Aberrant bone marrow vascularization patterns in untreated patients with Gaucher disease type 1.

Bone marrow (BM) in subjects with Gaucher disease (GD) displays accumulation of Gaucher cells (GC), i.e. glucocerebroside-laden macrophages. Following the assumption that macrophage proliferation and perturbation in GD modulates local inflammation-associated phenomena including angiogenesis, BM biopsies from 11 untreated GD patients and 36 controls were investigated for morphology and angiogenesis-associated features. These included microvascular density, (MVD), vessel structure and pericyte coverage, expression of VEGF-A and angiopoietins (ANGPT1 and 2). In GD BM, cellularity was higher, and GC clustered in cohesive but poorly demarcated areas, leaving irregular islands with normal hematopoiesis. MVD was 2.6-fold higher in GD marrows than in controls (p<0.001). In GC-rich areas, MVD was 1.4-fold higher (p=0.026), and vessel architecture was abnormal compared with GC-poor areas. MVD correlated with BM cellularity, particularly in GC-rich areas. Moreover, 30±17% of GD BM vessels were pericyte-coated, significantly fewer than in controls (48±16%; p<0.001). Expression of ANGPT1 and 2 was significantly higher in GD BM vessel walls than in controls (7.2- and 13.2-fold higher), whereas VEGF expression was 20-fold lower (p<0.05 for all). Thus, human GD BM shows increased angiogenesis with defective pericyte coating and skewed VEGF/ANGPT1 and 2 balances, presumably related to local accumulation of GC.

Serum betatrophin level increased in subjects with metabolic syndrome: a case-control study / Aumento del nivel de betatrofina en suero en sujetos con síndrome metabólico: estudio de casos y controles

Background: Betatrophin is a novel adipokine that provokes pancreatic β-cell proliferation and is involved in lipid metabolism. Aims: This study aims to evaluate the role of serum betatrophin in metabolic syndrome (MetS). Methods: A hospital-based, age-/gender-matched case control study was conducted. The serum betatrophin level was evaluated by enzymelinked immunosorbent assay. Serum concentrations of 12 adipokines were measured to assess their associations with serum betatrophin, using commercial Adipokine Magnetic Bead Panel kits. Statistical analyses included bivariate correlation, receiver operating characteristic (ROC) curve, and multivariate stepwise linear regression. Results: Serum betatrophin showed a higher level in MetS patients (997.36 ± 475.92 pg/ml, p = 0.001) compared with controls (735.35 ± 526.51 pg/ml). Compared with the lowest tertile, the highest tertile of serum betatrophin level indicated an association with higher risk of MetS (adjusted odds ratio = 3.521, 95% confidence interval [CI] [1.191-10.413], p = 0.023). ROC curve of betatrophin was developed to predict the presence of MetS (area under ROC = 0.682 [95% CI, 0.597-0.767], p < 0.001). Furthermore, betatrophin correlated with several parameters, e.g. age (r = 0.286, p < 0.001), body mass index (r = 0.160, p = 0.046), waist-to-hip ratio (r = 0.241, p = 0.002), high-density lipoprotein cholesterol (r = -0.167, p = 0.037), low-density lipoprotein cholesterol (r = -0.195, p = 0.015), fasting plasma glucose (r = 0.266, p = 0.001), hemoglobin A1C (r = 0.314, p < 0.001), homeostasis model assessment of insulin resistance (r = 0.272, p = 0.001), and various adipokines, e.g. resistin (r = 0.571, p < 0.001), interleukin-8 (r = 0.435, p < 0.001), tumor necrosis factor-α (r = 0.295, p = 0.011) and lipocalin-2 (r = 0.346, p = 0.003). Conclusions: This study supports that serum betatrophin plays an important role in MetS, involving the regulations of glucose and lipid metabolism and inflammation (AU)

