The Inflammasome-Dependent Dysfunction and Death of Retinal Ganglion Cells after Repetitive Intraocular Pressure Spikes.

The dysfunction and selective loss of retinal ganglion cells (RGCs) is a known cause of vision loss in glaucoma and other neuropathies, where ocular hypertension (OHT) is the major risk factor. We investigated the impact of transient non-ischemic OHT spikes (spOHT) on RGC function and viability in vivo to identify cellular pathways linking low-grade repetitive mechanical stress to RGC pathology. We found that repetitive spOHT had an unexpectedly high impact on intraocular homeostasis and RGC viability, while exposure to steady OHT (stOHT) of a similar intensity and duration failed to induce pathology. The repetitive spOHT induced the rapid activation of the inflammasome, marked by the upregulation of NLRP1, NLRP3, AIM2, caspases -1, -3/7, -8, and Gasdermin D (GSDMD), and the release of interleukin-1ß (IL-1ß) and other cytokines into the vitreous. Similar effects were also detected after 5 weeks of exposure to chronic OHT in an induced glaucoma model. The onset of these immune responses in both spOHT and glaucoma models preceded a 50% deficit in pattern electroretinogram (PERG) amplitude and a significant loss of RGCs 7 days post-injury. The inactivation of inflammasome complexes in Nlrp1-/-, Casp1-/-, and GsdmD-/- knockout animals significantly suppressed the spOHT-induced inflammatory response and protected RGCs. Our results demonstrate that mechanical stress produced by acute repetitive spOHT or chronic OHT is mechanistically linked to inflammasome activation, which leads to RGC dysfunction and death.

Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury.

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1ß, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1ß were detected within 6 h and peaked at 12-24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1-/-Casp4(11)del, Panx1-/- and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas.

Senescence-messaging secretome factors trigger premature senescence in human endometrium-derived stem cells.

Accumulating evidence suggests that the senescence-messaging secretome (SMS) factors released by senescent cells play a key role in cellular senescence and physiological aging. Phenomenon of the senescence induction in human endometrium-derived mesenchymal stem cells (MESCs) in response to SMS factors has not yet been described. In present study, we examine a hypothesis whether the conditioned medium from senescent cells (CM-old) may promote premature senescence of young MESCs. In this case, we assume that SMS factors, containing in CM-old are capable to trigger senescence mechanism in a paracrine manner. A long-term cultivation MESCs in the presence of CM-old caused deceleration of cell proliferation along with emerging senescence phenotype, including increase in both the cell size and SA-ß-Gal activity. The phosphorylation of p53 and MAPKAPK-2, a direct target of p38MAPK, as well as the expression of p21Cip1 and p16Ink4a were increased in CM-old treated cells with senescence developing whereas the Rb phosphorylation was diminished. The senescence progression was accompanied by both enhanced ROS generation and persistent activation of DNA damage response, comprising protein kinase ATM, histone H2A.X, and adapter protein 53BP1. Thus, we suggest that a senescence inducing signal is transmitted through p16/MAPKAPK-2/Rb and DDR-mediated p53/p21/Rb signaling pathways. This study is the first to demonstrate that the SMS factors secreted in conditioned medium of senescent MESCs trigger a paracrine mechanism of premature senescence in young cells.

Genotoxic and cytotoxic effects of the environmental pollutant 3-nitrobenzanthrone on bladder cancer cells.

3-Nitrobenzanthrone (3-NBA), a potential human carcinogen, is present in diesel exhaust. The main metabolite of 3-NBA, 3-aminobenzanthrone, was detected in urine of miners occupationally exposed to diesel emissions. Environmental and occupational factors play an important role in development of bladder cancer (BC), one of the most frequent malignancies. It is expected that exposure of urothelium to 3-NBA and its metabolites may induce BC initiation and/or progression. To test this hypothesis, we studied geno- and cytotoxicity of 3-NBA using an in vitro BC model. 3-NBA induced higher levels of DNA adducts, reactive oxygen species and DNA breaks in aggressive T24 cells than in more differentiated RT4 cells. To understand the nature of this difference we examined the role of several enzymes that were identified as 3-NBA bio activators. However, the difference in DNA adduct formation cannot be directly linked to the different activity of any of the examined enzymes. Conversely, the difference of tested cell lines in p53 status can partly explain the distinct levels of 3-NBA-DNA adducts and DNA damage induced by 3-NBA. Therefore, we assume that more aggressive T24 cells are more predisposed for DNA adduct formation, DNA damage and, possibly, mutations and as a result further tumorigenesis.

Definition of a direct extracellular interaction between Met and E-cadherin.

High levels of the Met tyrosine kinase receptor expression are associated with metastatic disease. Met activation by hepatocyte growth factor (HGF) is associated with decreased E-cadherin-dependent cell-cell contacts. The molecular mechanism underlying this process remains unclear. To better understand the relationship between E-cadherin and Met, we assessed Met localization in cells which form mature E-cadherin-dependent adhesion HT-29 and cells which have lost E-cadherin expression BT-549. Met colocalized with E-cadherin at the site of cell-cell adhesion in HT-29 cells, but Met was distributed in an intracellular compartment in BT-549 cells. Forced expression of E-cadherin in BT-549 cells recruited Met to the membrane. Cross-linking studies suggested that Met and E-cadherin interact in the extracellular domain in HT-29 cells. This is the first evidence of a physical interaction between Met and E-cadherin. We suggest that this receptor/cadherin pairing may be a mechanism for cellular presentation of receptors in a manner that localizes them optimally for interaction with ligand.