Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Investigation into the effect of platelet-released molecules on the brain

The brain is a highly fragile organ kept separated in its unique environment by specialised brain barriers, mainly the aptly named blood-brain barrier (BBB). Apart from being a physical barrier, the BBB also functions as a transport barrier to strictly govern the balance of ions and molecules that traverse between the blood and the brain. In a number of neuropathological conditions, particularly in the highly prevalent and debilitating conditions of stroke and traumatic brain injury (TBI), the integrity of the BBB becomes compromised leading to the dysregulated migration of blood constituents such as platelets, into the brain. Platelets are best known for their primary role in haemostasis. However, it is also known that platelets partake in several non-haemostatic events including immune regulation, angiogenesis, wound healing as well as in the propagation and migration of cancerous cells. The association of platelets with multiple roles has been attributed to the extensive inventory of bioactive molecules that are released by platelets or exposed on the platelet surface upon platelet activation. Due to its participation in wound repair, platelets in the form of “platelet concentrates” have been exogenously applied in clinical settings to accelerate the recovery of various organs such as skin, tendon, muscle and bone. Surprisingly, despite its widespread use, there is a clear lack of mechanistic insight into precisely how platelets aid in the recovery of these organs. With regard to the role of platelets in the brain, intriguingly, published studies to date have focussed solely on the thrombotic actions of platelets. Therefore, the purpose of this study was to assess the non-thrombotic roles of platelets in the brain. This project builds upon the understanding that upon BBB breakdown, platelets again unimpeded access into the brain where they become activated and release hundreds of bioactive molecules. These bioactive platelet-released molecules (PRMs) have direct contact with injured neurons and thereby may influence brain injury. To address this aim, we show that cerebrospinal fluid from human TBI patients contain elevated levels of soluble GPVI (a platelet marker) compared to cerebrospinal fluid from non-TBI patients; with the mean levels of soluble GPVI measured at 35.42ng/mL versus 0ng/mL, respectively. Interestingly, the amount of GPVI detected within cerebrospinal fluid of TBI patients is comparable to that observed in the plasma of patients with disseminated intravascular coagulation. Next, using a mouse model of TBI, we document that platelet deposition is not confined within cerebral vasculature but that platelets also extravasate in considerable number into the surrounding brain parenchyma. The deposition of platelets within the brain after TBI coincides with BBB breakdown and plasma extravasation. Moreover, we demonstrate for the first time that platelets that deposit in the brain after TBI become activated thereby intimating the presence of bioactive PRMs with the injured brain. Having established that PRMs do indeed come in contact with injured neurons, we used primary neuronal cultures to study the effects of PRMs on injured neurons. We find that PRMs profoundly protect neurons against injuries induced by etoposide or linsidomine. Contrastingly, PRMs have no influence on the viability of neurons that are challenged with glutamate or subjected to oxygen-glucose deprivation conditions. In our neuronal culture system, both etoposide and linsidomine induce apoptosis whereas glutamate and oxygen-glucose deprivation induce necrosis. Therefore, we show that PRMs selectively protect neurons from undergoing apoptosis but have no effect on necrotic injuries. We further demonstrate that the introduction of PRMs after the induction of apoptosis dampens neuronal injury, highlighting the presence of a therapeutic window for the cytoprotective capabilities of PRMs. This remarkable neuroprotective effect of PRMs has never been previously described. Altogether, our novel finding of a selective cytoprotective role of PRMs in neurons extends current understanding of the non-thrombotic role of platelets in the brain. Due to the paucity in efficacious cytoprotective therapies for neuropathological conditions, the isolation of neuroprotective molecule(s) coupled with the identification of how PRMs protect neurons will certainly be beneficial. Studies into these areas will open novel avenues to develop therapeutic strategies that may ultimately impact on the treatment and recovery of patients that sustain brain injury. Accordingly, we further pursued two avenues of investigation: 1) to determine which molecule(s) within the plethora of bioactive PRMs are responsible for its cytoprotective actions and 2) to determine the neuronal signal transduction events that mediate the cytoprotective actions of PRMs. To address the first objective, we separated PRMs into fractions using ion-exchange chromatography and assessed the capacity of these fractions to inhibit apoptosis. We uncovered several fractions that significantly prevent apoptosis. Within these fractions, we identified 6 candidate anti-apoptotic proteins namely: haemoglobin, profilin-I, GAPDH, aldolase A, immunoglobulin and CXCL7. Importantly, these identified molecules have no known thrombotic actions. Consistent with this notion, we show that 5 out of the 7 cytoprotective fractions do not cause platelet aggregation. Thus, we have not only narrowed the identities of cytoprotective molecules within PRMs to a number of candidates, but also uncoupled the cytoprotective function of platelets from their pro-thrombotic role; thereby substantially increasing the therapeutic potential of PRMs. To address the second objective, we used a phospho-protein microarray to show that in etoposide-injured neurons, PRMs cause differential up-regulation of 25 molecules and down-regulation of 44 molecules. Based on this, we find that neuronal activation of the following kinases: p38α, JNK1, JAK1 and DNA-PK, mediates the cytoprotective actions of PRMs. Interestingly, the anti-apoptotic action of PRMs is not restricted to neurons, as we find that PRMs also protect human U937 cells (a leukaemic monocyte lymphoma cell line) from chemotherapy-induced apoptosis. The ability of PRMs and its fractions to inhibit apoptosis in U937 cells denotes that in addition to the other known contributions of platelets to tumour growth and progression, platelets also confer significant protection to cancerous cells against chemotherapy. As such, efforts to counteract the anti-apoptotic effect of PRMs represent an interesting prospect to reduce chemoresistance of tumour cells and thereby improve cancer prognosis. Taken together, our findings of an anti-apoptotic role of platelets have implications not only for brain injury where there is a clear lack of neuroprotective therapies; but also bears substantial consequences to treatment of cancer whereby chemoresistance of cancer cells remains a major challenge. Given that platelet concentrates are currently used in clinical settings to aid recovery, this highlights the clinical applicability of our findings. Altogether, we provide novel evidence supporting the applicability of platelet concentrates in any situation where platelet activation and widespread apoptosis coincide

