The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

Increased expression of type 1 insulin-like growth factor receptor messenger RNA in rat hippocampal formation is associated with aging and behavioral impairment.

Insulin-like growth factor messenger RNAs are expressed in adult rat brain. However, little is known about the effects of aging on the expression of the insulin-like growth factors, their receptors, and their binding proteins in different regions of rat brain. The goal of the current study was to assess whether there is altered expression of the insulin-like growth factor system during normal aging in the hippocampal formation, a region particularly vulnerable to the aging process. A spatial learning task in the Morris water maze was used to assess the cognitive status of young (7-8-month-old) and aged (28-29-month-old) male Long-Evans rats. Sites of expression and abundance of insulin-like growth factor-I, type 1 insulin-like growth factor receptor, and insulin-like growth factor binding protein-4 messenger RNAs were then examined by in situ hybridization histochemistry and solution or northern blot hybridization assays. In situ hybridization histochemistry revealed no qualitative differences in the regional distribution of insulin-like growth factor-I, type 1 receptor, and insulin-like growth factor binding protein-4 messenger RNAs within the hippocampal formation of young and aged rats. However, quantitative analysis of messenger RNA abundance in hippocampal tissue homogenates showed a significant age-related increase in type 1 receptor messenger RNA (n = 25; t = -2.5; P < 0.02). Furthermore, linear regression analysis indicated that type 1 receptor messenger RNA abundance was significantly correlated with spatial learning impairment in the water maze (r = 0.44; P < 0.03) such that greater behavioral impairment was associated with higher type 1 receptor messenger RNA levels in the hippocampal formation. Neither insulin-like growth factor-I nor insulin-like growth factor binding protein-4 messenger RNA abundance was related to age or behavior. However, linear regression revealed a negative correlation between insulin-like growth factor-I messenger RNA abundance and type 1 receptor messenger RNA abundance in aged hippocampus (r = -0.72, P < 0.01). These data indicate that increased hippocampal expression of type 1 receptor messenger RNA is associated with aging and cognitive decline. The correlation between type 1 receptor and insulin-like growth factor-I messenger RNA abundance in the hippocampal formation of aged rats suggests that insulin-like growth factor availability may influence type 1 receptor expression. However, because no overall age difference was found in the amount of insulin-like growth factor-I messenger RNA in the hippocampal formation, decreased insulin-like growth factor from other sources such as the cerebrospinal fluid and the peripheral circulation may be involved in up-regulating type 1 receptor messenger RNA. Alternatively, type 1 receptor messenger RNA regulation may be part of a trophic response to the degenerative and regenerative events that occur within the hippocampal formation during aging.

Cloning and characterization of a cDNA encoding a novel fatty acid binding protein from rat brain.

We have adopted a polymerase chain reaction approach to identify and clone a cDNA that contains the complete coding sequence of a novel fatty acid binding protein (FABP) from a rat brain lambda gt10 library. Sequencing of the brain FABP (B-FABP) cDNA revealed an open reading frame coding for a protein with 132 amino acids and a predicted size of approximately 15,000 Da. This putative protein shares extensive sequence homology with other members of the FABP family. Northern blot analysis using the B-FABP cDNA as a probe established the presence of an abundant mRNA approximately 0.8 kb long in rat brain and in the MOCH-1 oligodendrocyte cell line. This transcript was also present in rat liver but not in other tissues examined. A developmental profile of this mRNA in rat brain demonstrated detectable expression in 15-day-old embryos with levels peaking in 1-day postnatal neonates and declining thereafter, reaching a low steady-state level at 3 weeks of age. In situ hybridization histochemistry revealed B-FABP mRNA in various brain regions, with the highest levels in fiber tracts. The B-FABP message was also detected at a lower level in several gray matter regions. The cloning approach used in this study would likely be useful in the identification and isolation of FABP-encoding genes from other tissues and species.

Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain.

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and 35S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain.