Microcirculatory perfusion disturbances following cardiac surgery with cardiopulmonary bypass are associated with in vitro endothelial hyperpermeability and increased angiopoietin-2 levels.

BACKGROUND: Endothelial hyperpermeability following cardiopulmonary bypass (CPB) contributes to microcirculatory perfusion disturbances and postoperative complications after cardiac surgery. We investigated the postoperative course of renal and pulmonary endothelial barrier function and the association with microcirculatory perfusion and angiopoietin-2 levels in patients after CPB. METHODS: Clinical data, sublingual microcirculatory data, and plasma samples were collected from patients undergoing coronary artery bypass graft surgery with CPB (n = 17) before and at several time points up to 72 h after CPB. Renal and pulmonary microvascular endothelial cells were incubated with patient plasma, and in vitro endothelial barrier function was assessed using electric cell-substrate impedance sensing. Plasma levels of angiopoietin-1,-2, and soluble Tie2 were measured, and the association with in vitro endothelial barrier function and in vivo microcirculatory perfusion was determined. RESULTS: A plasma-induced reduction of renal and pulmonary endothelial barrier function was observed in all samples taken within the first three postoperative days (P < 0.001 for all time points vs. pre-CPB). Angiopoietin-2 and soluble Tie2 levels increased within 72 h after CPB (5.7 ± 4.4 vs. 1.7 ± 0.4 ng/ml, P < 0.0001; 16.3 ± 4.7 vs. 11.9 ± 1.9 ng/ml, P = 0.018, vs. pre-CPB), whereas angiopoietin-1 remained stable. Interestingly, reduced in vitro renal and pulmonary endothelial barrier moderately correlated with reduced in vivo microcirculatory perfusion after CPB (r = 0.47, P = 0.005; r = 0.79, P < 0.001). In addition, increased angiopoietin-2 levels moderately correlated with reduced in vitro renal and pulmonary endothelial barrier (r = - 0.46, P < 0.001; r = - 0.40, P = 0.005) and reduced in vivo microcirculatory perfusion (r = - 0.43, P = 0.01; r = - 0.41, P = 0.03). CONCLUSIONS: CPB is associated with an impairment of in vitro endothelial barrier function that continues in the first postoperative days and correlates with reduced postoperative microcirculatory perfusion and increased circulating angiopoietin-2 levels. These results suggest that angiopoietin-2 is a biomarker for postoperative endothelial hyperpermeability, which may contribute to delayed recovery of microcirculatory perfusion after CPB. TRIAL REGISTRATION: NTR4212 .

Neuregulin-1 Promotes Myocardial Angiogenesis in the Rat Model of Diabetic Cardiomyopathy.

BACKGROUND/AIMS: Microvascular insufficiency takes a critical role in the development of diabetic cardiomyopathy (DCM). So this study was designed to investigate the effects of Neuregulin-1 (NRG-1) treatment on myocardial angiogenesis and the changes of VEGF/Flk1 and Ang-1/Tie-2 signaling in the rat model of DCM. METHODS: Diabetic rats were induced by a single intraperitoneal injection of Streptozotocin. 12 weeks after the diabetes induction, the rats with NRG-1 treatment were treated with tail vein injection of NRG-1 at the dose of 10µg/kg/d for consecutive 10 days. Cardiac function was assessed using catheter MPA cardiac function analysis system. Myocardial blood flow (MBF) was assessed with stable-isotope labeled microspheres. Capillary density was measured by CD31 immunohistochemistry. The protein expression and receptors phosphorylation were assessed using western blot. RESULTS: Left ventricular function, capillary density and MBF were significantly reduced in DCM group when compared with those in the control group (P< 0.01, P< 0.01 and P< 0.05 respectively). Left ventricular function and capillary density were significantly increased in NRG-1 treatment group when compared with those in the DCM group (P< 0.05 and P< 0.05 respectively). The expression of VEGF and Ang-1 and the phosphorylation of Flk1 and Tie-1 were significantly decreased in DCM group as compared with those in the control group. However, those in the NRG-1 treatment group were significantly increased as compared with those in the DCM group. In vitro, NRG-1 treatment increased significantly the expression of VEGF and Ang-1 in human coronary artery smooth muscle cells. CONCLUSIONS: NRG-1 can increase the myocardial angiogenesis of DCM, probably via the direct effects of NRG-1 and via the increasing expression of VEGF and Ang-1. These findings may contribute to developing a novel approach to reverse the impaired angiogenic responses in diabetes or coronary artery disease.

