Hemostasis and coagulation at a hematocrit level of 0.85: functional consequences of erythrocytosis

Abstract We have generated a transgenic mouse line that reaches a hematocrit concentration of 0.85 due to constitutive overexpression of human erythropoietin in an oxygen-independent manner. Unexpectedly, this excessive erythrocytosis did not lead to thrombembolic complications in all investigated organs at any age. Thus, we investigated the mechanisms preventing thrombembolism in this mouse model. Blood analysis revealed an age-dependent elevation of reticulocyte numbers and a marked thrombocytopenia that matched the reduced megakaryocyte numbers in the bone marrow. However, platelet counts were not different from wild-type controls, when calculations were based on the distribution (eg, plasma) volume, thereby explaining why thrombopoietin levels did not increase in transgenic mice. Nevertheless, bleeding time was significantly increased in transgenic animals. A longitudinal investigation using computerized thromboelastography revealed that thrombus formation was reduced with increasing age from 1 to 8 months in transgenic animals. We observed that increasing erythrocyte concentrations inhibited profoundly and reversibly thrombus formation and prolonged the time of clot development, most likely due to mechanical interference of red blood cells with clot-forming platelets. Transgenic animals showed increased nitric oxide levels in the blood that could inhibit vasoconstriction and platelet activation. Finally, we observed that plasmatic coagulation activity in transgenic animals was significantly decreased. Taken together, our findings suggest that prevention of thrombembolic disease in these erythrocytotic transgenic mice was due to functional consequences inherent to increased erythrocyte concentrations and a reduction of plasmatic coagulation activity, the cause of which remains to be elucidated.

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Expression of bovine β-lactoglobulin transgenic mice

Journal of Dairy Research ◽

10.1017/s0022029999003428 ◽

1999 ◽

Vol 66 (2) ◽

pp. 289-294 ◽

Cited By ~ 4

Author(s):

ALFONSO GUTIÉRREZ-ADÁN ◽

ELIZABETH A. MAGA ◽

ESMAIL BEHBOODI ◽

JANICE S. CONRAD-BRINK ◽

ANTHONY G. MACKINLAY ◽

...

Keyword(s):

Mammary Gland ◽

Transgenic Mice ◽

Transgenic Animals ◽

Milk Composition ◽

Developmentally Regulated ◽

Independent Manner ◽

Milk Protein Genes ◽

Dairy Animals ◽

Foreign Proteins ◽

Β Lactoglobulin

The use of transgenic animals to manipulate milk composition has considerable potential, both for the production of biomedical proteins and for the direct manipulation of milk composition for the improvement of dairy animals and their products (for reviews, see Wall et al. 1992; Yom & Bremel, 1993). Promoters from a number of milk protein genes from a variety of species have been tested for their ability to direct the expression of foreign proteins to the mammary gland (for review, see Maga & Murray, 1995).β-Lactoglobulin (β-lg) is the major whey protein produced in ruminant milk and is part of the normal milk composition of most mammals except humans and rodents (Pervaiz & Brew, 1985). It is expressed at high levels in the mammary gland and is developmentally regulated. Transgenic mice have been produced using the complete ovine (Simons et al. 1987; Shani et al. 1992) and caprine (Ibañez et al. 1997) β-lg genes. In general, high levels of expression were obtained with the ovine β-lg gene, and expression was also seen in a position-independent manner (Whitelaw et al. 1992). Lower levels of expression were reported using the caprine β-lg gene. Here we report the production of transgenic mice using the bovine β-lg gene. We describe high expression, position-dependent, and copy number-related expression of bovine β-lg protein in the milk of six lines of transgenic mice.

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Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

Biochemical Journal ◽

10.1042/bj3380311 ◽

1999 ◽

Vol 338 (2) ◽

pp. 311-316 ◽

Cited By ~ 17

Author(s):

Suvikki SUPPOLA ◽

Marko PIETILÄ ◽

Jyrki J. PARKKINEN ◽

Veli-Pekka KORHONEN ◽

Leena ALHONEN ◽

...

Keyword(s):

Enzyme Activity ◽

Transgenic Mice ◽

Transgenic Mouse ◽

Cell Injury ◽

Mrna Level ◽

Transgenic Animals ◽

Ultrastructural Changes ◽

Mouse Line ◽

Transgenic Mouse Line ◽

Polyamine Analogue

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT–SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and > 90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1–2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.

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Transgenic mice expressing the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter

Biochemical Journal ◽

10.1042/bj3140405 ◽

1996 ◽

Vol 314 (2) ◽

pp. 405-408 ◽

Cited By ~ 8

Author(s):

Leena ALHONEN ◽

Sami HEIKKINEN ◽

Riitta SINERVIRTA ◽

Maria HALMEKYTÖ ◽

Pekka ALAKUIJALA ◽

...

