Regorafenib therapy in metastatic colorectal cancer patients: markers and outcome in an actual clinical setting.

The oral multikinase inhibitor regorafenib had beneficial effects in randomized clinical phase III trials compared to the placebo in patients with metastatic colorectal cancer (mCRC) who progressed on standard therapies. The factors which influence regorafenib response and therapy sequence during treatment history are still highly discussed, and herein we analyzed the therapy algorithm, outcome and clinical markers following regorafenib application in a single center register study. Clinical data for 48 metastatic colorectal cancer patients were collected from 01.01.2013 to 31.12.2016. Treatment effects according to various patient and tumor characteristics were evaluated using univariate and multivariate Cox proportional hazard regression models. The 48 patients comprised 14 (29%) females and 34 (71%) males, with mean age 64.2±9 and ECOG 0-1. Progression free survival under regorafenib therapy was 2.9 months (quartiles 2.2; 4.4) and the overall response rate was 2 (4%) and disease control rate was 19 (40%). Overall survival (OS) and progression free survival (PFS) were investigated under regorafenib in the chemotherapy regimen given immediately before and afterthis treatment. Variables including tumor localization, Ras status, CEA and CA 19-9 plasma levels were analyzed for their impact on PFS, and the regorafenib-related adverse events were also observed. Our study confirms the efficacy of regorafenib in a real-life setting. We established that response rate and PFS in regorafenib treatment are independent of tumor localization, Ras status or biomarkers such as CEA and CA 19-9. Trifluridin/tripacil application or re-induction of chemotherapy +/- target therapy was effective following regorafenib therapy.

Age intrinsic loss of telomere protection via TRF1 reduction in endothelial cells.

Aging is a major factor predisposing for multiple diseases. Telomeres at the ends of chromosomes protect the integrity of chromosomal DNA. A specialized six-protein complex termed shelterin protects the telomere from unwanted interaction with DNA damage pathways. The aim of our study was to evaluate the integrity of telomeres and the stability of telomere protection during aging in endothelial cells (EC). We describe that aging EC can be characterized by an increased cell size (40%, p=0.02) and increased expression of PAI 1 (4 fold, p=0.02), MCP1 (10 fold, p=0.001) and GMCSF (15 fold, p=0.004). Telomeric state in aging cells is defined by an increased telomere oxidation (27%, p=0.01), reduced telomere length (62%, p=0.02), and increased DNA damage foci formation (5% in young EC versus 16% in aged EC, p=0.003). This telomeric dysfunction is accompanied by a reduction in the shelterin component TRF1 (33% mRNA, p=0.001; 24% protein, p=0.007). Overexpression of TRF1 in aging EC reduced telomere-associated DNA damage foci to 5% (p=0.02) and reduced expression levels of MCP1 (18% reduction, p=0.008). Aged EC have increased telomere damage and an intrinsic loss of telomere protection. Reestablishing telomere integrity could therefore be a target for rejuvenating endothelial cell function.

