Transpulmonary generation of cell-free hemoglobin contributes to vascular dysfunction in pulmonary arterial hypertension via dysregulated clearance mechanisms.

Circulating cell-free hemoglobin (CFH) is elevated in pulmonary arterial hypertension (PAH) and associated with poor outcomes but the mechanisms are unknown. We hypothesized that CFH is generated from the pulmonary circulation and inadequately cleared in PAH. Transpulmonary CFH (difference between wedge and pulmonary artery positions) and lung hemoglobin α were analyzed in patients with PAH and healthy controls. Haptoglobin genotype and plasma hemoglobin processing proteins were analyzed in patients with PAH, unaffected bone morphogenetic protein receptor type II mutation carriers (UMCs), and control subjects. Transpulmonary CFH was increased in patients with PAH (p = 0.04) and correlated with pulmonary vascular resistanc (PVR) (r s = 0.75, p = 0.02) and mean pulmonary arterial pressure (mPAP) (r s = 0.78, p = 0.02). Pulmonary vascular hemoglobin α protein was increased in patients with PAH (p = 0.006), especially in occluded vessels (p = 0.04). Haptoglobin genotype did not differ between groups. Plasma haptoglobin was higher in UMCs compared with both control subjects (p = 0.03) and patients with HPAH (p < 0.0001); patients with IPAH had higher circulating haptoglobin levels than patients with HPAH (p = 0.006). Notably, circulating CFH to haptoglobin ratio was elevated in patients with HPAH compared to control subjects (p = 0.02) and UMCs (p = 0.006). Moreover, in patients with PAH, CFH: haptoglobin correlated with PVR (r s = 0.37, p = 0.0004) and mPAP (r s = 0.25, p = 0.02). Broad alterations in other plasma hemoglobin processing proteins (hemopexin, heme oxygenase-1, and sCD163) were observed. In conclusion, pulmonary vascular CFH is associated with increased PVR and mPAP in PAH and dysregulated CFH clearance may contribute to PAH pathology. Further study is needed to determine whether targeting CFH is a viable therapeutic for pulmonary vascular dysfunction in PAH.

Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1ß, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

Blocking P2X7 receptor with AZ 10606120 exacerbates vascular hyperpermeability and inflammation in murine polymicrobial sepsis.

Sepsis is a devastating disease with high morbidity and mortality and no specific treatments. The pathophysiology of sepsis involves a hyperinflammatory response and release of damage-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), from activated and dying cells. Purinergic receptors activated by ATP have gained attention for their roles in sepsis, which can be pro- or anti-inflammatory depending on the context. Current data regarding the role of ATP-specific purinergic receptor P2X7 (P2X7R) in vascular function and inflammation during sepsis are conflicting, and its role on the endothelium has not been well characterized. In this study, we hypothesized that the P2X7R antagonist AZ 10606120 (AZ106) would prevent endothelial dysfunction during sepsis. As proof of concept, we first demonstrated the ability of AZ106 (10 µM) to prevent endothelial dysfunction in intact rat aorta in response to IL-1ß, an inflammatory mediator upregulated during sepsis. Likewise, blocking P2X7R with AZ106 (10 µg/g) reduced the impairment of endothelial-dependent relaxation in mice subjected to intraperitoneal injection of cecal slurry (CS), a model of polymicrobial sepsis. However, contrary to our hypothesis, AZ106 did not improve microvascular permeability or injury, lung apoptosis, or illness severity in mice subjected to CS. Instead, AZ106 elevated spleen bacterial burden and circulating inflammatory markers. In conclusion, antagonism of P2X7R signaling during sepsis appears to disrupt the balance between its roles in inflammatory, antimicrobial, and vascular function.

A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation.

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biological underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histological abnormalities, and physiological dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied the timing and dose of the CS injection, fluids and antibiotics, and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, C-X-C motif chemokine ligand 1 (CXCL-1) or by lung wet:dry weight ratio. Addition of hyperoxia [95% fraction of inspired oxygen ([Formula: see text])] to CS immediately after CS injection increased BAL cell counts, CXCL-1, and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12-h exposures (0-12 h and 24-36 h) after CS injection increased survival to 75% and caused significant lung injury compared with CS alone as measured by increased BAL total cell count (92,500 vs. 240,000, P = 0.0004), BAL protein (71 vs. 103 µg/mL, P = 0.0030), and lung wet:dry weight ratio (4.5 vs. 5.5, P = 0.0005), and compared with sham as measured by increased BAL CXCL-1 (20 vs. 2,372 pg/mL, P < 0.0001) and histological lung injury score (1.9 vs. 4.2, P = 0.0077). In addition, our final model showed evidence of lung epithelial [increased BAL and plasma receptor for advanced glycation end products (RAGE)] and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using intraperitoneal injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.