Investigation into the effect of platelet-released molecules on the brain

The brain is a highly fragile organ kept separated in its unique environment by specialised brain barriers, mainly the aptly named blood-brain barrier (BBB). Apart from being a physical barrier, the BBB also functions as a transport barrier to strictly govern the balance of ions and molecules that traverse between the blood and the brain. In a number of neuropathological conditions, particularly in the highly prevalent and debilitating conditions of stroke and traumatic brain injury (TBI), the integrity of the BBB becomes compromised leading to the dysregulated migration of blood constituents such as platelets, into the brain. Platelets are best known for their primary role in haemostasis. However, it is also known that platelets partake in several non-haemostatic events including immune regulation, angiogenesis, wound healing as well as in the propagation and migration of cancerous cells. The association of platelets with multiple roles has been attributed to the extensive inventory of bioactive molecules that are released by platelets or exposed on the platelet surface upon platelet activation. Due to its participation in wound repair, platelets in the form of “platelet concentrates” have been exogenously applied in clinical settings to accelerate the recovery of various organs such as skin, tendon, muscle and bone. Surprisingly, despite its widespread use, there is a clear lack of mechanistic insight into precisely how platelets aid in the recovery of these organs. With regard to the role of platelets in the brain, intriguingly, published studies to date have focussed solely on the thrombotic actions of platelets. Therefore, the purpose of this study was to assess the non-thrombotic roles of platelets in the brain. This project builds upon the understanding that upon BBB breakdown, platelets again unimpeded access into the brain where they become activated and release hundreds of bioactive molecules. These bioactive platelet-released molecules (PRMs) have direct contact with injured neurons and thereby may influence brain injury. To address this aim, we show that cerebrospinal fluid from human TBI patients contain elevated levels of soluble GPVI (a platelet marker) compared to cerebrospinal fluid from non-TBI patients; with the mean levels of soluble GPVI measured at 35.42ng/mL versus 0ng/mL, respectively. Interestingly, the amount of GPVI detected within cerebrospinal fluid of TBI patients is comparable to that observed in the plasma of patients with disseminated intravascular coagulation. Next, using a mouse model of TBI, we document that platelet deposition is not confined within cerebral vasculature but that platelets also extravasate in considerable number into the surrounding brain parenchyma. The deposition of platelets within the brain after TBI coincides with BBB breakdown and plasma extravasation. Moreover, we demonstrate for the first time that platelets that deposit in the brain after TBI become activated thereby intimating the presence of bioactive PRMs with the injured brain. Having established that PRMs do indeed come in contact with injured neurons, we used primary neuronal cultures to study the effects of PRMs on injured neurons. We find that PRMs profoundly protect neurons against injuries induced by etoposide or linsidomine. Contrastingly, PRMs have no influence on the viability of neurons that are challenged with glutamate or subjected to oxygen-glucose deprivation conditions. In our neuronal culture system, both etoposide and linsidomine induce apoptosis whereas glutamate and oxygen-glucose deprivation induce necrosis. Therefore, we show that PRMs selectively protect neurons from undergoing apoptosis but have no effect on necrotic injuries. We further demonstrate that the introduction of PRMs after the induction of apoptosis dampens neuronal injury, highlighting the presence of a therapeutic window for the cytoprotective capabilities of PRMs. This remarkable neuroprotective effect of PRMs has never been previously described. Altogether, our novel finding of a selective cytoprotective role of PRMs in neurons extends current understanding of the non-thrombotic role of platelets in the brain. Due to the paucity in efficacious cytoprotective therapies for neuropathological conditions, the isolation of neuroprotective molecule(s) coupled with the identification of how PRMs protect neurons will certainly be beneficial. Studies into these areas will open novel avenues to develop therapeutic strategies that may ultimately impact on the treatment and recovery of patients that sustain brain injury. Accordingly, we further pursued two avenues of investigation: 1) to determine which molecule(s) within the plethora of bioactive PRMs are responsible for its cytoprotective actions and 2) to determine the neuronal signal transduction events that mediate the cytoprotective actions of PRMs. To address the first objective, we separated PRMs into fractions using ion-exchange chromatography and assessed the capacity of these fractions to inhibit apoptosis. We uncovered several fractions that significantly prevent apoptosis. Within these fractions, we identified 6 candidate anti-apoptotic proteins namely: haemoglobin, profilin-I, GAPDH, aldolase A, immunoglobulin and CXCL7. Importantly, these identified molecules have no known thrombotic actions. Consistent with this notion, we show that 5 out of the 7 cytoprotective fractions do not cause platelet aggregation. Thus, we have not only narrowed the identities of cytoprotective molecules within PRMs to a number of candidates, but also uncoupled the cytoprotective function of platelets from their pro-thrombotic role; thereby substantially increasing the therapeutic potential of PRMs. To address the second objective, we used a phospho-protein microarray to show that in etoposide-injured neurons, PRMs cause differential up-regulation of 25 molecules and down-regulation of 44 molecules. Based on this, we find that neuronal activation of the following kinases: p38α, JNK1, JAK1 and DNA-PK, mediates the cytoprotective actions of PRMs. Interestingly, the anti-apoptotic action of PRMs is not restricted to neurons, as we find that PRMs also protect human U937 cells (a leukaemic monocyte lymphoma cell line) from chemotherapy-induced apoptosis. The ability of PRMs and its fractions to inhibit apoptosis in U937 cells denotes that in addition to the other known contributions of platelets to tumour growth and progression, platelets also confer significant protection to cancerous cells against chemotherapy. As such, efforts to counteract the anti-apoptotic effect of PRMs represent an interesting prospect to reduce chemoresistance of tumour cells and thereby improve cancer prognosis. Taken together, our findings of an anti-apoptotic role of platelets have implications not only for brain injury where there is a clear lack of neuroprotective therapies; but also bears substantial consequences to treatment of cancer whereby chemoresistance of cancer cells remains a major challenge. Given that platelet concentrates are currently used in clinical settings to aid recovery, this highlights the clinical applicability of our findings. Altogether, we provide novel evidence supporting the applicability of platelet concentrates in any situation where platelet activation and widespread apoptosis coincide

