Endothelial Adhesion Research Articles

Free fatty acids stabilize integrin β1via S-nitrosylation to promote monocyte–endothelial adhesion

Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin β1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin β1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin β1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages. To examine the effect of elevated levels of FFA on the modulation of integrin β1 expression, we treated the macrophages with a combination of oleic acid and palmitic acid (2:1) and found that FFA activated inducible nitric oxide synthase/nitric oxide and increased the integrin β1 protein level without altering the mRNA level. FFA promoted integrin β1 S-nitrosylation via inducible nitric oxide synthase/nitric oxide and prevented its degradation by decreasing binding to E3 ubiquitin ligase c-Cbl. Furthermore, we found that increased integrin α4β1 heterodimerization resulted in monocyte/macrophage adhesion to endothelium. In conclusion, these results provided novel evidence that FFA-stimulated N--O stabilizes integrin β1via S-nitrosylation, favoring integrin α4β1 ligation to promote vascular inflammation.

Extracellular Vesicles from Sickle Red Blood Cells Activate Endothelial Cells and Trigger Von Willebrand Factor-Mediated Sickle Red Cell Adhesion

Introduction: The sickle mutation of hemoglobin (HbS) afflicts millions of people worldwide and is characterized by hemolytic anemia, inflammation, painful vaso-occlusive crises, significant morbidity, and early mortality. In sickle cell disease (SCD), HbS polymerization under deoxygenation increases red blood cell (RBC) rigidity, adhesivity and susceptibility to lysis. Products of intravascular hemolysis including heme and RBC-released extracellular vesicles (REVs) promote acute and chronic inflammation triggering endothelial activation and abnormal RBC adhesion. Extracellular vesicles (EVs) are nano- or micro-particles composed of a lipid bilayer, comprised of transmembrane proteins, and enclosing intracellular remnants, including cytosolic proteins and miRNAs. EVs are known to exchange biomaterial between cells, and may serve as surrogate markers for the activated state of the parent cell thus are of high clinical significance. Quantitatively, REVs are more abundant from sickle RBCs (SS RBCs) compared with normal HbA-containing RBCs (AA RBCs). SS REVs are known to promote endothelial cell (EC) activation through cell signaling and transcriptional regulation at longer terms. However, the SS REV-mediated short term non transcriptional response of EC is unclear. Methods: Here, we have developed the SCD-EV-BioChip using microfluidic technology. SCD-EV-BioChip assesses RBC adhesion as a biomarker for REV-mediated human pulmonary microvascular endothelial cells (HPMECs) dysfunction using functional biomarker of RBC adhesion. Utilizing SCD-EV-BioChip, Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 hours. For in vitro studies, SS REVs and AA REVs were collected from pooled human SS RBCs and AA RBCs activated using calcium ionophore. The generated REVs were characterized using scanning electron microscopy (SEM) and nanoparticle tracking analyzer (NTA). HPMECs were cultured in microfluidic channels under controlled shear stress for more than 72 hours then treated with REVs at 37 ºC for 2 hours. Von Willebrand factor (vWF) expression on REVs-treated HPMECs were quantified. RBC adhesion tests were conducted with and without vWF protease ADAMTS13 and heme-binding protein hemopexin under shear stress at 1 dyne/cm2 followed by a rinse step and quantification of adhered RBCs. For in vivo study, HbSS-Townes mice and a dorsal skin-fold chamber model was used to determine if ADAMTS13 would reduce microvascular stasis in response to hemin. Results: Within 2 hours, SS REVs, but not AA REVs, promoted increased levels of vWF expression on HPMECs, indicating HPMEC activation (Fig. 1A-C). VWF is known to trigger SRBC adhesion and vaso-occlusion. Using SCD-EV-BioChip, we found significantly increased SS RBC adhesion to HPMECs exposed to SREVs, compared to SRBC adhesion to HPMECs exposed to AA REVs, and AA RBC adhesion to HPMECs exposed to SS REVs (Fig. 1D-G). SS RBC adhesion was significantly reduced on HPMECs treated with SS REVs pre-incubated with hemopexin. ADAMTS13 is a regulator which is responsible for cleaving ultra large vWF. In this study, SS RBC adhesion was significantly reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs (Fig. 1G). Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13 (Fig. 1F). SRBC adhesion was variable within a total of 15 test subjects with SCD, and was higher with SRBCs from patients with increased markers of hemolysis (LDH and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Conclusions: This work demonstrated the acute, non-transcriptional contribution made by REVs to the microvascular EC activation and vWF-mediated SRBC adhesion. Results from SCD-EV-BioChip indicated the effect of hemopexin in mitigating SREV-mediated HPMEC activation. Additionally, results from in vitro SCD-EV BioChip tests agreed with in vivo stasis tests using SS mice, both of which indicated the effect of ADAMTS13 in mitigating SS RBC adhesion and vaso-occlusion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reduction in Endothelial Adhesion of Red Blood Cells Under Crizanlizumab: Assessment of Standardized Microfluidic Platform in Sickle Cell Disease Population