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids.

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin-1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE-2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non-vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang-1 and Ang-2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE-2-expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang-1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation.

The role of angiopoietin-2 in nucleus pulposus cells during human intervertebral disc degeneration.

Although evidence shows that intervertebral disc degeneration is generally characterized by angiogenesis, the role of angiopoietin has not been investigated. This study examined the presence of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) within the native intervertebral disc (IVD) and elucidated their functions in the regulation of nucleus pulposus (NP) cells. Initial investigation of uncultured NP tissue revealed that Ang-1 and Ang-2 were expressed by native NP cells. Ang-2 expression was significantly increased in infiltrated and degenerate samples relative to normal samples. The ratio of Ang-2/Ang-1 in tissues from patients increased markedly with increasing age and level of degeneration of the IVD. The ratio of both Ang-2/Ang-1 mRNA and protein increased over time when cells were subjected to constant pressure at 1 Mpa in vitro. Our findings indicate that Ang-2 plays a role in suppressing cell adhesion and viability, and promotes the apoptosis of NP cells and that Ang-2 can inhibit the pathways stimulated by Ang-1 and fibronectin. Ang-2 release during IVD degeneration causes higher ratio of Ang-2 to Ang-1, further inhibits NP cell viability and adhesion, promoting apoptosis by blocking PI3K/Akt signaling. The present study therefore provides new insights into the role of the angiopoietin-Tie system in the pathogenesis of IVD degeneration.

Simvastatin ameliorates graft-vs-host disease by regulating angiopoietin-1 and angiopoietin-2 in a murine model.

Angiopoietins play an important role in vascular endothelial function. Endothelial damage is an important pathogenesis relating with acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), protecting endothelial cells (ECs) from damage may be a potent prophylaxis and therapeutic strategy of acute GVHD (aGVHD). In this study, we explored changes in Angiopoietin-1 (Ang-1) and Ang-2 expression in a aGVHD mouse model and determined whether simvastatin prevents GVHD through regulating Ang-1 and Ang-2 expression. In vitro simvastatin administration increased Ang-1 production and release but conversely inhibited Ang-2 release from EA.hy926 ECs. Simvastatin improved the survival of aGVHD mice, attenuated the histopathological GVHD grades and plasma levels of Ang-2, and elevated the plasma levels of Ang-1 as well as the aortic endothelial levels of Ang-1 and Ang-2. In summary, simvastatin represents a novel approach to combat GVHD by increasing Ang-1 production while suppressing Ang-2 release to stabilize endothelial cells.

Association of markers of endothelial dysregulation Ang1 and Ang2 with acute kidney injury in critically ill patients.