Selective inhibition of brain endothelial Rho-kinase-2 provides optimal protection of an <i>in vitro</i> blood-brain barrier from tissue-type plasminogen activator and plasmin

<div><p>Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an <i>in vitro</i> human model. ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.</p></div

Selective ROCK-2 inhibition blocks rt-PA-induced morphological changes and ROCK signalling in human and mouse astrocytes.

<p><b>(A)</b> Representative phase-contrast images (top panels) and double immunofluorescence images of the actin cytoskeleton (phalloidin; grey) and nuclei (Hoechst; red) (bottom panels) of SVG human astrocytes demonstrating blockade of morphology changes (arrows) by KD025 after 6 h treatment with DMSO (control) or with rt-PA+plasminogen (t-PA+Plgn; 25nM+100nM), in the presence or absence of KD025 (20μM). Scale bars = 50μm. <b>(B)</b> Representative phase-contrast images of primary mouse astrocytes treated overnight (13–16 h) as stipulated in A. n = 3. Scale bars = 200μm. Arrows depict changes in cell morphology. <b>(C)</b> Representative western blot analysis (left panel) and quantification (right panel) of phosphorylated myosin light chain levels (pMLC per total MLC) in primary mouse astrocytes treated for two hours with rt-PA+Plgn (50nM+100nM, respectively), with or without KD025 (20μM). Ratios obtained in the rt-PA+Plgn group were assigned a value of 100%. KD025 fully abolishes rt-PA- and plasmin-triggered MLC phosphorylation in mouse astrocytes. n = 3. Bars represent mean±SEM. ****<i>P</i><0.0001 compared to all other groups, *<i>P</i><0.05 compared to DMSO control by one-way ANOVA with Tukey’s post hoc analysis.</p

Selective ROCK-2 inhibition effectively blocks early, but not late rt-PA-induced increases in BBB permeability under normal conditions.

