Endothelial Activation Research Articles

Association between EASIX and acute kidney injury in critically ill cancer patients

BackgroundTo analyze the relationship between endothelial activation and stress index (EASIX) and the occurrence of acute kidney injury (AKI) in critically ill cancer patients. MethodsCritically ill cancer patients were selected from the Medical Information Mart for Intensive Care IV (MIMIC-IV). Multivariate logistic regression was used to analyze the association between EASIX and the occurrence of AKI in critically ill cancer patients.ResultsOne thousand forty-one cancer patients were retrospectively included, including 607 men and 434 women with mean age of 64.86 ± 13.67 years. Univariate analysis showed that high EASIX levels were associated with an increased risk of AKI occurrence in intensive care unit (ICU) cancer patients (OR: 1.47,95% CI: 1.13–1.91, P < 0.05). After adjusting for other confounders, high EASIX levels remained an independent risk factor predicting the development of AKI (OR: 1.42,95% CI: 1.08–1.88, P < 0.05). Trends in effect sizes were generally consistent across all subgroups in the prespecified subgroup analyses.ConclusionEASIX is an independent risk factor for AKI in critically ill cancer patients. More prospective studies are needed to validate the effect of EASIX on the occurrence of AKI in critically ill cancer patients in the future.

Navigating Hemorrhagic Shock: Biomarkers, Therapies, and Challenges in Clinical Care

Hemorrhagic shock remains a leading cause of preventable death worldwide, with mortality patterns varying significantly based on injury mechanisms and severity. This comprehensive review examines the complex pathophysiology of hemorrhagic shock, focusing on the temporal evolution of inflammatory responses, biomarker utility, and evidence-based therapeutic interventions. The inflammatory cascade progresses through distinct phases, beginning with tissue injury and endothelial activation, followed by a systemic inflammatory response that can transition to devastating immunosuppression. Recent advances have revealed pattern-specific responses between penetrating and blunt trauma, necessitating tailored therapeutic approaches. While damage control resuscitation principles and balanced blood product administration have improved outcomes, many molecular targeted therapies remain investigational. Current evidence supports early hemorrhage control, appropriate blood product ratios, and time-sensitive interventions like tranexamic acid administration. However, challenges persist in biomarker validation, therapeutic timing, and implementation of personalized treatment strategies. Future directions include developing precision medicine approaches, real-time monitoring systems, and novel therapeutic modalities while addressing practical implementation barriers across different healthcare settings. Success in hemorrhagic shock management increasingly depends on integrating multiple interventions across different time points while maintaining focus on patient-centered outcomes.

Splenic Irradiation Preceding Allogeneic Hematopoietic Stem Cell Transplantation as a Possible Risk Factor of Sinusoidal Obstruction Syndrome: A Report of Three Cases

Introduction: Splenomegaly is frequently encountered in patients with myeloproliferative neoplasms. Splenomegaly is associated with an increased incidence of engraftment failure during allogeneic hematopoietic stem cell transplantation (allo-HSCT), and some centers perform prior low-dose splenic irradiation (LDSI) which has been reported to be both safe and effective. However, we report conflicting results by presenting three allo-HSCT patients undergoing LDSI that subsequently developed sinusoidal obstruction syndrome (SOS). Case Presentation: The underlying diseases of the presented cases were atypical chronic myeloid leukemia, secondary myelofibrosis following essential thrombocythemia, and acute myeloid leukemia transforming from myeloproliferative neoplasm, unclassifiable. Endothelial Activation and Stress Index (EASIX) scores of the 3 patients were 0.40, 3.82, and 4.40, respectively. Conclusion: SOS is a potentially fatal complication of allo-HSCT, and the mortality rates of severe cases are reported to be above 80%. LDSI has not been recognized as a risk factor of SOS to date. Alternative management methods of splenomegaly during allo-HSCT such as ruxolitinib administration may be safer compared to LDSI.

