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Towards new therapeutic areas for HDL-targeted interventions
Plasma levels of high density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo), apo A-I, are inversely correlated with the incidence of coronary heart diseases. The Framingham Heart Study, the KuopioIschemic Heart Disease Factor Study, the Israel Ischemic Heart Disease Study, and other epidemiological prospective cohort studies have unequivocally demonstrated that lower HDL cholesterol levels are an independentrisk marker for ischemic heart diseases. A meta-analysis of 4 prospective studies has indicated that a 1 mg/dl increase of HDL cholesterol was associated with a 2% coronary risk reduction in men and a 3% risk reduction in women. Nevertheless, these epidemiological data do not prove a causal relationship since residual confounding factors may play a role. Proof of a causal relationship requires intervention studies. Till now, evaluation of the hypothesis that elevation of HDL cholesterol reduces atherosclerotic burden and/or decreases ischemic coronary events in humans has been hampered by the lack of drugs that selectively increase HDL cholesterol. Several recent trials that explored different raising HDL strategies (ILLUMINATE, ACCORD, dal-OUTCOMES, AIM-HIGH, HPS2-THRIVE) failed to show beneficial effects of HDL on hard clinical end-points. Moreover,Mendelian randomisation studies in humans have show that a genetic score for HDL cholesterol combining the HDL-cholesterol-raising alleles at each of 14 single nucleotide polymorphisms was not associated with the risk of myocardial infarction. Thus, despite a wealth of information on this intriguing lipoprotein, future research is imperative to investigate the HDL hypothesis. However, one should keep in mind that it might neverbe possible to prove or refute the HDL hypothesis. After all, as statedsupra, selective HDL raising drugs are currently not available for clinical use. Furthermore, progress in lipid lowering therapy, in particularthe ongoing evaluation of inhibition of proprotein convertase subtilisin/kexin type 9 in phase III clinical trials, may radically change the context for development of new lipidological therapies. Therefore, it is warranted to consider new therapeutic areas for HDL-targeted interventions.
A major mechanism postulated to contribute to the atheroprotective effects of HDL has been reverse cholesterol transport. However, HDL exerts divergent functions including antithrombotic properties and endothelial-protective properties. HDL enhances endothelial cell survival,nitric oxide production, endothelial cell migration, and endothelial progenitor cell (EPC)-mediated repair. Furthermore, HDL has anti-oxidative, anti-inflammatory, and anti-apoptotic properties. The actions of HDL extend to modulation of glucose metabolism and immunomodulation. This versatility of biological actions of HDL is an incentive to direct HDL-targeted interventions to new therapeutic areas that are outside the field of atherosclerosis and vascular biology. The coherent goal of this Ph.D. thesis is to experimentally demonstrate new paradigms concerning effectsof HDL and to form a solid basis for clinical translation in selected therapeutic areas.
In a series of two studies presented in this manuscript, the development of a topical HDL therapy was evaluated in a murine model of vein graft atherosclerosis and in a murine model of cutaneous wound healing.
In chapter 3.1, we evaluated topical HDL administrationon the adventitial side of vein grafts as a novel therapeutic modality to improve vein graft patency and function. Caval veins of C57BL/6 apolipoprotein E deficient (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) was administered in 20% pluronic F-127 gel, which is a biocompatible and non-toxic substance, and is characterized by thermoreversible gel formation at temperatures above 21 °C. Basedon the endothelial protective and anti-inflammatory effects of HDL, we hypothesized that topical HDL may increase incorporation of circulating EPCs in the vein graft endothelium, improve endothelial regeneration, and inhibit experimental vein graft atherosclerosis in apo E-/- mice.
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 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. The effect of topical HDL on vein graft atherosclerosis was equivalent to theeffect of systemic HDL elevation induced by human apo A-I gene transfer. Taken together, topical HDL administration on the adventitial side of vein grafts improved vein gaft patency and function via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation.
