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The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain–only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (T reg ) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral T reg s. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via T reg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity., The MANCO project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 101003651). This work made use of the Dutch national e infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) - 398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-04035-x.

Human coronavirus OC43 is a globally circulating common cold virus sustained by recurrent reinfections. How it persists in the population and defies existing herd immunity is unknown. Here we focus on viral glycoprotein S, the target for neutralizing antibodies, and provide an in-depth analysis of its antigenic structure. Neutralizing antibodies are directed to the sialoglycan-receptor binding site in S1

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses., The project was co-financed by a grant from the Zoonotic Anticipation and Preparedness Initiative [ZAPI project; Innovative Medicines Initiative (IMI) grant agreement no. 115760], with the assistance and financial support of IMI and the European Commission, and in-kind contributions from European Federation of Pharmaceutical Industries and Associations partners. The collaboration project is cofunded by the PPP Allowance made available by Health~Holland, Top Sector Life Sciences & Health, to stimulate public-private partnerships. This study was also partially financed by grants from the Ministry of Science and Innovation of Spain (BIO2016-75549-R AEI/FEDER, UE) and NIH (2PO1AIO6O699). The mice used to generate the mAbs produced in this study were provided by Harbour Antibodies BV, a daughter company of Harbour Biomed (http://www.harbourbiomed.com). Chunyan Wang was supported by a grant from the China Scholarship Council.

We use a high-pressure semicontinuous batch electrochemical reactor with a tin-based cathode to demonstrate that it is possible to efficiently convert CO2 to formic acid (FA) in low-pH (i.e., pH < pKa) electrolyte solutions. The effects of CO2 pressure (up to 50 bar), bipolar membranes, and electrolyte (K2SO4) concentration on the current density (CD) and the Faraday efficiency (FE) of formic acid were investigated. The highest FE (?80%) of FA was achieved at a pressure of around 50 bar at a cell potential of 3.5 V and a CD of ?30 mA/cm2. To suppress the hydrogen evolution reaction (HER), the electrochemical reduction of CO2 in aqueous media is typically performed at alkaline conditions. The consequence of this is that products like formic acid, which has a pKa of 3.75, will almost completely dissociate into the formate form. The pH of the electrolyte solution has a strong influence not only on the electrochemical reduction process of CO2 but also on the downstream separation of (dilute) acid products like formic acid. The selection of separation processes depends on the dissociation state of the acids. A review of separation technologies for formic acid/formate removal from aqueous dilute streams is provided. By applying common separation heuristics, we have selected liquid-liquid extraction and electrodialysis for formic acid and formate separation, respectively. An economic evaluation of both separation processes shows that the formic acid route is more attractive than the formate one. These results urge for a better design of (1) CO2 electrocatalysts that can operate at low pH without affecting the selectivity of the desired products and (2) technologies for efficient separation of dilute products from (photo)electrochemical reactors.

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.

A high pressure semicontinuous batch electrolyzer is used to convert CO2 to formic acid/formate on a tin-based cathode using bipolar membranes (BPMs) and cation exchange membranes (CEMs). The effects of CO2 pressure up to 50 bar, electrolyte concentration, flow rate, cell potential, and the two types of membranes on the current density (CD) and Faraday efficiency (FE) for formic acid/formate are investigated. Increasing the CO2 pressure yields a high FE up to 90% at a cell potential of 3.5 V and a CD of ∼30 mA/cm2. The FE decreases significantly at higher cell potentials and current densities, and lower pressures. Up to 2 wt % formate was produced at a cell potential of 4 V, a CD of ∼100 mA/cm2, and a FE of 65%. The advantages and disadvantages of using BPMs and CEMs in electrochemical cells for CO2 conversion to formic acid/formate are discussed.

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell – pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.

Objective Plasma phospholipid transfer protein (PLTP) plays a key role in lipoprotein metabolism. Its exact function in the development of atherosclerosis is still under debate however. We studied the effect of elevated PLTP expression in one of the most commonly used models of atherosclerosis, the ApoE deficient mouse. Methods Experiment 1: Plasma PLTP activity, total cholesterol, HDL cholesterol and atherosclerosis development was measured in ApoE deficient mice with or without elevated expression of PLTP. Experiment 2: The same parameters were measured in ApoE deficient mice after bone marrow transplantation from wild type mice or mice with elevated PLTP expression. Experiment 3: Similar to experiment 2, but using donor mice with an ApoE deficient background. Results Experiment 1: ApoE deficient mice have more than two times more atherosclerosis when overexpressing PLTP and a strongly decreased plasma level of HDL. Experiment 2: Bone marrow transplantation with ApoE proficient cells results in a strong reduction of plasma cholesterol in ApoE deficient acceptor mice. Still, elevated PLTP in bone marrow derived cells evoke a reduction of HDL cholesterol and increased atherosclerosis. Experiment 3: Bone marrow transplantation with ApoE deficient cells results in much higher cholesterol levels, but here too HDL cholesterol levels are reduced and atherosclerosis increased. Conclusion In all the models with ApoE deficiency, elevated PLTP expression causes higher levels of diet-induced atherosclerosis coinciding with decreased plasma levels of HDL cholesterol.

