Endothelial Cell Perturbation Research Articles

Ultrasound-Based Radiation Enhancement: Concepts, Mechanisms and Therapeutic Applications.

Microbubbles have emerged as versatile carriers used both for cancer diagnosis and therapy. Microbubbles in the presence of ultrasound waves undergo cavitation, generating bioeffects near the cell's vicinity. Studies have shown ultrasound-stimulated microbubbles (USMB) to cause mechanical perturbation of endothelial cells, resulting in acid sphingomyelinase (ASMase)-induced ceramide production. Disruption of endothelial cells further causes vascular deterioration, leading to secondary tumor cell death. These effects are known to be synergistically higher when USMB is combined with radiation. This paper provides insight into the use of USMB as a potential radioenhancer. The possible underlying mechanism and therapeutic effects of combining USMB and radiation therapy are also presented.

Characterization of Cell Type Abundance and Gene Expression Timeline from Burned Skin Bulk Transcriptomics by Deconvolution.

Currently, no timeline of cell heterogeneity in thermally injured skin has been reported. In this study, we proposed an approach to deconvoluting cell type abundance and expression from skin bulk transcriptomics with cell type signature matrix constructed by combining independent normal skin and peripheral blood scRNA-seq datasets. Using CIBERSORTx group mode deconvolution, we identified perturbed cell type fractions and cell type-specific gene expression in three stages postthermal injury. We found an increase in cell proportions and cell type-specific gene expression perturbation of neutrophils, macrophages, and endothelial cells and a decrease in CD4+ T cells, keratinocytes, melanocyte, and fibroblast cells, and cell type-specific gene expression perturbation postburn injury. Keratinocyte, fibroblast, and macrophage up regulated genes were dynamically enriched in overlapping and distinct Gene Ontology biological processes including acute phase response, leukocyte migration, metabolic, morphogenesis, and development process. Down-regulated genes were enriched in Wnt signaling, mesenchymal cell differentiation, gland and axon development, epidermal morphogenesis, and fatty acid and glucose metabolic process. We noticed an increase in the expression of CCL7, CCL2, CCL20, CCR1, CCR5, CCXL8, CXCL2, CXCL3, MMP1, MMP8, MMP3, IL24, IL6, IL1B, IL18R1, and TGFBR1 and a decrease in expression of CCL27, CCR10, CCR6, CCR8, CXCL9, IL37, IL17, IL7, IL11R, IL17R, TGFBR3, FGFR1-4, and IGFR1 in keratinocytes and/or fibroblasts. The inferred timeline of wound healing and CC and CXC genes in keratinocyte was validated on independent dataset GSE174661 of purified keratinocytes. The timeline of different cell types postburn may facilitate therapeutic timing.

A Review on COVID-19: Primary Receptor, Endothelial Dysfunction, Related Comorbidities, and Therapeutics

Since December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global pandemic named coronavirus disease-19 (COVID-19) and resulted in a worldwide economic crisis. Utilizing the spike-like protein on its surface, the SARS-CoV-2 binds to the receptor angiotensin-converting enzyme 2 (ACE2), which highly expresses on the surface of many cell types. Given the crucial role of ACE2 in the renin–angiotensin system, its engagement by SARS-CoV-2 could potentially result in endothelial cell perturbation. This is supported by the observation that one of the most common consequences of COVID-19 infection is endothelial dysfunction and subsequent vascular damage. Furthermore, endothelial dysfunction is the shared denominator among previous comorbidities, including hypertension, kidney disease, cardiovascular diseases, etc., which are associated with an increased risk of severe disease and mortality in COVID-19 patients. Several vaccines and therapeutics have been developed and suggested for COVID-19 therapy. The present review summarizes the relationship between ACE2 and endothelial dysfunction and COVID-19, also reviews the most common comorbidities associated with COVID-19, and finally reviews several categories of potential therapies against COVID-19.