Introducción: la betatrofina es una novedosa adipoquina que provoca la proliferación de células β pancreáticas e interviene en el metabolismo de los lípidos. Objetivos: el propósito de este estudio es evaluar el papel de la betatrofina en el síndrome metabólico. Método: se llevó a cabo un estudio hospitalario de casos y controles según sexo y edad. El nivel de betatrofina en suero fue evaluado mediante ensayo por inmunoabsorción ligado a enzimas. Se midieron las concentraciones en suero de 12 adipoquinas para evaluar las asociaciones con la betatrofina usando los kits comerciales Adipokine Magnetic Bead Panel. Los análisis estadísticos incluyeron correlación bivariada, análisis de curva ROC y análisis de regresión lineal multivariable. Resultados: el nivel de betatrofina en suero fue más elevado en pacientes con síndrome metabólico (997,36 ± 475,92 pg/ml, p = 0,001) que en los controles (735,35 ± 526,51 pg/ml). Frente al tercil más bajo, el tercil más alto del nivel de betatrofina mostró una asociación con mayor riesgo de síndrome metabólico (odds ratio ajustado = 3,521, intervalo de confianza [IC] 95% [1,191-10,413], p = 0,023). Se desarrolló la curva ROC de betatrofina para pronosticar la presencia de síndrome metabólico (área bajo la curva ROC = 0,682 [95% IC, 0,597-0,767], p < 0,001). Además, la betatrofina mostró correlación con distintos parámetros, como edad (r = 0,286, p < 0,001), índice de masa corporal (r = 0,160, p = 0,046), índice cintura-cadera (r = 0,241, p = 0,002), lipoproteína de alta densidad (r = -0,167, p = 0,037), lipoproteína de baja densidad (r = -0,195, p = 0,015), glucosa plasmática en ayunas (r = 0,266, p = 0,001), hemoglobina A1C (r = 0,314, p < 0,001), índice de resistencia a la insulina mediante HOMA (r = 0,272, p = 0,001) y diversas adipoquinas, entre ellas resistina (r = 0,571, p < 0,001), interleucina-8 (r = 0,435, p < 0,001), factor de necrosis tumoral alfa (r = 0,295, p = 0,011) y lipocalina-2 (r = 0,346, p = 0,003). Conclusiones: este estudio demuestra que la betatrofina en suero desempeña una importante labor en el síndrome metabólico, implicando la regulación del metabolismo de la glucosa y los lípidos y la inflamación (AU)

Acute High-Intensity Intermittent Aerobic Exercise Reduces Plasma Angiopoietin-Like 2 in Patients With Coronary Artery Disease.

BACKGROUND: Circulating levels of angiopoietin-like 2 (ANGPTL2), a proinflammatory and proatherogenic protein, are elevated in patients with coronary artery disease (CAD). We hypothesized that high-intensity intermittent exercise (HIIE), known to be beneficial in patients with CAD, would reduce circulating ANGPTL2 levels. METHODS: Plasma levels of ANGPTL2 were measured before and 20 minutes, 24 hours, and 72 hours after an acute exercise session in a crossover study comparing HIIE to moderate-intensity continuous exercise (MICE) in 14 patients with CAD and 20 age-matched and 20 young healthy controls. RESULTS: Pre-exercise ANGPTL2 levels were 3-fold higher in patients with CAD than in age-matched controls (P < 0.05) and correlated negatively with Vo2max/lean body mass (P < 0.0001). In healthy controls, ANGPTL2 levels were low and not affected by HIIE or MICE. In patients with CAD, ANGPTL2 levels decreased significantly by 41% after 20 minutes of HIIE, a reduction that was maintained after 24 and 72 hours (P < 0.05). In contrast, although ANGPTL2 levels decreased by 47% after 20 minutes of MICE, they increased by 104% after 24 hours and returned to baseline values after 72 hours (P < 0.05). A negative correlation was observed between this increase in ANGPTL2 levels and the mean rate-pressure product (heart rate × systolic blood pressure; index of myocardial O2 consumption) measured during MICE, suggesting that subclinical ischemia might promote ANGPTL2 expression. CONCLUSIONS: In patients with CAD, circulating ANGPTL2 levels are acutely reduced after HIIE and transiently increased after MICE. A sustained reduction in circulating ANGPTL2 levels could contribute to the chronic beneficial cardiometabolic effects of HIIE in patients with CAD.

Effect of living cellular sheets on the angiogenic potential of human microvascular endothelial cells.

