Etiology and treatment of cancer-related secondary lymphedema.

Lymphedema and specifically cancer-related lymphedema is not the main focus for both patients and physicians dealing with cancer. Its etiology is an unfortunate complication of cancer treatment. Although lymphedema treatments have gained an appreciable consensus, many practitioners have developed and prefer their own specific protocols and this is especially true for conventional (manual) versus surgical treatments. This collection of presentations explores the incidence and genetics of cancer-related lymphedema, early detection and monitoring techniques, both conventional and operative treatment options, and the importance and role of exercise for patients with cancer-related lymphedema. These assembled presentations provide valuable insights into the challenges and opportunities presented by cancer-related lymphedema including the latest research, treatments, and exercises available to improve patient outcomes and quality of life.

Secondary lymphedema from cancer therapy.

This manuscript is a summary of findings focusing on various aspects of secondary lymphedema specifically as a sequelae of treatment for cancer. The topic was addressed at a session held during the 8th International Congress on Cancer Metastasis that was unique a for the inclusion of patients with lymphedema and therapists joining physicians, healthcare professionals, and researchers in an effort to give an overview of secondary lymphedema following cancer therapy as well as highlighting the unknowns in the field. Lymphedema is defined and both diagnosis and incidence of cancer-related lymphedema are explored. Further, exploration of imaging options for lymphedema and information on the genetic research for patients with cancer-related secondary lymphedema are presented. Patient education and early detection methods are then explored followed by conservative treatment. Finally, an examination of surgical treatment methods available for patients with lymphedema is covered. Overall, this manuscript presents valuable information and updates for those not familiar with incidence, diagnosis, early detection, and rehabilitation of patients with cancer-related secondary lymphedema.

Digenic Inheritance of a FOXC2 Mutation and Two PIEZO1 Mutations Underlies Congenital Lymphedema in a Multigeneration Family.

BACKGROUND: The lymphatic system is essential for maintaining the balance of interstitial fluid in tissues and for returning protein-rich fluids (lymph) to the bloodstream. Congenital lymphatic defects lead to accumulation of lymph in peripheral tissues and body cavities, termed primary lymphedema. To date, only a limited number of individual genes have been identified in association with primary lymphedema. However, variability of age of onset and severity of lymphatic abnormalities within some families suggests that multiple mutations or genes may be responsible, thus hampering efforts to identify individual associated genes. METHODS: Whole exome sequencing (WES) was performed in 4 members of a large multigeneration family with highly variable lymphedema and followed by Sanger sequencing for identified mutations in 34 additional family members. Genotypes were correlated with clinical and lymphangioscintigraphic phenotypes. RESULTS: WES uncovered 2 different mechanotransducer PIEZO1 mutations and one FOXC2 transcription factor mutation in various combinations. Sanger sequencing confirmed the presence/absence of the 3 variants in affected and unaffected family members and co-segregation of one or more variants with disease. Genetic profiles did not clearly correlate with the highly variable severity of lymphatic abnormalities. CONCLUSIONS: WES in lymphedema families can uncover unexpected combinations of several lymphedema-associated mutations. These findings provide essential information for genetic counseling and reveal complex gene interactions in lymphatic developmental pathways. These can offer insights into the complex spectrum of clinical and lymphatic lymphedema phenotypes and potential targets for treatment.

Standardization of lower extremity quantitative lymphedema measurements and associated patient-reported outcomes in gynecologic cancers.

Practice changing standardization of lower extremity lymphedema quantitative measurements with integrated patient reported outcomes will likely refine and redefine the optimal risk-reduction strategies to diminish the devastating limb-related dysfunction and morbidity associated with treatment of gynecologic cancers. The National Cancer Institute (NCI), Division of Cancer Prevention brought together a diverse group of cancer treatment, therapy and patient reported outcomes experts to discuss the current state-of-the-science in lymphedema evaluation with the potential goal of incorporating new strategies for optimal evaluation of lymphedema in future developing gynecologic clinical trials.

Sex-limited penetrance of lymphedema to females with CELSR1 haploinsufficiency: A second family.