Introduction: Monoclonal P-selectin blocking antibody (Crizanlizumab, ADAKVEO®, Novartis, Switzerland) targets reduction of vaso-occlusive crises (VOCs) in adult and pediatric patients (≥16 years) with sickle cell disease (SCD). Patients with SCD have been reported with endothelial p-selectin to be initiatory for endothelial adhesion of sickle red blood cells (RBCs). Herein, we report the novel endothelium-on-a-chip (microfluidic platform - MFP) that enables assessment of Crizanlizumab on the adhesion of RBCs to perfusion-cultured human endothelial cells (ECs) in acute and chronic activation setup. Methods: Whole blood samples collected from SCD subjects (n=14), HbSS and in EDTA and sodium citrate. RBCs were isolated via centrifugation from whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were cultured within the microfluidic platform channels at 15 dyne/cm2 for at least 48 hours prior to experiments. To mimic the acute EC activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml Crizanlizumab and injected through the microfluidic channels for 15 minutes. Instead to mimic chronic pre-activation in SCD HUVECs were pretreated for 4 hours with TNFα (20 ng/ml) or 50% plasma of SCD patients in basal media before, +/- 100 µg/ml Crizanlizumab followed by injection of blood samples through the microfluidic channels. Non-adherent RBCs were removed via washing solution with or without Crizanlizumab and the remaining RBCs were imaged using an inverted microscope and the manual quantification was completed based on characteristics previous published. Paired t-test to evaluate the effects of the Crizanlizumab on RBCs adherent to activated EC has been used. Results: We observed a statistically significant reduction of RBC adhesion due to Crizanlizumab treatment in 15-minutes acutely heme activated ECs (135±40 vs 1513±617, p≤0.05). The effect of Crizanlizumab on decreasing RBC adhesion was also statistically significant when ECs were chronically pre-activated by TNF-α followed by a 15-minute heme activation (4404±1393 vs 2016±609, p≤0.05). To further elaborate on the effects of Crizanlizumab in the atherogenic SCD population, we activated the EC with subjects' autologous plasma for 4 hours followed by a 15-minute heme activation, (5876±2579 vs 2397±1381p≤0.05). Discussion and Conclusions: Per label use of Crizanlizumab to reduce VOC, application of novel MFP associated with Crizanlizumab use displayed a reduced RBC adhesion to acutely heme-activated EC. Effects were preserved in presence or absence of chronic TNFα or autologous plasma pre-activation. Endothelium-on-a-chip MFP has been shown as reliable in monitoring patient response to anti-adhesive therapies in SCD population. Fig 1. Endothelium-on-a-chip MFP set up (A). The phase-contrast image of HUVECs demonstrate a confluent layer of cells aligned with flow. (C) RBCs adhesion to activated HUVECs. Effects of Crizanlizumab on RBC adhesion levels to HUVECs activated for 15-minutes with heme (D); 4-hours with TNF-α and 15-minutes with heme (E); 4-hours with plasma and 15-minutes with heme(F). Microscope images at 10X. Scale bare 100um. Error bars represent the standard error of the mean (SEM). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13194: Towards Understanding the Pathogenesis of Covid-19: Acute and Long-Term Mouse Models