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 transgenic mice 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.

de Waard MC, van der Velden J, Boontje NM, Dekkers DH, van Haperen R, Kuster DW, Lamers JM, de Crom R, Duncker DJ. Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction. Am J Physiol Heart Circ Physiol 296: H1513-H1523, 2009; doi:10.1152/ajpheart.00485.2008.-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 (F-max). 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."

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.-Samyn, H., M. Moerland, T. van Gent, R. van Haperen, J. Metso, F. Grosveld, M. Jauhiainen, A. van Tol, and R. de Crom. Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study. J. Lipid Res. 2008. 49: 2504-2512.

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.

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.

Objective— Arteriogenesis is the major mechanism of vascular growth, which is able to compensate for blood flow deficiency after arterial occlusion. Endothelial nitric oxide synthase (eNOS) activity is essential for neovascularization, however its specific role in arteriogenesis remains unclear. We studied the role of eNOS in arteriogenesis using 3 mouse strains with different eNOS expression. Methods and Results— Distal femoral artery ligation was performed in eNOS-overexpressing mice (eNOStg), eNOS-deficient (eNOS −/− ) mice, and wild type (WT) controls. Tissue perfusion and collateral-dependent blood flow were significantly increased in eNOStg mice compared with WT only immediately after ligation. In eNOS −/− mice, although tissue perfusion remained significantly decreased, collateral-dependent blood flow was only decreased until day 7, suggesting normal, perhaps delayed collateral growth. Histology confirmed no differences in collateral arteries of eNOStg, eNOS −/− , and WT mice at 1 and 3 weeks. Administration of an NO donor induced vasodilation in collateral arteries of eNOS −/− mice, but not in WT, identifying the inability to vasodilate collateral arteries as main cause of impaired blood flow recovery in eNOS −/− mice. Conclusions— This study demonstrates that eNOS activity is crucial for NO-mediated vasodilation of peripheral collateral vessels after arterial occlusion but not for collateral artery growth.

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-gamma-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 alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha 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.

The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2(Akita) spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKβ) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2(Akita) diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2(Akita) cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.

Background— Atherosclerotic lesions are predominantly observed in curved arteries and near side branches, where low or oscillatory shear stress patterns occur, suggesting a causal connection. However, the effect of shear stress on plaque vulnerability is unknown because the lack of an appropriate in vivo model precludes cause-effect studies. Methods and Results— We developed a perivascular shear stress modifier that induces regions of lowered, increased, and lowered/oscillatory (ie, with vortices) shear stresses in mouse carotid arteries and studied plaque formation and composition. Atherosclerotic lesions developed invariably in the regions with lowered shear stress or vortices, whereas the regions of increased shear stress were protected. Lowered shear stress lesions were larger (intima/media, 1.38±0.68 versus 0.22±0.04); contained fewer smooth muscle cells (1.9±1.6% versus 26.3±9.7%), less collagen (15.3±1.0% versus 22.2±1.0%), and more lipids (15.8±0.9% versus 10.2±0.5%); and showed more outward vascular remodeling (214±19% versus 117±9%) than did oscillatory shear stress lesions. Expression of proatherogenic inflammatory mediators and matrix metalloproteinase activity was higher in the lowered shear stress regions. Spontaneous and angiotensin II–induced intraplaque hemorrhages occurred in the lowered shear stress regions only. Conclusions— Lowered shear stress and oscillatory shear stress are both essential conditions in plaque formation. Lowered shear stress induces larger lesions with a vulnerable plaque phenotype, whereas vortices with oscillatory shear stress induce stable lesions.

textabstractPlasma 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.

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, P0.005). Both the mean aortic pressure and the pulmonary artery pressure were reduced in eNOS-GFP mice (both approximately 30%, P0.05). Plasma cholesterol levels were also slightly reduced ( approximately 20%, P0.05). 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.

Objective— HDL plays a key role in protection against development of atherosclerosis by promoting reverse cholesterol transport from peripheral tissues to the liver for secretion into bile. Phospholipid transfer protein (PLTP) promotes the transfer of phospholipids between lipoproteins and modulates HDL size and composition, thereby having a crucial role in HDL metabolism. We investigated the effect of increased PLTP activity on removal of cholesterol from the body. Methods and Results— On a chow diet, transgenic mice overexpressing human PLTP have a 15-fold increased plasma PLTP activity compared with wild-type mice (572.4±59.2 versus 38.6±3.6 μmol/mL per h). Plasma cholesterol, mainly present in HDL, is strongly decreased (−92%), caused by a rapid clearance from the circulation by the liver and leading to a 1.8-fold increase in hepatic cholesteryl esters. This results in a 2-fold increase in biliary bile acid secretion without changing the bile saturation index. Consequently, the transgenic mice show a 1.4-fold increase in the amount of excreted fecal bile acids compared with wild-type mice, whereas fecal neutral sterol excretion is unchanged. Conclusions— Our data show that elevation of PLTP activity results in rapid disposal of cholesterol from the body via increased conversion into bile acids and subsequent excretion.

