Limitations of the hCMEC/D3 cell line as a model for Aß clearance by the human blood-brain barrier.

Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-ß (Aß) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Aß clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aß clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aß was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aß. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aß clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Inhibition of α-synuclein aggregation by small heat shock proteins.

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.

Small heat shock proteins induce a cerebral inflammatory reaction.

More than 80% of Alzheimer's disease (AD) patients have some degree of cerebral amyloid angiopathy (CAA). In addition to arteries and veins, capillaries can also be affected. Capillary CAA (capCAA), rather than CAA in larger vessels, is associated with flame-like amyloid-beta (Aß) deposits that may extend beyond the vessel wall and radiate into the neuropil, a phenomenon also known as "dyshoric angiopathy." Aß deposits in AD, parenchymal as well as (cap)CAA and dyshoric angiopathy, are associated with a local inflammatory reaction, including activation of microglial cells and astrocytes that, among others, produce cytokines and reactive oxygen species. This neuroinflammatory reaction may account for at least part of the cognitive decline. In previous studies we observed that small heat shock proteins (sHsps) are associated with Aß deposits in AD. In this study the molecular chaperones Hsp20, HspB8 and HspB2B3 were found to colocalize with CAA and capCAA in AD brains. In addition, Hsp20, HspB8 and HspB2B3 colocalized with intercellular adhesion molecule 1 (ICAM-1) in capCAA-associated dyshoric angiopathy. Furthermore, we demonstrated that Hsp20, HspB8 and HspB2B3 induced production of interleukin 8, soluble ICAM-1 and monocyte chemoattractant protein 1 by human leptomeningeal smooth muscle cells and human brain astrocytes in vitro and that Hsp27 inhibited production of transforming growth factor beta 1 and CD40 ligand. Our results suggest a central role for sHsps in the neuroinflammatory reaction in AD and CAA and thus in contributing to cognitive decline.

Do amyloid ß-associated factors co-deposit with Aß in mouse models for Alzheimer's disease?

Senile plaques and cerebral amyloid angiopathy in Alzheimer's disease (AD) patients not only consist of the amyloid-ß protein (Aß), but also contain many different Aß-associated factors, such as heparan sulfate proteoglycans, apolipoproteins, and complement factors. These factors may all influence Aß deposition, aggregation, and clearance and therefore seem important in the development of human Aß deposits. To study AD pathology and test new therapeutic agents, many different mouse models have been created. By transgenic expression of the amyloid-ß protein precursor, frequently in combination with other transgenes, these animals develop Aß deposits that morphologically resemble their human counterparts. Whether this resemblance also applies to the presence of Aß-associated factors is largely unclear. In this review, the co-deposition of factors known to associate with human Aß deposits is summarized for several different AD mouse models.

Enoxaparin treatment administered at both early and late stages of amyloid ß deposition improves cognition of APPswe/PS1dE9 mice with differential effects on brain Aß levels.

Enoxaparin (Enox), a low molecular weight heparin, has been shown to lower brain amyloid beta (A beta) load in a mouse model for Alzheimer's disease. However, the effect of Enox on cognition was not studied. Therefore, we examined the effect of peripheral Enox treatment on cognition and brain A beta levels in the APPswe/PS1dE9 mouse model by giving injections at an early (starting at 5 months of age) and late (starting at 10 and 12 months of age) stage of A beta accumulation for 3 months. Although Enox had no effect on behaviour in the open field at any age, it improved spatial memory in the Morris water maze in 5-, 10- and 12-month-old mice. Furthermore, Enox treatment seemed to decrease guanidine HCl-extracted brain A beta levels at 5 months of age, but significantly increased guanidine HCl-extracted A beta 42 and A beta 40 levels in both 10- and 12-month-old mice. In vitro, Enox increased aggregation of A beta, even when A beta was pre-aggregated. In conclusion, Enox treatment, either at an early or a late stage of A beta accumulation, could improve cognition in APPswe/PS1dE9 mice. However, since Enox treatment at an early stage of A beta accumulation decreased guanidine HCl-extracted A beta levels and Enox treatment at a late stage enhanced guanidine HCl-extracted A beta levels, it seems that Enox influences A beta deposition differently at different stages of A beta pathology. In any case, our study suggests that enoxaparin treatment has potential as a therapeutic agent for Alzheimer's disease.