SARS-CoV2 (CoV2) infection causes both acute and long-term health effects via damaging multiple organs including lung. The endothelial dysfunction associated with the infection may contribute to pathogenesis of acute COVID-19 and long COVID. However, the mechanisms underlying the endothelial dysfunction remain elusive. Development of mouse models for these diseases will help us better dissect these mechanisms and facilitate the development of therapeutics for treatment of the disease. Previously, we developed an acute COVID model by infecting human ACE2 transgenic (K18) mice with a lethal CoV2 dose. K18 mice developed severe COVID-19, including progressive body weight loss and fatality at days 7 post infection (DPI), severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. We detected CoV2 in capillary endothelial cells, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs in this model. These results indicate that CoV2 infection and infection-mediated immune activation caused endothelial dysfunction, which contributes to the pathogenesis of severe COVID-19. To further develop a model for long COVID, we infected K18 mice with sub lethal CoV2 dose, monitored the body weight and survival rate and characterized the lung and brain histological changes at 21 and 45 DPI. The infected mice progressively lost body weights from 5 to 7 DPI and started to rebound from 8 DPI and then returned to baseline at 13 DPI. Mice had extensive patchy inflammation in the lungs associated with collagen deposition and smooth muscle action expression. We also found moderate levels of total viral RNA in the lung but not brain while viral subgenomic RNA (a correlate of viral replication) was undetectable in lung or brain by qRT-PCR assay. Fluorescence staining showed co-localization of CoV2 spike protein and CD206 in lungs, suggesting macrophage engulfment CoV2 at late time points. Together, we have successfully established long-term COVID mouse models, which will be useful tools for further defining the role of endothelial dysfunction in pathogeneses of CoV2-related acute and long COVID.

Abstract 15421: Mice Deficient in Endothelial Cell-Selective Adhesion Molecule Display Coronary Microvascular Rarefaction and Left Ventricle Diastolic Dysfunction

Coronary microvascular dysfunction and chronic inflammation play a role in the development of left ventricular diastolic dysfunction (LVDD) in heart failure with preserved ejection fraction (HFpEF). Endothelial cell-selective adhesion molecule (ESAM) is part of the endothelial cell junction complex, which plays a role in the maintenance of cell barrier function, inflammatory cell transmigration and angiogenesis. Here, we examined the role of ESAM in affecting coronary microvascular density and left ventricle diastolic function by employing mice with genetic deletion of ESAM (ESAM -/- ). Using echocardiography, we found that ESAM -/- mice display LVDD, as indicated by a significantly (p<0.05) reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time and E wave deceleration time, with no change in the ejection fraction. Using an unbiased, automated tracing and 3D reconstruction of microvascular network revealed that ESAM -/- mice have a reduced coronary microvascular density, which was accompanied by myocardial accumulation of myeloperoxidase-positive neutrophils and elevated myocardial levels of inflammatory cytokines and chemokines. ESAM -/- mice exhibited a reduced endothelial sprout formation in the carotid artery, whereas in cultured endothelial cells siRNA-mediated ESAM knockdown impaired tube formation. Thus, ESAM deficiency in mice leads to coronary microvascular rarefaction, which likely via a myocardial hypoperfusion-related neutrophil accumulation and inflammatory mediator production impairs LV diastolic function.

Abstract 12912: Metabolic Labeling of Cardiomyocyte-Derived Small Extracellular-Vesicle (sEV) MiRNAs Identifies MiR-208a in Cardiac Regulation of Lung Gene Expression

Background & Hypothesis: T. gondii uracil phosphoribosyltransferase (UPRT) can convert 4-thiouracil(4TUc) into 4-thiouridine (4TUd), thus incorporating into nascent RNAs, and the thio-labeled RNAs can be identified by thio-linked-biotinylation and streptavidin-affinity isolation, we hypothesize that cardiomyocyte (CM) expression of UPRT ( CM UPRT) allows labeling and tracking of CM-derived miRNAs in PB sEV and peripheral organs. Methods & Results: We generated mice that expressed T. gondii UPRT only in cardiomyocytes ( CM UPRT mice) and tested our hypothesis that CM-derived miRNAs ( CM miRs) are transferred into remote organs after myocardial infarction (MI) by PB sEV. We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PB sEV of CM UPRT mice six hours after 4TUc injection. In PB sEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labeled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a ( CM miR-208a) levels peaked 12 hours after experimentally induced MI in PB sEV and 24 hours after MI in the lung. Notably, miR-208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PB sEV from mice that underwent MI (MI- PB sEV) or sham surgery (Sham- PB sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-κB pathway, was lower in the lungs of MI- PB sEV-treated animals than the lungs of animals treated with Sham- PB sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Conclusion: CM UPRT mice enables us to track PB sEV-mediated transport of CM miRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

KRIT1: A Traffic Warden at the Busy Crossroads Between Redox Signaling and the Pathogenesis of Cerebral Cavernous Malformation Disease.

Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell-cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell-cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496-528.

Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4.

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1+/−) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1+/− mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1+/− mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis.

Construction of functional magnetic scaffold with temperature control switch for long-distance vascular injury

Fusion of endothelial monolayer for rapid reendothelialization is the key to vascular transplantation, which is crucial for remodeling long-distance (≥10 mm) vascular injury (LDVJ) and reducing the long-term repair period. Although silk fibroin (SF) has been widely prepared as a scaffold to repair vascular injuries, it is still challenging to remodel LDVJs. The design and manufacture of functional scaffolds with a rapid reendothelialization enhancement effect are helpful to solve this problem. Here, we developed a well-designed functional magnetic scaffold (MDP) with a temperature-controlled switch, intelligently regulating the release of magnetic nanoparticles (MNPs) to induce macrophages migration and M2 polarization. The results show that the radial support force of MDP is appropriately five times higher than that of pure silk fibroin scaffold (SFC), and the degradation of MDP is delayed, which is beneficial to maintaining the integrity of the scaffold. MDP induces the migration of macrophages, upregulates the expression of repair cytokines such as TGF-β, IL-10 and VEGF, and promotes the proliferation of mouse aortic vascular smooth muscle (MOVAS) cells. More importantly, the enhanced migration of macrophages at the phase transition temperature is more conducive to the proliferation and accumulation of vascular repair-related cells, which is conducive to rapid reendothelialization. In addition, MDP also significantly regulates the polarization of macrophages to M2 type, which is conducive to the secretion of repair cytokines to further promote vascular recovery. The evaluation of endothelial cells' adhesion and function also proved that MDP could increase the expression of vascular endothelial cadherin (VE-Cad) and collagen IV (COL IV) and has the potential for rapid reendothelialization. The replacement model in vivo animal experiments further proved that MDP exhibited a better repair effect than SFC, providing a new option for the repair of LDVJ in the future.

Stent-retriever induces more endothelial damage than direct aspiration in endovascular therapy for acute ischemic stroke

Abstract Background Mechanical arterial recanalization is increasingly used to treat acute ischemic stroke. Known to inflict vascular injury, it might affect pro-thrombotic character and healing patterns. Stent-retriever thrombectomy (SR) and direct aspiration (DA) are both used for acute recanalization and not only lead to endothelial denudation, but also to platelet activation and local formation of microthrombi, potentially adding to the risk of distal embolization. Purpose We aimed to quantify and characterize endovascular damage, microthrombus formation, and vascular healing following SR and DA in a swine model of arterial thrombo-embolic occlusion. Methods Aged autologous thrombi, created under flow, were used to occlude swine extracranial arteries for two hours to mimic a thrombo-embolic occlusion. Arteries were randomized to SR, DA, or control (C). Injury was assessed acutely (SR n=8; DA n=7; C n=4), and at 3 days follow-up (SR n=8; DA n=7; C n=4) using Evans Blue (EB) dye-exclusion-testing to quantify injury, expressed as %EB-area in the treated area. Scanning electron microscopy (SEM) was used to characterize vascular injury and presence of microthrombi (<100 μm). Data are given as mean±SD or median (min-max). Results Regression analysis for EB-area (ANOVA p<0.001; Adj. R2=0.5) showed that SR induced more damage than DA (p=0.001). Both showed partial healing at 3 days (p<0.01) (Table 1). Only for DA, did more attempts lead to an increased EB-area (p=0.016), as for SR one attempt already resulted in almost complete denudation. For both SR and DA, SEM of acute injury showed endothelial denudation, microthrombi, and adhesion of leukocytes and activated platelets (Fig. 1A–C). At three days, SEM showed partial re-endothelialization with increased surface folds as a sign of endothelial activation (Fig. 1D). Microthrombi/cm2 decreased significantly from 7 (2–17) acutely to 1 (0–4) at 3 days (p<0.001). Conclusions Stent-retrievers induce more endothelial damage than direct aspiration. Both treatments yield microthrombi, platelet activation and leukocyte adhesion. After three days, the endothelium is significantly, but only partially recovered. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): We acknowledge the support of the Netherlands Cardiovascular Research Initiative which is supported by the Dutch Heart Foundation (CVON2015-01: CONTRAST), the support of the Brain Foundation Netherlands (HA2015.01.06), and the support of Health∼Holland, Top Sector Life Sciences & Health (LSHM17016), Stryker, Medtronic and Cerenovus. The collaboration project is additionally financed by the Ministry of Economic Affairs by means of the PPP Allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnerships.

