alignments, homology model of CXCR5 and structural assessment analysis were performed. Data and text mining were then performed to identify AMD-related risk genes and their interaction with CXCR5 using statistical and mathematical algorithms. Sequence alignment and phylogenetic tree analysis revealed that human CXCR5 was highly similar (85.4839%) to the rabbit. The least similarity (33.871%) was found to be in zebrafish compared to the other species. The CXCR5 model structural assessment and secondary structure analysis exhibited an excellent model. Network analysis revealed that IL10, TNF, ICAM1, CXCL1, CXCL8, APP, TLR4, SELL, C3, IL17A and CCR2 were the most connected genes CXCR5. These findings suggest that CXCR5 signaling may regulate the biological function of RPE and modulate AMD pathophysiology via GPCR signaling and interacting with identified AMD risk genes. In summary, the data presented here provide novel and crucial insights into the molecular mechanisms of CXCR5 involvement in AMD. Communicated by Ramaswamy H. Sarma

Purpose: To evaluate Nuclear Factor NF-kappa B (NF-kappa B) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). Methods: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 mu m, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 mu g) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1(gfp/wt) mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. Results: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-I kappa B alpha, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1(wt/gfp) mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-kappa B activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. Conclusion: The present data indicate that NF-kappa B activation in microglia and its migration capacity is, Funding Agencies|Northern Advances Center for Science & Technology (NOASTEC) Foundation [T-1-14]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01 EY027824]; Missouri University

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population. In our previous studies, we found that deficiency of CXCR5 causes AMD-like pathological phenotypes in mice, characterized by abnormalities and dysfunction of the retinal pigment epithelium (RPE) cells. The abnormalities included abnormal cellular shape and impaired barrier function. In the present study, primary RPE cells were derived separately from CXCR5 knockout (KO) mice and from C57BL6 wild type (WT). The isolated primary cells were cultured for several days, and then total RNA was isolated and used for library preparation, sequencing, and the resultant raw data analyzed. Relative to the WT, a total of 1392 differentially expressed genes (DEG) were identified. Gene ontology analysis showed various biological processes, cellular components, and molecular functions were enriched. Pathway enrichment analysis revealed several pathways, including the PI3K-Akt signaling, mTOR signaling, FoxO, focal adhesion, endocytosis, ubiquitin-mediated proteolysis, TNF alpha-NF-kappa B Signaling, adipogenesis genes, p53 signaling, Ras, autophagy, epithelial-mesenchymal transition (EMT), and mitochondrial pathway. This study explores molecular signatures associated with deficiency of CXCR5 in RPE cells. Many of these signatures are important for homeostasis of this tissue. The identified pathways and genes require further evaluation to better understand the pathophysiology of AMD., Funding Agencies|University of Missouri; National Eye Institute (NEI), Bethesda, MD, USA [EY027824]

To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) hetemdimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF hetemdimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naive animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF hetemdimers, VEGF, PlGF, NF kappa B, p-I kappa B alpha, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-I kappa B alpha, and colocalization of NF kappa B p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, P1GF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pI kappa B alpha. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NF kappa B activation., Funding Agencies|National Eye Institute, USAUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Eye Institute (NEI) [R01 EY027824]; Missouri University, USA; Project Sviluppo di Approcci Terapeutici INnovativi per patologie neoplastiche resistenti ai trattamenti (SATIN)-POR Campania FESR 2014/2020, Italy

Placental growth factor (PlGF or PGF) is a member of the VEGF (vascular endothelial growth factor) family. It plays a pathological role in inflammation, vascular permeability, and pathological angiogenesis. The molecular signaling by which PlGF mediates its effects in non-proliferative diabetic retinopathy (DR) remains elusive. This study aims to characterize the transcriptome changes of human retinal endothelial cells (HRECs) with the presence and the absence of PlGF signaling. Primary HRECs were treated with the PlGF antibody (ab) to block its activity. The total RNA was isolated and subjected to deep sequencing to quantify the transcripts and their changes in both groups. We performed transcriptome-wide analysis, gene ontology, pathway enrichment, and gene-gene network analyses. The results showed that a total of 3760 genes were significantly differentially expressed and were categorized into cell adhesion molecules, cell junction proteins, chaperone, calcium-binding proteins, and membrane traffic proteins. Functional pathway analyses revealed that the TGF-beta pathway, pentose phosphate pathway, and cell adhesion pathway play pivotal roles in the blood-retina barrier and antioxidant defense system. Collectively, the data provide new insights into the molecular mechanisms of PlGFs biological functions in HRECs relevant to DR and diabetic macular edema (DME). The newly identified genes and pathways may act as disease markers and target molecules for therapeutic interventions for the patients with DR and DME refractory to the current anti-VEGF therapy., Funding Agencies|MU start-up funds; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [EY027824]