BACKGROUND: The role of endothelial dysregulation with acute kidney injury (AKI) in critically ill patients is unclear. METHODS: We retrospectively assessed the associations of AKI with biomarkers of endothelial function and inflammation among 948 subjects admitted to the intensive care unit (ICU) at Harborview Medical Center (Seattle, WA, USA). From plasma obtained within 24 h of enrollment, we measured angiopoietin (Ang)-1 and Ang-2 alongside biomarkers of inflammation, including interleukin (IL)-6, IL-17 and granulocyte colony-stimulating factor. We tested for associations between standardized concentrations of biomarkers and AKI, defined by serum creatinine, from ICU admission to up to 7 days later. RESULTS: All biomarkers of inflammation and endothelial dysfunction were associated with AKI. After adjustment for demographics, comorbidities, and IL-6 concentration, every standard deviation of Ang-1 concentration was associated with a 19 % lower risk of AKI (relative risk (RR) = 0.85, 95 % confidence interval (CI) 0.77-0.93, p < 0.001). Conversely, higher Ang-2 concentration was associated with higher risk of AKI (RR per standard deviation = 1.17, 95 % CI 1.13-1.22, p < 0.001). CONCLUSIONS: In critically ill patients, plasma concentration of the endothelial growth factors Ang-1 and Ang-2 are associated with AKI, independently of inflammation.

Soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) Is Elevated in Bronchoalveolar Lavage Fluid of Patients with Acute Respiratory Distress Syndrome.

INTRODUCTION: Pulmonary vascular endothelial activation has been implicated in acute respiratory distress syndrome (ARDS), yet little is known about the presence and role of endothelial activation markers in the alveolar space in ARDS. We hypothesized that endothelial activation biomarkers would be differentially expressed in bronchoalveolar lavage fluid from patients with ARDS compared with healthy volunteers, and that biomarker concentrations would be associated with ARDS severity. METHODS: We performed a cross-sectional analysis of data from 26 intubated patients with ARDS undergoing evaluation for clinically suspected ventilator-associated pneumonia and five healthy volunteers. Patients underwent bronchoalveolar lavage a median of five days after intubation. Healthy volunteers also underwent bronchoalveolar lavage. Endothelial activation biomarkers (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble endothelial selectin [sESEL], angiopoietin-1 [Ang-1] and angiopoietin-2 [Ang-2]) were measured in bronchoalveolar lavage fluid. Clinically suspected ventilator-associated pneumonia was confirmed with microbiologic culture data. RESULTS: Patients with ARDS had significantly higher median sVCAM-1 concentrations in the bronchoalveolar lavage fluid compared with healthy volunteers (985 vs 119 pg/mL, p = 0.03). Additionally, there was a trend toward greater bronchoalveolar lavage fluid sVCAM-1 concentrations among patients with moderate/severe compared to mild ARDS (1395 vs 209 pg/mL, p = 0.06). We did not detect significant differences in bronchoalveolar lavage fluid levels of sESEL, Ang-1 or Ang-2 between patients with ARDS and healthy volunteers. Median bronchoalveolar lavage fluid biomarker levels did not differ between patients with and without microbiologically-confirmed ventilator-associated pneumonia. CONCLUSIONS: sVCAM-1 concentrations were significantly higher in the bronchoalveolar lavage fluid of patients with ARDS compared to healthy controls, and tended to be higher in moderate/severe ARDS compared to mild ARDS. Our findings add to the growing evidence supporting the concept that endothelial activation plays an important mechanistic role in the pathogenesis of ARDS. Further studies are necessary to characterize the role and/or clinical significance of sVCAM-1 and other endothelial activation markers present in the alveolar space in ARDS.

Association Between Angiopoietin-2 and Enterovirus 71 Induced Pulmonary Edema.

