Deciphering the impact of aging on splenic endothelial cell heterogeneity and immunosenescence through single-cell RNA sequencing analysis.

BACKGROUND: Aging is associated with significant structural and functional changes in the spleen, leading to immunosenescence, yet the detailed effects on splenic vascular endothelial cells (ECs) and their immunomodulatory roles are not fully understood. In this study, a single-cell RNA (scRNA) atlas of EC transcriptomes from young and aged mouse spleens was constructed to reveal age-related molecular changes, including increased inflammation and reduced vascular development and also the potential interaction between splenic endothelial cells and immune cells. RESULTS: Ten clusters of splenic endothelial cells were identified. DEGs analysis across different EC clusters revealed the molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the loss in vascular development function of aged ECs. Notably, four EC clusters with immunological functions were identified, suggesting an Endothelial-to-Immune-like Cell Transition (EndICLT) potentially driven by aging. Pseudotime analysis of the Immunology4 cluster further indicated a possible aging-induced transitional state, potentially initiated by Ctss gene activation. Finally, the effects of aging on cell signaling communication between different EC clusters and immune cells were analyzed. CONCLUSIONS: This comprehensive atlas elucidates the complex interplay between ECs and immune cells in the aging spleen, offering new insights into endothelial heterogeneity, reprogramming, and the mechanisms of immunosenescence.

Single-Cell Analysis Reveals the Heterogeneity of Monocyte-Derived and Peripheral Type-2 Conventional Dendritic Cells.

Dendritic cells (DCs) are critical for pathogen recognition and Ag processing/presentation. Human monocyte-derived DCs (moDCs) have been extensively used in experimental studies and DC-based immunotherapy approaches. However, the extent of human moDC and peripheral DCs heterogeneity and their interrelationship remain elusive. In this study, we performed single-cell RNA sequencing of human moDCs and blood DCs. We identified seven subtypes within moDCs: five corresponded to type 2 conventional DCs (cDC2s), and the other two were CLEC10A+CD127+ cells with no resemblance to any peripheral DC subpopulations characterized to date. Moreover, we defined five similar subtypes in human cDC2s, revealed the potential differentiation trajectory among them, and unveiled the transcriptomic differences between moDCs and cDC2s. We further studied the transcriptomic changes of each moDC subtype during maturation, demonstrating SLAMF7 and IL15RA as maturation markers and CLEC10A and SIGLEC10 as markers for immature DCs. These findings will enable more accurate functional/developmental analyses of human cDC2s and moDCs.

PDGFD switches on stem cell endothelial commitment.

The critical factors regulating stem cell endothelial commitment and renewal remain not well understood. Here, using loss- and gain-of-function assays together with bioinformatic analysis and multiple model systems, we show that PDGFD is an essential factor that switches on endothelial commitment of embryonic stem cells (ESCs). PDGFD genetic deletion or knockdown inhibits ESC differentiation into EC lineage and increases ESC self-renewal, and PDGFD overexpression activates ESC differentiation towards ECs. RNA sequencing reveals a critical requirement of PDGFD for the expression of vascular-differentiation related genes in ESCs. Importantly, PDGFD genetic deletion or knockdown increases ESC self-renewal and decreases blood vessel densities in both embryonic and neonatal mice and in teratomas. Mechanistically, we reveal that PDGFD fulfills this function via the MAPK/ERK pathway. Our findings provide new insight of PDGFD as a novel regulator of ESC fate determination, and suggest therapeutic implications of modulating PDGFD activity in stem cell therapy.

VEGF-B is a potent antioxidant.

VEGF-B was discovered a long time ago. However, unlike VEGF-A, whose function has been extensively studied, the function of VEGF-B and the mechanisms involved still remain poorly understood. Notwithstanding, drugs that inhibit VEGF-B and other VEGF family members have been used to treat patients with neovascular diseases. It is therefore critical to have a better understanding of VEGF-B function and the underlying mechanisms. Here, using comprehensive methods and models, we have identified VEGF-B as a potent antioxidant. Loss of Vegf-b by gene deletion leads to retinal degeneration in mice, and treatment with VEGF-B rescues retinal cells from death in a retinitis pigmentosa model. Mechanistically, we demonstrate that VEGF-B up-regulates numerous key antioxidative genes, particularly, Gpx1 Loss of Gpx1 activity largely diminished the antioxidative effect of VEGF-B, demonstrating that Gpx1 is at least one of the critical downstream effectors of VEGF-B. In addition, we found that the antioxidant function of VEGF-B is mediated mainly by VEGFR1. Given that oxidative stress is a crucial factor in numerous human diseases, VEGF-B may have therapeutic value for the treatment of such diseases.

Critical role of caveolin-1 in ocular neovascularization and multitargeted antiangiogenic effects of cavtratin via JNK.