A second multigeneration family with hereditary lymphedema (LE) secondary to a variant in the planar polarity gene, CELSR1, is described. Dominant inheritance of the variant was discovered using whole-exome sequencing and confirmed by Sanger sequencing. In contrast to heterozygous males, all heterozygous females showed LE during physical examination albeit variable in severity and age of onset. Lymphscintigraphy in affected females showed previously undescribed lymphatic abnormalities consistent with lymphangiectasia, valve dysfunction, and thoracic duct reflux.

Role of Lymphatic Deficiency in the Pathogenesis and Progression of Inflammatory Bowel Disease to Colorectal Cancer in an Experimental Mouse Model.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic inflammation, which can progress to colorectal cancer, with duration of disease being the most important risk factor. Although many factors are involved, the pathogenic link between inflammation and cancer and the role played by the lymphatic system have not been fully investigated. This project uses lymphatic-deficient mice (Angiopoietin-2 [Ang2] knockout) to examine the lymphatic system in the progression of IBD to colorectal cancer. METHODS: Angiopoietin-2 wild-type, heterozygote, and knockout mice received a single injection of the procarcinogen azoxymethane and had an IBD-promoting chemical irritant (dextran sodium sulfate) added to their drinking water over a 7-week period. We measured disease activity (weight loss, stool consistency, fecal occult blood) during the study and at sacrifice, collected blood for cytokine/biomarker (Ang2, interleukin [IL] 1-ß, IL-6, tumor necrosis factor α [TNFα], and VEGF-C) enzyme-linked immunosorbent assay analysis, measured colon length, and assessed tumor burden. RESULTS: Ang2 knockout (KO) mice exhibited reduced (55%) survival vs wild-type (100%) and heterozygotes (91%; P < 0.01 and P < 0.0001, respectively). Most (>89%) mice developed tumors, and the incidence of colorectal cancer did not differ among the genotypes (P = 0.32). The tumor area was significantly increased in KO mice (P = 0.004). Of the biomarkers measured in the serum, Ang2 and TNF-α concentrations were significantly different among the genotypes (P = 3.35e-08 and P = 0.003 respectively). Disease activity was significantly increased in KO mice compared with wild-type and heterozygote mice (P = 0.033). CONCLUSIONS: Lymphatic deficiency, defective lymphangiogenesis, and impaired lymphatic-generated inflammation did not protect against clinical IBD or progression to colorectal cancer in this experimental model.

Leukocyte expression profiles reveal gene sets with prognostic value for seizure-free outcome following stereotactic laser amygdalohippocampotomy.

Among patients with intractable epilepsy, the most commonly performed surgical procedure is craniotomy for amygdalohippocampectomy (AH). Stereotactic laser amygdalohippocampotomy (SLAH) has also been recently employed as a minimally invasive treatment for intractable temporal lobe epilepsy (TLE). Among patients treated with AH and SLAH approximately 65% and 54% of patients become seizure-free, respectively. Therefore, selection criteria for surgical candidates with improved prognostic value for post-operative seizure-free outcome are greatly needed. In this study, we perform RNA sequencing (RNA-Seq) on whole blood leukocyte samples taken from 16 patients with intractable TLE prior to SLAH to test the hypothesis that pre-operative leukocyte RNA expression profiles are prognostic for post-operative seizure outcome. Multidimensional scaling analysis of the RNA expression data indicated separate clustering of patients with seizure free (SF) and non-seizure-free (NSF) outcomes. Differential expression (DE) analysis performed on SF versus NSF groups revealed 24 significantly differentially expressed genes (≥2.0-fold change, p-value < 0.05, FDR <0.05). Network and pathway analyses identified differential activation of pathways involved in lipid metabolism, morphology of oligodendrocytes, inflammatory response, and development of astrocytes. These results suggest that pre-operative leukocyte expression profiles have prognostic value for seizure outcome following SLAH.

Lymphedema following cancer therapy: overview and options.

This summit focusing on lymphedema following cancer therapy was held during the 7th International Symposium on Cancer Metastasis through the Lymphovascular System. It was unique for the inclusion of patients with lymphedema joining physicians, therapists, healthcare professionals, and researchers to highlight what is known and more importantly what is unknown about the current state of research and treatment in the United States. The session opened with an introduction to lymphedema and then explored the incidence of multiple cancer-related lymphedemas, imaging tools and techniques useful for the diagnosis of lymphatic system abnormalities, and the new findings concerning the genetics of cancer-related lymphedema. It closed with a review of advocacy for patients and healthcare professionals and both conservative and surgical treatment options, followed by a panel discussion and questions. The session provided important information and updates which will be of value for improving the rehabilitation and overall support of patients with cancer-related lymphedema.

