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Biomedical subjects

A W Griffioen

Publications and source records attributed to A W Griffioen.

At least 19 recordsLinked to original sources
PubMed2006

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium.

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.

Adult↗
PubMed2004

Dynamic contrast-enhanced magnetic resonance imaging at 1.5 Tesla with gadopentetate dimeglumine to assess the angiostatic effects of anginex in mice.

The purpose of this study was to evaluate the effects of anginex on tumour angiogenesis assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) on a clinical 1.5 Tesla MR system and with the clinically available contrast agent gadopentetate dimeglumine. C57BL/6 mice carrying B16F10 melanomas were treated with anginex, TNP-470 or saline. Tumour growth curves and microvessel density (MVD) were recorded to establish the effects of treatment. DCE-MRI was performed on day 16 after tumour inoculation, and the endothelial transfer coefficients of the microvessel permeability surface-area product (K(PS)) were calculated using a two-compartment model. Both anginex and TNP-470 resulted in smaller tumour volumes (P<0.0001) and lower MVD (P <0.05) compared to saline. Treatment with anginex resulted in a 64% reduction (P<0.01) of tumour K(PS) and TNP-470 resulted in a 44% reduction (P=0.17), compared to saline. DCE-MRI with a clinically available, small-molecular contrast agent can therefore be used to evaluate the angiostatic effects of anginex and TNP-470 on tumour angiogenesis.

Angiogenesis Inhibitors↗
PubMed2002

Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment.

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.

Cell Count↗
PubMed2001

Anginex, a designed peptide that inhibits angiogenesis.

Novel beta-sheet-forming peptide 33-mers, betapep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences from the beta-sheet domains of anti-angiogenic proteins. One of these designed peptides (betapep-25), named anginex, was observed to be potently anti-angiogenic. Anginex specifically inhibits vascular endothelial cell proliferation and induces apoptosis in these cells, as shown by flow-cytometric detection of sub-diploid cells, TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-nick-end labelling) analysis and cell morphology. Anginex also inhibits endothelial cell adhesion to and migration on different extracellular matrix components. Inhibition of angiogenesis in vitro is demonstrated in the sprout-formation assay and in vivo in the chick embryo chorio-allantoic membrane angiogenesis assay. Comparison of active and inactive betapep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This is the first report of a designed peptide having a well-defined biological function as a novel cytokine, which may be an effective anti-angiogenic agent for therapeutic use against various pathological disorders, such as neoplasia, rheumatoid arthritis, diabetic retinopathy and restenosis.

Amino Acid Sequence↗
PubMed2001

Designed beta-sheet peptides that inhibit proliferation and induce apoptosis in endothelial cells.

Novel beta-sheet-forming peptide 33 mers, beta pep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences or proposed key residues from the beta-sheet domains of interleukin-8 (IL-8), platelet factor-4 (PF4) and bactericidal/permeability increasing protein (B/PI). Since PF4 and B/PI are anti-angiogenic and IL-8 is angiogenic, the library of 30 beta pep peptides was assayed for the ability to affect the growth of endothelial cells. Results indicate that five beta pep peptides (beta pep-2, 7, 8, 21 and 25) demonstrate greater than 50% anti-proliferative activity at 30 micrograms/ml, and one of those (beta pep-25) is similarly active at 10 micrograms/ml. Insight into the mechanism of action was probed in an apoptosis assay. Anti-proliferative activity was found to be correlated with the induction of apoptosis. For example, at 100 micrograms/ml beta pep-25 induces 85% of endothelial cells to undergo apoptosis within 2 days. These effects from beta pep peptides appear to be selective for endothelial cell (EC) because normal cells (fibroblasts and leukocytes) and various tumor cells are not significantly affected at peptide concentrations used in this study. Comparison of active and inactive beta pep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This research contributes to the development of novel anti-angiogenic peptides.

Amino Acid Sequence↗
PubMed2001

Angiostatic proteins and peptides.

