Non-Invasive Evaluation of Retinal Vascular Alterations in a Mouse Model of Optic Neuritis Using Laser Speckle Flowgraphy and Optical Coherence Tomography Angiography.

Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments.

Design, synthesis, and pharmacological evaluations of pyrrolo[1,2-a]quinoxaline-based derivatives as potent and selective sirt6 activators.

Sirt6 activation has emerged as a promising drug target for the treatment of various human diseases, while only limited Sirt6 activators have been reported. Herein, a series of novel pyrrolo[1,2-a]quinoxaline-based derivatives have been identified as potent and selective Sirt6 activators with low cytotoxicity. Sirt6-knockdown findings have validated the on-target effects of this class of Sirt6 activators. Docking studies indicate the protonated nitrogen on the side chain of 38 forms π-cation interactions with Trp188, further stabilizing it into this extended binding pocket. New compounds 35, 36, 38, 46, 47, and 50 strongly repressed LPS-induced proinflammatory cytokine/chemokine production, while 38 also significantly suppressed SARS-CoV-2 infection with an EC50 value of 9.3 µM. Moreover, compound 36 significantly inhibited the colony formation of cancer cells. These new molecules may serve as useful pharmacological tools or potential therapeutics against cancer, inflammation, and infectious diseases.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

Light-Sheet Microscopy of the Optic Nerve Reveals Axonal Degeneration and Microglial Activation in NMDA-Induced Retinal Injury.

Purpose: Optic nerve degeneration is a feature of neurodegenerative eye diseases and causes irreversible vision loss. Therefore, understanding the degenerating patterns of the optic nerve is critical to find the potential therapeutic target for optic neuropathy. However, the traditional method of optic nerve degeneration has the limitations of losing spatiotemporal tissue information. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique that allows capturing 3D images rapidly with a high spatial optical resolution. In this study, we evaluated the availability of LSFM on the optic nerve with NMDA injected Thy1-CFP mice. Methods: NMDA injected to both eyes of Thy1-CFP mice. After 7 days from the injection, the retina and optic nerve were collected and immunostained with anti-Iba1 antibody. NMDA excitotoxicity induced RGC, and its axon loss and microglial activation in the retina were observed using confocal microscopy. The immunostained optic nerve was completed the optical clearing process with TDE and mounted for LSFM imaging. Results: We found that retinal flatmounts confirmed significant loss of CFP-expressing RGC and axon degradation and loss in Thy1-CFP mice at 7 days after NMDA injection. Together with these data verifying that NMDA induces RGC and its axon loss, we confirmed that NMDA excitotoxicity induced microglia activation and leukostasis, such as increased microglia number, transform its morphology to ameboid or round, and increase in attached leukocytes in vessels. Using LSFM, we observed that CFP expressing nerve fiber was well organized and arranged parallel in vehicle treated optic nerve, whileas NMDA injected optic nerve showed axon swelling and fragmentation and loss of axon density from the anterior to the posterior regions. Furthermore, LSFM enabled the observation of microglia phenotype transformation in the entire optic nerve. Unlike microglia in vehicle injected optic nerve, microglia in NMDA injected optic nerve displayed larger soma and short process with high Iba1 expression through the entire optic nerve from the anterior to posterior. Conclusions: In summary, we examined the applicability of the modified optic clearing protocol for the optic nerve and verified it enabled to acquiring of the 3D images of the optic nerve successfully revealing the complex spatial relationships between the axons, microglia and vasculature throughout the entire organ with single acquisitions. With these optimized techniques, we successfully obtained the high-resolution 3D images of NMDA-induced optic neuropathy, including the clues for optic nerve degeneration such as axon swelling, axonal fragmentation, and microglia activation. Overall, we believe that our current study could help understand the pathology of the optic nerve in neurodegenerative diseases, and it will be the basis for translational research.

Peli1 signaling blockade attenuates congenital zika syndrome.

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.

