Homozygote Mice Research Articles

Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic overexpression of IL-1 receptor antagonist.

Inflammation is associated with both acute and chronic neurological disorders, including stroke and Alzheimer's disease (AD). Cytokines such as interleukin (IL)-1 have several activities in the brain both under physiological and pathophysiological conditions. The objective of this study was to evaluate consequences of the central blockade of IL-1 transmission in a previously developed transgenic mouse strain with brain-directed overexpression of human soluble IL-1 receptor antagonist (Tg hsIL-1ra). Effects on brain morphology and brain levels of the AD-related proteins β-amyloid precursor protein (APP) and presenilin 1(PS1), as well as the levels of IL-1β, IL-6 and tumour necrosis factor-α (TNF-α) were analysed in homozygotic and heterozygotic mice and wild type (WT) controls, of both genders and of young (30–40 days) and adult (13–14 months) age. A marked reduction in brain volume was observed in transgenic mice as determined by volumetry. Western blot analysis showed higher levels of APP, but lower levels of PS1, in adult animals than in young ones. In the cerebellum, heterozygotic (Tg hsIL-1ra+/−) mice had lower levels of APP and PS1 than WT mice. With one exception, there were no genotypic differences in the levels of IL-1β, IL-6 and TNF-α. The cytokine levels were generally higher in adult than in young mice. In conclusion, the chronic blockade of IL-1 signalling in the brain was associated with an atrophic phenotype of the brain, and with modified levels of APP and PS1. Brain-directed overexpression of hsIL-1ra was not followed by major compensatory changes in the levels of pro-inflammatory cytokines.

Sodium reduces renin secretion less in natriuretic peptide C receptor deficient mice

The impact of the natriuretic peptide C receptor (NPR-C) on renin secretion was assessed in mice possessing normal or mutated (nonfunctional) NPR-C’s. Heterozygous mice with a mutation of the NPR-C were bred to obtain either wild type, heterozygote or homozygote (NPR-C deficient) mice. The mice were tested at 2 months of age. Half of the mice of each genotype drank 0.9% saline and the other half drank tap water. After five days of saline or water consumption, the mice were anesthetized with pentobarbital sodium and 150 μl of blood were withdrawn from the inferior vena cava. Wild type control mice exhibited a decrease in renin activity from 2062 ± 364 to 1518 ± 178 ng angiotensin I/ml/hr after saline exposure. The decrease in heterozygotes was from 1663 ± 201 to 1366 ± 128, whereas homozygote (deficient) mice experienced a change from 2070 ± 284 to 1951 ± 314. The difference between renin activity while drinking water and saline averaged 544 ng angiotensin I/ml/hr in wildtype mice, 297 in heterozygote mice and 119 in homozygote mice. Thus, a linear relationship existed between the number of functional NPR-C copies and the ability of saline consumption to suppress renin secretion. Prior studies have identified the natriuretic peptide A receptor as the functional natriuretic peptide receptor suppressing renin release. These data suggest an involvement of the NPR-C as well. (This study was supported by the Minnesota Medical Foundation.)

Transcriptional profiling of the megabladder mouse: A unique model of bladder dysmorphogenesis

Recent studies in our lab identified a mutant mouse model of obstructive nephropathy designated mgb for megabladder. Homozygotic mgb mice (mgb-/-) develop lower urinary tract obstruction in utero due to a lack of bladder smooth muscle differentiation. This defect is the result of a random transgene insertion/translocation into chromosomes 11 and 16. Transcriptional profiling identified a significantly over-expressed cluster of gene products located on the translocated fragment of chromosome 16 including urotensin II-related peptide (Urp), which was shown to be preferentially over-expressed in developing mgb-/- bladders. Pathway analysis of mgb microarray data indicated dysregulation of at least 60 gene products associated with smooth muscle development. In conclusion, the results of this study indicate that the molecular pathways controlling normal smooth muscle development are severely altered in mgb-/- bladders, and provide the first evidence that Urp may play a critical role in bladder smooth muscle development.