Angiogenesis, or the formation of new vasculature out of preexisting capillaries, is a sequence of events that is essential in the normal physiological processes of tissue growth and in a broad spectrum of pathologies. The diseases in which angiogenesis plays a key role are divided into diseases that are characterized by hypoxia/ ischemia and diseases that are dependent on neovascularization. The formerpathologies may benefit from therapeutic angiogenesis stimulation. This review concentrates on the different strategies to inhibit angiogenesis in diseases that are characterized by excessive angiogenesis, for example, cancer, arthritis, diabetic retinopathy, and inflammatory diseases. These diseases are dependent on the development of newvasculature, and hence, a large variety of different strategies to inhibit angiogenesis are underwayin laboratories throughout the world. At present, over250 angiogenesis inhibitors are described, and approximately half of them display activity in in vivo models. A large percentage of these molecules are natural, nonnatural, or synthetic so-called small molecules. Others are of protein origin, either endogenous or exogenous by nature. The authors highlight the current knowledge on the development of angiostatic proteins and peptides and their potential in the treatment of disease.

Angiogenesis Inhibitors↗
PubMed2000

Bactericidal/permeability-increasing protein (BPI) inhibits angiogenesis via induction of apoptosis in vascular endothelial cells.

Bactericidal/permeability-increasing protein (BPI) has been known for some time to function in killing bacteria and in neutralizing the effects of bacterial endotoxin lipopolysaccharide. In the present study, BPI is found to be a novel endogenous inhibitor of angiogenesis. Within the sub-muM range, BPI shows a concentration-dependent inhibition of endothelial cell (EC) proliferation that is mediated by cell detachment and subsequent induction of apoptosis. As measured by flow cytometric analysis of the percentage of subdiploid cells, apoptosis induction was half-maximal at about 250 nmol/L BPI. Apoptosis was confirmed by quantification of cells with nuclear fragmentation. Apoptosis was found to be EC specific. In an in vitro collagen gel-based angiogenesis assay, BPI at 1.8 micromol/L inhibited tube formation by 81% after only 24 hours. Evidence for in vivo inhibition of angiogenesis was obtained, using the chorioallantoic membrane assay in which BPI was seen to be significantly effective at concentrations as low as 180 nmol/L. This newly discovered function of BPI might provide a possible therapeutic modality for the treatment of various pathologic disorders that depend on angiogenesis.

Allantois↗
PubMed2000

Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth.

Ovarian cancer is the leading cause of fatality among gynecological malignancies. Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease. Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system. Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast. Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice. These studies showed that both angiostatin and endostatin inhibited tumor growth. However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments. Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells. Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated. In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited. Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein. These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.

Angiostatins↗
PubMed2000

Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.

Tumor growth is angiogenesis dependent. As a consequence, strategies aimed at disrupting this mechanism are heavily investigated. Several angiogenesis assays are used to directly compare the efficacy of anti-angiogenic compounds. However, objective assessment of new vascular growth has been difficult to achieve. The aim of this study was to test and develop a computer-assisted image analysis method that would give an unbiased quantification of the microvessel density. Human tumors were grown in athymic mice and tumor biopsies were taken after a weeklong treatment with VEGF-toxin conjugate. Frozen tumor sections were prepared and stained with PE-conjugated anti-CD-31 antibodies and vessels were imaged with a fluorescence microscope. Vessel density was analyzed by quantifying PE-positive pixels per recorded field. In addition, images were further processed to investigate morphological differences by an automated binarization and skeletonization protocol. This procedure allowed the computer-assisted estimation of important angiogenic parameters such as total vessel number, length, and branch points. Based on these indices, differences in the angiogenic response between control tumors and those treated with VEGF-toxin conjugate were readily detected (P < 0.007 for all parameters). More importantly, computer-generated measurements correlated well with manual microvessel counts and showed significantly less variation. Our results suggest that computer-assisted image analysis represents a rapid, objective, and alternative method for the quantitative assessment of tumor angiogenesis and vessel architecture.

