Haperen, R. van

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins (Article)

2011-09-27T00:00:00Z

Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenicmice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies.

Endothelial nitric oxide synthase overexpression restores the efficiency of bone marrow mononuclear cell-based therapy (Article)

2011-01-01T00:00:00Z

Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. Transgenic eNOS overexpression in diabetic, atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increase in postischemic neovascularization compared with control. eNOS overexpression in diabetic or atherosclerotic BMMNCs restored their reduced proangiogenic potential in ischemic hind limb. This effect was associated with an increase in BMMNC ability to differentiate into cells with endothelial phenotype in vitro and in vivo and an increase in BMMNCs paracrine function, including vascular endothelial growth factor A release and NO-dependent vasodilation. Moreover, although wild-type BMMNCs treatment resulted in significant progression of atherosclerotic plaque in ischemic mice, eNOS transgenic atherosclerotic BMMNCs treatment even had antiatherogenic effects. Cell-based eNOS gene therapy has both proangiogenic and antiatherogenic effects and should be further investigated for the development of efficient therapeutic neovascularization designed to treat ischemic cardiovascular disease.

Beneficial effects of exercise training after myocardial infarction require full eNOS expression (Article)

2010-03-18T00:00:00Z

Exercise training attenuates left ventricular (LV) dysfunction after myocardial infarction (MI). It could be speculated that these effects of exercise are mediated by increased endothelial NO synthase (eNOS) activity. In the present study we tested the hypothesis that eNOS plays a critical role in the exercise-induced amelioration of LV dysfunction after MI. MI or sham was induced in eNOS-/-, eNOS+/-and eNOS+/+mice. After 8 weeks of voluntary wheel running (∼ 7 km/day in all groups) or sedentary housing, global cardiac function was determined in vivo and (immuno)histochemistry was performed to assess cardiomyocyte size, fibrosis, capillary density and apoptosis in remote myocardium. At baseline eNOS-/-mice had higher mean aortic pressure compared to eNOS+/-and eNOS+/+mice, but had normal global cardiac function. MI resulted in marked LV remodeling, including cardiomyocyte hypertrophy and a reduction in capillary density, increased fibrosis and apoptosis, as well as LV systolic and diastolic dysfunction to the same extent in all genotypes. In eNOS+/+MI mice exercise abolished fibrosis and apoptosis in the remote myocardium, attenuated LV systolic dysfunction and ameliorated pulmonary congestion. These beneficial effects were lost in eNOS+/-and eNOS-/-mice, while LV systolic dysfunction and pulmonary congestion in eNOS+/-mice were exacerbated by exercise. In conclusion, the beneficial effects of exercise after MI on LV remodeling and dysfunction depend critically on endogenous eNOS. The observation that the lack of one eNOS allele is sufficient to negate all beneficial effects of exercise, strongly suggests that exercise depends on full eNOS expression.

Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism (Article)

2009-10-01T00:00:00Z

Objective: Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency. Methods and results: HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL-/- mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL-/- background. Conclusions: HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice (Article)

2009-08-01T00:00:00Z

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1-/-). Parallel to the strong reduction in PLTP activity in plasma of ABCA1-/-mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1-/-mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1-/-mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1-/-, apoE-/-and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1-/-, apoE-/-and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4-, 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.

Elevation of systemic PLTP, but not macrophage-PLTP, impairs macrophage reverse cholesterol transport in transgenic mice (Article)

2009-06-01T00:00:00Z

Phospholipid transfer protein (PLTP) is a multifunctional protein synthesized by various cell types and secreted into the plasma. Plasma PLTP is able to transfer phospholipids between lipoproteins and modulate HDL particles. Mice with overexpression of human PLTP have an increased ability to generate preβ-HDL, reduced total HDL levels and an increased susceptibility to atherosclerosis. As the macrophage is a key component of the atherosclerotic lesion and an important site of PLTP expression, we investigated the role of systemic and peripheral PLTP in macrophage cholesterol efflux and reverse cholesterol transport (RCT) in vivo. We used an assay in which3H-labelled cholesterol-loaded macrophages were injected intraperitoneally into recipient mice, and radioactivity was quantified in plasma, liver and faeces. Firstly, wild type macrophages were injected into wild type, PLTP transgenic (PLTPtg) and apoAI transgenic (apoAItg) mice. While plasma3H-tracer levels in apoAItg mice were increased compared with wild type mice, they were reduced in PLTPtg mice. Moreover, overexpression of PLTP significantly decreased faecal3H-tracer levels compared with wild type and apoAItg mice. Secondly, wild type mice were injected with peritoneal macrophages derived from PLTPtg or wild type mice. No significant difference in the amount of3H-tracer in plasma, liver or faeces was found between the two groups of mice. Our findings demonstrate that macrophage cholesterol efflux and RCT to faeces is impaired in PLTP transgenic mice, and that elevation of macrophage-PLTP does not affect RCT, indicating that higher systemic PLTP levels may promote atherosclerosis development by decreasing the rate of macrophage RCT.

