Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B.

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

The pericyte: A critical cell in the pathogenesis of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary small vessel disease presenting with migraine, mood and cognitive disorders, focal neurological deficits, recurrent ischemic attacks, lacunar infarcts and brain white matter changes. As they age, CADASIL patients invariably develop cognitive impairment and subcortical dementia. CADASIL is caused by missense mutations in the NOTCH3 gene resulting in a profound cerebral vasculopathy affecting primarily arterial vascular smooth muscle cells, which target the microcirculation and perfusion. Based on a thorough review of morphological lesions in arteries, veins, and capillaries in CADASIL, we surmise that arteriolar and capillary pericyte damage or deficiency appears a key feature in the pathogenesis of the disease. This may affect critical pericyte-endothelial interactions causing stroke injury and vasomotor disturbances. Changes in microvascular permeability due to perhaps localized blood-brain barrier alterations and pericyte secretory dysfunction likely contribute to delayed neuronal as well as glial cell death. Moreover, pericyte-mediated cerebral venous insufficiency may explain white matter lesions and the dilatation of Virchow-Robin perivascular spaces typical of CADASIL. The postulated central role of the pericyte offers some novel approaches to the study and treatment of CADASIL and enable elucidation of other forms of cerebral small vessel diseases and subcortical vascular dementia.

Congruence between NOTCH3 mutations and GOM in 131 CADASIL patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary subcortical vascular dementia. It is caused by mutations in NOTCH3 gene, which encodes a large transmembrane receptor Notch3. The key pathological finding is the accumulation of granular osmiophilic material (GOM), which contains extracellular domains of Notch3, on degenerating vascular smooth muscle cells (VSMCs). GOM has been considered specifically diagnostic for CADASIL, but the reports on the sensitivity of detecting GOM in patients' skin biopsy have been contradictory. To solve this contradiction, we performed a retrospective investigation of 131 Finnish, Swedish and French CADASIL patients, who had been adequately examined for both NOTCH3 mutation and presence of GOM. The patients were examined according to the diagnostic practice in each country. NOTCH3 mutations were assessed by restriction enzyme analysis of specific mutations or by sequence analysis. Presence of GOM was examined by electron microscopy (EM) in skin biopsies. Biopsies of 26 mutation-negative relatives from CADASIL families served as the controls. GOM was detected in all 131 mutation positive patients. Altogether our patients had 34 different pathogenic mutations which included three novel point mutations (p.Cys67Ser, p.Cys251Tyr and p.Tyr1069Cys) and a novel duplication (p.Glu434_Leu436dup). The detection of GOM by EM in skin biopsies was a highly reliable diagnostic method: in this cohort the congruence between NOTCH3 mutations and presence of GOM was 100%. However, due to the retrospective nature of this study, exact figure for sensitivity cannot be determined, but it would require a prospective study to exclude possible selection bias. The identification of a pathogenic NOTCH3 mutation is an indisputable evidence for CADASIL, but demonstration of GOM provides a cost-effective guide for estimating how far one should proceed with the extensive search for a new or an uncommon mutations among the presently known over 170 different NOTCH3 gene defects. The diagnostic skin biopsy should include the border zone between deep dermis and upper subcutis, where small arterial vessels of correct size are located. Detection of GOM requires technically adequate biopsies and distinction of true GOM from fallacious deposits. If GOM is not found in the first vessel or biopsy, other vessels or additional biopsies should be examined.

Trisomy 19 ependymoma, a newly recognized genetico-histological association, including clear cell ependymoma.

Ependymal tumors constitute a clinicopathologically heterogeneous group of brain tumors. They vary in regard to their age at first symptom, localization, morphology and prognosis. Genetic data also suggests heterogeneity. We define a newly recognized subset of ependymal tumors, the trisomy 19 ependymoma. Histologically, they are compact lesions characterized by a rich branched capillary network amongst which tumoral cells are regularly distributed. When containing clear cells they are called clear cell ependymoma. Most trisomy 19 ependymomas are supratentorial WHO grade III tumors of the young. Genetically, they are associated with trisomy 19, and frequently with a deletion of 13q21.31-31.2, three copies of 11q13.3-13.4, and/or deletions on chromosome 9. These altered chromosomal regions are indicative of genes and pathways involved in trisomy 19 ependymoma tumorigenesis. Recognition of this genetico-histological entity allows better understanding and dissection of ependymal tumors.

Lessons from CADASIL.

