Pulmonary Vascular Signaling in Pulmonary Hypertension: Potential Role of Endothelial Ca2+ Signaling in Cellular Senescence and Inflammasome Activation.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

Ion channels in the lung vascular endothelial cells (LVECs) are essential in lung vascular function. Ca 2+ influx through Ca 2+ -permeable cation channels and K + efflux through K + -permeable non-selective cation channels (NSCC) and voltage-gated K + (K V ) channels have been implicated in regulating cell phenotypical transition, cell senescence and inflammasome activation. Single-cell RNAseq data reveals that multiple cation channels are expressed in LVECs. In this study, we aimed to characterize non-selective cation currents ( I NSCC ) and voltage-dependent inward ( I in ) and outward ( I out ) currents in human LVECs and examine the pharmacological properties of the channels expressed in LVECs. In the whole-cell recording configuration, we were able to record voltage-dependent I in and I out in LVECs bathed in extracellular solution containing 141 mM NaCl and 1.8 mM CaCl 2 (with the intracellular pipette solution containing 135 mM KCl, 5 mM Na 2 ATP and 10 mM EGTA) with the lineal currents subtracted using P/4 leakage-current subtraction protocol. Depolarizing the cells from a holding potential of -70 mV to a series of test potentials ranging from -40 to +20 mV elicited a rapidly inactivating I in and a rapidly inactivating component followed by a sustained component of I out . Approximately 13% of tested LVECs exhibited the rapidly inactivating voltage-dependent I in and I out , while all cells showed the sustained voltage-dependent I out . The voltage-dependent I in was present at -40 mV and maximized at 0 mV, similar to Ca 2+ currents through voltage-dependent Ca 2+ channels (VDCC). All cells showed sustained voltage-dependent I out (sensitive to 4-AP), likely K + currents through K V channels. These results indicate that human LVECs functionally express VDCC as responsible for rapidly inactivating I in and K V channels for at least two components of I out (rapidly inactivating and sustained currents). In these experiments, Ca 2+ -activated K + and ATP-sensitive K + currents were minimized by including 10 mM EGTA and 5 mM ATP in the Ca 2+ -free pipette (intracellular) solution. We also recorded I NSCC , elicited by both ramp and step voltage protocols, in LVECs held at 0 mV (which inactivates voltage-dependent channels) with no P/4 leakage current subtraction. Test potentials ranging from -80 to +80 mV in 20-mV increment from the holding potential of 0 mV elicited currents that reversed at 0 mV in LVECs with the extracellular solution containing 141 mM NaCl and 1.8 mM CaCl 2 and the intracellular (pipette) solution containing 135 mM KCl, 4 mM MgCl 2 , and 5 mM Na 2 ATP. The currents at -80, 0, and +80 mV were -154.8±20.8, 0.3±5.5, and 211.7±28.8 pA (n=38 cells), respectively. Both inward and outward currents were sensitive to 2-APB (100 µM). Given the Western blot and single-cell RNAseq data showing the expression of KCNA5 and TRPC6 channels in LVECs, we also examined the biophysical and pharmacological properties of these channels in KCNA5 - and TRPC6 -transfected HEK cells. In summary, our data indicate that human LVECs express multiple voltage-dependent and voltage-independent cation channels. Ca 2+ currents are potentially generated by, at least, Ca 2+ influx through VDCC and NSCC, while K + currents are carried by K + efflux through K V channels and NSCC. This study is supported in part by a grant from the NHLBI/NIH (HL171538). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Imbalance in lipid homeostasis is associated with discrepancies in immune signaling and is tightly linked to metabolic disorders. The diverse ways in which lipids impact immune signaling, however, remain ambiguous. The phospholipid phosphatidylinositol (PI), which is implicated in numerous immune disorders, is chiefly defined by its phosphorylation status. By contrast, the significance of the two fatty acid chains attached to the PI remains unknown. In this study, by using a mass spectrometry–based assay, we demonstrate a role for PI acyl group chains in regulating both the priming and activation steps of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in mouse macrophages. In response to NLRP3 stimuli, cells deficient in ABC transporter ATP Binding Cassette Subfamily B Member 1 (ABCB1), which effluxes lipid derivatives, revealed defective inflammasome activation. Mechanistically, Abcb1 deficiency shifted the total PI configuration exhibiting a reduced ratio of short-chain to long-chain PI acyl lipids. Consequently, Abcb1 deficiency initiated the rapid degradation of Toll/IL-1R domain–containing adaptor protein, the TLR adaptor protein that binds PI (4,5)-bisphosphate, resulting in defective TLR-dependent signaling, and thus NLRP3 expression. Moreover, this accompanied increased NLRP3 phosphorylation at the Ser291 position and contributed to blunted inflammasome activation. Exogenously supplementing wild-type cells with linoleic acid (LA), but not arachidonic acid, reconfigured PI acyl chains. Accordingly, LA supplementation increased Toll/IL-1R domain–containing adaptor protein degradation, elevated NLRP3 phosphorylation, and abrogated inflammasome activation. Furthermore, NLRP3 Ser291 phosphorylation was dependent on PGE2-induced protein kinase A signaling because pharmacological inhibition of this pathway in LA-enriched cells dephosphorylated NLRP3. Altogether, our study reveals, to our knowledge, a novel metabolic-inflammatory circuit that contributes to calibrating immune responses. ImmunoHorizons, 2022, 6: 642–659.

