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Background & Aims ER stress promotes liver fat accumulation and induction of inflammatory cytokines, which contribute to the development of steatohepatitis. Unresolved ER stress upregulates the pro-apoptotic CHOP. IL-1α is localized to the nucleus in apoptotic cells, but is released when these cells become necrotic and induce sterile inflammation. We investigated whether IL-1α is involved in ER stress-induced apoptosis and steatohepatitis. Methods We employed WT and IL-1α-deficient mice to study the role of IL-1α in ER stress-induced steatohepatitis. Results Liver CHOP mRNA was induced in a time dependent fashion in the atherogenic diet-induced steatohepatitis model, and was twofold lower in IL-1α deficient compared to WT mice. In the ER stress-driven steatohepatitis model, IL-1α deficiency decreased the elevation in serum ALT levels, the number of apoptotic cells (measured as caspase-3-positive hepatocytes), and the expression of IL-1β, IL-6, TNFα, and CHOP, with no effect on the degree of fatty liver formation. IL-1α was upregulated in ER-stressed-macrophages and the protein was localized to the nucleus. IL-1β mRNA and CHOP mRNA and protein levels were lower in ER-stressed-macrophages from IL-1α deficient compared to WT mice. ER stress induced the expression of IL-1α and IL-1β also in mouse primary hepatocytes. Recombinant IL-1α treatment in hepatocytes did not affect CHOP expression but upregulated both IL-1α and IL-1β mRNA levels. Conclusion We show that IL-1α is upregulated in response to ER stress and IL-1α deficiency reduces ER stress-induced CHOP expression, apoptosis and steatohepatitis. As a dual function cytokine, IL-1α may contribute to the induction of CHOP intracellularly, while IL-1α released from necrotic cells accelerates steatohepatitis via induction of inflammatory cytokines by neighboring cells.

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.

Objective: The aim of the present study was to compare the accumulation of 125I-labeled low-density lipoproteins (LDL), oxidized LDL (oxLDL), HDL and BSA in advanced atherosclerotic lesions of apoE-deficient mice. Methods:125I-lipoproteins or 125I-BSA were injected into the tail vein of apoE-deficient mice. Blood clearance of 125I-lipoproteins and 125I-BSA and their accumulation in atherosclerotic lesions were assayed. Results: Blood clearance of 125I-LDL and 125I-HDL was moderate, and approximately 30% of the injected lipoproteins were present in plasma 24 h following injection. oxLDL was removed much faster from plasma, and less than 10% of 125I-oxLDL was present in the circulation 30 min after 125I-oxLDL injection. The clearance of 125I-BSA from the circulation was slower than the lipoprotein clearance. The highest accumulation of LDL, oxLDL, HDL and BSA was detected in atherosclerotic lesions in the aortic arch and abdominal aorta, while lower accumulation was detected in the less atherosclerotic descending thoracic aorta. Conclusion: Our findings demonstrate that both 125I-HDL and 125I-BSA as well as 125I-LDL are accumulated in atherosclerotic plaques and that they can be used for the detection of atherosclerotic lesions.

Background: Autoimmune factors have been shown to play a role in atherosclerosis. The aim of this study is to correlate 5 autoantibodies (anticardiolipin, anti-CL, β2-glycoprotein-I, β2GPI, phosphatidylcholine, oxidized low-density lipoprotein, oxLDL, endothelial cell) with the presence of coronary heart disease, angiographic findings, and with coronary artery calcification. Methods: The levels of the 5 autoantibodies and a control antifibroblast line of 126 coronary heart disease patients and 20 healthy controls were measured. Fifty-one patients underwent coronary angiography, and 98 patients had coronary artery calcium determination using spiral computerized tomography (dual mode). Results: Levels of 3 autoantibodies (anti-CL, β2GPI, oxLDL) were significantly elevated in coronary heart disease patients compared with controls (p < 0.001,p = 0.001, p < 0.001, respectively). Within the subgroup of patients with significant coronary artery stenosis, anti-CL antibodies were also elevated (p = 0.008). No correlation was found between anti-CL, and anti-β2GPI autoantibody levels and coronary calcium scores as measured by spiral computerized tomography. However, anti-oxLDL antibodies were raised in patients with no calcification detected by spiral computerized tomography, compared with the patients with any coronary calcification (p = 0.046). Conclusion: Anti-CL, β2GPI and oxLDL antibodies are elevated in coronary heart disease patients regardless of coronary calcification.

15-Lipoxygenase (15-LOX) is a lipid-oxidizing enzyme that is involved in cell cycle regulation. To evaluate the effect of 15-LOX on reproduction, we studied transgenic mice that overexpress 15-LOX. The transgene was introduced over the genetic background of the low density lipoprotein receptor deficient mice (LDL-R–/–) and reproduction was compared to LDL-R–/– mice. We found a lower pregnancy rate in the 15-LOX/LDL-R–/– mice as compared to the LDL-R–/– mice (62.74 vs. 79.1%, p < 0.01). Additionally, a remarkably higher number of resorptions per pregnancy was found in the 15-LOX/LDL-R–/– mice (16.7 vs. 3.27%, p < 0.001) and it was accompanied by a significantly higher activity of the 15-LOX enzyme in these resorptions as compared to other tissues. These findings may implicate a role for 15-LOX in the development of spontaneous abortions.

Background —It has been proposed that autoimmune factors can influence the progression of atherosclerosis. We have previously shown that immunization of LDL receptor–deficient (LDL-RD mice) with β 2 -glycoprotein I (β2GPI; a principal target of “autoimmune” antiphospholipid antibodies) enhances early atherosclerosis. In the present study, we tested the hypothesis that adoptive transfer of β2GPI-reactive T cells can accelerate fatty streak formation in LDL-RD mice. Methods and Results —LDL-RD mice were immunized with human β2GPI. An additional group of mice were immunized with β2GPI and boosted with the same antigen 3 weeks later. Control mice with immunized with human serum albumin. Lymphocytes obtained from the draining lymph node cells or from splenocytes of β2GPI- or human serum albumin–immunized mice were stimulated in vitro with β2GPI or with the mitogen concavalin A, respectively. The cultured lymphocytes were transferred intraperitoneally to syngenic LDL-RD mice, and the mice were fed a high-fat “Western” diet for 5 weeks until death. Mice injected with lymphocytes from draining lymph nodes or spleens of β2GPI-immunized animals displayed larger fatty streaks than those induced by control treated animals. T-cell–depleted splenocytes from β2GPI were unable to promote lesion formation in the mice. Conclusions —The present study provides the first direct evidence for a role of antigen (β2GPI)-reactive T cells in the promotion of fatty streaks in mice.