Sulfation of heparan sulfate associated with amyloid-beta plaques in patients with Alzheimer's disease.

Alzheimer's disease (AD) is characterized by pathological lesions such as amyloid-beta (Abeta) plaques and cerebral amyloid angiopathy. Both these lesions consist mainly of aggregated Abeta protein and this aggregation is affected by macromolecules such as heparan sulfate (HS) proteoglycans. Previous studies demonstrated that HS enhances fibrillogenesis of Abeta and that this enhancement is dependent on the degree of sulfation of HS. In addition, it has been reported that these sulfation epitopes do not occur randomly but have a defined tissue distribution. Until now, the distribution of sulfation epitopes of HS has not yet been studied in human brain. We investigated whether a specific HS epitope is associated with Abeta plaques by performing immunohistochemistry on occipital neocortical and hippocampal tissue sections from AD patients using five HS epitope-specific phage display antibodies. Antibodies recognizing highly N-sulfated HS demonstrated the highest level of staining in both fibrillar Abeta plaques and non-fibrillar Abeta plaques, whereas antibodies recognizing HS regions with a lower degree of N-sulfate modifications were only immunoreactive with fibrillar Abeta plaques. Thus, our results suggest that a larger variety of HS epitopes is associated with fibrillar Abeta plaques, but the HS epitopes associated with non-fibrillar Abeta plaques seem to be more restricted, selectively consisting of highly N-sulfated epitopes.

Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Abeta40 (DAbeta(1-40)) are modulated by sulfate moieties of heparin.

Glycosaminoglycans (GAGs), in particular as part of heparan sulfate proteoglycans, are associated with cerebral amyloid angiopathy (CAA). Similarly, GAGs are also associated with the severe CAA found in patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), where the amyloid beta (Abeta) peptide contains the Dutch mutation (DAbeta(1-40)). This suggests a role for GAGs in vascular Abeta aggregation. It was the aim of this study to investigate the effect of different GAGs (heparin, chondroitin sulfate, heparan sulfate), the macromolecule dextran sulfate and, using desulfated heparins, the role of GAG sulfate moieties on the in vitro aggregation of CAA-associated DAbeta(1-40) and on DAbeta(1-40)-induced toxicity of cultured cerebrovascular cells. We also aimed to study the in vivo distribution of various sulfated heparan sulfate GAG epitopes in CAA. Of all GAGs tested, heparin was the strongest inducer of aggregation of DAbeta(1-40) in the different aggregation assays, with both heparin and heparan sulfate reducing Abeta-induced cellular toxicity. Furthermore, (partial) removal of the sulfate moieties of heparin partially abolished the effects of heparin on aggregation and cellular toxicity, suggesting an essential role for the sulfate moieties in heparin. Finally, we demonstrated the in vivo association of sulfated heparan sulfate (HS) GAGs with CAA. We conclude that sulfate moieties within GAGs, like heparin and HS, have an important role in Abeta aggregation in CAA and in Abeta-mediated toxicity of cerebrovascular cells.

Apolipoprotein E protects cultured pericytes and astrocytes from D-Abeta(1-40)-mediated cell death.