Neuronal Epac1 mediates retinal neurodegeneration in mouse models of ocular hypertension.

Progressive loss of retinal ganglion cells (RGCs) leads to irreversible visual deficits in glaucoma. Here, we found that the level of cyclic AMP and the activity and expression of its mediator Epac1 were increased in retinas of two mouse models of ocular hypertension. Genetic depletion of Epac1 significantly attenuated ocular hypertension-induced detrimental effects in the retina, including vascular inflammation, neuronal apoptosis and necroptosis, thinning of ganglion cell complex layer, RGC loss, and retinal neuronal dysfunction. With bone marrow transplantation and various Epac1 conditional knockout mice, we further demonstrated that Epac1 in retinal neuronal cells (especially RGCs) was responsible for their death. Consistently, pharmacologic inhibition of Epac activity prevented RGC loss. Moreover, in vitro study on primary RGCs showed that Epac1 activation was sufficient to induce RGC death, which was mechanistically mediated by CaMKII activation. Taken together, these findings indicate that neuronal Epac1 plays a critical role in retinal neurodegeneration and suggest that Epac1 could be considered a target for neuroprotection in glaucoma.

TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway.

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.

Recruitment of monocytes and epigenetic silencing of intratumoral CYP7B1 primarily contribute to the accumulation of 27-hydroxycholesterol in breast cancer.

Previous studies showed that intratumoral 27-Hydroxycholesterol (27-HC), a metabolite of cholesterol, promotes growth, invasion and migration of breast cancer cells and that tumor-associated macrophages (TAMs) in breast cancers are closely related to tumor growth and metastatic progression. However, the relationship between 27-HC and TAMs in breast cancer remains unclear. In the present study, we observed that CYP27A1, the 27-HC synthesizing enzyme, was expressed in a much higher level in THP1 monocytes and THP1-derived macrophages than in breast cancer cells, and the promoter of CYP7B1, the degrading enzyme for 27-HC, was highly methylated in breast tumor cells. In addition, THP-1 monocytes and murine bone marrow cells were differentiated toward M2 type macrophages after being co-cultured with breast cancer cells or being exposed to exosomes derived from breast cancer cells. M2 type macrophages produced higher amounts of 27-HC than M0 and M1 type macrophages. 27-HC not only stimulated ER+ cancer cell proliferation as reported, but also promoted the recruitment of CCR2- and CCR5-expressing monocytes by inducing macrophages to express multiple chemokines including CCL2, CCL3 and CCL4. Taken together, our data demonstrate that the hypermethylation of CYP7B1 and recruitment of monocytes likely contribute to the accumulation of 27-Hydroxycholesterol in breast cancer and that the interaction of 27-HC with macrophages further promote the development of breast cancer.

Colorimetric detection of glutathione in cells based on peroxidase-like activity of gold nanoclusters: A promising powerful tool for identifying cancer cells.

Glutathione (GSH), the most abundant biothiol in cells, not only plays a pivotal role in protective and detoxifying functions of the cell, but also serves as a very important mediator in many cellular functions. Especially, the difference of GSH level between cancer cells and normal cells is regarded as one of most important physiological parameters for cancer diagnosis. It is thereby extremely necessary to develop a simple, sensitive, and reliable analytical method for detection of GSH in cells. On the basis of the inhibition effect of GSH on the peroxidase-like activity of GSH stabilized gold nanoclusters, here a novel and facile strategy for colorimetric detection of cellular GSH level was well established. In this sensing system, GSH can effectively inhibit the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue colored product. Under the optimized conditions, the absorbance at 652 nm against GSH concentration shows a linear relationship within a range from 2 to 25 µM with detection limit of 420 nM. This excellent property allows our approach to be used to accurately evaluate the cellular GSH levels, and it is revealed that the overall GSH level in cancer cells was much higher than that in normal cells. The presented assay will enable a powerful tool for identifying cancer cells in a simple manner for biomedical diagnosis associated with GSH.