Vascular mechanisms and manifestations of COVID-19

Vascular mechanisms and manifestations of COVID-19

Fournier's gangrene during lenvatinib treatment: A case report

Fournier's gangrene is a rare and severe complication reported in patients with cancer treated with antiangiogenic drugs, most frequently with bevacizumab. The present report describes the case of an 80-year-old man with radioactive iodine-refractory metastatic thyroid cancer treated with lenvatinib (an oral multikinase inhibitor with antiangiogenic properties) who developed Fournier's gangrene in the absence of other known risk factors. To the best of our knowledge, this is the first case described during treatment with lenvatinib. The condition was likely due to a perturbation of vascular endothelial cells of the skin due to the inhibition of VEGF/VEGFR signaling. Fournier's gangrene may be a class effect of antiangiogenic treatment that clinicians should be aware of, as early diagnosis and treatment are associated with an improved outcome.

Prevalence of endothelial dysfunction in acquired immune deficiency syndrome patients and its correlation with degree of immunodeficiency

Background: Number of factors play a role in endothelial dysfunction observed in AIDS patients, which can lead to atherosclerosis along with cardiovascular mortality and morbidity. Human immunodeficiency virus (HIV), the etiologic agent of AIDS causes several vascular disorders characterized by an evident activation and perturbation of endothelial cells. Currently, there is lack of data in the Indian literature regarding study of endothelial dysfunction in HIV patients. The purpose of our research was to study the prevalence of endothelial dysfunction in HIV/AIDS patients.Methods: The study comprises a total number of 60 adult HIV positive patients of both sex (male and female) with confirmed HIV seropositivity. The patients were divided into two groups of 30 each, depending on the degree of immune dysfunction (CD 4 cell counts). Group I- patients with CD 4+T cell count>200/µl and group II-patients with CD 4+T cell count<200/µl. These patients were subjected to detailed clinical examination and markers of endothelial dysfunction-flow mediated vasodilatation (FMD) of brachial artery, S. nitrite and C-reactive protein (CRP) were performed.Results: The defect in endothelial function was most prevalent in patients with more severe immunosuppression. FMD of brachial artery was decreased in patients with CD 4+T cell count < 200/µl (7.07±2.89, p=0.00). S. nitrite was also significantly lower in group II patients (26.43±15.38), and these patients also showed more CRP positivity and higher CRP titres ranging from 1.2 mg/dl to 9.6 mg/dl.Conclusions: The defect in endothelial function was most prevalent in patients with more severe immunosuppression. FMD of brachial artery was decreased in patients with CD 4+T cell count <200/µl (7.07±2.89, p=0.00). S. nitrite was also significantly lower in group II patients (26.43±15.38), and these patients also showed more CRP positivity and higher CRP titres ranging from 1.2 mg/dl to 9.6 mg/dl.

Pathogenesis of systemic sclerosis-current concept and emerging treatments.

Systemic sclerosis (SSc) is an intractable multifaceted disease with high mortality. Although its pathogenesis is not fully understood, recent studies have advanced our knowledge on SSc. The cardinal pathological features of SSc are autoimmunity, vasculopathy, and fibrosis. The B cells in SSc are constitutively activated and lead to the production of a plethora of autoantibodies, such as anti-topoisomerase I and anti-centromere antibodies. In addition to these autoantibodies, which are valuable for diagnostic criteria or biomarkers, many other autoantibodies targeting endothelial cells, including endothelin type A receptor and angiotensin II type I receptor, are known to be functional and induce activation or apoptosis of endothelial cells. The autoantibody-mediated endothelial cell perturbation facilitates inflammatory cell infiltration, cytokine production, and myofibroblastic transformation of fibroblasts and endothelial cells. Profibrotic cytokines, such as transforming growth factor β, connective tissue growth factor, interleukin 4/interleukin 13, and interleukin 6, play a pivotal role in collagen production from myofibroblasts. Specific treatments targeting these causative molecules may improve the clinical outcomes of patients with SSc. In this review, we summarize recent topics on the pathogenesis (autoantibodies, vasculopathy, and fibrosis), animal models, and emerging treatments for SSc.

