Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.

Adiponectin (APN), a protein abundantly secreted from adipocytes, has been reported to possess beneficial effects on cardiovascular diseases in association with its accumulation on target organs and cells by binding to T-cadherin. However, little is known about the role of APN in the development of diabetic microvascular complications, such as diabetic retinopathy (DR). Here we investigated the impact of APN on the progression of early retinal vascular damage using a streptozotocin (STZ)-induced diabetic mouse model. Our immunofluorescence results clearly showed T-cadherin-dependent localization of APN in the vascular endothelium of retinal arterioles, which was progressively decreased during the course of diabetes. Such reduction of retinal APN accompanied the early features of DR, represented by increased vascular permeability, and was prevented by glucose-lowering therapy with dapagliflozin, a selective sodium-glucose co-transporter 2 inhibitor. In addition, APN deficiency resulted in severe vascular permeability under relatively short-term hyperglycemia, together with a significant increase in vascular cellular adhesion molecule-1 (VCAM-1) and a reduction in claudin-5 in the retinal endothelium. The present study demonstrated a possible protective role of APN against the development of DR.

As multiple spacefaring nations contemplate extended manned missions to Mars and the Moon, health risks could be elevated as travel goes beyond the Earth's protective magnetosphere into the more intense deep space radiation environment. The primary purpose of this study was to determine whether mortality rates due to cardiovascular disease (CVD), cancer, accidents and all other causes of death differ in (1) astronauts who never flew orbital missions in space, (2) astronauts who flew only in low Earth orbit (LEO), and (3) Apollo lunar astronauts, the only humans to have traveled beyond Earth's magnetosphere. Results show there were no differences in CVD mortality rate between non-flight (9%) and LEO (11%) astronauts. However, the CVD mortality rate among Apollo lunar astronauts (43%) was 4-5 times higher than in non-flight and LEO astronauts. To test a possible mechanistic basis for these findings, a secondary purpose was to determine the long-term effects of simulated weightlessness and space-relevant total-body irradiation on vascular responsiveness in mice. The results demonstrate that space-relevant irradiation induces a sustained vascular endothelial cell dysfunction. Such impairment is known to lead to occlusive artery disease, and may be an important risk factor for CVD among astronauts exposed to deep space radiation.

Although concern remains about the athero-thrombotic risk posed by cyclo-oxygenase (COX)-2-selective inhibitors, recent data implicates rofecoxib, while celecoxib appears equivalent to NSAIDs naproxen and ibuprofen. We investigated the hypothesis that celecoxib activates AMP kinase (AMPK) signalling to enhance vascular endothelial protection. In human arterial and venous endothelial cells (EC), and in contrast to ibuprofen and naproxen, celecoxib induced the protective protein heme oxygenase-1 (HO-1). Celecoxib derivative 2,5-dimethyl-celecoxib (DMC) which lacks COX-2 inhibition also upregulated HO-1, implicating a COX-2-independent mechanism. Celecoxib activated AMPKα(Thr172) and CREB-1(Ser133) phosphorylation leading to Nrf2 nuclear translocation. Importantly, these responses were not reproduced by ibuprofen or naproxen, while AMPKα silencing abrogated celecoxib-mediated CREB and Nrf2 activation. Moreover, celecoxib induced H-ferritin via the same pathway, and increased HO-1 and H-ferritin in the aortic endothelium of mice fed celecoxib (1000 ppm) or control chow. Functionally, celecoxib inhibited TNF-α-induced NF-κB p65(Ser536) phosphorylation by activating AMPK. This attenuated VCAM-1 upregulation via induction of HO-1, a response reproduced by DMC but not ibuprofen or naproxen. Similarly, celecoxib prevented IL-1β-mediated induction of IL-6. Celecoxib enhances vascular protection via AMPK-CREB-Nrf2 signalling, a mechanism which may mitigate cardiovascular risk in patients prescribed celecoxib. Understanding NSAID heterogeneity and COX-2-independent signalling will ultimately lead to safer anti-inflammatory drugs.

