Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)-deficient mice.

Mammalian female fertility depends on complex interactions between the ovary and the extraovarian environment (e.g., the hypothalamic-hypophyseal ovarian axis). The role of plasma lipoproteins in fertility was examined using HDL-receptor SR-BI knockout (KO) mice. SR-BI KO females have abnormal HDLs, ovulate dysfunctional oocytes, and are infertile. Fertility was restored when the structure and/or quantity of abnormal HDL was altered by inactivating the apoAI gene or administering the cholesterol-lowering drug probucol. This suggests that abnormal lipoprotein metabolism can cause murine infertility--implying a functional hepatic-ovarian axis--and may contribute to some forms of human female infertility.

Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium.

To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase (Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the beta-galactosidase (LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins.

Mice chimeric for integrins alpha(5), alpha(V), or alpha(4) were used to dissect the in vivo roles of these adhesion receptors during leukocyte development and traffic. No major defects were observed in the development of lymphocytes, monocytes, or granulocytes or in the traffic of lymphocytes to different lymphoid organs in the absence of alpha(5) or alpha(V) integrins. However, in agreement with previous reports, the absence of alpha(4) integrins produced major defects in development of lymphoid and myeloid lineages and a specific defect in homing of lymphocytes to Peyer's patches. In contrast, the alpha(4) integrin subunit is not essential for localization of T lymphocytes into intraepithelial and lamina propria compartments in the gut, whereas one of the partners of alpha(4), the beta(7) chain, has been shown to be essential. However, alpha(4)-deficient T lymphocytes cannot migrate properly during the inflammatory response induced by thioglycolate injection into the peritoneum. Finally, in vitro proliferation and activation of lymphocytes deficient for alpha(5), alpha(V), or alpha(4) integrins upon stimulation with different stimuli were similar to those seen in controls. These results show that integrins play distinct roles during in vivo leukocyte development and traffic.

Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo.

We investigated roles of alpha4 integrins during hematopoiesis using mutant and chimeric mice. Yolk sac erythropoiesis and migration of hematopoietic progenitors to fetal liver, spleen, and bone marrow can occur without alpha4 integrins. Although terminal differentiation of these progenitors is possible without alpha4 integrins, these receptors are essential to maintain normal hematopoiesis in fetal liver, spleen, and bone marrow microenvironments. Moreover, alpha4-deficient erythroid progenitors and pre-B cells neither transmigrate beneath the stroma nor expand-properly in vitro. In contrast, alpha4-null cells migrate and differentiate efficiently into T lymphocytes within the thymus. In summary, alpha4 integrins are essential for normal development of all hematopoietic lineages in fetal liver, bone marrow, and spleen, likely by regulating the proliferation/differentiation balance of hematopoietic progenitors.

Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds.

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.

Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation.

CD39, or vascular adenosine triphosphate diphosphohydrolase, has been considered an important inhibitor of platelet activation. Unexpectedly, cd39-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro. This platelet hypofunction was reversible and associated with purinergic type P2Y1 receptor desensitization. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in cd39-deficient mice and in transplanted cardiac grafts. Our data indicate a dual role for adenosine triphosphate diphosphohydrolase in modulating hemostasis and thrombotic reactions.

Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment.

We extend our previous analyses of mice deficient in selectins by describing the generation and comparative phenotype of mice lacking one, two, or three selectins after sequential ablation of the murine genes encoding P-, E-, and L-selectins. All mice deficient in selectins are viable and fertile as homozygotes. However, mice missing both P- and E-selectins (PE(-/-)), and mice missing all three selectins (ELP(-/-)) develop mucocutaneous infections that eventually lead to death. Mice deficient in multiple selectins display varying degrees of leukocytosis, resulting in part from alterations in leukocyte rolling and recruitment. PE(-/-) mice, ELP(-/-) mice, and mice missing both P- and L-selectins (PL(-/-)) show drastic reductions in leukocyte rolling and in extravasation of neutrophils in thioglycollate-induced peritonitis. In a separate inflammatory model (ragweed-induced peritoneal eosinophilia), we demonstrate P-selectin to be both necessary and sufficient for the recruitment of eosinophils. The phenotype of mice missing both E- and L-selectins (EL(-/-)) is less severe than those seen in the other double knockouts. Comparisons among the double knockouts suggest that P-selectin normally cooperates with both E- and L-selectins. Our results indicate a preeminent role for P-selectin in regulating leukocyte behavior in mice. Data from the ELP(-/-) mice indicate, however, that all three selectins are important to leukocyte homeostasis and efficient neutrophil recruitment.

Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

Intravascular coagulation activation in a murine model of thrombomodulin deficiency: effects of lesion size, age, and hypoxia on fibrin deposition.

