Genetic manipulation of vascular adhesion molecules in mice.
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Animals↗
Biomedical subjects
R O Hynes
Publications and source records attributed to R O Hynes.
Differential requirements for alpha4 integrins during fetal and adult hematopoiesis.
Mice chimeric for the expression of alpha4 integrins were used to dissect the roles of these receptors in development and traffic of lymphoid and myeloid cells. During fetal life, T cell development is alpha4 independent, but after birth further production of T cells becomes alpha4 dependent. Precursors for both T and B cells require alpha4 integrins for normal development within the bone marrow. In contrast, monocytes and natural killer cells can develop normally without alpha4 integrins. Thus, there are lymphocyte-specific, developmentally regulated requirements for alpha4 integrins in hematopoiesis in the bone marrow. We also show that alpha4 integrins are essential for T cell homing to Peyer's patches, but not to other secondary lymphoid organs, including spleen, lymph nodes, and intestinal epithelium.
Age Factors↗
Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization.
Cell adhesion to the extracellular matrix triggers a cascade of intracellular biochemical signals regulated by the integrin family of receptors. Recent evidence suggests that integrin engagement may activate a mitogen-activated protein (MAP) kinase cascade that may cooperate with more clearly defined mitogenic signaling pathways to regulate cell proliferation, adhesion, and migration. Here we report that the adhesion-dependent activation of the MAP kinase Erk2 (extracellular signal-regulated kinase 2) occurs in serum-starved NIH3T3 cells, and that this activation of Erk2 is preceded by the activation of the small GTP-binding protein Ras in fibronectin-adherent cells. Inhibition of Ras signaling by expression of a dominant-inhibitory mutant of Ras (N17Ras) in NIH3T3 cells blocked adhesion-dependent activation of Erk2, although the focal adhesion kinase (FAK) was still activated in these cells. Furthermore, activation of this Ras-MAP kinase pathway activated cytosolic phospholipase A2, leading to the release of arachidonic acid metabolites, and N17Ras also inhibited these events. However, N17Ras expression does not inhibit cell adhesion, spreading, or focal contact and stress fiber formation. These results suggest that, while integrin-dependent activation of this MAP kinase pathway is Ras-dependent, the integrin-dependent activation of FAK and several morphological events are Ras-independent. Thus, integrin-mediated signals involved in regulating cell morphology appear to diverge from those regulating MAP kinase activation at a level upstream of Ras activation.
3T3 Cells↗
Cytokine-induced meningitis is dramatically attenuated in mice deficient in endothelial selectins.
Leukocyte accumulation in cerebrospinal fluid and disruption of the blood-brain barrier are central components of meningitis and are associated with a poor prognosis. Genetically engineered deficiencies or functional inhibition of endothelial leukocyte adhesion receptors P-, or P- plus E-selectins, lead to deficits in leukocyte rolling and extravasation. However, their impact on meningeal inflammation has not been tested previously. An acute cytokine-induced meningitis model associated with significant cerebrospinal fluid leukocyte accumulation (averaging 14,000 leukocytes/microl as early as 4 h) and blood-brain barrier permeability was developed in adult mice. This model was applied to mice deficient in P-selectin and mice doubly deficient in P- and E-selectins. Partial inhibition of cerebrospinal fluid leukocyte influx and permeability was noted in P-selectin-deficient mice. Mice doubly deficient in P- and E-selectins displayed a near complete inhibition of these parameters. Our results suggest that P- and E-selectins cooperatively contribute to meningitis and that functional blocking of both endothelial selectins in conjunction with antibiotics may provide a therapeutic approach for treatment of bacterial meningitis.
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Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins.
We describe the phenotype of mice lacking both endothelial selectins after sequential ablation of the genes encoding P- and E-selectins. In contrast with the rather mild phenotypes observed in mice deficient in a single selectin gene, the doubly deficient mice present extreme leukocytosis, elevated cytokine levels, and alterations in hematopoiesis. Granulocytopoiesis is increased both in bone marrow and spleen, while erythropoiesis is partially translocated to the spleen. Virtual lack of leukocyte rolling and low extravasation at sites of inflammation make these animals susceptible to opportunistic bacterial infections, to which they succumb. Our results show that the absence of endothelial selectins severely affects leukocyte homeostasis and indicate that these two selectins are as important for normal leukocyte function as are the leukocyte beta2 integrins.