Activation of MMP8 and MMP13 by angiotensin II correlates to severe intra-plaque hemorrhages and collagen breakdown in atherosclerotic lesions with a vulnerable phenotype (Article)

2009-05-01T00:00:00Z

Angiotensin II (ATII)-mediated hypertension increases the risk for acute coronary events, which may be caused by augmented collagen degradation. Interstitial fibers of collagen type I in the plaque can be degraded by MMP8 and MMP13 specifically. Indeed high MMP8 levels have been correlated with ruptured plaques in patients. To study the contribution of ATII in plaque rupture, we evaluated its effect on MMP8 and MMP13 activity on the vulnerable lesions using an extravascular device that induces regions of pro-atherogenic shear stress in the carotid arteries of ApoE KO mice. This triggers the growth of lesions with a "vulnerable" macrophage-rich phenotype (referred to as upstream lesions) and lesions with a "stable" fibrotic phenotype (referred to as downstream lesions). ATII administration increased mean blood pressure, and increased the incidence of intra-plaque hemorrhages (IPH) from 30% to 73% of the animals in the upstream segments. The area of IPH was also increased by 5-fold. No IPHs were observed in the downstream lesions of the control group or the ATII group. In addition, ATII treatment doubled the size of upstream and downstream lesions. Upstream lesions in the ATII group were decreased in collagen content by 3-fold, contained 2-fold higher MMP8 and MMP13 levels, with a 2- and 3-fold increase in collagen type I degradation by MMP8 and MMP13 respectively compared to the upstream lesions in the control group. Gene expression analysis showed general increase in procollagens and TIMPs expression in response to ATII. However, ATII also decreased procollagen 5α3 expression in downstream lesions and decreased TIMP4 expression in upstream lesions. These data show that ATII promotes a "stable" fibrotic phenotype by inducing severe intra-plaque hemorrhages, characterized by increased degradation of interstitial collagen I via an MMP-mediated (MMP8 and MMP13) mechanism.

Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction (Article)

2009-05-01T00:00:00Z

It has been reported that exercise after myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be due to an exercise-induced upregulation of endothelial nitric oxide synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS expression using transgenic mice with overexpression of human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice and improved cardiomyocyte maximal force development (Fmax). However, only exercise training restored myofilament Ca2+-sensitivity and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a protein levels and improved the first derivative of LV pressure at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions, we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in Ca2+ homeostasis, as myofilament Fmax actually increased despite marked reductions in the phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were lost in eNOSTg mice. Antioxidant treatment with N-acetylcysteine or supplementation of tetrahydrobiopterin and L-arginine prevented these detrimental effects on LV function while partly restoring the phosphorylation status of myofilament proteins and further enhancing myofilament Fmax. In conclusion, the combination of exercise and elevated eNOS expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS "uncoupling."

Differentiation of bone marrow-derived endothelial progenitor cells is shifted into a proinflammatory phenotype by hyperglycemia (Article)