BACKGROUND: A fundamental issue limiting the efficacy of surgical approaches designed to correct periodontal mucogingival defects is that new tissues rely on limited sources of blood supply from the adjacent recipient bed. Accordingly, therapies based on tissue engineering that leverage local self-healing potential may represent promising alternatives for the treatment of mucogingival defects by inducing local vascularization. The aim of this study is to evaluate the effect of commercially available living cellular sheets (LCS) on the angiogenic potential of neonatal dermal human microvascular endothelial cells (HMVEC-dNeo). METHODS: The effect of LCS on HMVEC-dNeo proliferation, migration, capillary tube formation, gene expression, and production of angiogenic factors was evaluated over time. RESULTS: LCS positively influenced HMVEC-dNeo proliferation and migration. Moreover, HMVEC-dNeo incubated with LCS showed transcriptional profiles different from those of untreated cells. Whereas increased expression of angiogenic genes predominated early on in response to LCS, late-phase responses were characterized by up- and downregulation of angiostatic and angiogenic genes. However, this trend was not confirmed at the protein level, as LCS induced increased production of most of the angiogenic factors tested (i.e., epidermal growth factor [EGF], heparin-binding EGF-like growth factor, interleukin 6, angiopoietin, platelet-derived growth factor-BB, placental growth factor, and vascular endothelial growth factor) throughout the investigational period. Finally, although LCS induced HMVEC-dNeo proliferation, migration, and expression of angiogenic factors, additional factors and environmental pressures are likely to be required to promote the development of complex, mesh-like vascular structures. CONCLUSION: LCS favor initial mechanisms that govern angiogenesis but failed to enhance or accelerate HMVEC-dNeo morphologic transition to complex vascular structures.

Angiopoietin-Like Protein 2 Acts as a Novel Biomarker for Diagnosis and Prognosis in Patients with Esophageal Cancer.

BACKGROUND: Angiopoietin-like protein 2 (ANGPTL2) mediates chronic inflammation. Tumor cell-derived ANGPTL2 promotes tumor invasion and angiogenesis. Overexpression of ANGPTL2 in tumor cells is associated with tumor progression and has been recognized in lung, breast, colon, and gastric cancer. However, to our knowledge the functional and clinical significance of ANGPTL2 expression has not been investigated in patients with esophageal cancer (EC). METHODS: First, in vitro assays were performed for functional analysis of ANGPTL2 using small interfering RNA. Next, ANGPTL2 expression in EC tissues (n = 71) was evaluated by immunohistochemistry (IHC in patients with EC (n = 71). Finally, serum ANGPLT2 levels from patients with EC (n = 71) and healthy controls (n = 35) were evaluated using enzyme-linked immunosorbent assay. RESULTS: Knockdown of ANGPTL2 expression decreased the proliferative, invasive, and migration capacity in EC cell lines. ANGPTL2 expression in EC tissues was significantly elevated in patients with a high T stage, squamous cell carcinoma, and high TNM stage. Patients with high ANGPTL2 expression had significantly poorer overall and disease-free survival than those with low expression. Furthermore, high ANGPTL2 expression in EC tissues was an independent predictive marker for a poor prognosis. On the other hand, the serum ANGPTL2 level in patients with EC was significantly higher than that in healthy controls, and allowed for highly accurate discrimination between patients with and without EC. However, no significant association between serum ANGPTL2 levels and clinicopathological findings was observed in patients with EC. CONCLUSIONS: We have demonstrated novel evidence for the clinical significance of ANGPTL2 as a biomarker in patients with EC.

Clostridium butyricum reduce lipogenesis through bacterial wall components and butyrate.

Intervention strategies for obesity are global issues that require immediate attention. The objective of this study was to assess the possibility that Clostridium butyricum and its potential components could reduce lipogenesis. Co-culture experiments of Caco-2 cells and 1 × 10(6), 1 × 10(7), and 1 × 10(8) CFU/ml of C. butyricum were set up to monitor the cytotoxicity of C. butyricum and the changes of angiopoietin-like protein 4 (ANGPTL4) mRNA expression. It was found that cell viability was not affected by C. butyricum, and ANGPTL4 mRNA expression in Caco-2 cells was highly induced by 1 × 10(7) CFU/ml of C. butyricum. Co-culture experiment of Caco-2 cells and potential components of C. butyricum were set up to monitor any ensuing alteration in ANGPTL4. It was observed that bacterial wall components and potentially secreted factors from C. butyricum could induce ANGPTL4 mRNA expression and protein secretion. To determine whether butyrate could affect the ANGPTL4 production in Caco-2 cells, the role of monocarboxylate transporter 1 (MCT1) in mediating potentially secreted factors from C. butyricum-induced ANGPTL4 production in Caco-2 cells and the effect of 0.1 mM of butyrate on ANGPTL4 production in Caco-2 cells were investigated. It is confirmed that butyrate was the factor secreted by C. butyricum to stimulate ANGPTL4 production. Besides, the soluble factors secreted by live C. butyricum-Caco-2 cells interaction, bacterial wall components-Caco-2 cells interaction, and the main metabolites butyrate-Caco-2 cells interaction reduced lipogenic gene expression in HepG2 cells. In conclusion, 1 × 10(7) CFU/ml of C. butyricum could reduce lipogenesis through the bacterial wall components and the metabolites such as butyrate.