Endothelial molecule levels (VCAM-1, ICAM-1) in uncomplicated malaria cases in Lagos, Nigeria

Background. Elevated endothelial activation markers are linked to malaria syndromes due to the sequestration of Plasmodium falciparum-infected red blood cells in deep tissue vessels. This study evaluated vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) levels in uncomplicated malaria and their association with host factors. Methods. A cross-sectional study was conducted in four health facilities in Ikorodu, Lagos State, Nigeria. Blood samples from children and adults were screened for malaria via microscopy, and plasma ICAM-1 and VCAM-1 levels were measured using ELISA. Results. ICAM-1 (1.03 × 106 ± 20,689.2 pg/ml) and VCAM-1 (1.11 × 106 pg/ml; range: 3,725–6,273,725 pg/ml) levels were significantly higher in malaria-positive cases compared to controls (p &lt; 0.01). The geometric mean parasite density was 11,183 parasites/μl, but ICAM-1 and VCAM-1 levels did not correlate with parasite density (p &gt; 0.1). Packed cell volume (PCV) was significantly lower in malaria-positive cases (p = 0.042), with ICAM-1 and VCAM-1 negatively correlating with PCV (p &lt; 0.05). Both markers were also inversely associated with age (p &lt; 0.05). Conclusion. VCAM-1 and ICAM-1 levels are elevated in uncomplicated malaria, independent of parasite density but negatively associated with PCV and age, highlighting the role of endothelial activation in malaria pathology.

Association between endothelial activation and stress index and 30-day mortality risk in acute myocardial infarction patients: a study based on the medical information mart for intensive care-IV database

ObjectiveThis study aimed to evaluate the association between the Endothelial Activation and Stress Index (EASIX) and 30-day mortality risk in acute myocardial infarction (AMI) patients.MethodsUsing a retrospective cohort design, data were extracted from the Medical Information Mart for Intensive Care-IV (MIMIC-IV) database between 2008 and 2019. Patients diagnosed with AMI at intensive care unit (ICU) admission were included. EASIX score was calculated as follows: lactate dehydrogenase (LDH) level (U/L) × creatinine level (mg/dL)/platelet count (109/L). Cox regression models assessed the association between EASIX and 30-day mortality, with subgroup analyses based on age, gender, AMI subtype, and sepsis status.ResultsA total of 1,036 patients were analyzed, among whom 323 did not survive beyond 30 days post-ICU admission. Higher EASIX scores were associated with increased 30-day mortality in AMI patients [Hazard ratio (HR): 1.70, 95% confidence interval (CI): 1.17–2.46, P = 0.005). Subgroup analyses supported these findings and revealed significant interactions between EASIX and variables such as gender and AMI subtype (P < 0.05).ConclusionElevated EASIX scores are significantly correlated with increased 30-day mortality risk in AMI patients, suggesting EASIX as a valuable prognostic tool that may inform clinical management strategies to improve outcomes in AMI.Clinical trial numberNot applicable.

Endothelial TLR4 Activation by Endogenous Ligands Contributes to Small Blood Vessels Formation in Angiolipoma

Endothelial TLR4 Activation by Endogenous Ligands Contributes to Small Blood Vessels Formation in Angiolipoma

Platelet count and hypertension as indicators of height loss in the general population: A prospective study

Circulating CD34-positive cell count is inversely associated with height loss. It acts as an indicator of endothelial repair activity. In conjunction with CD34-positive cells, platelets contribute to endothelial repair. The presence of hypertension increases the demand for endothelial repair. Therefore, platelet count could be associated with height loss among individuals with hypertension. A retrospective study of 2,343 individuals aged 40 to 79 years was conducted. Height loss was defined as being in the highest quartile of annual height decrease (1.6 mm/year for men and 2.0 mm/year for women). A significant inverse association between platelet count and height loss was observed only among participants with hypertension. After adjusting for known cardiovascular risk factors, the odds ratio (95% confidence interval) for height loss per 1 standard deviation increment in platelet count (5.09×104/μL for men and 5.03×104/μL for women) was 0.83 (0.70, 0.98) for participants with hypertension and 1.02 (0.90, 1.16) for participants without hypertension. Independent of known cardiovascular risk factors, platelets could prevent accelerated height loss among individuals with hypertension. Unlike CD34-positive cell count, platelet count and blood pressure, which are easy to assess in daily clinical practice, influence height loss.

Brain endothelial cell activation and dysfunction associate with and contribute to the development of enlarged perivascular spaces and cerebral small vessel disease.

