Prevalence of Atherosclerotic Cardiovascular Disease, Heart Failure, and Chronic Kidney Disease in Patients with Type 2 Diabetes Mellitus: A Primary Care Research Network-based Study.

AIMS: This study aims to assess the prevalence of atherosclerotic cardiovascular disease (ASCVD), heart failure (HF), chronic kidney disease (CKD), and their combined presence in type 2 diabetes (T2D) patients in primary care for whom the 2019 ADA/EASD consensus update "Management of Hyperglycemia in Type 2 Diabetes" recommends GLP-1 receptor agonists (GLP-1RA) or sodium-glucose cotransporter-2 inhibitors (SGLT-I) as first-line medications after metformin. METHODS: Data were obtained in 2015 from Intego, a morbidity registration network of 111 general practitioners (GPs) working in 48 practices and including 123 261 registered patients. RESULTS: Of 123 261 patients, 9616 had T2D. Of these patients, 4200 (43.7%) presented with ASCVD and/or CKD and/or HF. Specifically, 3348 (34.8%) patients had ASCVD, 388 (4.0%) had heart failure, and 1402 (14.6%) had CKD. Compared to patients without any of these comorbidities, patients with at least 1 of these conditions were older (69.7 ±12.6 vs. 63.1±12.5 years), had higher LDL-C values (104.2±35.8 mg/dl vs. 97.2±37.7) and less frequently achieved the systolic blood pressure target of 140 mm Hg (53 vs. 61%) (all p<0.001). Comorbid patients also had significantly more other comorbidities, such as dementia or cancer; received more recommended medications, such as statins; and received less metformin. Most patients with HF (325; 3.4%) had ASCVD (114; 1.2%), CKD (76; 0.8%), or both (135; 1.4%). In total, 478 patients with CKD (5.0%) also had ASCVD. CONCLUSIONS: At the primary care level, 44% of T2D patients suffer from ASCVD, CKD, and/or HF, and thus qualify for GLP-1RA or SGLT2-I therapy.

Six-year outcomes in neovascular age-related macular degeneration with ranibizumab.

AIM: To evaluate the outcomes of ≥6y ranibizumab therapy in neovascular age-related macular degeneration (AMD). METHODS: HELIX was a retrospective, observational effectiveness study using medical records of patients treated in three clinics in Belgium. Patients had neovascular AMD and were initially treated with intravitreal ranibizumab (0.5 mg) between November 1, 2007 and October 31, 2008, had ≥6y of data available, and were treated on an ongoing, as-needed basis. Outcomes included best-corrected visual acuity (BCVA) and central retinal thickness (CRT). RESULTS: The sample consisted of 88 eyes from 69 patients. Mean age was 76.4±6.5y, most patients were female (62.3%). Most eyes (62.5%) were treatment-naive, 33 previously treated eyes had received predominantly other anti-vascular endothelial growth factor agents and verteporfin. Mean baseline BCVA was 57.4±12.7 ETDRS letters and CRT was 291.5±86.1 µm. On average, patients received 20.6±11.9 ranibizumab injections over the ≥6y. Intervals between injections were on average 12.7±16.1wk. Mean change in BCVA from baseline to last observation for the sample was less than one letter (-0.9±17.3 letters), with an average loss of -3.2±15.6 letters in previously treated eyes versus a gain of 0.6±18.4 letters in treatment-naïve eyes. When considering a loss of <15 letters over 6y as stabilization of disease, 75.9% of all eyes showed a positive (improvement or stabilization) outcome. Mean change in CRT from baseline to last observation for the sample was -26.9±148.4 µm with the greatest reduction observed in treatment-naive eyes. CONCLUSION: This retrospective study of 69 neovascular AMD patients treated for ≥6y with ranibizumab demonstrates long-term visual stabilization. In light of the natural evolution of the disease, these data confirm that ranibizumab is effective long-term under real-world conditions of heterogeneity of patients, clinicians, and centers.

Lipid lowering and HDL raising gene transfer increase endothelial progenitor cells, enhance myocardial vascularity, and improve diastolic function.