Bax ablation protects against hepatic ischemia/reperfusion injury in transgenic mice

Apoptosis appears to be a central mechanism of cell death following reperfusion of the ischemic liver. The aim of this study was to determine the effect of decreased expression of the proapoptotic Bax gene on hepatic apoptotic warm ischemia/reperfusion (I/R) injury. Three groups of mice were studied: homozygotic knockout mice (Bax-/-); heterozygotic (Bax+/-); and wild type (Bax+/+). Isolated mouse livers were subjected to 90 minutes of ischemia (37 degrees C) followed by 15 minutes of reperfusion. Bax and Bcl-2 expression in liver tissue homogenates was measured by Western blot. Serum liver enzyme levels were measured and intrahepatic caspase-3 activity was determined by fluorimetric assay. Oil red O (ORO) staining was performed for fat detection. Apoptotic cells were identified by morphological criteria, immunohistochemistry for caspase-3, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay. At 1 minute of reperfusion, the ischemic (Bax-/-) livers were characterized by statistically significantly lower liver enzyme levels and lower caspase-3 activity than the ischemic (Bax+/+) livers (P<0.05 for both). The reduction in postischemic apoptotic hepatic injury in the ischemic Bax-/- livers group was confirmed morphologically, by the significantly reduced microvesicular steatosis as determined by ORO staining, fewer apoptotic hepatocyte cells detected (P<0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (P<0.05); and by TUNEL assay (P<0.05). Similar levels of antiapoptotic Bcl-2 protein expression were detected in all 3 groups of ischemic livers on Western blots. Bax protein was not expressed in Bax-deficient livers and was detected in Bax+/+ normal livers. In the Bax+/- livers, levels of the damage markers were moderate. In conclusion, The better tolerance of Bax knockout livers to I/R injury suggests that the Bax gene may serve as a potential target for therapeutic intervention in hepatic I/R injury.

Knockout of the mu opioid receptor enhances the survival of adult-generated hippocampal granule cell neurons

Recent evidence suggests that mu opioid receptors (MOR) are key regulators of hippocampal structure and function. For example, exogenous MOR agonists morphine and heroin negatively impact hippocampal function and decrease adult hippocampal neurogenesis. Here we explored the role of MOR in the birth and survival of hippocampal progenitor cells by examining adult neurogenesis in mice that lack MOR. Adult male mice lacking exon 1 of MOR were injected with the S phase marker bromodeoxyuridine (BrdU) and killed either 2 hours or 4 weeks later to evaluate proliferating and surviving BrdU-immunoreactive (IR) cells, respectively, in the adult hippocampal granule cell layer. Wild-type (WT), heterozygote, and homozygote mice did not differ in the number of BrdU-IR cells at a proliferation time point. However, 4 weeks after BrdU injection, heterozygote and homozygote mice had 57% and 54% more surviving BrdU-IR cells in the hippocampal granule cell layer as compared with WT mice. A decrease in apoptosis in the heterozygote and homozygote mice did not account for the difference in number of surviving BrdU-IR cells since there were no alterations in number of pyknotic, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive, or activated caspase 3-IR cells compared with WT. In concordance with the increased numbers of granule cells maturing into neurons, heterozygote and homozygote mice had larger hippocampal granule cell layers and increased numbers of granule cells. These findings indicate that MOR may play a role in regulating progenitor cell survival and more generally encourage further exploration of how MOR activation can influence hippocampal structure and function.

TNFalpha mediates the skeletal effects of thyroid-stimulating hormone.

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNFalpha, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR-/- and TSHR+/- mice is abrogated in compound TSHR-/-/TNFalpha-/- and TSHR+/-/TNFalpha+/- mice, respectively. In parallel studies, we find that TSH directly inhibits TNFalpha production, reduces the number of TNFalpha-producing osteoclast precursors, and attenuates the induction of TNFalpha expression by IL-1, TNFalpha, and receptor activator of NF-kappaB ligand. TSH also suppresses osteoclast formation in murine macrophages and RAW-C3 cells. The suppression is more profound in cells that overexpress the TSHR than those transfected with empty vector. The overexpression of ligand-independent, constitutively active TSHR abrogates osteoclast formation even under basal conditions and in the absence of TSH. Finally, IL-1/TNFalpha and receptor activator of NF-kappaB ligand fail to stimulate AP-1 and NF-kappaB binding to DNA in cells transfected with TSHR or constitutively active TSHR. The results suggest that TNFalpha is the critical cytokine mediating the downstream antiresorptive effects of TSH on the skeleton.