Alleles↗
Defects in hemostasis in P-selectin-deficient mice.
Recently, our laboratory showed that platelets, like leukocytes, roll on activated endothelium expressing P-selectin, thus suggesting a role for P-selectin in hemostasis (Frenette et at, Proc Natl Acad Sci USA 92:7450, 1995). We report here that the P-selectin--deficient mice show a 40% prolongation of the bleeding time on amputation of the tip of the tail. Moreover, defective hemostasis was observed in a local Shwartzman-like reaction induced by skin injections of lipopolysaccharide followed by tumor necrosis factor-alpha in the P-selectin--deficient mice. The hemorrhagic lesions, quantitated both macroscopically and microscopically, were twofold larger in the P-selectin--deficient mice. This was also confirmed by measuring the radioactivity in the skin using chromium-labeled red blood cells. Therefore, it is evident that P-selectin plays a role in hemostasis as suggested by its support of platelet rolling.
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Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.
The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.
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Mesodermal development in mouse embryos mutant for fibronectin.
Three independent mutations were made by homologous recombination in two different regions of the fibronectin (FN) gene; all three appeared to be functional null mutations. The embryonic lethal phenotypes of these mutations were indistinguishable; all three FN mutant strains show mesodermal defects and fail to develop notochord or somites. Nevertheless analysis with lineage markers (Brachyury, sonic hedgehog, Notch-1, and mox-1) showed that both the notochord and the somite lineages were induced at the correct times and places. Furthermore, notochord precursor cells showed extensive cell migration in the absence of FN. However, neither notochord nor somites condensed properly in the absence of FN. These results show that specification of notochordal and somitic mesodermal lineages and significant cell migration are independent of fibronectin but that correct morphogenesis of these structures is FN-dependent.
Animals↗
Double knockout highlights value of endothelial selectins.
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Animals↗
Tumour suppressor genes and angiogenesis: the role of TP53 in fibroblasts.
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Animals↗
Acute passive anti-glomerular basement membrane nephritis in P-selectin-deficient mice.
P-selectin present on surfaces of activated endothelium and platelets mediates neutrophil-endothelial and neutrophilplatelet interactions. The role of P-selectin in vivo was examined in a model of acute passive anti-GBM nephritis in P-selectin-deficient and wild-type mice which was induced by intravenous injection of anti-GBM serum. There were two major differences between P-selectin-deficient and wild-type mice. Firstly, mutant mice had approximately two fold more glomerular PMNs and albuminuria than wild-type animals at the peak of neutrophil influx and proteinuria. Secondly, Lipoxin A4 (LXA4), an eicosanoid which inhibits leukocyte-endothelial adhesion in vitro, and is generated primarily by transcellular biosynthetic routes during P-selectin-mediated platelet-PMN interaction [1], was approximately 60% of wild type levels in nephritic kidneys of P-selectin-deficient mice. Injection of wild-type platelets into P-selectin-null mice restored LXA4 to wild-type levels. The corresponding PMN influx approximated PMN levels in wild-type mice receiving platelets but urine albuminuria remained higher. Although these two P-selectin-dependent events cannot be directly linked, our results point to the importance of considering both platelet and endothelial P-selectin in determining the cellular events in inflammation.
Acute Disease↗
Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle.
It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.
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Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.
alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.
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Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.
We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.
Actins↗
Fibronectin isoform distribution in the mouse. I. The alternatively spliced EIIIB, EIIIA, and V segments show widespread codistribution in the developing mouse embryo.