Cerebral amyloid angiopathy (CAA) is a common pathological finding in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type; in this latter condition it is caused by deposition of mutated amyloid beta protein (Abeta Glu22Gln; D-Abeta(1-40)). Previously, we found a dependence of the Abeta-mediated toxicity and apolipoprotein E (apoE) production by cultured pericytes on apoE genotype. Given their close association with the cerebrovascular wall both astrocytes and pericytes may be involved in CAA development, a process that includes Abeta deposition and clearance and that may be affected by interaction with locally produced apolipoprotein E (apoE). Although astrocytes are regarded as the major source of apolipoprotein E (apoE) in the brain, also pericytes produce apoE. In this study we compared the apoE production capacity, the effects of apoE on D-Abeta(1-40) internalization, D-Abeta(1-40) cell surface accumulation and the vulnerability for D-Abeta(1-40)-induced toxicity of either cell type in order to quantify the relative contributions of astrocytes and pericytes in the various processes that contribute to CAA formation. Strikingly, cultured astrocytes produced only 3-10% of the apoE amounts produced by pericytes. Furthermore, pericytes with the apoE epsilon4 allele produced three times less apoE and were more vulnerable to D-Abeta(1-40) treatment than pericytes without an epsilon4 allele. Such relations were not observed with astrocytes in vitro. Both pericytes and astrocytes, however, were protected from Abeta-induced cytotoxicity by high levels of pericyte-derived apoE, but not recombinant apoE. In addition, pericyte-derived apoE dose-dependently decreased both internalization of Abeta and Abeta accumulation at the cell surface in either cell type. The present data suggest that apoE produced by pericytes, rather than astrocyte-produced apoE, modulates Abeta cytotoxicity and Abeta removal near the vasculature in the brain. Furthermore, since apoE production in pericytes is genotype dependent, this may contribute to the apoE genotype-dependent development of CAA in vivo.

Small heat shock proteins associated with cerebral amyloid angiopathy of hereditary cerebral hemorrhage with amyloidosis (Dutch type) induce interleukin-6 secretion.

In hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), severe cerebral amyloid angiopathy (CAA) is associated with an inflammatory reaction. Small heat shock proteins (sHsps) are molecular chaperones and association of HspB8 with CAA in HCHWA-D has been observed. The aims of this study were to investigate (1) if other sHsps are associated with the pathological lesions in HCHWA-D brains, (2) if the amyloid-beta protein (A beta) increases production of sHsps in cultured cerebral cells and (3) if sHsps are involved in the cerebral inflammatory processes in both Alzheimer's disease (AD) and HCHWA-D. We conclude that Hsp20, HspB8 and HspB2 are present in CAA in HCHWA-D, and that A beta did not affect cellular sHsps expression in cultured human brain pericytes and astrocytes. In addition, we demonstrated that Hsp20, HspB2 and HspB8 induced interleukin-6 production in cultured pericytes and astrocytes, which could be antagonized by dexamethasone, whereas other sHsps and A beta were inactive, suggesting that sHsps may be among the key mediators of the local inflammatory response associated with HCHWA-D and AD lesions.

Metastatic dormancy imposed by the primary tumor: does it exist in humans?

BACKGROUND: In cancer patients, occult micrometastases may become apparent shortly after removal of the primary tumor. Animal experiments have shown that metastatic dormancy is maintained by apoptosis, and that primary tumor removal induces a flare-up of angiogenesis, leading to metastatic outgrowth. This phenomenon has led to the hypothesis that the primary tumor generates certain factors that inhibit angiogenesis at distant sites. It is still unknown whether such a phenomenon is operative in human cancer as well. Should it occur, it might have important therapeutic consequences. MATERIALS AND METHODS: Evidence for such a mechanism may be obtained from studies that analyze a series of tissue samples of metastases, taken before or after surgical removal of the primary lesion. RESULTS: Data from our laboratory on colorectal cancer have shown that, in the absence of the primary tumor, vascular density in the metastases is increased as well as their metabolic activity, as measured by (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Mitotic activity is increased mildly, while levels of apoptosis are collapsed. CONCLUSION: These data indicate that a mechanism of primary-tumor-induced inhibition of angiogenesis exists, maintaining metastatic dormancy. We now suggest that this mechanism may be exploited to avoid the use of exogenous, potentially harmful angiogenesis inhibitors such as bevacizumab in a neoadjuvant setting. Treatment of patients with the primary tumor still in situ could thus be restricted to chemotherapy, since the synergistic effect of an angiogenesis inhibitor would be generated by the primary tumor itself. In the present paper the clinical relevance and possible consequences of our findings and suggestions are discussed.

Lipoprotein receptor-related protein-1 mediates amyloid-beta-mediated cell death of cerebrovascular cells.