Distribution of co‐activators CBP and p300 during mouse oocyte and embryo development

cAMP response element binding protein (CREB)-binding protein (CBP) and p300 are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes. Current dogma would suggest that these transcriptional co-activators have similar mechanisms of transcription regulation. Studies of CBP or p300 homozygotic mouse mutants indicate that normal embryogenesis requires the existence of both factors. However, whether this is indicative of a dosage effect of these two proteins, or whether these proteins play different roles in mouse embryo development is not clear. Here we demonstrated that both factors are first found in the cytoplasm of oocytes within primordial follicles, and that they enter into the oocyte nucleus at different stages of oocyte growth, suggesting that they may play different roles in gene expression during oocyte growth and development. Consistent with this model, in the pre-implantation mouse embryos, from the two-cell stage to the blastocyst stage, the localizations of CBP and p300 are different, at times opposite, indicating that CBP and p300 also have different functions in early mouse embryogenesis.

Cardiac Norepinephrine Contents are Influenced by Natriuretic Peptide Receptors

The purpose of the present study was to define the effect of natriuretic peptide receptor (NPR) alterations on tissue catecholamine levels. Transgenic mice lacking functional natriuretic peptide A receptors (NPR-A) were obtained from Dr. D. Garbers (UT Southwestern) and NPR-C deficient mice were purchased from Jackson Research Laboratories. Mice were anesthetized with pentobarbital sodium at 25 to 35 days of age and tissues were removed. Catecholamines were extracted and analyzed by high performance liquid chromatography with electrochemical detection. Cardiac norepinephrine contents were suppressed with decreasing functional NPR-A levels (1.65 ± 0.24 ng/mg in homozygotes and 2.81 ± 0.63 ng/mg in wild type) although these differences did not reach statistical significance (p = 0.15). The hearts from homozygote mice were hypertrophied at this age (8.0 ± 0.4 mg/g vs 6.5 ± 0.2 mg/g in homozygote vs wild-type hearts) and this difference was significant (p = 0.003). Alterations in NPR-C levels produced the opposite effect on cardiac catecholamine concentrations. Ablation of functional NPR-C produced elevated cardiac catecholamine concentrations (6.1 ± 1.5 ng/mg) compared to wild-type mice (2.97 ± 0.4 ng/mg) and these differences were statistically significant (p = 0.03). There was no influence of NPR-C alterations on cardiac size. These data indicate that sympathetic innervation is influenced by the natriuretic peptide system with the presence of NPR-A being associated with greater cardiac catecholamine content and the presence of NPR-C tending to suppress cardiac catecholamine content. (Study supported by a grant-in-aid from the Minnesota Medical Foundation).

DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.

Anatomic modifications in the enteric nervous system of piebald mice and physiological consequences to colonic motor activity

It has been assumed that in piebald lethal mice that develop megacolon, impaired colonic motor activity is restricted to the aganglionic distal colon. Peristaltic mechanical recordings, immunohistochemistry, and quantitative PCR were used to investigate whether regions of the colon, other than the aganglionic segment, may also show anatomical modifications and dysfunctional colonic motor activity. Contrary to expectations, colonic migrating motor complexes (MMCs) were absent along the whole colon of piebald lethal homozygote mice and severely impaired in heterozygote siblings. Aganglionosis was detected not only in the distal colon of piebald homozygote lethal mice (mean length: 20.4 +/- 2.1 mm) but also surprisingly in their heterozygote siblings (mean length: 12.4 +/- 1.1 mm). Unlike homozygote lethal mice, piebald heterozygotes showed no signs of megacolon. Interestingly, mRNA expression for PGP 9.5 was also dramatically reduced (by 71-99%) throughout the entire small and large bowel in both homozygote lethal and heterozygous littermates (by 67-87%). Histochemical staining confirmed a significant reduction in myenteric ganglia along the whole colon. In summary, the piebald mutation in homozygote lethal and heterozygote siblings is associated with dramatic reductions in myenteric ganglia throughout the entire colon and not limited to the distal colon as originally thought. Functionally, this results in an absence or severe impairment of colonic MMC activity in both piebald homozygote lethal and heterozygote siblings, respectively. The observation that piebald heterozygotes have an aganglionic distal colon (mean length: 12 mm) but live a normal murine life span without megacolon suggests that aganglionosis >12 mm and the complete absence of colonic MMCs may be required before any symptoms of megacolon arise.

