Single-cell transcriptomics analysis of proliferative diabetic retinopathy fibrovascular membranes reveals AEBP1 as fibrogenesis modulator.

The management of preretinal fibrovascular membranes, a devastating complication of advanced diabetic retinopathy (DR), remains challenging. We characterized the molecular profile of cell populations in these fibrovascular membranes to identify potentially new therapeutic targets. Preretinal fibrovascular membranes were surgically removed from patients and submitted for single-cell RNA-Seq (scRNA-Seq). Differential gene expression was implemented to define the transcriptomics profile of these cells and revealed the presence of endothelial, inflammatory, and stromal cells. Endothelial cell reclustering identified subclusters characterized by noncanonical transcriptomics profile and active angiogenesis. Deeper investigation of the inflammatory cells showed a subcluster of macrophages expressing proangiogenic cytokines, presumably contributing to angiogenesis. The stromal cell cluster included a pericyte-myofibroblast transdifferentiating subcluster, indicating the involvement of pericytes in fibrogenesis. Differentially expressed gene analysis showed that Adipocyte Enhancer-binding Protein 1, AEBP1, was significantly upregulated in myofibroblast clusters, suggesting that this molecule may have a role in transformation. Cell culture experiments with human retinal pericytes (HRP) in high-glucose condition confirmed the molecular transformation of pericytes toward myofibroblastic lineage. AEBP1 siRNA transfection in HRP reduced the expression of profibrotic markers in high glucose. In conclusion, AEBP1 signaling modulates pericyte-myofibroblast transformation, suggesting that targeting AEBP1 could prevent scar tissue formation in advanced DR.

Limited Hyperoxia-Induced Proliferative Retinopathy (LHIPR) as a Model of Retinal Fibrosis, Angiogenesis, and Inflammation.

The progression to fibrosis and traction in retinopathy of prematurity (ROP) and other ischemic retinopathies remains an important clinical and surgical challenge, necessitating a comprehensive understanding of its pathogenesis. Fibrosis is an unbalanced deposition of extracellular matrix components responsible for scar tissue formation with consequent tissue and organ impairment. Together with retinal traction, it is among the main causes of retinal detachment and vision loss. We capitalize on the Limited Hyperoxia Induced Retinopathy (LHIPR) model, as it reflects the more advanced pathological phenotypes seen in ROP and other ischemic retinopathies. To model LHIPR, we exposed wild-type C57Bl/6J mouse pups to 65% oxygen from P0 to P7. Then, the pups were returned to room air to recover until later endpoints. We performed histological and molecular analysis to evaluate fibrosis progression, angiogenesis, and inflammation at several time points, from 1.5 months to 9 months. In addition, we performed in vivo retinal imaging by optical coherence tomography (OCT) or OCT Angiography (OCTA) to follow the fibrovascular progression in vivo. Although the retinal morphology was relatively preserved, we found a progressive increase in preretinal fibrogenesis over time, up to 9 months of age. We also detected blood vessels in the preretinal space as well as an active inflammatory process, altogether mimicking advanced preretinal fibrovascular disease in humans.

SARA suppresses myofibroblast precursor transdifferentiation in fibrogenesis in a mouse model of scleroderma.

We previously reported that Smad anchor for receptor activation (SARA) plays a critical role in maintaining epithelial cell phenotype. Here, we show that SARA suppressed myofibroblast precursor transdifferentiation in a mouse model of scleroderma. Mice overexpressing SARA specifically in PDGFR-ß+ pericytes and pan-leukocytes (SARATg) developed significantly less skin fibrosis in response to bleomycin injection compared with wild-type littermates (SARAWT). Single-cell RNA-Seq analysis of skin PDGFR-ß+ cells implicated pericyte subsets assuming myofibroblast characteristics under fibrotic stimuli, and SARA overexpression blocked the transition. In addition, a cluster that expresses molecules associated with Th2 cells and macrophage activation was enriched in SARAWT mice, but not in SARATg mice, after bleomycin treatment. Th2-specific Il-31 expression was increased in skin of the bleomycin-treated SARAWT mice and patients with scleroderma (or systemic sclerosis, SSc). Receptor-ligand analyses indicated that lymphocytes mediated pericyte transdifferentiation in SARAWT mice, while with SARA overexpression the myofibroblast activity of pericytes was suppressed. Together, these data suggest a potentially novel crosstalk between myofibroblast precursors and immune cells in the pathogenesis of SSc, in which SARA plays a critical role.

R-spondin 1/dickkopf-1/beta-catenin machinery is involved in testicular embryonic angiogenesis.

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.

Cytoskeleton modifications and autophagy induction in TCam-2 seminoma cells exposed to simulated microgravity.

The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one.