Multiple injurious stimuli to the brain's endothelium results in brain endothelial cell activation and dysfunction (BECact/dys) with upregulation of inflammatory signaling cascades and a decrease in bioavailable nitric oxide respectively. These injurious stimuli initiate a brain injury and a response to injury wound healing genetically programed cascade of events, which result in cellular remodeling of the neurovascular unit and blood-brain barrier with increased inflammation and permeability. These remodeling changes also include the perivascular spaces that become dilated to form enlarged perivascular spaces (EPVS) that may be identified noninvasively by magnetic resonance imaging. These EPVS are associated with and considered to be a biomarker for cerebral small vessel disease (SVD) and a dysfunctional glymphatic system with impaired removal of neurotoxic waste, which ultimately results in neurodegeneration with impaired cognition and dementia. The penultimate section discusses the understudied role of venous cerebral circulation in relation to EPVS, SVD, and the vascular contribution to cognitive impairment (VCID). The focus of this review will be primarily on BECact/dys that associates with and contributes to the development of EPVS, SVD, and impaired glymphatic system efflux. Importantly, BECact/dys may be a key piece of the puzzle to unlock this complicated story of EPVS and SVD. Multiple transmission electron micrographs and illustrations will be utilized to depict anatomical ultrastructure and allow for the discussion of multiple functional molecular cascades.

CLEC14A facilitates angiogenesis and alleviates inflammation in diabetic wound healing

BackgroundDelayed wound healing is a serious complication of diabetic wounds, posing a significant challenge to the treatment of patients with diabetes. Diabetic wound healing is a complex dynamic process involving angiogenesis and inflammatory responses. Currently, there are limited targeted therapies to promote diabetic wound healing. This study aimed to reveal the role of CLEC14A in the process of diabetic wound healing, with the hope of identifying new therapeutic targets to accelerate the healing of diabetic wounds. MethodsIn vivo, diabetic mice were generated by combined streptozotocin (STZ) and high-fat diet treatment. The wound healing model was established in wild-type and Clec14a−/− diabetic mice. The degree of wound healing, as well as angiogenesis and inflammation during the healing process, were evaluated through Hematoxylin and Eosin (H&E) staining, immunohistochemical staining, and immunofluorescence staining. In vitro, the angiogenic activities of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed following treatment with high glucose and adenoviruses overexpressing CLEC14A, using scratch assays and tube formation assays. Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were utilized to evaluate the levels of inflammation in HUVECs. ResultsCLEC14A expression was suppressed in diabetic wounds. Deletion of the Clec14a inhibited angiogenesis and activated inflammatory responses in vivo. High-glucose treatment led to decreased CLEC14A expression, impaired angiogenic capacity, and elevated inflammatory levels in vitro. However, adenoviral-mediated overexpression of CLEC14A reversed the response induced by high glucose. ConclusionCLEC14A accelerates diabetic wound healing by promoting angiogenesis and reducing wound inflammation.

Dynamic conditioning of porcine kidney grafts with extracellular vesicles derived from urine progenitor cells: A proof-of-concept study.

: Among strategies to limit ischemia/reperfusion (IR) injuries in transplantation, cell therapy using stem cells to condition/repair transplanted organs appears promising. We hypothesized that using a cell therapy based on extracellular vesicles (EVs) derived from urine progenitor cells (UPCs) during hypothermic and normothermic machine perfusion can prevent IR-related kidney damage. We isolated and characterized porcine UPCs and their extracellular vesicles (EVs). Then these were used in an ex vivo porcine kidney preservation model. Kidneys were subjected to warm ischemia (32min) and then preserved by hypothermic machine perfusion (HMP) for 24h before 5h of normothermic machine perfusion (NMP). Three groups were performed (n=5-6): Group 1 (G1): HMP/vehicle + NMP/vehicle, Group 2 (G2): HMP/EVs + NMP/vehicle, Group 3 (G3): HMP/EVs + NMP/EVs. Porcine UPCs were successfully isolated from urine and fully characterized as well as their EVs which were found of expected size/phenotype. EVs injection during HMP alone, NMP alone, or both was feasible and safe and did not impact perfusion parameters. However, cell damage markers (LDH, ASAT) were decreased in G3 compared with G1, and G3 kidneys displayed a preserved tissue integrity with reduced tubular dilatation and inflammation notably. However, renal function indicators such as creatinine clearance measured for 5h of normothermic perfusion or NGAL perfusate's level were not modified by EVs injection. Regarding perfusate analysis, metabolomic analyses and cytokine quantification showed an immunomodulation signature in G3 compared with G1 and highlighted potential metabolic targets. In vitro, EVs as well as perfusates from G3 partially recovered endothelial cell metabolic activity after hypoxia. Finally, RNA-seq performed on kidney biopsies showed different profiles between G1 and G3 with regulation of potential IR targets of EVs therapy. We showed the feasibility/efficacy of UPC-EVs for hypothermic/normothermic kidney conditioning before transplantation, paving the way for combining machine perfusion with EVs-based cell therapy for organ conditioning. HIGHLIGHTS: ·UPCs from porcine urine can be used to generate a cell therapy product based on extracellular vesicles (pUPC-EVs). ·pUPC-EVs injection during HMP and NMP decreases cell damage markers and has an immunomodulatory effect. ·pUPC-EVs-treated kidneys have distinct biochemical, metabolic, and transcriptomic profiles highlighting targets of interest. ·Our results pave the way for combining machine perfusion with EV-based cell therapy for kidney conditioning.