Ultrasound-stimulated microbubble enhanced low-dose radiation response

We have recently demonstrated that mechanical perturbation of endothelial cells from ultrasound-stimulated microbubbles (USMB) results in enhanced tumor radiosensitivity at low 2 Gy doses of radiation. Our hypothesis is that USMB-based endothelial membrane perturbations produce ceramide via a sphingomyelinase (ASMase) pathway, and act synergistically with radiation to enhance overall tumor response. Here, we investigate the role of the SMase-ceramide pathway on USMB-based endothelial radiosensitization. Experiments were carried out in wild type (C57BL/6) and ASMase knockout mice, implanted with a fibrosarcoma line (MCA-129). Animals were treated with radiation doses varying from 0 to 8 Gy alone, or in combination with ultrasound-stimulated microbubbles. Treatment response was assessed with Doppler ultrasound vascularity index acquired at 3, 24, and 72 hrs using a VEVO770 preclinical ultrasound system. Staining using ISEL, ceramide, and CD31 immunohistochemistry of tumor sections was used to complement res...

Toll-like receptors 2 and 4 mediate hyperglycemia induced macrovascular aortic endothelial cell inflammation and perturbation of the endothelial glycocalyx

ObjectivesHyperglycemia-induced inflammation is central to the vascular complications in diabetes. Toll-like receptors (TLRs) are key players in regulating inflammatory responses. There are sparse data on the role of TLR2 and TLR4 in regulating human macrovascular aortic endothelial cells (HMAECs) inflammation and glycocalyx dysfunction under hyperglycemia. We examined the role of TLR2/4 in the above dysfunctions in HMAEC under high glucose (HG) conditions. MethodsHMAECs were treated with high or normal glucose and TLR-2, TLR-4, MyD88, IRF3, TRIF, nuclear NF-κB p65, IL-8, IL-1β, TNF-α, MCP-1, ICAM-1, sVCAM-1, monocyte adhesion to HMAECs, heparan sulfate and hyaluronic acid were measured. ResultsHG upregulated TLR2 and TLR4 mRNA and protein and increased both MyD88 and non-MyD88 pathways, NF-κB p65, inflammatory biomediators, and monocyte adhesion to HMAECs. Heparan sulfate protein expression was reduced and hyaluronic acid secretion was increased on HG exposure. Inhibition of TLR2 and TLR4 signaling by inhibitory peptides and knockdown of TLR-2 and TLR-4 gene expression by siRNA attenuated HG induced inflammation, leukocyte adhesion and glycocalyx dysfunction. An increase in ROS paralleled the increase in TLR-2/4 and antioxidants treatment reduced TLR-2/4 expression and downstream inflammatory biomediators. ConclusionThus hyperglycemia induces HMAEC inflammation and glycocalyx dysfunction through TLR-2/4 pathway activation via increased ROS.

