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R M De Waal

Publications and source records attributed to R M De Waal.

17 recordsLinked to original sources
PubMed2000

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators.

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.

Angiostatins↗
PubMed2000

Angiogenic balance in human melanoma: expression of VEGF, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor.

Angiostatins↗
PubMed2000

Amyloid-beta-induced degeneration of human brain pericytes is dependent on the apolipoprotein E genotype.

Amyloid-beta (A beta) deposition in cerebral vessels (cerebral amyloid angiopathy, CAA) is accompanied by degeneration of vascular cells, including pericytes and smooth muscle cells. Previous studies indicated that specific A beta protein isoforms are toxic for cultured human brain pericytes and smooth muscle cells. In particular, A beta 1-40 carrying the E22Q mutation, as in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), is toxic. We investigated the effects of the A beta-binding protein apolipoprotein E (ApoE) on the toxicity of A beta for cultured human brain pericytes. We compared the toxicity of HCHWA-D A beta 1-40 for pericyte cultures with different ApoE genotypes, studied the accumulation of A beta and ApoE in these different cell cultures, and investigated the effects of exogenous ApoE. Pericyte cultures with an ApoE epsilon 2/epsilon 3 genotype were more resistant to HCHWA-D A beta 1-40 treatment than cultures with a epsilon 3/epsilon 3 or epsilon 3/epsilon 4 genotype. Cell death was highest in cultures homozygous for ApoE epsilon 4. The extent to which both A beta ApoE accumulated at the cell surface was parallel to the degree of toxicity. The addition of purified ApoE resulted in a decrease in cell death. These data suggest that ApoE4 may direct A beta more efficiently than other ApoE isoforms into a pathological interaction with the HBP cell surface. The results of this study are in line with the observations that inheritance of the ApoE epsilon 4 allele increases the risk of developing Alzheimer's disease, and that the ApoE epsilon 2 allele has a relatively protective effect.

Aged↗
PubMed1999

3-Nitropropionic acid induces a spectrum of Huntington's disease-like neuropathology in rat striatum.

Systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) to rats results in selective striatal lesions and serves as an experimental model of Huntington's disease (HD). However, the effects of the 3-NP treatment are unpredictable and result in lesions of variable severity. The present study was aimed at further characterizing the variability of the striatal lesions induced by systemic administration of 3-NP using osmotic pumps. Hematoxylin-eosin (HE) and Nissl stains as well as immunohistochemical labelling of astrocytes and striatal neurones were performed to analyse the neurotoxic effects of 3-NP. In general, chronic systemic administration of 3-NP resulted in obvious bilateral striatal lesions, which ranged from mild to severe, together with a subtle, but detectable behavioural lesion. Severe type lesions showed marked neuronal loss and an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes surrounding the lesion area, whereas in the core of the lesion GFAP-immunoreactivity was absent. The mild type lesion was characterized by a substantial loss of striatal neurones and an increased expression of GFAP-positive astrocytes throughout the lesion. In a number of 3-NP-treated animals, neither type of lesion was observed, although these animals demonstrated behavioural changes in the paw test compared to controls. In the striatum of these tested 3-NP-treated animals, compromised rk' neurones were detected, suggestive of subtle and early 3-NP-induced neuronal injury. Similar dark neurones were also detected in mild and severe lesions and were immunocytochemically characterized as gamma-aminobutyric acid (GABA) and substance P containing spiny neurones, which belong to the neuronal population that is affected in early HD. These results indicate that systemic administration of 3-NP to rats may result in a spectrum of striatal pathology of which the morphology of the mild type lesion resembles the characteristic HD neuropathology most closely.

Animals↗
PubMed1998

Distribution of A beta-associated proteins in cerebrovascular amyloid of Alzheimer's disease.

Senile plaques and cerebrovascular amyloidosis (CA) are two of the major neuropathological lesions in brains of patients with dementia of the Alzheimer type. We studied the expression of a number of amyloid beta (A beta)-associated proteins in CA, which have previously been identified in senile plaques and which were suggested to play an important role in the pathogenesis of these lesions. Our findings show that involvement of inflammatory components in CA is restricted to activation of the complement system, resulting in deposition of the complement factors C1q, C3c, C4d and the membrane attack complex C5b-9 as well as of the complement inhibitor clusterin. Furthermore, we observed expression of apolipoprotein E, amyloid P component and heparan sulfate proteoglycans in CA, whereas expression of lactoferrin was almost absent. Other inflammatory proteins, known to be present in senile plaques, such as alpha1-antichymotrypsin, alpha2-macroglobulin and intercellular adhesion molecule-1, were absent or detectable only in small amounts. These data suggest that an incomplete inflammatory response occurs in CA as compared to senile plaques. This was confirmed by the finding that the number of cells of the monocyte/macrophage lineage around CA was not increased compared to unaffected vessels. Based on their expression patterns, complement factors, apolipoprotein E and heparan sulfate proteoglycans may be produced early in the process of CA formation and may play an important role in the formation of A beta fibrils in CA. The absence of a number of A beta-associated proteins in CA in comparison to senile plaques is in support of a different pathogenesis for these two lesions.