We have made consecutive studies to prove that autoimmune factors can influence the progression of atherosclerosis in inbred and transgenic mice. C57BL/6 as well as LDL-receptor deficient mice were immunized with heat shock protein 65. LDL-RD and apolipoprotein E knockout (apoE KO) mice were immunized with human B-glycoprotein I. ApoE KO mice were immunized with oxidized LDL. In all immunized mice, a sustained humoral response to the provided antigen was elicited evident by high titers of antibodies by ELISA. A primary cellular immune response was also shown by thymidine incorporation studies employing the antigens in vitro. Immunization with hsp-65 and with beta 2-GPI served to enhance the progression atherosclerosis and led to an increase in the infiltration of CD3 in the subendothelial regions of the early plaques. Transfer of hsp-65 and beta 2-GPI reactive lymphocytes to syngenic mice led to enhancement of fatty streak formation. However, immunization with homologous oxLDL in apoE KO mice led to attenuation of lesion progression concomitant with the production of anti-oxLDL antibodies. Thus, autoimmune factors appear to influence early artherosclerosis progression in mice. If proven in humans these antigen specific responses may be harnessed for selective immunomodulation of the atherosclerotic plaque.

Abstract —Atherosclerotic lesions can be induced in rabbits and mice immunized with heat shock protein 65 (HSP65). In the current study, we investigated the role of interleukin (IL)-4 in the HSP65- and Mycobacterium tuberculosis (MT)–induced models that exhibit an inflammatory phenotype. Fatty streak formation in IL-4–knockout (IL-4 KO) mice immunized with HSP65 or MT was significantly reduced when compared with lesions in wild-type C57BL/6 mice. However, when injected with control (HSP-free) adjuvant, no differences were evident in the lesion size between wild-type and the IL-4 KO mice. Next, we studied comparatively the extent of humoral and cellular immune responses to HSP65 in the IL-4 KO and wild-type mice, as those are thought to be influential in murine atherosclerosis. Anti-HSP65 antibody levels were reduced in the HSP65-immunized IL-4 KO mice as compared with their wild-type littermates, whereas no differences were evident between the groups with respect to the primary cellular immune response to HSP65. Other than the absence of IL-4 in the knockout mice, the pattern of secreting cytokines interferon-γ and IL-10 in concanavalin A–primed splenocytes was similar between the groups. HSP65-primed inguinal lymphocytes from IL-4 KO mice immunized with HSP65 secreted higher levels of interferon-γ (previously shown to be proatherogenic in vivo) as compared with their wild-type controls. 12-/15-Lipoxygenase expression, known to be regulated by IL-4 and to contribute to murine atherosclerosis, in the lesions was not influenced by the immunization protocol used or by IL-4 disruption. Thus, IL-4 may prove a principal cytokine in the progression of early “inflammatory” atherosclerotic lesions and may serve as a target for immunomodulation.

Diabetes and atherosclerosis have been proposed to be influenced by immune and autoimmune mechanisms. A common incriminated antigen in both disorders is the heat shock protein (HSP)-60/65. In the current study, we established a model combining hyperglycemia with hyperlipidemia in LDL receptor-deficient (LDL-RD) mice and assessed its possible influences on lipid profile, HSP60/65, and atherogenesis. LDL-RD mice were injected either with streptozotocin to induce hyperglycemia or with citrate buffer (control). When hyperglycemia was induced, both study groups were challenged with a high-fat (Western) diet for 6 weeks. Plasma fasting glucose, lipid profile, and antibody levels to HSP65 and oxidized LDL were assessed. At death, the spleens from both groups were evaluated for their proliferative response to HSP65 and the consequent cytokine production. The extent of atherosclerosis was assessed at the aortic sinus. Plasma glucose, cholesterol, and triglyceride levels were elevated in mice injected with streptozotocin compared with control mice. Atherosclerotic lesions were significantly larger in the streptozotocin-injected hyperglycemic LDL-RD mice (132 +/- 23 x 10(5) microm2) in comparison to their normoglycemic litter-mates (20 +/- 6.6 x 10(5) microm2; P < 0.0001). Both humoral and cellular immune response to HSP65 was more pronounced in streptozotocin-injected mice. When challenged with HSP65 in vitro, splenocytes from streptozotocin-injected mice favored the production of the T-helper (TH)-1 cytokine gamma-interferon. In conclusion, we have established a mouse model that combines hyperglycemia with diet-induced hyperlipidemia in LDL-RD mice and studied its effect on atherosclerosis progression. The accelerated atherosclerotic process is associated with heightened immune response to HSP65 and a shift to a TH1 cytokine profile.

The modern view of atherosclerosis is of a chronic inflammatory disorder. In accord with this paradigm, the process of uninhibited influx of fat to the vessel wall results from an ‘adequate’ response to various forms of injury (i.e. turbulence, infections, modified lipoproteins). This idea has been further extended by several groups, to assume that the atherosclerotic lesion can be the target of an autoimmune mediated attack. According to this hypothesis, the site of initiation of the plaque should bear/express the target autoantigen, whereas concomitantly a respective immune response is generated in the periphery. The examples illuminating this notion are β2GPI as a target autoantigen, HSP60/65 an oxidized-LDL. Herein we present evidence to support the involvement of autoimmune mechanisms in atherogenesis based on the experience from experimental models and human studies.

Objective: The renin-angiotensin system may play a role in the development of atherosclerosis. Nevertheless, different results from studies attempting to attenuate the process by inhibiting the converting enzyme were equivocal, and in those who succeeded, blood pressure was lowered and/or the lipid profile was improved in addition to the inhibition of the renin-angiotensin axis. The aim of this study is to investigate the effect of low doses of fosinopril, a converting enzyme inhibitor, on the development of atherosclerosis in LDL-receptor-deficient mice. Methods: Three groups of 15 mice were fed a high-fat, high-cholesterol western diet. The three study groups received either distilled water (control group), or water supplemented with fosinopril 0.01 mg/kg/day (low-dose group) or with 0.1 mg/kg/day (high-dose group). Plasma aldosterone levels and lipid profiles were measured at the beginning and at the end of the study. After 10 weeks, the mice were sacrificed and the extent of atherosclerosis was assessed at the aortic sinus. Results: Plasma aldosterone levels did not change in the control group, but decreased significantly in both treated groups from 74.7 to 39.3 ng/ml in the low-dose group (p < 0.003) and from 70.7 to 33.6 ng/ml in the high-dose group (p < 0.001). The lipid profile at the end of the study showed significantly lower levels of cholesterol and triglycerides in the high-dose group as compared to the low-dose group (p < 0.05). There was no difference between the three groups regarding the area of atherosclerosis at the aortic sinus: 157,000 ± 34,000, 130,000 ± 58,000 and 145,000 ± 26,000 µm2 in the control, low-dose and high-dose groups, respectively. Conclusion: Inhibition of the renin-angiotensin-aldosterone axis by itself does not prevent the development of early atherosclerosis in LDL-receptor-deficient mice.

