The synergistic action of a VEGF-receptor tyrosine-kinase inhibitor and a sensitizing PDGF-receptor blocker depends upon the stage of vascular maturation

OBJECTIVE: To investigate the effects of tyrosine-kinase inhibitors of vascular endothelial growth factor (VECF) and platelet-derived growth factor (PDCF)-receptors on non-malignant tissue and whether they depend upon the stage of vascular maturation. MATERIALS AND METHODS: PTK787/ZK222584 and CGP53716 (VEGF- and PDGF-receptor inhibitor respectively), both alone and combined, were applied on chicken chorioallantoic membrane (CAM). RESULTS: On embryonic day of CAM development (E)8, only immature microvessels, which lack coverage of pericytes, are present: whereas the microvessels on E12 have pericytic coverage. This development was reflected in the expression levels of pericytic markers (alpha-smooth muscle actin, PDGF-receptor beta and desmin), which were found by immunoblotting to progressively increase between E8 and E12. Monotherapy with 2 microg of PTK787/ZK222584 induced significant vasodegeneration on E8, but not on E12. Monotherapy with CGP53716 affected only pericytes. When CGP53716 was applied prior to treatment with 2 microg of PTK787/ZK222584, vasodegeneration occurred also on E12. The combined treatment increased the apoptotic rate. as evidenced by the cDNA levels of caspase-9 and the TUNEL-assay. CONCLUSION: Anti-angiogenic treatment strategies for non-neoplastic disorders should aim to interfere with the maturation stage of the target vessels: monotherapy with VEGF-receptor inhibitor for immature vessels, and combined anti-angiogenic treatment for well developed mature vasculature.

Angiopoietin-2 deficiency decelerates age-dependent vascular changes in the mouse retina

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.

Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy

OBJECTIVE: The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS: Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS: Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (-20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS: Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction

The central nervous system (CNS) is tightly sealed from the changeable milieu of blood by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). While the BBB is considered to be localized at the level of the endothelial cells within CNS microvessels, the BCSFB is established by choroid plexus epithelial cells. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Combined with the absence of fenestrae and an extremely low pinocytotic activity, which inhibit transcellular passage of molecules across the barrier, these morphological peculiarities establish the physical permeability barrier of the BBB. In addition, a functional BBB is manifested by a number of permanently active transport mechanisms, specifically expressed by brain capillary endothelial cells that ensure the transport of nutrients into the CNS and exclusion of blood-borne molecules that could be detrimental to the milieu required for neural transmission. Finally, while the endothelial cells constitute the physical and metabolic barrier per se, interactions with adjacent cellular and acellular layers are prerequisites for barrier function. The fully differentiated BBB consists of a complex system comprising the highly specialized endothelial cells and their underlying basement membrane in which a large number of pericytes are embedded, perivascular antigen-presenting cells, and an ensheathment of astrocytic endfeet and associated parenchymal basement membrane. Endothelial cell morphology, biochemistry, and function thus make these brain microvascular endothelial cells unique and distinguishable from all other endothelial cells in the body. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier. Besides its barrier function, choroid plexus epithelial cells have a secretory function and produce the CSF. The barrier and secretory function of the choroid plexus epithelial cells are maintained by the expression of numerous transport systems allowing the directed transport of ions and nutrients into the CSF and the removal of toxic agents out of the CSF. In the event of CNS pathology, barrier characteristics of the blood-CNS barriers are altered, leading to edema formation and recruitment of inflammatory cells into the CNS. In this review we will describe current knowledge on the cellular and molecular basis of the functional and dysfunctional blood-CNS barriers with focus on CNS autoimmune inflammation.

Switching of vascular phenotypes within a murine breast cancer model induced by angiopoietin-2

Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.

Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

Chondroitin sulfate proteoglycan CSPG4 as a novel hypoxia-sensitive marker in pancreatic tumors.