Vascular dementia (VaD) includes several different vascular mechanisms and changes in the brain. Among VaD, CADASIL is an inherited angiopathy caused by mutations in the Notch3 gene. The pathological hallmark of CADASIL is a granular osmiophilic material deposit (GOM) that is not only found in the brain, but also in the peripheral vascular tree. Consequently, a window into the brain was opened from a strictly neurological disease with tremendous consequences thanks to a skin biopsy. The latter was and continues to be used as a diagnostic tool for CADASIL, despite an immunohistochemical test that is now available. The skin biopsy first used as a diagnostic tool revealed the existence of numerous other VaDs presenting systemic vascular changes. Later, skin biopsy became a research tool, and a morphological skin vessel change classification was proposed on 300 patients. Interestingly, similar skin vessel lesions appear to be related to the same biological modifications. In addition, an early destruction of the medial muscle cells was noticed in 74% of cases. Because vascular smooth muscle cells secrete a powerful endothelial permeability factor (VEGF), their destruction could lead to a decrease in vascular permeability. Cocultures of endothelial cells with vascular muscle cells showed that their presence doubled vascular permeability. Thus, alteration or the loss of vascular muscle cells likely results in hypopermeability, in addition to vessel wall hypotonia and a watershed hypoperfusion. The wealth of information brought forth by knowledge of CADASIL provided new tools for research and clues for understanding the consequences of vascular impairments in dementia.

Characterization of the levels of expression of retinoic acid receptors, galectin-3, macrophage migration inhibiting factor, and p53 in 51 adamantinomatous craniopharyngiomas.

OBJECT: Craniopharyngiomas are histopathologically defined as benign tumors that can behave very aggressively at the clinical level. They can originate from different types of embryonal epithelial tissue in which correct spatiotemporal regulation has been disrupted at the effector production level. The goal of this study was to determine the efficacy of using selected biological markers to distinguish between recurring and nonrecurring craniopharyngiomas. METHODS: The authors used computer-assisted microscopy to determine quantitatively the immunohistochemical levels of expression of selected markers, including retinoic acid receptors (RARs), as response elements to retinoic acid in a series of 51 adamantinomatous craniopharyngiomas. These tumors may also originate as the result of physiological defects in the apoptosis-mediated elimination of embryological remnants of epithelial tissue. Galectin-3, p53, and the macrophage migration inhibiting factor (MIF) are known to play crucial roles in these processes. The authors quantitatively determined the levels of expression of these substances in this series of 51 craniopharyngiomas. The data show that all craniopharyngiomas were immunoreactive for RARalpha, whereas their immunoreactivity for RARbeta and RARgamma varied dramatically from one case to another. Craniopharyngiomas with low levels of RARbeta and high levels of RARgamma are more likely to recur than those with higher levels of RARbeta and lower levels of RARgamma. Rapidly recurring craniopharyngiomas also show significantly lower levels of expression of galectin-3 and MIF than nonrecurring or slowly recurring cases. Few tumors exhibited p53 immunopositivity. CONCLUSIONS: The data indicate that even in the so-called adamantinomatous group of craniopharyngiomas, several subgroups with different clinical behavior patterns can be identified on the basis of differentiation markers relating mainly to the presence or absence of RARbeta and RARgamma.

Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro.

BACKGROUND: The function of pericytes remains questionable but with improved cultured technique and the use of genetically modified animals, it has become increasingly clear that pericytes are an integral part of blood-brain barrier (BBB) function, and the involvement of pericyte dysfunction in certain cerebrovascular diseases is now emerging. The porcine stress syndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH) in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms, including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to the human syndrome. Symptoms are due to an enhanced calcium release from intracellular stores. These conditions are associated with a point mutation in ryr1/hal gene, encoding the ryanodine receptor, a calcium channel. Important blood vessel wall muscle modifications have been described in PSS, but potential brain vessel changes have never been documented in this syndrome. METHODS: In the present work, histological and ultrastructural analyses of brain capillaries from wild type and ryr1 mutated pigs were conducted to investigate the potential impairment of pericytes, in this pathology. In addition, brain pericytes were isolated from the three porcine genotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/hal alleles, respectively), and tested in vitro for their influence on the permeability of BBB endothelial monolayers. RESULTS: Enlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to a partial or total detachment of the astrocytic endfeet. These spaces were electron lucent and sometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level, brain pericytes did not seem to be affected because they showed regular morphology and characteristics, so we aimed to check their ability to maintain BBB properties in vitro. Our results indicated that pericytes from the three genotypes of pigs had differing influences on the BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able to maintain low BBB permeability. CONCLUSIONS: Electron microscopy observations demonstrated brain capillary modifications in PSS condition, but no change in pericyte morphology. Results from in vitro experiments suggest that brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition at the ultrastructural level, are not able to maintain BBB integrity in comparison with pericytes from wild-type animals.

Atypical BSE (BASE) transmitted from asymptomatic aging cattle to a primate.