Atherosclerosis is a multifactorial process, the hallmark of which is fat deposition in the vessel wall. Autoimmune factors have recently been shown to play an important role in the initiation and progression of atherosclerosis; candidate autoantigens are oxidized lipids and heat shock proteins. beta2-glycoprotein I (beta2-GPI) is a highly glycosylated plasma protein that serves as a major antigenic target for autoimmune type antiphospholipid antibodies. Its major relevant property is binding to negatively charged phospholipids/surfaces. In the set of studies presented in this paper, we provide evidence pointing towards beta2-GPI as an influential determinant in murine and human atherogenesis. Thus, immunization of transgenic atherosclerosis-prone mice (apolipoprotein E and low-density lipoprotein receptor knockouts) with human beta2-GPI results in a brisk and sustained respective response that extends to cross-react with the 'self' murine beta2-GPI. Atherosclerosis is accelerated in both strains concomitant with the infiltration of CD4 lymphocytes in the aortic sinus of the mice. When human plaques were studied, it was found that beta2-GPI resides in the subendothelial regions and co-localizes with CD4 lymphocytes. Thus, the immune response towards beta2-GPI may play an important role in atherogenesis, serving as a possible target for antigen specific therapies.

Abstract —Although lipid oxidation plays a major role in atherogenesis, the role of antioxidants in the prevention and treatment of the process is not clear. Apolipoprotein (apo) E–deficient mice develop spontaneous atherosclerotic lesions in major arteries. The presence of oxidized lipoprotein epitopes in the lesion suggests that oxidation reactions are involved in atherogenesis in this mouse model, but the inhibitory effect of antioxidants on atherogenesis in the model is controversial. To test the effect of dietary antioxidants on atherogenesis, male apoE-deficient mice (n=15) were fed a standard chow diet supplemented with 0.05% α-tocopherol and 0.05% all-trans β-carotene. A control group (n=15) received no antioxidant supplement. At the end of the trial, mice consuming vitamins had 5× more plasma vitamin E but undetectable β-carotene levels. However, liver levels of the β-carotene metabolite, retinyl palmitate, were higher in antioxidant-treated mice compared with control mice. The antioxidants had no effect on lipoprotein or on plasma anti–oxidatively modified low density lipoproteins (anti-oxLDL) antibody levels. The vitamins had a small but insignificant effect on lipoprotein resistance to ex vivo oxidation, determined by a longer lag period of conjugated diene formation. Atherosclerosis, determined by the lesion size at the aortic sinus, was insignificantly suppressed in antioxidant-treated mice (mean area±SE, 20 000±7129 versus 13 281±5861 μm 2 ; P =0.40). The aortic atherosclerotic lesion area was similar in both experimental groups (2.55±0.65% and 2.08±0.5% of total aortic area in the control and antioxidant group, respectively; P =0.58). The results of the current study suggest that moderate levels of synthetic antioxidant vitamins have no effect on atherogenesis in apoE-deficient mice.

Background —Immunization with β 2 -glycoprotein I (β2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of β2GPI immunization on the progression of atherosclerosis. Methods and Results —In the first experiment, 3 groups of LDL receptor–deficient (LDL-RD) mice (n=15 per group) were immunized with either β2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All β2GPI-immunized mice developed high titers of anti-β2GPI antibodies as well as a specific lymph node proliferation to β2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the β2GPI-immunized mice (mean aortic lesion, 26 000±5700 μm 2 ) in comparison with their ovalbumin-immunized (mean, 3000±1099 μm 2 ; P 2 ; P 2 ) was larger than the ovalbumin-immunized mice (mean, 81 250±12 933 μm 2 ; P =NS) or the nonimmunized controls (mean, 75 625±7281 μm 2 ; P =NS). The atherosclerotic plaques in the β2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice. Conclusions —The results of the present study provide the first direct evidence for the proatherogenic effect of β2GPI immunization and establish a new model for immune-mediated atherosclerosis.

Background The role of Kupffer cell interleukin (IL)-1 in non-alcoholic steatohepatitis development remains unclear. Aims To evaluate the role of Kupffer cell IL-1α, IL-1β or IL-1 receptor type-1 (IL-1R1) in steatohepatitis. Methods C57BL/6 mice were irradiated and transplanted with bone marrow-derived cells from WT, IL-1α−/−, IL-1β−/− or IL-1R1−/− mice combined with Kupffer cell ablation with Gadolinium Chloride, and fed atherogenic diet. Plasma and liver triglycerides and cholesterol, serum alanine aminotransferase (ALT), liver histology and expression levels of inflammatory genes were assessed. Results The ablation and replacement of Kupffer cells with bone marrow-derived cells was confirmed. The atherogenic diet elevated plasma and liver cholesterol, reduced plasma and liver triglycerides and increased serum ALT levels in all groups. Steatosis and steatohepatitis were induced, but without liver fibrosis. A reduction in the severity of portal inflammation was observed only in mice with Kupffer cell deficiency of IL-1α. Accordingly, liver mRNA levels of inflammatory genes encoding for IL-1α, IL-1β, TNFα, SAA1 and IL-6 were significantly lower in mice with Kupffer cell deficiency of IL-1α compared to WT mice. Conclusion Selective deficiency of IL-1α in Kupffer cells reduces liver inflammation and expression of inflammatory cytokines, which may implicate Kupffer cell-derived IL-1α in steatohepatitis development.

Introduction.β-Carotene-rich diet has been shown to be inversely associated with the risk of coronary heart disease. However, clinical trials using synthetic all-trans-β-carotene failed to demonstrate a beneficial effect. We therefore sought to study the effect of natural source ofβ-carotene, the algaDunaliella, containing both all-trans and 9-cis-β-carotene on atherosclerosis. In a previous study we showed that 9-cis-β-carotene-rich powder of the algaDunaliellainhibits early atherogenesis in low-density lipoprotein receptor knockout mice.Aims. The aims of the current work were to study whether diet enriched withDunaliellapowder would inhibit the progression of established atherosclerosis in old male apoE-deficient mice and to compare the effect ofDunaliellaon lipid profile and atherosclerosis in a low-versus high-fat diet fed mice.Methods. In the first experiment, young mice (12 weeks old) were allocated into 3 groups: (1) low-fat diet; (2) low-fat diet + Dunaliellapowder (8%); (3) low-fat diet + β-carotene-deficientDunaliella. In the second experiment, old mice (7 months old) with established atherosclerotic lesions were allocated into 4 groups: (1) low-fat diet; (2) low-fat diet + Dunaliella; (3) high fat-diet; (4) high-fat diet + Dunaliella.Results. In young mice fed a low-fat diet, a trend toward lower atherosclerotic lesion area in the aortic sinus was found in theDunaliellagroup compared with the control group. In old mice with established atherosclerotic lesion,Dunaliellainhibited significantly plasma cholesterol elevation and atherosclerosis progression in mice fed a high-fat diet.Conclusion. The results of this study suggest that a diet containing natural carotenoids, rich in 9-cis-β-carotene, has the potential to inhibit atherosclerosis progression, particularly in high-fat diet regime.

