Randal J. Westrick

Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P2 synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P2, is critical for the survival of neural cells.

Mvwf, a Dominant Modifier of Murine von Willebrand Factor, Results from Altered Lineage-Specific Expression of a Glycosyltransferase

We have identified altered lineage-specific expression of an N-acetylgalactosaminyltransferase gene, Galgt2, as the gain-of-function mechanism responsible for the action of the Mvwf locus, a major modifier of plasma von Willebrand factor (VWF) level in RIIIS/J mice. A switch of Galgt2 gene expression from intestinal epithelial cell-specific to a pattern restricted to the vascular endothelial cell bed leads to aberrant posttranslational modification and rapid clearance of VWF from plasma. Transgenic expression of Galgt2 directed to vascular endothelial cells reproduces the low VWF phenotype, confirming this switch in lineage-specific gene expression as the likely molecular mechanism for Mvwf. These findings identify alterations in glycosyltransferase function as a potential general mechanism for the genetic modification of plasma protein levels.

Murine Models of Vascular Thrombosis

Thrombotic complications of vascular disease are the leading cause of morbidity and mortality in most industrialized countries. Despite this, safe and effective drugs targeting these complications are limited, especially in the chronic setting. This is because of the complexity of thrombosis in both arteries and veins, which is becoming increasingly evident as numerous factors are now known to affect the fate of a forming thrombus. To fully characterize thrombus formation in these settings, in vivo models are necessary to study the various components and intricate interactions that are involved. Genetic manipulations in mice are greatly facilitating the dissection of relevant pro- and antithrombotic influences. Standardized models for the study of thrombosis in mice as well as evolving techniques that allow imaging of molecular events during thrombus formation are now available. This review will highlight some of the recent developments in the field of thrombosis using mouse models and how these studies are expanding our knowledge of thrombotic disease.

Heme Oxygenase-1 Deficiency Accelerates Formation of Arterial Thrombosis Through Oxidative Damage to the Endothelium, Which Is Rescued by Inhaled Carbon Monoxide

Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage. Because intravascular thrombosis forms at sites of tissue inflammation, we hypothesized that HO-1 protects against arterial thrombosis during oxidant stress. To investigate the direct function of HO-1 on thrombosis, we used photochemical-induced vascular injury in Hmox1−/− and Hmox1+/+ mice. Hmox1−/− mice developed accelerated, occlusive arterial thrombus compared with Hmox1+/+ mice, and we detected several mechanisms accounting for this antithrombotic effect. First, endothelial cells in Hmox1−/− arteries were more susceptible to apoptosis and denudation, leading to platelet-rich microthrombi in the subendothelium. Second, tissue factor, von Willebrand Factor, and reactive oxygen species were significantly elevated in Hmox1−/− mice, consistent with endothelial cell damage and loss. Third, following transplantation of Hmox1−/− donor bone marrow into Hmox1+/+ recipients and subsequent vascular injury, we observed rapid arterial thrombosis compared with Hmox1+/+ mice receiving Hmox1+/+ bone marrow. Fourth, inhaled carbon monoxide and biliverdin administration rescued the prothrombotic phenotype in Hmox1−/− mice. Fifth, using a transcriptional analysis of arterial tissue, we found that HO-1 determined a transcriptional response to injury, with specific effects on cell cycle regulation, coagulation, thrombosis, and redox homeostasis. These data provide direct genetic evidence for a protective role of HO-1 against thrombosis and reactive oxygen species during vascular damage. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with vascular oxidant stress and inflammation.