Preeclampsia (PE) is a pregnancy syndrome characterized by hypertension and organ damage manifesting after 20 gestational weeks. The etiology is of multifactorial origin, where placental stress causes increased levels of placenta-derived extracellular vesicles (STBEVs) in the maternal circulation, shown to cause inflammation, endothelial activation, vasoconstriction, and anti-angiogenic activity. General endothelial dysfunction is believed to be initiated by endothelial insult during pregnancy that alters vascular function resulting in increased arterial stiffness, cardiac dysfunction, and increased risk of cardiovascular disease later in life. We compared the effect of normal and PE derived STBEVs in vitro on vascular contractility of human subcutaneous arteries using wire myography. Cellular structures of exposed vessels were investigated by transmission electron microscopy. We explored strategies to pharmacologically block the effects of the STBEVs on human vessels. The PE STBEVs caused significantly stronger angiotensin II-mediated contractions and extended structural damage to human subcutaneous arteries compared to normal STBEVs. These negative effects could be reduced by blocking vesicle uptake by endothelial cells, using chlorpromazine or specific antibodies towards the LOX-1 receptor. The therapeutic potential of blocking vesicle uptake should be further explored, to reduce the permanent damage caused on the vasculature during PE pregnancy to prevent future cardiovascular risk.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

Various anticoagulant properties have been associated with hydroxyethyl starch (HES). However, the mechanism remains unclear and it has not been fully considered whether these properties are beyond the dilutional effect itself. The aim of this study was to reproduce the coagulopathy induced by HES and to test the hypothesis that the coagulopathy is caused by endothelial or glycocalyx damage due to localization of HES on the endothelium, which is caused by the high shear viscosity of dilutional blood. Using a rat model, we compared blood coagulability measured by Sonoclot, levels of endothelial and glycocalyx damage markers and coagulation factors, and blood shear viscosity when hemodilution was performed with physiological saline (PS), 6% HES 130/0.4 in PS, and 10% HES 200/0.5 in PS. We also evaluated the localization rates of fluorescently labeled HES on endothelium in the isolated aorta. HES decreased the fibrin gel formation rate more than did PS. HES was shown to cover the endothelium, possibly due to its high shear viscosity, and this mechanism potentially acted to protect, rather than damage, the endothelium and glycocalyx. However, this covering effect may be the cause of coagulopathy due to inhibition of von Willebrand factor secretion from the endothelium.

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was fivefold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia.

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether β1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6 J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of β1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for β1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that β1 integrin-blocking antibody or endothelial cell-specific depletion of β1 integrin attenuated FMD of the femoral artery, and blocking of β1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial β1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS.

There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease.

Activated factor X (FXa) plays a central role in the coagulation cascade, while it also mediates vascular function through activation of protease-activated receptors (PARs). Here, we examined whether inhibition of FXa by rivaroxaban, a direct FXa inhibitor, attenuates endothelial dysfunction in streptozotocin (STZ)-induced diabetic mice. Induction of diabetes increased the expression of a major FXa receptor, PAR2, in the aorta (P < 0.05). Administration of rivaroxaban (10 mg/kg/day) to diabetic wild-type (WT) mice for 3 weeks attenuated endothelial dysfunction as determined by acetylcholine-dependent vasodilation compared with the control (P < 0.001), without alteration of blood glucose level. Rivaroxaban promoted eNOSSer1177 phosphorylation in the aorta (P < 0.001). Induction of diabetes to PAR2-deficient (PAR2-/-) mice did not affect endothelial function and eNOSSer1177 phosphorylation in the aorta compared with non-diabetic PAR2-/- mice. FXa or a PAR2 agonist significantly impaired endothelial function in aortic rings obtained from WT mice, but not in those from PAR2-/- mice. FXa promoted JNK phosphorylation (P < 0.01) and reduced eNOSSer1177 phosphorylation (P < 0.05) in human coronary artery endothelial cells (HCAEC). FXa-induced endothelial dysfunction in aortic rings (P < 0.001) and eNOSSer1177 phosphorylation (P < 0.05) in HCAEC were partially ameliorated by a JNK inhibitor. Rivaroxaban ameliorated diabetes-induced endothelial dysfunction. Our results suggest that FXa or PAR2 is a potential therapeutic target.