Fibronectins (FNs) are extracellular matrix glycoproteins that are essential for embryonic development. In order to gain clues to possible developmental roles played by the particular isoforms of FN, we used indirect immunofluorescence microscopy to examine and compare the distributions of the alternatively spliced EIIIB, EIIIA, and V segments, as well as the total pool of FNs, in serial sections from mouse embryos. Antibodies to each of these segments produced staining patterns that colocalized during gastrulation (E7.5) and during early morphogenesis of somites and notochord (E9.5). During the period of continuing organogenesis in the latter half of gestation (E10.5 to E16.5), the antibodies generally continued to produce similar staining patterns localized to epithelial basement membranes, stromal connective tissues, blood vessel walls, and muscles. However, as development proceeded, there was a gradual decline in the intensity of staining for the spliced segments relative to the total pool of FN, with a particularly noticeable decline in staining for EIIIB and EIIIA segments in certain glandular organs, including the liver. A specific reduction in expression of these latter two segments was also evident in the uterus and placenta at early timepoints in gestation. However, the most dramatic difference in the expression of the spliced segments occurred in developing hyaline cartilage, which showed a selective reduction in staining for the EIIIA segment that was evident in the axial skeletal precursors by E12.5 and complete throughout the embryo by E15.5. Our findings suggest that the alternatively spliced EIIIB, EIIIA, and V segments are included in the FN that is required for the morphogenesis of "FN dependent" structures, including somites, notochord, and the vasculature. Conversely, these segments would appear to play divergent, and sometimes exclusive, biological roles in specific tissues such as liver, cartilage, and placenta.
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Fibronectin isoform distribution in the mouse. II. Differential distribution of the alternatively spliced EIIIB, EIIIA, and V segments in the adult mouse.
The alternatively spliced EIIIB, EIIIA, and V segments of fibronectin (FN) show widespread codistribution in the mouse embryo, suggesting that EIIIB+, EIIIA+, and V+ isoforms serve to facilitate morphogenesis and organogenesis (Peters, JH, and Hynes, RO, 1996, this issue). To gain further clues to functions of these segments, we have used segment-specific anti-FN antibodies to perform immunofluorescence microscopy on tissue sections obtained from mice aged 9 to 15 weeks. Staining for each of the three spliced segments, relative to that for the total FN pool, was reduced in the adult as compared with the embryo. Anti-V antibodies produced patterns which were most similar to those obtained with anti-total FN antibodies, localizing to basement membranes, connective tissues subjacent to epithelia, walls of blood vessels, and cartilage. Anti-EIIIA antibodies produced the next most widespread pattern, which included prominent staining of the walls of blood vessels of all sizes, the lung interstitium, and smooth muscle associated with the gastrointestinal (GI), genitourinary (GU), and respiratory tracts. Although anti-EIIIB antibodies produced the faintest and most restricted pattern of staining, EIIIB+ FN could be detected in the walls of some smaller blood vessels, smooth muscle of the GI, GU, and respiratory tracts, as well as within cartilaginous structures, and eye. There were quantitative and/or qualitative differences in the staining patterns produced by the three segment-specific antibodies in a variety of tissues, including liver, cartilage, synovium, cornea, muscle, peripheral nerve, and lymph node. These findings suggest that each of the spliced segments of the FN molecule may occupy unique physical or functional positions within the extracellular matrix of the adult mouse.
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Rolling in P-selectin-deficient mice is reduced but not eliminated in the dorsal skin.
P-selectin-mediated rolling is believed to be important in the recruitment of leukocytes to tissue after ischemia-reperfusion injury. The dorsal skin chamber was used to examine differences in the rolling and stable adhesion of circulating leukocytes in subcutaneous (SC) vessels of P-selectin-deficient and age-matched wild-type mice, both under basal conditions and after ischemia-reperfusion. Rolling in the postcapillary venules in SC tissue of P-selectin-deficient mice was significantly lower than that in wild-type mice under the basal conditions and post-ischemia-reperfusion (P < .05), but was not eliminated by the deletion of the P-selectin gene. No significant difference between P-selectin-deficient and wild-type mice in shear rate or leukocyte-endothelial adhesion was observed up to 24 hours after ischemia-reperfusion. These results show that P-selectin-mediated rolling is not a prerequisite for ischemia-reperfusion-induced leukocyte-endothelial adhesion in the skin.
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Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin.
P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin.
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