Robert M.W. de Waal

Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

Antiangiogenic therapy of cerebral melanoma metastases results in sustained tumor progression via vessel co-option

Purpose: In the brain, tumors may grow without inducing angiogenesis, via co-option of the dense pre-existent capillary bed. The purpose of this study was to investigate how this phenomenon influences the outcome of antiangiogenic therapy. Experimental Design: Mice carrying brain metastases of the human, highly angiogenic melanoma cell line Mel57-VEGF-A were either or not treated with different dosages of ZD6474, a vascular endothelial growth factor (VEGF) receptor 2 tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor. Effect of treatment was evaluated using contrast-enhanced magnetic resonance imaging (CE- MRI) and (immuno)morphologic analysis. Results: Placebo-treated Mel57-VEGF-A brain metastases evoked an angiogenic response and were highlighted in CE-MRI. After treatment with ZD6474 (100 mg/kg), CE-MRI failed to detect tumors in either prevention or therapeutic treatment regimens. However, (immuno)histologic analysis revealed the presence of numerous, small, nonangiogenic lesions. Treatment with 25 mg/kg ZD6474 also resulted in efficient blockade of vessel formation, but it did not fully inhibit vascular leakage, thereby still allowing visualization in CE-MRI scans. Conclusions: Our data show that, although angiogenesis can be effectively blocked by ZD6474, in vessel-dense organs this may result in sustained tumor progression via co-option, rather than in tumor dormancy. Importantly, blocking VEGF-A may result in undetectability of tumors in CE-MRI scans, leading to erroneous conclusions about therapeutic efficacy during magnetic resonance imaging follow-up. The maintenance of VEGF-A-induced vessel leakage in the absence of neovascularization at lower ZD6474 doses may be exploited to improve delivery of chemotherapeutic agents in combined treatment regimens of antiangiogenic and chemotherapeutic compounds.

Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders

Proteoglycans are associated with all kinds of amyloid deposits in the human body. These complex macromolecules, in particular heparan sulphate proteoglycans, have also been implicated in several features of the pathogenesis of Alzheimers disease (AD), including the genesis of senile plaques, cerebrovascular amyloid, and neurofibrillary tangles. In this review we focus on the role of proteoglycans and glycosaminoglycans in amyloidogenesis in general and in AD in particular. Heparan sulphate proteoglycans may promote amyloid-beta peptide (Abeta) or tau fibrillisation on the one hand, and provide resistance against proteolytic breakdown on the other. Knowledge about the role of proteoglycans in AD pathology may eventually be of therapeutic use, because small polysulphated compounds, which can interfere with the interaction between proteoglycan and Abeta, have been shown to stop or even prevent amyloidogenesis.

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. Int. J. Cancer 86:768–776, 2000.

Agrin Is a Major Heparan Sulfate Proteoglycan Accumulating in Alzheimer's Disease Brain

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimers type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.

Outgrowth of human liver metastases after resection of the primary colorectal tumor: a shift in the balance between apoptosis and proliferation.

Rapid outgrowth of metastases after removal of the primary tumor has been described in several mouse models. Loss of primary tumor‐induced inhibition of angiogenesis in the metastases has been suggested as the underlying cause. Accordingly, we recently demonstrated that vascular density in human colorectal liver metastases increases after primary tumor resection. Here, we investigate whether this increase in vascular density has, in its turn, effects on the tumor growth of the liver metastases. We analyzed tumor growth in synchronous liver metastases from patients with the primary tumor in place, in synchronous metastases from patients with the primary tumor resected and in metachronous metastases. Tumor growth was studied by assessing the percentage of cells undergoing apoptosis by activated caspase‐3 staining, and the percentage of proliferating cells by Ki‐67 staining. While the percentage of proliferating cells within the metastases showed a modest increase after primary tumor resection, a significant decrease in the percentage of apoptotic cells was observed. Taken together, an increased net tumor growth of the metastases occurred after primary tumor resection. This acceleration of tumor growth could be confirmed by studying biopsies taken from the same patient before and after tumor resection. Our data show that in human cancer patients, a primary tumor may inhibit the growth of its liver metastases.

Lymphangiogenesis in malignant tumours: Does it occur?

