The role of caspase family in acute brain injury: the potential therapeutic targets in the future

: The caspase family is commonly involved in the pathophysiology of acute brain injury (ABI) through complex apoptotic, pyroptotic, and inflammatory pathways. Current translational strategies for caspase modulation in ABI primarily focus on caspase inhibitors. Because there are no caspase-inhibiting drugs approved for clinical use on the market, the development of caspase inhibitors remains an attractive challenge for researchers and clinicians. Therefore, we conducted the present review with the aim of providing a comprehensive introduction of caspases in ABI. In this review, we summarized the available evidence and potential mechanisms regarding the biological function of caspases. We also reviewed the therapeutic effects of caspase inhibitors on ABI and its subsequent complications. However, various important issues remain unclear, prompting further verification of the efficacy and safety regarding clinical application of caspase inhibitors. We believe that our work will be helpful to further understand the critical role of the caspase family, and will provide novel therapeutic potential for ABI treatment.

NSAIDs Immunomodulation in COVID-19 Might Inhibit SARS CoV-2 ORF Proteins Induced Caspase Activation, Necroptosis and Endoplasmic Reticulum Stress.

We have previously suggested numerous immunomodulatory and anti-inflammatory benefits when NSAIDs are administered to manage COVID-19 and in this commentary, we add other potential benefits related to SARS CoV-2 ORF proteins dependent activation of caspases with subsequent mitochondrial dysfunction, endoplasmic reticulum stress and necroptosis that were described with complicated COVID-19 as NSAIDs are known to be caspase inhibitors. Moreover, NSAIDs might independently inhibit other COVID-19 associated downstream pathological signaling mechanisms. We also postulate that CARD-14, a caspase recruitment domain-containing protein, polymorphisms might play a role in development of severe and critical COVID-19. We believe that it is very unfortunate that for more than one year of relentless struggle, our recommendation to adopt NSAIDs as first choice COVID-19 therapy has not adopted while lives are lost are succumbed every day.

NSAIDs Immunomodulation in COVID-19 Might Inhibit SARS CoV-2 ORF Proteins Induced Caspase Activation, Necroptosis and Endoplasmic Reticulum Stress.

We have previously suggested numerous immunomodulatory and anti-inflammatory benefits when NSAIDs are administered to manage COVID-19 and in this commentary, we add other potential benefits related to SARS CoV-2 ORF proteins dependent activation of caspases with subsequent mitochondrial dysfunction, endoplasmic reticulum stress and necroptosis that were described with complicated COVID-19 as NSAIDs are known to be caspase inhibitors. Moreover, NSAIDs might independently inhibit other COVID-19 associated downstream pathological signaling mechanisms. We also postulate that CARD-14, a caspase recruitment domain-containing protein, polymorphisms might play a role in development of severe and critical COVID-19. We believe that it is very unfortunate that for more than one year of relentless struggle, our recommendation to adopt NSAIDs as first choice COVID-19 therapy has not adopted while lives are lost are succumbed every day.

NSAIDs Immunomodulation in COVID-19 Might Inhibit SARS CoV-2 ORF Proteins Induced Caspase Activation, Necroptosis and Endoplasmic Reticulum Stress.

We have previously suggested numerous immunomodulatory and anti-inflammatory benefits when NSAIDs are administered to manage COVID-19 and in this commentary, we add other potential benefits related to SARS CoV-2 ORF proteins dependent activation of caspases with subsequent mitochondrial dysfunction, endoplasmic reticulum stress and necroptosis that were described with complicated COVID-19 as NSAIDs are known to be caspase inhibitors. Moreover, NSAIDs might independently inhibit other COVID-19 associated downstream pathological signaling mechanisms. We also postulate that CARD-14, a caspase recruitment domain-containing protein, polymorphisms might play a role in development of severe and critical COVID-19. We believe that it is very unfortunate that for more than one year of relentless struggle, our recommendation to adopt NSAIDs as first choice COVID-19 therapy has not adopted while lives are lost are succumbed every day.

Endothelial cell death subroutines at physiological and supraphysiological concentrations of calcium phosphate bions