Aged↗
PubMed1997

Immunohistochemical analysis of integrin alpha vbeta3 expression on tumor-associated vessels of human carcinomas.

Expression of the alpha vbeta3 integrin is upregulated on sprouting endothelia. Systemic application of antibody or peptidic inhibitors of alpha vbeta3 function disrupts tumor angiogenesis and reduces growth and invasiveness of human tumors in animal models. We systematically investigated alpha vbeta3 expression on tumor-associated vessels of 4 different human epithelial tumors and the corresponding normal tissues by means of immunohistochemistry on frozen sections using the alpha vbeta3-complex specific monoclonal antibody LM609. Variable levels of LM609 staining were found in all carcinoma lesions. A considerable number of tumor tissues (35/50) expressed alpha vbeta3 on more than 50% of their vessels. Inflammatory infiltrates and the possibly hypoxic conditions near necrotic areas of tumors were accompanied by an increased alpha vbeta3 expression. Remarkably, the vasculature in apparently normal tissue also stained for alpha vbeta3. However, the percentages of stained vessels and their staining intensity were lower than in neoplastic tissues. Besides the vascular alpha vbeta3 expression, several extravascular cell types stained positive, in both normal and tumor specimens. Taken together, our findings show a considerable number of colon, pancreas, lung and breast carcinoma lesions with many alpha vbeta3-expressing vessels that could be targets for anti-alpha vbeta3-therapy.

Adenocarcinoma↗
PubMed1996

Pathology of malignant melanoma, including new markers and techniques in diagnosis and prognosis.

Although good prognostic markers are already available for patients with cutaneous melanoma, there still is a need for additional markers that are helpful to assess prognosis in individual patients. These so-called progression markers are likely to be found in molecules that play a role in the process of metastasis. Here, emphasis is put on the potential clinical implications of studies on the adhesion molecules from the integrin and CD44 families, proteases of the plasminogen activation system, and components involved in angiogenesis. Based on their differential expression in melanocytic tumor progression, several molecules of these categories appear promising for prognostic and diagnostic purposes. In this respect, the parallelism between key processes in the pathogenesis of metastasis and of angiogenesis is noteworthy. As technical developments in molecular pathology are relevant for diagnostic and prognostic purposes, some recent applications are discussed. An integrated molecular approach in a proper clinicopathologic context is advocated, including proper quality control measures.

Biomarkers, Tumor↗
PubMed1995

VWF release and platelet aggregation in human melanoma after perfusion with TNF alpha.

Twenty-nine stage IIIA/B melanoma patients treated by isolated limb perfusion (ILP) with a high dose of recombinant human tumour necrosis factor alpha (rHuTNF alpha), interferon gamma (IFN gamma), and melphalan were histologically documented with emphasis on therapy-induced changes of the tumour vasculature. Sequential biopsies were taken at various intervals before and after the treatment to compare the morphological change. In order to visualize microvascular changes, immunostaining was performed for von Willebrand factor (VWF), type IV collagen, alpha-smooth muscle actin, endothelial antigen PAL-E, tissue factor, CD41 (thrombocyte marker), and fibrin. In biopsies prior to perfusion, necrosis, haemorrhage, and fibrin thrombi were not found. Within 3 h following triple combination therapy, a change in the distribution of VWF staining occurred, from a discrete endothelial pattern in the untreated lesions to a fuzzy perivascular and subepidermal pattern in the treated lesions. Within 24 h, this was accompanied by intravascular thrombocyte aggregation and erythrostasis, in the absence of tissue factor and fibrin deposits. These findings indicate that the thrombocyte aggregation observed is not caused by local procoagulant activity, but is rather the result of the therapy-associated vascular damage or haemostasis. Although it is difficult to derive the dynamics of this process from static images, we assume that TNF alpha induced endothelial cell damage, leading to VWF release. Release VWF may play a role in the adhesion between thrombocytes and the damaged endothelium or the denuded subendothelium. As a consequence, the blood flow is impaired, leading to congestion and oedema, compatible with an early stage of haemorrhagic infarction.