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas.

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.

Protective effects of angiotensin-(1-7) administrated with an angiotensin-receptor blocker in a rat model of chronic kidney disease.

AIM: Angiotensin-(1-7) (Ang-(1-7)) opposes angiotensin-II-induced cell growth, matrix accumulation and fibrosis in cardiac tissue. However, the role of Ang-(1-7) in the pathogenesis of renal fibrosis is uncertain. This study observed the effects of Ang-(1-7), on its own or in combination with losartan, an angiotensin-receptor blocker, on five-sixths nephrectomized rats. METHODS: Male Sprague-Dawley rats underwent five-sixths nephrectomy, and then were either untreated, treated with Ang-(1-7), treated with losartan, or treated with a combination therapy of Ang-(1-7) and losartan. After 8 weeks, renal function was assessed by measuring systolic blood pressure, serum creatinine and proteinuria. The effect of nephrectomy on the renin-angiotensin system was examined by measuring plasma levels of Ang-II and Ang-(1-7). The extent of glomerulosclerosis and tubulointerstitial fibrosis was assessed by periodic acid-Schiff staining and Masson-trichrome staining. The expression of plasminogen activator inhibitor-1, fibronectin and angiopoietins-Tie-2 was investigated by immunohistochemistry and western blot. RESULTS: In the groups of treated rats, serum creatinine, proteinuria and markers of glomerulosclerosis, such as fibronectin and plasminogen activator inhibitor-1, were ameliorated compared with the untreated, nephrectomized rats. Plasma Ang-(1-7) levels were elevated in all treatment groups, but the plasma Ang-II levels were reduced in the Ang-(1-7)-treated group and the combination therapy group. The ratio of Ang-1/Ang-2 was increased in the combination therapy group compared with two other treatment groups. CONCLUSION: Ang-(1-7) ameliorated the renal injury of nephrectomized rats. The combination of Ang-(1-7) treatment alongside losartan exerted a superior effect to that of Ang-(1-7) alone on regression of glomerulosclerosis.

Relationship between levels of angiogenic and lymphangiogenic factors and the endoscopic, histological and clinical activity, and acute-phase reactants in patients with inflammatory bowel disease.

BACKGROUND: Angiogenic and lymphangiogenic factors (ALFs) may play an important role in inflammatory bowel disease (IBD). Our aims were to evaluate levels of ALFs in serum and the colonic mucosa culture supernatant (MCS) of patients with active and quiescent IBD and healthy subjects and to correlate them with the endoscopic, clinical and histological activity and with acute-phase reactants. METHODS: This is a prospective study of 28 controls and 72 IBD patients. Serum and MCS concentration of VEGFA, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, PlGF, Ang1, Ang2 and Tie2 were measured by ELISA. Activity was established by specific indexes (CDAI, Mayo score, SES-CD, D'Haens scale and Riley index). Acute-phase reactants were routinely measured. RESULTS: MCS levels of all ALFs except VEGFR3 were higher in patients with endoscopic (p<0.05), clinical (p<0.05) and histological (p<0.01) activity than in those without it. In serum, VEGFA, VEGFC and Ang1 and VEGFA and Ang1 levels were lower in patients in remission than in patients with clinical and histological activity, respectively (p<0.05). There was a correlation between serum and MCS concentrations for VEGFD, VEGFR3, PlGF and Tie2 (r=0.25, r=0.48, r=-0.45 and r=0.36; p<0.05). Ang2 in MCS was the best predictor for the diagnosis of endoscopic, histological and clinical activity (area under ROC curve>0.8). CONCLUSIONS: MCS determination suggests a local increase in ALFs that correlates with IBD activity. Although the correlation between ALFs in serum and MCS was not good, the study of some of these factors as possible targets of new drugs for IBD constitutes a key new line of research.

