The 5-yr longitudinal study tested the hypothesis that serum and urinary markers of type II colla... more The 5-yr longitudinal study tested the hypothesis that serum and urinary markers of type II collagen metabolism would be associated with radiological progression of disease in patients with mild-to-moderate knee osteoarthritis (OA). Synthesis of type IIA collagen and degradation of total type II collagen were assessed in 135 patients with mild-to-moderate knee OA over 5 yrs using serum concentration of the N-propeptide of collagen type IIA (PIIANP) and urinary excretion of crosslinked C-telopeptide (CTX-II), respectively. The markers were measured at baseline, 2, 3 and 5 yrs' follow-up corresponding to X-ray time points. Analysis of variance (ANOVA) was performed to determine longitudinal changes over 5 yrs in the biomarkers in all patients and between progressors and non-progressors. Complete X-ray progression data over 5 yrs, serum PIIANP and urinary CTX-II were available for 84/135 patients. There were 24 progressors and 60 non-progressors. Overall, over the 5-yr study period average PIIANP and CTX-II levels were higher in progressors compared with non-progressors (P < 0.05 for both, ANOVA). The patients with serum PIIANP in the highest quartile of 5-yr levels of PIIANP had a significantly higher risk of progression than the other patients [relative risk (95% CI): 3.2 (1.1-9.2)]. Increased levels of urinary CTX-II were also associated with a higher risk of progression with a relative risk (95% CI) of 3.4 (1.2-9.4) in patients with 5-yr levels above the median. The risk of progression was highest in patients with 5-yr levels of PIIANP in the highest quartile and/or CTX-II in the two highest quartiles with a relative risk (95% CI) of progression, 11.8 (2.5-54). The data presented here suggest that progression of knee OA is associated with alterations of systemic levels of biological markers of type II collagen metabolism. The data also suggest that the combined measurement of serum PIIANP and urinary CTX-II may be useful to identify patients with knee OA at increased risk of disease progression.
The balance between proliferation and apoptosis within a tissue is important in controlling its o... more The balance between proliferation and apoptosis within a tissue is important in controlling its overall growth. When either or both are altered, uncontrolled cell proliferation can contribute to cancer. The aim of this study was to investigate apoptosis and proliferation in the progression from Barrett's oesophagus to adenocarcinoma. Fifty-one paraffin sections of Barrett's mucosa with both intestinal and gastric-type Barrett's mucosa, dysplasia, and adenocarcinoma, from 28 patients, were examined for apoptosis using haematoxylin and eosin (H&E)-stained sections counterstained immunohistochemically with CD45 to distinguish leucocytes from apoptotic bodies. Proliferation was detected by immunohistochemistry using the MIB-1 (Ki-67) antibody. There was an increase in proliferation in dysplastic and carcinomatous tissue compared with metaplastic tissue (p=0.0001). In dysplasia, proliferation was distributed throughout the basal-luminal axis, whereas in metaplasia, cell division was compartmentalized to the lower crypt (p<0.001). Conversely, there was a decrease in apoptosis in the upper crypt and luminal surface in dysplasia and adenocarcinoma compared with metaplasia (p<0.0008). There was a significant increase in apoptotic activity in intestinal-type Barrett's mucosa compared with gastric-type. There was a highly significant increase in the glandular proliferation to apoptosis ratio (GPAR) in the progression of metaplasia to dysplasia to adenocarcinoma (p=0.001). The shift in the GPAR in the progression of neoplastic change suggests that it may be a useful and sensitive marker of neoplastic change in Barrett's oesophagus, especially if the assessment of both apoptotic and proliferative activity in the mucosa can be made easier by more sophisticated technical methods.