The development of a vascular bed is essential for solid tumour growth and metastasis. In many tumours, mean vascular density can be related to the rate of metastasis and, therefore, to prognosis. In other tumour types, such as cutaneous melanoma and head‐and‐neck squamous cell carcinoma, this relation is absent. Until now, the reason for this has been unclear, but since these particular tumour types are also known for their propensity to spread via the lymphatic system, it may be speculated that the presence of a pre‐existing lymphatic bed and the formation of new lymphatics (lymphangiogenesis) are important factors. Growth factors involved in lymphangiogenesis during embryogenesis have been recently identified and these are also expressed in many tumour types, but the existence of tumour‐induced lymphangiogenesis has not so far been reported. Partly, this could be due to the lack of reliable endothelial markers, thereby hampering a consistent evaluation of lymphatic vasculature. This editorial discusses the role of the lymphatic bed in mediating the metastasis of solid tumours, summarizes known methods to detect lymphatics, and proposes a hypothetical mechanism of tumour‐induced lymphangiogenesis. Copyright

Heat shock proteins and amateur chaperones in amyloid-Beta accumulation and clearance in Alzheimer's disease.

The pathologic lesions of Alzheimer’s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-β (Aβ) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. “Professional chaperones”, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. “Amateur” chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of Aβ, and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood–brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention.

Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

Alzheimer’s disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-β precursor protein (APP), the amyloid-β peptide (Aβ). CAA is also the major pathological lesion in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), caused by a mutation in the gene coding for the Aβ peptide. Several members of the small heat shock protein (sHsp) family, such as αB-crystallin, Hsp27, Hsp20 and HspB2, are associated with the pathological lesions of AD, and the direct interaction between sHsps and Aβ has been demonstrated in vitro. HspB8, also named Hsp22 of H11, is a recently discovered member of the sHsp family, which has chaperone activity and is observed in neuronal tissue. Furthermore, HspB8 affects protein aggregation, which has been shown by its ability to prevent formation of mutant huntingtin aggregates. The aim of this study was to investigate whether HspB8 is associated with the pathological lesions of AD and HCHWA-D and whether there are effects of HspB8 on Aβ aggregation and Aβ-mediated cytotoxicity. We observed the expression of HspB8 in classic SPs in AD brains. In addition, HspB8 was found in CAA in HCHWA-D brains, but not in AD brains. Direct interaction of HspB8 with Aβ1–42, Aβ1–40 and Aβ1–40 with the Dutch mutation was demonstrated by surface plasmon resonance. Furthermore, co-incubation of HspB8 with D-Aβ1–40 resulted in the complete inhibition of D-Aβ1–40-mediated death of cerebrovascular cells, likely mediated by a reduction in both the β-sheet formation of D-Aβ1–40 and its accumulation at the cell surface. In contrast, however, with Aβ1–42, HspB8 neither affected β-sheet formation nor Aβ-mediated cell death. We conclude that HspB8 might play an important role in regulating Aβ aggregation and, therefore, the development of classic SPs in AD and CAA in HCHWA-D.

Heparan sulfate proteoglycan expression in cerebrovascular amyloid β deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains

Abstract.Cerebrovascular deposition of amyloid β protein (Aβ) is a characteristic lesion of Alzheimers disease (AD) and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). Besides Aβ, several other proteins and proteoglycans accumulate in cerebral amyloid angiopathy (CAA). We have now analyzed the expression of the heparan sulfate proteoglycan (HSPG) subtypes agrin, perlecan, glypican-1, syndecans 1–3 and HS glycosaminoglycan (GAG) side chains in CAA in brains of patients with AD and HCHWA-D. Hereto, specific well-characterized antibodies directed against the core protein of these HSPGs and against the GAG side chains were used for immunostaining. Glypican-1 was abundantly expressed in CAA both in AD and HCHWA-D brains, whereas perlecan and syndecans-1 and -3 were absent in both. Colocalization of agrin with vascular Aβ was clearly observed in CAA in HCHWA-D brains, but only in a minority of the AD cases. Conversely, syndecan-2 was frequently associated with vascular Aβ in AD, but did not colocalize with vascular Aβ deposits in HCHWA-D. The three different syndecans, agrin, glypican-1 and HS GAG, but not perlecan, were associated with the majority of senile plaques (SPs) in all brains. Our results suggest a role for agrin in the formation of SPs and of CAA in HCHWA-D, but not in the pathogenesis of CAA in AD. Both syndecan-2 and glypican, but not perlecan, may be involved in the formation of CAA. We conclude that specific HSPG species may be involved in the pathogenesis of CAA in both AD and HCHWA-D, and that the pathogenesis of CAA and SPs may differ with regard to the involvement of HSPG species.