Neutrophil gelatinase associated lipocalin (NGAL) is elevated in type 2 diabetics with carotid artery stenosis and reduced under metformin treatment.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), an acute phase protein released by neutrophils, has been described as biomarker of inflammatory states. Type 2 diabetes mellitus (T2DM) is characterized by increased inflammation and an elevated risk for embolization of carotid artery stenosis (CAS). We aimed to explore the role of NGAL systemically and in plaques of diabetics undergoing carotid endarterectomy. Moreover, the potential anti-inflammatory effect of metformin on NGAL was addressed in diabetics. METHODS: Serum NGAL and matrix metalloproteinase (MMP)-9/NGAL levels were measured in 136 patients (67 with T2DM vs. 69 non-diabetics) by specific ELISA. Endarterectomy samples were graded histologically according to the American Heart Association´s classification. NGAL mRNA expression was detected using RealTime-PCR in carotid endarterectomy specimens. RESULTS: Serum NGAL [median 107.4 ng/ml (quartiles: 75.2-145.0) vs. 64.4 (50.4 -81.3), p < 0.0001] and MMP-9/NGAL [41.5 ng/ml (20.8-63.9) vs. 27.6 (16.0-42.4), p = 0.017] were significantly elevated in diabetics compared to non-diabetics, as were leukocytes, neutrophils, C-reactive protein and fibrinogen (all p < 0.05). In patients with symptomatic and asymptomatic CAS diabetics had higher NGAL levels compared to non-diabetics [128.8 ng/ml (100.8-195.6) vs. 64.8 (48.9-82.2] and [101.6 ng/ml (70.1-125.3) vs. 63.8 (51.0-81.3), respectively, both p < 0.0001]. Presence of T2DM and type VI plaques (with surface defect, hemorrhage or thrombus) had a profound impact on NGAL levels (both p < 0.01) in multiple linear regression analysis. NGAL mRNA was detectable in 95% of analyzed carotid artery lesions of diabetics compared to 5% of non-diabetics (p < 0.0001). Accordingly, cerebral embolization was more frequent in diabetics (52.2% vs. 29%, p = 0.006). Metformin treatment was associated with decreased NGAL [60.7 ng/ml (51.9-69.2) vs. 121.7 (103.7-169.9), p < 0.0001] and MMP-9/NGAL [20.8 ng/ml (12.1-26.5) vs. 53.7 (27.4-73.4), p = 0.007] in diabetics and reduced leukocyte infiltration in carotid lesions of diabetics. CONCLUSIONS: Higher NGAL levels in serum and plaques are associated with T2DM in patients with CAS. Metformin significantly reduced the inflammatory burden including NGAL in diabetics. Early treatment of these patients may be recommended, as elevated NGAL levels were linked with vulnerable plaques prone for embolization.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues ex vivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells in vitro. METHODS: NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. RESULTS: Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n = 19) compared with asymptomatic patients (in 37%; n = 20, p < .001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (p < 0.05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. CONCLUSION: By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.

The Influence of Simvastatin on NGAL, Matrix Metalloproteinases and Their Tissue Inhibitors in Human Intraluminal Thrombus and Abdominal Aortic Aneurysm Tissue.

OBJECTIVE/BACKGROUND: Matrix metalloproteinases (MMPs) play a pivotal role in the development and progression of abdominal aortic aneurysms (AAAs). The action of MMPs depends on a balance between tissue inhibitors of MMPs (TIMPs) and compounds that may prolong protease activity, such as neutrophil gelatinase-associated lipocalin (NGAL). METHODS: The study was designed to analyse gene expression and protein concentration of MMPs, TIMPs, and NGAL in AAA walls and intraluminal thrombi (ILTs) of patients on simvastatin (n = 10) and not on statins (n = 10). The patients were matched by age, sex, and AAA diameter. Expression of MMP2, MMP9, TIMP1, TIMP2, and NGAL was investigated by real time polymerase chain reaction, and MMP2, MMP9, MMP9/TIMP1, MMP9/TIMP2, and MMP9/NGAL protein levels by enzyme-linked immunosorbent assay. RESULTS: MMP2 and MMP9 protein and mRNA levels were comparable in the simvastatin and non-statin groups (p > .05); however, there was a significant decrease in TIMP1 mRNA in AAA tissue (p = .04). Moreover, a significant increase in MMP9/TIMP2 complex concentration in ILTs of patients on simvastatin was noted (median 94.71 ng/mL in the simvastatin group vs. 36.80 ng/mL in the non-statin group; p = .01). No significant difference was observed for NGAL mRNA or protein content in AAA and ILT. CONCLUSION: Simvastatin treatment in patients with AAAs may influence the concentration of proteases and their inhibitors (TIMPs) in aneurysmal wall tissue and ILTs. Thus, further studies should be undertaken to understand the different influence of statin therapy on the components of the MMP/TIMP system in AAAs and ILTs.

Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue.

