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Biomedical subjects

C Willam

Publications and source records attributed to C Willam.

14 recordsLinked to original sources
PubMed2006

Expression of hypoxia-inducible transcription factors in developing human and rat kidneys.

Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.

Animals↗
PubMed2001

Independent function of two destruction domains in hypoxia-inducible factor-alpha chains activated by prolyl hydroxylation.

Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF-1 alpha that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non-redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal.

Amino Acid Motifs↗
PubMed2000

Tie2 receptor expression is stimulated by hypoxia and proinflammatory cytokines in human endothelial cells.

The tyrosine kinase receptor Tie2 (also known as Tek) plays an important role in the development of the embryonic vasculature and persists in adult endothelial cells (ECs). Tie2 was shown to be upregulated in tumors and skin wounds, and its ligands angiopoietin-1 and -2, although they are not directly mitogenic, modulate neovascularization. To gain further insight into the regulation of Tie2, we have studied the effect of hypoxia and inflammatory cytokines, two conditions frequently associated with neoangiogenic processes, on Tie2 expression in human ECs. Exposure to 1% O(2) led to a time-dependent significant rise of Tie2 protein levels in human coronary microvascular endothelial cells (HCMECs) and dermal microvascular ECs (HMEC-1) (3.2- and 2.5-fold within 24 hours), which was reversible after reoxygenation, and induced a less marked increase in human umbilical vein ECs (HUVECs; 1.7-fold). Hypoxia-conditioned medium and D-deoxyglucose did not change Tie2 expression, but desferrioxamine and cobalt, which are known to mimic hypoxia-sensing mechanisms, induced Tie2 at ambient oxygen tensions. Tumor necrosis factor-alpha induced Tie2 in a time- and dose-dependent fashion in all 3 EC types (HUVEC, 2.3-fold; HMEC-1, 2. 8-fold; and HCMEC, 3.0-fold; 10 ng/mL, 24 hours). Enhanced expression was also found after exposure to interleukin-1beta (1 ng/mL). Changes in Tie2 protein levels were paralleled by changes in mRNA expression. In accordance with these in vitro findings, immunohistochemistry revealed focal upregulation of Tie2 in capillaries at the border of infarcted human and rat myocardium. In conclusion, the data show that hypoxia and inflammatory cytokines upregulate Tie2, which may contribute to the angiogenic response in ischemic tissues.

Animals↗
PubMed2000

Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets.

Low oxygen (O(2)) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O(2)). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.

Adult↗
PubMed1999

Vascularization of reactively enlarged lymph nodes analyzed by color duplex sonography.

PURPOSE: This study involved a prospective evaluation of the reliability of sonography, and especially duplex sonography, in confirmation of the benign nature of enlarged cervical lymph nodes. PATIENTS AND METHODS: In 53 untreated patients with enlarged cervical lymph nodes, B-mode, plain, and d-galactose-enhanced color duplex sonography were performed. The B-mode sonomorphology was analyzed for the structure of vascularization. Quantitative parameters such as maximum flow velocity, pulsatility index, and resistive index were also assessed. The benignity of the lymph nodes was confirmed by microscopic analysis. RESULTS: The B-mode showed 20 homogeneous lymph nodes, 23 with a central echogenoic line covering less than one third, and 10 with a distinct hilus sign extending to more than one third of the lymph node diameter. Microscopically, the least fibrotic or chronic inflammatory changes in the parenchyma were observed in the homogeneous lymph nodes, whereas those with central echogeneoity had fibrotic and lipoid hilus changes. Histologically, all lymph nodes had normal afferent and efferent hilus vessels. In 37 lymph nodes, the vessel structure could be reliably visualized by both plain and enhanced color duplex sonography, whereas in 16 it could only be demonstrated after the use of signal enhancement. Nine of 53 lymph nodes had Solbiati-(L/T-) indices below 2, which were suggestive of malignancy. Quantitative flow parameters did not provide useful information. CONCLUSION: Color duplex analysis of enlarged lymph nodes is a useful method for assessment of benignity, especially after application of a signal-enhancing agent.

Adult↗
PubMed1999

Increases in oxygen tension stimulate expression of ICAM-1 and VCAM-1 on human endothelial cells.