Abstract 1812: Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide

Abstract We have recently demonstrated that mechanical perturbation of endothelial cells from ultrasound-stimulated microbubbles (USMB) results in enhanced tumour radiosensitivity. When exposed to high-power ultrasound, microbubbles release energy in the form of local mechanical forces on the membranes of surrounding endothelial cells. Upon irradiation, rapid vascular shutdown occurs followed by extensive secondary tumour cell death. Our hypothesis is that USMB-based endothelial membrane perturbations produce ceramide via a sphingomyelinase (ASMase) pathway, and act synergistically with radiation to enhance overall tumour response. Here we investigate the role of the SMase-ceramide pathway on USMB-based endothelial radiosensitization. Experiments were carried out in wild type (C57BL/6) and ASMase knockout mice, implanted with a fibrosarcoma line (MCA-129). Animals were treated with radiation doses varying from 0-8 Gy alone, or in combination with USMB. In addition, we pre-treated a subset of animals with sphingosine-1-phosphate (S1P). Treatments with USMB consisted of a 16-cycle tone burst at a 500 kHz center frequency and 570 kPa using a 2.86 cm element diameter ultrasound transducer. The total insonification time was 750 ms over 5 minutes. Microbubbles (Lantheus Medical) were injected through the tail-vein resulting in a blood volume concentration of 1% or 3% (v/v). Treatment response was assessed with Doppler ultrasound acquired at 3, 24 and 72 hrs using a VEVO770 preclinical ultrasound system. The vascularity index (VI) was used to quantify power Doppler data. Staining using ISEL, ceramide and CD31 immunohistochemistry of tumour sections was used to complement ultrasound images, and to confirm results. Results suggest a link between USMB-based radiosensitization and ASMase. We observed an average decrease of 30% in the VI by 3 hrs in wild type tumours receiving 8 Gy radiation alone. In contrast, those receiving 8 Gy and USMB resulted in a VI decrease of up to 50%. Similarly, while 2 Gy alone induced minimal effects on the tumour vasculature, combining it with USMB resulted in up to 40% decrease in the VI by 24 hrs. Vascular effects were significant (p < 0.05) and sustained for up to 72 hrs after combined treatments. These resulted in significant cell death. In contrast to wild type animals, ASMase knockout mice, or wild-type mice receiving S1P, were found to be generally resistant to the anti-vascular effects of radiation and USMB. We noted minimal cell death and no vascular shutdown following any of the treatments in those experimental groups. Overall conclusions drawn from this work point to a mechanotransduction-like effect that results in endothelial radiosensitization. Citation Format: Gregory Jan Czarnota, Ahmed El Kaffas, Anoja Giles, Azza Al Mahrouki, Amr Hashim. Ultrasound-stimulated microbubble based biomechanical enhancement of radiation cell death: Role of acid sphingomyelinase and ceramide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2015-1812

Toll-like receptor 4 and β2 glycoprotein I interaction on endothelial cells.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombotic events and/or pregnancy morbidity in the persistent presence of antiphospholipid antibodies (aPL). aPL targeting β2 glycoprotein I (anti-β2GPI Abs) provide the main pathogenic autoantibody subset. In monocytes, platelets and endothelial cells (ECs), the interaction of circulating aPL with membrane-bound β2GPI results in cell activation, and EC perturbation provides an important player in clotting. Several receptors have been suggested to mediate β2GPI/EC binding. AnnexinA2 provides a high-affinity binding site for β2GPI but, since it does not span the cell membrane, an adaptor protein is required to trigger signal. Consistent evidence supports the role of Toll-like receptor (TLR) 4 as co-receptor for β2GPI on ECs. β2GPI was recently reported to behave as lipopolysaccharide (LPS) scavenger. In monocytes, TLR4 activation was shown to be apparent, due to LPS/β2GPI complexes. Conversely, our findings in ECs demonstrate that β2GPI interacts directly with TLR4, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. LPS enhanced anti-β2GPI Ab binding to EC only at cell-activating concentrations, able to up-regulate TLR4. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in vivo.

Evaluation of von Willebrand Factor and von Willebrand Factor Propeptide in Models of Vascular Endothelial Cell Activation, Perturbation, and/or Injury

Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ∼33% while in control rats VWF increased ∼5%. In dogs, VWF levels transiently increased ∼30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.

Markers of Vascular Perturbation Correlate with Airway Structural Change in Asthma

Air trapping and ventilation defects on imaging are characteristics of asthma. Airway wall thickening occurs in asthma and is associated with increased bronchial vascularity and vascular permeability. Vascular endothelial cell products have not been explored as a surrogate to mark structural airway changes in asthma. Determine whether reporters of vascular endothelial cell perturbation correlate with airway imaging metrics in patients with asthma of varying severity. Plasma from Severe Asthma Research Program subjects was analyzed by ELISAs for soluble von Willebrand factor mature protein (VWF:Ag) and propeptide (VWFpp), P-selectin, and platelet factor 4. Additional subjects were analyzed over 48 hours after whole-lung antigen challenge. We calculated ventilation defect volume by hyperpolarized helium-3 magnetic resonance imaging and areas of low signal density by multidetector computed tomography (less than -856 Hounsfield units [HU] at functional residual capacity and -950 HU at total lung capacity [TLC]). VWFpp and VWFpp/Ag ratio correlated with and predicted greater percentage defect volume on hyperpolarized helium-3 magnetic resonance imaging. P-selectin correlated with and predicted greater area of low density on chest multidetector computed tomography less than -950 HU at TLC. Platelet factor 4 did not correlate. Following whole-lung antigen challenge, variation in VWFpp, VWFpp/Ag, and P-selectin among time-points was less than that among subjects, indicating stability and repeatability of the measurements. Plasma VWFpp and P-selectin may be useful as surrogates of functional and structural defects that are evident on imaging. The results raise important questions about why VWFpp and P-selectin are associated specifically with different imaging abnormalities.