Expression of angiopoietin-TIE system components in angiosarcoma.

Angiosarcoma is an aggressive malignancy of endothelial differentiation. Potential roles of the endothelial angiopoietin-tunica interna endothelial cell kinase (ANGPT-TIE) system in angiosarcoma diagnosis, pathogenesis, prognosis and treatment are undefined. To examine the expression and prognostic significance of angiopoietin-1, angiopoietin-2, TIE1 and TEK (TIE2) proteins in angiosarcoma, we immunohistochemically evaluated clinically annotated human angiosarcoma samples. Correlations of protein expression with overall survival and pathological features were explored. The cohort included 51 patients diagnosed with angiosarcoma at the age of 30-86 years (median 67). The 5-year overall survival was 45% with a median of 26 months. Moderate to strong expression of angiopoietin-1, TIE1 and TEK (TIE2) was identified in the majority of angiosarcomas and moderate to strong expression of angiopoietin-2 was observed in 42% of angiosarcomas. Increased angiopoietin-1 expression correlated with improved survival. Non-significant trends toward longer survival were also observed with increased TIE1 and TEK (TIE2) expression. Increased expression of angiopoietin-2, TIE1 and TEK (TIE2) was associated with vasoformative architecture. No differences in expression of these proteins were observed when patients were segregated by age, gender, presence or absence of metastases at diagnosis, primary tumor location, radiation association or the presence of necrosis. We conclude that components of the ANGPT-TIE system are commonly expressed in angiosarcomas. Reduced expression of these proteins is associated with non-vasoformative and clinically more aggressive lesions.

Angiogenic growth factors in rheumatoid arthritis.

We investigated whether the angiogenic profile, which is based on the local expression and systemic levels of angiogenic growth factors (VEGF, Ang-1, Ang-2, and the corresponding receptors), differs between rheumatoid arthritis (RA) and osteoarthritis (OA) patients. We determined the expression of VEGF, Ang-1, and Ang-2 together with its receptors (VEGFR-1/-2 and Tie2) in synovium tissue (ST) and muscular tissue (MT) from patients with RA and OA using quantitative PCR. Tissue samples were obtained from 15 RA and 19 OA patients during total knee arthroplasty. Control MT samples (n = 10) were obtained during spinal surgery. Results are correlated to VEGF and angiopoietin serum levels via ELISA measurements. The VEGF expressions in ST and serum levels were significantly higher in RA patients than in OA patients (P < 0.05). Furthermore, the VEGFR-1 and VEGFR-2 expression in ST from RA patients were significantly higher than in OA patients (P < 0.001 and P < 0.05). The relative concentration of angiopoietins (Ang-1/Ang-2 ratio) was significantly increased in RA (P < 0.01). Serum levels for Ang-2 showed no significant differences. Statistical analysis showed a significant higher level of Tie2 in RA patients (P < 0.001). Analysis of local levels of VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, and Tie2 in the muscular tissue showed no significant difference between RA and OA patients. These results underline the importance of pro-angiogenic growth factor levels for RA corroborating the assumption that VEGF and angiopoietins play an important role in the pathogenesis of RA.

1,3,5,8-tetrahydroxyxanthone regulates ANGPTL3-LPL pathway to lessen the ketosis in mice.

Ketosis is a metabolic disorder closely associated with both lipid and carbohydrate metabolism. Recent studies show that angiopoietin-like protein 3 (ANGPTL3) contributes to the development of metabolic disorder. The objective of this study was to explore the inhibitory effect of 1,3,5,8-tetrahydroxyxanthone (Xan), a naturally occurring flavonoid compound, on ketosis and the mechanisms involved in this regulation. After 4weeks, Xan (10 or 30mg/kg, intragastrically) treatment decreased plasma total ketone bodies, malondialdehyde, 8-isoprostane, triglyceride, total cholesterol levels, and hepatic ANGPTL3 expression concomitantly with increased plasma glucose concentration and adipose lipoprotein lipase (LPL) expression in ketosis murine. The present results suggest that Xan regulates ANGPTL3-LPL pathway to lessen the ketosis in mice.