S2643 Rare Gastrointestinal Bleeding in a Patient With Severe Plasmodium Falciparum Malaria

Introduction: Malaria clinically presents as a systemic febrile illness; the most severe form is caused by Plasmodium Falciparum. In addition to hematologic manifestations such as severe anemia, thrombocytopenia, and coagulopathy, malaria patients uncommonly experience gastrointestinal complications such as GI bleeds, splenic rupture and subacute intestinal obstruction. We report a severe case of P. falciparum malaria complicated by a GI bleed. Case Description/Methods: A 66-year-old male with recent travel to Nigeria and a medical history of hypertension was brought to the ER with altered mental status, abdominal pain and a 25lb weight loss in the last month. In the ED he spiked a fever to 102F, prompting a thorough infectious workup. An LP was remarkable for many RBCs, glucose 81 and protein 47. CSF was negative for meningitis/encephalitis; he did not have EBV, CMV or HIV. Blood and fungal cultures were negative. Due to his recent travel history, our patient had a 5-thin and 2-thick malarial smear which showed P. falciparum malaria ring forms. He was started on cefepime, vancomycin, metronidazole, atovaquone/proguanil. Over the next 12-hours, the patient rapidly deteriorated with a large coffee ground emesis, a hemoglobin drop from 12.1 to 8.6, and a thrombocytopenia from 135 to 15. Hemolysis labs were positive: haptoglobin < 30, LDH 607, reticulocyte 14.6%, fibrinogen 112. Upper endoscopy showed Grade-C esophagitis, an oozing duodenal ulcer and hematin all throughout the stomach wall. Abdominal ultrasound and CT showed no acute intra-abdominal pathologies. With new ongoing hematemesis and melena, he went into progressive septic vs hemorrhagic shock and required vasopressors and stress-dose steroids. Discussion: The hematologic manifestations often seen in malaria rarely lead to massive GI bleeds. Our patient did not use NSAIDS, he was not tested for H. Pylori, and despite the stress of his acute severe illness, he had no other risk factors for duodenal ulceration. High levels of free oxygen radicals and tumor necrosis factor may have played a role in the etiology of his peptic ulcer formation. Malarial hemolysis results from the release of cytokines, macrophage recruitment, causing a cytokine storm and activation of endothelial adhesion molecule type 1, E-selectin, enhancing cytoadherence of parasitized cells, mediating lactic acidemia, shock, gut mucosal damage and increased permeability. Despite aggressive measures, our patient ultimately developed shock and died.

Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression.

Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4‐thiouracil (4TUc) into 4‐thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin‐affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CMUPRT mice) and tested our hypothesis that CM‐derived miRNAs (CMmiRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PBsEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PBsEV of CMUPRT mice 6 h after 4TUc injection. In PBsEV, 4TUd was incorporated into CM‐specific/enriched miRs including miR‐208a, but not into miRs with other organ or tissue‐type specificities. 4TUd‐labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM‐derived miR‐208a (CMmiR‐208a) levels peaked 12 h after experimentally induced MI in PBsEV and 24 h after MI in the lung. Notably, miR‐208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PBsEV from mice that underwent MI (MI‐PBsEV) or sham surgery (Sham‐PBsEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR‐208a and cooperatively regulate inflammation via the NF‐κB pathway, was lower in the lungs of MI‐PBsEV–treated animals than the lungs of animals treated with Sham‐PBsEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro‐inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR‐208a antagomirs prior to MI surgery. Thus, CMUPRT mice enables us to track PBsEV‐mediated transport of CMmiRs and identify an miR‐208a‐mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

Beneficial Effects of Insulin on Ischemia Reperfusion Injury in Human Skeletal Muscle.