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.

Correction of barrier dysfunction and inflammation in acute lung injury (ALI) represents an important problem. Previous studies demonstrate barrier-protective and anti-inflammatory effects of bioactive lipid prostacyclin and its stable analog iloprost (ILO). We generated a phospholipase resistant synthetic phospholipid with iloprost attached at the sn-2 position (ILO-PC) and investigated its biological effects. In comparison to free ILO, ILO-PC caused sustained endothelial cell (EC) barrier enhancement, linked to more prolonged activation of Rap1 and Rac1 GTPases and their cytoskeletal and cell junction effectors: cortactin, PAK1, p120-catenin and VE-cadherin. ILO and ILO-PC equally efficiently suppressed acute, Rho GTPase-dependent EC hyper-permeability caused by thrombin. However, ILO-PC exhibited more sustained barrier-protective and anti-inflammatory effects in the model of chronic EC dysfunction caused by bacterial wall lipopolysacharide (LPS). ILO-PC was also more potent inhibitor of NFκB signaling and lung vascular leak in the murine model of LPS-induced ALI. Treatment with ILO-PC showed more efficient ALI recovery over 3 days after LPS challenge than free ILO. In conclusion, this study describes a novel synthetic phospholipid with barrier-enhancing and anti-inflammatory properties superior to existing prostacyclin analogs, which may be used as a prototype for future development of more efficient treatment for ALI and other vascular leak syndromes.

Vascular endothelial cells play a vital role in the health and maintenance of vascular homeostasis, but hyperglycemia disrupts their function by increasing cellular oxidative stress. Resveratrol, a plant polyphenol, possesses antioxidant properties that can mitigate oxidative stress. Addressing the challenges of its limited solubility and stability, gold nanoparticles (GNps) were utilized as carriers. A microfluidic chip (MFC) with dynamic flow conditions was designed to simulate body vessels and to investigate the antioxidant properties of resveratrol gold nanoparticles (RGNps), citrate gold nanoparticles (CGNps), and free Resveratrol on human umbilical vein endothelial cells (HUVEC). The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to measure the extracellular antioxidant potential, and cell viability was determined using the Alamar Blue test. For assessing intracellular oxidative stress, the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was conducted, and results from both the cell culture plate and MFC were compared. Free Resveratrol demonstrated peak DPPH scavenging activity but had a cell viability of about 24-35%. RGNPs, both 3.0 ± 0.5 nm and 20.2 ± 4.7 nm, consistently showed high cell viability (more than about 90%) across tested concentrations. Notably, RGNPs (20 nm) exhibited antioxidative properties through DPPH scavenging activity (%) in the range of approximately 38-86% which was greater than that of CGNps at about 21-32%. In the MFC,the DCFH-DA analysis indicated that RGNPs (20 nm) reduced cellular oxidative stress by 57-82%, surpassing both CGNps and free Resveratrol. Morphologically, cells in the MFC presented superior structure compared to those in traditional cell culture plates, and the induction of hyperglycemia successfully led to the formation of multinucleated variant endothelial cells (MVECs). The MFC provides a distinct advantage in observing cell morphology and inducing endothelial cell dysfunction. RGNps have demonstrated significant potential in alleviating oxidative stress and preventing endothelial cell disorders.

