Effect of isoflavone extracts from Glycine max on human endothelial cell damage and on nitric oxide production

Objective: In this study, we determined the protective effect of isoflavones from Glycine max on human umbilical vein endothelial cell (ECV304) damage induced by hydrogen peroxide (H(2)O(2)) and on nitric oxide (NO) production. Methods: We studied the regulation of NO synthesis in cultured human endothelial cells by phytoestrogens contained in soy extracts in the presence or absence of ICI 182,780 or N(omega)-nitro-L-arginine methyl esther and determined the protective effect of these isoflavones on ECV304 damage induced by H(2)O(2). Results: We show that soy extracts activate NO synthesis in endothelial cells and protect against cell damage. Conclusions: In conclusion, soy isoflavones markedly protect ECV304 cells against H(2)O(2) damage and promote NO synthesizing. Therefore, these isoflavones call potentially act as an NO promoter and as an antioxidant.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells

Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95 kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Immunohistochemical study of stromal and vascular components of tonsillar polyps: high endothelial venules as participants of the polyp`s lymphoid tissue

Tonsillar polyps are nonneoplastic lesions usually composed of variable amounts of lymphoid and vascular and connective tissues. All of them are generally assumed to be hamartomatous proliferations, but the profile of vascular and connective components has yet to be explored. The vascular system of the tonsils is complex and includes highly specialized structures (i.e., high endothelial venules (HEVs)) involved in lymphocyte homing into lymphoid tissues. In 14 tonsillar polyps and 26 control tonsils, an immunohistochemical study was performed using CD34 (blood vessels and HEVs), MECA-79 (HEVs), D2-40 (lymphatic vessels), Ki-67, collagens I and III, fibronectin, and tenascin-C. The polyps showed increased total lymphatic area, whereas the number of blood vessels and lymphatics and the blood vascular area did not differ significantly from those of control tonsils. Rare Ki-67+ endothelial cells were found. In the polyps, we detected, possibly for the first time, HEVs amid lymphoid tissue, and that the amount of the latter correlated positively with HEV density. The polyps also presented lesser amounts of fibronectin and collagens I and III than in normal tonsils, which were distributed in a disorganized fashion. Tenascin-C expression was uncommon in the polyps and control tonsils. Tonsillar polyps are composed of disorganized connective tissue and lymphatic channels which can be considered hamartomatous proliferations. However, the lymphoid component is possibly reactive due to its relationship with the HEVs. The highly differentiated phenotype of the HEVs and their complex biology are not in agreement with what would be expected for a component of hamartomatous nature.

Diagnostic implications of oral intravascular papillary endothelial hyperplasia

This study examined the clinical, histological, and immunohistochemical features as well as the differential diagnoses of oral intravascular papillary endothelial hyperplasia (IPEH) to aid clinicians and pathologists in its diagnosis. Clinical features of five oral IPEH cases were obtained from medical records, and all histopathological diagnoses were reviewed. Immunohistochemical reactions, including anti-CD-34, laminin, vimentin, estrogen receptor alpha, and Ki-67, were assessed. Microscopically, a reactive proliferation of vascular cells composed of small papillary structures with hypocellular and hyalinized cores arising in an organized thrombus was seen. CD-34, vimentin, and laminin staining were strongly positive, while estrogen receptor alpha was negative in all cases. A low percentage of cells were positive for Ki-67 in four of five lesions, but one case was strongly positive. A diagnosis of angiosarcoma was investigated and rejected. IPEH presents specific microscopic characteristics that along with clinical data lead to an accurate diagnosis. The general dentist, the first to participate in the diagnostic process, must share the responsibility for diagnosis with the pathologist, and they must work together to determine the correct diagnosis and management. Oral lesions of IPEH are uncommon. Their main significance is that they show a microscopic resemblance to angiosarcoma. Thus, clinicians should have more information regarding this benign entity. Finally, we suggest that in recurrent cases exhibiting strong immunolabeling of proliferative markers the possibility of angiosarcoma should be investigated.