Coagulation and inflammation in scrub typhus and murine typhus—a prospective comparative study from Laos

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.

Markers of Endothelial Angiogenic Dysfunction in Patients with Antiphospholipid Antibodies

Abstract 2300 Background:The antiphospholipid syndrome (APS) is characterised by presence of persistent antiphospholipid antibodies in association with thrombosis and/or pregnancy morbidity and mortality. Activation of endothelial cells by aPL has been proposed to pay a role in the pathogenesis of APS;antiphospholipid antibodies(aPL) activate endothelial cells in vitro and some evidence of endothelial cell perturbation has been found in patients with aPL.Vascular endothelial growth factor(VEGF) promotes endothelial cell growth and angiogenesis and has been shown to upregulate tissue factor(TF), and elevated VEGF levels have been found to correlate with elevated TF levels in APS. Soluble FMS like tyrosine kinase −1(SLFT-1) and soluble Endoglin(sENG) are antiangiogenic proteins. sFLT1 is a variant of the VEGF receptor released by endothelial cells and monocytes, binds VEGF causing endothelial dysfunction and decreased angiogenesis. sENG is a TGFβ co-receptor which impairs TGF β1 receptor binding and its downstream signalling effects. sFLT-1 and sENG are implicated in the pathogenesis of pre-eclampsia, and are elevated in disorders associated with endothelial dysfunction such as sickle cell disease, chronic kidney disease and coronary artery disease, but have not been studied in patients with aPL. Aims:The aim of this study was to measure sFLT1, sENG, sTF and VEGF in patients with aPL. Materials & methods:Local ethics committee approval was obtained and samples were taken from 182 patients (175 females, 7 males, median age 42 (range 19–73) years) who had PAPS, or had persistent aPL without associated complications. 28 healthy controls (28 females, median age (range 20–58) years) were included. Patients with PAPS included 95 with thrombotic complications, 48 with obstetric complications and 39 with isolated aPL. Patients with intercurrent infection or malignancy were excluded and the control group were not known to have aPL.Blood was drawn into Vacutainers containing EDTA 0.105M and processed within 3 hours of collection. ELISA assays measuring sFLT1, sENG, VEGF and sTF levels were performed according to manufacturer's protocol (Quidel, Pathway diagnostics ltd., Dorking, UK). Intra-assay CV for sFLT1,sENG, VEGF and sTF was 3.0,3.3,4.8 and 6.0 respectively. Inter assay CV for sFLT1,sENG, VEGF and sTF was 6.5,7.6, 7.4 and 5.0 respectively. Statistical analysis of results was performed using the Stata-11 software statistical package. Logarithmic transformation of data was performed and differences between groups were compared using linear regression methods, adjusting for age, ethnicity and medications. Results:Results are shown below in table 1 and are described as means and 95% confidence intervals (adjusted for age, sex, ethnicity and medications).Plasma levelsAPL/PAPS Overall (n=182)Thrombotic APS (n=92)Obstetric APS (n=47)Isolated aPL (n=39)Controls (n=28)VEGF pg/ml64 58–7169 59–8158 47–7359 47–7667 51–85TF Pg/ml170 153–188249 217–28597 79–117129 105–160205 155–268sFLT1 Pg/ml153 133–176172 140–211148 112–198117 86–16093 65–132sENG Ng/ml11.3 10.8–11.912.5 12–1311.3 10–128.8 8–109.7 8.6–11.1sFLT1 and sENG levels were significantly higher in patients with aPL/PAPS compared to healthy controls(p<0.05). TF levels were significantly lower in patients with aPL/PAPS compared to healthy controls(p<0.05). There were no significant differences in VEGF levels in patients with aPL compared to controls(regardless of complication).When patients were categorised according to aPL related complication (thrombotic APS, obstetric aPS, isolated aPL), sFLT1 and sENG levels were found to be significantly higher in patients with thrombotic APS(p<0.05) compared to controls, sFLT1 levels were also significantly elevated in obstetric APS. TF levels were significantly lower in patients with obstetric APS and isolated aPL compared to controls. There were no differences between patients with aPL and controls when means were adjusted for age, ethnicity or medications. sFLT1 was associated with the presence of aPL/PAPS(area under ROC=0.76), whereas sENG had a weaker association (area under ROC: 0.65). Conclusions:We have demonstrated evidence of increased levels of sENG and sFLT1 in patients with aPL/PAPS.This suggests that there is underlying endothelial dysfunction in patients with APS.The role of sENG and sFLT1 in the pathogenesis of APS requires further exploration. Disclosures:No relevant conflicts of interest to declare.