Актуальность. Формирующиеся при перенасыщении крови ионами кальция и фосфора и циркулирующие в кровотоке кальций-фосфатные бионы (КФБ) вызывают дисфункцию эндотелия вследствие гибели части артериальных эндотелиальных клеток (ЭК). Цель исследования. Оценить типы гибели первичных артериальных ЭК человека под воздействием физиологических и супрафизиологических концентраций сферических КФБ (СКФБ) и игольчатых КФБ (ИКФБ). Материалы и методы. К конфлюэнтным культурам первичных ЭК коронарной и внутренней грудной артерии человека в 96-луночных планшетах были добавлены равные объемы (10 мкл на лунку) суспензий СКФБ и ИКФБ с оптической плотностью 0,08-0,10 (физиологическая концентрация) или 0,42-0,45 (супрафизиологическая концентрация) на длине волны 650 нм. Во всех экспериментальных группах также производилось селективное ингибирование каспазы-3 (Z-D(OMe)E(OMe)VD(OMe)-FMK, 100 мкмоль/л) или ингибирование всех каспаз (Z-VAD(OMe)-FMK, 100 мкмоль/л) для оценки типа клеточной гибели (регулируемая или моментальная). Жизнеспособность клеток определялась посредством последовательного колориметрического определения их метаболической активности через 4, 24 и 48 часов после добавления КФБ. Результаты. При добавлении супрафизиологических концентраций КФБ уже на первой временной точке большинство (60-85%) эндотелиальных клеток погибало вне зависимости от типа добавленных КФБ и воздействия ингибиторов каспаз, при этом через 24 и 48 часов экспозиции ингибиторы каспаз оказывали некоторое цитопротективное действие на незначительное количество выживших клеток. При добавлении физиологических концентраций КФБ ингибиторы каспаз оказывали выраженное цитопротективное действие через 24 и 48 часов экспозиции, при этом ИКФБ демонстрировали существенно более высокую токсичность для ЭК в сравнении с СКФБ. Независимо от временной точки пан-каспазный ингибитор оказывал значительно более выраженное цитопротективное действие по сравнению с селективным ингибитором каспазы-3, что свидетельствует о кумулятивном эффекте ингибирования каспаз, возникающем, вероятно, вследствие запуска внутреннего пути апоптоза. Заключение. В супрафизиологических концентрациях КФБ вызывают моментальную гибель абсолютного большинства ЭК, однако в физиологических концентрациях ингибиторы каспаз существенно повышают выживаемость ЭК, что свидетельствует о регулируемом направлении их клеточной гибели. Дальнейшие исследования в этом направлении должны расшифровать молекулярные пути регулируемой клеточной гибели ЭК под воздействием физиологических концентраций КФБ. Background. Calcium phosphate bions (CPB) formed and circulating in the blood at its supersaturation with calcium and phosphate provoke endothelial dysfunction by causing the demise of arterial endothelial cells (ECs). Aim. To examine cell death subroutines of human primary arterial ECs exposed to physiological and supraphysiological concentrations of spherical CPB (CPB-S) and needle-shaped CPB (CPB-N). Materials and methods. Equal volumes (10 μL) of CPB-S and CPB-N at physiological concentration (optical density at 650 nm wavelength = 0.08-0.10) or supraphysiological amounts (optical density at 650 nm wavelength = 0.42-0.45) were added to the confluent primary human coronary artery and internal thoracic artery ECs cultured in 96-well plates. In all experimental groups, we selectively inhibited caspase-3 by adding Z-D(OMe)E(OMe)VD(OMe)-FMK (100 μmol/L) or all caspases (Z-VAD(OMe)-FMK, 100 μmol/L) to assess whether the CPB-induced cell death is regulated or accidental. Cell viability was evaluated by sequential colorimetric determination of metabolic activity at 4, 24, and 48 hours of incubation with CPB. Results. At supraphysiological CPB concentrations, the majority (60-85%) of ECs died regardless of CPB type and caspase inhibitors, albeit at 24- and 48-hour time points the latter had minor cytoprotective action. However at physiological CPB levels, caspase inhibitors rescued a considerable proportion of ECs after 24 or 48 hours of exposure, and CPB-N had significantly higher toxicity than CPB-S. Regardless of the time point, the cytoprotective effect of the pan-caspase inhibitor was significantly higher than that of the selective caspase-3 inhibitor indicating a cumulative caspase inhibition and suggesting that cell death was precipitated by an intrinsic apoptosis pathway. Conclusion. At supraphysiological concentrations, CPB cause instant cell death; yet at physiological amounts, caspase inhibitors rescue the majority of ECs testifying to the regulated cell death. Further studies in this field should decipher the molecular pathways of CPB-induced regulated cell death of ECs.

Inhibition of Caspases Improves Non-Viral T Cell Receptor Editing

T cell receptor (TCR) knockout is a critical step in producing universal chimeric antigen receptor T cells for cancer immunotherapy. A promising approach to achieving the knockout is to deliver the CRISPR/Cas9 system into cells using electrotransfer technology. However, clinical applications of the technology are currently limited by the low cell viability. In this study, we attempt to solve the problem by screening small molecule drugs with an immortalized human T cell line, Jurkat clone E6-1, for inhibition of apoptosis. The study identifies a few caspase inhibitors that could be used to simultaneously enhance the cell viability and the efficiency of plasmid DNA electrotransfer. Additionally, we show that the enhancement could be achieved through knockdown of caspase 3 expression in siRNA treated cells, suggesting that the cell death in electrotransfer experiments was caused mainly by caspase 3-dependent apoptosis. Finally, we investigated if the caspase inhibitors could improve TCR gene-editing with electrotransferred ribonucleoprotein, a complex of Cas9 protein and a T cell receptor-α constant (TRAC)-targeting single guide RNA (sgRNA). Our data showed that inhibition of caspases post electrotransfer could significantly increase cell viability without compromising the TCR disruption efficiency. These new findings can be used to improve non-viral T cell engineering.