Toxic effects of cell-free hemoglobin on the microvascular endothelium: implications for pulmonary and nonpulmonary organ dysfunction.

Levels of circulating cell-free hemoglobin are elevated during hemolytic and inflammatory diseases and contribute to organ dysfunction and severity of illness. Though several studies have investigated the contribution of hemoglobin to tissue injury, the precise signaling mechanisms of hemoglobin-mediated endothelial dysfunction in the lung and other organs are not yet completely understood. The purpose of this review is to highlight the knowledge gained thus far and the need for further investigation regarding hemoglobin-mediated endothelial inflammation and injury to develop novel therapeutic strategies targeting the damaging effects of cell-free hemoglobin.

Cell-free hemoglobin-mediated human lung microvascular endothelial barrier dysfunction is not mediated by cell death.

Circulating cell-free hemoglobin (CFH) contributes to endothelial injury in several inflammatory and hemolytic conditions. We and others have shown that CFH causes increased endothelial permeability, but the precise mechanisms of CFH-mediated endothelial barrier dysfunction are not fully understood. Based on our previous study in a mouse model of sepsis demonstrating that CFH increased apoptosis in the lung, we hypothesized that CFH causes endothelial barrier dysfunction through this cell death mechanism. We first confirmed that CFH causes human lung microvascular barrier dysfunction in vitro that can be prevented by the hemoglobin scavenger, haptoglobin. While CFH caused a small but significant decrease in cell viability measured by the membrane impermeable DNA dye Draq7 in human lung microvascular endothelial cells, CFH did not increase apoptosis as measured by TUNEL staining or Western blot for cleaved caspase-3. Moreover, inhibitors of apoptosis (Z-VAD-FMK), necrosis (IM-54), necroptosis (necrostatin-1), ferroptosis (ferrostatin-1), or autophagy (3-methyladenine) did not prevent CFH-mediated endothelial barrier dysfunction. We conclude that although CFH may cause a modest decrease in cell viability over time, cell death does not contribute to CFH-mediated lung microvascular endothelial barrier dysfunction.

Standardization of methods for sampling the distal airspace in mechanically ventilated patients using heat moisture exchange filter fluid.

Noninvasive sampling of the distal airspace in patients with acute respiratory distress syndrome (ARDS) has long eluded clinical and translational researchers. We recently reported that fluid collected from heat moisture exchange (HME) filters closely mirrors fluid directly aspirated from the distal airspace. In the current study, we sought to determine fluid yield from different HME types, optimal HME circuit dwell time, and reliability of HME fluid in reflecting the distal airspace. We studied fluid yield from four different filter types by loading increasing volumes of saline and measuring volumes of fluid recovered. We collected filters after 1, 2, and 4 h of dwell time for measurement of fluid volume and total protein from 13 subjects. After identifying 4 h as the optimal dwell time, we measured total protein and IgM in HME fluid from 42 subjects with ARDS and nine with hydrostatic pulmonary edema (HYDRO). We found that the fluid yield varies greatly by filter type. With timed sample collection, fluid recovery increased with increasing circuit dwell time with a median volume of 2.0 mL [interquartile range (IQR) 1.2-2.7] after 4 h. Total protein was higher in the 42 subjects with ARDS compared with nine with HYDRO [median 708 µg/mL (IQR 244-2017) vs. 364 µg/mL (IQR 136-578), P = 0.047], confirming that total protein concentration in HME is higher in ARDS compared with hydrostatic edema. These studies establish a standardized HME fluid collection protocol and confirm that HME fluid analysis is a novel noninvasive tool for the study of the distal airspace in ARDS.

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

OBJECTIVES: We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvß3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation. METHODS AND RESULTS: Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition. CONCLUSION: The FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Cell-free hemoglobin increases inflammation, lung apoptosis, and microvascular permeability in murine polymicrobial sepsis.

Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.

AKT2 maintains brain endothelial claudin-5 expression and selective activation of IR/AKT2/FOXO1-signaling reverses barrier dysfunction.