Ocular neovascularization is a devastating pathology of numerous ocular diseases and is a major cause of blindness. Caveolin-1 (Cav-1) plays important roles in the vascular system. However, little is known regarding its function and mechanisms in ocular neovascularization. Here, using comprehensive model systems and a cell permeable peptide of Cav-1, cavtratin, we show that Cav-1 is a critical player in ocular neovascularization. The genetic deletion of Cav-1 exacerbated and cavtratin administration inhibited choroidal and retinal neovascularization. Importantly, combined administration of cavtratin and anti-VEGF-A inhibited neovascularization more effectively than monotherapy, suggesting the existence of other pathways inhibited by cavtratin in addition to VEGF-A. Indeed, we found that cavtratin suppressed multiple critical components of pathological angiogenesis, including inflammation, permeability, PDGF-B and endothelial nitric oxide synthase expression (eNOS). Mechanistically, we show that cavtratin inhibits CNV and the survival and migration of microglia and macrophages via JNK. Together, our data demonstrate the unique advantages of cavtratin in antiangiogenic therapy to treat neovascular diseases.

Novel function of VEGF-B as an antioxidant and therapeutic implications.

Oxidative stress, due to insufficiency of antioxidants or over-production of oxidants, can lead to severe cell and tissue damage. Oxidative stress occurs constantly and has been shown to be involved in innumerable diseases, such as degenerative, cardiovascular, neurological, and metabolic disorders, cancer, and aging, thus highlighting the vital need of antioxidant defense mechanisms. Vascular endothelial growth factor B (VEGF-B) was discovered a long time ago, and is abundantly expressed in most types of cells and tissues. VEGF-B remained functionally mysterious for many years and later on has been shown to be minimally angiogenic. Recently, VEGF-B is reported to be a potent antioxidant by boosting the expression of key antioxidant enzymes. Thus, one major role of VEGF-B lies in safeguarding tissues and cells from oxidative stress-induced damage. VEGF-B may therefore have promising therapeutic utilities in treating oxidative stress-related diseases. In this review, we discuss the current knowledge on the newly discovered antioxidant function of VEGF-B and the related molecular mechanisms, particularly, in relationship to some oxidative stress-related diseases, such as retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, glaucoma, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

Novel multi-targeted inhibitors suppress ocular neovascularization by regulating unique gene sets.

Neovascular diseases, such as many cancers and ocular disorders, are life threatening and devastating. Although anti-vascular endothelial growth factor A (VEGF-A) therapy is available, many patients are not responsive and drug resistance can develop. To try to overcome these problems, combination therapy targeting VEGF-A and platelet-derived growth factor B (PDGF-B) was tested. However, one obvious drawback was that the other VEGF and PDGF family members were not inhibited and therefore could compensate. Indeed, this was, at least to some extent, demonstrated by the disappointing outcomes. To this end, we designed novel multi-targeted inhibitors that can block most of the VEGF and PDGF family members simultaneously by making a fusion protein containing the ligand-binding domains of vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor beta (PDGFRß), which can therefore act as a decoy blocker for most of the VEGF and PDGF family members. Indeed, in cultured cells, the novel inhibitors suppressed the migration and proliferation of both vascular endothelial cells and smooth muscle cells, and abolished VEGFR2 and PDGFRß activation. Importantly, in a choroidal neovascularization model in vivo, the novel inhibitor inhibited ocular neovascularization more efficiently than the mono-inhibitors against VEGFR or PDGFR alone respectively. Mechanistically, a genome-wide microarray analysis unveiled that the novel inhibitor regulated unique sets of genes that were not regulated by the mono-inhibitors, further demonstrating the functional uniqueness and superiority of the novel inhibitor. Together, we show that the multi-targeted inhibitors that can block VEGFR1, VEGFR2 and PDGFRß simultaneously suppress pathological angiogenesis more efficiently than monotherapy, and may therefore have promising therapeutic value for the treatment of neovascular diseases.

Nintedanib inhibits TGF-ß-induced myofibroblast transdifferentiation in human Tenon's fibroblasts.