article i nfo Objective: The acute effect of heparin on lipoprotein clearance is well characterized. Yet, the effect of prolonged low-molecular-weight-heparin (LMWH) administration on post-prandial lipemia has remained so far unex- plored. Recent reports suggest that LMWH could modify lipid and carbohydrate metabolism by diminishing TNFα-mediated inflammatory response. This, together with the known negative effect of TNF-α on insulin sen- sitivity, prompted us to hypothesize that LMWH would favorably affect post-prandial lipoprotein disposal. Methods: Twenty four patients were given a vitamin A-fat loading meal at the end of 6-week enoxaparin treat- ment and after 3-month washout period. Post-prandial lipemia was assessed by measuring retinyl-palmitate (RP) during 8 hours following the meal. Insulin sensitivity index (ISI), plasma lipolytic activity and plasma TNF-α were measured. Results: Enoxaparin did not impact fasting plasma lipids and lipoproteins levels. Enoxaparin increased RP clear- ance in the chylomicron remnant (CMR) fraction by 32% (P b 0.01). Additionally, enoxaparin decreased plasma TNF-α by 22% (P b 0.01), increased hepatic lipase (HL) activity by 81% (P b 0.01), along with a 2-fold increase in ISI (P b 0.01). The decrease in CMR correlated with the reduction in TNFα and the increase in ISI and HL activity (R = 0.48, -0.68, -0.56, respectively, p b 0.05). Significant correlations were also found between the reduction in TNFα and both the increase in ISI and increase in HL activity (R = -0.43, -0.54, respectively, P b 0.05). Conclusions: The association of the effect on post-prandial metabolism, plasma TNFα level and HL activity during prolonged enoxaparin treatment may support the hypothesis that the beneficial outcome of enoxaparin may possibly be linked to anti-inflammatory and lipase-potentiating impact.

Importance Retinitis pigmentosa (RP) is the leading cause of incurable inherited blindness in the developed world, with an estimated prevalence of 1 in 3500 individuals. Therefore, it is important to develop new treatments for this disease. Objective To determine the effect of oral treatment with 9- cis β-carotene on visual function of patients with RP. Design Randomized, double-masked, placebo-controlled, crossover clinical trial. Setting University tertiary medical facility. Participants Thirty-four patients with RP who were at least 18 years of age. Twenty-nine patients completed the study and were included in the analysis. Interventions Patients were treated daily for 90 days with capsules containing 300 mg of 9- cis β-carotene–rich alga Dunaliella bardawil (β-carotene, approximately 20 mg) or placebo (starch). Following a 90-day washout period, they were treated for 90 days with the other capsules. Main Outcomes and Measures The primary outcome was the change for both eyes from baseline to the end of each treatment in dark-adapted maximal electroretinographic b-wave amplitude. The secondary outcomes were the changes in light-adapted maximal b-wave amplitude, dark- and light-adapted visual field, and best-corrected visual acuity. Results The mean change in dark-adapted maximal b-wave amplitude relative to initial baseline was +8.4 μV for 9- cis β-carotene vs −5.9 μV for placebo ( P = .001). Ten participants (34.5%) had an increase of more than 10 μV for both eyes (range, 11-42 μV) after 9- cis β-carotene treatment compared with no participants after placebo treatment. The percentage change in light-adapted b-wave response was +17.8% for 9- cis β-carotene vs −3.0% for placebo ( P = .01). No significant differences were found between the groups for visual field and best-corrected visual acuity. No adverse effects were observed. Conclusions and Relevance Treatment with 9- cis β-carotene significantly increased retinal function in patients with RP under the tested conditions. The optimal therapeutic regimen will be determined in future, larger clinical trials. 9- cis β-Carotene may represent a new therapeutic approach for some patients with RP. Trial Registration clinicaltrials.gov Identifier:NCT01256697

Purpose: VB-111 is an antiangiogenic agent consisting of a nonreplicating adenovirus vector (Ad-5) with a modified murine pre-proendothelin promoter leading to apoptosis of tumor vasculature by expressing a Fas-chimera transgene in angiogenic endothelial cells. In a phase I dose-escalation study, pharmacokinetics, pharmacodynamics, safety, and efficacy of a single dose of VB-111 in patients with advanced solid tumors were evaluated. Experimental Design: VB-111 was administered as a single i.v. infusion at escalating doses from 1 × 1010 (cohort 1) to 1 × 1013 (cohort 7) viral particles (VP) in successive cohorts. Assessments included pharmacokinetic and pharmacodynamic profiles, tumor response, and overall survival. Results: Thirty-three patients were enrolled. VB-111 was safe and well-tolerated; self-limited fever and chills were seen at doses above 3 × 1011 VPs. Transgene expression was not detected in blood but was detected in an aspirate from a subcutaneous metastasis after treatment. One patient with papillary thyroid carcinoma had a partial response. Conclusions: VB-111 was safe and well tolerated in patients with advanced metastatic cancer at a single administration of up to 1 × 1013 VPs. Evidence of transgene expression in tumor tissue and tumor response was observed. Clin Cancer Res; 19(14); 3996–4007. ©2013 AACR.

Atherosclerosis is a histopathological process of a multifactorial origin. Whereas the genetic and biochemical causes received considerable attention, the involvement of the immune system has generally been considered negligible. In recent years evidence has been presented to support the dominant role played by the immune system in atherosclerosis. Two major antigenic determinants against which the immune response may be triggered have been suggested, namely the heat shock protein 60/65 and oxidized low density lipoprotein. The current paper reviews the data regarding the involvement of the immune system in atherogenesis with respect to the antigenic candidates mentioned above.

Atherosclerosis is one of the main causes of morbidity and mortality world-wide and specifically in Israel. These guidelines update the previous guidelines of the Israeli Society for Research, Prevention and Treatment of Atherosclerosis, published in 2005. The need for an update is based on new scientific data published in recent years necessitating changes in the recommendations for preventing and treating atherosclerosis. These guidelines were written in collaboration between all the societies outlined here and the content of this statement was approved by the delegates of these societies. The recommendations were written taking into consideration guidelines published by other international medical societies and also the specific needs of the Israeli medical system. Due to limitations of space, in the current paper we present: assessment of cardiovascular risk, smoking cessation and the treatment of dyslipidemia. Other sections including: recommendations to the general population, nutritional and physical activity recommendations, treatment of hypertension, prevention of ischemic stroke and the metabolic syndrome are available at http://www.ima.org.il/harefuah.

Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow characterized by abnormalities in maturation of hematopoietic cells of all lineages. MDS patients frequently have lower lipids and high rates of apoptosis and p53 (TP53) expression. An association between the reduced lipids in MDS and the expression of lipid-related genes was sought. We further evaluated whether 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGcoAR) and low-density lipoprotein receptor (LDL-R) are regulated by TP53 in vivo and in vitro. Gene expression was measured using real-time reverse transcription polymerase chain reaction on RNA extracted from bone marrow and peripheral blood from eight newly diagnosed MDS patients and eight controls and from mice livers. Serum lipid profile was measured using colorimetric enzymatic procedures. Total- and LDL cholesterol were lower in MDS patients in comparison to controls (p = 0.04 and p = 0.01, respectively). HMGcoAR messenger RNA increased in peripheral blood and bone marrow of MDS patients compared to controls (p = 0.04 and p = 0.01, respectively). LDL-R messenger RNA was higher only in the peripheral blood of MDS patients (p = 0.05). Comparable results were obtained in vivo. The transcription of these genes correlates with TP53 activation as documented by p21 messenger RNA elevation, a surrogate for TP53 activation and by using TP53 temperature-sensitive cells treated with adriamycin. To conclude, an association between reduced lipids in MDS and expression of HMGcoAR and LDL-R genes was documented. The transcription of these genes can be regulated by TP53.

VB-111 is an engineered antiangiogenic adenovirus that expresses Fas-c in angiogenic blood vessels and has previously been shown to have significant antitumor activity in vitro and in vivo in Lewis lung carcinoma, melanoma, and glioblastoma models. To evaluate the efficacy of VB-111 in thyroid cancer, we conducted in vivo xenograft nude mouse studies using multiple thyroid cancer-derived cell lines models. VB-111 treatment resulted in 26.6% (P = 0.0596), 34.4% (P = 0.0046), and 37.6% (P = 0.0249) inhibition of tumor growth in follicular, papillary and anaplastic thyroid cancer models, respectively. No toxicity was observed in any model. All tumor types showed a consistent and significant reduction of CD-31 staining (P < 0.05), reflecting a reduction of angiogenic activity in the tumors, consistent with the intended targeting of the virus. A phase 2 clinical trial of VB-111 in patients with advanced differentiated thyroid cancer is ongoing.

It is well accepted that high levels of low-density lipoprotein (LDL) cholesterol in the plasma are associated with increased risk of atherosclerosis. The cellular and molecular mechanisms linking the two however, have not been fully resolved. One of the processes involved in atherogensis that has been intensively studied in this regard is the oxidation of LDL. Oxidation may convert LDL into an atherogenic form, which incites an inflammatory and proliferative response characteristic of the atherosclerotic lesion. One of the potential mediators in this process is the lipid peroxidating enzyme 15-lipoxygenase, which has been shown to be induced in the atherosclerotic lesion and is capable of oxidizing LDL. In this article, we review the motivation for looking at mechanisms of LDL oxidation and the proposed involvement of 15-lipoxygenase in the pathogenesis of the disease.

Item does not contain fulltext BACKGROUND & AIMS: The identification of the cellular and molecular pathways that mediate the development of non-alcoholic steatohepatitis is of crucial importance. Cytokines produced by liver-resident and infiltrating inflammatory cells, play a pivotal role in liver inflammation. The role of the proinflammatory cytokines IL-1alpha and IL-1beta in steatohepatitis remains elusive. METHODS: We employed IL-1alpha and IL-1beta-deficient mice and transplanted marrow cells to study the role of liver-resident and bone marrow-derived IL-1 in steatosis and its progression to steatohepatitis. RESULTS: Atherogenic diet-induced steatohepatitis in wild-type mice was associated with 16 and 4.6 fold-elevations in mRNA levels of hepatic IL-1alpha and IL-1beta, respectively. In mice deficient in either IL-1alpha or IL-1beta the transformation of steatosis to steatohepatitis and liver fibrosis was markedly reduced. This protective effect in IL-1alpha-deficient mice was noted despite increased liver cholesterol levels. Deficiency of IL-1alpha markedly reduced plasma serum amyloid A and steady-state levels of mRNA coding for inflammatory genes (P-selectin, CXCL1, IL-6, and TNFalpha) as well as pro-fibrotic genes (MMP-9 and Collagen) and particularly a 50% decrease in TGFbeta levels (p = 0.004). IL-1alpha mRNA levels were two-folds lower in IL-1beta-deficient mice, and IL-1beta transcripts were three-folds lower in IL-1alpha-deficient compared to wild-type mice. Hepatic cell derived IL-1alpha rather than from recruited bone marrow-derived cells was required for steatohepatitis development. CONCLUSIONS: These data demonstrate the critical role of IL-1alpha and IL-1beta in the transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. Therefore, the potential of neutralizing IL-1alpha and/or IL-1beta to inhibit the development of steatohepatitis should be explored. 01 november 2011

The European Atherosclerosis Society Workshop on the Immune System in Atherosclerosis was held at the Hotel Intercontinental, Geneva, Switzerland, from 8–11 March 2001.

Item does not contain fulltext OBJECTIVE: Interleukin (IL)-1alpha and IL-1beta are products of macrophages, endothelial cells and vascular smooth muscle cells; moreover, each of these cell types is affected by the pro-inflammatory properties of both IL-1's. Whereas several studies demonstrate the proatherogenic properties of IL-1beta, the role of IL-1alpha in atherogenesis remains unclear. We assessed whether IL-1alpha and IL-1beta from tissue resident vascular cells or emigrating bone marrow-derived cells promote the development of atherosclerosis in apoE-/- mice and determined the effect of selective macrophage IL-1alpha or IL-1beta deficiency on degradation of LDL and cytokine production. METHODS: We generated strains of double knock-out (KO) mice (apoE-/-/IL-1alpha-/- and apoE-/-/IL-1beta-/-) and created chimeras consisting of apoE-/- mice reconstituted with bone marrow-derived cells from apoE-/-/IL-1+/+, apoE-/-/IL-1alpha-/- and apoE-/-/IL-1beta-/-. RESULTS: The areas of aortic sinus lesions were lower in either double KO mice compared to solely apoE-/- mice, despite higher non-HDL cholesterol levels. Importantly, selective deficiency of IL-1alpha or IL-1beta in bone marrow-derived cells inhibited atherogenesis to the same extent as in double KO mice without affecting plasma lipids. Aortic sinus lesions in apoE-/- mice transplanted with IL-1beta-/- or IL-1alpha-/- cells were 32% and 52% lower, respectively, than in IL-1+/+ transplanted mice. Ex vivo, isolated IL-1alpha-/- macrophages from atherosclerotic mice degraded LDL and secreted IL-6, TNFalpha and IL-12 similarly to IL-1+/+ macrophages; however, IL-1alpha deficient macrophages secreted reduced levels of IL-1beta (-50%) and 2-3-fold higher levels of the anti-inflammatory cytokine IL-10. CONCLUSION: We show for the first time that it is IL-1alpha from bone marrow-derived cells that accelerates atherogenesis in apoE-deficient mice rather than constitutive IL-1alpha in vascular cells, possibly by increasing the inflammatory cytokine profile of macrophages.