BACKGROUND: Hypercholesterolemia and low high density lipoprotein (HDL) cholesterol contribute to coronary heart disease but little is known about their direct effects on myocardial function. Low HDL and raised non-HDL cholesterol levels carried increased risk for heart failure development in the Framingham study, independent of any association with myocardial infarction. The objective of this study was to test the hypothesis that increased endothelial progenitor cell (EPC) number and function after lipid lowering or HDL raising gene transfer in C57BL/6 low density lipoprotein receptor deficient (LDLr(-/-)) mice may be associated with an enhanced relative vascularity in the myocardium and an improved cardiac function. METHODOLOGY/PRINCIPAL FINDINGS: Lipid lowering and HDL raising gene transfer were performed using the E1E3E4-deleted LDLr expressing adenoviral vector AdLDLr and the human apolipoprotein A-I expressing vector AdA-I, respectively. AdLDLr transfer in C57BL/6 LDLr(-/-) mice resulted in a 2.0-fold (p<0.05) increase of the circulating number of EPCs and in an improvement of EPC function as assessed by ex vivo EPC migration and EPC adhesion. Capillary density and relative vascularity in the myocardium were 28% (p<0.01) and 22% (p<0.05) higher, respectively, in AdLDLr mice compared to control mice. The peak rate of isovolumetric relaxation was increased by 12% (p<0.05) and the time constant of isovolumetric relaxation was decreased by 14% (p<0.05) after AdLDLr transfer. Similarly, HDL raising gene transfer increased EPC number and function and raised both capillary density and relative vascularity in the myocardium by 24% (p<0.05). The peak rate of isovolumetric relaxation was increased by 16% (p<0.05) in AdA-I mice compared to control mice. CONCLUSIONS/SIGNIFICANCE: Both lipid lowering and HDL raising gene transfer have beneficial effects on EPC biology, relative myocardial vascularity, and diastolic function. These findings raise concerns over the external validity of studies evaluating myocardial biology and cardiac repair in normocholesterolemic animals.

Circulating apoptotic endothelial cells and apoptotic endothelial microparticles independently predict the presence of cardiac allograft vasculopathy.

OBJECTIVES: Maintenance of endothelial homeostasis may prevent the development of cardiac allograft vasculopathy (CAV). This study investigated whether biomarkers related to endothelial injury and endothelial repair discriminate between CAV-negative and CAV-positive heart transplant recipients. BACKGROUND: CAV is the most important determinant of cardiac allograft survival and a major cause of death after heart transplantation. METHODS: Fifty-two patients undergoing coronary angiography between 5 and 15 years after heart transplantation were recruited in this study. Flow cytometry was applied to quantify endothelial progenitor cells (EPCs), circulating endothelial cells (CECs), and endothelial microparticles. Cell culture was used for quantification of circulating EPC number and hematopoietic progenitor cell number and for analysis of EPC function. RESULTS: The EPC number and function did not differ between CAV-negative and CAV-positive patients. In univariable models, age, creatinine, steroid dose, granulocyte colony-forming units, apoptotic CECs, and apoptotic endothelial microparticles discriminated between CAV-positive and CAV-negative patients. The logistic regression model containing apoptotic CECs and apoptotic endothelial microparticles as independent predictors provided high discrimination between CAV-positive and CAV-negative patients (C-statistic 0.812; 95% confidence interval: 0.692 to 0.932). In a logistic regression model with age and creatinine as covariates, apoptotic CECs (p = 0.0112) and apoptotic endothelial microparticles (p = 0.0141) were independent predictors (C-statistic 0.855; 95% confidence interval: 0.756 to 0.953). These 2 biomarkers remained independent predictors when steroid dose was introduced in the model. CONCLUSIONS: The high discriminative ability of apoptotic CECs and apoptotic endothelial microparticles is a solid foundation for the development of clinical prediction models of CAV.

A critical reassessment of murine and rabbit models of atherosclerosis: focus on lesion progression and remodelling.