Effect of Bimatoprost on Intraocular Pressure in Prostaglandin FP Receptor Knockout Mice

To determine the effect of bimatoprost on intraocular pressure in the prostaglandin FP receptor knockout mouse. The IOP response to a single 1.2-microg (4 microL) dose of bimatoprost was measured in the treated and untreated fellow eyes of homozygote (FP+/+, n = 9) and heterozygote (FP+/-, n = 10) FP-knockout mice, as well as in wild-type C57BL/6 mice (FP+/+, n = 20). Serial IOP measurements were also performed after topical bimatoprost in a separate generation of homozygous FP-knockout mice and wild-type littermate control animals (n = 4 per group). Aqueous humor protein concentrations were measured to establish the state of the blood-aqueous barrier. Tissue, aqueous humor and vitreous concentrations of bimatoprost, latanoprost, and their C-1 free acids were determined by liquid chromatography and tandem mass spectrometry. A significant reduction in IOP was observed in the bimatoprost-treated eye of wild-type mice at 2 hours, with a mean difference and 95% confidence interval (CI) of the difference in means of -1.33 mm Hg (-0.81 to -1.84). Bimatoprost did not lead to a significant reduction in IOP in either the heterozygous knockout -0.36 mm Hg (-0.82 to +0.09) or homozygous FP-knockout mice 0.25 mm Hg (-0.38 to +0.89). The lack of an IOP response in the FP-knockout mice was not a consequence of blood-aqueous barrier breakdown, as there was no significant difference in aqueous humor protein concentration between treated and fellow eyes. Tissue and aqueous humor concentrations of bimatoprost, latanoprost, and their C-1 free acids indicate that latanoprost, but not bimatoprost, is hydrolyzed in the mouse eye after topical administration. An intact FP receptor gene is critical to the IOP response to bimatoprost in the mouse eye.

Characterisation of SCL DNA-Binding Functions In Vivo in Haematopoiesis.

The basic Helix-Loop-Helix (bHLH) transcription factor SCL is required for specification of haematopoietic stem cells (HSCs) and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. We challenged this concept by showing that SCL DNA-binding activity was dispensable for some of its functions using SCL−/− ES cells rescued with a DNA-binding defective SCL mutant (SCL-RER) (Porcher et al. Development,1999). We have now studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line SCL-RER mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of primitive erythroid progenitors was observed in SCL RER/RER mutant yolk sacs. At day E12.5 and E13.5, homozygote mutant SCL RER/RER embryos were smaller and paler than wild-type (wt) and heterozygote littermates but presented at the expected mendelian frequency. Lethality was first observed at day E14.5. However, 7% of homozygote mice were born from heterozygous crosses. Surviving adult mice presented with mild microcytic hypochromic anemia. Assessment of progenitor replating potential showed qualitative defects with poor haemoglobinisation of homozygote-derived fetal and adult CFU-Es compared to controls. To understand the cause of the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for either early progenitors or late normoblasts. We observed decreased or increased mRNA expression of most genes tested in mutant-derived erythroid populations when compared to controls matched for differentiation stage, thereby showing that SCL DNA-binding activities are required for both activation and repression of target genes. As examples, mRNA for red cell membrane protein Band 4.2 was dramatically decreased; in contrast, alpha-globin expression was up-regulated in early progenitors, indicating that SCL DNA-binding activity might be required for repression of alpha-globin levels in this setting. We then pursued the analysis of SCL-mediated alpha-globin gene regulation by chromatin immunoprecipitation (ChIP) analysis and tested 4 DNase I hypersensitive sites (DHS) previously shown to bind SCL. From material derived from mutant fetal liver cells, we observed slight variations of SCL binding on 3 out of the 4 cis-acting elements when compared to controls. Importantly, there was a dramatic decrease in SCL binding on the fourth DHS site (HS-12). We concluded that SCL DNA-binding activity was likely to be directly required for repression of alpha-globin levels in erythroid progenitors. Interestingly, we have recently characterised the interaction of SCL with a co-repressor complex comprising the oncoprotein ETO-2 in erythroid cells. We have now shown by ChIP that ETO-2 occupies the alpha-globin locus on HS-12 in wt erythroid progenitors, but not in more mature cells, therefore suggesting a role for the SCL/ETO-2 complex in repression of erythroid-specific genes in the early stages of erythroid maturation. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of HSCs and allows characterisation of DNA-binding requirements throughout development. Combined with studies of the dynamic of SCL-containing multiprotein complexes during erythroid maturation, this model will help define the molecular pathways involved in erythropoiesis.