Keyword(s):

Transgenic Mice ◽

Ornithine Decarboxylase ◽

Mammalian Cells ◽

Fold Increase ◽

Transgenic Animals ◽

Decarboxylase Activity ◽

Transgenic Mouse Line ◽

Regulatory Machinery ◽

Dividing Cells ◽

Very High

We have generated a transgenic mouse line harbouring the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter. Even in the absence of an exposure to heavy metals, ornithine decarboxylase was over-expressed in heart, testis, brain, and especially in liver, of the transgenic animals. An exposure of the transgenic mice to zinc further enhanced the enzyme activity to a level which in liver represented up to 8000-fold increase in comparison with non-transgenic animals. The striking stimulation of liver ornithine decarboxylase activity upon treatment of the transgenic mice with zinc was accompanied by a nearly 150-fold increase in the hepatic putrescine content as compared with similarly treated non-transgenic animals. Even though the liver putrescine concentration reached that of spermidine and spermine in the transgenic animals, the contents of the higher polyamines only transiently increased upon zinc administration and then returned to the basal level. These findings once again indicate that mammalian cells possess extremely powerful regulatory machinery to prevent an over-accumulation of spermidine and spermine in non-dividing cells, and that very high tissue putrescine concentrations can be tolerated, at least for periods of a few days, with seemingly no phenotypic changes.

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Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice.

Journal of Experimental Medicine ◽

10.1084/jem.180.3.861 ◽

1994 ◽

Vol 180 (3) ◽

pp. 861-872 ◽

Cited By ~ 139

Author(s):

R Cibotti ◽

J P Cabaniols ◽

C Pannetier ◽

C Delarbre ◽

I Vergnon ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Transgenic Mice ◽

Transgenic Animals ◽

Cell Receptor ◽

T Cell Response ◽

Terminal Deoxynucleotidyl Transferase ◽

Blood Levels ◽

Transgenic Mouse Line ◽

Hen Egg

We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.

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Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

Thrombosis and Haemostasis ◽

10.1160/th06-05-0277 ◽

2007 ◽

Vol 97 (01) ◽

pp. 99-108 ◽

Cited By ~ 74

Author(s):

Yuxi Feng ◽

Franziska vom Hagen ◽

Frederick Pfister ◽

Snezana Djokic ◽

Sigrid Hoffmann ◽

...

Keyword(s):

Transgenic Mice ◽

Transgenic Animals ◽

Mouse Retina ◽

Vascular Density ◽

Wild Type ◽

Transgenic Mouse Line ◽

Oxygen Induced Retinopathy ◽

Genes Encoding ◽

Angiopoietin 2 ◽

The Impact

SummaryAngiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p<0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26 %; p<0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

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Transgenic mice over-expressing galanin exhibit pituitary adenomas and increased secretion of galanin, prolactin and growth hormone

Journal of Endocrinology ◽

10.1677/joe.0.1790145 ◽

2003 ◽

Vol 179 (2) ◽

pp. 145-154 ◽

Cited By ~ 23

Author(s):

P Perumal ◽

ME Vrontakis

Keyword(s):

Transgenic Mice ◽

Secretory Granules ◽

Cell Lineage ◽

Transgenic Animals ◽

Serum Levels ◽

Pituitary Hormones ◽

Biologically Active ◽

Serum Prolactin ◽

Independent Manner ◽

Pituitary Hyperplasia

Galanin is a biologically active 29 amino acid peptide, widely distributed in the central and peripheral nervous system, and most abundantly in the hypothalamus where it may serve in the regulation of anterior pituitary hormones. We herein report that mice carrying the rat preprogalanin cDNA specifically targeted to the somatomammotroph cell lineage, under the control of the rat GH promoter, over-express and over-secrete galanin. Galanin peptide is localised within the GH and prolactin secretory granules. GH and prolactin release is increased as well, predominantly in males, while older transgenic animals develop pituitary hyperplasia and adenoma. In both male and female transgenic mice there is a significant increase in serum galanin (P<0.00003 and P<0.001 respectively) and prolactin (P<0.002 and P<0.05 respectively) levels, while only in male transgenic mice is there a significant increase in the serum levels of GH. Furthermore, in male transgenic mice serum prolactin levels are significantly correlated with the serum galanin levels (P<0.03). We conclude that galanin plays a key role in the process of pituitary hyperplasia, acting as a growth factor to promote pituitary cell proliferation, and participates in pituitary adenoma formation not necessarily dependent on oestrogens. Targeted over-expression and over-secretion of galanin in the somatomammotroph cell lineage stimulates predominantly hyperprolactinaemia in an oestrogen-independent manner.