BACKGROUND: Experimental animal atherosclerosis models are frequently regarded as an adequate surrogate for human vascular disease. The external validity of these models should be approached critically. OBJECTIVES: The current study provides a direct comparison of atherosclerosis progression in four different animal models: C57BL/6 apolipoprotein (apo) E(-/-) mice, C57BL/6 low density lipoprotein receptor deficient mice (LDLr(-/-) mice), heterozygous LDL receptor deficient rabbits (LDLr(+/-) rabbits), and homozygous LDL receptor deficient rabbits (LDLr(-/-) rabbits). The main objective was to perform a longitudinal analysis of arterial remodelling and of the evolution of the medial area during atherosclerosis progression. Secondary objectives were to analyse sex differences in atherosclerosis progression and to determine intersite correlations. RESULTS: Progression of atherosclerosis in all models was accompanied by expansive (overcompensatory) remodelling leading not only to the absence of luminal narrowing but also to an increase of the absolute lumen size. Atherosclerosis progression in mice and rabbits is often accompanied by an increase of the medial area. Female mice are more susceptible or equally susceptible to atherosclerosis development compared to male mice notwithstanding lower plasma cholesterol levels. However, this sex difference was not reiterated in both rabbit models. Whereas cholesterol-fed LDLr(-/-) mice show a moderate or strong correlation between the extent of advanced atherosclerosis in the aortic root and the brachiocephalic artery, no such correlation was observed in apo E(-/-) mice. CONCLUSION: The extensive morphometric data in the current study provide a framework to critically reassess the potential and limitations of animal models of atherosclerosis.

Correlation of atherosclerosis between different topographic sites is highly dependent on the type of hyperlipidemia.

There is a surprising paucity of studies that provide quantitative correlative data on the extent of atherosclerosis between different topographic sites. The impact of cardiovascular risk factors is dependent on the vascular bed, which underlies site-selective effects on progression of atherosclerosis. Therefore, the intraindividual correlation of atherosclerosis between different topographic sites may be dependent on the specific cardiovascular risk profile. The focused objective of the current study is to evaluate whether the correlation of the extent of atherosclerosis between different topographic sites is dependent on the type of hyperlipidemia. Atherosclerosis was quantified at four different topographic locations in the aorta of rabbits with type II or type III hyperlipidemia. Correlation coefficients and semi-partial correlation coefficients adjusted for plasma lipoproteins and sex were determined to compare the degree of atherosclerosis at different topographic sites. Semi-partial correlations adjusted for total plasma cholesterol, plasma triglycerides, and sex of the intima/media ratio between different topographic sites were highly dependent on the type of hyperlipidemia. E.g., the semi-partial correlation coefficient between the intima/media ratio at the level of the ascending aorta and at the level of the descending thoracic aorta was 0.87 (p < 0.0001) in the model of type II hyperlipidemia and was only 0.10 (p = NS) in the model of type III hyperlipidemia. This divergent pattern was also observed for other intersite correlations. Semi-partial Pearson correlation coefficients were very similar to unadjusted Pearson correlation coefficients. Correlation of atherosclerosis between different topographic sites may vary importantly in relation to the type of hyperlipidemia.

Gene transfer for inherited metabolic disorders of the liver: immunological challenges.

Hepatocytes are a key target for gene transfer directed at correction of inborn errors of metabolism. The theoretical potential of hepatocyte-directed gene transfer contrasts with the hurdles for clinical translation of this technology. Innate immune responses following gene transfer are initiated by recognition of pathogen-associated molecular patterns by pattern recognition receptors like Toll-like receptors. Adaptive immune responses may constitute the most significant hurdle for efficient gene transfer. Besides the challenge imposed by adaptive immune responses against the vector and the potential problem of pre-existing immunity, immune responses against the transgene product may also constitute an obstacle. The liver is a tolerogenic organ. Naive T cells encounter liver antigens initially in the liver, rather than in lymphoid tissue. Lymph nodes and the spleen are anatomical compartments that provide a particular microarchitecture and microenvironment for the induction of immunity. In contrast, antigen presentation in the liver takes place in a completely different microarchitecture and microenvironment. This is a key aspect of the hepatic adaptive immune tolerance induction. Consistent with the tolerogenic nature of the liver microenvironment, the risk of antibody formation against the transgene product may be limited in the setting of hepatocyte-directed gene transfer and specifically by restricting transgene expression to hepatocytes by use of hepatocyte-specific expression cassettes. However, it is unclear to which extent animal experimental data following gene transfer predict immune responses in humans. Extrapolations from animals to humans are required but should be performed with sufficient insight into the dramatic species differences of the immune system.