Intravascular papillary endothelial hyperplasia: Report of 4 cases with immunohistochemical findings

Intravascular papillary endothelial hyperplasia (IPEH) is a benign endothelial proliferation, usually intravascular, that may mimic angiosarcoma. In this report, four new cases of IPEH involving the oral region are described. The affected sites were the lower lip, labial comissure and the submandibular region. After clinical evaluation, the complete removal of the lesions showed a circumscribed and soft mass. Histologically, the major feature was a reactive proliferation of endothelial cells composed of small papillary structures with hypocellular and hyalinized cores arising in an organized thrombus. Immunohistochemical staining for CD34 was strongly positive in endothelial cells. Vimentin and laminin immunolabelling were also consistent with a vascular origin. In order to verify the proliferative potential of the lesions, the Ki-67 antibody was used, revealing low percentage of labeled cells (<20%). No immunoreactivity for GLUT-1 was observed. Since the complete removal is curative, no additional treatment was necessary, and no signs of recurrence had been observed until now. Due to the particular features of IPEH, it is important for pathologists and clinicians to become familiar with this lesion. Additionally, the specific histological arrangement, including the absence of cellular pleomorphism, mitotic activity and necrosis, represents a guide to help in the differential diagnosis. Moreover, the vascular origin and the proliferative index should be assessed by immunohistochemistry in order to provide an accurate diagnosis.

Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

Corneal endothelial response to induced myopia in the chicken

Purpose: To investigate the role of corneal endothelial surface enlargement in the chicken myopia model in inducing corneal endothelial changes. Methods: Lid suture was performed on one eye of 1-day-old cockerels. Five chickens were killed at 1 week, and four chickens killed at each of 3 weeks, 6 weeks, and 10 weeks postnatal. The endothelial morphology was obtained by flat mounting the endothelial surface and the subsequent digitisation. Comparisons were undertaken between the control unsutured eye and the lid-sutured eye endothelium, and between the central endothelial areas compared to the peripheral endothelial areas in both the myopic and the normal corneas. Calculation of the contribution to the endothelial change by hypertrophy and mitosis were calculated using Bahn's formula. Results: Total endothelial surface area increased significantly over time in the myopic model compared to control eyes but the mean cell area of endothelial cells remained the same for both the enlarged myopic endothelial surface area and in the normal controls. Sampling from the central and the peripheral corneal endothelial surface also disclosed no difference. The mean cell area did increase steadily with age but was the same for both normal and myopic corneas. Conclusions: It would appear that there are equal contributions from hypertrophy and mitosis in the myopic group and the normal corneal group with a slightly increasing trend towards mitotic activity in the myopic corneal endothelial layer.

SOX18 directly interacts with MEF2C in endothelial cells

Recently, we demonstrated that mutations in the Sry-related HMG box gene Sox18 underlie vascular and hair follicle defects in the mouse allelic mutants ragged (Ra) and RaJ. Ra mice display numerous anomalies in the homozygote including, oedema, peritoneal secretions, and are almost completely naked. Sox18 and the MADS box transcription factor, Mef2C, are expressed in developing endothelial cells. Null mutants in Sox18 and Mef2c display overlapping phenotypic abnormalities, hence, we investigated the relationship between these two DNA binding proteins. We report here the direct interaction between MEF2C and SOX18 proteins, and establish that these proteins are coexpressed in vivo in endothelial cell nuclei. MEF2C expression potentiates SOX18-mediated transcription in vivo and regulates the function of the SOX18 activation domain. Interestingly, MEF2C fails to interact or co-activate transcription with the Ra or RaJ mutant SOX18 proteins. These results suggest that MEF2C and SOX18 may be important partners directing the transcriptional regulation of vascular development. (C) 2001 Academic Press.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 mum) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P

Cold Storage Preservation and Warm Ischaemic Injury to Isolated Arterial Segments: Endothelial Cell Injury

Injury to endothelial calls is thought to be important to the development of the vascular lesion of chronic rejection. It was the aim of this study to develop a semiquantitative method to assess endothelial injury in arterial grafts and to document the injury produced by cold storage preservation and additional warm ischaemia. Twelve- and 24-h cold preservation of rat aortic segments, together with an additional 1 h of warm ischaemia, were assessed. Electron micrographs of representative endothelial cells were scored for cytoplasmic, nuclear and mitochondrial injury. The overall injury score was obtained by addition of the individual scores. Storage for up to 24 h in University of Wisconsin (UW) and Terasaki did not produce any injury. Twenty-four hours of storage in Euro-Collins resulted in endothelial cell death. Injury occurred after 12 h of storage in Ross, Collins and normal saline, and the injury increased following 24 h of storage. One hour of warm ischaemia did not increase the injury. Injury to endothelial cells varies with the preservation solution used and the time of cold storage, so that both the type of solution and the storage time should be taken into account in clinical studies looking at the influence of cold ischaemia time and graft outcome.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.