OBJECTIVE: Obesity is associated with chronic inflammation of the adipose tissue, which contributes to obesity-associated complications such as insulin resistance and type 2 diabetes. Interleukin (IL)-33 acts via its receptor ST2 and is involved in the pathogenesis of inflammatory disorders including atherosclerosis and heart disease. IL-33 has been demonstrated to promote endothelial cell inflammatory response, but also anti-inflammatory and protective actions such as TH2 and M2 polarization of T cells and macrophages, respectively. IL-33 and ST2 have been shown to be expressed in human and murine adipose tissue. Our objective was to investigate alterations in obesity and a possible role of IL-33 in adipose tissue inflammation. SUBJECTS AND METHODS: We investigated severely obese patients (BMI>40 kg m(-2), n=20) and lean to overweight controls (BMI<30 kg m(-2); n=20) matched for age and sex, as well as diet-induced obese and db/db mice, in order to determine the impact of obesity on IL-33 and ST2 gene and protein expression levels in adipose tissue and blood, and their correlation with inflammatory and metabolic parameters. Furthermore, we examined the cellular source and location of IL-33 and ST2 in situ. RESULTS: IL-33 and ST2 expression levels were markedly elevated in omental and subcutaneous adipose tissue of severely obese humans and in diet-induced obese mice, but not in leptin receptor-deficient db/db mice. In addition, soluble ST2, but not IL-33 serum levels, were elevated in obesity. The main source for IL-33 in adipose tissue were endothelial cells, which, in humans, exclusively expressed ST2 on their surface. IL-33 expression strongly correlated with leptin expression in human adipose tissue. CONCLUSIONS: Expression of IL-33 and its receptor ST2 in human adipose tissue is predominantly detectable in endothelial cells and increased by severe obesity indicating an autocrine action. Thus, the adipose tissue microvasculature could participate in obesity-associated inflammation and related complications via IL-33/ST2.

Decreased tissue levels of cyclophilin A, a cyclosporine a target and phospho-ERK1/2 in simvastatin patients with abdominal aortic aneurysm.

BACKGROUND: Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown. MATERIAL AND METHODS: The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot. RESULTS: The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression. CONCLUSION: Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.

Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB.

OBJECTIVES: Statins have been reported to suppress the progression of abdominal aortic aneurysm (AAA). However, the effects of statins on inflammatory processes and free radicals generation are poorly understood. METHODS: Wall samples from 51 patients (simvastatin patients, n = 34; non-statin patients, n = 17; matched by sex, age and aneurysm size) subjected to elective open AAA repair were analysed. We examined the effects of simvastatin on lipid peroxidation (4-hydroxy-trans-2-nonenal (4-HNE)), hydrogen peroxide (H(2)O(2)), tumour necrosis factor alpha (TNF-α) concentration, superoxide dismutase (SOD) and catalase (CAT) activity as well as nuclear factor kappa B (NF-κB) pathway activation in human AAA wall samples. RESULTS: Treatment with simvastatin resulted in a decrease in 4-HNE and TNF-α concentration (median 4.18 µg/mg protein vs. 4.75, p = 0.012; median 10.33 pg/ml vs. 11.81, p = 0.026, respectively). CAT activity was higher in the simvastatin group (median 3.98 U ml vs. 3.19, p = 0.023). NF-κB expression was lower (p = 0.018) in the simvastatin group. However, simvastatin had little effect on H(2)O(2) concentration (p = 0.832) and SOD activity (p = 0.401). CONCLUSION: Simvastatin inhibits free radicals and TNF-α generation and improves antioxidant capacity of human AAA wall tissue, possibly through the suppression of NF-κB activity. This may be one possible explanation how statins can inhibit AAA oxidative stress.

Cardiac biomarkers for risk stratification of arrhythmic death in patients with heart failure and reduced ejection fraction.