Gene therapy for familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an inherited metabolic disorder characterized by high levels of plasma low density lipoproteins (LDL) and an increased risk of premature atherosclerosis and coronary heart disease. LDL receptor (LDLr) deficiency is the most prevalent cause of FH. Therefore, hepatocyte-directed LDLr gene transfer constitutes an important strategy for the treatment of this monogenetic disease. Nowadays, homozygous FH patients are treated with lipid-lowering drugs complemented by plasma or LDL apheresis. Liver transplantation can restore metabolism of apolipoprotein B containing lipoproteins, but requires lifelong immunosuppression to prevent organ rejection. Recently, significant progress in gene transfer technology has encouraged investigators to further develop LDLr gene transfer approaches for the treatment of FH. In experimental animal models of FH, LDLr overexpression following viral vector-based gene transfer has been shown to be associated with long-term stable correction of hyperlipidemia, with attenuation of atherosclerosis progression, and in certain cases even with lesion regression. The first part of this review provides a thorough overview of familial hypercholesterolemia including its diagnosis, lipoprotein metabolism, and current management. In the second part, we critically review experimental LDLr gene transfer studies demonstrating the progress that has been made from the initial proof of principle studies to recent investigations showing dramatic regression of atherosclerosis in experimental models.

Correction of endothelial dysfunction after selective homocysteine lowering gene therapy reduces arterial thrombogenicity but has no effect on atherogenesis.

Hyperhomocysteinemia is an independent risk factor for ischemic cardiovascular diseases, but its causal role in atherothrombosis remains controversial. Proatherogenic and/or prothrombotic effects may underlie the potential causal relation between hyperhomocysteinemia and cardiovascular events. Here, the effects of selective lowering of plasma homocysteine, plasma cholesterol, or both on endothelial function and on atherogenesis in male hyperlipidemic and hyperhomocysteinemic C57BL/6 low-density lipoprotein receptor (LDLr)(-/-)/cystathionine-ß-synthase (CBS)(+/-)-deficient mice were investigated. Second, we evaluated whether selective homocysteine lowering has anti-thrombotic effects in a model of arterial thrombosis. A hyperhomocysteinemic and atherogenic diet was started at the age of 12 weeks. Three weeks later, gene transfer was performed with E1E3E4-deleted adenoviral vectors for hepatocyte-restricted overexpression of CBS (AdCBS) or of the LDLr (AdLDLr), or with the control vector Adnull. In a fourth group, AdCBS and AdLDLr were co-administered. Selective homocysteine lowering but not selective cholesterol lowering restored endothelial function at 6 weeks after gene transfer. Intimal area in the aortic root and in the brachiocephalic artery at 13 weeks was more than 100-fold (p < 0.001) smaller in AdLDLr and AdCBS/AdLDLr mice than in control mice and AdCBS mice. No differences in intimal area were observed between control mice and AdCBS mice. In a model of carotid artery thrombosis, the average time to first occlusion and to stable occlusion were 1.9-fold (p < 0.01) and 2.1-fold longer (p < 0.01), respectively, in AdCBS-treated mice than in control mice. Taken together, these data show that correction of endothelial dysfunction following selective homocysteine lowering has anti-thrombotic but no anti-atherogenic effects.

Regression and stabilization of advanced murine atherosclerotic lesions: a comparison of LDL lowering and HDL raising gene transfer strategies.