Preeclampsia and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that share a common pathophysiology. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that plays an important role in placental development. HIF-1α is elevated in preeclamptic placentas and induces soluble vascular endothelial growth factor receptor-1 (sFLT-1), a central factor in preeclampsia and IUGR pathogenesis. Our objective was to investigate the effects of HIF-1α overexpression on pregnancy in mice. C57BL/6J pregnant mice were systemically administered either adenovirus expressing stabilized HIF-1α (cytomegalovirus [CMV]-HIF), luciferase control (CMV-Luc), or saline on gestational day 8. Pregnant mice overexpressing HIF-1α had significantly elevated blood pressure and proteinuria compared with pregnant controls. HIF-1α mice showed fetal IUGR, decreased placental weights, and histopathological placental abnormalities compared with control mice. Glomerular endotheliosis, the hallmark lesion of preeclampsia, was demonstrated in the kidneys of these mice relative to the normal histology in control mice. Moreover, liver enzyme levels were significantly elevated, whereas complete blood counts revealed significant anemia and thrombocytopenia in CMV-HIF mice compared with controls. Blood smears confirmed microangiopathic hemolytic anemia in CMV-HIF mice, consistent with HELLP (hemolysis, elevated liver enzymes, and low platelets)-like syndrome. CMV-HIF mice showed elevation in serum sFLT-1 and soluble endoglin, providing a mechanistic explanation for the observations. Collectively, our results suggest a possible role for HIF-1α in the pathogenesis of both preeclampsia and IUGR.

The aim of the current study was to evaluate the therapeutic effects of omega-3 plant sterol esters (n-3-PSE) on lipid profile and other coronary heart disease risk factors in subjects with mixed hyperlipidemia.Ninety-one patients with mixed hyperlipidemia were randomized in a double blind fashion to receive either placebo (corn oil) or n-3-PSE. Twenty four patients dropped out or were excluded from the efficacy analysis due to protocol violation. The primary efficacy endpoint was mean change in plasma low-density lipoprotein cholesterol (LDL-C) levels after 12 weeks of treatment. Other efficacy measures included plasma lipids, lipoproteins, and high-sensitivity C-reactive protein (hsCRP) levels. Participants who completed the double-blind study were given the option to continue into an open-label, 12-weeks follow up phase.n-3-PSE treatment did not result in a significant change in LDL-C levels. Triglyceride levels were reduced significantly by 19% (51 mg/dL, p 0.0001) in the n-3-PSE group in comparison with the placebo group (p = 0.025). Diastolic blood pressure and hsCRP were reduced by 7% (5.9 mmHg) and 7.8% (0.6 mg/L), respectively, and were significantly different from the placebo group (p = 0.036 and p = 0.018, respectively).In patients with mixed hyperlipidemia, n-3-PSE treatment may offer a safe and effective therapy for triglyceride level reduction while avoiding the typical increase in LDL-C levels associated with n-3 fatty acid treatment. The observed reduction in blood pressure and inflammation markers warrants further evaluation. The positive effect of n-3-PSE treatment was preserved at the end of the follow up phase.

Background and Aims Suppression of stearoyl-coenzyme A desaturase (SCD) activity leads to reduction of obesity, fatty liver as well as of insulin resistance. It was, however, recently reported to enhance atherogenesis. The aim of the present study was to investigate whether inhibition of SCD by Aramchol, a fatty acid bile conjugate with known hypocholesterolemic effects, will affect atherogenesis and how. Methods Aramchol was tested in vitro in cultured cells and in vivo in rodents. Results Aramchol, at very low concentrations, reduced SCD activity in liver microsomes of mice. Aramchol enhanced cholesterol efflux from macrophages more than twofold. In vivo it increased fecal sterol output and decreased markedly plasma cholesterol levels in mice. In ApoE −/− , LDRL –/– and C57Bl6 mice, the effects of Aramchol on atherogenesis were non-atherogenic. Conclusions Aramchol reduces SCD activity and is non-atherogenic. It may offer a means to obtain the desirable hepatic metabolic effects of SCD inhibition without the deleterious atherogenic effect.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.

To test the hypothesis that B-type natriuretic peptide (BNP) acts as a potent vasculogenic agent by enhancing the number, proliferation, adhesion, and migration of endothelial progenitor cells (EPCs).BNP is a neurohormonal peptide that predicts outcome and used for treatment in chronic heart failure patients. It has been shown to promote angiogenesis in experimental animals. EPCs have been demonstrated to contribute to postnatal angiogenesis and vasculogenesis.The number of EPC colony forming units (CFU) and levels of N-terminal ProBNP were assayed in patients with severe, yet controlled, New York Heart Association (NYHA) II-IV heart failure. The in vitro effects of BNP on early EPC-CFU numbers, proliferation, migration, adhesive, and vascular tube formation capacities were studied using human and murine systems. The effects of in vivo BNP administration on Sca-1/Flk-1 progenitors and on vasculogenesis in the hindlimb ischemia model were then assayed in wild-type mice.A significant correlation was found between circulating N-terminal ProBNP levels and EPC-CFU numbers. We observed a dose-dependent effect of BNP on the numbers of CFU and proliferation capacity of human EPCs as well as on their adhesive, migratory, and tube formation properties, in vitro. Systemic BNP administration to mice led to a significant increase in bone marrow Sca-1/Flk-1 EPCs and improvement in blood flow and capillary density in the ischemic limbs of mice.BNP promotes vessel growth by increasing the number of endothelial progenitors and enhancing their functional properties. These provasculogenic properties of BNP could account for some of its beneficial effects in chronic heart failure patients and may be harnessed for the purpose of improving collateral formation in ischemic subjects.