In chapter 3.2, the same technology, namely topical HDL administration, was used to promote cutaneous wound healing. Wound healing results from complex interactions between extracellular matrix molecules, soluble mediators, resident skin cells, and infiltrating leukocytes as well as infiltrating EPCs. Rather artificially, wound healing can be divided in an inflammation phase, a phase of tissue formation with accumulation of granulation tissue and re-epithelialization, and finally a phase of tissue remodelling. Diabetes is a predisposing factor for chronic, non-healing wounds.Several factors contribute to deficient wound healing in patients with diabetes: deficient growth factor production, deficient neovascularization, attenuated keratinocyte and fibroblast proliferation and migration, and an altered balance between extracellular matrix accumulation and remodelling of the extracellular matrix by matrix metalloproteinases. Whether dyslipidemia in diabetics is a contributing factor to impaired wound healing was not investigated till now. The objective of this project was to investigate whether hypercholesterolemia in C57BL/6 apo E-/- mice negatively affects cutaneous wound healing and to test the hypothesis that topical HDL therapy enhances wound healing in C57BL/6 apo E-/- mice and in C57BL/6 mice with streptozotocin-induced diabetes mellitus. We first compared wound healing in normocholesterolemic C57BL/6 and in dyslipidemic C57BL/6 apolipoprotein E deficient (apo E-/-) mice. Wound coverage with newly formed epithelial tissue was 1.89-fold (p<0.0001), 1.75-fold (p<0.05), 1.47-fold (p<0.001), 1.47-fold (p<0.001), and 1.18-fold (p<0.01)higher in C57BL/6 mice compared to C57BL/6 apo E-/- mice at day 2, day 4, day 6, day 8, and day 10 after wound application, respectively. In C57BL/6 apo E-/- mice, topical administration of HDL in 20% pluronic F-127gel (pH 7.2) increased wound re-epithelialization by 1.35-fold (p<0.05), 1.44 fold (p<0.05), and 1.21-fold (p<0.01) at day 6, day 8, and day 10, respectively. The rate of re-epithelialization was not significantly different between untreated control wounds and control wounds treated with pluronic F-127 gel (pH 7.2). Wound healing was delayed in streptozotocin-induced diabetic C57BL/6 mice compared to non-diabetic littermates. In C57BL/6 mice with streptozotocin-induced diabetes, wound coverage withnew epithelium was 1.93-fold (p<0.01), 1.22-fold (p=NS), 1.37-fold (p<0.01), and 1.11-fold (p=NS) increased at day 4, day 6, day 8, and day 10 after wound application, respectively, by topical HDL therapy. Wound coverage by granulation tissue data showed a similar pattern compared to re-epithelialization data in the different experimental conditions.
In summary, this study demonstrates that topical HDL accelerates wound healing in dyslipidemic mice and in diabetic mice to the rate observed in normocholesterolemic, non-diabetic mice and thereby provides the first foundations for a new treatment paradigm of cutaneous ulcers: topical HDL therapy.
The next two studies of this manuscript entail investigations to evaluate the effect of systemically raised HDL induced by human apo A-I gene transfer on myocardial function and structure in the absence or presence of a myocardial infarction.
It is well established that hypercholesterolemia and low plasma HDL cholesterol levels are risk factors for coronary heart disease but little is known about their direct effects on myocardial function. In patients with low HDL cholesterol levels, post-myocardial infarction (MI) ejection fraction is decreased. In Framingham Heart Study participants free of coronary heart diseaseat baseline, low HDL cholesterol levels were independently associated with heart failure incidence after adjustment for interim MI and clinical covariates. Moreover, low HDL cholesterol levels and low levels of apo A-I indicate an unfavourable prognosis in patients with heart failure independent of the etiology. Direct cellular effects of HDL have been demonstrated in isolated cardiomyocytes in vitro as evidenced by increased phosphorylation of extracellular signal-regulated kinases, of the transcription factor STAT3, and of the pro-survival kinase Akt. Furthermore, it has previously been shown that HDL inhibits cardiomyocyte apoptosis underconditions of hyperglycemia. Finally, the development of diabetic cardiomyopathy is inhibited by human apo A-I gene transfer in rats. Taken together, epidemiological and experimental studies provide converging linesof evidence that HDL may exert direct protective effects on the myocardium.