Transgenic expression of human heme oxygenase‐1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys

The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically. These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation.

Phospholipolyzed LDL induces an inflammatory response in endothelial cells through endoplasmic reticulum stress signaling

Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2alpha, IRE1alpha, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.

Severe Plasmodium Falciparum Malaria Is Associated with Circulating Ultra-Large Von Willebrand Multimers and ADAMTS13 Inhibition

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparum infection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation.To address this question, we collected plasma samples from a cohort of African children presenting with cerebral malaria (CM), or non-cerebral severe malaria (SM). In children with CM and SM, plasma VWF:Ag levels were significantly elevated (medians 3.1 and 3.4 IU/ml) as before. VWF collagen binding (VWF:CB) levels were also markedly increased (medians 7.6 and 7.0 IU/ml). Furthermore, the relative rise in VWF:CB was much greater, so that the ratio of CB:Ag was consistently increased. VWF multimer analysis demonstrated abnormal circulating ULVWF multimers in children with CM and SM, consistent with acute, regulated VWF secretion. To characterize mechanisms responsible for the markedly increased VWF:CB and circulating ULVWF in malarial plasma, we investigated ADAMTS13 antigen and activity levels in CM and SM plasma. Plasma ADAMTS13 activity levels (FRETS-VWF73 assay) and antigen levels were both significantly reduced (medians = 0.63 U/ml and 0.56 U/ml; p< 0.001) in children with CM and SM compared to controls. Classical mixing studies of malaria and normal plasma demonstrated no evidence of immediate ADAMTS13 inhibition. However, ADAMTS13 activity in normal plasma was significantly reduced (~60%) following 30 min incubation with malarial plasma (75%:25% mix). This inhibitory ability of malaria plasma was further confirmed by spiking malarial plasmas with recombinant human ADAMTS13. Significant time-dependent inhibition of FRETS-VWF73 activity was again observed in the plasmas of children with severe P. falciparum, but not in normal control plasmas. Potential inhibitors of ADAMTS13 in vivo include interleukin-6 (IL-6), thrombospondin-1 (TSP-1), thrombin and plasmin, free plasma haemoglobin, and reduced FVIII levels. Although plasma IL-6 levels were significantly elevated in children with either CM (mean 240 pg/ml; p<0.001) or SM (mean 217 pg/ml; p=0.01) compared to normal controls, levels did not approach those previously reported necessary to inhibit ADAMTS13 activity. Disseminated intravascular coagulation (DIC) is associated with enhanced thrombin generation, and consumption of FVIII, both of which can inhibit VWF proteolysis by ADAMTS13. However, in children with CM or SM respectively, we observed significantly increased plasma FVIII:C levels. Although intravascular haemolysis is also a recognised complication of malarial infection, we observed only minor increased plasma haemoglobin concentrations, again well below that previously described to significantly inhibit ADAMTS13 activity. Finally, in contrast to the increased plasma levels of IL-6 and FVIII:C, we found that plasma TSP-1 levels were not significantly elevated in children with either CM or SM compared to pooled normal plasma.In conclusion, we demonstrate that the presence of ULVWF multimers in the plasma of children with severe P. falciparum malaria is the result ofacute EC activation and release of ULVWF from WP bodies;significantly reduced plasma ADAMTS13 antigen levelsa circulating but unidentified inhibitor of human ADAMTS13 activity.In view of the critical role played by VWF in mediating platelet adhesion/aggregation, and the accumulating evidence suggesting that platelet adhesion/aggregation also facilitate cytoadhesion of IE, we propose a novel role for ULVWF multimers in the pathophysiology of severe P. falciparum malaria.