Purpose: Angiogenesis is an essential process for solid tumor development. To interfere with angiogenesis, AdPPE3x-E1, an adenovirus that is transcriptionally targeted to replicate in angiogenic endothelial cells, was constructed, by replacing the E1 promoter with the modified preproendothelin-1 promoter, PPE-1-3x, previously shown to induce specific transcription in angiogenic endothelial cells. Experimental Design: The specificity of AdPPE3x-E1 to endothelial cells was shown by quantitative PCR and immunostaining, and its antiangiogenic effect was evaluated in Matrigel models. The in vivo efficacy of AdPPE3x-E1 was also tested in a cotton rat lung metastases model. Results: The replication rate of AdPPE3x-E1 in endothelial cells was similar to that of AdCMV-E1, a nonselective replicating adenovector, but the replication rate was reduced up to 60-fold in nonendothelial cells. Moreover, AdPPE3x-E1 reduced endothelial cell viability by 90% whereas nonendothelial cells were not affected. In in vitro and in vivo Matrigel models, endothelial cells infected with AdPPE3x-E1 did not develop capillary-like structures. The systemic administration of AdPPE3x-E1 reduced the lung metastases burden in a cotton rat model by 55%, compared with saline-treated rats, without significant evidence of toxicity. Quantitative PCR analysis showed that the viral copy number of AdPPE3x-E1 was increased 3-fold in the lung metastases but not in the liver, compared with a nonreplicating adenovector control. Conclusions: We have shown here for the first time an antimetastatic effect induced by an angiogenesis-transcriptionally targeted adenovirus following systemic administration. Because adenovirus replication is more efficient in humans than in cotton rats, we assume a significant effect for AdPPE3x-E1 treatment in fighting human solid tumors and metastases.

Background: Atherosclerosis is a complex histopathologic process that is analogous to chronic inflammatory conditions. Several factors have been shown to correlate with the extent of atherosclerosis. Whereas hypertension, obesity, hyperlipidemia, diabetes, smoking, and family history are all well documented, recent literature points to additional associated factors. Thus, antibodies to oxidized low-density lipoprotein (oxLDL), cytomegalovirus (CMV), Chlamydia pneumonia, Helicobacter pylori, as well as homocysteine and C-reactive protein (CRP) levels have all been implicated as independent markers of accelerated atherosclerosis. Hypothesis: In the current study we attempted to formulate a system by which to predict the extent of coronary atherosclerosis as assessed by angiographic vessel occlusion. Methods: The 81 patients were categorized as having single-, double-, triple-, or no vessel involvement. The clinical data concerning the “classic” risk factors were obtained from clinical records, and sera were drawn from the patients for determination of the various parameters that are thought to be associated with atherosclerosis. Results: Using four artificial neural networks, we have found the most effective parameters predictive of coronary vessel involvement were (in decreasing order of importance) antibodies to oxLDL, to cardiolipin, to CMV, to Chlamydia pneumonia, and to β2-glycoprotein I (β2GPI). Although important in the prediction of vessel occlusion, hyperlipidemia, hypertension, CRP levels, and diabetes were less accurate. Conclusion: The results of the current study, if reproduced in a larger population, may establish an integrated system based on the creation of artificial neural networks by which to predict the extent of atherosclerosis in a given subject fairly and noninvasively.

Our aim was to study the effect of 9-cis beta-carotene-rich powder of the alga Dunaliella bardawil on lipid profile, atherogenesis, and liver steatosis in high-fat diet-fed LDL receptor knockout mice. In 4 sets of experiments, mice were distributed into the following groups: control, fed an unfortified diet; Dunaliella 50, fed a diet composed of 50% 9-cis and 50% all-trans beta-carotene; Dunaliella 25, fed a diet containing 25% 9-cis and 75% all-trans beta-carotene; beta-carotene-deficient Dunaliella, fed beta-carotene-deficient Dunaliella powder; and all-trans beta-carotene, fed a synthetic all-trans beta-carotene. All fortified diets contained 0.6% total beta-carotene. Algal 9-cis beta-carotene was absorbed by the mice and accumulated in the liver. Synthetic all-trans beta-carotene was not converted to 9-cis beta-carotene. Dunaliella 50 inhibited high-fat diet-induced plasma cholesterol elevation by 40-63% and reduced cholesterol concentrations in the atherogenic VLDL and LDL. Atherosclerotic lesion area in mice treated with Dunaliella 50 was 60-83% lower compared with mice fed the high-fat diet alone. beta-Carotene-deficient Dunaliella did not influence plasma cholesterol and atherogenesis, suggesting that beta-carotene is essential for a Dunaliella protective effect. Moreover, by administrating Dunaliella powder containing different levels of 9-cis and all-trans beta-carotene isomers, we found that the effect on plasma cholesterol concentration and atherogenesis is 9-cis-dependent. Dunaliella 50 also inhibited fat accumulation and inflammation in the livers of mice fed a high-fat diet, which was accompanied by reduced mRNA levels of inflammatory genes. These results in mice suggest that 9-cis beta-carotene may have the potential to inhibit atherogenesis in humans.

Atherosclerotic cardiovascular disease (CVD) is the universal leading cause of mortality and a major cause of morbidity. Additionally, the global epidemic of diabetes is associated with considerable cardiovascular mortality risk due to accelerated premature atherosclerosis. Development of effective therapies for atherosclerosis is dependent upon improved tools to assess atherosclerotic lesion progression in animal models. We present a novel technique that utilises scanning electron microscopy (SEM) for imaging wet biological specimens, thus enabling rapid and high-resolution imaging of atherosclerotic lesions. This wet SEM technique was used in an apoEdeficient mice model for morphological characterisation of early and advanced atherosclerotic lesions. Further demonstration of lipid-rich atherosclerotic lesions was carried out with osmium tetroxide staining for cholesterol. Gold immunolabelling of specific epitopes was applied in identification of the cellular and molecular components within the atherosclerotic lesions, namely foam cells, smooth muscle cells and collagen. The wet SEM technique demonstrates an accurate and detailed structural evaluation of the pathological process of atherosclerosis. Understanding the mechanisms that precipitate the atherosclerotic process, utilising this novel technique, may assist in the development of innovative therapeutic interventions for CVD management and prevention in the general population and in those with diabetes. Diabetes Vasc Dis Res 2008;5:44‐7 doi:10.3132/dvdr.2008.008

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

The metabolic syndrome (MS) is a common risk factor for cardiovascular disease and type-2 diabetes. Recently, telmisartan, an angiotensin II receptor antagonist that has an antihypertensive effect, has been reported to be a partial peroxisome proliferator-activated receptor gamma (PPARgamma) agonist. The anti-diabetic hormone adiponectin has been recognized as a marker of in vivo PPARgamma activation. Therefore, we studied telmisartan's effect on the metabolic profile and adiponectin levels in a fructose-induced hypertensive, hyperinsulinemic, hyperlipidemic rat model. Twenty-four male Sprague-Dawley rats were divided into three groups (eight in each). One group of control rats was fed standard chow for 5 weeks while a second was fed a fructose-enriched diet. A third group was fed a fructose-enriched diet for 5 weeks and treated with telmisartan 5 mg/kg/day during the last 2 weeks. Fructose feeding increased systolic blood pressure (mean+/-SEM), from 130+/-1 to 148+/-2 mmHg, insulin from 0.26+/-0.03 to 0.68+/-0.08 ng/mL, and triglycerides from 102+/-6 to 285+/-23 mg/dL (p

Antiphospholipid antibodies (aPL) are found in a variety of autoimmune diseases, and are thought to predispose to arterial and venous thrombosis. These antibodies, when investigated in different assays in vitro, activate endothelial cells and promote uptake of modified LDL to macrophages. These observations suggest that aPL can contribute to atheroma development by targeting some of the sequential steps that constitute early atherogenesis. If substantiated by large-scale clinical trials, the pro-atherogenic properties of aPL may merit screening and intervention programs in selected populations.