Differential expression of two NMDA receptor interacting proteins, PSD‐95 and SynGAP during mouse development

Patterns of neural activity mediated by N-methyl-D-aspartate (NMDA) receptors are known to play important roles in development of the central nervous system. However, the signalling pathways downstream from NMDA receptors that are critical for normal neuronal development are not yet clearly understood. NMDA receptors interact with various signalling proteins via scaffolding proteins, which are important in adult neuronal and behavioural plasticity. For example, the NR2B subunits of the NMDA receptor interact with postsynaptic density 95 (PSD-95), which in turn binds to synaptic ras GTPase-activating protein (SynGAP). Interestingly, the developmental phenotype of mice carrying null mutations in these genes differ. NR2B and SynGAP homozygote mice die within the first week of birth whereas PSD-95 homozygote mice survive to adulthood. We therefore examined the expression patterns of PSD-95 and SynGAP genes from embryonic stages to adult using lacZ (beta-galactosidase) marker gene knock-in mice. Dramatic changes of expression were observed throughout development in brain and other tissues. Although SynGAP binds PSD-95, both genes had distinct, as well as overlapping expression. SynGAP expression peaked at times of synaptogenesis and developmental plasticity in contrast to PSD-95, which was expressed throughout the brain from early embryonic stages. Furthermore, SynGAP showed a more spatially restricted pattern as illustrated by its restriction to forebrain in contrast to PSD-95, which was also found in mid- and hindbrain. These data support the model that synaptic signalling complexes are heterogeneous and individual components show temporal and spatial specificity during development.

ENU Mutagenesis Screen Identifies a Novel Regulator of Platelet Production.

The genes that regulate variation in steady-state platelet count are largely unknown. We have attempted to identify novel genes that regulate platelet production by creating heritable models of thrombocytosis in mice by randomly inducing point mutations in the genome with chemical mutagenesis.Mice with a high platelet count were identified in the third generation of an ENU mutagenesis screen and several different lines of thrombocytotic mice have been established. Subsequent breeding of one line has demonstrated that this mutation, called Plt2, is inherited in an autosomal recessive manner. Automated platelet counts for Plt2 homozygote mice were 1997±642x109/L (mean±2SD; n=51), 140% of those observed for wild-type mice (1487±372; n=56) and Plt2 heterozygote mice (1402±554; n=76). Analysis of Plt2 animals has shown increased numbers of megakaryocyte precursors (as measured by meg-CFC/5x105 cells to maximal cytokine stimulation) in the bone marrow (Plt2 40±20 compared to wild-type (WT) animals 28±8; n=7) and spleen (Plt2 16±6 compared to WT 8±3; n=7, p=0.01). Mature megakaryocytes were increased in frequency in bone marrow sections (Plt2 86.8±28.4 compared to WT 56.8±18.5) and demonstrate a similar maturation profile to control animals when their nuclear ploidy is measured by flow cytometry.Reciprocal bone marrow transplantation experiments demonstrate that the thrombocytotic phenotype was observed in Plt2 homozygous recipients of either wild type or Plt2 homozygous bone marrow, but not observed in wild type recipients of Plt2 homozygous bone marrow. This data indicates that the Plt2 mutation is acting extrinsically on the haemopoietic system. Despite having an elevated platelet count, Plt2 mutant mice have inappropriately elevated serum thrombopoietin levels (Plt2 5420±2842; n=15) compared to controls (2947 ±2310; n=13; p<0.0001).Mapping experiments have excluded both thrombopoietin and the thrombopoietin receptor, c-mpl, as the mutated gene. Further experiments will aim to map this mutation, which may be a novel regulator of thrombopoietin.