Tumor cell-derived angiopoietin-like protein ANGPTL2 is a critical driver of metastasis.

Strategies to inhibit metastasis have been mainly unsuccessful in part due to insufficient mechanistic understanding. Here, we report evidence of critical role for the angiopoietin-like protein 2 (ANGPTL2) in metastatic progression. In mice, Angptl2 has been implicated in inflammatory carcinogenesis but it has not been studied in human tumors. In patients with lung cancer, elevated levels of ANGPTL2 expression in tumor cells within the primary tumor were associated with a reduction in the period of disease-free survival after surgical resection. Transcription factors NFATc, ATF2, and c-Jun upregulated in aggressive tumor cells promoted increased Angptl2 expression. Most notably, tumor cell-derived ANGPTL2 increased in vitro motility and invasion in an autocrine/paracrine manner, conferring an aggressive metastatic tumor phenotype. In xenograft mouse models, tumor cell-derived ANGPTL2 accelerated metastasis and shortened survival whereas attenuating ANGPTL2 expression in tumor cells-blunted metastasis and extended survival. Overall, our findings showed that tumor cell-derived ANGPTL2 drives metastasis and provided an initial proof of concept for blockade of its action as a strategy to antagonize the metastatic process.

Prognostic impacts of angiopoietins in NSCLC tumor cells and stroma: VEGF-A impact is strongly associated with Ang-2.

INTRODUCTION: Angiopoietins and their receptor Tie-2 are, in concert with VEGF-A, key mediators in angiogenesis. This study evaluates the prognostic impact of all known human angiopoietins (Ang-1, Ang-2 and Ang-4) and their receptor Tie-2, as well as their relation to the prognostic expression of VEGF-A. METHODS: 335 unselected stage I-IIIA NSCLC-patients were included and tissue samples of respective tumor cells and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression of markers in duplicate tumor and stroma cores. PRINCIPAL FINDINGS: In univariate analyses, low tumor cell expression of Ang-4 (P = 0.046) and low stromal expressions of Ang-4 (P = 0.009) and Ang-2 (P = 0.017) were individually associated with a poor survival. In the multivariate analysis, low stromal Ang-2 (HR 1.88; CI 95% 1.15-3.08) and Ang-4 (HR 1.47, CI 95% 1.02-2.11, P = 0.04) expressions were independently associated with a poor prognosis. In patients with high tumor cell expression of Ang-2, a concomitantly high tumor VEGF-A expression mediated a dramatic survival reduction (P<0.001). In the multivariate analysis of patients with high Ang-2 expression, high tumor VEGF-A expression appeared an independent poor prognosticator (HR 6.43; CI 95% 2.46-16.8; P<0.001). CONCLUSIONS: In tumor cells, only Ang-4 expression has prognostic impact in NSCLC. In tumor stroma, Ang-4 and Ang-2 are independently associated with survival. The prognostic impact of tumor cell VEGF-A in NSCLC appears strongly associated with a concomitantly high tumor cell expression of Ang-2.

Angiopoietin-like 4 (ANGPTL4) gene polymorphisms and risk of brain arteriovenous malformations.

BACKGROUND: Brain arteriovenous malformations (BAVM) are high-flow vascular lesions prone to intracranial hemorrhage (ICH). Abnormal angiogenesis is a key characteristic of BAVM tissue. Angiopoietin-like 4 (ANGPTL4), a secreted glycoprotein, is thought to be involved in angiogenesis and required for proper postnatal blood vessel partitioning. We investigated whether common single nucleotide polymorphisms (SNPs) in ANGPTL4 were associated with risk of BAVM or ICH. METHODS AND RESULTS: We conducted a case-control study of 216 Caucasian BAVM cases and 246 healthy controls, and a secondary case-only analysis, comparing 83 ruptured (ICH) with 133 unruptured BAVM cases at presentation. Four tagSNPs in ANGPTL4 captured variation over a 10-kb region (rs2278236, rs1044250, rs11672433, and rs1808536) and were tested for association with BAVM or ICH. The minor allele (A) of rs11672433 (exon 6, Pro389Pro) was associated with an increased risk of BAVM (p = 0.006), which persisted after adjusting for multiple comparisons (p = 0.03). After adjustments for age and sex, carriers of the minor allele (A) remained at higher risk for BAVM compared to noncarriers (odds ratio, OR = 1.56; 95% confidence interval, CI = 1.01-2.41; p = 0.046) and risk of BAVM was increased with increasing copy of the minor A allele (OR = 1.49, 95% CI = 1.03-2.15; p(trend) = 0.03). Five common haplotypes (frequency >1%) were inferred; overall haplotype distribution differed between BAVM cases and controls (χ(2) = 12.2, d.f. = 4, p = 0.02). Neither SNPs (p > 0.05) nor haplotype distribution (χ(2) = 1.1, d.f. = 4, p = 0.89) were associated with risk of ICH among BAVM cases. CONCLUSION: A synonymous SNP in ANGPTL4 and haplotypes carrying it are associated with risk of BAVM but not with ICH presentation in BAVM cases.

