Citations

Previous studies have established that cerebral blood vessels possess high capacity to synthesize prostacyclin (PGI 2 ). Substantial evidence indicates that amyloid precursor protein (APP) and soluble APPα (sAPPα) have neurotrophic and nueroprotective properties. We hypothesized that activation of IP-cAMP pathway stimulates non-amyloidogenic processing of APP in cerebral endothelial cells. Western blotting was used to detect protein expression; real time RT-PCR was employed to measure mRNA levels of APP, and a disintegrin and metalloprotease 10 (ADAM10, an α processing enzyme); sAPPα secretion was detected in condition medium derived from culturing human brain microvascular endothelial cells (BMVECs), by ELISA. After human BMVECs were treated with PGI 2 analog iloprost (0.01, 0.05, 0.1, or 0.5 μM) for 3 days, protein levels of ADAM10 (n=7-9, P<0.05) and APP (n=6, P<0.05) were significantly increased in a concentration dependent manner, while β amyloidogenic processing enzyme BACE1, and other α processing enzymes such as ADAM9 and ADAM17 were not changed. mRNA levels of APP (n=7, P<0.05) and ADAM10 (n=4, P<0.05), as well as sAPPα production in human BMVECs (n=6, P<0.05) were also significantly augmented in response to iloprost treatment. Moreover, stimulation of human BMVECs with forskolin (40μM) significantly enhanced APP and ADAM10 protein expression. Most notably, iloprost significantly augmented protein expression of peroxisome proliferation-activated receptor-δ (PPARδ) and SIRT1. We further explored the role of PPARδ in APP processing. ADAM10 expression (n=6, P<0.05), but not APP and BACE1, was significantly increased in human BMVECs treated with PPARδ ligand GW501516 (30 or 100 nM). GW501516 (100 nM) also significantly increase sAPPα production (n=9-10, P<0.05). siRNA against PPARδ significantly suppressed basal levels of ADAM10, and blocked iloprost-induced ADAM10 expression (n=4, P<0.05). These findings suggest that activation of IP-cAMP pathway enhances non-amyloidogenic processing of APP and consequently increases production of neurotrophic molecule sAPPα in human BMVECs. Activation of PPARδ in part mediates iloprost-induced α processing of APP.

Background: Cardiomyopathy-like phenotype has been recently reported in adult zebrafish stressed by either anemia or doxorubicin. However, it still remains unclear whether gene-based manipulation in this emerging animal model can faithfully recapitulate cardiomyopathy phenotypes in mammals. Here, we aim to address this question by generating the first transgenic model for cardiomyopathy in zebrafish. Results: We generated a panel of three transgenic fish lines, including Tg(cmlc2:Tnnt2) that overexpress wild type Tnnt2 in the whole heart, Tg(cmlc2:Tnnt2c) that drive a C-terminal amino acid truncated Tnnt2 (Tnnt2C) in the whole heart, and Tg(vmhc:Tnnt2c) that drives Tnnt2c expression only in the ventricle. Upon overexpression of the Tnnt2C, but not the full-length control, in the heart caused reduced ventricular size but enlarged atrium. A same morphological phenotype was noted in Tg(vmhc:Tnnt2c). At the cellular level, we found significantly reduced size of cardiomyocyte and increased cell proliferation. The morphological remodeling is accompanied by hallmarks of cardiomyopathy including reduced cardiac function and myofibril disarray. Conclusion: Transgenic overexpression of Tnnt2C in zebrafish faithfully recapitulates unique cardiomyopathy phenotypes noted in both human patients harboring the similar mutation and mice transgenic models, underscoring zebrafish as a conserved vertebrate model for cardiomyopathy. Our study further suggested that the dysfunction of Tnnt2C in ventricle is primarily responsible for cardiac phenotypes, while the enlarged atrium is a consequence of a defective ventricle.