BACKGROUND: Human variant Creutzfeldt-Jakob Disease (vCJD) results from foodborne transmission of prions from slaughtered cattle with classical Bovine Spongiform Encephalopathy (cBSE). Atypical forms of BSE, which remain mostly asymptomatic in aging cattle, were recently identified at slaughterhouses throughout Europe and North America, raising a question about human susceptibility to these new prion strains. METHODOLOGY/PRINCIPAL FINDINGS: Brain homogenates from cattle with classical BSE and atypical (BASE) infections were inoculated intracerebrally into cynomolgus monkeys (Macacca fascicularis), a non-human primate model previously demonstrated to be susceptible to the original strain of cBSE. The resulting diseases were compared in terms of clinical signs, histology and biochemistry of the abnormal prion protein (PrPres). The single monkey infected with BASE had a shorter survival, and a different clinical evolution, histopathology, and prion protein (PrPres) pattern than was observed for either classical BSE or vCJD-inoculated animals. Also, the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine. CONCLUSION/SIGNIFICANCE: Our results point to a possibly higher degree of pathogenicity of BASE than classical BSE in primates and also raise a question about a possible link to one uncommon subset of cases of apparently sporadic CJD. Thus, despite the waning epidemic of classical BSE, the occurrence of atypical strains should temper the urge to relax measures currently in place to protect public health from accidental contamination by BSE-contaminated products.

Morphometric analysis of ultrastructural vascular changes in CADASIL: analysis of 50 skin biopsy specimens and pathogenic implications.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a systemic vascular disease caused by Notch 3 gene mutations. On electron microscopy a specific granular osmiophilic material (GOM) is found surrounding the vascular smooth muscle cells. In 1993, we first proposed the use of skin biopsy to diagnose patients and to identify relatives of patients with CADASIL. We analyze here our experience with skin biopsies from 50 patients with CADASIL and compare the findings with those of 20 normal skin biopsy specimens. A morphometric analysis of skin vessel morphology on electron microscopy was performed by systematic measurements of several blood vessel diameters, as well as of areas of lumen, endothelial cell and smooth muscle cell cross-sectional areas, vessel wall area, arterial media and extracellular matrix areas. We found relative absence of stenosis but marked destruction of smooth muscle cells, resulting in decrease of vessel wall thickness and loss of extracellular matrix area, producing vessel wall weakness. Similar changes were also observed in brain arterioles from 5 patients with CADASIL. Our results suggest that hypotonicity of the arteriolar tree may constitute an important pathogenetic mechanism in CADASIL. Other than hypotonicity, the early and severe destruction of smooth muscle cells may potentially result in decreased secretion of vascular endothelial growth factor, loss of vascular permeability and damaging hemodynamic consequences. Blood vessel morphology of skin vessels correlated well with changes in brain arterioles. Vascular morphology in skin biopsy samples contributes to our understanding of the pathogenesis of CADASIL. It could be important to perform skin biopsies in future therapeutic trials of CADASIL as a direct measure of therapeutic effectiveness.

Phosphorylated serine 199 of microtubule-associated protein tau is a neuronal epitope abundantly expressed in youth and an early marker of tau pathology.

Microtubule-associated protein tau is abnormally phosphorylated in many neurodegenerative disorders, and is the major component of neurofibrillary degeneration, a degenerating process with many biochemical phenotypes. The serine 199 (S199) residue of tau is phosphorylated at early and late stages of Alzheimer's disease (AD). We studied the immunohistochemical distribution of this phosphorylated epitope in AD and other neurodegenerative disorders, as well as in controls of different ages. The phosphorylated S199 (S199P) epitope was observed in tau lesions from numerous diseases with neurofibrillary degeneration. This epitope was found to be abundantly expressed in the hippocampus formation in childhood and in young adult brain samples, and more specifically in subsets of neurons vulnerable to neurodegeneration. Interestingly, our data suggests that S199P is particularly resistant to phosphatase activity occurring during post-mortem delays. We suggest a peculiar and important role of the S199 residue as a qualitative indicator of the normal and pathological phosphorylation status of tau proteins.

Retinal involvement in dementia with Lewy bodies: a clue to hallucinations?

Visual hallucinations are a core feature of dementia with Lewy bodies. Their pathophysiology is not well understood, because neither clinical nor histological data have shown their basic mechanisms. Here, we report the presence of pale inclusions in the outer plexiform layer of the retina in a patient with dementia with Lewy bodies. These inclusions are related to cytoskeletal disorganization of the cones at ultrastructural level and modifications of the immunohistochemical pattern of distribution of synucleins in the retina. These modifications may participate in the visual impairment in dementia with Lewy bodies.