Adenocarcinoma↗
PubMed2000

Tumor angiogenesis factors reduce leukocyte adhesion in vivo.

Leukocyte-endothelium interactions are diminished in tumors. It is reported here that, in a tumor-free in vivo model, angiogenic factors can down-regulate leukocyte adhesion to endothelium. Slow releasing pellets were loaded with either basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF) or vehicle alone and were placed in the scrotum of mice. After 3 days, a single intrascrotal injection of 1 microg/kg IL-1beta was given 4 h before vessels of the cremaster muscle were investigated for leukocyte rolling and adhesion by means of intravital microscopy. Exposure of normal tissue to either bFGF or VEGF resulted in markedly decreased levels of cytokine-induced leukocyte adhesion. Suppression of leukocyte rolling was not observed. Instead a moderate enhancement of rolling by VEGF was found. The observed differences could not be explained by differences in fluid dynamic parameters or systemic leukocyte counts. In conclusion, evidence is presented that, in vivo, angiogenic factors significantly reduce leukocyte adhesion, the final step preceding leukocyte infiltration. This observation may explain why tumors escape from immune surveillance.

Angiogenesis Inducing Agents↗
PubMed2000

Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.

Angiogenesis, or the formation of new blood vessels out of pre-existing capillaries, is a sequence of events that is fundamental to many physiologic and pathologic processes such as cancer, ischemic diseases, and chronic inflammation. With the identification of several proangiogenic molecules such as the vascular endothelial cell growth factor, the fibroblast growth factors (like in FGFs), and the angiopoietins, and the recent description of specific inhibitors of angiogenesis such as platelet factor-4, angiostatin, endostatin, and vasostatin, it is recognized that therapeutic interference with vasculature formation offers a tool for clinical applications in various pathologies. Whereas inhibition of angiogenesis can prevent diseases with excessive vessel growth such as cancer, diabetes retinopathy, and arthritis, stimulation of angiogenesis would be beneficial in the treatment of diseases such as coronary artery disease and critical limb ischemia in diabetes. In this review we highlight the current knowledge on angiogenesis regulation and report on the recent findings in angiogenesis research and clinical studies. We also discuss the potentials, limitations, and challenges within this field of research, in light of the development of new therapeutic strategies for diseases in which angiogenesis plays an important role.

Angiogenesis Inhibitors↗
PubMed2000

AG-3340 (Agouron Pharmaceuticals Inc).

Agouron Pharmaceuticals is developing AG-3340 (prinomastat), the lead compound in a series of structurally related metalloproteinase inhibitors, for the potential treatment of cancer and age-related macular degeneration. AG-3340, an oral, non-peptide inhibitor of gelatinase types A and B (MMP-2 and -9), MT1-MP (MMP-14) and collagenase III [234058], was selected following demonstration of activity in a variety of in vivo preclinical models upon oral dosing. In May 1999, phase III trials for lung and prostate cancers of AG-3340 in front-line combination with chemotherapy was begun in the US and Canada [286380,326640]. The tested dose for these trials is 5 to 15 mg bid. Following demonstration of the enhanced efficacy of chemotherapy when supplemented with AG-3340 in preclinical tumor models, pilot combination studies and double-blinded, placebo-controlled phase III trials in 700 patients are in progress for the treatment of non-small cell lung cancer or advanced hormone-refractory prostate cancer [302584,327014]. In August 1999, Agouron initiated a second, confirmatory phase III trial of AG-3340 in combination with chemotherapy in patients with advanced non-small cell lung cancer [337253]. Pharmacokinetic studies have been conducted in healthy male volunteers and single agent dose-escalation studies in patients demonstrated toxicities (grade 1 or 2; joint related) were not doselimiting [302238]. At the 10th European Organization for Research and Treatment of Cancer (EORTC) meeting in Amsterdam (June 1998), Agouron released encouraging results from two phase I studies and one preclinical study of AG-3340 [289688]. In a further 15- patient, phase I study of AG-3340 with paclitaxel and carboplatin, the combination was safe and well tolerated [326268]. AG-3340 has demonstrated significant antimetastatic and antitumor activity in animal models, as well as oral bioavailability and a favorable pharmacokinetic profile. Daily doses of 50 mg/kg completely halted growth of Lewis lung carcinomas in two thirds of test animals and reduced the formation of lung metastasis (0.5 mm in diameter) by 90% [205708]. In December 1997, Roche announced it would discontinue its cancer R&D collaborations with Agouron on AG-3340 as it did not fulfill its scientific and business objectives [271723]. Agouron is investigating analogs of AG-3340 which are undergoing animal studies [332841].