HIF-1 is a transcription factor that regulates genes involved in oxygen homeostasis. In normoxia, degradation of the HIF-1 alpha subunit is enabled by two prolyl hydroxylations at residues P402 and P564, while inactivation occurs through asparaginyl hydroxylation at residue N803 within its C-transactivation domain (C-TAD). For therapeutic angiogenesis purposes, HIF-1 alpha stabilization was previously achieved by either deleting its oxygen-dependent degradation domains, or introducing two proline point mutations at residues P402 and P564. We assessed the hypothesis that constitutive activation of HIF-1 alpha in addition to its stabilization would result in greater HIF-1 alpha transcriptional activity and angiogenic effects than mere stabilization of the molecule. For this, we constructed a Triple mutant HIF-1 alpha (TM), bearing mutations P402A and P564G N803A. Transient co-transfections with hypoxia-responsive element-luciferase construct revealed 2 to 2.5-fold increase in transcriptional activity of TM compared with P402A P564G double mutant and wild-type HIF-1 alpha. In-vitro angiogenesis assay using transfected human umbilical vein endothelial cells (HUVEC) showed that TM stimulated tube formation to a greater extent than both P402A P564G mutant and wild-type HIF-1 alpha. Accordingly, ELISA revealed that VEGF levels within the transfected HUVEC were about 10-fold greater with the TM.Constitutive activation of the HIF-1 alpha C-TAD, and not merely stabilization of the HIF-1 alpha molecule, is essential for optimal HIF-mediated transcriptional and angiogenic effects. This finding could have important implications for therapeutic angiogenesis using HIF-1 alpha.

The antioxidative effect of the carotenoids phytoene and phytofluene in biological systems has not yet been studied. We therefore sought to investigate the effect of these carotenoids, isolated from the alga Dunaliella bardawil, in a biological system and used the in vitro low density lipoprotein (LDL) oxidation method to assay their antioxidative effects. We found that similar to beta-carotene and alpha-tocopherol, a carotenoid algal preparation containing phytoene and phytofluene inhibited LDL oxidation. These findings and the presence of phytoene and phytofluene in human tissues suggest that they can be part of the defense system against oxidative stress.

Background: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. Objectives: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). Results: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. Conclusions: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

International audience; BACKGROUND/AIMS: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point. METHODS: Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR. RESULTS: r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry. CONCLUSIONS: LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.

In industrialized societies atherosclerosis is still a major cause of morbidity and mortality, in spite of advances in development of cholesterol lowering drugs such as statins and ezetimibe. The detection of procoagulant proteases within atherosclerotic plaques, and the observation that patients with severe factor XI (FXI) deficiency have a reduced tendency to develop ischemic stroke, lead us to investigate whether absence of FXI would affect the process of atherogenesis. For this purpose, we created mice that are homozygous for null alleles for apolipoprotein E (apoE) and FXI (Double knock out [DKO] mice), and compared development of atherosclerosis in these animals to mice lacking apoE alone. At 24 weeks of age, there was a 32% reduction in atherosclerotic plaque area in the aortic sinus in apoE/FXI DKO mice compared to apoEKOmice (p=0.004). At age 42 weeks, apoE/FXI DKO mice had 25% smaller atherosclerotic plaque area in the aortic sinus compared to apoEKO mice (p=0.024), and aortic plaque area was reduced by 49% (p=0.028). Plasma cholesterol and triglycerides levels and lipoprotein profiles were similar in apoE/FXI DKO and apoE KO mice. In some murine infection models, FXI deficiency or FXI inhibition is associated with reduced markers of inflammation and improved survival. Based on this observation, we explored the possibility that reduced atherogenesis in apoE/FXI DKO mice is associated with reduced inflammation. There was a significant reduction in macrophages within atherosclerotic plaque in apoE/FXI DKO mice compared to apoE KO mice as determined by CD68 immunostaining. In conclusion, this work indicates for the first time that factor XI deficiency significantly reduces early, as well as advanced, atherosclerotic burden in an established animal model. We hypothesize that the effect of FXI on atherosclerosis is related to a proinflammatory effect within the atherosclerotic plaque. and propose that therapeutic targeting of FXI may have a role in preventing development of atherosclerosis. As FXI serves a minor role in hemostasis, treatment directed at this protein should not be associated with a high risk of major bleeding. Disclosures No relevant conflicts of interest to declare.

Background: Several epidemiological studies have shown that diets rich in carotenoids are associated with a reduced risk of cardiovascular disease. However, administration of synthetic all- trans b-carotene was reported to have no effect on cardiovascular disease. We previously showed that the 9- cis b-carotene-rich powder of the alga Dunaliella bardawil inhibits atherogenesis and reduces plasma non-HDL cholesterol levels in mice. Context and purpose of this study: We sought to study whether isolated 9- cis b-carotene inhibits atherogenesis in a murine model of atherosclerosis. Results: Twelve-week-old female LDL receptor knockout mice (LDLR-/-) were pretreated for 2 weeks with regular chow diet fortified with the alga Dunaliella powder, 9- cis β-carotene isomer, all- trans β-carotene isomer, or 9- cis retinoic acid, followed by 10 weeks of a high-fat diet with the same fortifications. In contrast to Dunaliella , 9- cis β-carotene did not inhibit the high fat diet-induced elevation of plasma cholesterol. In addition, diet fortification with Dunaliella powder, β-carotene isomers, or 9- cis retinoic acid did not change the plasma retinol or retinoic acid levels. Nevertheless, 9- cis β-carotene significantly inhibited atherogenesis compared to the control mice (39% reduction). Conclusions: The results suggest that 9- cis β-carotene should be considered as an anti-atherogenic agent in the human diet. Key words: Atherosclerosis, Dunaliella , 9CBC, LDLR-/- mice