Chemotherapy, Cancer, Regional Perfusion↗
PubMed1994

Anti-endothelial cell antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis.

In some patients suffering from rheumatoid arthritis (RA), vasculitis is a clear clinical manifestation, mentioned as rheumatoid vasculitis (RV). Autoantibodies directed against endothelial cells (AEA) have been implicated in the pathogenesis of this disorder, and it has been suggested in a number of studies that testing for AEA should be included in diagnosing RV. To test this hypothesis, we have evaluated the presence of AEA in sera of patients suffering from various autoimmune diseases, employing an ELISA with fixed cultured endothelial cells (EC). In all the groups of patients ELISA-positive sera were present. A significant difference in percentage of positivity was found between the RA and RV group (P < 0.05). In addition, our results indicated that not only antibodies directed against antigens on the EC membrane were detected, but also antibodies directed against intracellular components like DNA, histones and cytoskeletal components. Therefore, we also tested all these patient sera on unfixed intact EC using indirect immunofluorescence followed by FACS analysis. Whereas in the total patient population 34 out of 65 patients were AEA-positive as determined in the ELISA, only seven patients were weakly positive when examined by flow cytometry. We conclude that: (i) an ELISA on fixed EC does not specifically detect AEA. A positive test result is, however, to some extent correlated with the occurrence of vasculitis, and may therefore be helpful in diagnosing this disease; (ii) FACS analysis is a more suitable method than ELISA to measure the presence of membrane-specific AEA in patient sera; (iii) specific IgG-AEA are less common in patients suffering from autoimmune disorders than was assumed previously.

Adult↗
PubMed1993

Endothelial cells promote anti-CD3-induced T-cell proliferation via cell-cell contact mediated by LFA-1 and CD2.

T-cell activation requires not only T-cell receptor (TCR) engagement and subsequent TCR/CD3 cross-linking, but also one or more secondary activation signals generated by accessory cells (AC). We investigated the accessory function of endothelial cells (EC) in an in vitro model for T-cell activation where the first cross-linking signal was delivered to peripheral human T lymphocytes by either immobilized anti-CD3 monoclonal antibody (MoAb) or by PHA. In a previous report, we showed that EC provided a potent costimulatory signal in this model system. We have now analysed the nature of the EC-derived costimulatory signal by testing whether EC could be substituted by cytokines, by studying the effect of EC fixation and by testing the involvement of a number of adhesion molecules. Our findings indicate that EC accessory function is mediated mainly by membrane-bound factors. The nature of these membrane-bound factors was analysed by studying the inhibitory properties of a series of MoAbs directed against several adhesion molecules. Antibodies directed against CD44, E-selectin, CD31, CD26, B7/BB1, VLA-4 or VCAM-1 were not inhibitory. However, an inhibition, was clearly observed with antibodies against LFA-1 and CD2. Remarkably, this inhibition was not found with MoAbs to their respective counterstructures ICAM-1 and LFA-3. In summary, we postulate that both LFA-1/ICAM-1, and CD2/LFA-3 interactions are involved in EC accessory function, although the role of the EC-associated adhesion partners is not fully understood.

Antibodies, Monoclonal↗
PubMed1993

Angiogenesis in wound healing and tumor metastasis.

Formation of new blood vessels is essential for several physiological and pathological events, e.g. embryogenesis, wound healing and tumor growth and metastasis. In order to increase the insight into the mechanisms of angiogenesis we have visualized the different components of the microvasculature in human wounds and tumors by immunohistochemistry on the light and electronmicroscopic level. For this purpose, antibodies recognizing distinct markers for human endothelial cells, pericytes and basal lamina were used on freshly frozen or paraformaldehyde-fixed tissue samples. In terms of efficacy, the PAL-E antigen is highly specific for blood vessel endothelium. Its sensitivity is less than other endothelial markers, such as von Willebrand factor and CD 31, as it is not expressed in arterioles. Within the context of the microvasculature alpha-smooth muscle actin and the HMW-MAA chondroitin sulphate proteoglycan are useful markers for pericytes. Type IV Collagen and Laminin can be visualized consistently in the microvascular basal lamina. During the formation of granulation tissue in wound healing a heterogeneity of the expression of endothelial and pericyte markers is found. In the least matured zone in granulation tissue of decubitus lesions and experimental skin wounds microvessels already contained both endothelial cells and pericytes, suggesting a role for both cell types in the early steps of angiogenesis. Regarding the tumor microvasculature, antibodies to von Willebrand factor often failed to stain capillaries, that did show expression of the other endothelial markers studied. Broad staining in pericytes was found for the HMW-MAA chondroitin sulphate proteoglycan. In contrast, these cells only locally expressed alpha-smooth muscle actin. Staining of the basal lamina components Type IV Collagen and Laminin within tumors was not restricted to the microvasculature. Therefore, antibodies recognizing endothelial markers, particularly PAL-E and BMA 120, are preferable as tools to visualize the tumor microvasculature. In accordance with the situation in granulation tissue of wound healing the broad presence of pericytes in the microvasculature of human tumor suggests an involvement of this cell type in tumor angiogenesis. Recent immunohistochemical studies on human tumor lesions indicated that a high number of microvessels adjacent to the tumor as a measure of tumor angiogenesis is an unfavorable prognostic factor in cutaneous melanoma, mammary carcinoma and non-small cell pulmonary carcinoma. This new application of immunohistochemistry represents a valuable, clinically relevant adjunct to the repertoire of the surgical pathologist.