We consecutively inactivated both alleles of the thrombomodulin (TM) gene in murine embryonic stem (ES) cells and generated TM-deficient (TM-/-) chimeric mice. Quantitation of an ES-cell marker and protein C cofactor activity indicates that up to 50% of pulmonary endothelial cells are ES-cell derived and therefore TM deficient. Infusions of 125I-fibrinogen into mice show a significant increase (fourfold, P <.005) in radiolabeled cross-linked fibrin in TM-/- chimeric mouse lung as compared with wild-type mice. However, only chimeric mice that exhibit at least a 30% reduction in protein C cofactor activity and are at least 15 months old display this phenotype. Immunocytochemical localization of TM in chimeras shows a mosaic pattern of expression in both large and small blood vessels. Colocalization of cross-linked fibrin and neo (used to replace TM) reveals that fibrin is deposited in TM-/- regions. However, the fibrin deposits were largely restricted to pulmonary vessels with a lumenal area greater than 100 micrometer2. The hypercoagulable phenotype can be induced in younger chimeric mice by exposure to hypoxia, which causes a fivefold increase in beta-fibrin levels in lung. Our findings show that TM chimerism results in spontaneous, intravascular fibrin deposition that is dependent on age and the magnitude of the TM deficiency.

Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins.

alphav integrins have been implicated in many developmental processes and are therapeutic targets for inhibition of angiogenesis and osteoporosis. Surprisingly, ablation of the gene for the alphav integrin subunit, eliminating all five alphav integrins, although causing lethality, allows considerable development and organogenesis including, most notably, extensive vasculogenesis and angiogenesis. Eighty percent of embryos die in mid-gestation, probably because of placental defects, but all embryos develop normally to E9.5, and 20% are born alive. These liveborn alphav-null mice consistently exhibit intracerebral and intestinal hemorrhages and cleft palates. These results necessitate reevaluation of the primacy of alphav integrins in many functions including vascular development, despite reports that blockade of these integrins with antibodies or peptides prevents angiogenesis.

Dystrophic muscle in mice chimeric for expression of alpha5 integrin.

alpha5-deficient mice die early in embryogenesis (). To study the functions of alpha5 integrin later in mouse embryogenesis and during adult life we generated alpha5 -/-;+/+ chimeric mice. These animals contain alpha5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that alpha5 -/- cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (). Histological analysis of alpha5 -/-;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of alpha5 -/- ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is alpha5-integrin-dependent.

A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.

von Willebrand factor (vWf) deficiency causes severe von Willebrand disease in humans. We generated a mouse model for this disease by using gene targeting. vWf-deficient mice appeared normal at birth; they were viable and fertile. Neither vWf nor vWf propolypeptide (von Willebrand antigen II) were detectable in plasma, platelets, or endothelial cells of the homozygous mutant mice. The mutant mice exhibited defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events in approximately 10% of neonates. As in the human disease, the factor VIII level in these mice was reduced strongly as a result of the lack of protection provided by vWf. Defective thrombosis in mutant mice was also evident in an in vivo model of vascular injury. In this model, the exteriorized mesentery was superfused with ferric chloride and the accumulation of fluorescently labeled platelets was observed by intravital microscopy. We conclude that these mice very closely mimic severe human von Willebrand disease and will be very useful for investigating the role of vWf in normal physiology and in disease models.

PDGF mediates cardiac microvascular communication.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state.

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.

The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.

A test of the role of alpha5 integrin/fibronectin interactions in tumorigenesis.

Published data show that reduction or loss of fibronectin or its receptor, alpha5beta1 integrin, occurs frequently in tumors and transformed cells. Furthermore, restoration of these adhesion proteins has been reported to reduce tumorigenesis. These results suggest that fibronectin/alpha5beta1 interactions may act to suppress tumor development or progression. To test this hypothesis in the context of spontaneous tumor formation, we have analyzed tumor development in mice genetically altered in the genes for fibronectin or alpha5 integrin. Our results show that heterozygosity for either does not lead to an increased incidence of tumors, alteration in tumor spectrum, or increased levels of metastasis, even when the fibronectin or alpha5 mutations are combined with mutations in the p53 tumor suppressor gene that lead to spontaneous tumor formation and could also cause loss of heterozygosity. Furthermore, loss of heterozygosity for alpha5 was not a common concomitant of tumorigenesis or metastasis. Finally, chimeric animals containing high proportions of alpha5-null cells did not show an increased incidence of tumors or a change in tumor progression. We conclude that, in the genetic backgrounds studied here, loss of fibronectin or alpha5beta1 integrin does not contribute to tumorigenesis or metastasis.

Precocious mammary gland development in P-cadherin-deficient mice.

To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin- deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin-null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin-null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin-mediated adhesion and/or signals derived from cell-cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by approximately 31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.