Transgenic overexpression of interleukin-1 receptor antagonist in the CNS influences behaviour, serum corticosterone and brain monoamines

The effects of brain-directed overexpression of human soluble interleukin-1 receptor antagonist (hsIL-1ra) on behaviour, serum corticosterone (CST) levels and concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites in different brain regions, were investigated in six months old homozygotic transgenic male mice (Tg hsIL-1ra +/+). The transgenic and age-matched wild type (WT) mice were subjected to a battery of behavioural tests for analysis of open field (OF) behaviours, anxiety in elevated plus maze (EPM), and motor performance in rotarod. One week after the last behavioural test, half of the mice from each genotype were subjected to a mild stress, while the remaining mice served as controls for the determination of serum CST levels and monoamine concentrations in different brain regions. Tg hsIL-1ra +/+ mice had higher locomotor scores and showed less habituation in the OF test, spent more time in the open arms of the EPM and had similar motor performance as compared to WT mice. The serum CST levels were comparable, both in basal conditions and upon stress, in the two genotypes. Tg hsIL-1ra +/+ mice had lower concentrations of DA, 5-HT and their metabolites in several brain regions, with different effects on monoamine turnover upon stress. In conclusion, brain-directed overexpression of hsIL-1ra resulted in increased locomotion and decreased habituation, an anxiolytic effect, but did not influence motor performance. Finally, the activation of hypothalamo-pituitary-adrenal (HPA)-axis was comparable in the two genotypes, however Tg hsIL-1ra +/+ mice had a modified metabolism of brain monoamines as compared to WT mice.

"Lumen digestion" technique for isolation of aortic endothelial cells from heme oxygenase-1 knockout mice.

Endothelial cell dysfunction plays a critical role in the pathogenesis of cardiovascular diseases. Gene targeted mutant, knockout. or transgenic mice are widely used in the laboratory investigation of these disorders. We describe a simple and reproducible "lumen digestion" technique to isolate aortic endothelial cells from mice that would be useful for researchers in endothelial cell biology. We used wild-type, homozygote, or heterozygote heme oxygenase-1 null mice from which the aorta is isolated and removed under anesthesia. After cauterizing all the branches, both ends of the aorta are cannulated using an Intramedic PE-20 tube. After flushing the aorta with phosphate-buffered saline (PBS), the lumen is repeatedly instilled (five times) with 50 microL 0.25% trypsin in PBS, incubated for 2 min, and flushed with PBS. The outflow is collected in endothelial cell media with 20% fetal bovine serum. After centrifugation, the endothelial cells in the pellet are resuspended in media and plated in a 24-well tissue culture dish. Following culture for 2 to 3 weeks, the cells demonstrate typical cobblestone appearance, stain positive for the endothelial marker CD31, and are capable of low-density lipoprotein uptake. Following challenge with oxidized lipids, heme oxygenase-1 deficient endothelial cells demonstrate increased susceptibility to cell injury.

An ENU-induced mutation in AP-2alpha leads to middle ear and ocular defects in Doarad mice.

One of the advantages of N-ethyl- N-nitrosourea (ENU)-induced mutagenesis is that, after randomly causing point mutations, a variety of alleles can be generated in genes leading to diverse phenotypes. For example, transcription factor AP-2alpha ( Tcfap2a) null homozygote mice show a large spectrum of developmental defects, among them missing middle ear bones and tympanic ring. This is the usual occurrence, where mutations causing middle ear anomalies usually coincide with other abnormalities. Using ENU-induced mutagenesis, we discovered a new dominant Tcfap2a mutant named Doarad ( Dor) that has a missense mutation in the PY motif of its transactivation domain, leading to a misshapen malleus, incus, and stapes without any other observable phenotype. Dor homozygous mice die perinatally, showing prominent abnormal facial structures and ocular defects. In vitro assays suggest that this mutation causes a "gain of function" in the transcriptional activation of AP-2alpha. These mice enable us to address more specifically the developmental role of Tcfap2a in the eye and middle ear and are the first report of a mutation in a gene specifically causing middle ear abnormalities, leading to conductive hearing loss.

Mutant huntingtin directly increases susceptibility of mitochondria to the calcium-induced permeability transition and cytochrome c release.

