Hemostasis in the mouse (Mus musculus): a review.
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Biomedical subjects
R O Hynes
Publications and source records attributed to R O Hynes.
Insights and questions arising from studies of a mouse model of Glanzmann thrombasthenia.
In summary, studies of beta 3-deficient mice demonstrate that they provide an excellent model of the human bleeding disorder, Glanzmann thrombasthenia (GT), and replicate essentially all features of the human disease. Furthermore, these mice reveal additional defects consequent upon the absence of integrins alpha v beta 3 and alpha IIB beta 3 that had not become evident from studies of the human patients. These include defects in placental development and in bone resorption. In contrast, despite published evidence suggesting a role for alpha v beta 3 in angiogenesis, beta 3-null mice have not yet shown any defects in this process. The availability of these mice will allow extensive further studies of GT pathogenesis and therapy and of the roles of beta 3 integrins in different forms of angiogenesis, including the pathologically important angiogenic processes in the retina and in response to tumors. Furthermore, they open the way to studies of a variety of other processes in which roles for beta 3 integrins have been suggested.
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Cell adhesion: old and new questions.
Metazoans clearly need cell adhesion to hold themselves together, but adhesion does much more than that. Adhesion receptors make transmembrane connections, linking extracellular matrix and adjacent cells to the intracellular cytoskeleton, and they also serve as signal transducers. In this article, I briefly summarize our present understanding of the molecular basis and biological consequences of cell adhesion and discuss how our current knowledge sheds light on questions of specificity of cell adhesion. I offer some thoughts and speculations about the evolution of cell-adhesion molecules and processes, consider their inter-relationships with other forms of cell-cell communication and discuss unresolved questions ripe for investigation as we enter the postgenomic era.
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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.
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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.
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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.
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Layilin, a novel talin-binding transmembrane protein homologous with C-type lectins, is localized in membrane ruffles.
Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten-amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin's amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.
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Overlapping functions of E- and P-selectin in neutrophil recruitment during acute inflammation.
Selectin adhesion molecules mediate leukocyte rolling on activated endothelium, a prerequisite to leukocyte accumulation at sites of inflammation. The precise role of each selectin (E-, P-, and L-) in this process is unclear and may vary depending on the particular inflammatory stimulus, vascular bed, leukocyte subset, and species; most data suggest discrete functional roles for each selectin. To define the relative roles of E- and P-selectin in mediating neutrophil accumulation in acute dermal inflammation, mice genetically deficient in E-selectin, P-selectin, or both E- and P-selectin were injected intradermally with zymosan. Luminal endothelial expression of E- and P-selectin in response to zymosan was documented in wild-type mice by intravenous administration of fluorochrome-labeled anti-E- and anti-P-selectin antibodies. In mice deficient in E- or P-selectin, neutrophil accumulation was unchanged or only subtly reduced relative to wild-type control mice. In mice deficient in both E- and P-selectin, neutrophil accumulation was significantly reduced (87% at 4 hours and 79% at 8 hours). These data demonstrate that, in this model of acute inflammation, there is considerable overlap in the functions of E- and P-selectin; loss of both selectins was required to impair neutrophil accumulation.
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Novel roles for alpha3beta1 integrin as a regulator of cytoskeletal assembly and as a trans-dominant inhibitor of integrin receptor function in mouse keratinocytes.
Previously we found that alpha3beta1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of alpha3beta1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of alpha3beta1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of alpha3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and alpha-actinin at focal contact sites in the alpha3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to alpha3beta1 deficiency. Apart from the loss of alpha3beta1 there is no change in expression of the other integrins expressed by the alpha3-null keratinocytes. However, in functional assays, alpha3beta1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of alpha3beta1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.
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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.
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Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.
We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by alpha4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.
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Integrin-mediated signals regulated by members of the rho family of GTPases.
The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.
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The combined role of P- and E-selectins in atherosclerosis.