RATIONALE: Endothelial-to-mesenchymal transition (EndMT) is a cellular process in which endothelial cells (ECs) transition into mesenchymal cells, a mechanism recently linked to pulmonary vascular remodeling in pulmonary hypertension (PH). However, the regulatory mechanisms driving EndMT in PH remain largely undefined. MicroRNA-153 (miR-153), a small non-coding RNA, regulates gene expression by targeting the 3'-untranslated region (3'-UTR) of mRNA, affecting mRNA translation and stability. This study investigates miR-153's role in modulating EndMT in PH. METHODS: Human lung vascular endothelial cells (LVECs) were isolated from healthy individuals and patients with idiopathic pulmonary arterial hypertension (IPAH). In vitro, normal LVECs were exposed to hypoxia (3% O₂) for 72 hours or treated with TGF-β1 (10 ng/mL) for 7 days to induce EndMT. Levels of miR-153 and EndMT markers were quantified by qPCR, Western blotting, or immunostaining. Proliferation and apoptosis of ECs were assessed using EdU and TUNEL assays, respectively. LVECs were transfected with miR-153 inhibitor or mimic, then exposed to normoxic or hypoxic conditions, respectively for 72 hours. In vivo, mice were subjected to either hypoxia (10% O₂ for 6 weeks, Hx-PH) or Sugen5416/hypoxia (20 mg/kg i.p. plus 10% O₂ for 6 weeks, SuHx-PH) to induce PH. The PH phenotype in mice was evaluated by hemodynamic measurements and histological analysis of H&E-stained lung tissues. RESULTS:In silico analysis identified miR-153 as a direct regulator of human SNAI1 and SNAI2 genes via binding to their 3'-UTR. Downregulation of miR-153 in IPAH LVECs was correlated with elevated SNAI1/2 expression, increased EndMT, and higher proliferation compared to normal LVECs. Hypoxia or TGF-β1 treatment reduced miR-153 expression, increased EndMT transcription factors (SNAI1/2), mesenchymal markers (SM-22, vimentin), and reduced EC markers (CD31, VE-cadherin). In normoxic LVECs, miR-153 inhibition significantly upregulated SNAI1/2, increased EndMT and EC proliferation, and reduced apoptosis. Conversely, miR-153 mimic abolished hypoxia- and TGF-β1-induced EndMT. Overexpression of miR-153 attenuated hypoxia-induced EC survival. In PH mouse models, reduced miR-153 levels were associated with activated SNAI1/2, EndMT, and pulmonary vascular remodeling. CONCLUSIONS: Downregulation of miR-153 drives phenotypic transition of LVECs to proliferative mesenchymal cells via SNAI-mediated EndMT, contributing to pulmonary vascular remodeling and PH. Therefore, miR-153 could be a potential therapeutic target for PH treatment.