Endothelial cell Ass1 inhibits arteriosclerotic calcification in diabetes mellitus

Endothelial cell (EC) dysfunction is an important pathological feature of early calcification in diabetic plaques. Argininosuccinic synthase 1 (Ass1) is important in protecting EC activity. Therefore, this study aimed to explore the effect of endothelial Ass1 on calcification in diabetic plaques and its potential regulatory mechanism. In this study, serum Ass1 levels were measured in 84 patients, and the study showed that the serum Ass1 level in patients with diabetes was significantly decreased compared with the non-diabetic group, and the serum Ass1 level in patients with coronary artery calcification was significantly decreased compared with the non-coronary artery calcification group. The ApoE-/- mouse diabetic plaque calcification model and the mouse aortic endothelial cell (MAEC) calcification model were constructed, and the influence of endothelial cell Ass1 on diabetic plaque calcification was further investigated by adeno-associated virus and plasmid intervention. Molecular biology studies have shown that endothelial Ass1 overexpression can reduce plaque calcification and inhibit MAEC osteogenic differentiation in diabetic mice, and Ass1 has protective effects on EC and blood vessels in mice. 4D-label-free proteomic sequencing, bioinformatics analysis, and IP experiments were performed on ApoE-/- mouse aorta after adeno-associated virus intervention. It was found that the differential protein Ptk2b was closely related to vascular calcification (VC) and interacted with the target protein Ass1. The above studies indicate that endothelial Ass1 affects calcification formation in diabetic plaques, and the mechanism may be related to Ptk2b. Ass1 may be a new target for the treatment of diabetic VC.

Bisphenol A and Its Emergent Substitutes: State of the Art of the Impact of These Plasticizers on Oxidative Stress and Its Role in Vascular Dysfunction

The “One Health approach” has evidenced the significant impact of xenobiotic exposure to health, and humans are a relevant target for their toxic effects. Bisphenol A (BPA) exerts a ubiquitous exposure source in all ecosystems. Given its endocrine-disrupting and harmful consequences on health, several countries have enforced new regulations to reduce exposure to BPA. Cardiovascular diseases (CVDs) are complex conditions that lead to higher mortality worldwide, where family history, lifestyle, and environmental factors, like BPA exposure, have a remarkable contribution. This chemical compound is the most widely used in plastic and epoxy resin manufacturing and has been associated with effects on human health. Therefore, new-generation bisphenols (NGBs) are replacing BPA use, arguing that they do not harm health. Nonetheless, the knowledge about whether NGBs are secure options is scanty. Although BPA’s effects on several organs and systems have been documented, the role of BPA and NGBs in CVDs has yet to be explored. This review’s goals are focused on the processes of endothelial activation (EA)–endothelial dysfunction (ED), a cornerstone of CVDs development, bisphenols’ (BPs) effects on these processes through oxidant and antioxidant system alteration. Despite the scarce evidence on pro-oxidant effects associated with NGBs, our review demonstrated a comparable harmful effect on BPA. The results from the present review suggest that the biological mechanisms to explain BPs cardiotoxic effects are the oxidant stress ↔ inflammatory response ↔ EA ↔ ED → atherosclerotic plate → coagulation promotion. Other effects contributing to CVD development include altered lipid metabolism, ionic channels, and the activation of different intracellular pathways, which contribute to ED perpetuation in a concerted manner.

Soluble P-selectin as an inflammatory mediator potentially influencing endothelial activation in people living with HIV in sub-rural areas of Limpopo, South Africa.