Animals↗
PubMed1993

Involvement of LFA-1/ICAM and CD2/LFA-3 in human endothelial cell accessory function.

Interactions of endothelial cells with T cells occur during the process of lymphocyte migration and during leukocyte extravasation in the course of an inflammatory response. Adhesion molecules are crucially involved in these interactions. It has been assumed that, in this way, endothelial cells play an important role in the recruitment of immune cells to sites of inflammation. In vitro experiments indicate that, in addition to this function in leukocyte recirculation, endothelial cells might also be involved as accessory cells in the stimulation of T cells. In this overview, the possible role of adhesion molecules in this process will be discussed.

Antigens, CD↗
PubMed1992

Accessory function of endothelial cells in anti-CD3-induced T-cell proliferation: synergism with monocytes.

Monoclonal antibodies to CD3 can induce proliferation of resting T cells. In vitro this effect is dependent on the presence of monocytes. They serve as accessory cells providing a co-stimulatory signal after cross-linking of the antibody-coated TcR/CD3 complex by the Fc receptor on the monocytes. We have studied whether endothelial cells can replace monocytes with regard to this function. Highly purified T-cell preparations were cultured in the presence of anti-CD3 antibody, purified monocytes, and human umbilical vein endothelial cells. Anti-CD3 and endothelial cells alone were unable to support T-cell proliferation, due to lack of FcR expression. Addition, however, of as few as 1000 FcR+ monocytes (0.8% of the number of T cells present) to a coculture of T cells and endothelial cells (EC) in the presence of soluble anti-CD3 resulted in a strong proliferation of T cells. When anti-CD3 was presented in an immobilized form (coated to the culture well or to Sepharose beads), or when phytohaemagglutinin was added to the culture as a cross-linking agent, EC could support T-cell proliferation in the absence of any monocytes. We conclude that EC by themselves cannot support the proliferation of pure T cells induced by soluble anti-CD3, but are potent generators of the co-stimulatory signal(s). They provide a suitable starting material to further define this co-stimulatory activity.

Antigen-Presenting Cells↗
PubMed1988

An improved sensitive and simple microassay of mouse complement.

A simple and fast hemolytic microassay was developed for the determination of classical pathway complement activity in mouse serum. The assay is based on hemolysis of sheep red blood cells (SRBC) that are sensitized with polyclonal mouse antibodies. The degree of hemolysis was measured in the reaction supernatants by photometric reading in an ELISA plate scanner at 405 nm wavelength. It was found that some batches of unpurified mouse anti-SRBC antibodies gave insufficient hemolysis. Analysis of two antibody preparations indicated that this might be caused by anti-complementary factors in the ascites fluid, and by an excess of non-complement fixing IgG1 antibodies. For optimal and standardized results, removal of anticomplementary factors and enrichment for complement fixing IgG2 antibodies was required and was achieved using protein A purified anti-SRBC IgG. In our assay it is possible to determine CH50 titers in triplicate in 80 microliters samples of individual mouse sera with high sensitivity. Using this rapid one-step method large numbers of tests could be performed in 1 day.

Animals↗
PubMed1977

Procedure for the isolation of mouse anti H-2 antibodies from complex alloantisera.

B6AF1-anti-B10.D2 ascites fluid was incubated with donor strain red blood cells to absorb the anti-H-2 antibodies. The antibody-coated RBC were then lysed, and from the ghosts the antibodies were eluted. This anti-H-2 preparation was further purified by affinity chromatography, using an immobilized anti IgG2 antiserum. Thus a pure anti-H-2 IgG2 was obtained for in vitro and in vivo testing.

Animals↗