2009-05-01T00:00:00Z

Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to vascular maintenance by participating in angio- genesis, re-endothelialization, and remodeling. Myeloid progenitor cells in the BM are functionally and quantitatively an important precursor pool for cells that contribute to these processes. However, these precursor pools in the BM also give rise to important effector cells of the innate immune system, such as macrophages and dendritic cells. We hypothesized that the disturbed repair responses that are being observed in diabetes mellitus are also related to an effect on functional and differentiation characteristics at the level of this bone marrow precursor pool. Indeed, we observed that bone marrow differentiation cultures for EPC, macrophages (Mph), or dendritic cells (DC) from hyperglycemic BM yielded 40% fewer EPC and 50% more Mph compared with control BM. These changes were directly related to the hemoglobin A1Clevels of the donor mice. BM-derived DC numbers were not affected by hyperglycemia.The composition of the BM was not altered; in particular, the numbers of CD31+/Ly6C+cells, which serve as common progenitors for EPC,Mph,and DC,were unaffected. In addition,BM-derived EPC from hyperglycemic mice were less angiogenic and more proinflammatory in regards to endocytosis, T-cell activation, and interleukin 12 production. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibition by statin supplementation of the culture medium counteracted these hyperglycemia-induced changes. Our study results show that hyperglycemia alters the differentiation fate of BM precursor cells, reducing the potential to generate vascular regenerative cells and favoring the development of proinflammatory cells.

Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: A mutation-inactivation study (Article)

2008-12-01T00:00:00Z

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration. Copyright

Acute elevation of plasma PLTP activity strongly increases pre-existing atherosclerosis (Article)

2008-07-01T00:00:00Z

Objective - A transgenic mouse model was generated that allows conditional expression of human PLTP, based on the tetracycline-responsive gene system, to study the effects of an acute increase in plasma PLTP activity as may occur in inflammation. Methods and Results - The effects of an acute elevation of plasma PLTP activity on the metabolism of apolipoprotein B-containing lipoproteins and on diet-induced pre-existing atherosclerosis were determined in mice displaying a humanized lipoprotein profile (low-density lipoprotein receptor knockout background). Induced expression of PLTP strongly increases plasma VLDL levels in LDL receptor knockout mice, whereas VLDL secretion is not affected. The elevation in plasma triglyceride levels is explained by a PLTP-dependent inhibition of VLDL catabolism, which is caused, at least partly, by a decreased lipoprotein lipase activity. Together with the decreased plasma HDL levels, the acutely increased PLTP expression results in a highly atherogenic lipoprotein profile. Induction of PLTP expression leads to a further increase in size of pre-existing atherosclerotic lesions, even on a chow diet. In addition, the lesions contain more macrophages and less collagen relative to controls, suggesting a less stable lesion phenotype. Conclusion - In conclusion, acute elevation of PLTP activity destabilizes atherosclerotic lesions and aggravates pre-existing atherosclerosis.

Elevated expression of phospholipid transfer protein in bone marrow derived cells causes atherosclerosis (Article)

2008-05-28T00:00:00Z

Background: Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerosis lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP. Methods and Findings: Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR-/-) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt → LDLR-/ - mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR-/ - mice (4.5-fold) compared with the control PLTPwt/wt→LDLR-/- mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages. Conclusions: We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.

Rapamycin modulates the eNOS vs. shear stress relationship (Article)

2008-04-01T00:00:00Z

Aims: Studies in animals and patients indicate that rapamycin affects vasodilatation differently in outer and inner curvatures of blood vessels. We evaluated in this study whether rapamycin affects endothelial nitric oxide synthase (eNOS) responsiveness to shear stress under normo- and hypercholesteraemic conditions to explain these findings. Methods and results: Shear stress levels were varied over a large range of values in carotid arteries of transgenic mice expressing human eNOS fused to enhanced green fluorescence protein. The mice were divided into control, low-dose rapamycin (3 μg/kg/day), and high-dose rapamycin (3 mg/kg/day) groups and into normocholesteraemic and hypercholesteraemic (ApoE-/- on high cholesterol diet for 3-4 weeks) groups. The effect of rapamycin treatment on eNOS was evaluated by quantification of eNOS expression and of intracellular protein levels by en face confocal microscopy. A sigmoid curve fit was used to described these data. The efficacy of treatment was confirmed by measurement of rapamycin serum levels (2.0 ± 0.5 ng/mL), and of p27kip1expression in vascular tissue (increased by 2.4 ± 0.5-fold). In control carotid arteries, eNOS expression increased by 1.8 ± 0.3-fold in response to rapamycin. In the treated vessels, rapamycin reduced maximal eNOS expression at high shear stress levels (>5 Pa) in a dose-dependent way and shifted the sigmoid curve to the right. Hypercholesteraemia had a tendency to increase the leftward shift and the reduction in maximal eNOS expression (P = 0.07). Conclusion: Rapamycin is associated with high eNOS in low shear regions, i.e. in atherogenic regions, protecting these regions against atherosclerosis, and is associated with a reduction of eNOS at high shear stress affecting vasomotion in these regions.