In the past years, a new approach towards highly porous metal components has been under developme... more In the past years, a new approach towards highly porous metal components has been under development at the Dresden based Department of Powder Metallurgy and Composite Materi- als of the Fraunhofer Institute for Manufacturing and Advanced Materials (IFAM). The com- ponents are made from melt extracted metal fibers, where the consolidation of the porous structure is achieved by sintering. Interest
Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in man... more Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in many pathological conditions including cancer, arthritis, and diabetes. VEGF activates VEGF-Receptor 1(VEGF-R1) and VEGF-Receptor 2 (VEGF-R2), which autophosphorylate to initiate a signaling cascade resulting in angiogenesis and increased microvascular permeability. Here we describe a novel VEGF-R2 selective inhibitor, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), that is a potent and selective inhibitor of VEGF-R2 tyrosine kinase in vitro (IC(50) < 2 nM), compared with other receptor tyrosine kinases, including VEGF-R1 (IC(50) > 50 microM). Endothelial cell proliferation was assayed by (3)H-thymidine incorporation in response to VEGF-A +/- ZM323881. The effect of ZM323881 on VEGF-mediated permeability was measured in frog microvessels using the Landis Michel technique. To ensure that ZM323881 was effective in frogs, western analysis was performed on protein extracted from frog lungs incubated in the presence or absence of VEGF-A or VEGF-A with ZM323881. ZM323881 inhibits VEGF-A-induced endothelial cell proliferation (IC(50) = 8 nM) and VEGF-R2 tyrosine phosphorylation in vitro. VEGF-A-mediated increases in vascular permeability in perfused mesenteric microvessels in vivo were reversibly abolished by both ZM323881 and the class III receptor tyrosine kinase inhibitor PTK787/ZK222584. These data suggest that VEGF-R2 phosphorylation is necessary for VEGF-A-mediated increases in microvascular permeability in vivo.
Vascular endothelial growth factors (VEGFs) are known to increase vascular permeability. VEGF-A a... more Vascular endothelial growth factors (VEGFs) are known to increase vascular permeability. VEGF-A acts on two receptor tyrosine kinases, VEGF receptor-1 (VEGF-R1 or flt-1) and VEGF receptor-2 (VEGF-R2, flk-1 or KDR). VEGF-C acts only on VEGF-R2 on vascular endothelial cells, whereas placental growth factor-1 (PlGF-1) acts only on VEGF-R1. The effects of perfusion of these receptor-specific proteins on hydraulic conductivity (L(p)) was measured in frog mesenteric capillaries. The effect of PlGF on L(p) was not conclusive, and overall fluid flux did not increase during that time. VEGF-C acutely and transiently increased L(p) (4.5 +/- 0.9-fold), which was more obvious in a subset of vessels, in a similar manner to that reported for VEGF-A. In the subset of vessels in which VEGF-C significantly increased L(p) acutely, there was a sustained 12-fold increase in L(p) 20 min after perfusion, but this was not seen in those vessels which did not respond acutely to VEGF-C, or in vessels exposed to PlGF-1. L(p) was also increased 24 h after perfusion with VEGF-C, but not with PlGF-1. Western blot analysis showed that VEGF-R1 and VEGF-R2 are both present in frog tissue. These data show that the VEGFs that stimulate VEGF-R2 chronically increase L(p), but not those that stimulate VEGF-R1 only. This supports the hypothesis that chronic increases in microvascular permeability induced by VEGF are mediated via activation of VEGF-R2 rather than VEGF-R1.
To investigate whether there are any variations in chondrocyte susceptibility to an apoptotic sti... more To investigate whether there are any variations in chondrocyte susceptibility to an apoptotic stimulus between cells of articular cartilage (AC) from equine joints that differ in prevalence of osteoarthritis (OA). Cartilage from macroscopically normal equine metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints was used. Prior to culture, chondrocyte viability was assessed using the fluorescein diacetate (FDA) and propidium iodide paravital staining method. AC explants were subsequently treated with tumour necrosis factor-α (TNF-α) in combination with Actinomycin D to induce apoptosis. Apoptosis of chondrocytes in cartilage sections was assessed by expression of active caspase-3 using indirect immunohistochemistry and sections also histologically graded using a 'modified' Mankin scoring system. Prior to culture (mean ± standard deviation) chondrocyte viability was 80.7% (3.5). The extent of chondrocyte apoptosis induced by TNF-α/Actinomycin D varied markedly according to the joint type that the cartilage was sampled from. For MCP joints, the extent of overall chondrocyte apoptosis was significantly higher (P < 0.001) in stimulated explants (26.7%, 10.3) than that observed in unstimulated control samples (9.6%, 7.5). Conversely, chondrocytes from PIP and DIP joint cartilage did not respond significantly to apoptotic stimulation (P > 0.05). Significant variations in cellularity and thickness were also evident between cartilages of different joint types. Data in this study demonstrate that chondrocytes from three equine joint types with varying prevalences of OA differ significantly in terms of susceptibility to apoptosis induction. This may provide a possible explanation for the joint-specific nature of the disease.