Calbindin-d(28k): a marker of recurrence for medulloblastomas.

BACKGROUND: The expression of the Ca(2+)-binding protein calbindin-D(28k) was analyzed in medulloblastomas in relation to clinical features and other biologic markers related to cell proliferation, differentiation, p53, and cerebellar developmental regulated gene expression. METHODS: Immunohistochemistry was carried out on histologic slides from a first retrospective series of 29 nonmetastatic and 10 metastatic medulloblastoma formalin-fixed, paraffin-embedded tissues, using specific antibodies against calbindin-D(28k), calretinin, alpha-parvalbumin and beta-parvalbumin, and S100 proteins. Informed consent was obtained from the subjects and/or guardians. Other biologic markers for differentiation, cell proliferation, the expression of the p53 tumor suppressor gene protein, and cerebellar developmental regulated genes were similarly investigated. A second series of 16 medulloblastomas from young patients (younger than 15 years) was added in order to validate the results obtained in the first series. RESULTS: Of all the markers investigated, only calbindin-D(28k) was significantly associated with prognosis. Survival and remission (i.e. recurrence free) time analysis performed on all the cases (n = 55) confirmed a high risk of death (P = 0.004) and recurrence (P = 0.003) associated with calbindin-positivity. As calbindin-positivity was predominantly observed in tumors from young patients, the authors confirmed its prognostic value in the subgroup of patients younger than 15 years (n = 37). Cox regression analysis showed a significant and independent prognostic value for calbindin expression and, to a lesser extent, the type of surgery (total or subtotal). Three risk groups were thus identified, distinguishing among the cases characterized by a total resection and calbindin-negativity (good prognosis), by a subtotal resection and calbindin-negativity (intermediary), and by calbindin-positivity (bad prognosis). CONCLUSIONS: The current study suggests that calbindin-positive medulloblastomas represent a subclass of aggressive tumors more frequently seen in younger patients.

Transgenic mice expressing mutant Notch3 develop vascular alterations characteristic of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an increasingly recognized adult-onset autosomal dominant vascular dementia, caused by highly stereotyped mutations in the Notch3 receptor. CADASIL is a widespread angiopathy characterized by a degeneration of vascular smooth muscle cells (VSMCs) and the abnormal accumulation of electron-dense granular material called GOM and Notch3 protein, because of an impaired clearance. Evidence that VSMCs are the primary target of the pathogenic process is supported by the restricted expression of Notch3 in these cells but mechanisms of their degeneration remain essentially unknown. We generated transgenic mice in which the SM22alpha promoter drove, in VSMCs, the expression of a full-length human Notch3 carrying the Arg90Cys mutation, a CADASIL archetypal mutation. Transgenic mice showed no evidence of prominent brain parenchyma damage but demonstrated the two hallmarks of the CADASIL angiopathy, GOM deposits and Notch3 accumulation, within both the cerebral and peripheral arteries. Of interest, arteries of the tail were more severely affected with prominent signs of VSMC degeneration. Time-course analysis of vessel changes revealed that disruption of normal VSMC anchorage to adjacent extracellular matrix and cells, VSMC cytoskeleton changes as well as starting signs of VSMC degeneration, which were detected around 10 months of age, preceded Notch3 and GOM accumulation appearance, which were observed only by 14 to 16 months of age. In conclusion, we have generated transgenic mice that recapitulate the characteristic vascular lesions observed in CADASIL. Our results indicate that Notch3 or GOM accumulation are unlikely to be the prerequisites for the induction of VSMC degeneration and suggest that degeneration of VSMCs may rather be triggered by the disruption of their normal anchorage, based on the important role of adhesion for cell survival.

Diffuse form of argyrophilic grain disease: a new variant of four-repeat tauopathy different from limbic argyrophilic grain disease.

Argyrophilic grain disease (AGD) is characterized by the occurrence of argyrophilic grains and coiled bodies in brain tissue, mainly in limbic areas located in the temporal lobe. Recent biochemical data have shown that inclusions in AGD consist of aggregates of pathological microtubule-associated tau protein isoforms of 64/69 kDa. We report here a study on two AGD patients, belonging to a series of demented patients affected by several tauopathies, prospectively followed until death. In both patients, clinical, neuropathological and biochemical investigations clearly demonstrated AGD. Diffuse tau pathology was shown by Gallyas' silver stain, tau immunohistochemistry and tau protein variant biochemical analysis, not only in temporal lobes but also in all cortical and subcortical areas that were assessed. Primary motor, primary sensory, and associative brain cortices were involved, as well as brain stem, but not cerebellum. We suggest that "diffuse" AGD might be a subgroup of AGD, the specific profile of which is different from that of "limbic" AGD.