Automation paves the way to improved gas monitoring.
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Air Pollutants↗
Biomedical subjects
D D Wagner
Publications and source records attributed to D D Wagner.
How much intrinsic safety is enough?
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Electronics↗
Welding essentials.
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Accidents, Occupational↗
ApoE deficiency compromises the blood brain barrier especially after injury.
BACKGROUND: Apolipoprotein E (apoE) mediates lipoprotein uptake by receptors such as the LDL receptor (LDLR). The isoform apoE4 has been linked to Alzheimer's disease and to poor outcomes after brain injury. Astrocytes that induce blood brain barrier (BBB) properties in endothelium also produce apoE. We decided to investigate the role of apoE in BBB function and in the restoration of BBB after brain injury. MATERIALS AND METHODS: Wild-type (WT) mice and mice deficient in apoE or LDLR were fed normal chow or diets rich in fat and cholesterol. The BBB leakage was determined through injection of Evans blue dye and measurement of the amount of dye extravasated in the brains 3 hours later. Brain injury was induced by applying dry ice directly onto the excised parietal region of the brain. The mice were given 7 days to recover. In some experiments, peroxidase was infused to observe the site of leakage by histology. RESULTS: We found 70% more spontaneous leakage of injected Evans blue dye in the brains of apoE-/- mice than in wild type. This increase in permeability appeared selective for the brain. The leaky BBB in apoE-/- mice may provide an explanation for the neurological deficits seen in these animals. In an established model of BBB leakage induced by trauma (cold injury), the apoE-/- mice showed even more compromised BBB function, compared with WT mice, suggesting that apoE is important for BBB recovery. No deficit in BBB was observed in injured LDLR-/- mice, even on Western Diet. In contrast, higher plasma cholesterol levels in apoE-/- mice further increased BBB leakage after injury. We extracted 5x more Evans blue from these brains than from WT. In the injury model, injection of peroxidase resulted in prominent retention of this protein in the cortex of apoE-/- but not in WT. CONCLUSIONS: Our results show that the combination of loss of apoE function with high plasma cholesterol and especially brain injury results in dramatic BBB defects in the cortex and may explain in part the importance of apoE in Alzheimer's disease and in successful recovery from brain injury.
Animals↗
Pro-coagulant state resulting from high levels of soluble P-selectin in blood.
The plasma concentration of soluble adhesion receptors is increased under pathological circumstances, but their function remains enigmatic. Soluble P-selectin (sP-sel) is shed from activated platelets and endothelial cells. Mice genetically engineered to express P-selectin without the cytoplasmic tail (DeltaCT) constitutively show a 3- to 4-fold increase of sP-sel in plasma. We observed that the DeltaCT mice formed fibrin very readily. In an ex vivo perfusion chamber, there was more fibrin deposited at the site of platelet thrombus formation than in wild type (WT), whereas no fibrin deposits were detected using P-selectin-deficient blood during the same interval. Similarly, in vivo, the hemorrhage produced by local Shwartzman reaction was smaller in the DeltaCT mice than in WT. In contrast, we previously showed hemorrhage to be more prominent in P-selectin knock-out mice. Infusion of mouse P-sel-Ig chimera produced the same protective effect in WT mice as seen in the DeltaCT mice, indicating that the effect was due to increased levels of sP-sel. Mice infused with P-sel-Ig showed significantly more fibrin deposited on the luminal face of the injured vessels than control mice. Plasma from DeltaCT mice or mice infused with P-sel-Ig contained higher concentration of pro-coagulant microparticles and clotted one minute faster than WT. This pro-coagulant phenotype of DeltaCT mice could be reversed by a 4-day treatment with PSGL-Ig, a P-selectin inhibitor. We propose that sP-sel should no longer be considered only as a marker of inflammation or platelet activation, but also as a direct inducer of pro-coagulant activity associated with vascular and thrombotic diseases.
Animals↗
Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins.
With the use of intravital microscopy, a new type of platelet-endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds(-1)) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm(2).s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded with A23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in beta3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet-endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibalpha (GPIbalpha) because the adhesion of murine platelets expressing exclusively the human GPIbalpha could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbalpha. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbalpha. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.
Animals↗
Prominent role of P-selectin in the development of advanced atherosclerosis in ApoE-deficient mice.