Dengue Virus (DENV) induces assembly of the NOD-like receptor (NLR) family pyrin domain containing-3 (NLRP3) inflammasome and autophagy, which are closely interconnected processes playing crucial roles in lipid metabolism and DENV replication. However, the autophagy–NLRP3 activation interplay during DENV infection in human endothelial cells remains incompletely understood. We aimed to elucidate effects of NLRP3 activation on autophagy during DENV-2 infection. We investigated how autophagy-related molecules are altered by NLRP3 inhibition and how this regulation affects lipid metabolism, through the master lipid transcription factors SREBP-1 and 2, which increase the expression of their target lipid-synthesizing genes such as fatty acid synthase (FAS) in a model of microvascular endothelial cells (HMEC-1). We demonstrated a dynamic interplay between inflammasome activity and autophagy in DENV-infected HMEC-1 cells: autophagy increases early during infection and decreases as inflammasome activity increases. NLRP3 inflammasome inhibition affects viral replication. Glyburide (an inflammasome inhibitor) treatment partially inhibited DENV-induced NLRP3 inflammasome activation. Non-structural viral protein expression (NS3 and NS5) and infectious viral-particle formation were significantly reduced. NLRP3 inhibition also downregulated SREBP-1 and SREBP-2 activation. These findings provide new insights into the modulation of the interconnected NLRP3 inflammasome, autophagy, and lipid metabolism pathways, presenting a promising therapeutic strategy for severe clinical forms of dengue.

Cannabinoid Receptor 1/miR-30b-5p Axis Governs Macrophage NLRP3 Expression and Inflammasome Activation in Liver Inflammatory Disease

The NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome is a multiprotein complex that orchestrates innate immune responses to infection and cell stress through activation of caspase-1 and maturation of inflammatory cytokines pro-interleukin-1β (pro-IL-1β) and pro-IL-18. Activation of the inflammasome during infection can be protective, but unregulated NLRP3 inflammasome activation in response to non-pathogenic endogenous or exogenous stimuli can lead to unintended pathology. NLRP3 associates with mitochondria and mitochondrial molecules, and activation of the NLRP3 inflammasome in response to diverse stimuli requires cation flux, mitochondrial Ca(2+) uptake, and mitochondrial reactive oxygen species accumulation. It remains uncertain whether NLRP3 surveys mitochondrial integrity and senses mitochondrial damage, or whether mitochondria simply serve as a physical platform for inflammasome assembly. The structure of the active, caspase-1-processing NLRP3 inflammasome also requires further clarification, but recent studies describing the prion-like properties of ASC have advanced the understanding of how inflammasome assembly and caspase-1 activation occur while raising new questions regarding the propagation and resolution of NLRP3 inflammasome activation. Here, we review the mechanisms and pathways regulating NLRP3 inflammasome activation, discuss emerging concepts in NLRP3 complex organization, and expose the knowledge gaps hindering a comprehensive understanding of NLRP3 activation.

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1EC-/-), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1EC-/- mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

In the NLR family, pyrin domain containing 3 (NLRP3) is an intracellular pattern recognition receptor that activates pro-caspase-1, leading to IL-1β and IL-18 processing and activation in a large complex called the NLRP3 inflammasome. Since various pathogens or endogenous metabolites have been reported to stimulate NLRP3 inflammasome, the interaction between NLRP3 and ASC induced by these stimulants may be an attractive drug target for NLRP3-related diseases, called inflammasomopathies. However, the endogenous ligand that directly interacts with NLRP3, leading to binding to ASC, remains unclear. Therefore, we developed a cell-free system consisting of NLRP3, ASC, and pro-caspase-1 or ASC and NLRP3 with an amplified luminescent proximity homogeneous assay (ALPHA). ALPHA signals of the interaction between NLRP3 and ASC were not enhanced following an incubation without any ligand, whereas strong ALPHA signals for the interaction between NLRP3 and ASC and between NLRP3 and pro-caspase-1 with the adaptor ASC were observed upon an incubation with poly (I:C) and hyaluronic acid (HA). Poly (I:C) and HA both directly interacted with NLRP3 within a specific concentration. These results suggest that NLRP3 directly interacts with intrinsic RNA and HA, which is followed by the activation of NLRP3 inflammasome, and the cell-free system consisting of NLRP3 and ASC, or NLRP3, ASC, and pro-caspase-1 may be a useful tool for elucidating the pathogenesis of inflammasomopathies and developing target therapeutics.