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Restriction of neuroblastoma to the prostate gland in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4518-4527.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4518-4527

Author(s):

D G Skalnik ◽

D M Dorfman ◽

D A Williams ◽

S H Orkin

Keyword(s):

Transgenic Mice ◽

Prostate Gland ◽

Simian Virus 40 ◽

Transgenic Animals ◽

Simian Virus ◽

Biochemical Properties ◽

T Antigen ◽

Cell Of Origin ◽

Independent Manner ◽

Early Region

Male transgenic mice that carry a construct containing 5'-flanking sequences of the gp91-phox gene linked to the early region of the simian virus 40 (SV40) genome reproducibly develop tumors arising from the prostate gland. As gp91-phox is expressed exclusively in terminally differentiating hematopoietic cells of the myelomonocytic lineage, the induction of tumors arising from the prostate gland was unexpected. These lesions appear to be due to a novel transcription signal that was generated during the construction of the transgene. Surprisingly, the histopathological and biochemical properties of the tumor are diagnostic of neuroblastoma rather than of adenocarcinoma of the prostate gland. Tumors produce SV40 T antigen and isoforms of neural cell adhesion molecule characteristic of neuronal cells, and they occur in a testosterone-independent manner. Microscopic examination of prostate glands from young transgenic mice reveals the presence of small lesions arising outside of the prostate gland epithelium, which is consistent with the diagnosis of neuroblastoma and further distinguishes this tumor from prostatic adenocarcinoma. Prostate gland tumors occur in all male animals of susceptible lines carrying the gp91-phox promoter/SV40 early-region transgene. However, variability in the time at which gross tumors appear and the presence of cells expressing T antigen prior to tumorigenesis suggest that somatic events in addition to T-antigen production are required for the development of a malignancy. The extraordinary restriction of the site of tumorigenesis in these animals indicates the presence in the prostate gland of a novel, tissue-specific neuroectodermal cell of origin. These transgenic animals provide a model system for the study of neuroectodermal malignancies.

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Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N1-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice

Biochemical Journal ◽

10.1042/bj3580343 ◽

2001 ◽

Vol 358 (2) ◽

pp. 343-348 ◽

Cited By ~ 12

Author(s):

Suvikki SUPPOLA ◽

Sami HEIKKINEN ◽

Jyrki J. PARKKINEN ◽

Mikko UUSI-OUKARI ◽

Veli-Pekka KORHONEN ◽

...

Keyword(s):

Transgenic Mice ◽

Ornithine Decarboxylase ◽

Transgenic Animals ◽

Transgenic Mouse Line ◽

Polyamine Catabolism ◽

Permanent Loss ◽

Organ Specific ◽

Polyamine Analogue ◽

Efflux Mechanism ◽

Ssat Activity

We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N1,N11-diethylnorspermine. In tracer experiments with [14C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.

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Restriction of neuroblastoma to the prostate gland in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4518 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4518-4527 ◽

Cited By ~ 26

Author(s):

D G Skalnik ◽

D M Dorfman ◽

D A Williams ◽

S H Orkin

Keyword(s):

Transgenic Mice ◽

Prostate Gland ◽

Simian Virus 40 ◽

Transgenic Animals ◽

Simian Virus ◽

Biochemical Properties ◽

T Antigen ◽

Cell Of Origin ◽

Independent Manner ◽

Early Region

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Transgenic mice over-expressing the human spermidine synthase gene

Biochemical Journal ◽

10.1042/bj2930513 ◽

1993 ◽

Vol 293 (2) ◽

pp. 513-516 ◽

Cited By ~ 13

Author(s):

L Kauppinen ◽

S Myöhänen ◽

M Halmekytö ◽

L Alhonen ◽

J Jänne

Keyword(s):

Transgenic Mice ◽

Reverse Transcriptase ◽

Human Gene ◽

Transgenic Animals ◽

Chromosome 1 ◽

Pcr Analysis ◽

Spermidine Synthase ◽

Mouse Line ◽

Transgenic Mouse Line ◽

Adenosylmethionine Decarboxylase

We have generated a transgenic mouse line harbouring the functional (chromosome-1-derived) human spermidine synthase (EC 2.5.1.16) gene in their genome. The transgenic animals expressed the human gene-derived mRNA, as revealed by reverse-transcriptase/PCR analysis, in all tissues studied and displayed tissue spermidine synthase activity that was 2-6 times that in their syngenic littermates. The elevated spermidine synthase activity, however, had virtually no effect on tissue putrescine, spermidine or spermine levels. The view that the accumulation of spermidine and spermine is possibly controlled by S-adenosylmethionine decarboxylase was further supported by the finding that tissue spermidine and spermine contents also remained practically normal in hybrid transgenic mice over-expressing both human ornithine decarboxylase and spermidine synthase genes.

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