Advanced Oxidation Protein Products Activate Vascular Endothelial Cellsviaa RAGE-Mediated Signaling Pathway

The accumulation of advanced oxidation protein products (AOPPs) has been linked to vascular lesions in diabetes, chronic renal insufficiency, and atherosclerosis. However, the signaling pathway involved in AOPPs-induced endothelial cells (ECs) perturbation is unknown and was investigated. AOPPs modified human serum albumin (AOPPs-HSA) bound to the receptor for advanced glycation end products (RAGE) in a dose-dependent and saturable manner. AOPPs-HSA competitively inhibited the binding of soluble RAGE (sRAGE) with its preferential ligands advanced glycation end products (AGEs). Incubation of AOPPs, either prepared in vitro or isolated from uremic serum, with human umbilical vein ECs induced superoxide generation, activation of NAD(P)H oxidase, ERK 1/2 and p38, and nuclear translocation of NF-kappaB. Activation of signaling pathway by AOPPs-ECs interaction resulted in overexpression of VCAM-1 and ICAM-1 at both gene and protein levels. This AOPPs-triggered biochemical cascade in ECs was prevented by blocking RAGE with either anti-RAGE IgG or excess sRAGE, but was not affected by the neutralizing anti-AGEs IgG. These data suggested that AOPPs might be new ligands of endothelial RAGE. AOPPs-HSA activates vascular ECs via RAGE-mediated signals.

Tissue factor pathway inhibitor relates to fibrin degradation in patients with acute deep venous thrombosis

Reduced concentration of tissue factor pathway inhibitor is a risk factor for development of deep venous thrombosis, whereas elevated concentrations of tissue factor pathway inhibitor are observed in patients with acute myocardial infarction and disseminated intravascular coagulation. Presently, we studied the association between inflammation, endothelial cell perturbation, fibrin degradation and the concentration of tissue factor pathway inhibitor in patients suspected for acute deep venous thrombosis. We determined the tissue factor pathway inhibitor -33T/C polymorphism, free and total tissue factor pathway inhibitor, C-reactive protein, von Willebrand factor and D-Dimer in 160 consecutive patients admitted to hospital with a tentative diagnosis of acute deep venous thrombosis. Deep venous thrombosis was identified in 57 patients (18 distal and 39 proximal). The distribution of the tissue factor pathway inhibitor genotypes between patients with and without deep venous thrombosis showed a trend toward significant deviation (P = 0.08). The concentrations of free and total tissue factor pathway inhibitor, C-reactive protein, von Willebrand factor and D-Dimer were significantly higher in patients with deep venous thrombosis than in patients without deep venous thrombosis (P < 0.001 for all quantities). The significant relationship between free and total tissue factor pathway inhibitor and deep venous thrombosis persisted when adjusted for the tissue factor pathway inhibitor -33T/C polymorphism, C-reactive protein, von Willebrand Factor and potentially confounding clinical conditions (P < or = 0.004), but disappeared when adjusted for D-Dimer (P > or = 0.10). We conclude that patients suffering from acute deep venous thrombosis express significantly higher concentrations of tissue factor pathway inhibitor than patients without deep venous thrombosis. The significant relationship is not associated with the -33T/C polymorphism, inflammation or endothelial cell perturbation, but is most likely related to release of tissue factor pathway inhibitor from fibrin deposits.