Characterization of TCP-1 probes for molecular imaging of colon cancer.

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.

Cortical gene expression: prognostic value for seizure outcome following temporal lobectomy and amygdalohippocampectomy.

Whole genome analyses were performed to test the hypothesis that temporal cortical gene expression differs between epilepsy patients rendered seizure-free versus non-seizure-free following anterior temporal lobectomy with amygdalohippocampectomy (ATL/AH). Twenty four patients underwent ATL/AH to treat medically intractable seizures of temporal lobe origin (mean age 35.5 years, mean follow-up 42.2 months); they were then dichotomized into seizure-free and non-seizure-free groups. Tissue RNA was isolated from the lateral temporal cortex and gene expression analysis was performed. Whole genome data were analyzed for prognostic value for seizure-free outcome following ATL/AH by logistic regression. Genes that could distinguish seizure outcome groups were identified based on providing an accuracy of >0.90 judging by area under the receiver operating characteristic curve, AUC, with a P value of the slope coefficient of <0.05. Four genes and seven RNA probes were with prognostic value for post-operative seizure-free outcome. Gene expression associated with seizure-free outcome included relative down-regulation of zinc finger protein 852 (ZNF852), CUB domain-containing protein 2 (CDCP2), proline-rich transmembrane protein 1 (PRRT1), hypothetical LOC440200 (FLJ41170), RNA probe 8047763, RNA probe 8126238, RNA probe 8113489, RNA probe 8092883, RNA probe 7935228, RNA probe 806293, and RNA probe 8104131. This study describes the predictive value of temporal cortical gene expression for seizure-free outcome after ATL/AH. Four genes and seven RNA probes were found to predict post-operative seizure-free outcome. Future prospective investigation of these genes and probes in human brain tissue and blood could establish new biomarkers predictive of seizure outcome following ATL/AH.

Cortical gene expression correlates of temporal lobe epileptogenicity.

INTRODUCTION: Despite being one of the most common neurological diseases, it is unknown whether there may be a genetic basis to temporal lobe epilepsy (TLE). Whole genome analyses were performed to test the hypothesis that temporal cortical gene expression differs between TLE patients with high vs. low baseline seizure frequency. METHODS: Baseline seizure frequency was used as a clinical measure of epileptogenicity. Twenty-four patients in high or low seizure frequency groups (median seizures/month) underwent anterior temporal lobectomy with amygdalohippocampectomy for intractable TLE. RNA was isolated from the lateral temporal cortex and submitted for expression analysis. Genes significantly associated with baseline seizure frequency on likelihood ratio test were identified based on >0.90 area under the ROC curve, P value of <0.05. RESULTS: Expression levels of forty genes were significantly associated with baseline seizure frequency. Of the seven most significant, four have been linked to other neurologic diseases. Expression levels associated with high seizure frequency included low expression of Homeobox A10, Forkhead box A2, Lymphoblastic leukemia derived sequence 1, HGF activator, Kelch repeat and BTB (POZ) domain containing 11, Thanatos-associated protein domain containing 8 and Heparin sulfate (glucosamine) 3-O-sulfotransferase 3A1. CONCLUSIONS: This study describes novel associations between forty known genes and a clinical marker of epileptogenicity, baseline seizure frequency. Four of the seven discussed have been previously related to other neurologic diseases. Future investigation of these genes could establish new biomarkers for predicting epileptogenicity, and could have significant implications for diagnosis and management of temporal lobe epilepsy, as well as epilepsy pathogenesis.

Downregulation of FoxC2 Increased Susceptibility to Experimental Colitis: Influence of Lymphatic Drainage Function?