Heparin cofactor II inhibits arterial thrombosis after endothelial injury

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate, heparan sulfate, or heparin. HCII has been proposed to regulate coagulation or to participate in processes such as inflammation, atherosclerosis, and wound repair. To investigate the physiologic function of HCII, about 2 kb of the mouse HCII gene, encoding the N-terminal half of the protein, was deleted by homologous recombination in embryonic stem cells. Crosses of F1 HCII(+/-) animals produced HCII(-/-) offspring at the expected mendelian frequency. Biochemical assays confirmed the absence of dermatan sulfate-dependent thrombin inhibition in the plasma of HCII(-/-) animals. Crosses of HCII(-/-) animals produced litters similar in size to those obtained from heterozygous matings. At 1 year of age, HCII-deficient animals were grossly indistinguishable from their wild-type littermates in weight and survival, and they did not appear to have spontaneous thrombosis or other morphologic abnormalities. In comparison with wild-type animals, however, they demonstrated a significantly shorter time to thrombotic occlusion of the carotid artery after photochemically induced endothelial cell injury. This abnormality was corrected by infusion of purified HCII but not ovalbumin. These observations suggest that HCII might inhibit thrombosis in the arterial circulation.

Hyperlipidemia Promotes Thrombosis After Injury to Atherosclerotic Vessels in Apolipoprotein E–Deficient Mice

The increased risk of hyperlipidemia on the development of complications of atherosclerosis is well established. Cholesterol-lowering therapies lead to a decrease in the incidence of vascular thrombotic events that is out of proportion to the reduction in plaque size. This suggests that the occurrence of acute thrombosis overlying a disrupted plaque is influenced by changes in lipid levels. The influence of acute hyperlipidemia on the development of thrombosis overlying an atherosclerotic plaque in vivo has not been extensively studied. We used a murine model of vascular injury induced by a photochemical reaction to elicit thrombus formation overlying an atherosclerotic plaque. Fifteen apolipoprotein E-deficient mice were maintained on normal chow until the age of 30 weeks. Five days before the induction of thrombosis, 6 mice were started on a high fat diet, and 9 mice were continued on normal chow. Mice then underwent photochemical injury to the common carotid artery immediately proximal to the carotid bifurcation, where an atherosclerotic plaque is consistently present. Mice maintained on normal chow developed occlusive thrombi, determined by cessation of blood flow, 44+/-5 minutes (mean+/-SEM) after photochemical injury, whereas mice fed a high fat chow developed occlusive thrombosis at 27+/-3 minutes (P<0.02). Histological analysis confirmed the presence of acute thrombus formation overlying an atherosclerotic plaque. These studies demonstrate a useful model for assessing the determinants of thrombosis in the setting of atherosclerosis and show that acute elevations in plasma cholesterol facilitate thrombus formation at sites of atherosclerosis after vascular injury.

Atherosclerosis Progression in LDL Receptor–Deficient and Apolipoprotein E–Deficient Mice Is Independent of Genetic Alterations in Plasminogen Activator Inhibitor-1

Impaired fibrinolysis has been linked to atherosclerosis in a number of experimental and clinical studies. Plasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of plasminogen activation and has been proposed to promote atherosclerosis by facilitating fibrin deposition within developing lesions. We examined the contribution of PAI-1 to disease progression in 2 established mouse models of atherosclerosis. Mice lacking apolipoprotein E (apoE-/-) and mice lacking the low density lipoprotein receptor (LDLR-/-) were crossbred with transgenic mice overexpressing PAI-1 (resulting in PAI-1 Tg(+)/apoE-/- and PAI-1 Tg(+)/LDLR-/-, respectively) or were crossbred with mice completely deficient in PAI-1 gene expression (resulting in PAI-1-/-/apoE-/- and PAI-1-/-/LDLR-/-, respectively). All animals were placed on a western diet (21% fat and 0.15% cholesterol) at 4 weeks of age and analyzed for the extent of atherosclerosis after an additional 6, 15, or 30 weeks. Intimal and medial areas were determined by computer-assisted morphometric analysis of standardized microscopic sections from the base of the aorta. Atherosclerotic lesions were also characterized by histochemical analyses with the use of markers for smooth muscle cells, macrophages, and fibrin deposition. Typical atherosclerotic lesions were observed in all experimental animals, with greater severity at the later time points and generally more extensive lesions in apoE-/- than in comparable LDLR-/- mice. No significant differences in lesion size or histological appearance were observed among PAI-1-/-, PAI-1 Tg(+), or PAI-1 wild-type mice at any of the time points on either the apoE-/- or LDLR-/- genetic background. We conclude that genetic modification of PAI-1 expression does not significantly alter the progression of atherosclerosis in either of these well-established mouse models. These results suggest that fibrinolytic balance (as well as the potential contribution of PAI-1 to the regulation of cell migration) plays only a limited role in the pathogenesis of the simple atherosclerotic lesions observed in the mouse.