Background: Reduced left atrial (LA) compliance (LAC) contributes to pulmonary venous hypertension in heart failure with preserved ejection fraction (HFpEF); however the underlying mechanisms are poorly defined. We hypothesized that microvascular inflammation with rarefaction and impaired nitric oxide (NO) signaling, LA titin hypophosphorylation and fibrosis may each contribute to reduced LAC in HFpEF. Methods: Invasive LA pressure-volume (PV) analyses (open-chest; pericardiotomy; admittance catheter) were performed in 9 aged dogs with experimental hypertension (bilateral renal wrap + DOCA; HFpEF) compared to 13 sham-operated young adult dogs (control). LAC was measured at end-LA reservoir during acute preload reduction (cava occlusion). LA tissue and LA microvascular function were analyzed. Results: Compared to controls, the curvilinear end-LA reservoir PV relationship ( Fig A ) was shifted upward/leftward in HFpEF (LA diastolic stiffness constant 0.08 vs 0.16 mmHg/mL in controls, p=0.005). LA microvascular density (MVD; CD31 stain) tended to be lower in HFpEF than controls (2154±578 vs 2650±523 vessels/mm 2 , p=0.07) and LA MVD negatively correlated with the LA diastolic stiffness constant (r=-0.66, p=0.006). LA microvessels from HFpEF dogs showed impaired endothelium-dependent shear stress-mediated dilation compared to controls, consistent with impaired NO signaling ( Fig B ). Titin isoform expression was similar between groups, but isoform N2B (stiffer) was (p=0.03) and N2BA (compliant) tended to be (p=0.09) hypophosphorylated in HFpEF LA appendage (LAA) tissue. Fibrosis (Picrosirius Red) was similar between groups in the LA free wall and LAA, but increased in areas adjacent to the pulmonary veins in HFpEF (27±12% vs controls 15±8%, p=0.01). Conclusion: In experimental HFpEF, LAC was reduced in association with evidence of LA microvascular rarefaction and impaired NO signaling, titin hypophosphorylation and regional increases in LA fibrosis.

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.

Increasing rates of infection by antibiotic resistant bacteria have led to a resurgence of interest in bacteriophage (phage) therapy. Several phage therapy studies in animals and humans have been completed over the last two decades. We conducted a systematic review of safety and toxicity data associated with phage therapy in both animals and humans reported in English language publications from 2008–2021. Overall, 69 publications met our eligibility criteria including 20 animal studies, 35 clinical case reports or case series, and 14 clinical trials. After summarizing safety and toxicity data from these publications, we discuss potential approaches to optimize safety and toxicity monitoring with the therapeutic use of phage moving forward. In our systematic review of the literature, we found some adverse events associated with phage therapy, but serious events were extremely rare. Comprehensive and standardized reporting of potential toxicities associated with phage therapy has generally been lacking in the published literature. Structured safety and tolerability endpoints are necessary when phages are administered as anti-infective therapeutics.

Epoxyeicosatrienoic acids (EETs) potently and stereospecifically activate the cardiac ATP‐sensitive K + (Kir6.2) channels by reducing channel sensitivity to ATP. In this study, we determined the effects of EETs on the binding of ATP to Kir6.2 by using 2,4,6‐trinitrophenyl (TNP)‐ATP, which increases fluorescence emission >8‐fold with binding to protein, and by using GST fusion protein containing Kir6.2. TNP‐ATP binds to the GST fusion protein containing the Kir6.2C‐terminus but not to that with the Kir6.2N‐terminus. Binding of TNP‐ATP to GST‐Kir6.2C (K D 5.0 μ M) was unchanged by 5 μ M 11,12‐EET (K D 4.4 μ M) but B max was reduced by half, suggesting non‐competitive inhibition of ATP binding. The effect of 11,12‐EET was dose‐dependent and 8,9‐ and 14,15‐EETs were equipotent inhibitors of TNP‐ATP binding to GST‐Kir6.2C. Arachidonic acid and 11,12‐DHET were not effective inhibitors, whereas the methyl ester of 11,12‐EET was as effective as 11,12‐EET in inhibiting TNP‐ATP binding to GST‐Kir6.2C. R195A mutation in GST‐Kir6.2C abolished the EET effects on TNP‐ATP binding, while the K185A mutation, which reduced ATP binding to Kir6.2, had no effect. These findings are consistent with previous patch clamp results that showed the epoxide group in EET and R195 in Kir6.2 are critical for K ATP channel activation by EET. We conclude that EETs bind to the C‐terminus of the K ATP channel and allosterically alter the ATP binding site, reducing the channel sensitivity to ATP.