In chapter 3.3, we examined the effect of lipid lowering gene transfer and selective HDL raising gene transfer on EPC number and EPC function and on cardiac structure and function in hypercholesterolemic C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice. 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 circulatingnumber 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 AdLDLrtransfer. Similarly, HDL raising gene transfer increased EPC number andfunction 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. In conclusion, 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 animal models that are often characterized by a predominance of HDL.
In chapter 3.4 we assessed effects of selective HDL raising gene transfer in a murine model of ischemic cardiomyopathy. Specifically, the objective of this study was to examine whether human apo A-I gene transfer in hypercholesterolemic C57BL/6 LDLr-/- mice affects survival, infarct size, and cardiac function after permanent ligation of the left anterior descending coronary artery. In order to focus on the effect of HDL on myocardial biology, a model of permanent ligation was usedand not a model of ischemia-reperfusion injury. Gene transfer in C57BL/6 LDLr-/- mice was performed with the E1E3E4-deleted adenoviral vector AdA-I, inducing hepatocyte-specific expression of human apolipoprotein (apo A-I), or with the control vector Adnull. A ligation of the left anterior descending coronary artery was performed two weeks after transfer orsaline injection. HDL cholesterol levels were persistently 1.5-times (p<0.0001) higher in AdA-I mice compared to controls. Survival was increased (p<0.01) in AdA-I MI mice compared to control MI mice during the 28-day follow-up period (hazard ratio for mortality 0.42; 95% CI 0.24 to 0.76). Longitudinal morphometric analysis demonstrated attenuated infarct expansion and inhibition of left ventricular dilatation in AdA-I MI mice compared to 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. Inconclusion, selective HDL raising gene transfer exerts cardioprotectiveeffects following myocardial infarction.
Taken together, the data of this Ph.D. thesis provide a solid base for future pre-clinical development of new therapeutic modalities for HDL-targeted interventions. Topical HDL therapy may constitute a future innovative and clinically feasible therapeutic strategy to enhance healing of diabetic foot ulcers. Clinical translation of apo A-I gene transfer as a means to systemically increase HDL levels will depend on the development of potent and safe gene transfer vectors and on progress made in the adeno-associated viral gene therapy field.
A major mechanism postulated to contribute to the atheroprotective effects of HDL has been reverse cholesterol transport. However, HDL exerts divergent functions including antithrombotic properties and endothelial-protective properties. HDL enhances endothelial cell survival,nitric oxide production, endothelial cell migration, and endothelial progenitor cell (EPC)-mediated repair. Furthermore, HDL has anti-oxidative, anti-inflammatory, and anti-apoptotic properties. The actions of HDL extend to modulation of glucose metabolism and immunomodulation. This versatility of biological actions of HDL is an incentive to direct HDL-targeted interventions to new therapeutic areas that are outside the field of atherosclerosis and vascular biology. The coherent goal of this Ph.D. thesis is to experimentally demonstrate new paradigms concerning effectsof HDL and to form a solid basis for clinical translation in selected therapeutic areas.
In a series of two studies presented in this manuscript, the development of a topical HDL therapy was evaluated in a murine model of vein graft atherosclerosis and in a murine model of cutaneous wound healing.
In chapter 3.1, we evaluated topical HDL administrationon the adventitial side of vein grafts as a novel therapeutic modality to improve vein graft patency and function. Caval veins of C57BL/6 apolipoprotein E deficient (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) was administered in 20% pluronic F-127 gel, which is a biocompatible and non-toxic substance, and is characterized by thermoreversible gel formation at temperatures above 21 °C. Basedon the endothelial protective and anti-inflammatory effects of HDL, we hypothesized that topical HDL may increase incorporation of circulating EPCs in the vein graft endothelium, improve endothelial regeneration, and inhibit experimental vein graft atherosclerosis in apo E-/- mice.