Inflammation-induced blood-brain barrier (BBB) dysfunction and microvascular leakage are associated with a host of neurological disorders. The tight junction protein claudin-5 (CLDN5) is a crucial protein necessary for BBB integrity and maintenance. CLDN5 is negatively regulated by the transcriptional repressor FOXO1, whose activity increases during impaired insulin/AKT signaling. Owing to an incomplete understanding of the mechanisms that regulate CLDN5 expression in BBB maintenance and dysfunction, therapeutic interventions remain underdeveloped. Here, we show a novel isoform-specific function for AKT2 in maintenance of BBB integrity. We identified that AKT2 during homeostasis specifically regulates CLDN5-dependent barrier integrity in brain microvascular endothelial cells (BMVECs) and that intervention with a selective insulin-receptor (IR) agonist, demethylasterriquinone B1 (DMAQ-B1), rescued IL-1ß-induced AKT2 inactivation, FOXO1 nuclear accumulation, and loss of CLDN5-dependent barrier integrity. Moreover, DMAQ-B1 attenuated preclinical CLDN5-dependent BBB dysfunction in mice subjected to experimental autoimmune encephalomyelitis. Taken together, the data suggest a regulatory role for IR/AKT2/FOXO1-signaling in CLDN5 expression and BBB integrity during neuroinflammation.

Endothelial microvesicles carrying Src-rich cargo impair adherens junction integrity and cytoskeleton homeostasis.

AIMS: Microvesicles (MVs) conduct intercellular communication and impact diverse biological processes by transferring bioactive cargos to other cells. We investigated whether and how endothelial production of MVs contribute to vascular dysfunction during inflammation. METHODS AND RESULTS: We measured the levels and molecular properties of endothelial-derived MVs (EC-MVs) from mouse plasma following a septic injury elicited by cecal ligation and puncture, as well as those from supernatants of cultured endothelial cells stimulated by inflammatory agents including cytokines, thrombin, and complement 5a. The mouse studies showed that sepsis caused a significant increase in total plasma vesicles and VE-cadherin+ EC-MVs compared to sham control. In cultured ECs, different inflammatory agents caused diverse patterns of EC-MV production and cargo contents. When topically applied to endothelial cells, EC-MVs induced a cytoskeleton-junction response characterized by myosin light chain phosphorylation, contractile fibre reorganization, VE-cadherin phosphorylation, and adherens junction dissociation, functionally measured as increased albumin transendothelial flux and decreased barrier resistance. The endothelial response was coupled with protein tyrosine phosphorylation promoted by MV cargo containing c-Src kinase, whereas MVs produced from c-Src deficient cells did not exert barrier-disrupting effects. Additionally, EC-MVs contribute to endothelial inflammatory injury by promoting neutrophil-endothelium adhesion and release of neutrophil extracellular traps containing citrullinated histones and myeloperoxidase, a response unaltered by c-Src knockdown. CONCLUSION: Endothelial-derived microparticles cause endothelial barrier dysfunction by impairing adherens junctions and activating neutrophils. The signalling mechanisms underlying the endothelial cytoskeleton-junction response to EC-MVs involve protein phosphorylation promoted by MV cargo carrying c-Src. However, EC-MV-induced neutrophil activation was not dependent on c-Src.

Thrombin-cleaved syndecan-3/-4 ectodomain fragments mediate endothelial barrier dysfunction.

OBJECTIVE: The endothelial glycocalyx constitutes part of the endothelial barrier but its degradation leaves endothelial cells exposed to transmigrating cells and circulating mediators that can damage the barrier or promote intercellular gaps. Syndecan proteins are key components of the endothelial glycocalyx and are shed during disease states where expression and activity of proteases such as thrombin are elevated. We tested the ability of thrombin to cleave the ectodomains of syndecans and whether the products could act directly on endothelial cells to alter barrier function. APPROACH AND RESULTS: Using transmission electron microscopy, we illustrated the presence of glycocalyx in human lung microvasculature. We confirmed expression of all syndecan subtypes on the endothelial surface of agarose-inflated human lungs. ELISA and western blot analysis suggested that thrombin can cleave syndecan-3/-4 ectodomains to produce fragments. In vivo, syndecan-3 ectodomain fragments increased extravasation of albumin-bound Evans blue in mouse lung, indicative of plasma protein leakage into the surrounding tissue. Syndecan-3/-4 ectodomain fragments decreased transendothelial electrical resistance, a measure of cell-cell adhesive barrier integrity, in a manner sensitive to a Rho kinase inhibitor. These effects were independent of glycosylation and thrombin receptor PAR1. Moreover, these cleavage products caused rapid VE-cadherin-based adherens junction disorganization and increased F-actin stress fibers, supporting their direct effect on endothelial paracellular permeability. CONCLUSIONS: We suggest that thrombin can cleave syndecan-3/4 ectodomain into fragments which interact with endothelial cells causing paracellular hyperpermeability. This may have important implications in the pathogenesis of vascular dysfunction during sepsis or thrombotic disease states where thrombin is activated.

