The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other actin genes, the beta-actin gene lacks the codon for Cys between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

We previously localized a quantitative trait locus (QTL) on chromosome 6 affecting milk fat and protein concentration to a 4-cM confidence interval, centered on the microsatellite BM143. We characterized the genes and sequence variation in this region and identified common haplotypes spanning five polymorphic sites in the genes IBSP, SPP1, PKD2, and ABCG2 for two sires heterozygous for this QTL. Expression of SPP1 and ABCG2 in the bovine mammary gland increased from parturition through lactation. SPP1 and all the coding exons of ABCG2 and PKD2 were sequenced for these two sires. The single nucleotide change capable of encoding a substitution of tyrosine-581 to serine (Y581S) in the ABCG2 transporter was the only polymorphism corresponding to the segregation status of all 3 heterozygous and 15 homozygous sires for the QTL in the Israeli and U.S. Holstein populations. The allele substitution fixed effects on the genetic evaluations of 335 Israeli sires were −341 kg milk, +0.16% fat, and +0.13% protein (F-value = 200). No other polymorphism gave significant effect for fat and protein concentration in models that also included Y581S. The allele substitution effects on the genetic evaluations of 670 cows, daughters of two heterozygous sires, were −226 kg milk, 0.09% fat, and 0.08% protein (F-value = 394), with partial dominance towards the 581S homozygotes. We therefore propose that Y581S in ABCG2 is the causative site for this QTL.

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic signi®cance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte de®-ciency, prenatal lethality has so far precluded analysis of postnatal consequences in the retina. We now report that endothelium-restricted ablation of platelet-derived growth factor-B generates viable mice with extensive inter-and intra-individual variation in the density of pericytes throughout the CNS. We found a strong inverse correlation between pericyte density and the formation of a range of retinal microvascular abnormalities strongly reminiscent of those seen in diabetic humans. Proliferative retinopathy invariably developed when pericyte density was <50% of normal. Our data suggest that a reduction of the pericyte density is suf®cient to cause retinopathy in mice, implying that pericyte loss may also be a causal pathogenic event in human diabetic retinopathy.

The essential role of VEGF in blood vessel formation in the embryo and in the adult is well established (1, 2). A single VEGF gene gives rise, by alternative splicing, to multiple isoforms (VEGF 121 , VEGF 165 , and VEGF 189 in humans versus VEGF 120 , VEGF 164 , and VEGF 188 in the mouse) that differ in molecular mass, solubility, and receptor binding (3). VEGF 120 lacks exons 6 and 7, encoding extracellular matrix binding structures, and is therefore the most soluble. VEGF 188 contains all exons and avidly binds to the cell surface and extracellular matrix. VEGF 164 lacks only exon 6 and has intermediate properties. The VEGF isoforms bind to several receptors: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1 (NP-1) (3). NP-1 is a semaphorin receptor involved in neuron guidance. As a VEGF 164-specific coreceptor for VEGFR-2, NP-1 also affects angiogenesis (4) and enhances the angiogenic activity of VEGF 164 (5). To define the differential role of the VEGF isoforms in vivo, mice expressing single VEGF isoforms were generated. Impaired myocardial angiogenesis has been demonstrated in VEGF 120/120 mice (expressing VEGF 120) (6). Here we report that loss of VEGF 164 (in VEGF 120/120 and VEGF 188/188 mice) impaired retinal arterial development, whereas loss of VEGF 164 and VEGF 188 (in VEGF 120/120 mice) led to dysregulated vessel outgrowth and patterning in the retina. These observations suggest possible mechanisms for the distinct roles of the VEGF isoforms in retinal vascular patterning and arterial endothelial cell specification. Methods Generation of transgenic mice. Targeted mutagenesis was achieved by homologous and Cre/lox P-mediated sitespecific recombination in embryonic stem (ES) cells. The strategy to generate VEGF 120/120 mice (deletion of exons 6 and 7) has been described previously (6). Targeting vectors used to generate VEGF 164/164 mice and VEGF 188/188 mice were constructed by replacing the genomic sequence with a cDNA containing the fused

Pericytes provide vascular stability and control endothelial proliferation. Pericyte loss, microaneurysms, and acellular capillaries are characteristic for the diabetic retina. Platelet-derived growth factor (PDGF)-B is involved in pericyte recruitment, and brain capillaries of mice with a genetic ablation of PDGF-B show pericyte loss and microaneurysms. We investigated the role of capillary coverage with pericytes in early diabetic retinopathy and the contribution to proliferative retinopathy using mice with a single functional allele of PDGF-B (PDGF-B ؉/؊ mice). As assessed by quantitative morphometry of retinal digest preparations, pericyte numbers in nondiabetic PDGF-B ؉/؊ mice were reduced by 30% compared with wild-type mice, together with a small but significant increase in acellular capillaries. Pericyte numbers were reduced by 40% in diabetic wild-type mice compared with nondiabetic wild-type controls. Pericyte numbers were decreased by 50% in diabetic PDGF-B ؉/؊ mice compared with nondiabetic wild-type littermates, and the incidence of acellular capillaries was increased 3.5-fold when compared with nondiabetic PDGF-B ؉/؊ mice. To investigate the effect of pericyte loss in the context of ongoing angiogenesis, we subjected mice to hypoxia-induced proliferative retinopathy. As a result, PDGF-B ؉/؊ mice developed twice as many new blood vessels as their wild-type littermates. We conclude that retinal capillary coverage with pericytes is crucial for the survival of endothelial cells, particularly under stress conditions such as diabetes. At high vascular endothelial growth factor levels, such as those in the retinopathy of prematurity model, pericyte deficiency leads to reduced inhibition of endothelial proliferation in vivo. Diabetes 51:3107-3112, 2002

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 Ͻ 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P Ͻ 0.05) and was predominantly observed in LacZpositive pericytes (Ϫ20%, P Ͻ 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P Ͻ 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P Ͻ 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P Ͻ 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wildtype littermates.CONCLUSIONS-Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

Background-Tumor blood vessels are both structurally and functionally abnormal compared with normal vessels. A limited support of mural cells may contribute to these abnormalities. Here, we characterized mural cell recruitment in 2 mouse tumor models and addressed the question of why tumor vessels fail to recruit a proper coat of mural cells. Methods and Results-We studied mural cell recruitment to the vasculature of 2 transplantable mouse tumor models, T241 fibrosarcoma and KRIB osteosarcoma. We found that both tumors formed a vessel network with heterogeneous and highly abnormal organization of mural cells.