A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration.

Neurodegenerative disorders are characterized by the progressive loss of structure and function of neurons, often including the death of the neuron. Previously, we reported that, by removing the cell death stimulus, dying/injured neurons could survive and recover from the process of regulated cell death, even if the cells already displayed various signs of cellular damage. Now we investigated the role of mitochondrial dynamics (fission/fusion, biogenesis, mitophagy) in both degeneration and in recovery of neuronal cells. In neuronal PC12 cells, exposure to ethanol (EtOH) induced massive neurite loss along with widespread mitochondrial fragmentation, mitochondrial membrane potential loss, reduced ATP production, and decreased total mitochondrial volume. By removing EtOH timely all these mitochondrial parameters recovered to normal levels. Meanwhile, cells regrew neurites and survived. Study of the mitochondrial dynamics showed that autophagy was activated only during the cellular degeneration phase (EtOH treatment) but not in the recovery phase (EtOH removed), and it was not dependent on the Parkin/PINK1 mediated mitophagy pathway. Protein expression of key regulators of mitochondrial fission, phospho-Drp1Ser616 and S-OPA1, increased during EtOH treatment and recovered to normal levels after removing EtOH. In addition, the critical role of PGC-1α mediated mitochondrial biogenesis in cellular recovery was revealed: inhibition of PGC-1α using SR-18292 after EtOH removal significantly impeded recovery of mitochondrial damage, regeneration of neurites, and cell survival in a concentration-dependent manner. Taken together, our study showed reversibility of mitochondrial morphological and functional damage in stressed neuronal cells and revealed that PGC-1α mediated mitochondrial biogenesis played a critical role in the cellular recovery. This molecular mechanism could be a target for neuroprotection and neurorescue in neurodegenerative diseases.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

OBJECTIVE AND DESIGN: Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. METHODS: We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. RESULTS: LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. CONCLUSION: ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Collagen-Targeting Self-Assembled Nanoprobes for Multimodal Molecular Imaging and Quantification of Myocardial Fibrosis in a Rat Model of Myocardial Infarction.

Currently, inadequate early diagnostic methods hinder the prompt treatment of patients with heart failure and myocardial fibrosis. Magnetic resonance imaging is the gold standard noninvasive diagnostic method; however, its effectiveness is constrained by low resolution and challenges posed by certain patients who cannot undergo the procedure. Although enhanced computed tomography (CT) offers high resolution, challenges arise owing to the unclear differentiation between fibrotic and normal myocardial tissue. Furthermore, although echocardiography is real-time and convenient, it lacks the necessary resolution for detecting fibrotic myocardium, thus limiting its value in fibrosis detection. Inspired by the postinfarction accumulation of collagen types I and III, we developed a collagen-targeted multimodal imaging nanoplatform, CNA35-GP@NPs, comprising lipid nanoparticles (NPs), encapsulating gold nanorods (GNRs) and perfluoropentane (PFP). This platform facilitated ultrasound/photoacoustic/CT imaging of postinfarction cardiac fibrosis in a rat model of myocardial infarction (MI). The surface-modified peptide CNA35 exhibited excellent collagen fiber targeting. The strong near-infrared light absorption and substantial X-ray attenuation of the nanoplatform rendered it suitable for photoacoustic and CT imaging. In the rat model of MI, our study demonstrated that CNA35-GNR/PFP@NPs (CNA35-GP@NPs) achieved photoacoustic, ultrasound, and enhanced CT imaging of the fibrotic myocardium. Notably, the photoacoustic signal intensity positively correlated with the severity of myocardial fibrosis. Thus, this study presents a promising approach for accurately detecting and treating the fibrotic myocardium.

A novel insight into neurological disorders through HDAC6 protein-protein interactions.

Due to its involvement in physiological and pathological processes, histone deacetylase 6 (HDAC6) is considered a promising pharmaceutical target for several neurological manifestations. However, the exact regulatory role of HDAC6 in the central nervous system (CNS) is still not fully understood. Hence, using a semi-automated literature screening technique, we systematically collected HDAC6-protein interactions that are experimentally validated and reported in the CNS. The resulting HDAC6 network encompassed 115 HDAC6-protein interactions divided over five subnetworks: (de)acetylation, phosphorylation, protein complexes, regulatory, and aggresome-autophagy subnetworks. In addition, 132 indirect interactions identified through HDAC6 inhibition were collected and categorized. Finally, to display the application of our HDAC6 network, we mapped transcriptomics data of Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis on the network and highlighted that in the case of Alzheimer's disease, alterations predominantly affect the HDAC6 phosphorylation subnetwork, whereas differential expression within the deacetylation subnetwork is observed across all three neurological disorders. In conclusion, the HDAC6 network created in the present study is a novel and valuable resource for the understanding of the HDAC6 regulatory mechanisms, thereby providing a framework for the integration and interpretation of omics data from neurological disorders and pharmacodynamic assessments.

The intrinsic coagulation pathway plays a dominant role in driving hypercoagulability in ANCA-associated vasculitis.

ABSTRACT: The risk of a venous thrombotic event (VTE) is increased in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV); however, a detailed understanding of the underlying mechanisms of hypercoagulability is limited. We assessed prospectively different coagulation parameters in 71 patients with active AAV at baseline and after 6 months of follow-up. D-dimers and fibrinogen were increased in most patients at presentation and remained elevated in half of the patients. Particularly, thrombin-antithrombin (T:AT) complex and activated coagulation factors in complex with their natural inhibitors of the intrinsic coagulation pathway (ie, activated FXII:C1 esterase inhibitor [FXIIa:C1Inh], FXIa:AT, and FXIa:alpha1-antitrypsin [FXIa:α1AT]) were profoundly elevated in patients at baseline. Thrombin formation was dominantly correlated with coagulation factors of the intrinsic pathway (ie, FXIIa:AT, FXIa:AT, FXIa:α1AT, and FXIa:C1Inh) compared to the extrinsic pathway (ie, FVIIa:AT). Hypercoagulability correlated with higher disease activity, ANCA levels, C-reactive protein, serum creatinine, and proteinuria. VTEs were observed in 5 out of 71 (7%) patients within 1 month (interquartile range, 1-5) after inclusion. Baseline T:AT levels were significantly higher in patients with VTE than in those without VTE (P = .044), but other clinical or laboratory markers were comparable between both groups. Hypercoagulability is dominantly characterized by activation of the intrinsic coagulation pathway and elevated D-dimers in active AAV. The driving factors of hypercoagulability are yet to be studied but are most likely related to an interplay of increased disease activity, vascular inflammation, and endothelial damage. Future targets for intervention could include inhibitors of the intrinsic coagulation pathway and compounds specifically reducing the hyperinflammatory state.