Role of Neutrophil Extracellular Traps and Vesicles in Regulating Vascular Endothelial Permeability.

The microvascular endothelium serves as the major barrier that controls the transport of blood constituents across the vessel wall. Barrier leakage occurs during infection or sterile inflammation, allowing plasma fluid and cells to extravasate and accumulate in surrounding tissues, an important pathology underlying a variety of infectious diseases and immune disorders. The leak process is triggered and regulated by bidirectional communications between circulating cells and vascular cells at the blood-vessel interface. While the molecular mechanisms underlying this complex process remain incompletely understood, emerging evidence supports the roles of neutrophil-endothelium interaction and neutrophil-derived products, including neutrophil extracellular traps and vesicles, in the pathogenesis of vascular barrier injury. In this review, we summarize the current knowledge on neutrophil-induced changes in endothelial barrier structures, with a detailed presentation of recently characterized molecular pathways involved in the production and effects of neutrophil extracellular traps and extracellular vesicles. Additionally, we discuss the therapeutic implications of altering neutrophil interactions with the endothelial barrier in treating inflammatory diseases.

Citrullinated histone 3 causes endothelial barrier dysfunction.

Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.

A disintegrin and metalloproteinase 15-mediated glycocalyx shedding contributes to vascular leakage during inflammation.

Aims: Endothelial hyperpermeability exacerbates multiple organ damage during inflammation or infection. The endothelial glycocalyx, a protective matrix covering the luminal surface of endothelial cells (ECs), undergoes enzymatic shedding during inflammation, contributing to barrier hyperpermeability. A disintegrin and metalloproteinase 15 (ADAM15) is a sheddase capable of cleaving the ectodomains of membrane-bound molecules. Herein, we tested whether and how ADAM15 is involved in glycocalyx shedding and vascular leakage during sepsis. Methods and results: Dextran-150kD exclusion assay revealed lipopolysaccharide (LPS) significantly reduced glycocalyx thickness in mouse cremaster microvessels. Consistently, shedding products of glycocalyx constituents, including CD44 ectodomain, were detected with an increased plasma level after cecal ligation and puncture (CLP)-induced sepsis. The direct effects of CD44 ectodomain on endothelial barrier function were evaluated, which revealed CD44 ectodomain dose-dependently reduced transendothelial electrical resistance (TER) and caused cell-cell adherens junction disorganization. Furthermore, we examined the role of ADAM15 in CD44 cleavage and glycocalyx shedding. An in vitro cleavage assay coupled with liquid chromatography-tandem mass spectrometry confirmed ADAM15 cleaved CD44 at His235-Thr236 bond. In ECs with ADAM15 knockdown, LPS-induced CD44 cleavage and TER reduction were greatly attenuated, whereas, ADAM15 overexpression exacerbated CD44 cleavage and TER response to LPS. Consistently, ADAM15 knockout in mice attenuated CLP-induced increase in plasma CD44. Intravital and electron microscopic images revealed ADAM15 deficiency prevented LPS-induced glycocalyx injury in cremaster and pulmonary microvasculatures. Functionally, ADAM15-/- mice with better-preserved glycocalyx exhibited resistance to LPS-induced vascular leakage, as evidenced by reduced albumin extravasation in pulmonary and mesenteric vessels. Importantly, in intact, functionally vital human lungs, perfusion of LPS induced a significant up-regulation of ADAM15, accompanied by elevated CD44 in the effluent and increased vascular permeability to albumin. Conclusion: Together, our data support the critical role of ADAM15 in mediating vascular barrier dysfunction during inflammation. Its mechanisms of action involve CD44 shedding and endothelial glycocalyx injury.

Neutrophil-mediated vascular barrier injury: Role of neutrophil extracellular traps.

Neutrophils play an essential role in host defense against infection or injury. While neutrophil activation is necessary for pathogen clearance and tissue repair, a hyperactive response can lead to tissue damage and microcirculatory disorders, a process involving complex neutrophil-endothelium cross talk. This review highlights recent research findings about neutrophil-mediated signaling and structural changes, including those induced by neutrophil extracellular traps, which ultimately lead to vascular barrier injury.

Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome.

Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCß1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCß1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCß1 palmitoylation in endothelial inflammation.