PurposeExaggerated leucocyte activity is a crucial step in the pathophysiology of skeletal muscle ischemia-reperfusion injury (IRI). We tested the hypothesis that insulin, via its’ anti-leukocyte activity, attenuates skeletal muscle IRI in humans.MethodsMaterials and This randomized, blinded, placebo-controlled trial was conducted in patients with skeletal muscle ischemia who required revascularization. Treatment protocols were similar among them except for the insulin group, which received an infusion of insulin at 2.5 U/h. The degree of endothelial adhesiveness; leukocyte activity and pro-inflammatory status via P-selectin, tumor necrosis factor (TNF)-alpha, and myeloperoxidase (MPO) levels in the venous effluent; and clinical outcomes were measured.ResultsTwenty-four consenting patients were randomized to the insulin or control group. There were no significant differences between the two groups except for the median serum insulin level, which was higher in the insulin group (P<0.01). No serious intervention-related adverse events were observed. P-selectin (55.04-99.86 pg/mL; P<0.001), MPO (110.8-160.6 pg/mL; P<0.001), and TNF-alpha (12.16-36.01 pg/mL; P<0.001) levels demonstrated a significant increase post-reperfusion in the ‘control’ group, reaching a peak value at 2 hours post-reperfusion. The increase in all three markers from baseline was significantly diminished in the insulin group at the two-hour (P-selectin, P=0.001; MPO, P=0.001; TNF-alpha, P=0.005) and four-hour (P-selectin, P=0.003; MPO, P=0.002; TNF-alpha, P=0.01) intervals. The differences in clinical outcomes between the insulin and control groups were not statistically significant.ConclusionIn clinical practice, insulin has the potential to attenuate the severity of skeletal muscle IRI inhibiting P-selectin, MPO, and TNF-alpha levels.

The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis.

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis.In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

Silymarin reduces retinal microvascular damage in streptozotocin-induced diabetic rats

Diabetic retinopathy is a severe microvascular problem in diabetes mellitus. Silymarin is a flavonoid compound, and according to previous studies, it is a bioactive compound with potent antioxidant and anti-inflammatory properties. This investigation aims to peruse the impact of silymarin against diabetic retinopathy in streptozotocin (STZ)-provoked rats. Thirty-two adult male Wistar rats were randomly allocated into the control group, STZ group, STZ + silymarin (50 mg/kg), and STZ + silymarin (100 mg/kg). STZ rats received silymarin every day until 2 months after diabetes induction. The serum and retinal tissues were collected 2 months after silymarin treatment to determine biochemical and molecular analyses. Silymarin markedly lowered the serum glucose concentration in diabetic rats. Silymarin reduced the increased levels of advanced glycosylated end products (AGEs), the receptors for AGEs (RAGE), and reactive oxygen species (ROS) in diabetic rats. Silymarin also attenuated the phosphorylation of p38 MAP kinase and nuclear factor (NF)-κB p65 and diminished diabetes-induced overexpression of inflammatory cytokines, vascular endothelial growth factor (VEGF), adhesion molecules, and extracellular matrix proteins in STZ rats. Our data suggested that silymarin has protective effects against diabetic retinopathy, which might be related to the inhibition of the AGEs/RAGE axis and its antioxidant and anti-inflammatory activities.

SARS-CoV-2 Infection of Airway Epithelium Triggers Pulmonary Endothelial Cell Activation and Senescence Associated with Type I IFN Production.

Airway epithelial cells represent the main target of SARS-CoV-2 replication but several pieces of evidence suggest that endothelial cells (ECs), lining pulmonary blood vessels, are key players in lung injury in COVID-19 patients. Although in vivo evidence of SARS-CoV-2 affecting the vascular endothelium exists, in vitro data are limited. In the present study, we set up an organotypic model to dissect the crosstalk between airway epithelium and pulmonary endothelial cells during SARS-CoV-2 infection. We showed that SARS-CoV-2 infected airway epithelium triggers the induction of endothelial adhesion molecules in ECs, suggesting a bystander effect of dangerous soluble signals from the infected epithelium. The endothelial activation was correlated with inflammatory cytokines (IL-1β, IL-6, IL-8) and with the viral replication in the airway epithelium. Interestingly, SARS-CoV-2 infection determined a modulation of endothelial p21, which could be partially reversed by inhibiting IFN-β production from ECs when co-cultured with HAE. Altogether, we demonstrated that SARS-CoV-2 infected epithelium triggers activation/senescence processes in ECs involving type I IFN-β production, suggesting possible antiviral/damage mechanisms occurring in the endothelium.