BACKGROUND: Although inflammation-induced expansion of the intestinal lymphatic vasculature (lymphangiogenesis) is known to be a crucial event in limiting inflammatory processes, through clearance of interstitial fluid and immune cells, considerably less is known about the impact of an impaired lymphatic clearance function (as seen in inflammatory bowel diseases) on this cascade. We aimed to investigate whether the impaired intestinal lymphatic drainage function observed in FoxC2 mice would influence the course of disease in a model of experimental colitis. METHODS: Acute dextran sodium sulfate colitis was induced in wild-type and haploinsufficient FoxC2 mice, and survival, disease activity, colonic histopathological injury, neutrophil, T-cell, and macrophage infiltration were evaluated. Functional and structural changes in the intestinal lymphatic vessel network were analyzed, including submucosal edema, vessel morphology, and lymphatic vessel density. RESULTS: We found that FoxC2 downregulation in FoxC2 mice significantly increased the severity and susceptibility to experimental colitis, as displayed by lower survival rates, increased disease activity, greater histopathological injury, and elevated colonic neutrophil, T-cell, and macrophage infiltration. These findings were accompanied by structural (dilated torturous lymphatic vessels) and functional (greater submucosal edema, higher immune cell burden) changes in the intestinal lymphatic vasculature. CONCLUSIONS: These results indicate that sufficient lymphatic clearance plays a crucial role in limiting the initiation and perpetuation of experimental colitis and those disturbances in the integrity of the intestinal lymphatic vessel network could intensify intestinal inflammation. Future therapies might be able to exploit these processes to restore and maintain adequate lymphatic clearance function in inflammatory bowel disease.

A five generation family with a novel mutation in FOXC2 and lymphedema worsening to hydrops in the youngest generation.

We describe a five generation family with dominantly inherited lymphedema, but no distichiasis, in which 3/3 affected offspring in the fifth generation have died of fetal hydrops and related birth defects. Mutational analysis disclosed a novel mutation in FOXC2 (R121C) in affected members. We searched for possible genetic influences on the greater severity of lymphedema (hydrops) in the fifth generation. Karyotypes disclosed an extra band in Xp in one affected fetus, but this was also found in the mother. Copy number variation (CNV) studies on four members of the pedigree (mother of the three severely affected fetuses/infants; one severely affected; a full, and a half, unaffected sibs) did not detect the source of the Xp band or a possible influence on the severe phenotype. However, use of SNP arrays did allow identification of the portion of the maternal proximal Xp shared by a hydrops-affected daughter and son which was not shared by an unaffected daughter from the same sibship.

Role of angiopoietin-2 in corneal lymphangiogenesis.

PURPOSE: Lymphatic research has progressed rapidly in recent years. Lymphatic dysfunction has been found in myriad disorders from cancer metastasis to transplant rejection; however, effective treatment for lymphatic disorders is still limited. This study investigates the role of angiopoietin-2 (Ang-2) in corneal inflammatory lymphangiogenesis (LG) in vivo and in lymphatic endothelial cell (LEC) functions in vitro. METHODS: Standard suture placement model was used to study Ang-2 expression in inflamed cornea, and corneal LG and hemangiogenesis (HG) responses in Ang-2 knockout mice. Moreover, human LEC culture system was used to examine the effect of Ang-2 gene knockdown on LEC functions using small interfering RNAs (siRNAs). The effect of siRNA treatment on corneal LG was also assessed in vivo. RESULTS: Angiopoietin-2 was expressed on lymphatic vessels and macrophages in inflamed cornea. While corneal LG response was abolished in Ang-2 knockout mice, the HG response was also significantly suppressed with disorganized patterning. Moreover, anti-Ang-2 treatment inhibited LEC proliferation and capillary tube formation in vitro and corneal LG in vivo. CONCLUSIONS: Angiopoietin-2 is critically involved in lymphatic processes in vivo and in vitro. Further investigation of the Ang-2 pathway may provide novel insights and therapeutic strategies for lymphatic-related disorders, which occur both inside and outside the eye.

Curcuminoids limit neutrophil-mediated reperfusion injury in experimental stroke by targeting the endothelium.

OBJECTIVE: We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion. METHODS: Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed. RESULTS: Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC. CONCLUSION: Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.

Assessment and risk reduction in lymphedema.

OBJECTIVES: To inform oncology nurses about current concepts and practices for the diagnosis and assessment of patients with cancer-related lymphedema and to examine risk-reduction strategies. DATA SOURCES: Peer reviewed literature. CONCLUSION: Although there is not an established, standardized protocol for the assessment of these patients, multiple diagnostic and assessment tools are available and in use. In a clinical setting for comprehensive lymphedema care, development of assessment protocols that are reproducible and utilized over the course of care is necessary. Risk-reduction strategies for lymphedema are an emerging focus in oncology care. IMPLICATIONS FOR NURSING PRACTICE: Established methods for diagnosis and assessment are available and should be incorporated into practice. Risk-reduction strategies should be considered.

Chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy.

To identify the upstream signals of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy (TLE), we evaluated by immunohistochemistry and confocal microscopy brain tissues of 13 TLE patients and 5 control patients regarding expression of chemokines and cell-cycle proteins. The chemokine RANTES (CCR5) and other CC-chemokines and apoptotic markers (caspase-3, -8, -9) were expressed in lateral temporal cortical and hippocampal neurons of TLE patients, but not in neurons of control cases. The chemokine RANTES is usually found in cytoplasmic and extracellular locations. However, in TLE neurons, RANTES was displayed in an unusual location, the neuronal nuclei. In addition, the cell-cycle regulatory transcription factor E2F1 was found in an abnormal location in neuronal cytoplasm. The pro-inflammatory enzyme cyclooxygenase-2 and cytokine interleukin-1ß were expressed both in neurons of patients suffering from temporal lobe epilepsy and from cerebral trauma. The vessels showed fibrin leakage, perivascular macrophages and expression of IL-6 on endothelial cells. In conclusion, the cytoplasmic effects of E2F1 and nuclear effects of RANTES might have novel roles in neuronal apoptosis of TLE neurons and indicate a need to develop new medical and/or surgical neuroprotective strategies against apoptotic signaling by these molecules. Both RANTES and E2F1 signaling are upstream from caspase activation, thus the antagonists of RANTES and/or E2F1 blockade might be neuroprotective for patients with medically intractable temporal lobe epilepsy. The results have implications for the development of new medical and surgical therapies based on inhibition of chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy.

Best Practice Guidelines in Assessment, Risk Reduction, Management, and Surveillance for Post-Breast Cancer Lymphedema.

Breast cancer-related lymphedema (LE) is a progressive, chronic disease that affects millions of cancer survivors and primarily results from surgical lymphatic vessel/node removal and radiation therapy. Patient education and support for importance of early detection is essential in helping health care providers detect lymphedema early, when there is the best chance to prevent progression. Improved imaging and surgical techniques have decreased the incidence of LE; however, effective risk-reduction and treatment have historically lacked the level of evidence necessary to standardize effective treatment. The purpose of this article is to report an extensive review of literature, including highlighted multidisciplinary studies within the past three years, in order to update best practice guidelines in assessment, risk reduction, management, and surveillance for post-breast cancer lymphedema.

Dysmorphogenesis of lymph nodes in Foxc2 haploinsufficient mice.

Dysmorphogenesis of lymph nodes displayed in a fork head transcription factor Foxc2 haploinsufficient mice--a model for lymphedema-distichiasis syndrome--was studied by immunohistochemistry and electron microscopy. The Foxc2 heterozygous mice manifested lymph node hyperplasia composed of conspicuous proliferation of endothelial cells forming the lymphatic sinus and α-smooth muscle actin (SMA)-immunopositive fibroblast-like cells in the lymphatic pulp, particularly around the sinus. The hyperplastic sinus endothelial cells and the SMA-positive cells demonstrated distinct immunolocalization of platelet-derived growth factor (PDGF)-B, a crucial chemoattractant for vascular mural cell recruitment, and its receptor, PDGFR-ß, respectively. The observations suggest that the sinus endothelial cells elicit abnormal recruitment of the fibroblast-like cells as a type of vascular mural cells via PDGF-B/PDGFR-ß signaling in lymph nodes of the Foxc2 heterozygotes. Furthermore, in Foxc2 heterozygous lymph nodes, recruited SMA-positive cells displayed an intense immunoreaction for vascular endothelial growth factor (VEGF)-C, a highly specific lymphangiogenic factor, and its receptor, VEGFR-3, was preferentially distributed in the lymphatic sinus endothelial cells. These findings suggest that an interactive cycle between lymphatic sinus endothelial cells and the fibroblast-like cells, which involves PDGF-B/PDGFR-ß and VEGF-C/VEGFR-3 signaling, is essential for aberrant hyperplasia of the lymphatic sinus and the fibroblast-like cells in Foxc2 haploinsufficiency.

Elevated levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity.

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 µM UCB, in the presence of 100 µM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 µM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of ß-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and ß-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.