BACKGROUND: Adhesive interactions between leukocytes and endothelial cells are characteristic of the development of atherosclerotic lesions, but the receptors involved remain to be defined. P-selectin is an adhesion receptor expressed on activated endothelial cells or platelets and was shown to be involved in fatty streak formation in LDL receptor-deficient mice on an atherogenic diet. The main purpose of this study is to examine the role of P-selectin in the spontaneous development of advanced atherosclerosis in apoE-deficient mice. METHODDS AND RESULTS: We intercrossed P-selectin-deficient mice with mice lacking apoE and compared lesion development in apoE-deficient mice with P-selectin (apoE(-/-) P(+/+)) and without P-selectin (apoE(-/-) P(-/-)) that were fed normal mouse chow. At 4 months of age, apoE(-/-) P(-/-) mice had 3. 5-fold smaller aortic sinus lesions than apoE(-/-) P(+/+) mice. These were limited to fatty streaks in the apoE(-/-) P(-/-) mice, whereas 70% of apoE(-/-) P(+/+) lesions contained smooth muscle cells. Significantly more of the aortic sinus circumference was covered by lesions in the apoE(-/-) P(+/+) animals. The P-selectin genotype affected macrophage recruitment, because twice as many mononuclear cells were present in the P-selectin-positive lesions. At 15 months, the lesions progressed to the fibrous plaque stage in both genotypes and spread throughout the aorta, but this process was delayed in apoE(-/-) P(-/-) mice. In the aortic sinus, the lesions of the apoE(-/-) P(-/-) mice were 2.6-fold smaller and less calcified. CONCLUSIONS: P-selectin appears to be a key adhesion receptor mediating leukocyte recruitment into lesions and promoting advanced atherosclerosis in apoE-deficient mice.
Animals↗
P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo.
The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
Animals↗
Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen.
We used intravital microscopy to observe the formation of platelet plugs in ferric chloride-injured arterioles of live mice. With this model, we evaluated thrombus growth in mice lacking von Willebrand factor (vWF) and fibrinogen (Fg), the two key ligands known to mediate platelet adhesion and aggregation. In vWF(-/-) mice, despite the presence of arterial shear, delayed platelet adhesion occurred and stable thrombi formed. In many mice, a persisting high-shear channel never occluded. Abundant thrombi formed in Fg(-/-) mice, but they detached from the subendothelium, which ultimately caused downstream occlusion in all cases. Surprisingly, mice deficient in both vWF and Fg successfully formed thrombi with properties characteristic of both mutations, leading to vessel occlusion in the majority of vessels. Platelets of these doubly deficient mice specifically accumulated fibronectin in their alpha-granules, suggesting that fibronectin could be the ligand supporting the platelet aggregation.
Afibrinogenemia↗
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.
Alleles↗
P- and E-selectin-deficient mice are susceptible to cerebral ischemia-reperfusion injury.
We examined brain sections from P- and E-selectin-deficient mice (-/-) and their nontransgenic littermates (+/+) after focal cerebral ischemia and reperfusion (I/R) tissue injury. There was no difference in the subsequent infarct volume after 3 h of ischemia and 21 h of reperfusion. These data indicate that selectin-independent mechanisms mediate tissue injury after a prolonged period of transient focal ischemia.
Animals↗
Insights from von Willebrand disease animal models.
von Willebrand disease is a genetic bleeding disorder that arises from abnormalities in von Willebrand factor, an adhesive glycoprotein involved in both primary hemostasis and coagulation. It is the most common inherited bleeding disorder in humans, and over the years several animal species have also been described as suffering from this disease whether through a spontaneous mutation (pigs, dogs) or a genetically engineered one (mouse). These different animal models are extremely useful in exploring the characteristics of von Willebrand disease and in testing new treatments. This review provides an update of the various von Willebrand disease models and the contribution that these models can make to a better understanding of human von Willebrand disease.
Animals↗
Carboxypeptidase E does not mediate von Willebrand factor targeting to storage granules.