Leukocyte infiltration plays a major role in ischemia-associated organ dysfunction and damage. A crucial step for extravasation of white blood cells is binding of leukocyte beta-integrins to endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). To test for direct effects of oxygen on this process we studied ICAM-1 and VCAM-1 expression in human dermal microvascular and umbilical vein endothelial cells (EC) exposed to different oxygen tensions in the absence or presence of tumor necrosis factor-alpha (TNF-alpha). Hypoxia (95% N2-5% CO2) resulted in a downregulation of basal but not TNF-alpha-induced expression of ICAM-1 and VCAM-1. Subsequent rises in oxygen (21, 40, or 95% O2) led to marked increase of ICAM-1 and VCAM-1 cell surface and mRNA expression in both EC types, which after 16 h amounted to about one-third to one-half of maximal TNF-alpha-induced expression. This increase was greatest after 0.5-h hypoxia and was blunted with prolonged hypoxic preincubation. Exposure of cells preincubated under "normoxic" (21% O2) conditions to hyperoxia (40 or 95% O2) also enhanced expression of both adhesion molecules, but the increase was lower than in cells preexposed to hypoxia. The nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) enhanced ICAM-1 and VCAM-1 expression under basal and hypoxic conditions, but in the presence of L-NAME, levels in reoxygenated cells were not higher than basal levels. Moreover, the oxygen-induced rise could be mimicked by addition of H2O2 to normoxic cells, and the oxygen-induced expression of VCAM-1 but not of ICAM-1 was inhibited by addition of the free radical scavengers superoxide dismutase, N-acetyl-L-cysteine, and pyrrolidinedithiocarbamate. These data indicate that an increase in oxygen availability stimulates ICAM-1 and VCAM-1 expression on micro- and macrovascular EC, which may contribute to adhesion and transmigration of different leukocyte populations in ischemia-reperfusion injuries.

Cells, Cultured↗
PubMed1998

Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1alpha.

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.

Animals↗
PubMed1998

[Signal-enhanced color-coded duplex sonography of reactively and metastatically enlarged lymph nodes].

PURPOSE: Prospective comparison of B-mode-, plain and signal-enhanced colour Doppler ultrasound to evaluate new criteria for assessment of the tumour status of enlarged cervical lymph nodes. METHODS: Colour Doppler sonography was performed before and after application of d-galactose in 35 patients with suspected lymph node metastases of the neck. Sonomorphology was analysed qualitatively, maximal flow rate, pulsatility index and resistance index were determined quantitatively. RESULTS: Signal enhanced Doppler revealed a typical vessel morphology of the lymph nodes facilitating differential diagnosis. In 23%, vascularisation of the lymph nodes was only visible after application of the signal-enhancing agent (benign: 44%, malignant: 5%). The measurement of flow velocity and the calculation of RI and PI did not lead to a reliable differentiation between malignant and benign lymph nodes whereas B-mode sonography yielded highly valid results. CONCLUSION: The application of d-galactose facilitates differentiation between malignant and benign lymph nodes.

Aged↗
PubMed1998

Acidosis activates complement system in vitro.

We investigated the in vitro effect of different forms of acidosis (pH 7.0) on the formation of anaphylatoxins C3a and C5a. Metabolic acidosis due to addition of hydrochloric acid (10 micromol/ml blood) or lactic acid (5.5 micromol/ml) to heparin blood (N=12) caused significant activation of C3a and C5a compared to control (both p=0.002). Respiratory acidosis activated C3a (p=0.007) and C5a (p=0.003) compared to normocapnic controls. Making blood samples with lactic acidosis hypocapnic resulted in a median pH of 7.37. In this respiratory compensated metabolic acidosis, C3a and C5a were not increased. These experiments show that acidosis itself and not lactate trigger for activation of complement components C3 and C5.

Acidosis↗
PubMed1998

Assessment of vascularity in reactive lymph nodes by means of D-galactose contrast-enhanced Doppler sonography.

RATIONALE AND OBJECTIVES: A prospective study in signal-enhanced Doppler sonography of lymph nodes that were assumed pretherapeutically to be benign was performed to investigate characteristic sonomorphologic features and vascularity of reactively enlarged lymph nodes. METHODS: Thirty-four patients with enlarged superficial lymph nodes of the neck were examined first by B-scan sonography then by Doppler sonography before and after administration of an ultrasound signal-enhancing agent. In B-scan sonography, lymph nodes were classified into three groups according to their sonomorphologic features: (1) homogeneous parenchyma, (2) a centrally located echogenoic line, and (3) a echogenoic "hilus reflex." In conventional and contrast-enhanced Doppler sonography, peak flow rate, pulsatility index, and resistive index were assessed. Sonomorphologic criteria were compared with histologic findings. RESULTS: Reactively enlarged lymph nodes showed characteristic sonomorphologic patterns correlating to their histologic features. Echogenicity of the hilus corresponded to fibrosis (centrally located echogenoic line in 13 nodes; 38.2%) or fatty involution of the hili (echogenoic hilus reflex in 15 nodes; 44.1%). Administration of the galactose-based ultrasound contrast enhancer facilitated the assessment of hilar vessels, which projected to the echogenoic hili, respectively, actually additionally visualized a hilar vascularity in 10 of the 34 lymph nodes compared with conventional Doppler. Measured Doppler indices gave not a significant clue for identifying reactive lymph nodes or for differential diagnosis. CONCLUSIONS: Qualitative sonomorphologic assessment of characteristic sonomorphologic features of reactive lymph nodes may serve as a valuable tool for examining reactively enlarged lymph nodes. Administration of an ultrasound echo enhancer allows the assessment of a characteristic nodal vascularity in reactive lymph nodes and were superior to conventional B-mode and conventional Doppler sonography.