Vascular dysfunction is an important phenomenon in hypertension. We hypothesized that angiotensin II (AngII) affects transcriptome in the vasculature in a region-specific manner, which may help to identify genes related to vascular dysfunction in AngII-induced hypertension. Mesenteric artery and aortic transcriptome was profiled using Illumina WG-6v2.0 chip in control and AngII infused (490 ng/kg/min) hypertensive mice. Gene set enrichment and leading edge analyses identified Sphingosine kinase 1 (Sphk1) in the highest number of pathways affected by AngII. Sphk1 mRNA, protein and activity were up-regulated in the hypertensive vasculature. Chronic sphingosine-1-phosphate (S1P) infusion resulted in a development of significantly increased vasoconstriction and endothelial dysfunction. AngII-induced hypertension was blunted in Sphk1-/- mice (systolic BP 167 ± 4.2 vs. 180 ± 3.3 mmHg, p < 0.05), which was associated with decreased aortic and mesenteric vasoconstriction in hypertensive Sphk1-/- mice. Pharmacological inhibition of S1P synthesis reduced vasoconstriction of mesenteric arteries. While Sphk1 is important in mediating vasoconstriction in hypertension, Sphk1-/- mice were characterized by enhanced endothelial dysfunction, suggesting a local protective role of Sphk1 in the endothelium. S1P serum level in humans was correlated with endothelial function (arterial tonometry). Thus, vascular transcriptome analysis shows that S1P pathway is critical in the regulation of vascular function in AngII-induced hypertension, although Sphk1 may have opposing roles in the regulation of vasoconstriction and endothelium-dependent vasorelaxation.

Disturbed fluid flow or modulated shear stress is associated with vascular conditions such as atherosclerosis, thrombosis, and aneurysm. In vitro simulation of the fluid flow around the plaque micro-environment remains a challenging approach. Currently available models have limitations such as complications in protocols, high cost, incompetence of co-culture and not being suitable for massive expression studies. Hence, the present study aimed to develop a simple, versatile model based on Computational Fluid Dynamics (CFD) simulation. Current observations of CFD have shown the regions of modulated shear stress by the disturbed fluid flow. To execute and validate the model in real sense, cell morphology, cytoskeletal arrangement, cell death, reactive oxygen species (ROS) profile, nitric oxide production and disturbed flow markers under the above condition were assessed. Endothelium at disturbed flow region which had been exposed to low shear stress and swirling flow pattern showed morphological and expression similarities with the pathological disturbed flow environment reported previously. Altogether, the proposed model can serve as a platform to simulate the real time micro-environment of disturbed flow associated with eccentric plaque shapes and the possibilities of studying its downstream events.

The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF-α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.

Acute kidney injury (AKI) is a common condition in hospitalized patients who often requires kidney support therapy (KST). However, predicting the need for KST in critically ill patients remains challenging. This study aimed to analyze endothelium-related biomarkers as predictors of KST need in critically ill patients with stage 2 AKI. A prospective observational study was conducted on 127 adult ICU patients with stage 2 AKI by serum creatinine only. Endothelium-related biomarkers, including vascular cell adhesion protein-1 (VCAM-1), angiopoietin (AGPT) 1 and 2, and syndecan-1, were measured. Clinical parameters and outcomes were recorded. Logistic regression models, receiver operating characteristic (ROC) curves, continuous net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were used for analysis. Among the patients, 22 (17.2%) required KST within 72 h. AGPT2 and syndecan-1 levels were significantly greater in patients who progressed to the KST. Multivariate analysis revealed that AGPT2 and syndecan-1 were independently associated with the need for KST. The area under the ROC curve (AUC-ROC) for AGPT2 and syndecan-1 performed better than did the constructed clinical model in predicting KST. The combination of AGPT2 and syndecan-1 improved the discrimination capacity of predicting KST beyond that of the clinical model alone. Additionally, this combination improved the classification accuracy of the NRI and IDI. AGPT2 and syndecan-1 demonstrated predictive value for the need for KST in critically ill patients with stage 2 AKI. The combination of AGPT2 and syndecan-1 alone enhanced the predictive capacity of predicting KST beyond clinical variables alone. These findings may contribute to the early identification of patients who will benefit from KST and aid in the management of AKI in critically ill patients.