Background: Myocardial infarction (MI), or “heart attack”, remains a leading cause of death worldwide. MI is caused by prolonged coronary artery ischemia (I; blockage of blood flow) leading to cell death. While reperfusion (R) is therapeutic, continued activation of cell death pathways through distinct and overlapping mechanisms exacerbates damage beyond that due to I. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is activated during I/R, leading to pyroptosis. However, NLRP3 inhibition beyond 2 hr R fails to reduce MI size. Therefore, we sought to determine whether limiting NLRP3 priming prior to I is sufficient to reduce I/R injury and MI size. Purpose: To investigate the protective efficacy of NLRP3 gene knockout (KO) and loss of NLRP3 priming on MI size in male vs female rats. Methods: NLRP3 KO DAHL/SS- Nlrp3 em2Mcwi rats were generated via CRISPR-Cas9 (14 base pair deletion in exon 1 of NLRP3 gene) in Dahl salt sensitive rats. Wildtype (WT) and NLRP3 KO rats received Teklad 7034 low salt diet (0.12% NaCl) ad libitum to delay the development of hypertension. In-vivo coronary artery ligation (CAL; 31 min I) was used to induce MI in male (n=7) and female (n=13) WT (n=8) and NLRP3 KO (n=12) rats aged 90-135 days, with sham surgery as a non-ischemic control. Cardiac puncture was used to collect blood following 3 hr R to assess IL-18 levels and complete blood count (CBC). The left ventricle was harvested to determine area at risk (AAR) and infarct size using Evans Blue dye perfusion and 2,3,5-Triphenyltetrazolium chloride (TTC) staining, respectively. AAR and infarct size quantification was conducted using ImageJ, with data analyzed via 2-way ANOVA (Minitab). Data were presented with mean ± SEM. Results: Left ventricle weight (LVW)/right tibia length (RTL) was increased in NLRP3KO (male: 246.41 ± 5.51mg/cm; female: 201.98 ± 4.49 mg/cm) vs. WT (male: 233.26 ± 4.34 mg/cm; female: 191.11 ± 3.69 mg/cm; p < 0.05). Surprisingly, baseline plasma IL-18 levels were elevated in NLRP3 KO (male: 21.54 ± 2.00 pg/mL; female: 19.36 ± 1.80 pg/mL) vs. WT (male: 9.06 ± 2.01 pg/mL; female: 5.13 ± 1.55 pg/mL; p < 0.05). Following MI, NLRP3 KO rats had lower IL-18 levels (male: 148.26 ± 16.81 pg/mL; female: 170.18 ± 59.04 pg/mL) vs. WT (male: 252.01 ± 30.92 pg/mL; female: 330.39 ± 51.18 pg/mL; p < 0.05). Infarct size was reduced in NLRP3 KO vs WT females (KO vs. WT: 26.27% vs. 19.08%), but not males (KO vs. WT: 27.72% vs. 27.41%). Conclusion: IL-18 levels were reduced in NLRP3 KO groups with I/R as expected. The absence of NLPR3 may be cardioprotective in female but not male rats. Elevated IL-18 levels at baseline in NLRP3 KO require further investigation, but may suggest dysregulated Interferon-γ expression and/or alternative inflammasome activation, such as AIM2. Understanding divergent roles of NLRP3 inflammasome activation may inform sex-specific therapeutic interventions for the treatment of cardiac inflammation and MI. Our data also suggest a possible direct role for NLRP3 in the regulation of cardiac growth. This work was supported by NIH R21 AG070723-01 (DHK) and NIH training program T32GM108563 and T32GM154124. Animal protocols were approved by the Penn State IACUC #43534. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

Neuroinflammation associated with HIV-1 infection is a problem affecting ∼50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose- and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1β levels and enhanced the IL-1β secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1β mRNA expression and inflammasome activation (ASC oligomers and mature IL-1β). Intriguingly, LPS potentiated upregulation of IL-1β mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1β secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.SIGNIFICANCE STATEMENT Despite successful suppression of viremia with increased longevity in the era of combined antiretroviral therapy, chronic inflammation with underlying neurocognitive impairment continues to afflict almost 50% of infected individuals. Viral, bacterial, and cellular products have all been implicated in promoting the chronic inflammation found in these individuals. Understanding the molecular mechanism(s) by which viral proteins such as HIV-1 Transactivator of Transcription (Tat) protein can activate microglia is thus of paramount importance. Herein, we demonstrate a novel role of Tat in priming and activating NLR family pyrin domain containing 3 (NLRP3) inflammasomes in microglial cells and in HIV-Tg rats administered lipopolysaccharide. Targeting NLRP3 inflammasome pathway mediators could thus be developed as therapeutic interventions to alleviate or prevent neuroinflammation and subsequent cognitive impairment in HIV-positive patients.

Broxyquinoline targets NLRP3 to inhibit inflammasome activation and alleviate NLRP3-associated inflammatory diseases.

BackgroundWhile immune-based therapies induce durable remissions in subsets of patients across a wide range of malignancies, efficacy in metastatic castrate-resistant prostate cancer (mCRPC) is limited.1–3 One hypothesis for immunotherapy failure...