Purpose: This study aimed to investigate the effect of nintedanib on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts and reveal the molecular mechanisms involved. Methods: Primary cultured HTFs were incubated with transforming growth factor ß1 (TGF-ß1) alone or combined with nintedanib, and cell proliferation and migration were measured by cell counting kit-8 (CCK8) and the scratch wound assay, respectively. HTF contractility was evaluated with a 3D collagen contraction assay. The mRNA and protein levels of α smooth muscle actin (α-SMA) and Snail and the phosphorylation levels of Smad2/3, p38 mitogen-activated protein kinase (p38MAPK), and extracellular signal-regulated kinase ½ (ERK1/2) were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunofluorescence staining. Results: Nintedanib inhibited the proliferation and migration of HTFs in a dose-dependent manner. Furthermore, nintedanib prevented HTF myofibroblast differentiation via downregulation of mRNA and protein expression of α-SMA and Snail. A three-dimensional (3D) collagen gel contraction assay demonstrated that nintedanib effectively inhibits myofibroblast contraction induced by TGF-ß1. Mechanistically, we revealed that nintedanib reduces the TGF-ß1-induced phosphorylation of Smad2/3, p38MAPK, and ERK1/2, suggesting that nintedanib acts through both classic and nonclassic signaling pathways of TGF-ß1 to prevent HTF activation. Conclusions: Our study provides new evidence that nintedanib has potent antifibrotic effects in HTFs and suggests that it may be used as a potential therapeutic agent for subconjunctival fibrosis.

CD169+ classical monocyte as an important participant in Graves' ophthalmopathy through CXCL12-CXCR4 axis.

Patients with Graves' disease (GD) can develop Graves' ophthalmopathy (GO), but the underlying pathological mechanisms driving this development remain unclear. In our study, which included patients with GD and GO, we utilized single-cell RNA sequencing (scRNA-seq) and multiplatform analyses to investigate CD169+ classical monocytes, which secrete proinflammatory cytokines and are expanded through activated interferon signaling. We found that CD169+ clas_mono was clinically significant in predicting GO progression and prognosis, and differentiated into CD169+ macrophages that promote inflammation, adipogenesis, and fibrosis. Our murine model of early-stage GO showed that CD169+ classical monocytes accumulated in orbital tissue via the Cxcl12-Cxcr4 axis. Further studies are needed to investigate whether targeting circulating monocytes and the Cxcl12-Cxcr4 axis could alleviate GO progression.

Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro.

PURPOSE: To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. METHODS: Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFßS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFßS, FN, and Snail1 were analyzed using reverse transcriptase-polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. RESULTS: PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFß1, TGFß2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFß1, and TGFß2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. CONCLUSIONS: PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity.

PDGF-D-induced immunoproteasome activation and cell-cell interactions.

Platelet-derived growth factor-D (PDGF-D) is abundantly expressed in ocular diseases. Yet, it remains unknown whether and how PDGF-D affects ocular cells or cell-cell interactions in the eye. In this study, using single-cell RNA sequencing (scRNA-seq) and a mouse model of PDGF-D overexpression in retinal pigment epithelial (RPE) cells, we found that PDGF-D overexpression markedly upregulated the key immunoproteasome genes, leading to increased antigen processing/presentation capacity of RPE cells. Also, more than 6.5-fold ligand-receptor pairs were found in the PDGF-D overexpressing RPE-choroid tissues, suggesting markedly increased cell-cell interactions. Moreover, in the PDGF-D-overexpressing tissues, a unique cell population with a transcriptomic profile of both stromal cells and antigen-presenting RPE cells was detected, suggesting PDGF-D-induced epithelial-mesenchymal transition of RPE cells. Importantly, administration of ONX-0914, an immunoproteasome inhibitor, suppressed choroidal neovascularization (CNV) in a mouse CNV model in vivo. Together, we show that overexpression of PDGF-D increased pro-angiogenic immunoproteasome activities, and inhibiting immunoproteasome pathway may have therapeutic value for the treatment of neovascular diseases.

Single-cell chromatin accessibility and transcriptomic characterization of Behcet's disease.

Behect's disease is a chronic vasculitis characterized by complex multi-organ immune aberrations. However, a comprehensive understanding of the gene-regulatory profile of peripheral autoimmunity and the diverse immune responses across distinct cell types in Behcet's disease (BD) is still lacking. Here, we present a multi-omic single-cell study of 424,817 cells in BD patients and non-BD individuals. This study maps chromatin accessibility and gene expression in the same biological samples, unraveling vast cellular heterogeneity. We identify widespread cell-type-specific, disease-associated active and pro-inflammatory immunity in both transcript and epigenomic aspects. Notably, integrative multi-omic analysis reveals putative TF regulators that might contribute to chromatin accessibility and gene expression in BD. Moreover, we predicted gene-regulatory networks within nominated TF activators, including AP-1, NF-kB, and ETS transcript factor families, which may regulate cellular interaction and govern inflammation. Our study illustrates the epigenetic and transcriptional landscape in BD peripheral blood and expands understanding of potential epigenomic immunopathology in this disease.

Single-cell analysis of immune cells on gingiva-derived mesenchymal stem cells in experimental autoimmune uveitis.