Large variations in absolute wall shear stress levels within one species and between species (Article)

2007-12-01T00:00:00Z

Wall shear stress (WSS), the frictional force between blood and endothelium, is an important determinant of vascular function. It is generally assumed that WSS remains constant at a reference value of 15 dyn/cm2. In a study of small rodents, we realized that this assumption could not be valid. This review presents an overview of recent studies in large and small animals where shear stress was measured, derived from velocity measurements or otherwise, in large vessels. The data show that large variations exist within a single species (human: variation of 2-16 N/m2). Moreover, when we compared different species at the same location within the arterial tree, an inverse relationship between animal size and wall shear stress was noted. When we related WSS to diameter, a unique relationship was derived for all species studied. This relationship could not be described by the well-known r3law of Murray, but by the r2law introduced by Zamir et al. in 1972. In summary, by comparing data from the literature, we have shown that: (i) the assumption of a physiological WSS level of ∼15 dyn/cm2for all straight vessels in the arterial tree is incorrect; (ii) WSS is not constant throughout the vascular tree; (iii) WSS varies between species; (iv) WSS is inversely related to the vessel diameter. These data support an "r2law" rather than Murray's r3law for the larger vessels in the arterial tree.

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice (Article)

2007-12-01T00:00:00Z

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages. Copyright

Appraisal of hepatic lipase and lipoprotein lipase activities in mice (Article)

2007-12-01T00:00:00Z

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required. Copyright

Vasomotor control in mice overexpressing human endothelial nitric oxide synthase (Article)

2007-08-01T00:00:00Z

Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K+channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K+and voltage-dependent K+channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways. Copyright

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: Acute effects of PLTP on lipoprotein metabolism (Article)

2007-08-01T00:00:00Z

One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to diet-induced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.

High pressure promotes monocyte adhesion to the vascular wall (Article)

2007-04-01T00:00:00Z

Hypertension is a known risk factor for the development of atherosclerosis. To assess how mechanical factors contribute to this process, mouse carotid arteries were maintained in organ culture at normal (80 mm Hg) or high (150 mm Hg) intraluminal pressure for 1, 6, 12, or 24 hours. Thereafter, fluorescent human monocytic cells (U937) were injected intraluminally and allowed to adhere for 30 minutes before washout. U937 adhesion was increased in vessels kept at 150 mm Hg 12 hours (23.5±5.7 versus 9.9±2.2 cells/mm at 80 mm Hg; P<0.05) or 24 hours (26.7±5.7 versus 8.8±1.5 cells/mm; P<0.05). At 24 hours, high pressure was associated with increased mRNA expression of monocyte chemoattractant protein-1, interleukin-6, keratinocyte-derived chemokine, and vascular cell adhesion molecule-1 (6.9±2.1, 4.4±0.1, 9.8±2.8, and 2.4±0.1-fold respectively; P<0.05), as assessed by quantitative RT-PCR and corroborated by immunohistochemistry, which also revealed an increase in intracellular adhesion molecule-1 expression. Nuclear factor κB inhibition using SN50 peptide abolished the overexpression of chemokines and adhesion molecules and reduced U937 adhesion in vessels at 150 mm Hg. Moreover, treatment of vessels and cells with specific neutralizing antibodies established that monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine released from vessels at 150 mm Hg primed the monocytes, increasing their adhesion to vascular cell adhesion molecule-1 but not intracellular adhesion molecule-1 via α4β1 integrins. The additive effect of chemokines on the adhesion of U937 cells to vascular cell adhesion molecule-1 was confirmed by in vitro assay. Finally, pressure-dependent U937 adhesion was blunted in arteries from mice overexpressing endothelial NO synthase. Hence, high intraluminal pressure induces cytokine and adhesion molecule expression via nuclear factor κB, leading to monocytic cell adhesion. These results indicate that hypertension may directly contribute to the development of atherosclerosis through nuclear factor κB induction.

Shear stress-induced changes in atherosclerotic plaque composition are modulated by chemokines (Article)

2007-03-01T00:00:00Z

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-γ-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene α (GRO-α) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-α and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.

Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins. (Article)

2004-05-01T00:00:00Z

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and mediates HDL conversion. PLTP-overexpressing mice have increased atherosclerosis. However, mice do not express cholesteryl ester transfer protein (CETP), which is involved in the same metabolic pathways as PLTP. Therefore, we studied atherosclerosis in heterozygous LDL receptor-deficient (LDLR(+/-)) mice expressing both human CETP and human PLTP. We used two transgenic lines with moderately and highly elevated plasma PLTP activity. In LDLR(+/-)/huCETPtg mice, cholesterol is present in both LDL and HDL. Both are decreased in LDLR(+/-)/huCETPtg/huPLTPtg mice (>50%). An atherogenic diet resulted in high levels of VLDL+LDL cholesterol. PLTP expression caused a strong PLTP dose-dependent decrease in VLDL and LDL cholesterol (-26% and -69%) and a decrease in HDL cholesterol (-70%). Surprisingly, atherosclerosis was increased in the two transgenic lines with moderately and highly elevated plasma PLTP activity (1.9-fold and 4.4-fold, respectively), indicating that the adverse effect of the reduction in plasma HDL outweighs the beneficial effect of the reduction in apolipoprotein B (apoB)-containing lipoproteins. The activities of the antiatherogenic enzymes paraoxonase and platelet-activating factor acetyl hydrolase were both PLTP dose-dependently reduced ( approximately -33% and -65%, respectively). We conclude that expression of PLTP in this animal model results in increased atherosclerosis in spite of reduced apoB-containing lipoproteins, by reduction of HDL and of HDL-associated antioxidant enzyme activities.

Functional expression of endothelial nitric oxide synthase fused to green fluorescent protein in transgenic mice (Article)

2003-01-01T00:00:00Z

The activity of endothelial nitric oxide synthase (eNOS) is subject to complex transcriptional and post-translational regulation including the association with several proteins and variations in subcellular distribution. In the present study we describe a transgenic mouse model expressing eNOS fused to green fluorescent protein (GFP), which allows the study of localization and regulation of eNOS expression. We tested the functionality of eNOS in the eNOS-GFP mice. Expression of eNOS was restricted to the endothelial lining of blood vessels in various tissues tested, without appreciable expression in non-endothelial cells. Activity of the enzyme was confirmed by assaying the conversion of L-arginine to L-citrulline. NO production in isolated vessels was increased in transgenic mice when compared to non-transgenic control animals (4.88 +/- 0.59 and 2.48 +/- 0.47 micro mol/L NO, respectively, P < 0.005). Both the mean aortic pressure and the pulmonary artery pressure were reduced in eNOS-GFP mice (both approximately 30%, P < 0.05). Plasma cholesterol levels were also slightly reduced ( approximately 20%, P < 0.05). In conclusion, eNOS-GFP mice express functional eNOS and provide a unique model to study regulation of eNOS activity or eNOS-mediated vascular events, including response to ischemia, response to differences in shear stress, angiogenesis and vasculogenesis, and to study the subcellular distribution in relation with functional responses to these events.

Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. (Article)

2002-12-13T00:00:00Z

In the vascular system, nitric oxide is generated by endothelial NO synthase (eNOS). NO has pleiotropic effects, most of which are believed to be atheroprotective. Therefore, it has been argued that patients suffering from cardiovascular disease could benefit from an increase in eNOS activity. However, increased NO production can cause oxidative damage, cell toxicity, and apoptosis and hence could be atherogenic rather than beneficial. To study the in vivo effects of increased eNOS activity, we created transgenic mice overexpressing human eNOS. Aortic blood pressure was approximately 20 mm Hg lower in the transgenic mice compared with control mice because of lower systemic vascular resistance. The effects of eNOS overexpression on diet-induced atherosclerosis were studied in apolipoprotein E-deficient mice. Elevation of eNOS activity decreased blood pressure ( approximately 20 mm Hg) and plasma levels of cholesterol (approximately 17%), resulting in a reduction in atherosclerotic lesions by 40%. We conclude that an increase in eNOS activity is beneficial and provides protection against atherosclerosis.

Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein. (Article)

2002-12-13T00:00:00Z

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (

Elevation of plasma phospholipid transfer protein in transgenic mice increases VLDL secretion (Article)

2002-01-01T00:00:00Z

Two lipid transfer proteins are active in human plasma, cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). Mice by nature do not express CETP. Additional inactivation of the PLTP gene resulted in reduced secretion of VLDL and subsequently in decreased susceptibility to diet-induced atherosclerosis. The aim of this study is to assess possible effects of differences in PLTP expression on VLDL secretion in mice that are proficient in CETP and PLTP. We compared human CETP transgenic (huCETPtg) mice with mice expressing both human lipid transfer proteins (huCETPtg/huPLTPtg). Plasma cholesterol in huCETPtg mice was 1.5-fold higher compared with huCETPtg/huPLTPtg mice (P < 0.001). This difference was mostly due to a lower HDL level in the huCETPtg/huPLTPtg mice, which subsequently could lead to the somewhat decreased CETP activity and concentration that was found in huCETPtg/huPLTPtg mice (P < 0.05). PLTP activity was 2.8-fold increased in these animals (P < 0.001). The human PLTP concentration was 5 microg/ml. Moderate overexpression of PLTP resulted in a 1.5-fold higher VLDL secretion compared with huCETPtg mice (P < 0.05). The composition of nascent VLDL was similar in both strains. These results indicate that elevated PLTP activity in huCETPtg mice results in an increase in VLDL secretion. In addition, PLTP overexpression decreases plasma HDL cholesterol as well as CETP.

Evaluation of phospholipid transfer protein and cholesteryl ester transfer protein as contributors to the generation of pre beta-high-density lipoproteins (Article)

2001-01-01T00:00:00Z

High-density lipoproteins (HDLs) are considered anti-atherogenic because they mediate peripheral cell cholesterol transport to the liver for excretion and degradation. An important step in this reverse cholesterol-transport pathway is the uptake of cellular cholesterol by a specific subclass of small, lipid-poor apolipoprotein A-I particles designated pre beta-HDL. The two lipid-transfer proteins present in human plasma, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), have both been implicated in the formation of pre beta-HDL. In order to investigate the relative contribution of each of these proteins, we used transgenic mouse models. Comparisons were made between human CETP transgenic mice (huCETPtg), human PLTP transgenic mice (huPLTPtg) and mice transgenic for both lipid-transfer proteins (huCETPtg/huPLTPtg). These animals showed elevated plasma levels of CETP activity, PLTP activity or both activities, respectively. We evaluated the generation of pre beta-HDL in mouse plasma by immunoblotting and crossed immuno-electrophoresis. Generation of pre beta-HDL was equal in huCETPtg and wild-type mice. In contrast, in huPLTPtg and huCETPtg/huPLTPtg mice, pre beta-HDL generation was 3-fold higher than in plasma from either wild-type or huCETPtg mice. Our findings demonstrate that, of the two plasma lipid-transfer proteins, PLTP rather than CETP is responsible for the generation of pre beta-HDL. These data support the hypothesis of a role for PLTP in the initial stage of reverse cholesterol transport.

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice (Article)

2000-01-01T00:00:00Z

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

A Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver. (Article)

1999-03-25T00:00:00Z

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

The Murine CYLN2 Gene: genomic organization, chromosome localization and comparison to the human gene that is located within the 7q11.23 Williams Syndrome Critical Region. (Article)

1998-11-01T00:00:00Z

Cytoplasmic linker proteins (CLIPs) have been proposed to mediate the interaction between specific membranous organelles and microtubules. We have recently characterized a novel member of this family, called CLIP-115. This protein is most abundantly expressed in the brain and was found to associate both with microtubules and with an organelle called the dendritic lamellar body. CLIP-115 is highly homologous to CLIP-170, or restin, which is a protein involved in the binding of endosomes to microtubules. Using the rat cDNA as a probe we have isolated overlapping cosmids containing the complete murine and part of the humanCYLN2(cytoplasmic linker-2) genes, which encode CLIP-115. The murine gene spans 60 kb and consists of 17 exons, and its promoter is embedded in a CpG island. MurineCYLN2maps to the telomeric end of mouse chromosome 5. The humanCYLN2gene is localized to a syntenic region on chromosome 7q11.23, which is commonly deleted in Williams syndrome. It spans at least 140 kb at the 3′ end of the deletion. HumanCYLN2is very likely identical to the previously characterized, incompleteWSCR4andWSCR3transcription units