Inefficient clearance of A beta, caused by impaired blood-brain barrier crossing into the circulation, seems to be a major cause of A beta accumulation in the brain of late-onset Alzheimer's disease patients and hereditary cerebral hemorrhage with amyloidosis Dutch type. We observed association of receptor for advanced glycation end products, CD36, and low-density lipoprotein receptor (LDLR) with cerebral amyloid angiopathy in both Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis Dutch type brains and increased low-density lipoprotein receptor-related protein-1 (LRP-1) expression by perivascular cells in cerebral amyloid angiopathy. We investigated if these A beta receptors are involved in A beta internalization and in A beta-mediated cell death of human cerebrovascular cells and astrocytes. Expression of both the LRP-1 and LDLR by human brain pericytes and leptomeningeal smooth muscle cells, but not by astrocytes, increased on incubation with A beta. Receptor-associated protein specifically inhibited A beta-mediated up-regulation of LRP-1, but not of LDLR, and receptor-associated protein also decreased A beta internalization and A beta-mediated cell death. We conclude that especially LRP-1 and, to a minor extent, LDLR are involved in A beta internalization by and A beta-mediated cell death of cerebral perivascular cells. Although perivascular cells may adapt their A beta internalization capacity to the levels of A beta present, saturated LRP-1/LDLR-mediated uptake of A beta results in degeneration of perivascular cells.

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-beta (Abeta) 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 Abeta, and in the clearance of Abeta 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 Abeta and tau and in other Abeta-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention.

Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators.

BACKGROUND: Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs). Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD) agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. METHODS: In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. RESULTS: Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. CONCLUSION: Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (pre)clinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy.

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.

Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity.

Small heat shock proteins Hsp20 and HspB2/B3 co-localize with Abeta deposition in senile plaques and cerebral amyloid angiopathy in Alzheimer's disease brains, respectively. It was the aim of our study to investigate if these and other sHsps bind to wild-type Abeta1-42 or the more toxic Abeta1-40 carrying the 'Dutch' mutation (22Glu-->Gln) (D-Abeta1-40), affect Abeta aggregation and thereby influence Abeta cytotoxicity. Binding affinity between sHsps and Abeta was investigated by surface plasmon resonance. Abeta aggregation was studied by using circular dichroism spectroscopy and electron microscopy. Furthermore, we used cultured cerebrovascular cells to investigate the effects of sHsps on Abeta-mediated cytotoxicity. Hsp20, Hsp27 and alphaB-crystallin, but not HspB2/B3, bound to Abeta (both D-Abeta1-40 and Abeta1-42) and reduced or completely inhibited aggregation of D-Abeta1-40 into mature fibrils but did not affect Abeta1-42 aggregation. Furthermore, these sHsps were effective inhibitors of the cerebrovascular toxicity of Abeta (both D-Abeta1-40 and Abeta1-42) in vitro. Binding affinity of the sHsps to D-Abeta1-40 correlated to the degree of inhibition of Abeta-mediated cytotoxicity and the potential to reduce Abeta beta-sheet and fibril formation. With Abeta1-42, a similar correlation between binding affinity and cytotoxicity was observed, but not with its aggregation state. In conclusion, sHsps may regulate Abeta aggregation and serve as antagonists of the biological action of Abeta, but the extent of their interaction depends on the type of sHsp and Abeta peptide.

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-beta precursor protein (APP), the amyloid-beta peptide (Abeta). 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 Abeta peptide. Several members of the small heat shock protein (sHsp) family, such as alphaB-crystallin, Hsp27, Hsp20 and HspB2, are associated with the pathological lesions of AD, and the direct interaction between sHsps and Abeta 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 Abeta aggregation and Abeta-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 Abeta(1-42), Abeta(1-40) and Abeta(1-40) with the Dutch mutation was demonstrated by surface plasmon resonance. Furthermore, co-incubation of HspB8 with D-Abeta(1-40) resulted in the complete inhibition of D-Abeta(1-40)-mediated death of cerebrovascular cells, likely mediated by a reduction in both the beta-sheet formation of D-Abeta(1-40) and its accumulation at the cell surface. In contrast, however, with Abeta(1-42), HspB8 neither affected beta-sheet formation nor Abeta-mediated cell death. We conclude that HspB8 might play an important role in regulating Abeta aggregation and, therefore, the development of classic SPs in AD and CAA in HCHWA-D.