We examined the role of H 2 S on shear stress‐mediated dilation of isolated mouse coronary arteries. Na 2 S produced concentration‐dependent dilation in these vessels, which was significantly inhibited by iberiotoxin. BK currents in mouse coronary smooth muscle cells were directly activated by Na 2 S, suggesting H 2 S produced vasodilation through BK channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm 2 ), producing graded dilatory responses, but such effects were diminished in the presence of 100 μM Na 2 S. Pre‐incubation with the cystathionine γ‐lyase inhibitor, D,L‐propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress‐mediated vasodilation. However, in the presence of L‐NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress‐mediated vasodilation, suggesting an interaction between NO and H 2 S signaling. Using cultured bovine aortic endothelial cells, eNOS activity was inhibited by Na 2 S. These results suggest that both NO and H 2 S are important shear stress‐mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H 2 S through inhibition of NO generation. (Supported by grants from NIH)

Abstract Background Pain is the highest prioritized patient-reported outcome in people with autosomal dominant polycystic kidney disease (ADPKD) but remains infrequently and inconsistently measured in clinical trials and poorly managed in clinical settings. A recently completed systematic review of pain in ADPKD identified 26 different outcome measures. None of these measures were considered appropriate as a core outcome measure due to the lack of patient-important dimensions, inadequate content, relatively long duration of completion time and limited evidence to support psychometric robustness. Methods We convened an international Standardized Outcomes in Nephrology–Polycystic Kidney Disease consensus workshop involving 21 patients/caregivers and 40 health professionals (clinicians, nurses, researchers, policy makers and industry representatives) from 18 countries to discuss the identification or development of a core outcome measure for pain. Results Four themes were identified highlighting fundamental issues for the measurement of pain in ADPKD: distressing and disrupting life participation; variability and ambiguity in defining pain; stigma, frustration and adaptation to pain; and ensuring validity and feasibility of pain measures. Conclusions Existing measures were found to be insufficient in capturing pain as a core outcome and there was consensus on the need for a new validated measure that is simple, succinct and addresses the impact of pain on life participation. This measure will facilitate the appropriate prioritization of pain in all trials and guide clinical decision making in people with ADPKD.

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3′-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. Significance: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR–tyrosine kinase inhibitor resistant-related MET amplification. This article is highlighted in the In This Issue feature, p. 2659

AbstractFerroptosis is a newly identified cell death mechanism and potential biomarker for hepatocellular carcinoma (HCC) therapy; however, its clinical relevance and underlying mechanism remain unclear. In this study, transcriptome and methylome data from 374 HCC cases were investigated for 41 ferroptosis‐related genes to identify ferroptosis activity‐associated subtypes. These subtypes were further investigated for associations with clinical and pathological variables, gene mutation landscapes, deregulated pathways and tumour microenvironmental immunity. A gene expression signature and predictive model were developed and validated using an additional 232 HCC cases from another independent cohort. Two distinct ferroptosis phenotypes (Ferroptosis‐H and Ferroptosis‐L) were identified according to ferroptosis gene expression and methylation in the patients with HCC. Patients with the Ferroptosis‐H had worse overall and disease‐specific survival, and the molecular subtypes were significantly associated with different clinical characteristics, mRNA expression patterns, tumour mutation profiles and microenvironmental immune status. Furthermore, a 15‐gene ferroptosis‐related prognostic model (FPM) for HCC was developed and validated which demonstrated accurate risk stratification ability. A nomogram included the FPM risk score, ECOG PS and hepatitis B status was developed for eventual clinical translation. Our results suggest that HCC subtypes defined by ferroptosis gene expression and methylation may be used to stratify patients for clinical decision‐making.