Abstract IA024: Identification of a “hypoxic secretome” and its role in metastasis

Abstract Only a small percentage of disseminating tumor cells are capable of forming lethal metastatic foci. Though advances in sequencing and genomics have dramatically enhanced our understanding of primary tumor biology and novel target identification, these techniques alone have not proven sufficient to identify the factors that permit metastasis of this small cellular fraction. For example, we observed that in some tumors the major molecular predictor of metastatic potential is HIF1α protein stabilization in response to low intratumoral O2 tension (hypoxia). These observations clearly show that in-depth understanding of environmental signals and subsequent cellular responses is necessary to fully characterize metastatic potential. Consistent with our earlier observations, we discovered that the collagen-modifying enzyme PLOD2, a direct transcriptional target of HIF1α, dramatically enhances both early (cell migration/invasion) and late (extravasation/lung colonization) steps of the metastatic cascade. We previously reported the role of PLOD2 in modulating the primary tumor microenvironment to facilitate cell migration and intravasation. However, the mechanisms by which PLOD2 and tumor associated collagen impact later metastatic stages (i.e., endothelial adherence, extravasation) are unknown- in part because these processes are particularly difficult to simulate in vitro and to visualize in vivo. Therefore, we have developed new models and tools including a zebrafish embryo xenograft system to image migrating and extravasating tumor cells in vivo. This approach allows us to investigate the late metastatic cascade and define the microenvironmental cues that promote tumor cell dissemination. We also observed that PLOD2-modified collagen is secreted into the extracellular milieu during dissemination and weakens endothelial barrier function. We are investigating the role of tumor collagen and PLOD2 in endothelial adherence and extravasation for the purpose of therapeutically targeting the molecular underpinnings of metastases. Our work focuses on soft tissue sarcomas. However, recent studies by other groups have linked PLOD2 and modified collagen to carcinoma metastasis as well, suggesting a broader applicability for our work. Ultimately, we believe these studies will transform our ability to treat and even prevent metastasis, a unique possibility in sarcoma patients due to the relatively long interval between primary tumor diagnosis and metastatic outgrowth in some patients (5-10 years). The tools we have developed and the mechanisms we are pursuing will open new avenues of research that were once inaccessible and lead to novel therapeutic opportunities for the treatment of metastatic disease in multiple cancer contexts. Citation Format: T.S. Karin Eisinger, Ying Liu, Ileana Murazzi. Identification of a “hypoxic secretome” and its role in metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA024.

7-Methoxyisoflavone suppresses vascular endothelial inflammation by inhibiting the expression of endothelial adhesion molecules

Endothelial cells (ECs) are vital regulators of inflammatory processes, there is the potential for inhibition of EC inflammation to be a therapeutic target in chronic inflammatory diseases. This study aimed to investigate the effect of 7-methoxyisoflavone (7-Mif) on endothelial inflammation. Our results showed that 7-Mif have no cytotoxicity on HUVECs. Pretreatment with 5 μM, 10 μM and 50 μM 7-Mif significantly reduced IL-1β-induced ICAM-1 (28.1% ± 4.1%, 25.9 ± 2.5% and 32.0% ± 3.2%, respectively) and VCAM-1 (48.0% ± 5.6%, 40.1 ± 3.1% and 39.6% ± 3.1%, respectively) mRNA expression. And pretreatment with 10 μM and 50 μM 7-Mif significantly reduced IL-1β-induced ICAM-1 (45.1% ± 4.4% and 33.6 ± 4.4%, respectively) and VCAM-1 (53.0% ± 3.7% and 53.7 ± 5.1%, respectively) protein levels. Furthermore, pretreatment with 50 μM 7-Mif inhibited monocyte-endothelial cell adhesion (50.2% ± 4.2%). Mechanistically, our results showed that 7-Mif reversed IL-1β-induced NF-κB activation and p65 translocation to the nucleus, therefore inhibiting endothelial cell inflammation. In addition, we confirmed that 7-Mif 10 mg/kg and 20 mg/kg reduced LPS-induced ICAM-1 (47.3% ± 1.3% and 39.0% ± 3.2%, respectively) and VCAM-1 (56.5 ± 2.8% and 47.8 ± 4.3%, respectively) expression and attenuated inflammatory injury in mice. In conclusion, we showed the inhibitory effect of 7-Mif on endothelial inflammation by suppressing the expression of endothelial adhesion molecules and monocyte adhesion. Our data illustrated that 7-Mif could positively regulate the process of endothelial inflammation.