Gingiva-derived mesenchymal stem cells (GMSCs) have shown astonishing efficacy in the treatment of various autoimmune diseases. However, the mechanisms underlying these immunosuppressive properties remain poorly understood. Here, we generated a lymph node single-cell transcriptomic atlas of GMSC-treated experimental autoimmune uveitis mice. GMSC exerted profound rescue effects on T cells, B cells, dendritic cells, and monocytes. GMSCs rescued the proportion of T helper 17 (Th17) cells and increased the proportion of regulatory T cells. In addition to globally altered transcriptional factors (Fosb and Jund), we observed cell type-dependent gene regulation (e.g., Il17a and Rac1 in Th17 cells), highlighting the GMSCs' cell type-dependent immunomodulatory capacity. GMSCs strongly influenced the phenotypes of Th17 cells, suppressing the formation of the highly inflammatory CCR6-CCR2+ phenotype and enhancing the production of interleukin (IL) -10 in the CCR6+CCR2+ phenotype. Integration of the glucocorticoid-treated transcriptome suggests a more specific immunosuppressive effect of GMSCs on lymphocytes.

VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Insights gained from Single-Cell analysis of immune cells on Cyclosporine A treatment in autoimmune uveitis.

Cyclosporine A (CsA) is a widely known immunosuppressive agent that is clinically important in autoimmune diseases owing to its selective suppression of T lymphocytes. Although it has long been recognized to inhibit T cell responses by blocking calcineurin, the potential targets and specific downstream mechanisms remain elusive. Herein, we built a comprehensive single-cell transcriptomic landscape of immune cells in the blank, untreated experimental autoimmune uveitis (EAU), and CsA-treated EAU mice. CsA reversed EAU-associated changes in cell type composition, genomic expression, cell trajectory, and cell-cell communication. We found that CsA reverses the proportion change of disease-related immune cells; regulates several crucial pathogenic factors (eg. IL1r1, CD48, and Bhlhe40) in T helper 17 cells (Th17), the transcription factor Bhlhe40 was also rescued in T helper 1 cells (Th1); and may differentiate Tregs into a state of enhanced immunosuppression. In addition, we revealed the rescued impact of CsA on all immune cell types, especially on plasma B cells differentiation and immunoglobulin secretion. Furthermore, comparisons with glucocorticoids showed that CsA might have a more premium rescue effect involved in attenuating the pathogenicity of autoreactive T cells. Our work provides a comprehensive single-cell transcriptional atlas of immune cells under CsA therapy, providing advanced insights into the mechanisms underlying CsA and a reference for developing new therapeutic strategies for autoimmune diseases.

Corrigendum: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization.

[This corrects the article DOI: 10.3389/fcell.2021.686886.].

Pharmacokinetics of pirfenidone after topical administration in rabbit eye.

PURPOSE: Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a new, broad-spectrum agent that has an inhibition effect on the proliferation, migration, and collagen contraction of human Tenon's fibroblasts, and thus modulating the wound healing process of glaucoma filtering surgical site. This study investigated the pharmacokinetics of topically administered pirfenidone (0.5%) in rabbit eyes. METHODS: Pirfenidone solution (50 µl) was instilled into the rabbit's conjunctival sac. The rabbits were quickly sacrificed at 2, 5, 8, 10, 15, 20, 30, 60, 90, and 120 min after the administration and ocular tissues were obtained. The concentrations of pirfenidone in conjunctiva, sclera, cornea, aqueous humor, and vitreous were determined by high performance liquid chromatography. RESULTS: After topical administration, there was wide distribution and fast clearance of pirfenidone among the various ocular tissues. The mean maximum concentrations (C(max)) of pirfenidone in cornea, conjunctiva, sclera, aqueous humor, and vitreous were 9.64 mg/g, 9.62 mg/g, 2.13 mg/g, 34.88 mg/l and 0.52 mg/l, respectively. The half-life for these tissues was 18.26, 34.16, 15.71, 70.91, and 39.48 min, respectively. CONCLUSIONS: Measurable concentrations of pirfenidone are achieved in ocular tissues after topical application in rabbit model. Topical administration of pirfenidone may be an effective approach for modulation of wound healing responses in glaucoma filtration surgical site.

Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization.

Platelet-derived growth factor-D (PDGF-D) is highly expressed in immune cells. However, the potential role of PDGF-D in immune system remains thus far unclear. Here, we reveal a novel function of PDGF-D in activating both classical and alternative complement pathways that markedly increase chemokine and cytokine responses to promote macrophage polarization. Pharmacological targeting of the complement C3a receptor using SB290157 alleviated PDGF-D-induced neuroinflammation by blocking macrophage polarization and inhibited pathological choroidal neovascularization. Our study thus suggests that therapeutic strategies targeting both PDGF-D and the complement system may open up new possibilities for the treatment of neovascular diseases.

Mitogen-Inducible Gene 6 Inhibits Angiogenesis by Binding to SHC1 and Suppressing Its Phosphorylation.

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.