There is a growing need to understand the potential role of soluble platelet selectin (sP-selectin) in sustained endothelial activation through increased levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion-1 (sVCAM-1) in people living with HIV (PLWH) on highly active antiretroviral therapy (HAART). This was a cross-sectional study involving PLWH on HAART (n = 55), in comparison to PLWH not on treatment (HAART-naïve) (n = 29), and (iii) HIV negative controls (n = 48) from the Mankweng area in the Limpopo province, South Africa. We quantified serum levels of sP-selectin, together with sICAM-1 and sVCAM-1. Most of the HAART-exposed group were on treatment for <5 years. We further performed frequency distribution and descriptive statistics for categorical variables. Soluble P-selectin was positively correlated with sVCAM-1 (r = 0.469; p<0.001) in PLWH on HAART, even after adjusting for confounding factor such as age, BMI, and total cholesterol (r = 0.467; p<0.001). Moreover, in PLWH on HAART sP-selecting was independently associated with the release of sVCAM-1 (β = 0.445; p<0.001), even after adjusting for confounders (β = 0.475; p = 0.001). Serum levels of low-density lipoprotein cholesterol (LDL-C) (p = 0.004) and total cholesterol (p<0.001) were significantly higher in PLWH on HAART as compared to the HAART-naïve group. There is a need for more studies to investigate the role of sP-selectin in promoting endothelial activation and CVD-risk in PLWH on HAART, especially within the sub-Saharan Africa region.

Efficacy of 6% Hydroxyethyl Starch and 2% Lidocaine in Reducing the Pain of Propofol Injection: A randomised, placebo-controlled trial.

Propofol administration is associated with pain, mediated by the activation of vascular endothelium. Hydroxyethyl starch (HES) inhibits endothelial membrane activation by various nociceptive substances. Thus, this study hypothesised that pre-administration of HES can reduce pain on propofol administration. This study aimed to compare the proportion of patients with no pain on propofol administration and to compare the severity of pain and any change in pre- and post-induction haemodynamic variables. This prospective, randomised, placebo-controlled clinical trial was conducted at Chirayu Medical College & Hospital, Bhopal, India, between August 2023 and December 2023 and included patients undergoing elective surgery under general anaesthesia. Patients were randomly assigned to 3 groups to receive either 100 mL of 6% HES followed by propofol (Group HES), 100 mL normal saline (NS) followed by propofol premixed with 2% lidocaine (Group L) or 100 mL NS followed by propofol induction (Group P). A total of 339 patients were included. The proportion of patients with no pain on propofol injection was significantly higher in the HES group (n = 75) than in the lignocaine (n = 33) and placebo (n = 13) groups (P <0.0001 each). The median pain scores were 0 (interquartile range [IQR]: 0-1), 1 (IQR: 0-1) and 2 (IQR: 2-3) in the HES, lignocaine and placebo groups, respectively. The proportion of patients with moderate (n = 44) and severe (n = 48) pain scores was significantly higher in the placebo group than in the HES and lignocaine groups (P <0.0001 each). The proportion of patients experiencing pain on propofol injection was found to be significantly less with the pre-administration of 100 mL 6% HES compared to the pre-administration of lidocaine.

Epigenetic mechanisms mediate cytochrome P450 1A1 expression and lung endothelial injury caused by MRSA invitro and invivo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of severe pneumonia and acute respiratory distress syndrome (ARDS). To advance our mechanistic understanding of this important pathogen, we characterized the effects of MRSA-induced epigenetic modification of histone 3 lysine 9 acetylation (H3K9ac), an activator of gene transcription, on lung endothelial cells (EC), a critical site of ARDS pathophysiology. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis revealed that MRSA induces H3K9ac in the promoter regions of multiple genes, with the highest ranked peak annotated to the CYP1A1 gene. Subsequent experiments confirm that MRSA increases CYP1A1 protein and mRNA expression, and its enzymatic activity in EC. Epigenetic inhibitors (C646, RVX-208) reduce MRSA-induced CYP1A1 expression and inflammatory responses, including cytokine release and adhesion molecule expression. Inhibition of the Aryl hydrocarbon receptor (Ahr), a known mediator of CYP1A1 expression, blocks MRSA-induced upregulation of CYP1A1 mRNA and protein expression, enzyme activity, and cytokine release. Reduction of CYP1A1 protein expression by siRNA or inhibition of its activity by rhapontigenin attenuated MRSA-induced EC permeability and inflammatory responses. In a mouse model of MRSA-induced acute lung injury (ALI), inhibition of CYP1A1 activity by rhapontigenin improved multiple indices of ALI, including bronchoalveolar lavage (BAL) protein concentration, cytokine levels, and markers of endothelial damage. Analysis of publicly available data suggests upregulation of CYP1A1 expression in ARDS patients compared to ICU controls. In summary, these studies provide new insights into MRSA-induced lung injury and identify a novel functional role for epigenetic upregulation of CYP1A1 in lung EC during ARDS pathogenesis.