Dextran sulfate sodium leads to chronic colitis and pathological angiogenesis in Endoglin heterozygous mice.

BACKGROUND: Pathological angiogenesis is an intrinsic component of chronic intestinal inflammation, which results in remodeling and expansion of the gut microvascular bed. Endoglin is essential for endothelial cell function and physiological angiogenesis. In this study we investigated its potential role in the regulation of inflammation by testing the response of Endoglin heterozygous (Eng(+/-)) mice to experimental colitis. METHODS: C57BL/6 Eng(+/-) and littermate control mice drank water supplemented with 3% dextran sulfate sodium (DSS) for 5 days and were monitored for up to 26 days for clinical signs of colitis. Inflammation, crypt damage, and angiogenic index were scored on histological sections of distal colon. Levels of the vascular endothelial growth factor (VEGF) and angiopoietins were measured by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blots. Vascular permeability was assessed using Evans Blue. RESULTS: Eng(+/-) and control mice developed acute colitis, which peaked at day 9. While control mice recovered by days 19-26, Eng(+/-) mice progressed to chronic colitis and showed numerous vascular protrusions penetrating into the serosa of the inflamed distal colon. Prior to DSS induction, VEGF levels and vascular permeability were higher in the distal colon of Eng(+/-) mice, while angiopoietin 1 and 2 levels were unchanged. In the chronic phase of colitis, VEGF levels were increased in both groups of mice and remained significantly higher in the Eng(+/-) mice. CONCLUSIONS: Higher VEGF levels and increased vascular permeability in the distal colon may predispose Eng(+/-) mice to progress to chronic and persistent bowel inflammation, associated with pathological angiogenesis.

Analysis of soluble angiogenic factors in Crohn’s disease: a preliminary study / Análisis de los factores angiogénicos solubles en la Enfermedad de Crohn: un estudio preliminar

Aims: Soluble angiogenic factors could be altered in patients with Crohn’s disease (CD), since inflammation and angiogenesis may play a critical pathogenic role. Methods: Serum samples were collected from 30 patients with CD (50% female; median age 44 ± 14 yrs) grouped according to their phenotypic behavior into equal subgroups: inflammatory, fibrostenotic and fistulizing; and 30 healthy controls (50% female; median age 43 ± 14 yrs). Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), angiopoietins (Ang) and receptors (VEGFR1, VEGFR2 and Tie2) in sera were assayed by ELISA. Results: Serum levels of VEGF (494 ± 247 pg/ml), PlGF (36 ± 11 pg/ml), VEGFR1 (1.9 ± 0.3 ng/ml), Ang2 (6.0 ± 2.3 ng/ml) and Tie2 (36 ± 5 ng/ml) in CD patients were significantly higher than those found in healthy controls (335 ± 118 pg/ml; 23 ± 9 pg/ml; 1.0 ± 0.3 ng/ml; 3.9 ± 2.0 ng/ml; 22 ± 7 ng/ml, respectively). Conversely, CD patients showed significantly lower serum levels of Ang1 than healthy controls (46 ± 11 versus 67 ± 23 pg/ml). In the case of VEGFR2 serum levels, no differences between groups were found. Finally, patients with fibrostenotic CD were characterized by elevated VEGF and Ang2 levels, while patients with an inflammatory phenotype by elevated Tie2 and PlGF levels. Conclusions: An activated «pro-angiogenic» profile of angiogenesis soluble markers was observed in CD patients, in comparison with healthy controls. According to the phenotypic behavior, these patients showed differences in serum levels of angiogenic factors