Both reductions in atherogenic lipoproteins and increases in high-density lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative potential of low-density lipoprotein (LDL) lowering and HDL raising gene transfer strategies to induce regression of complex murine atherosclerotic lesions was directly compared. Male C57BL/6 LDL receptor (LDLr)(-/-) mice were fed an atherogenic diet (1.25% cholesterol and 10% coconut oil) to induce advanced atherosclerotic lesions. A baseline group was killed after 6 months and remaining mice were randomized into a control progression (Adnull or saline), an apolipoprotein (apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdA-I/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks with continuation of the atherogenic diet. Gene transfer with AdLDLr decreased non-HDL cholesterol levels persistently by 95% (p < 0.001) compared with baseline. This drastic reduction of non-HDL cholesterol levels induced lesion regression by 28% (p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery at 12 weeks after transfer. Change in lesion size was accompanied by enhanced plaque stability, as evidenced by increased collagen content, reduced lesional macrophage content, a drastic reduction of necrotic core area, and decreased expression of inflammatory genes. Elevated HDL cholesterol following AdA-I transfer increased collagen content in lesions, but did not induce regression. Apo A-I gene transfer on top of AdLDLr transfer resulted in additive effects, particularly on inflammatory gene expression. In conclusion, drastic lipid lowering induced by a powerful gene transfer strategy leads to pronounced regression and stabilization of advanced murine atherosclerosis.

Topical HDL administration reduces vein graft atherosclerosis in apo E deficient mice.

OBJECTIVE: Use of autologous vein grafts for surgical revascularisation is limited by vein graft failure. Topical high-density lipoprotein (HDL) administration on the adventitial side of vein grafts was evaluated as a new therapeutic modality to improve vein graft patency and function. METHODS: Caval veins of C57BL/6 apo E(-/-) mice were grafted to the right carotid arteries of recipient 3 month-old C57BL/6 TIE2-LacZ/apo E(-/-) mice. HDL (200 µg/ml; 50 µl) in 20% pluronic F-127 gel was applied on the adventitial side of vein grafts. RESULTS: Topical HDL application reduced intimal area by 55% (p < 0.001) at day 28 compared to control mice. Blood flow quantified by micro magnetic resonance imaging at day 28 was 2.8-fold (p < 0.0001) higher in grafts of topical HDL treated mice than in control mice. Topical HDL potently reduced intimal inflammation and resulted in enhanced endothelial regeneration as evidenced by a 1.9-fold (p < 0.05) increase in the number of CD31 positive endothelial cells. HDL potently enhanced migration and adhesion of endothelial colony-forming cells (ECFCs) in vitro, and these effects were dependent on signaling via scavenger receptor-BI, extracellular signal-regulated kinases, and NO, and on increased ß1 integrin expression. Correspondingly, the number of CD31 ß-galactosidase double positive cells, reflecting incorporated circulating progenitor cells, was 3.9-fold (p < 0.01) higher in grafts of HDL treated mice than in control grafts. CONCLUSIONS: Topical HDL administration on the adventitial side of vein grafts attenuates vein graft atherosclerosis via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation.

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation.

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p<0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p<0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p<0.05) and 2.1-fold (p<0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A-I transfer. In vitro, supplementation of HDL or apo A-I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality.

The diameter of liver sinusoidal fenestrae is not a major determinant of lipoprotein levels and atherosclerosis in cholesterol-fed rabbits.