The 5-yr longitudinal study tested the hypothesis that serum and urinary markers of type II colla... more The 5-yr longitudinal study tested the hypothesis that serum and urinary markers of type II collagen metabolism would be associated with radiological progression of disease in patients with mild-to-moderate knee osteoarthritis (OA). Synthesis of type IIA collagen and degradation of total type II collagen were assessed in 135 patients with mild-to-moderate knee OA over 5 yrs using serum concentration of the N-propeptide of collagen type IIA (PIIANP) and urinary excretion of crosslinked C-telopeptide (CTX-II), respectively. The markers were measured at baseline, 2, 3 and 5 yrs' follow-up corresponding to X-ray time points. Analysis of variance (ANOVA) was performed to determine longitudinal changes over 5 yrs in the biomarkers in all patients and between progressors and non-progressors. Complete X-ray progression data over 5 yrs, serum PIIANP and urinary CTX-II were available for 84/135 patients. There were 24 progressors and 60 non-progressors. Overall, over the 5-yr study period average PIIANP and CTX-II levels were higher in progressors compared with non-progressors (P < 0.05 for both, ANOVA). The patients with serum PIIANP in the highest quartile of 5-yr levels of PIIANP had a significantly higher risk of progression than the other patients [relative risk (95% CI): 3.2 (1.1-9.2)]. Increased levels of urinary CTX-II were also associated with a higher risk of progression with a relative risk (95% CI) of 3.4 (1.2-9.4) in patients with 5-yr levels above the median. The risk of progression was highest in patients with 5-yr levels of PIIANP in the highest quartile and/or CTX-II in the two highest quartiles with a relative risk (95% CI) of progression, 11.8 (2.5-54). The data presented here suggest that progression of knee OA is associated with alterations of systemic levels of biological markers of type II collagen metabolism. The data also suggest that the combined measurement of serum PIIANP and urinary CTX-II may be useful to identify patients with knee OA at increased risk of disease progression.
The balance between proliferation and apoptosis within a tissue is important in controlling its o... more The balance between proliferation and apoptosis within a tissue is important in controlling its overall growth. When either or both are altered, uncontrolled cell proliferation can contribute to cancer. The aim of this study was to investigate apoptosis and proliferation in the progression from Barrett's oesophagus to adenocarcinoma. Fifty-one paraffin sections of Barrett's mucosa with both intestinal and gastric-type Barrett's mucosa, dysplasia, and adenocarcinoma, from 28 patients, were examined for apoptosis using haematoxylin and eosin (H&E)-stained sections counterstained immunohistochemically with CD45 to distinguish leucocytes from apoptotic bodies. Proliferation was detected by immunohistochemistry using the MIB-1 (Ki-67) antibody. There was an increase in proliferation in dysplastic and carcinomatous tissue compared with metaplastic tissue (p=0.0001). In dysplasia, proliferation was distributed throughout the basal-luminal axis, whereas in metaplasia, cell division was compartmentalized to the lower crypt (p<0.001). Conversely, there was a decrease in apoptosis in the upper crypt and luminal surface in dysplasia and adenocarcinoma compared with metaplasia (p<0.0008). There was a significant increase in apoptotic activity in intestinal-type Barrett's mucosa compared with gastric-type. There was a highly significant increase in the glandular proliferation to apoptosis ratio (GPAR) in the progression of metaplasia to dysplasia to adenocarcinoma (p=0.001). The shift in the GPAR in the progression of neoplastic change suggests that it may be a useful and sensitive marker of neoplastic change in Barrett's oesophagus, especially if the assessment of both apoptotic and proliferative activity in the mucosa can be made easier by more sophisticated technical methods.
