Ilayaraja Muthuramu

The Liver as a Target Organ for Gene Therapy: State of the Art, Challenges, and Future Perspectives

The liver is a target for gene therapy of inborn errors of metabolism, of hemophilia, and of acquired diseases such as liver cancer and hepatitis. The ideal gene transfer strategy should deliver the transgene DNA to parenchymal liver cells with accuracy and precision in the absence of side effects. Liver sinusoids are highly specialized capillaries with a particular endothelial lining: the endothelium contains open fenestrae, whereas a basal lamina is lacking. Fenestrae provide a direct access of gene transfer vectors to the space of Disse, in which numerous microvilli from parenchymal liver cells protrude. The small diameter of fenestrae in humans constitutes an anatomical barrier for most gene transfer vectors with the exception of adeno-associated viral (AAV) vectors. Recent studies have demonstrated the superiority of novel AAV serotypes for hepatocyte-directed gene transfer applications based on enhanced transduction, reduced prevalence of neutralizing antibodies, and diminished capsid immune responses. In a landmark clinical trial, hemophilia B was successfully treated with an AAV8 human factor IX expressing vector. Notwithstanding significant progress, clinical experience with these technologies remains very limited and many unanswered questions warrant further study. Therefore, the field should continue to progress as it has over the past decade, cautiously and diligently.

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.

Beneficial effects of selective HDL-raising gene transfer on survival, cardiac remodelling and cardiac function after myocardial infarction in mice.

Post-myocardial infarction (MI) ejection fraction is decreased in patients with low high-density lipoprotein (HDL) cholesterol levels, independent of the degree of coronary atherosclerosis. The objective of this study is to evaluate whether selective HDL-raising gene transfer exerts cardioprotective effects post MI. Gene transfer in C57BL/6 low-density lipoprotein receptor (LDLr)−/− mice was performed with the E1E3E4-deleted adenoviral vector AdA-I, inducing hepatocyte-specific expression of human apo A-I, or with the control vector Adnull. A ligation of the left anterior descending coronary artery was performed 2 weeks after transfer or saline injection. HDL cholesterol levels were persistently 1.5-times (P<0.0001) higher in AdA-I mice compared with controls. Survival was increased (P<0.01) in AdA-I MI mice compared with control MI mice during the 28-day follow-up period (hazard ratio for mortality 0.42; 95% confidence interval 0.24–0.76). Longitudinal morphometric analysis demonstrated attenuated infarct expansion and inhibition of left ventricular (LV) dilatation in AdA-I MI mice compared with controls. AdA-I transfer exerted immunomodulatory effects and increased neovascularisation in the infarct zone. Increased HDL after AdA-I transfer significantly improved systolic and diastolic cardiac function post MI, and led to a preservation of peripheral blood pressure. In conclusion, selective HDL-raising gene transfer may impede the development of heart failure.

The Impact of Lipoproteins on Wound Healing: Topical HDL Therapy Corrects Delayed Wound Healing in Apolipoprotein E Deficient Mice

Chronic non-healing wounds lead to considerable morbidity and mortality. Pleiotropic effects of high density lipoproteins (HDL) may beneficially affect wound healing. The objectives of this murine study were: (1) to investigate the hypothesis that hypercholesterolemia induces impaired wound healing and (2) to study the effect of topical HDL administration in a model of delayed wound healing. A circular full thickness wound was created on the back of each mouse. A silicone splint was used to counteract wound contraction. Coverage of the wound by granulation tissue and by epithelium was quantified every 2 days. Re-epithelialization from day 0 till day 10 was unexpectedly increased by 21.3% (p < 0.05) in C57BL/6 low density lipoprotein (LDLr) deficient mice with severe hypercholesterolemia (489 ± 14 mg/dL) compared to C57BL/6 mice and this effect was entirely abrogated following cholesterol lowering adenoviral LDLr gene transfer. In contrast, re-epithelialization in hypercholesterolemic (434 ± 16 mg/dL) C57BL/6 apolipoprotein (apo) E−/− mice was 22.6% (p < 0.0001) lower than in C57BL/6 mice. Topical HDL gel administered every 2 days increased re-epithelialization by 25.7% (p < 0.01) in apo E−/− mice. In conclusion, topical HDL application is an innovative therapeutic strategy that corrects impaired wound healing in apo E−/− mice.

Permanent ligation of the left anterior descending coronary artery in mice: a model of post-myocardial infarction remodelling and heart failure.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure. Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling. Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Pleiotropic Effects of HDL: Towards New Therapeutic Areas for HDL-Targeted Interventions

Plasma levels of high density lipoprotein (HDL) cholesterol levels and of apolipoprotein A-I are inversely correlated with the incidence of coronary heart disease. According to the HDL hypothesis, raising HDL cholesterol is expected to lead to a decrease of coronary heart disease risk. The stringent requirement for proving or refuting this hypothesis is that the causal pathway between the therapeutic intervention and a hard clinical end-point obligatory passes through HDL. The lack of positive clinical results in several recent HDL trials should be interpreted in light of the poor HDL specificity of the drugs that were investigated in these trials. Nevertheless, the results of Mendelian randomization studies further raise the possibility that the epidemiological relationship between HDL cholesterol and coronary artery disease might reflect residual confounding. HDL are circulating multimolecular platforms that exert divergent functions: reverse cholesterol transport, antiinflammatory effects, anti-oxidative effects, immunomodulatory effects, improved endothelial function, increased endothelial progenitor cell number and function, antithrombotic effects, and potentiation of insulin secretion and improvement of insulin sensitivity. Pleiotropic effects of HDL might be translated in clinically significant effects in strategically selected therapeutic areas that are not directly related to native coronary artery disease. In this review, four new therapeutic areas for HDL-targeted diseases are presented: critical illness, allograft vasculopathy and vein graft atherosclerosis, type 2 diabetes mellitus, and heart failure. The strategic selection of these therapeutic areas is not only based on specific functional properties of HDL but also on significant pre-clinical and clinical data that support this choice.

Selective Homocysteine Lowering Gene Transfer Improves Infarct Healing, Attenuates Remodelling, and Enhances Diastolic Function after Myocardial Infarction in Mice

Background and aims Homocysteine levels predict heart failure incidence in prospective epidemiological studies and correlate with severity of heart failure in cross-sectional surveys. The objective of this study was to evaluate whether a selective homocysteine lowering intervention beneficially affects cardiac remodelling and cardiac function after myocardial infarction (MI) in a murine model of combined hypercholesterolemia and hyperhomocysteinemia. Methodology and principal findings A selective homocysteine lowering gene transfer strategy was evaluated in female C57BL/6 low density lipoprotein receptor (Ldlr)−/− cystathionine-ß-synthase (Cbs)+/− deficient mice fed a hyperhomocysteinemic and high saturated fat/high cholesterol diet using an E1E3E4-deleted hepatocyte-specific adenoviral vector expressing Cbs (AdCBS). MI was induced by permanent ligation of the left anterior descending coronary artery 14 days after saline injection or gene transfer. AdCBS gene transfer resulted in a persistent more than 5-fold (p<0.01) decrease of plasma homocysteine levels and significantly improved endothelial progenitor cell function. Selective homocysteine lowering enhanced infarct healing as indicated by a 21% (p<0.01) reduction of infarct length at day 28 after MI and by an increased number of capillaries and increased collagen content in the infarct zone. Adverse remodelling was attenuated in AdCBS MI mice as evidenced by a 29% (p<0.05) reduction of left ventricular cavity area at day 28, by an increased capillary density in the remote myocardium, and by reduced interstitial collagen. The peak rate of isovolumetric relaxation was increased by 19% (p<0.05) and the time constant of left ventricular relaxation was reduced by 21% (p<0.05) in AdCBS MI mice compared to control MI mice, indicating improved diastolic function. Conclusion/significance Selective homocysteine lowering gene transfer improves infarct healing, attenuates remodelling, and significantly enhances diastolic function post-MI in female C57BL/6 Ldlr −/− Cbs +/− mice. The current study corroborates the view that hyperhomocysteinemia exerts direct effects on the myocardium and may potentiate the development of heart failure.

Coconut Oil Aggravates Pressure Overload-Induced Cardiomyopathy without Inducing Obesity, Systemic Insulin Resistance, or Cardiac Steatosis