Huntington's disease (HD) is initiated by an abnormally expanded polyglutamine stretch in the huntingtin protein, conferring a novel property on the protein that leads to the loss of striatal neurons. Defects in mitochondrial function have been implicated in the pathogenesis of HD. Here, we have examined the hypothesis that the mutant huntingtin protein may directly interact with the mitochondrion and affect its function. In human neuroblastoma cells and clonal striatal cells established from HdhQ7 (wild-type) and HdhQ111 (mutant) homozygote mouse knock-in embryos, huntingtin was present in a purified mitochondrial fraction. Subfractionation of the mitochondria and limited trypsin digestion of the organelle demonstrated that huntingtin was associated with the outer mitochondrial membrane. We further demonstrated that a recombinant truncated mutant huntingtin protein, but not a wild-type, directly induced mitochondrial permeability transition (MPT) pore opening in isolated mouse liver mitochondria, an effect that was prevented completely by cyclosporin A (CSA) and ATP. Importantly, the mutant huntingtin protein significantly decreased the Ca2+ threshold necessary to trigger MPT pore opening. We found a similar increased susceptibility to the calcium-induced MPT in liver mitochondria isolated from a knock-in HD mouse model. The mutant huntingtin protein-induced MPT pore opening was accompanied by a significant release of cytochrome c, an effect completely inhibited by CSA. These findings suggest that the development of specific MPT inhibitors may be an interesting therapeutic avenue to delay the onset of HD.

Cathepsin L-deficient mice exhibit abnormal skin and bone development and show increased resistance to osteoporosis following ovariectomy.

The role of cathepsin L in normal physiological processes was assessed using cathepsin L homozygous knockout mice (B6;129-Ctsl(tm1Alpk)). These mice were generated using gene targeting in embryonic stem cells. Null mice fail to express mRNA and protein to cathepsin L. They developed normally and were fertile. The distinct phenotypic change exhibited was a progressive hair loss, culminating in extensive alopecia by 9 months of age. Histological analysis of the skin from homozygous mice revealed diffuse epithelial hyperplasia, hypotrichosis, hair shaft fragmentation and utricle formation. These findings provide evidence that cathepsin L is involved in the regulation of epithelial cell proliferation and differentiation in the skin. In addition, the role of cathepsin L in bone remodelling was evaluated. Using bone histomorphometric measurements, trabecular, but not cortical, bone volume was found to be significantly decreased in the cathepsin L heterozygote and homozygote mice compared to the wild-type mice. Following ovariectomy, it was observed that loss of trabecular bone, the most metabolically active component of bone, occurred to a lesser extent in homozygote, and heterozygote mice, than was seen in wild-type mice. These observations suggest that cathepsin L is likely to have a role in controlling bone turnover during normal development and in pathological states.

The effect of vascular smooth muscle cell-targeted expression of tissue factor pathway inhibitor in a murine model of arterial thrombosis.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates the extrinsic pathway of coagulation by inhibiting the factor VIIa/tissue factor (TF) catalytic complex. TFPI is expressed by both endothelial and smooth muscle cells in the vasculature and circulates at low levels. The role of local vascular TFPI in thrombosis and the development of vascular disease is unknown. To establish an experimental animal model to directly modulate smooth muscle cell-derived TFPI on the development of arterial thrombosis, transgenic mice in which a cDNA encoding murine TFPI is expressed from the murine SM22alpha promoter were generated. Expression of transgenic mRNA was 4-fold higher than the level of endogenous TFPI mRNA in arteries from transgenic mice. In situ hybridization confirmed that expression of the transgene was limited to medial vascular smooth muscle cells. Vascular TFPI activity was increased to 2 to 3-fold in carotid homogenates. There was no difference in plasma TFPI levels or hemostatic measures (PT, aPTT and tail vein bleeding times) between these mice and their wildtype littermates. In a ferric chloride-induced model of carotid thrombosis, homozygotic transgenic mice demonstrated resistance to thrombotic occlusion compared to wildtype littermates. In transgenic mice 22% occluded within 30 minutes of application while 84% of wild type mice occluded within the same time frame (p<0.01). Heterozygotic transgenic mice had an intermediate thrombotic phenotype. Taken together, these data indicated that local VSMC-specific TFPI overexpression attenuated ferric chloride-induced thrombosis without systemic or hemostatic effects. Furthermore, this transgenic mouse model should prove useful for studying the role of TFPI in the development and progression of vascular disease.