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 (/=1.5-fold). The mice were given a high fat, high cholesterol diet, which resulted in hypercholesterolemia in all animals. HDL concentrations were dramatically reduced in PLTP-overexpressing animals when compared with LDL receptor controls, whereas VLDL + LDL cholesterol levels were identical. Susceptibility to atherosclerosis was increased in a PLTP dose-responsive manner. We conclude that PLTP increases susceptibility to atherosclerosis by lowering HDL concentrations, and therefore we suggest that an increase in PLTP is a novel, long term risk factor for atherosclerosis in humans.

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 μg/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.

Endothelial dysfunction due to deficient nitric oxide (NO) generation cause progressive vascular complications in diabetes. Our earlier findings revealed that Inhibitor kappa B kinase beta (IKK-beta) -an upstream regulator of an inflammatory mediator, nuclear factor kappa-B (NF-kB), competitively inhibits endothelial nitric oxide synthase (eNOS), the chief source of NO in blood vessels. Therefore, we investigated the influence of an inhibitor of IKK beta, salsalate in augmenting eNOS activity. The co-operative effect of L-arginine supplementation (substrate of eNOS) with salsalate intake in enhancing eNOS activity has been evaluated. Both in vitro cell cultures and diabetic/transgenic animal models were used. Human aortic endothelial cells incubated with 25 mM glucose with or without L-arginine (2mM), the cofactor sepiapterin (100uM) and salsalate(50uM) were examined for NO production. Combined L-arginine and salsalate treatment enhanced NO production, phospho eNOS expression and eNOS-dimer vs monomer ratio. For in vivo assays, diabetic Ins2Akita mice were fed with L-arginine and salsalate for 15 weeks. Aortae of these mice exhibited increased phospho eNOS expression and increased eNOS dimer/monomer ratio. Arterial ring assays showed improved acetylcholine mediated vasodialation. Transgenic-Akita mice that overexpress human eNOS in endothelial cells (Tie-2eNOS-Ins2 Akita) generated in-house showed improved vasodialation. In conclusion, enhancing eNOS activity in diabetic mice either by genetic modification as seen in Tie-2 eNOS-Akita mice or by nutritional approach (supplementation of L-arginine with simultaneous inhibition of IKK beta) restores normal vasodialatory function.

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.

textabstractPURPOSE. In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O2 -), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina. METHODS. Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA. RESULTS. In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH4) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels. CONCLUSIONS. In hyperoxia, reduced BH4 bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia.

Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to vascular maintenance by participating in angiogenesis, 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 A(1C) levels 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.

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 atherosclerotic 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

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 hypercholesteratemic 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 mu g/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 p27(kiP1) expression 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.

Alteration in post-ischemic neovascularization is a common complication of atherosclerotic disease. This results, at least in part, from abrogation of bone-marrow mononuclear cells (BM-MNC) pro-angiogenic potential. Overexpression of eNOS has been shown to promote vessel growth in the setting of ischemia. We hypothesized that eNOS overexpression could restore impaired neovascularization in atherosclerotic (ApoE KO) mice. Hind limb ischemia was induced in mice by right femoral artery ligation. After two weeks we evaluated tissue perfusion of the foot by Laser Doppler, vessel density in the hind limb by micro-angiography and histology, and atherosclerotic plaque size. In vitro BM-MNC cell culture assays were performed. Tissue perfusion and vessel density were 1.5-fold increased in transgenic mice overexpressing eNOS (eNOStg) as compared to wild type (WT) (P Conclusion: eNOS overexpression in the endothelium improves post-ischemic neovascularization in both physiological as atherosclerotic settings. Furthermore, eNOS overexpression in the bone marrow restores the impaired pro-angiogenic potential of atherosclerotic BM-MNC without adverse effects on plaque size. Therefore, overexpression of eNOS could play a vital part in the development of therapeutic angiogenesis for atherosclerotic disease.

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, p0.001). 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.

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 P P 4 β 1 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.

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.

We thank Dr Jankowski for his interest in our article.1 The major conclusions from our work are that both reduced and oscillating shear stress are proatherogenic but that they induce different plaque compositions. Reduced shear stress induces vulnerable plaques, and oscillating shear stress induces stable plaques. In addition, we found that high shear stress is atheroprotective. Dr Jankowski challenges our last conclusion, referring to the …