OBJECTIVE: To characterize pulmonary edema (PE) fluid induced by enterovirus 71 (EV71) infection, elucidate the relationship between angiopoietin-2 (Ang-2) and PE, and explore the pathogenesis of PE. METHODS: Clinical data were collected from critical infants with EV71 infection. The infants were grouped into PE, non-PE, and control groups. The control group included infants in the preoperative period of elective inguinal hernia surgery. Biochemical changes in PE fluid were evaluated, and Ang-2 levels in serum and PE fluid were measured. Human pulmonary microvascular endothelial cells (HPMECs) were incubated with serum from the control and PE groups and human recombinant Ang-2 or serum from the PE group and human recombinant Ang-1, and changes in the intercellular junctions were recorded via immunofluorescence. RESULTS: Of the 161 infants with critical EV71 infection admitted to the hospital, 39 had PE. PE fluid was collected from 18 of these infants. The PE fluid-to-serum (P/S) ratio of total protein was 0.9 ± 0.2, and all P/S ratios of albumin were 1.0 ± 0.3. The Ang-2 level was higher in the non-PE group (333.2 ± 79.7 pg/ml) than in the control group (199.9 ± 26.7 pg/ml), although without statistical significance (P = 0.115). The Ang-2 level in the PE group (2819.2 ± 908.7 pg/ml) was higher than those in both the non-PE and the control groups (both, P < 0.001). Serum samples from the PE group had damaged cell junctions of confluent HPMEC monolayers that were reversed by Ang-1. CONCLUSIONS: The PE fluid of infants with EV71-induced PE was protein-rich, and elevated Ang-2 expression was associated with PE. The mechanism through which PE develops may be related to Ang-2-induced cell junction damage.

Role of the P38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro.

INTRODUCTION: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. METHODS: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. RESULTS: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 ± 3.07, 315.03 ± 5.80, and 319.95 ± 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. CONCLUSIONS: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement.

Serum levels of angiopoietin-2, but not angiopoietin-1, are elevated in patients with erythrodermic cutaneous T-cell lymphoma.

Angiogenesis is a crucial process in the growth and progression of cancer, correlating with the metastatic potential of tumour cells. Angiopoietins are ligands for the endothelium-specific tyrosine kinase Tie2 receptor, which comprise 4 structurally related proteins, termed angiopoietin (Ang)-1, Ang-2, Ang-3 and Ang-4. The roles of Ang-1 and Ang-2 have recently been clarified as crucial in angiogenesis. In this report, we measured serum Ang-1 and Ang-2 levels in patients with cutaneous T-cell lymphoma (CTCL). Serum levels of Ang-2, but not Ang-1, in patients with Sézary syndrome were significantly higher than those in patch mycosis fungoides (MF), plaque/tumour MF, and healthy controls. In patients with CTCL, serum Ang-2 correlated with disease activity. Moreover, the numbers of Ang-2+ cells in lesional skin of CTCL were significantly larger than those in normal skin. These results suggest that Ang-2 may have important roles in the development of CTCL.

Expression and function of Angiopoietins and their tie receptors in human basophils and mast cells.

The Angiopoietin/Tie system is a key regulator of vascular remodeling, maturation, angiogenesis and lymphangiogenesis. In humans there are three angiopoietins: Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2), and Angiopoietin-4 (Ang4). Ang1 and Ang2 are the best characterized angiopoietins. The angiopoietin receptor system consists of two type I tyrosine kinase receptors (Tie1 and Tie2). Tie2 binds all known angiopoietins. We sought to characterize Ang1, Ang2, Tie1 and Tie2 expression and functions in human basophils and mast cells. Basophils, LAD-2 cells and Human Lung Mast Cells (HLMCs) constitutively express Ang1 and Ang2 mRNA. Intracellular staining for Ang1 and Ang2 was stronger in basophils than in mast cells. Immunoelectron microscopy demonstrated Ang1 in cytoplasmic vesicles of basophils. The protein kinase C activators phorbol diester (PMA) and bryostatin 1 (Bryo1) stimulated basophils to rapidly release a large amount of Ang1. PMA-induced Ang1 release was inhibited by brefeldin A. Tie1 and Tie2 mRNAs were expressed in basophils, LAD-2 and HLMCs. Basophils, LAD-2 and HLMCs expressed Tie1 on the cell surface. HLMCs and LAD-2 expressed Tie2 on the cell surface, whereas basophils did not. Ang1, but not Ang2, induced migration of mast cells through the engagement of Tie2. Neither Ang1 nor Ang2 induced basophil chemotaxis. We have identified a novel mechanism of cross-talk between human basophils and mast cells mediated by the Ang1/Tie2 system that might be relevant in the orchestration of inflammatory and neoplastic angiogenesis.