Lethal Perinatal Thrombosis in Mice Resulting From the Interaction of Tissue Factor Pathway Inhibitor Deficiency and Factor V Leiden

Background—Factor V Leiden (FVL) is a common genetic risk factor for thrombosis in humans. The incomplete penetrance of FVL suggests important contributions from other genetic or environmental modifying factors. Variation in the expression of tissue factor pathway inhibitor (TFPI) has also been proposed as a risk factor for venous thrombosis and has been shown to enhance the prothrombotic effect of FVL in vitro. Methods and Results—To examine the potential in vivo interaction between Tfpi and FvL, we analyzed crosses between mice carrying FvL and a deficiency of TFPI. The FvQ/Q, Tfpi+/− genotype was nearly completely fatal in the early perinatal period. Increased fibrin deposition was observed in multiple organs from the FvQ/Q, Tfpi+/− fetuses, suggesting disseminated thrombosis. Conclusions—These observations demonstrate the prothrombotic effect of modest variations in the level of TFPI expression and suggest that TFPI could be an important genetic modifier for the thrombosis associated with FVL in humans.

Homozygosity for Factor V Leiden Leads to Enhanced Thrombosis and Atherosclerosis in Mice

Background—Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial. Methods and Results—To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type (Fv+/+), heterozygous FVL (FvQ/+), and homozygous FVL (FvQ/Q) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. FvQ/Q mice formed occlusive thromboses 27±3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv+/+ mice (56±7 minutes, n=9, P<0.01), whereas FvQ/+ mice (41±7 minutes, n=5) were intermediate (P=0.5, compared with Fv+/+). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv+/+ and FvQ/Q mice. FvQ/Q mice transplanted with Fv+/+ bone marrow formed occlusive thromboses at 35±5 minutes (n=7, P<0.05 compared with Fv+/+ mice), whereas Fv+/+ mice transplanted with FvQ/Q bone marrow occluded at 59±7 minutes (n=6, P<0.001 compared with FvQ/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, FvQ/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)–deficient strain (ApoE−/−) to generate FvQ/Q,ApoE−/− mice. By 52 weeks of age, FvQ/Q,ApoE−/− mice (n=8) had developed more aortic atherosclerosis (40±6% lesion area) compared with Fv+/+,ApoE−/− mice (15±3% lesion area; n=12, P<0.02). Conclusions—In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non–platelet-derived plasma FVL.

Plasminogen activator inhibitor-1 in vascular thrombosis.

Thrombotic complications of vascular disease constitute the leading cause of morbidity and mortality in much of the developed world. Current drug therapies available to treat the thrombotic component of arterial and venous vascular complications remain limited. Novel safe and effective treatment strategies to reduce formation of occlusive thrombosis will likely have a major impact on reducing the economic burden of vascular disease on the healthcare system. Enhancing endogenous fibrinolysis by targeting plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of circulating plasminogen activators, has been shown to be effective in markedly attenuating the formation of arterial and venous occlusive thrombosis in animal models. In addition, animal and human studies of PAI-1 deficiency indicate that spontaneous bleeding complications associated with even complete PAI-1 deficiency would be rare. Patients most likely to benefit from PAI-1 inhibition would be those at high risk for vascular events where PAI-1 is elevated, such as is observed in obesity, diabetes and the metabolic syndrome. Since obesity and metabolic syndrome are now epidemic, and will likely have a major adverse impact on vascular thrombotic events, it may be time to test the clinical effectiveness of PAI-1 inhibition in a patient population at high risk for vascular thrombosis.