Post-ischemic inactivation of HIF Prolyl Hydroxylases in endothelium promotes maladaptive kidney repair by inducing glycolysis.

Ischemic acute kidney injury (AKI) is common in hospitalized patients and increases the risk for chronic kidney disease (CKD). Impaired endothelial cell (EC) functions are thought to contribute in AKI to CKD transition, but the underlying mechanisms remain unclear. Here, we identify a critical role for endothelial oxygen sensing prolyl hydroxylase domain (PHD) enzymes 1-3 in regulating post-ischemic kidney repair. In renal endothelium, we observed compartment-specific differences in the expression of the three PHD isoforms in both mice and humans. Post-ischemic concurrent inactivation of endothelial PHD1, PHD2, and PHD3 but not PHD2 alone promoted maladaptive kidney repair characterized by exacerbated tissue injury, fibrosis, and inflammation. Single-cell RNA-seq analysis of the post-ischemic endothelial PHD1, PHD2 and PHD3 deficient (PHDTiEC) kidney revealed an endothelial hypoxia and glycolysis related gene signature, also observed in human kidneys with severe AKI. This metabolic program was coupled to upregulation of the SLC16A3 gene encoding the lactate exporter monocarboxylate transporter 4 (MCT4). Strikingly, treatment with the MCT4 inhibitor syrosingopine restored adaptive kidney repair in PHDTiEC mice. Mechanistically, MCT4 inhibition suppressed pro-inflammatory EC activation reducing monocyte-endothelial cell interaction. Our findings suggest avenues for halting AKI to CKD transition based on selectively targeting the endothelial hypoxia-driven glycolysis/MCT4 axis.

Endothelial cell phenotype is linked to endothelial dysfunction in individuals with a family history of type 2 diabetes.

The patient's family history of type 2 diabetes (FH-DM2) has been negatively associated with the functionality of endothelial cells (ECs). Our objectives in this work were to use human umbilical vein endothelial cells (HUVECs) as a model, to substantiate whether FH-DM2 influences endothelial phenotype and impairs NO and ROS synthesis, cell metabolism, and mitochondrial activity of ECs from individuals with FH-DM2. In this study were evaluated the synthesis of reactive oxygen species (ROS) and nitric oxide (NO), mitochondrial membrane potential (MMP), mRNA of eNOS, glucose consumption, and lactate synthesis in HUVECs from newborns with FH-DM2. Furthermore, we also evaluated EC complexity and cell size through flow cytometry. Our results showed significant differences in HUVECs with FH-DM2, regarding their complexity and cell size, in the synthesis of ROS (p<0.01), and NO (p<0.05); they also reflected diminished glucose consumption and slight changes in the lactate levels. In conclusion, our results showed that HUVECs from children with FH-DM2 have a reduced capability of synthesizing ROS and NO, which might be linked to the metabolism of endothelial cells. These results are relevant since early endothelial dysfunction has been reported in individuals with FH-DM2, and could be used to establish preventive measures to reduce the risk of developing atherosclerosis or cardiovascular diseases in healthy individuals, but with this family background.

First-in-class mitogen-activated protein kinase (MAPK) p38a: MAPK-activated protein kinase 2 dual signal modulator with anti-inflammatory and endothelial-stabilizing properties

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38a mitogen-activated protein kinase (MAPK) near the binding site of its proinflammatory substrate, mitogen-activated protein kinase–activated protein kinase (MK)2. UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a-binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by surface plasmon resonance, 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells in vitro, and greater lung protection in vivo. GEn-1124 improved survival from 10%–40% in murine acute lung injury induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide instillation and febrile-range hyperthermia and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in tumor necrosis factor α–treated human pulmonary artery endothelial cells showed that the gene-modifying effects of GEn-1124 were much more restricted to tumor necrosis factor α–inducible genes than those of the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38a and MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex and dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance StatementWe describe a novel analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream proinflammatory and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in acute respiratory distress syndrome.