Effect of Helicobacter pylori eradication according to the IL-8-251 polymorphism in Koreans.

Previous studies suggested that polymorphisms of proinflammatory cytokine genes are important host genetic factors in Helicobacter pylori infection. The present study evaluated whether IL-8-251 polymorphism affected H. pylori eradication rate and to investigate the effect of H. pylori eradication on angiogenesis and the inflammatory process according to the IL-8-251 polymorphism. A total of 250 H. pylori-positive patients treated by endoscopic resection of the gastric neoplasm were classified into 3 groups (134 H. pylori-eradicated group, 19 H. pylori-eradication failure group, and 97 H. pylori-infected group). H. pylori status, histology, and angiogenic factor levels were evaluated at baseline, 6 months, and 18 months. H. pylori eradication rate was 92.9% in AA genotype, 85.7% in AT genotype and 88.4% in TT genotype (P value = 0.731). Elevated IL-8 and matrix metalloproteinase-9 concentrations in H. pylori-infected gastric mucosa were reversible by successful eradication of H. pylori, independent of the IL-8-251 polymorphism. It is suggested that elevated IL-8 and MMP-9 concentrations in H. pylori-infected gastric mucosa are altered significantly after successful eradication and these conditions continue for 18 months. However, IL-8-251 polymorphism does not affect H. pylori eradication rate and the sequential changes of related angiogenic factors after H. pylori eradication in Koreans.

Vascular endothelial growth factors and their receptors and regulators in gestational trophoblastic diseases and normal placenta.

OBJECTIVE: To study the expression of vascular endothelial growth factors (VEGFs), placental growth factor (PLGF) and their receptors (VEGFR-1, -2, -3) and their regulators (IL-6, CD147) in normal placenta and gestational trophoblastic disease (GTD) in order to evaluate their potential role in the biology of GTD. STUDY DESIGN: Paraffin sections of 10 normal, first-trimester placentas, 10 partial moles, 10 complete moles, 5 choriocarcinomas and 5 placental site trophoblastic tumors (PSTTs) were studied immunohistochemically for expression of VEGFR-1, VEGFR-2, VEGFR-3, IL-6, PLGF and CD147. Immunolocalization of VEGF, Angiopoietin-1 and Angiopoietin-2 was performed on 5 choriocarcinomas and 5 PSTTs. The levels of VEGF and VEGFR-2 were determined in supernatants and lysates of normal trophoblast, JEG-3 and JAR choriocarcinoma cells with electrochemiluminescence assays. RESULTS: The normal placenta had significantly stronger expression of VEGFR-2 than did those of partial and complete mole (p = 0.001, p = 0.003). VEGF, Angiopoietin-1 and Angiopoietin-2 expression in PSTT were significantly higher than those in choriocarcinoma (p = 0.002, p= 0.01, p = 0.038). Choriocarcinoma showed stronger intensity of staining for VEGFR-3 than did normal placenta, partial and complete mole (p = 0.036, p = 0.038, p = 0.05). Choriocarcinoma had significantly stronger staining of CD147 than did partial and complete mole (p<0.01, p<0.01). PSTT exhibited significantly stronger staining for IL-6 than did choriocarcinoma (p = 0.03). CONCLUSION: PSTTs exhibited strong staining for VEGF, and choriocarcinoma showed strong staining for VEGFR-3. Agents that inhibit the activity of VEGF and VEGF receptors may prove to be useful in the therapy of gestational trophoblastic neoplasia.

Increased levels of angiopoietins 1 and 2 in sputum supernatant in severe refractory asthma.