Objectives. Patients with heart failure and reduced left ventricular ejection fraction (HFrEF) are prone to ventricular tachyarrhythmias. We tested whether biomarkers C-terminal Endothelin 1 (CT-ET1), midregional pro atrial natriuretic peptide (MR-proANP) and midregional pro adrenomedullin (MR-proADM) might improve risk stratification for arrhythmic death.Methods: This prospective observational study included 160 heart failure patients with ischaemic cardiomyopathy (ICM) or non-ischaemic, dilated cardiomyopathy (DCM) and 30 control patients without heart disease. Primary endpoint was arrhythmic death (ArD) or resuscitated cardiac arrest (resCA).Results: A total of 61 patients died during the median follow-up of 7.0 [5.2-8.4] years. An ArD or resCA was observed in 48 patients. Plasma levels of CT-ET1 (p = 0.002), MR-proANP (p < 0.001) and MR-proADM (p = 0.013) were significantly higher in ICM or DCM patients compared to controls. MR-proANP levels in ICM patients were associated with a significantly increased risk for ArD or resCA (hazard ratio (HR) = 1.42, [95%CI: 1.08-1.85], p = 0.011) in a multivariable Cox regression model. Plasma levels of CT-ET1 (HR = 1.07 [0.98-1.17], p = 0.113) and MR-proADM (HR = 1.80 [0.92-3.55], p = 0.087) were not associated with ArD or resCA in ICM patients. No significant association with ArD or resCA was found in DCM patients. Multivariable Cox regression showed that CT-ET1 (HR = 1.14 [1.07-1.22], p < 0.001), MR-proANP (HR = 1.64 [1.29-2.08], p < 0.001) and MR-pro ADM (HR = 2.06 [1.12-3.77], p = 0.020) were associated with a higher risk for overall mortality.Conclusion: Patients with HFrEF had elevated levels of CT-ET1, MR-proANP and MR-proADM. Plasma levels of MR-proANP are useful as predictor for arrhythmic death in patients with ICM.

Roux-en-Y-Bariatric Surgery Reduces Markers of Metabolic Syndrome in Morbidly Obese Patients.

BACKGROUND: Obesity is closely linked to increased markers of metabolic syndrome and development of diabetes. Roux-en-Y bariatric surgery reduces hyperinsulinemia and improves insulin sensitivity and hence benefits morbidly obese patients. AIM: To determine changes in markers of metabolic syndrome, pancreatic function, and hepatic insulin sensitivity in patients before and 1 year after undergoing Roux-en-Y gastric bypass surgery. METHODS: We enrolled 43 consecutive patients in a single center. Markers for metabolic syndrome included proinsulin, insulin, C-peptide, liver enzymes, and serum levels of selected microRNAs hsa-miR-122, hsa-miR-130, hsa-miR-132, and hsa-miR-375. RESULTS: After surgery, all patients showed a significant 37% drop of body mass index (p < 0.001). Furthermore, proinsulin (59% reduction, p < 0.001), insulin (76% reduction, p < 0.001), and C-peptide (56% reduction, p < 0.001) were all reduced 1 year after surgery. Using the hepatic insulin clearance score, we determined a significant increase in hepatic insulin clearance after surgery (76% increase, p < 0.001). Especially diabetic patients showed a marked 2.1-fold increase after surgery. Hepatic enzymes ALT (35% reduction, p = 0.002) and γGT (48% reduction, p < 0.001) were significantly reduced in all patients with similar improvement in diabetic and non-diabetic patients. miRNAs hsa-miR-122, hsa-miR-130, and hsa-miR-132 were all significantly reduced whereas hsa-miR-375 was increased after gastric bypass surgery (p < 0.001 for all miRNAs). CONCLUSION: Both liver and pancreatic stress parameters were reduced significantly 1 year after Roux-en-Y gastric bypass surgery suggesting an overall amelioration of the metabolic syndrome in all patients regardless of previous health status.

Vascular endothelial growth factor is induced by the inflammatory cytokines interleukin-6 and oncostatin m in human adipose tissue in vitro and in murine adipose tissue in vivo.

OBJECTIVES: It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. METHODS AND RESULTS: We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1beta synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. CONCLUSION: We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.

Reduction of Premature Aging Markers After Gastric Bypass Surgery in Morbidly Obese Patients.