Studies evaluating the effects of high-saturated fat diets on cardiac function are most often confounded by diet-induced obesity and by systemic insulin resistance. We evaluated whether coconut oil, containing C12:0 and C14:0 as main fatty acids, aggravates pressure overload-induced cardiomyopathy induced by transverse aortic constriction (TAC) in C57BL/6 mice. Mortality rate after TAC was higher (p < 0.05) in 0.2% cholesterol 10% coconut oil diet-fed mice than in standard chow-fed mice (hazard ratio 2.32, 95% confidence interval 1.16 to 4.64) during eight weeks of follow-up. The effects of coconut oil on cardiac remodeling occurred in the absence of weight gain and of systemic insulin resistance. Wet lung weight was 1.76-fold (p < 0.01) higher in coconut oil mice than in standard chow mice. Myocardial capillary density (p < 0.001) was decreased, interstitial fibrosis was 1.88-fold (p < 0.001) higher, and systolic and diastolic function was worse in coconut oil mice than in standard chow mice. Myocardial glucose uptake was 1.86-fold (p < 0.001) higher in coconut oil mice and was accompanied by higher myocardial pyruvate dehydrogenase levels and higher acetyl-CoA carboxylase levels. The coconut oil diet increased oxidative stress. Myocardial triglycerides and free fatty acids were lower (p < 0.05) in coconut oil mice. In conclusion, coconut oil aggravates pressure overload-induced cardiomyopathy.

New perspectives on biological HDL-targeted therapies

High-density lipoproteins (HDL) consist of various subclasses, which share the abundance of apolipoprotein (apo) A-I, phospholipids, and cholesterol but are distinct by the variable presence of one or more representatives of at least 85 proteins and hundreds of lipid species [1,2]. HDL are circulating multimolecular platforms that exert divergent functions: reverse cholesterol transport, anti-inflammatory effects, antioxidative properties, immunomodulatory effects, improvement of endothelial function, antithrombotic effects, and potentiation of insulin secretion and improvement of insulin sensitivity [2]. Plasma HDL-cholesterol levels and plasma levels of its major apo, apo A-I, are inversely correlated with the incidence of ischemic cardiac diseases. A meta-analysis of four prospective studies indicated that a 1 mg/dL increase in HDLcholesterol was associated with a 2% risk reduction of coronary heart disease in men and a 3% risk reduction in women [3]. In a more recent analysis of The Emerging Risk Factors Collaboration, the adjusted hazard ratio for coronary heart disease associated with a one standard deviation increase of HDL-cholesterol (15 mg/dL) was 0.78 (95% confidence interval, 0.74–0.82) [4]. In contrast, Mendelian randomization studies have demonstrated that genetic mechanisms that raise plasma HDL-cholesterol do not appear to lower the risk of myocardial infarction [5]. The possibility that the epidemiological relationship between HDL-cholesterol and coronary artery disease in classical epidemiological studies reflects residual confounding and/or unmeasured confounding cannot be excluded. Low HDL-cholesterol could be an integrated biomarker of adverse metabolic processes including abnormal metabolism of triglyceride-rich lipoproteins, insulin resistance, and ongoing tissue inflammation [2]. Based on the biological potential of HDL and on epidemiological evidence, the development of HDL-targeted therapies has been an important objective for several decades. According to the original HDL hypothesis, raising HDL-cholesterol was expected to lead to a decrease in coronary heart disease risk. However, HDL-cholesterol is a very poor proxy to analyze biological action and clinical effects of HDL. Scavenger receptor class B type I is the major receptor for HDL-cholesterol, and a rare variant abrogates selective HDL-cholesterol uptake, raises HDL-cholesterol, and increases the risk of coronary heart disease [6]. In general, genetic or pharmacological modifications of HDL metabolism and associated compositional changes of the proteome or lipidome of HDL particles may lead to an impaired function of these lipoproteins. Reduced HDL function may also be due to post-translational modifications of proteins or occur as a result of ongoing inflammation [7]. HDL function encompasses several dimensions: cholesterol efflux capacity, vasculoprotective function, anti-inflammatory potential, and antioxidative capacity. Rohatgi et al. [8] demonstrated in a seminal prospective cohort study that HDL-cholesterol efflux capacity predicted incident cardiovascular events independent of traditional risk factors, HDL-cholesterol level, and HDLparticle concentration. According to a modified version of the HDL hypothesis, improving HDL function will lead to a decrease of coronary events [2].

Role of lipids and lipoproteins in myocardial biology and in the development of heart failure

Abstract As the population ages, heart failure will continue to be a growing public health problem. Metabolic homeostasis in the heart requires a fine-tuning of metabolism of different substrates. Notwithstanding a retro control of fatty acid and glucose utilization, the heart functions best when it oxidizes both substrates simultaneously. Mismatch between the uptake and oxidation of long-chain fatty acids in the myocardium induces lipotoxicity characterized by the accumulation of triglycerides, diacylglycerols, ceramides and other lipids. Lipotoxicity may result in cardiomyocyte apoptosis, interstitial fibrosis and cardiac dysfunction, and may promote insulin resistance. In this review, we will highlight the impact of lipids and lipoproteins on myocardial biology and on the development of heart failure independent of their effects on coronary heart disease.