BACKGROUND: Airway and vascular remodeling may play a prominent role in the clinical severity of severe refractory asthma (SRA). Angiopoietin-1 (Ang-1) is an essential mediator of angiogenesis by establishing vascular integrity, whereas angiopoietin-2 (Ang-2) acts as its natural inhibitor. OBJECTIVE: We aimed to determine the levels of angiopoietins in sputum supernatants of patients with SRA and to investigate the possible associations with mediators and cells involved in both the inflammatory and the vascular remodeling processes. METHODS: Thirty-eight patients with SRA, 35 patients with moderate asthma, and 20 healthy subjects were studied. All participants underwent lung function tests, bronchial hyperresponsiveness assessment and sputum induction for cell count identification and Ang-1, Ang-2, VEGF, TGF-ß1, Cys-LTs, MMP-2, IL-13, ECP, and IL-8 measurement in supernatants. Airway vascular permeability (AVP) index was also assessed. RESULTS: Ang-1 (ng/ml) and Ang-2 (pg/ml) levels were significantly elevated in patients with SRA compared with patients with moderate asthma and control subjects [median, interquartile ranges: 30 (17-39) vs 7.5 (5-11) vs 4.7 (3.8-5.9) respectively, P < 0.001; and 506 (400-700) vs 190 (146-236) vs 96 (89-120) respectively, P < 0.001]. Regression analysis showed a significant positive association between Ang-2 and AVP index, MMP-2, Ang-1, and VEGF in SRA. A weak association was also observed between Ang-1 and sputum eosinophils% in SRA. CONCLUSION: Our results indicate that both angiopoietins levels are higher in SRA compared with moderate asthma and healthy subjects. In SRA, Ang-2 is associated with mediators involved in both the inflammatory and the vascular remodeling processes.

Angiopoietin 1 and angiopoietin 2 in follicular fluid of women undergoing a long protocol.

OBJECTIVE: To determine the concentrations of angiopoietin 1 (ANGPT1) and ANGPT2 in individual human preovulatory follicles in relation to their diameter or volume to clarify the role of these molecules in folliculogenesis. DESIGN: Prospective study. SETTING: Research laboratory at Kansai Medical University, Osaka, Japan. PATIENT(S): Twenty-three women undergoing IVF. INTERVENTION(S): On the day of oocyte retrieval, serum samples and follicular fluid (FF) from individual follicles were collected. We analyzed 348 follicles. MAIN OUTCOME MEASURE(S): ANGPT1 and ANGPT2 concentrations in FF and serum and oocyte recovery rates. RESULT(S): On average, ANGPT1 concentrations in FF were 150 times lower than those in serum, whereas ANGPT2 concentrations in FF were 8 times higher than those in serum. The concentrations of ANGPT1 in follicles with a diameter ≤17 mm were significantly higher than those in follicles with a diameter ≥18 mm. On the other hand, the concentrations of ANGPT2 in follicles with a diameter ≤17 mm were significantly lower than those in follicles with a diameter ≥18 mm. The ANGPT2/ANGPT1 ratio increased with enlargement of follicular diameter. ANGPT1 concentrations in FF decreased with follicular volume. ANGPT2 concentrations and the ANGPT2/ANGPT1 ratio in FF rose with follicular volume. The ANGPT2/ANGPT1 ratio in FF from the oocyte recovery group was significantly higher than that from the nonrecovery group. CONCLUSION(S): Our data suggested that the change in ANGPT1 and ANGPT2 levels may be associated with follicular growth and angiogenesis during the preovulatory period.

Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis?

The endothelial integrity, as mechanical barrier against microorganisms and as natural "anticoagulant", is crucial for physiologic organ function. Systemic activation of the endothelium upon inflammation, sepsis, and septic shock is always ending in blood-tissue barrier disruption. With increasing dysfunction, uncontrolled clotting activation, capillary microthrombi formation, tissue edema, local hypoxia, and ischemia are initiated. This in turn enhances a vicious circle leading to multiple organ failure and death. Therefore, biomarkers reflecting this special compartment may help in the early detection of systemic inflammation and its complications. This review provides an overview of the most important endothelial biomarkers and their possible use in sepsis.