BACKGROUND: The liver is a key organ in lipid and lipoprotein metabolism. It has been postulated that a small diameter of sinusoidal fenestrae retards clearance of chylomicron remnants, resulting in hypercholesterolemia and atherosclerosis. However, this hypothesis has not been rigorously tested hitherto. METHODS: In the current study, we compared plasma levels of proatherogenic lipoproteins and assessed the development of atherosclerosis at distinct locations throughout the arterial tree in heterozygous New Zealand White and Dutch Belt rabbits that are deficient in low-density lipoprotein receptor and with an average fenestrae size of 103 and 124 nm, respectively. RESULTS: Feeding of a 0.15% cholesterol diet for 4 months resulted in similar total plasma cholesterol levels in New Zealand White (420±20 mg/dl) and Dutch Belt (380±30 mg/dl) rabbits. Following isolation of lipoproteins by ultracentrifugation, no biologically significant differences in very-low-density lipoprotein, intermediate-density lipoprotein, and low-density lipoprotein cholesterol levels were observed between cholesterol-fed New Zealand White and Dutch Belt rabbits. Furthermore, the relative amount of intestinally derived apolipoprotein-B48-containing lipoproteins did not differ significantly between both strains (7.3±0.42% vs. 8.0±0.54%). Atherosclerosis was more pronounced in the thoracic aorta in New Zealand White rabbits than in Dutch Belt rabbits, but the reverse was observed with the abdominal aorta. These topographic differences cannot be explained by circulating lipoprotein levels. CONCLUSIONS: The data presented in this study do not support the hypothesis that the diameter of fenestrae is an important determinant of chylomicron remnant levels, diet-induced hypercholesterolemia, and atherosclerosis in cholesterol-fed rabbits.

Hepatocyte-specific ABCA1 transfer increases HDL cholesterol but impairs HDL function and accelerates atherosclerosis.

AIMS: The ATP-binding cassette transporter A1 (ABCA1) lipidates apolipoprotein (apo) A-I. The hypothesis that hepatocyte-specific ABCA1 overexpression results in high-density lipoprotein (HDL) dysfunction was evaluated by comparing the effects of murine ABCA1 (AdABCA1) and human apo A-I (AdA-I) transfer on lipoprotein profile, HDL function, and progression of atherosclerosis. METHODS AND RESULTS: Gene transfer in male and female C57BL/6 apo E(-/-) mice was performed at the age of 3 months with E1E3E4-deleted adenoviral vectors containing hepatocyte-specific expression cassettes. Atherosclerosis was quantified at baseline and 56 days later in AdABCA1, AdA-I, and control mice. HDL cholesterol after AdA-I transfer was 1.7-fold (P < 0.001) and 1.8-fold (P < 0.001) higher in male and female mice, respectively, and potently inhibited atherosclerosis progression compared with respective controls. Notwithstanding a 1.4-fold (P < 0.01) and a 1.7-fold (P < 0.01) increase of HDL cholesterol in male and female mice, respectively, after AdABCA1 transfer, the intima was 2.2-fold (P < 0.001) larger in male and 1.3-fold (P = NS) larger in female mice compared with respective controls. HDL isolated from control and AdA-I mice but not from AdABCA1 mice enhanced endothelial progenitor cell (EPC) migration in vitro and reduced endothelial cell death in vitro after serum and growth factor withdrawal. Scavenger receptor class B type I (SR-BI) protein level in the liver was significantly lower in AdABCA1 mice than in control and AdA-I mice. CONCLUSION: Hepatocyte-specific ABCA1 transfer decreases SR-BI protein level in the liver and abrogates beneficial effects of HDL on EPCs and endothelial cells. Decreased HDL function may underlie accelerated atherosclerosis in AdABCA1 apo E(-/-)mice.

Impact of HDL on adipose tissue metabolism and adiponectin expression.