BACKGROUND: Obesity is considered to be a major comorbidity. Obese patients suffer from an increased proinflammatory state associated with a premature aging phenotype including increased secretion of senescence-associated secretory proteins (SASP) and reduced telomere length. Micro-ribonucleic acids (miRNAs) are non-coding RNA molecules that could modify the post-transcriptional process. Several studies have reported associations between miRNAs and metabolic unhealthy conditions. AIM: To determine if bariatric surgery and the resulting weight loss could reverse the premature aging phenotype. METHODS: We enrolled 58 morbidly obese patients undergoing bariatric surgery. Markers of premature aging including the SASP IL-6, CRP and PAI-1, 7 miRNAs, as well as telomere length and telomere oxidation in mononuclear cells were evaluated. RESULTS: Patients showed a significant drop of body mass index (BMI; 43.98 ± 3.5 versus 28.02 ± 4.1, p < 0.001). We observed a significant reduction in SASP including a reduction of 55% of plasma IL-6 levels (p = 0 < 0.001), 83% of CRP levels (p = 0.001) and 15% of plasma PAI-1 levels (p < 0.001). Telomere length doubled in the patient cohort (p < 0.001) and was accompanied by a reduction in the telomere oxidation index by 70% (p < 0.001). Telomere length was inversely correlated with telomere oxidation. The aging-associated miRNA miR10a_5p was upregulated significantly (p = 0.039), while the other tested miRNAs showed no difference. CONCLUSION: Our data indicate a significant reduction of the proinflammatory SASP after bariatric surgery. We observed an increase in telomere length and reduced oxidative stress at telomeres. miR10a_5p which is downregulated during aging was upregulated after surgery. Overall, bariatric surgery ameliorated the premature aging phenotype.

Inflammatory cytokines interleukin-6 and oncostatin m induce plasminogen activator inhibitor-1 in human adipose tissue.

BACKGROUND: Adipose tissue is a prominent source of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of plasminogen activation. Increased PAI-1 expression acts as a cardiovascular risk factor, and plasma levels of PAI-1 strongly correlate with body mass index (BMI). Elevated serum levels of interleukin-6 (IL-6), an inflammatory cytokine and a member of the glycoprotein 130 (gp130) ligand family, are found in obese patients and might indicate low-grade systemic inflammation. Another gp130 ligand, oncostatin M (OSM), upregulates PAI-1 in cardiac myocytes, astrocytes, and endothelial cells. We used tissue explants and primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether IL-6 and OSM affect PAI-1 expression in fat. METHODS AND RESULTS: Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in PAI-1 production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte differentiation was induced by hormone supplementation. All cell types expressed receptors for IL-6 and OSM and produced up to 12-fold increased levels of PAI-1 protein and up to 9-fold increased levels of PAI-1 mRNA on stimulation with IL-6 and OSM. AG-490, a janus kinase/signal transducer and activator of transcription inhibitor, abolished the OSM-dependent PAI-1 induction almost completely. CONCLUSIONS: We have for the first time established a link between the gp130 ligands, the proinflammatory mediators IL-6 and OSM, and the expression of PAI-1 in human adipose tissue. Thus, we speculate that IL-6 and OSM, by upregulating PAI-1 in adipose tissue, can contribute to the increased cardiovascular risk of obese patients.

Interleukin-33 induces urokinase in human endothelial cells--possible impact on angiogenesis.

BACKGROUND: Urokinase-type plasminogen activator (u-PA) plays a pivotal role in extracellular proteolysis and is thought to be critically involved in the modulation of angiogenesis. Interleukin (IL)-33 is a member of the IL-1 cytokine family, which is thought to act as danger signal that is released from cells after injury. IL-33 is involved in the pathogenesis of various inflammatory diseases and previously was shown to induce angiogenesis and inflammatory activation of endothelial cells. OBJECTIVE: We investigated the impact of IL-33 on u-PA in endothelial cells as a new possible function for IL-33. METHODS AND RESULTS: We could demonstrate that IL-33 upregulated u-PA mRNA expression and protein production in human coronary artery and human umbilical vein endothelial cells in a time- and concentration-dependent manner via interaction with its receptor ST2 and activation of the nuclear factor-κB pathway but independent of autocrine IL-1-induced effects. The hydroxymethylglutaryl-coenzyme A reductase inhibitor simvastatin abrogated the IL-33-induced increase in u-PA, thus providing further evidence for pleiotropic effects of statins. IL-33 induced u-PA-dependent capillary-like tube formation and vessel sprouting. In human carotid atherosclerotic plaques (n = 16), u-PA mRNA positively correlated with IL-33 mRNA expression (r = 0.780, P < 0.001). Furthermore, IL-33 and u-PA protein were detected in endothelial cells in these samples using fluorescence immunohistochemistry. CONCLUSIONS: We hypothesize that IL-33, representing a danger signal that is released after tissue damage, in addition to its role in the inflammatory activation of endothelial cells, is involved in u-PA-driven angiogenesis, a process that has been shown before to be linked to inflammation in various pathologies.