Sorting of von Willebrand factor precursor (pro-vWf) from the trans-Golgi network to secretory granules (Weibel-Palade bodies) is critical for its conversion to the biologically active highly multimeric form, as well as for regulated secretion by the endothelial cells. When expressed in hormone-secretory cells, vWf is also recognized as a stored protein and is directed to storage granules. Recently, carboxypeptidase E (CPE) was proposed as a granular sorting receptor for prohormones (Cool et al., Cell 88: 73, 1997). To explore whether CPE is also involved in pro-vWf sorting, we initially examined its expression in human umbilical vein endothelial cells. A specific message for CPE and the protein itself were detected making it a plausible candidate as a targeting receptor for vWf in endothelium. To investigate this possibility, we used mice lacking CPE. The highly multimeric forms, subunit composition and plasma levels of vWf in CPE-deficient mice were similar to those of their wild-type littermates. vWf was also found in alpha-granules of platelets and in Weibel-Palade bodies of endothelial cells obtained from the CPE-deficient mice. Furthermore, vWf was released from the cultured CPE-deficient endothelial cells after stimulation with a secretagogue. We conclude that CPE is not essential for sorting vWf to the regulated secretory pathway. Thus, a CPE-independent mechanism must exist for protein sorting to storage granules.
Animals↗
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.
Adenosine Triphosphatases↗
Donor MHC and adhesion molecules in transplant arteriosclerosis.
Transplant-associated arteriosclerosis remains an obstacle to long-term graft survival. To determine the contribution to transplant arteriosclerosis of MHC and adhesion molecules from cells of the donor vasculature, we allografted carotid artery loops from six mutant mouse strains into immunocompetent CBA/CaJ recipients. The donor mice were deficient in either MHC I molecules or MHC II molecules, both MHC I and MHC II molecules, the adhesion molecule P-selectin, intercellular adhesion molecule (ICAM)-1, or both P-selectin and ICAM-1. Donor arteries in which ICAM-1, MHC II, or both MHC I and MHC II were absent showed reductions in neointima formation of 52%, 33%, and 38%, respectively, due primarily to a reduction in smooth muscle cell (SMC) accumulation. In P-selectin-deficient donor arteries, neointima formation did not differ from that in controls. In donor arteries lacking both P-selectin and ICAM-1, the size of the neointima was similar to that in those lacking ICAM-1 alone. In contrast, neointima formation increased by 52% in MHC I-deficient donor arteries. The number of CD4-positive T cells increased by 2.8-fold in MHC I-deficient arteries, and that of alpha-actin-positive SMCs by twofold. These observations indicate that ICAM-1 and MHC II molecules expressed in the donor vessel wall may promote transplant-associated arteriosclerosis. MHC I molecules expressed in the donor may have a protective effect.
Animals↗
New discoveries with mice mutant in endothelial and platelet selectins.
Genetically engineered mice bring animal studies to the molecular level in that they help establish the role of a particular molecule, or its portion, in a complex biological process. In recent years, several discoveries were made using the selectin-mutant mice. For example, it was shown that these molecules not only mediate leukocyte rolling, but also platelet rolling on the vessel wall. The functional significance of platelet rolling has yet to be uncovered. The process could be important for hemostasis leading to firm platelet adhesion at sites of denuded endothelium and/or in inflammation. After activation, platelets may help in leukocyte recruitment as shown by studies of lymphocyte homing to peripheral lymph nodes. Surprisingly, work with the P-selectin mutant mice has also revealed an anti-inflammatory aspect of platelet P-selectin. P-selectin binding to leukocytes promoted the transcellular production of an anti-inflammatory mediator limiting the extent of acute glomeluronephritis. In addition, soluble P-selectin was shown recently to be shed from both activated platelets and endothelium and there are strong indications that it too could have an attenuating effect on inflammatory disease progression. Another discovery made with the selectin-deficient mice is on the crucial role of P- and E-selectins in the homing of hematopoietic progenitor cells to the bone marrow. This observation could perhaps be further exploited by use of selectin inhibitors when liberating the progenitors from the marrow for transplant purposes. The use of selectin inhibitors could also be evaluated in two major disease processes where selectins were recently shown to play a role: cancer and atherosclerosis. Thus the selectin mutant mice have taught us a great deal about the role of selectins in normal physiology and in pathology. Further studies are needed to explore the regulation of shedding of the selectins and the function of soluble selectins in vivo. Exploring new territories of selectin-mediated interactions may provide a basis for developing new interventions and treatments for diseases in which the role of selectins has not yet been suspected.
Animals↗
P-Selectin and platelet clearance.
P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin-deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin-deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4 degreesC caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.
Aging↗
Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.
The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E-/-), reconstituted with minimal numbers (</=5 x 10(4)) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P/E-/- mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P/E-/- animals, either splenectomized or spleen-intact. Homing into the BM of P/E-/- recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P/E-/- mice treated with anti-VCAM-1 compared with P/E-/- mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.
Animals↗