OBJECTIVE: The objective of the current study was to investigate the hypothesis that high-density lipoprotein (HDL) influences adipocyte metabolism and adiponectin expression. Therefore, HDL was increased in vivo via apolipoprotein (apo) A-I gene transfer and in vitro via supplementation of HDL to partly differentiated adipocytes, in the presence or absence of lipopolysaccharide (LPS), known to decrease HDL cholesterol and adiponectin levels in vivo. METHODS AND RESULTS: Apo A-I transfer resulted in a significant increase of HDL cholesterol in control and LPS-injected C57BL/6 mice, which was paralleled by an increase in plasma adiponectin levels and adiponectin expression in abdominal fat. Triglyceride and free fatty acids levels after LPS administration were 2.2-fold (p<0.05) and 1.3-fold (p<0.05) lower, respectively, in Ad.hapoA-I-LPS than in Ad.Null-LPS mice. In parallel, the LPS-induced mRNA expression of hormone sensitive lipase was 3.5-fold (p=0.05) decreased in the Ad.hapoA-I-LPS group. On the other hand, apo A-I transfer abrogated the LPS-mediated reduction in lipin-1 and CD36 mRNA expression by 8.2-fold (p<0.05) and 18-fold (p<0.05), respectively. Concomitantly, the phosphorylation state of Akt was 2.0-fold (p<0.05) increased in the Ad.hapoA-I-LPS compared to the Ad.Null-LPS group. Pre-incubation of partly differentiated adipocytes with HDL (50 microg protein/ml) increased adiponectin expression by 1.5-fold under basal conditions (p<0.05) and could abrogate LPS-induced down-regulation of adiponectin, both in a phosphatidylinositol-3-kinase-dependent manner. CONCLUSIONS: HDL affects adipocyte metabolism and adiponectin expression.

Gene therapy to improve high-density lipoprotein metabolism and function.

Plasma levels of high-density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo), apo A-I, are inversely correlated with the incidence of ischemic cardiovascular diseases. Till now, evaluation of the hypothesis that elevation of HDL cholesterol reduces atherosclerotic burden and/or decreases ischemic cardiovascular events in humans has been hampered by the lack of drugs that selectively increase HDL cholesterol. In contrast to the lack of clinical data, evidence for a direct causal role of HDL in modulating atherogenesis in experimental models has been provided by investigations in human apo A-I transgenic mice and rabbits. The development of gene transfer technologies with a sufficiently high therapeutic index may pave the road for a selective and effective HDL raising therapeutic intervention. The goal of a therapeutic strategy that modulates HDL metabolism is not an increase of HDL cholesterol as such, but an enhancement of HDL function. The value of HDL cholesterol as a surrogate end-point to predict reduced atherosclerosis or a decrease in clinical events may be highly dependent on the mechanism leading to an increased level of HDL cholesterol. In the case of gene transfer, this implies that beneficial effects of increasing HDL cholesterol will be dependent on the transgene that is expressed. Here, we critically review HDL metabolism and HDL function in relation to the development of HDL raising gene transfer, advances and drawbacks of different gene transfer technologies, and experimental gene transfer studies evaluating the effect of raised HDL on histological and functional outcomes in animal models.

The relative atherogenicity of VLDL and LDL is dependent on the topographic site.

To evaluate whether the relative atherogenicity of VLDL and LDL is dependent on the topographic site, atherosclerosis was compared at four topographic sites in homozygous LDL receptor (LDLr)-deficient rabbits fed normal chow and in heterozygous LDLr-deficient rabbits with the same genetic background fed a 0.15% cholesterol diet to match cholesterol levels. VLDL cholesterol was significantly higher and LDL cholesterol significantly lower in LDLr(+/-) diet rabbits compared with LDLr(-/-) rabbits. Intimal area in the ascending thoracic aorta and in the abdominal aorta at the level of the renal arteries was 1.4-fold (P < 0.05) and 1.5-fold (P < 0.05) higher, respectively, in LDLr(-/-) rabbits than in LDLr(+/-) diet rabbits, whereas no significant difference occurred in the descending thoracic aorta and in the abdominal aorta just above the bifurcation. Differences remained statistically significant after adjustment for plasma cholesterol, triglycerides, and sex. Compared with LDLr(+/-) diet rabbits, higher intimal lipoprotein lipase (LPL) and apolipoprotein (apo) B levels were observed in LDLr(-/-) rabbits only at the level of the descending thoracic aorta. Intimal apo E levels in LDLr(-/-) rabbits were significantly lower in sites with a larger intima than in LDLr(+/-) diet rabbits. In conclusion, the relative atherogenicity of VLDL and LDL is dependent on the topographic site.

Species differences in hepatocyte-directed gene transfer: implications for clinical translation.