Journal Article↗
PubMed1999

[Angiogenesis and the regulation of antitumor defense].

Formation of new blood vessels is a prerequisite for outgrowth of solid tumours and metastasis. Leukaemia and lymphoma are also dependent on angiogenesis. Inhibition of angiogenesis is therefore a promising therapy for all cancer types. Angiogenic factors reduce the expression of tumour endothelial adhesion molecules for leukocytes, which enables tumours to escape the inflammation response. Antiangiogenic factors induce not only starvation of the tumour owing to deprivation of vasculature, but also re-expression of adhesion molecules, resulting in increased leukocyte infiltration. On the basis of de novo design of chemokines with antiangiogenic properties, novel inhibitors of angiogenesis are developed and selected for their ability to induce a tumour inflammatory reaction.

Angiogenesis Inducing Agents↗
PubMed1999

Angiogenesis inhibitors overcome tumor induced endothelial cell anergy.

We report here that tumor angiogenesis-mediated endothelial cell (EC) anergy can be overcome by inhibitors of angiogenesis. We found previously that tumor growth, known to be dependent on angiogenesis, results in down-regulation of endothelial adhesion molecules and tumor EC anergy to inflammatory signals. We hypothesized that counteracting angiogenesis induces re-expression of adhesion molecules and normalizes responses to inflammatory cytokines. Here, we present data to show that the angiogenesis inhibitor platelet factor-4 (PF4) is able to prevent basic fibroblast growth factor (bFGF)-induced down-regulation of intercellular adhesion molecule-1 (ICAM-1). Furthermore, PF4 restores ICAM-1 expression following bFGF-induced down-regulation of ICAM-1. This PF4 effect occurs at the protein level and the RNA level and it has functional impact on leukocyte adhesion. In addition, PF4 overcomes the tumor-induced EC anergy to inflammatory signals such as tumor necrosis factor alpha (TNF alpha). Our findings may be the basis of new cancer therapies by combining anti-angiogenic therapy and immunotherapy to decrease blood vessel formation and to increase the effectiveness of inflammatory reactions against tumors.

Animals↗
PubMed1998

Angiogenic potential of malignant and non-malignant human breast tissues in an in vivo angiogenesis model.

Tumors need to acquire an angiogenic phenotype for outgrowth and metastasis formation. Limited information on the angiogenic potential of specific tissues, especially human breast tissues is available. Here we describe an in vivo model, using the dorsal skin fold chamber in immunodeficient nude mice, where various tissues of human breast origin were xenografted and evaluated for their angiogenesis-inducing potential. We found that angiogenesis was abundantly induced by all breast carcinoma tissue samples. Similar angiogenesis was induced by tissue samples from breasts with hyperplasia and apocrine metaplasia. Histologically normal tissues adjacent to the tumor induced angiogenesis in 66% of the cases. Angiogenesis was not induced by control tissues from normal healthy breasts, obtained after cosmetic breast reduction. Angiogenesis induction parallelled VEGF production by the tumor cells. The tissue induced neovascularization, found both around and in the human tissue, was functional since a tail vein injection of albumin-FITC revealed positive tumor microcirculation within 5 min, while the tumor tissue still consisted of vital human epithelial cells after 14 days.

Animals↗
PubMed1998

Effect of culture conditions on endothelial cell growth and responsiveness.

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions.

Apoptosis↗