Adult↗
PubMed1997

Evaluation of metastases and reactive lymph nodes in Doppler sonography using an ultrasound contrast enhancer.

RATIONALE AND OBJECTIVES: The authors conducted a prospective study in D-galactose signal-enhanced Doppler sonography of lymph nodes to investigate new aspects in differentiating malignant from reactive lymph nodes of patients with suspected malignancy of the neck. METHODS: Twenty-one patients with suspected squamous epithelial cell carcinoma metastases of the neck were examined by Doppler sonography before and after administration of an ultrasound signal-enhancing agent, consisting of D-galactose microbubbles. Qualitative sonomorphology, peak flow rates, and pulsatility and resistive indices were assessed. RESULTS: Compared with conventional Doppler, enhanced Doppler sonography gave detailed additional information about vascularization of metastases or reactive lymph nodes. Signal-enhanced Doppler of metastases showed a relatively characteristic pattern of vascularity, therefore facilitating differential diagnoses and allowing better discrimination from surrounding tissue, demonstrated by the infiltration of neighboring vessels in the neck. Concerning reactive lymph nodes, vascularization could be stated and measured in many cases only after signal enhancement. Evaluating peak velocities and pulsatility and resistive indices could not differentiate significantly malignant from reactive lymph nodes. CONCLUSIONS: Administration of a D-galactose-based signal-enhancer helps to differentiate malignant from reactive lymph nodes of the neck. It is superior to conventional Doppler by improving evaluation of the vascularity and could be of use for staging procedures.

Aged↗
PubMed1996

Keratinization and necrosis. Morphologic aspects of lymphatic metastases in ultrasound.

RATIONALE AND OBJECTIVE: The authors performed a retrospective study in ultrasound to investigate new aspects in the sonomorphology of lymph node metastases of the neck. In this study, it could be demonstrated the first time that the histologic characteristics of the metastases determine the sonographic appearance. In addition to criteria such as the longitudinal/ transversal quotient, sonomorphology could support a more precise differential diagnosis of neck lymph nodes. METHODS: In 105 of 145 patients with histologically proved head and neck carcinomas, 187 lymph node metastases were detected by ultrasound. Sonomorphology was compared with the corresponding histology. RESULTS: Five sonomorphologic groups could be differentiated. (1) Thirty-one percent of the metastases were homogenous. (2) Concerning the more complex morphology of lymph node metastases in ultrasound, echolucent forms could be differentiated from echogenic textures: low- or nondifferentiated and nonkeratinizing metastases appeared echolucent and cyst-like, with dorsal signal amplification. (3) Nonkeratinizing lymphomas with necrosis showed single or multiple echolucent intranodal lesions. (4) In correlation with an increasing keratinization, the echogenecity of the lymph nodes increased and intranodal echogenic inclusions appeared. (5) An extended keratinization correlated with a central echogenecity. CONCLUSIONS: The morphologic assessment of lymph nodes in ultrasound allows for primary histologic and prognostic evaluation of lymph node metastases.

Aged↗
PubMed1996

[Differential diagnosis of cervical lymph node enlargements: ultrasound and histomorphology of reactive lymph nodes].

The purpose of this study was to investigate the morphology of reactive lymph nodes of the neck in ultrasound and its histological implications. By revealing a characteristic sonomorphology of reactive lymph nodes we would like to contribute to the differential diagnosis of cervical lymph node enlargements. In 81 patients the morphology and the longitudinal/ transverse axis ratio (L/T ratio) of 257 reactively enlarged lymph nodes were examined. Besides 6 false-positive cases the L/T ratio turned out to be a reliable instrument in detecting reactive lymph nodes. Concerning the nodal texture, 89 lymph nodes (34.6%) appeared homogenously. Those lymph nodes which demonstrated a more complex nodal texture could be assigned to three sonomorphological groups: In 134 lymph nodes (52.1%) a centrally located echogenoic line ("central echogenoic line', CEL) was observed. Its maximal diameter was defined to be maximally up to one third of the lymph node width. In the histological examination this CEL could be identified as a widened hilus with an increased fibrosis. An echogenoic nodal area measuring between one third up to one half of the lymph node width demonstrated histologically a fatty replacement of the hilar tissue. This "echogenoic hilus reflex' occurred in 28 lymph nodes (10.9%). A nearly complete lipomatous atrophy of the lymph node parenchyma was found in 6 lymph nodes (2.4%) demonstrating a "central echogenicity' with just a small peripheral, less echogenoic border corresponding to the remaining parenchyma.

Adult↗