Endogenous vascular endothelial growth factor and angiopoietin-2 expression in critical limb ischemia.

AIM: The aim of this paper was to contribute to a better understanding of the angiogenesis in peripheral arterial disease (PAD); we evaluated the expression of vascular endothelial growth factor (vegf) and angiopoietin-2 (Ang-2) in critical limb ischemia (CLI). METHODS: Skin and muscle biopsies were collected from 12 patients submitted to major amputation for CLI, proximal samples from amputation level and distal ones from the more ischemic region. Three controls were obtained from orthopedic patients. Capillary density was determined in random selected high-power fields. Expression pattern of VEGF and Ang-2 was studied by immunohistochemistry and quantification was performed by enzyme-linked immunosorbent assay. RESULTS: In skin, capillary density and levels of VEGF and Ang-2 were higher in distal samples when compared to proximal (capillary density, P=0.003, VEGF, P=0.008, Ang-2, P=0.041). Distal muscle had also elevated capillary number (P=0.005) and Ang-2 concentration (P=0.023). VEGF concentration in distal muscle was found to be similar to proximal muscle (P=1). Immunohistochemical expression of VEGF was clearly more evident in distal samples and was predominantly present in epidermis and skeletal myocytes. Ang-2 was essentially detected distally and only observed in endothelial cells. CONCLUSION: The capillary density is enhanced in distal samples, suggesting an effective angiogenic drive in CLI. In addition, the observed increase of VEGF expression in ischemic skin and Ang-2 in ischemic skin and muscle may contribute to clarify the potential role of VEGF and Ang-2 supplementation for therapeutic angiogenesis in CLI.

Serum levels of angiopoietin-1 in patients with pulmonary hypertension due to mitral stenosis.

The molecular basis and pathophysiology of pulmonary hypertension (PH) are rapidly evolving areas. Recently discovered angiopoietins (Ang) constitute a family of growth factors, and whether they play a causal or protective role in pulmonary hypertension has not been fully elucidated. Since left heart disease probably represents the most frequent cause of PH, we sought to determine whether there was a relationship between serum Ang-1 levels and pulmonary hypertension caused by mitral stenosis (MS). The study population was composed of 49 patients with isolated MS. These patients were then divided into group 1 [31 patients with severe MS: mitral valve area (MVA) ≤1.1 cm(2)] and group 2 (18 patients with mild-moderate MS: MVA 1.2-2.0 cm(2)). Twenty-one healthy volunteers comprised the control group (group 3). All of the subjects underwent complete transthoracic echocardiography with determination of systolic pulmonary artery pressure (PAPs). Ang-1 levels were determined in serum. Serum levels of Ang-1 were significantly higher in the control group compared to patients with severe (group 1) and mild-moderate (group 2) MS (p < 0.001). Ang-1 levels were found to have moderate inverse correlation with PAPs and left atrial (LA) diameter (r: -0.620, p < 0.001 and r: -0.489, p < 0.001, respectively). The AUC for the ROC curve for predicting PAPs <50 mmHg by serum Ang-1 level was 0.824 (95% CI 0.722-0.926, p < 0.001). A serum level of Ang-1 above 34,656 pg/ml has 74% sensitivity and 80% specificity for predicting that PH is not severe (PAPs <50 mmHg). In conclusion, the findings of this study are distinctive in the sense that they clearly demonstrate a negative correlation between serum Ang-1 levels and the degree of PH.

Real-time PCR study of Ang1, Ang2, Tie-2, VEGF, and KDR expression in human erectile tissue during aging.