In the past years, a new approach towards highly porous metal components has been under developme... more In the past years, a new approach towards highly porous metal components has been under development at the Dresden based Department of Powder Metallurgy and Composite Materi- als of the Fraunhofer Institute for Manufacturing and Advanced Materials (IFAM). The com- ponents are made from melt extracted metal fibers, where the consolidation of the porous structure is achieved by sintering. Interest
Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in man... more Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis in many pathological conditions including cancer, arthritis, and diabetes. VEGF activates VEGF-Receptor 1(VEGF-R1) and VEGF-Receptor 2 (VEGF-R2), which autophosphorylate to initiate a signaling cascade resulting in angiogenesis and increased microvascular permeability. Here we describe a novel VEGF-R2 selective inhibitor, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), that is a potent and selective inhibitor of VEGF-R2 tyrosine kinase in vitro (IC(50) < 2 nM), compared with other receptor tyrosine kinases, including VEGF-R1 (IC(50) > 50 microM). Endothelial cell proliferation was assayed by (3)H-thymidine incorporation in response to VEGF-A +/- ZM323881. The effect of ZM323881 on VEGF-mediated permeability was measured in frog microvessels using the Landis Michel technique. To ensure that ZM323881 was effective in frogs, western analysis was performed on protein extracted from frog lungs incubated in the presence or absence of VEGF-A or VEGF-A with ZM323881. ZM323881 inhibits VEGF-A-induced endothelial cell proliferation (IC(50) = 8 nM) and VEGF-R2 tyrosine phosphorylation in vitro. VEGF-A-mediated increases in vascular permeability in perfused mesenteric microvessels in vivo were reversibly abolished by both ZM323881 and the class III receptor tyrosine kinase inhibitor PTK787/ZK222584. These data suggest that VEGF-R2 phosphorylation is necessary for VEGF-A-mediated increases in microvascular permeability in vivo.
Vascular endothelial growth factors (VEGFs) are known to increase vascular permeability. VEGF-A a... more Vascular endothelial growth factors (VEGFs) are known to increase vascular permeability. VEGF-A acts on two receptor tyrosine kinases, VEGF receptor-1 (VEGF-R1 or flt-1) and VEGF receptor-2 (VEGF-R2, flk-1 or KDR). VEGF-C acts only on VEGF-R2 on vascular endothelial cells, whereas placental growth factor-1 (PlGF-1) acts only on VEGF-R1. The effects of perfusion of these receptor-specific proteins on hydraulic conductivity (L(p)) was measured in frog mesenteric capillaries. The effect of PlGF on L(p) was not conclusive, and overall fluid flux did not increase during that time. VEGF-C acutely and transiently increased L(p) (4.5 +/- 0.9-fold), which was more obvious in a subset of vessels, in a similar manner to that reported for VEGF-A. In the subset of vessels in which VEGF-C significantly increased L(p) acutely, there was a sustained 12-fold increase in L(p) 20 min after perfusion, but this was not seen in those vessels which did not respond acutely to VEGF-C, or in vessels exposed to PlGF-1. L(p) was also increased 24 h after perfusion with VEGF-C, but not with PlGF-1. Western blot analysis showed that VEGF-R1 and VEGF-R2 are both present in frog tissue. These data show that the VEGFs that stimulate VEGF-R2 chronically increase L(p), but not those that stimulate VEGF-R1 only. This supports the hypothesis that chronic increases in microvascular permeability induced by VEGF are mediated via activation of VEGF-R2 rather than VEGF-R1.
To investigate whether there are any variations in chondrocyte susceptibility to an apoptotic sti... more To investigate whether there are any variations in chondrocyte susceptibility to an apoptotic stimulus between cells of articular cartilage (AC) from equine joints that differ in prevalence of osteoarthritis (OA). Cartilage from macroscopically normal equine metacarpophalangeal (MCP), proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints was used. Prior to culture, chondrocyte viability was assessed using the fluorescein diacetate (FDA) and propidium iodide paravital staining method. AC explants were subsequently treated with tumour necrosis factor-α (TNF-α) in combination with Actinomycin D to induce apoptosis. Apoptosis of chondrocytes in cartilage sections was assessed by expression of active caspase-3 using indirect immunohistochemistry and sections also histologically graded using a 'modified' Mankin scoring system. Prior to culture (mean ± standard deviation) chondrocyte viability was 80.7% (3.5). The extent of chondrocyte apoptosis induced by TNF-α/Actinomycin D varied markedly according to the joint type that the cartilage was sampled from. For MCP joints, the extent of overall chondrocyte apoptosis was significantly higher (P < 0.001) in stimulated explants (26.7%, 10.3) than that observed in unstimulated control samples (9.6%, 7.5). Conversely, chondrocytes from PIP and DIP joint cartilage did not respond significantly to apoptotic stimulation (P > 0.05). Significant variations in cellularity and thickness were also evident between cartilages of different joint types. Data in this study demonstrate that chondrocytes from three equine joint types with varying prevalences of OA differ significantly in terms of susceptibility to apoptosis induction. This may provide a possible explanation for the joint-specific nature of the disease.
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