Elucidating novel mechanism of action of spiperone for drug repurposing to prevent and treat murine colitis and sepsis

AimsWhile Ca2+ signaling plays a vital role in maintaining normal endothelial function and vascular activity, aberrant Ca2+ signaling in endothelial dysfunction is involved in the pathogenesis of inflammation. As a safe anti-psychotic drug to mobilize Ca2+ signaling, we repurposed spiperone as a potential drug for two intestinal epithelial injury related diseases, colitis and sepsis. Materials and methodsSpiperone-induced vasorelaxation of human submucosal arterioles and mesenteric arterioles from wide-type and TRPV4 KO mice was determined by Mulvany-style wire myograph. The action of spiperone in HUVEC was tested by Ca2+ imaging and patch clamp, and its action on murine mesenteric arterioles was measured in vivo. LPS- and CLP-induced septic mice and DSS-induced colitic mice were used to examine the anti-inflammatory effects of spiperone. Key findingsSpiperone induced endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation of healthy arterioles with EC50 of ∼50 nM predominately via PLC/IP3/IP3R pathway to induce endoplasmic reticulum (ER) Ca2+ release and further to promote Ca2+ entry via TRPV4-constituted SOCE. In both LPS- and CLP-induced septic mice, spiperone effectively prevented and treated sepsis by reducing serum proinflammatory factors, alleviating multiple organ dysfunction, rescuing the impaired EDH-mediated vasorelaxation and improving murine survival rate. Similarly, spiperone could also protect against murine colitis. SignificanceWe reveal new action mode and mechanism of spiperone to induce EDH-mediated vasorelaxation of both human and murine arterioles to protect against colitis and sepsis by innovatively inducing PLC/IP3R/Ca2+ signaling rather than canonically antagonizing GPCR. Spiperone could be repurposed as a potential new drug for the prevention/treatment of colitis and sepsis.

Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway

Background: Pyroptosis, an inflammatory cell death, is involved in the progression of atherosclerosis. Pyroptosis in endothelial cells (ECs) and its underlying mechanisms in atherosclerosis are poorly understood. Here, we investigated the role of a caspase-4/5-NF-κB pathway in pyroptosis in palmitic acid (PA)-stimulated ECs and EVs as players in pyroptosis. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in an endothelial cell medium, treated with Ox-LDL, PA, caspase-4/5 inhibitor, NF-κB inhibitor, and sEV release inhibitor for 24 h, respectively. The cytotoxicity of PA was determined using an MTT assay, cell migration using a scratch-wound-healing assay, cell morphology using bright field microscopy, and lipid deposition using oil red O staining. The mRNA and protein expression of GSDM-D, CASP4, CASP5, NF-κB, NLRP3, IL-1β, and IL-18 were determined with RT-PCR and Western blot. Immunofluorescence was used to determine NLRP3 and ICAM-1 expressions. Extracellular vesicles (EVs) were isolated using an exosome isolation kit and were characterized by Western blot and scanning electron microscopy. Results: PA stimulation significantly changed the morphology of the HUVECs characterized by cell swelling, plasma membrane rupture, and increased LDH release, which are features of pyroptosis. PA significantly increased lipid accumulation and reduced cell migration. PA also triggered inflammation and endothelial dysfunction, as evidenced by NLRP3 activation, upregulation of ICAM-1 (endothelial activation marker), and pyroptotic markers (NLRP3, GSDM-D, IL-1β, IL-18). Inhibition of caspase-4/5 (Ac-FLTD-CMK) and NF-κB (trifluoroacetate salt (TFA)) resulted in a significant reduction in LDH release and expression of caspase-4/5, NF-κB, and gasdermin D (GSDM-D) in PA-treated HUVECs. Furthermore, GW4869, an exosome release inhibitor, markedly reduced LDH release in PA-stimulated HUVECs. EVs derived from PA-treated HUVECs exacerbated pyroptosis, as indicated by significantly increased LDH release and augmented expression of GSDM-D, NF-κB. Conclusions: The present study revealed that inflammatory, non-canonical caspase-4/5-NF-κB signaling may be one of the crucial mechanistic pathways associated with pyroptosis in ECs, and pyroptotic EVs facilitated pyroptosis in normal ECs during atherosclerosis.