The liver is a key organ in numerous metabolic pathways, in cholesterol metabolism, and in production of coagulation factors. Therefore, gene transfer to hepatocytes has been extensively pursued. There are numerous biological parameters that may affect the outcome of hepatocyte-directed gene transfer. Species or strain variation of any of these multiple determinants hinders the process of clinical translation. This review specifically focuses on functional aspects of liver histology that are pertinent for gene transfer to parenchymal liver cells. We discuss the reticulo-endothelial cells of the liver and the spleen, and their impact on innate immune responses after adenoviral transfer and on vector clearance. Liver sinusoidal endothelial cells contain pores, called fenestrae, and have no basal lamina. Fenestrae are clustered in sieve plates and may provide direct access for circulating gene transfer vectors to the space of Disse, in which microvilli of parenchymal liver cells protrude. We present multiple lines of evidence that the species differences in the diameter of sinusoidal fenestrae are a critical determinant of transgene expression after adenoviral transfer. The small diameter of fenestrae in humans should be considered in any rational design of gene transfer technologies for hepatocyte-directed transfer. Hydrodynamic gene transfer is highly successful in rodents. The significantly lower efficacy in higher species may also partially be due to species differences in liver architecture. Finally, we discuss species differences in adaptive immune responses against the transgene product that may constitute one of the most significant hurdles for clinical translation.

Critical role of scavenger receptor-BI-expressing bone marrow-derived endothelial progenitor cells in the attenuation of allograft vasculopathy after human apo A-I transfer.

Allograft vasculopathy is the leading cause of death in patients with heart transplantation. Accelerated endothelial regeneration mediated by enhanced endothelial progenitor cell (EPC) incorporation may attenuate the development of allograft vasculopathy. We investigated the hypothesis that modulation of EPC biology and attenuation of allograft vasculopathy by increased high-density lipoprotein cholesterol after human apo A-I (AdA-I) transfer requires scavenger receptor (SR)-BI expression in bone marrow-derived EPCs. After AdA-I transfer, the number of circulating EPCs increased 2.0-fold (P < .001) at different time points in C57BL/6 mice transplanted with SR-BI(+/+) bone marrow but remained unaltered in mice with SR-BI(-/-) bone marrow. The effect of high-density lipoprotein on EPC migration in vitro requires signaling via SR-BI and extracellular signal-regulated kinases and is dependent on increased nitric oxide (NO) production in EPCs. Human apo A-I transfer 2 weeks before paratopic artery transplantation reduced intimal area at day 21 3.7-fold (P < .001) in mice with SR-BI(+/+) bone marrow but had no effect in mice with SR-BI(-/-) bone marrow. AdA-I transfer potently stimulated EPC incorporation and accelerated endothelial regeneration in chimeric SR-BI(+/+) mice but not in chimeric SR-BI(-/-) mice. In conclusion, human apo A-I transfer accelerates endothelial regeneration mediated via SR-BI expressing bone marrow-derived EPCs, thereby preventing allograft vasculopathy.

Wild-type apo A-I and apo A-I(Milano) gene transfer reduce native and transplant arteriosclerosis to a similar extent.

Apolipoprotein (apo) A-I(Milano) is an apo A-I mutant characterized by a cysteine for arginine substitution at position 173. Apo A-I(Milano) carriers have much less atherosclerosis than expected from their low plasma high-density lipoprotein cholesterol levels, suggesting that this mutant may have superior atheroprotective properties. Here, we compare the effect of hepatocyte-directed gene transfer of wild-type human apo A-I and human apo A-I(Milano) on endothelial progenitor cell (EPC) biology and on the progression of native atherosclerosis and allograft vasculopathy in C57BL/6 apo E(-/-) mice. Human apo A-I and apo A-I(Milano) transfer resulted in an equivalent increase of EPC number and function as well as EPC incorporation and endothelial regeneration in allografts and inhibited the progression of native atherosclerosis and allograft vasculopathy to a similar extent. In conclusion, the current head-to-head comparison indicates that human apo A-I(Milano) transfer is not superior compared to wild-type human apo A-I transfer.