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissue(high)/sera(low)-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic 'drop and restoration' alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Towards personalized medicine: Chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform

Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clinical tests. In addition to genetic mutation analysis, predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amount of patient's cells is essential to compare the responses under various conditions (compound and concentration). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for additional chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncology field.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

The physiological roles of ICAM-1 and ICAM-2 in neutrophil migration into tissues

PURPOSE OF REVIEW Neutrophil extravasation from the blood into tissues is initiated by tethering and rolling of neutrophils on endothelial cells, followed by neutrophil integrin activation and shear resistant arrest, crawling, diapedesis and breaching the endothelial basement membrane harbouring pericytes. Endothelial intercellular cell adhesion molecule (ICAM)-1 and ICAM-2, in conjunction with ICAM-1 on pericytes, critically contribute to each step. In addition, epithelial ICAM-1 is involved in neutrophil migration to peri-epithelial sites. The most recent findings on the role of ICAM-1 and ICAM-2 for neutrophil migration into tissues will be reviewed here. RECENT FINDINGS Signalling via endothelial ICAM-1 and ICAM-2 contributes to stiffness of the endothelial cells at sites of chronic inflammation and junctional maturation, respectively. Endothelial ICAM-2 contributes to neutrophil crawling and initiation of paracellular diapedesis, which then proceeds independent of ICAM-2. Substantial transcellular neutrophil diapedesis across the blood-brain barrier is strictly dependent on endothelial ICAM-1 and ICAM-2. Endothelial ICAM-1 or ICAM-2 is involved in neutrophil-mediated plasma leakage. ICAM-1 on pericytes assists the final step of neutrophil extravasation. Epithelial ICAM-1 rather indirectly promotes neutrophil migration to peri-epithelial sites. SUMMARY ICAM-1 and ICAM-2 are involved in each step of neutrophil extravasation, and have redundant but also distinct functions. Analysis of the role of endothelial ICAM-1 requires simultaneous consideration of ICAM-2.

Bone marrow-derived stem/progenitor cells play an important role in the growth of Ewing's sarcoma tumors

Both angiogenesis and vasculogenesis contribute to the formation and expansion of tumor neovasculature. We demonstrated that bone marrow (BM)-derived cells migrated to TC71 Ewing's tumors and differentiated into endothelial cells lining perfused, functional tumor neovessels. In addition, a substantial fraction of recruited, BM-derived cells resided in the vessel vicinity but did not demonstrate endothelial differentiation. Rather, these perivascular cells expressed desmin and PDGFR-β, implying pericyte-like/vascular smooth muscle cell differentiation. No defined, consensus set of markers exists for endothelial progenitor cells (EPCs) and the specific subsets of BM cells that participate in vessel formation are poorly understood. We used a functional in vivo assay to investigate the roles performed by specific human- and murine-derived stem/progenitor subpopulations within Ewing's sarcoma tumors. CD34 +45+, CD34+38-, VEGFR2 + and Sca1+Gr1+ cells were demonstrated to establish residence within the expanding tumor vascular network and differentiate into endothelial cells and pericytes. By constrast, CD34-45 + and Sca1-Gr1+ cells predominantly localized to sites outside the Ewing's tumor vasculature, and differentiated into macrophages. Cytokines, such as VEGF, influence the recruitment of BM cells and their incorporation into the tumor vasculature. VEGF165-inhibited TC/siVEGF7-1 Ewing's tumors showed delayed in vivo tumor growth, decreased vessel density, and reduced infiltration of BM progenitor cells. We tested whether another chemoattractant, Stromal Cell-Derived Factor-1 (SDF-1), could augment the growth of these VEGF165-inhibited TC/siVEGF 7-1 tumors by enhancing the recruitment of BM cells and stimulating neovasculature expansion. SDF-1 promoted progenitor cell chemotaxis and retainment of BM-derived pericyte precursors in close association with functional, perfused tumor blood vessels. Treatment of TC/siVEGF7-1 tumors with adenovirus-SDF-1α resulted in augmented tumor size, enhanced pericyte coverage of tumor neovessels, remodeling of vascular endothelium into larger, functional structures, and upregulation of PDGF-BB, with no effect on VEGF165. Taken together, these findings suggest that the recruitment of BM stem/progenitor cells plays an important role in the growth of Ewing's tumors. ^

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding α-N-acetylglucosaminidase

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding α-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu−/− mice were healthy and fertile while young and could survive for 8–12 mo. They were totally deficient in α-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides GM2 and GM3 in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu−/− mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the α-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.