INTRODUCTION: Aging is a recognized risk factor for erectile dysfunction (ED), contributing independently to vascular damage of penile tissue. Vascular maintenance depends on angiogenic balance in tissues. Vascular endothelial growth factor (VEGF) is a modulator of endothelial cells functions, after engagement to specific receptor kinase domain region (KDR). Other factors, such as angiopoietins, cross talk with VEGF, modulating its effects. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) compete for binding to Tie-2 and, while Ang1 promotes vascular stabilization, Ang2 acts as a partial agonist or antagonist of Ang1 signaling, depending on VEGF bioavailability. AIMS: To quantify the expression of Ang1, Ang2, Tie-2, VEGF, and KDR by real-time polymerase chain reaction (PCR) in human corpus cavernosum (CC) from young and aged healthy individuals. METHODS: Human CC fragments were obtained from organ donors without known risk factors to ED and divided in two groups: young (16-35 years) and aged (59-74 years). RNA was extracted and converted to cDNA. Real-time PCR reactions employed appropriate primers. KDR, Tie-2, Akt, and phospho-Akt protein levels were also assessed by Western blotting (WB). Computer-assisted evaluation of vascular areas was performed. MAIN OUTCOME MEASURES: Study of angiopoietins-Tie-2 and VEGF-KDR systems in human CC during aging by real-time PCR and WB. The ratios Ang1/Tie-2 and VEGF/KDR and Akt levels were also determined. RESULTS: Real-time PCR results showed a sixfold significant reduction in the Ang1/Tie-2 ratio during aging. Ang2, VEGF, and KDR expression results were highly variable. Nevertheless, the ratio VEGF/KDR was significantly higher in the aged individuals. Akt and phospho-Akt levels were similar in both groups. Immunohistological evaluation revealed a significant decrease in vascular areas and endothelial surface in CC with aging, despite no differences found in vessel number. CONCLUSIONS: The obtained results suggest an aging-associated downregulation of angiopoietins/Tie-2 system and an apparent compensatory upregulation of the VEGF/KDR system.

Preoperative angiopoietin-2 serum levels: a marker of malignant potential in ovarian neoplasms and poor prognosis in epithelial ovarian cancer.

INTRODUCTION: The aims of the study were to explore the levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in patients with benign, borderline, or malignant epithelial ovarian tumors and to compare them to those of healthy controls. In addition, we aimed to study how Ang-1 and Ang-2 levels predict the clinical course and survival of patients with epithelial ovarian cancer. METHODS: We enrolled 150 patients with ovarian neoplasms and 34 women with healthy ovaries in this study. Furthermore, we measured the levels of Ang-1 and Ang-2 in patients having an ovarian metastasis or another cancer (n = 29). Serum samples were collected preoperatively at the time of diagnosis, and Ang-1 and Ang-2 levels were measured with an enzyme-linked immunosorbent assay. RESULTS: Angiopoietin-1 and Ang-2 levels were significantly elevated in serum samples of patients with ovarian carcinoma compared with healthy controls (P = 0.0005 and P < 0.0005, respectively). In addition, Ang-2 levels were significantly higher in patients with ovarian carcinoma compared with patients with benign (P < 0.0005) or borderline ovarian tumors (P = 0.011). In receiver operating characteristic analysis, the area under the curve for serum Ang-2 (0.77) was greater than Ang-1 (0.60) but lower than for cancer antigen 125 (0.95) to differentiate ovarian cancer from healthy control. High serum levels of Ang-1 and Ang-2 were associated with primary residual tumor more than 1 cm after debulking surgery, and high Ang-2 levels correlated positively with an advanced tumor stage (P = 0.042). Elevated Ang-2 level (>2.7 ng/mL) was a significant predictor of poor overall and recurrence-free survival (P = 0.043 and P = 0.033, respectively) when assessing Kaplan-Meier curves by a log-rank test. CONCLUSIONS: Patients with ovarian cancer have higher serum levels of angiopoietins than patients with benign or borderline tumors reflecting the increased angiogenesis. These results also suggest that Ang-2 may serve as an angiogenic marker of decreased patient survival in ovarian cancer.