Objetivos: Los factores angiogénicos solubles podrían estar alterados en los pacientes con enfermedad de Crohn (EC), dado que que la inflamación y la angiogénesis pueden desempeñar un papel de carácter crítico en la patogenia de esta enfermedad. Métodos: Se obtuvieron muestras de suero en 30 pacientes con EC (50% mujeres; edad media, 44 ± 14 años) agrupados subgrupos equivalentes en función de su comportamiento fenotípico: inflamatorio, fibroestenosante y fistulizante, así como en 30 controles sanos (50% mujeres; edad media, 43 ± 14 años). Mediante análisis de inmunoabsorción ligada a enzimas (Elisa, enzime-linked immunoadsorbent assay) se evaluaron en el suero el factor de crecimiento endotelial vascular (VEGF, vascular endothelial growth factor), el factor de crecimiento placentario (PlGF, placental growth factor), las angiopoyetinas (Ang) y los receptores VEGFR1, VEGFR2 y Tie2. Resultados: Las concentraciones séricas de VEGF (494 ± 247 pg/ml), PlGF (36 ± 11 pg/ml), VEGFR1 (1,9 ± 0,3 ng/ ml), Ang2 (6,0 ± 2,3 ng/ml) y Tie2 (36 ± 5 ng/ml) en los pacientes con EC fueron significativamente mayores que las observadas en los controles sanos (± 118 pg/ml; 23 ± 9 pg/ ml; 1,0 ± 0,3 ng/ml; 3,9 ± 2,0 ng/ml; 22 ± 7 ng/ml, respectivamente). Por el contrario, los pacientes con EC presentaron concentraciones séricas significativamente inferiores de Ang1, en comparación con los controles sanos (46 ± 11 y 67 ± 23 pg/ml, respectivamente). En el caso de las concentraciones séricas de VEGFR2, no se observaron diferencias entre los 2 grupos. Finalmente, los pacientes con EC fibroestenosante se caracterizaron por una elevación de las concentraciones de VEGF y Ang2, mientras aquellos que tenían el fenotipo inflamatorio presentaron concentraciones elevadas de Tie2 y PlGF. Conclusiones: En los pacientes con EC se detectó un perfil «proangiogénico» activado en lo relativo a los marcadores solubles de la angiogénesis, en comparación con lo observado en los controles sanos. Según el comportamiento fenotípico, estos pacientes presentaron diferencias en las concentraciones séricas de los factores angiogénicos (AU)

Expression of angiopoietin-1, 2 and 4 and Tie-1 and 2 in gastrointestinal stromal tumor, leiomyoma and schwannoma.

AIM: To investigate the role of angiopoietin (Ang) -1, -2 and -4 and its receptors, Tie-1 and -2, in the growth and differentiation of gastrointestinal stromal tumors (GISTs). METHODS: Thirty GISTs, seventeen leiomyomas and six schwannomas were examined by immunohistochemistry in this study. RESULTS: Ang-1, -2 and -4 proteins were expressed in the cytoplasm of tumor cells, and Tie-1 and -2 were expressed both in the cytoplasm and on the membrane of all tumors. Immunohistochemical staining revealed that 66.7% of GISTs (20 of 30), 76.5% of leiomyomas (13 of 17) and 83.3% of schwannomas (5 of 6) were positive for Ang-1. 83.3% of GISTs (25 of 30), 82.4% of leiomyomas (14 of 17) and 100% of schwannomas (6 of 6) were positive for Ang-2. 36.7% of GISTs (11 of 30), 58.8% of leiomyomas (10 of 17) and 83.3% of schwannomas (5 of 6) were positive for Ang-4. 60.0% of GISTs (18 of 30), 82.4% of leiomyomas and 100% of schwannomas (6 of 6) were positive for Tie-1. 10.0% of GISTs (3 of 30), 94.1% of leiomyomas (16 of 17) and 33.3% of schwannomas (2 of 6) were positive for Tie-2. Tie-2 expression was statistically different between GISTs and leiomyomas (P < 0.001). However, there was no correlation between expression of angiopoietin pathway components and clinical risk categories. CONCLUSION: Our results suggest that the angiopoietin pathway plays an important role in the differentiation of GISTs, leiomyomas and schwannomas.