Cerebral amyloid angiopathy with severe secondary vascular pathology: a histopathological study.

Cerebral amyloid angiopathy (CAA) is a common neuropathological finding and is characterized by deposition of fibrillar amyloid in cortical and leptomeningeal vessels. In this study we describe the macroscopic and microscopic neuropathological findings of 5 patients with severe CAA-associated secondary vascular changes, including smooth muscle cell degeneration, hyalinization, 'double-barreling' phenomenon, macrophage infiltration, and aneurysmal dilatation of the vessel wall. In 3 of the 5 patients these vascular changes were associated with multiple small hemorrhages, whereas in 2 patients areas of ischemic necrosis were observed. However, none of these patients suffered from large (lobar) hemorrhagic accidents. Nevertheless, severe CAA, particularly when associated with secondary vascular pathology, may lead to vascular dementia-like ischemic changes. Hence, the distinction between patients with severe CAA and secondary vascular abnormalities from those suffering from vascular dementia can be difficult. We speculate that CAA, particularly when associated with secondary vascular pathology, although not resulting in large hemorrhages, may contribute to cognitive decline. The functional impact of CAA and CAA-related secondary vascular changes on cognitive performance warrants further exploration.

Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy?

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.

Functional and morphologic analysis of the fluid-conducting meshwork in xenografted cutaneous and primary uveal melanoma.

PURPOSE: In primary uveal and cutaneous melanoma lesions, extracellular matrix (ECM) is often deposited in arcs, loops, and network patterns. Based on prognostic relevance, these patterns appear to play a significant role in facilitating metastasis. It has been demonstrated that these patterns were capable of transmitting fluid. The current study was undertaken to elucidate further the functional role of these patterns in tumor perfusion and to examine the composition of the patterns by immunohistochemistry. METHODS: To study the role of these patterns in perfusion, fluorochrome-labeled bovine serum albumin, bovine insulin, and dextrans of different molecular sizes were injected intravenously into nude mice bearing subcutaneous human cutaneous melanoma xenografts. Distribution of the human melanoma cells and murine host cells was analyzed by DNA in situ hybridization. To elucidate the composition of these patterns, human uveal melanoma tissues were analyzed for expression of ECM components by immunohistochemistry. RESULTS: Small molecules (Stokes' radius <4.4 nm) crossed the vessel wall and spread along the ECM patterns within 2 to 10 minutes, whereas larger molecules (Stokes' radius approximately 5.8 nm) required 30 to 45 minutes to enter. Murine host cells were found exclusively in the ECM pattern compartment. In primary uveal melanoma, different types of collagen, ECM-associated heparan sulfate proteoglycans, and different types of cells were present in the patterns. CONCLUSIONS: The data suggest that the ECM deposited as arcs, loops, and network patterns, accommodate the transport of plasma-derived molecules, (e.g., nutrients), to the tumor lesion, thus enhancing tumor growth and progression, and facilitating infiltration of tumor tissue by host-derived cells.

Apolipoprotein E genotype regulates amyloid-beta cytotoxicity.

The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the epsilon2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-beta1-40 peptide (Abeta1-40) with the 22Glu--> Gln "Dutch" mutation caused pericyte death in vitro by a mechanism that involves Abeta fibril-like assembly at the cell surface. It is known that ApoE binds to Abeta and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Abeta-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an epsilon4/epsilon4 genotype were more vulnerable to Abeta than cultures with an epsilon3/epsilon3 or epsilon3/epsilon4 genotype. The one cell culture with the epsilon2/epsilon3 genotype was relatively resistant to Abeta compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Abeta-mediated toxicity of cerebrovascular cells and, in addition, ApoE epsilon2/epsilon3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with epsilon4/epsilon4 cells. Thus, the disparity between ApoE genotype and Abeta-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Abeta cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to Abeta and thereby modify Abeta-mediated cell death.