Senescent endothelial cells: a potential target for diabetic retinopathy.

Diabetic retinopathy (DR) is a diabetic complication that results in visual impairment and relevant retinal diseases. Current therapeutic strategies on DR primarily focus on antiangiogenic therapies, which particularly target vascular endothelial growth factor and its related signaling transduction. However, these therapies still have limitations due to the intricate pathogenesis of DR. Emerging studies have shown that premature senescence of endothelial cells (ECs) in a hyperglycemic environment is involved in the disease process of DR and plays multiple roles at different stages. Moreover, these surprising discoveries have driven the development of senotherapeutics and strategies targeting senescent endothelial cells (SECs), which present challenging but promising prospects in DR treatment. In this review, we focus on the inducers and mechanisms of EC senescence in the pathogenesis of DR and summarize the current research advances in the development of senotherapeutics and strategies that target SECs for DR treatment. Herein, we highlight the role played by key factors at different stages of EC senescence, which will be critical for facilitating the development of future innovative treatment strategies that target the different stages of senescence in DR.

Transcriptomics and metabolomics reveal the role of CYP1A2 in psoralen/isopsoralen-induced metabolic activation and hepatotoxicity.

Psoralen and isopsoralen are the pharmacologically important but hepatotoxic components in Psoraleae Fructus. The purpose of this study was to reveal the underlying mechanism of psoralen/isopsoralen-induced hepatotoxicity. Initially, we applied integrated analyses of transcriptomic and metabolomic profiles in mice treated with psoralen and isopsoralen, highlighting the xenobiotic metabolism by cytochromes P450 as a potential pathway. Then, with verifications of expression levels by qRT-PCR and western blot, affinities by molecular docking, and metabolic contributions by recombinant human CYP450 and mouse liver microsomes, CYP1A2 was screened out as the key metabolic enzyme. Afterwards, CYP1A2 induction and inhibition models in HepG2 cells and mice were established to verify the role of CYP1A2, demonstrating that induction of CYP1A2 aggravated the hepatotoxicity, and conversely inhibition alleviated the hepatotoxic effects. Additionally, we detected glutathione adducts with reactive intermediates of psoralen and isopsoralen generated by CYP1A2 metabolism in biosystems of recombinant human CYP1A2 and mouse liver microsomes, CYP1A2-overexpressed HepG2 cells, mice livers and the chemical reaction system using UPLC-Q-TOF-MS/MS. Ultimately, the high-content screening presented the cellular oxidative stress and relevant hepatotoxicity due to glutathione depletion by reactive intermediates. In brief, our findings illustrated that CYP1A2-mediated metabolic activation is responsible for the psoralen/isopsoralen-induced hepatotoxicity.

Integrin α5ß1 promotes BMCs mobilization and differentiation to exacerbate choroidal neovascularization.

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-ß and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV.

SNAI1, an endothelial-mesenchymal transition transcription factor, promotes the early phase of ocular neovascularization.

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target.

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca2+, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

Microglial Galectin3 enhances endothelial metabolism and promotes pathological angiogenesis via Notch inhibition by competitively binding to Jag1.

Microglia were considered as immune cells in inflammation until their angiogenic role was widely understood. Although the pro-inflammatory role of microglia in retinal angiogenesis has been explored, little is known about its role in pro-angiogenesis and the microglia-endothelia interaction. Here, we report that galectin-3 (Gal3) released by activated microglia functions as a communicator between microglia and endothelia and competitively binds to Jag1, thus inhibiting the Notch signaling pathway and enhancing endothelial angiogenic metabolism to promote angiogenesis. These results suggest that Gal3 may be a novel and effective target in the treatment of retinal angiogenesis.

Repression of rRNA gene transcription by endothelial SPEN deficiency normalizes tumor vasculature via nucleolar stress.

Human cancers induce a chaotic, dysfunctional vasculature that promotes tumor growth and blunts most current therapies; however, the mechanisms underlying the induction of a dysfunctional vasculature have been unclear. Here, we show that split end (SPEN), a transcription repressor, coordinates rRNA synthesis in endothelial cells (ECs) and is required for physiological and tumor angiogenesis. SPEN deficiency attenuated EC proliferation and blunted retinal angiogenesis, which was attributed to p53 activation. Furthermore, SPEN knockdown activated p53 by upregulating noncoding promoter RNA (pRNA), which represses rRNA transcription and triggers p53-mediated nucleolar stress. In human cancer biopsies, a low endothelial SPEN level correlated with extended overall survival. In mice, endothelial SPEN deficiency compromised rRNA expression and repressed tumor growth and metastasis by normalizing tumor vessels, and this was abrogated by p53 haploinsufficiency. rRNA gene transcription is driven by RNA polymerase I (RNPI). We found that CX-5461, an RNPI inhibitor, recapitulated the effect of Spen ablation on tumor vessel normalization and combining CX-5461 with cisplatin substantially improved the efficacy of treating tumors in mice. Together, these results demonstrate that SPEN is required for angiogenesis by repressing pRNA to enable rRNA gene transcription and ribosomal biogenesis and that RNPI represents a target for tumor vessel normalization therapy of cancer.

Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear. AIM OF STUDY: The aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo. MATERIALS AND METHODS: Psoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 µM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. RESULTS: Psoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2. CONCLUSIONS: Hepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.

Precise identification of Celosia argentea seed and its five adulterants by multiple morphological and chemical means.

Celosia argentea seed (CAS) has been used as a traditional Chinese medicine for the treatment of liver damage and eye diseases. CAS is easily falsely harvested and misused, five species from Amaranthaceae family have frequently found to be involved in the adulteration and misapplication, namely Celosia cristata seed (CCS), Amaranthus tricolor seed (ATS), Amaranthus retroflexus seed (ARS), Amaranthus cruentus seed (ACS), and Amaranthus spinosus seed (ASS). For the purpose of identification, multiple morphological means including stereomicroscopy, scanning electron microscopy, normal light and polarized light microscopy were comprehensively employed. As a result, micromorphological and microscopic characteristics were extracted and a diagnostic key to CAS and its five adulterants was proposed for the first time. With respect to the genetically closely related species, viz. CAS and CCS, chemical means were developed to achieve the goal of precise identification. Firstly, triterpenoid saponins in CAS and CCS were fully characterized by an HPLC-QTOF-MS/MS method, a total of 20 triterpenoid saponins including 9 novel members were identified. Secondly, the HPLC-ELSD specific chromatogram was established, in which 12 common peaks were assigned. Finally, after a careful comparison, the peak area ratio of two triterpenoid saponins was discovered as interspecies discriminant marker. In conclusion, CAS and its five adulterants can be precisely identified by multiple morphological and chemical means.

Long-term oral administration of Epimedii Folium induced cholestasis in mice by interfering with bile acid transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedii Folium (EF) is a common traditional Chinese medicine that functions as a tonifying kidney yang to strengthen bones and muscles and dispel wind dampness (limb pain, lethargy, nausea, anorexia, and loose stools). Several studies have reported the potential risk of cholestatic liver damage from EF use; however, there have been few investigations of EF-induced cholestasis, particularly the underlying mechanisms. AIMS OF THE STUDY: The purpose of this study was to evaluate the risk of EF-induced cholestasis in vivo and to explore the mechanisms of action. MATERIALS AND METHODS: ICR mice were orally administered a water extract of EF (WEF) in doses of 6.5 and 19.5 g/kg/day for 14 weeks. Liver-to-body weight ratios, body weight, histopathological examination, and biochemical analyses were performed to assess WEF-induced cholestasis in the mice. Genes associated with bile acid (BA) metabolism and transport, including sodium taurocholate cotransporting polypeptide (NTCP), cytochrome P450 8B1 (CYP8B1), bile-salt export pump (BSEP), multidrug resistance P-glycoproteins 1 (MDR1), and farnesoid X receptor (FXR), were measured at the transcript and protein levels to investigate the potential mechanisms through which cholestasis is aroused by EF. RESULTS: After administration of WEF for 14 weeks, mice in the high-dose WEF group showed poor health with an increased liver-to-body weight ratio as well as higher serum aminotransferase, alkaline phosphatase, direct bilirubin, and total BA levels. Compared with the control group, mRNA expression of NTCP and cholesterol 7a-hydroxylase (CYP7A1) increased, and levels of BSEP, MDR1, multidrug resistance-associated protein 2, and multidrug resistance-associated protein 3 decreased in the WEF-treated group. NTCP, BSEP, MDR1, and CYP8B1 showed similar mRNA and protein expression trends. CONCLUSION: We demonstrated that the long-term oral administration of WEF causes cholestatic liver injury in mice, which is consistent with reported clinical cases. Furthermore, we found that the destruction of BA metabolism and transport is involved in WEF-induced cholestasis. The fine-scale molecular mechanisms of WEF-induced cholestasis and the active compounds of EF need further study.