Contrasting effects of sleep fragmentation and angiotensin-II treatment upon pro-inflammatory responses of mice

Disordered sleep promotes inflammation in brain and peripheral tissues, but the mechanisms that regulate these responses are poorly understood. One hypothesis is that activation of the sympathetic nervous system (SNS) from sleep loss elevates blood pressure to promote vascular sheer stress leading to inflammation. As catecholamines produced from SNS activation can directly regulate inflammation, we pharmacologically altered blood pressure using an alternative approach-manipulation of the renin-angiotensin system (RAS). Male C57BL6/J mice were treated with angiotensin or captopril to elevate and reduce blood pressure, respectively and then exposed to 24-h of sleep fragmentation (SF) or allowed to sleep (control). Pro- and anti-inflammatory cytokine gene expression and as endothelial adhesion gene expression as well as serum glucocorticoids (corticosterone) were measured. RAS manipulation elevated cytokines and endothelial adhesion expression in heart and aorta while SF increased cytokine expression in peripheral tissues, but not brain. However, there were interactive effects of angiotensin-II and SF upon cytokine gene expression in hippocampus and hypothalamus, but not prefrontal cortex. SF, but not RAS manipulation, elevated serum corticosterone concentration. These findings highlight the contrasting effects of RAS manipulation and SF, implying that inflammation from SF is acting on different pathways that are largely independent of RAS manipulation.

PHACTR1, a coronary artery disease risk gene, mediates endothelial dysfunction.

Genome-wide association studies (GWAS) have recently identified phosphatase and actin regulator-1 (PHACTR1) as a critical risk gene associated with polyvascular diseases. However, it remains largely unclear how PHACTR1 is involved in endothelial dysfunction. Here, by mining published datasets of human stable and vulnerable/ruptured plaque tissues, we observed upregulated expression of PHACTR1 in vulnerable/ruptured plaques. Congruent with these data, we demonstrated increased Phactr1 gene expression in aortic endothelium from ApoE-/- mice fed a western type diet compared with that in normal C57BL/6J mice. Relevantly, PHACTR1 gene expression was upregulated by pro-inflammatory and pro-atherogenic stimuli, including TNF-α, IL-1β and oxidized LDL (oxLDL). By employing next-generation RNA sequencing, we demonstrate that PHACTR1 overexpression disrupts pathways associated with endothelial homeostasis. Cell biological studies unravel that PHACTR1 mediates endothelial inflammation and monocyte adhesion by activating NF-κB dependent intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) expression. In addition, overexpression of PHACTR1 also reduces the generation of nitric oxide (NO) by inhibiting Akt/eNOS activation. In-house compound screening of vasoprotective drugs identifies several drugs, including lipid-lowering statins, decreases PHACTR1 gene expression. However, PHACTR1 gene expression was not affected by another lipid-lowering drug-fenofibrate. We also performed a proteomic study to reveal PHACTR1 interacting proteins and validated that PHACTR1 can interact with heat shock protein A8 (HSPA8) which was reported to be associated with coronary artery disease and eNOS degradation. Further studies are warranted to confirm the precise mechanism of PHACTR1 in driving endothelial dysfunction. In conclusion, by using systems biology approach and molecular validation, we disclose the deleterious effects of PHACTR1 on endothelial function by inducing endothelial inflammation and reducing NO production, highlighting the potential to prevent endothelial dysfunction and atherosclerosis by targeting PHACTR1 expression. The precise role of endothelial cell PHACTR1 in polyvascular diseases remains to be validated in diseased conditions.