<p><b>(A, B)</b> Permeability changes in the <i>in vitro</i> human BBB 6 h (<b>A</b>; n = 4) or 24 h (<b>B</b>; n = 4–10) post stimulation under normal conditions with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (2 and 20μM, added to both luminal and abluminal chambers). <b>(C)</b> Comparison of selective ROCK-2 inhibition by KD025 (20μM) <i>versus</i> non-selective ROCK inhibition by fasudil (HA1077; 20μM) against rt-PA+plasminogen (25nM+100nM, respectively) 6 h and 24 h after treatment under normal conditions. KD025, but not HA1077, displays strong protective capacity at 6 h, but not at 24 h. n = 3–4. Bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by one-way ANOVA with Tukey’s post hoc analysis. # <i>P</i><0.05 compared to rt-PA+Plgn, ## <i>P</i><0.01 compared to DMSO control and specified <i>P</i> values are by two-tailed paired t-test.</p

ROCK-2 is the primary isoform expressed in cells of the neurovascular unit.

<p>Real-time RT-PCR (<b>A</b>; n = 4 for each cell type) and western blot analysis <b>(B)</b> showing higher steady-state levels of ROCK-1 and similar levels of ROCK-2 (per equal quantities of total RNA and proteins) in unstimulated human SVG astrocytes compared to human brain microvascular endothelial cells (hBECs). <b>(C)</b> Comparative qPCR analysis (by ΔΔCt, normalising to HPRT) in hBECs and SVGs (n = 4 each) demonstrating that ROCK-2 is the principal ROCK transcript expressed in each human cell type. <b>(D)</b> Real-time RT-PCR in equal quantities of RNA from unstimulated primary mouse neurons (mNeuro; n = 4) and primary mouse astrocytes (mAstro; n = 4) relative to primary mouse brain endothelial cells (mBECs; n = 3). mBECs are enriched in ROCK-1 and ROCK-2 transcripts while mouse neurons contains the least. <b>(E)</b> Western blot analysis of ROCK-1 and ROCK-2 protein levels in unstimulated mouse BECs, astrocytes and neurons. <b>(F)</b> Comparative qPCR analysis of ROCK-1 <i>versus</i> ROCK-2 mRNA relative to HPRT within primary mouse BECs (n = 3), astrocytes (n = 4) and neurons (n = 4). The ROCK-2 isoform is dominant also within each of the mouse brain cell types. In all panels bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001 compared to the respective reference column (on the left) by two-tailed unpaired (A) or paired (B, D) t-tests. # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001, #### <i>P</i><0.0001 (compared to all other groups if unspecified) by one-way ANOVA with Tukey’s post hoc analysis. In panels (B) and (E) the letters A and B represent independent cultures.</p

Selective ROCK-2 inhibition by KD025 blocks rt-PA-induced effects on BBB permeability under stroke-like conditions.

<p><b>(A)</b> Permeability changes in the <i>in vitro</i> human BBB 7.5 h post stimulation under oxygen-glucose deprivation (OGD) with DMSO as control or with rt-PA (25nM) and human plasminogen (plgn; 100nM), with or without KD025 (0.2, 2 and 20μM, added to both luminal and abluminal chambers). Data is presented relative to DMSO control under normoxia. n = 3, Bars represent mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01 compared to rt-PA+Plgn by one-way ANOVA with Newman–Keuls post hoc analysis. #<i>P</i><0.05 compared to DMSO OGD control by two-tailed t-test. <b>(B)</b> Comparison of selective ROCK-2 inhibition by KD025 (20μM) <i>versus</i> non-selective ROCK inhibition by fasudil (HA1077; 20μM) against rt-PA+plasminogen (25nM+100nM, respectively) 6 h and 7.5 h after treatment under OGD (relative to DMSO control under OGD). n = 3 for 6 h, n = 4 for 7.5 h. Data points represent mean±SEM. *<i>P</i><0.05 by one-way ANOVA with Tukey’s post hoc analysis. #<i>P</i><0.05 compared to rt-PA+Plgn by one-tailed paired t-test.</p

Selective ROCK-2 inhibition blocks rt-PA-induced MMP-2 activation in SVG human astrocytes, but not in BECs.

<p>Representative gelatine zymogram (left panels) and quantitation by densitometry (right panels) of active MMP-2, pro-MMP-2 (<b>A</b>) or their ratio (<b>B</b>) in media harvested from the luminal (endothelial; <b>A</b>) or the abluminal (astrocytic; <b>B</b>) chambers of the <i>in vitro</i> BBB 6 h post stimulation with DMSO (control) or with rt-PA+plasminogen (t-PA+Plgn; 25nM+100nM, respectively), in the presence or absence of KD025 (20μM). In (B) ratios obtained in the t-PA+Plgn group were assigned a value of 100%. KD025 reduces MMP activation in SVG, but not in BECs. n = 4. ****<i>P</i><0.0001 against all other groups by one-way ANOVA, **<i>P</i><0.01 against control or KD025 by two-way ANOVA with Tukey’s post hoc analysis.</p