Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory–fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.

Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck (Hck) and p58c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2 and β3 integrins. To determine if the defective function of hck−/−fgr−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck−/−fgr−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

Prothrombin deficiency results in embryonic and neonatal lethality in mice

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII−/−) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII−/− embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII−/− embryos within this gestational time frame. However, at least one-quarter of the FII−/− mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice

Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2−/− embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2−/− embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2−/− neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.

Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.

Doxycycline control of prion protein transgene expression modulates prion disease in mice

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at ≈50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

Nrf2 is essential for protection against acute pulmonary injury in mice

Nrf2 is a member of the “cap ‘n’ collar” family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2−/− mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2−/− mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2−/− mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2−/− mice. The Nrf2−/− mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+ T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit, in vivo, peptides other than CLIP.

Increased B-lymphopoiesis by interleukin 7 induces bone loss in mice with intact ovarian function: Similarity to estrogen deficiency

Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss due to stimulated bone resorption by osteoclasts. During our investigations of the pathogenesis of bone loss in estrogen deficiency, we found that OVX selectively stimulates B-lymphopoiesis which results in marked accumulation of B220-positive pre-B cells in mouse bone marrow. To examine the possible correlation between stimulated B-lymphopoiesis and bone loss, 8-week-old female mice were treated with interleukin (IL) 7, which stimulates B-lymphopoiesis in bone marrow. We also examined bone mass in IL-7 receptor-knockout mice that exhibit marked suppression of B-lymphopoiesis in the bone marrow. The increased B-lymphopoiesis induced by IL-7 administration resulted in marked bone loss by stimulation of osteoclastic bone resorption in mice with intact ovarian function. The changes in both B-lymphopoiesis and bone mass in IL-7-treated female mice were similar to those in age-matched OVX mice. In contrast, the trabecular bone volume of the femur was greatly increased in both female and male IL-7 receptor-knockout mice when compared with the respective wild-type and heterozygous littermates. These results show that the perturbation of B-lymphopoiesis in the bone marrow is closely linked to the change in bone mass. We propose here that the increased B-lymphopoiesis due to estrogen deficiency is involved in the mechanism of stimulated bone resorption.

Lipoprotein lipase controls fatty acid entry into adipose tissue, but fat mass is preserved by endogenous synthesis in mice deficient in adipose tissue lipoprotein lipase

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the import of triglyceride-derived fatty acids by muscle, for utilization, and adipose tissue (AT), for storage. Relative ratios of LPL expression in these two tissues have therefore been suggested to determine body mass composition as well as play a role in the initiation and/or development of obesity. To test this, LPL knockout mice were mated to transgenics expressing LPL under the control of a muscle-specific promoter (MCK) to generate induced mutants with either relative (L2-MCK) or absolute AT LPL deficiency (L0-MCK). L0-MCK mice had normal weight gain and body mass composition. However, AT chemical composition indicated that LPL deficiency was compensated for by large increases in endogenous AT fatty acid synthesis. Histological analysis confirmed that such up-regulation of de novo fatty acid synthesis in L0-MCK mice could produce normal amounts of AT as early as 20 h after birth. To assess the role of AT LPL during times of profound weight gain, L0-MCK and L2-MCK genotypes were compared on the obese ob/ob background. ob/ob mice rendered deficient in AT LPL (L0-MCK-ob/ob) also demonstrated increased endogenous fatty acid synthesis but had diminished weight and fat mass. These findings reveal marked alterations in AT metabolism that occur during LPL deficiency and provide strong evidence for a role of AT LPL in one type of genetic obesity.

Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis

A critical link between hemostatic factors and atherosclerosis has been inferred from a variety of indirect observations, including the expression of procoagulant and fibrinolytic factors within atherosclerotic vessels, the presence of fibrin in intimal lesions, and the cellular infiltration of mural thrombi leading to their incorporation into developing plaques. To directly examine the role of the key fibrinolytic factor, plasminogen, in atherogenesis, plasminogen-deficient mice were crossed to hypercholesterolemic, apolipoprotein E-deficient mice predisposed to atherosclerosis. We report that the loss of plasminogen greatly accelerates the formation of intimal lesions in apolipoprotein E-deficient animals, whereas plasminogen deficiency alone does not cause appreciable atherosclerosis. These studies provide direct evidence that circulating hemostatic factors strongly influence vessel wall disease in the context of a disorder in lipid metabolism.