P- and E-selectins are adhesion molecules mediating the first step in leukocyte extravasation. Because their function in leukocyte adhesion is overlapping, we hypothesized that there might be a combined effect of these selectins on the development of atherosclerotic lesions. We bred P- and E-selectin-double-deficient mice onto the low-density lipoprotein receptor (LDLR)-deficient background (LDLR-/- P/E-/-) and compared lesion development in these mice to that in mice wild type for both selectins (LDLR-/- P/E+/+). After 8 wk on atherogenic diet, the LDLR-/- P/E-/- mice developed fatty streaks in the aortic sinus that were five times smaller than those in LDLR-/- P/E+/+ mice. The density of macrophages in the fatty streaks was comparable between LDLR-/- P/E+/+ and LDLR-/- P/E-/- mice. After 22 wk on the diet, the lesions spread throughout the aorta but this process was delayed in LDLR-/- P/E-/- mice. At 37 wk on diet, the lesions progressed to the fibrous plaque stage in both genotypes. However, the lesions in the aortic sinus in LDLR-/- P/E-/- mice were 40% smaller and less calcified than those of LDLR-/- P/E +/+ mice. Our results suggest that P- and E-selectins together play an important role in both early and advanced stages of atherosclerotic lesion development.
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Thrombospondin-1 is a major activator of TGF-beta1 in vivo.
The activity of TGF-beta1 is regulated primarily extracellularly where the secreted latent form must be modified to expose the active molecule. Here we show that thrombospondin-1 is responsible for a significant proportion of the activation of TGF-beta1 in vivo. Histological abnormalities in young TGF-beta1 null and thrombospondin-1 null mice were strikingly similar in nine organ systems. Lung and pancreas pathologies similar to those observed in TGF-beta1 null animals could be induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-beta1 by thrombospondin-1. Although these organs produced little active TGF-beta1 in thrombospondin null mice, when pups were treated with a peptide derived from thrombospondin-1 that could activate TGF-beta1, active cytokine was detected in situ, and the lung and pancreatic abnormalities reverted toward wild type.
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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.
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Platelet-endothelial interactions in inflamed mesenteric venules.
The selectins are membrane glycoproteins promoting adhesive events between leukocytes, platelets, and endothelial cells. We have previously demonstrated that platelets roll on P-selectin expressed on stimulated endothelium. In this study, we wished to examine the function of both the platelet and endothelial selectins, P- and E-selectins, in mediating platelet-endothelial interactions during inflammation. We demonstrate, using intravital microscopic examination of venules inflamed with tumor necrosis factor-alpha (TNF-alpha), that resting platelets interact with both P- and E-selectins and that the leukocyte alpha(1,3)fucosyltransferases FucT IV and FucT VII do not provide platelets with selectin ligand activity. We also show that after thrombin activation of wild-type (+/+) platelets, platelet P-selectin can mediate interactions on a TNF-alpha-inducible endothelial ligand. To evaluate the potential role of platelet P-selectin in the recruitment of leukocytes to inflammatory sites, we reconstituted the bone marrow of mice deficient in both P- and E-selectins (P/E-/-) with wild-type (+/+) or P-selectin-deficient (P-/-) bone marrow containing megakaryocytic precursors. Providing +/+ platelets to P/E-/- mice by bone marrow transplantation did not rescue the immunodeficient phenotype, suggesting that platelet P-selectin does not have an active function in the recruitment of leukocytes into inflammatory sites. To participate in inflammatory or hemostatic responses, platelets may use the endothelial selectins.
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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.
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E and P selectins are not required for resistance to severe murine lyme arthritis.
Borrelia burgdorferi-induced arthritis in mice is characterized by tendonitis, synovitis, and inflammatory-cell infiltrate, predominantly of neutrophils. Because genetic deficiency in E and P selectins results in delayed recruitment of neutrophils to sites of inflammation, mice with this deficiency were tested for their response to infection with B. burgdorferi. E and P selectins were not required for the control of B. burgdorferi numbers, nor did deficiency in E and P selectins result in alteration of arthritis severity.
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