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 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. The effect of topical HDL on vein graft atherosclerosis was equivalent to theeffect of systemic HDL elevation induced by human apo A-I gene transfer. Taken together, topical HDL administration on the adventitial side of vein grafts improved vein gaft patency and function via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation.
In chapter 3.2, the same technology, namely topical HDL administration, was used to promote cutaneous wound healing. Wound healing results from complex interactions between extracellular matrix molecules, soluble mediators, resident skin cells, and infiltrating leukocytes as well as infiltrating EPCs. Rather artificially, wound healing can be divided in an inflammation phase, a phase of tissue formation with accumulation of granulation tissue and re-epithelialization, and finally a phase of tissue remodelling. Diabetes is a predisposing factor for chronic, non-healing wounds.Several factors contribute to deficient wound healing in patients with diabetes: deficient growth factor production, deficient neovascularization, attenuated keratinocyte and fibroblast proliferation and migration, and an altered balance between extracellular matrix accumulation and remodelling of the extracellular matrix by matrix metalloproteinases. Whether dyslipidemia in diabetics is a contributing factor to impaired wound healing was not investigated till now. The objective of this project was to investigate whether hypercholesterolemia in C57BL/6 apo E-/- mice negatively affects cutaneous wound healing and to test the hypothesis that topical HDL therapy enhances wound healing in C57BL/6 apo E-/- mice and in C57BL/6 mice with streptozotocin-induced diabetes mellitus. We first compared wound healing in normocholesterolemic C57BL/6 and in dyslipidemic C57BL/6 apolipoprotein E deficient (apo E-/-) mice. Wound coverage with newly formed epithelial tissue was 1.89-fold (p<0.0001), 1.75-fold (p<0.05), 1.47-fold (p<0.001), 1.47-fold (p<0.001), and 1.18-fold (p<0.01)higher in C57BL/6 mice compared to C57BL/6 apo E-/- mice at day 2, day 4, day 6, day 8, and day 10 after wound application, respectively. In C57BL/6 apo E-/- mice, topical administration of HDL in 20% pluronic F-127gel (pH 7.2) increased wound re-epithelialization by 1.35-fold (p<0.05), 1.44 fold (p<0.05), and 1.21-fold (p<0.01) at day 6, day 8, and day 10, respectively. The rate of re-epithelialization was not significantly different between untreated control wounds and control wounds treated with pluronic F-127 gel (pH 7.2). Wound healing was delayed in streptozotocin-induced diabetic C57BL/6 mice compared to non-diabetic littermates. In C57BL/6 mice with streptozotocin-induced diabetes, wound coverage withnew epithelium was 1.93-fold (p<0.01), 1.22-fold (p=NS), 1.37-fold (p<0.01), and 1.11-fold (p=NS) increased at day 4, day 6, day 8, and day 10 after wound application, respectively, by topical HDL therapy. Wound coverage by granulation tissue data showed a similar pattern compared to re-epithelialization data in the different experimental conditions.
In summary, this study demonstrates that topical HDL accelerates wound healing in dyslipidemic mice and in diabetic mice to the rate observed in normocholesterolemic, non-diabetic mice and thereby provides the first foundations for a new treatment paradigm of cutaneous ulcers: topical HDL therapy.
The next two studies of this manuscript entail investigations to evaluate the effect of systemically raised HDL induced by human apo A-I gene transfer on myocardial function and structure in the absence or presence of a myocardial infarction.
It is well established that hypercholesterolemia and low plasma HDL cholesterol levels are risk factors for coronary heart disease but little is known about their direct effects on myocardial function. In patients with low HDL cholesterol levels, post-myocardial infarction (MI) ejection fraction is decreased. In Framingham Heart Study participants free of coronary heart diseaseat baseline, low HDL cholesterol levels were independently associated with heart failure incidence after adjustment for interim MI and clinical covariates. Moreover, low HDL cholesterol levels and low levels of apo A-I indicate an unfavourable prognosis in patients with heart failure independent of the etiology. Direct cellular effects of HDL have been demonstrated in isolated cardiomyocytes in vitro as evidenced by increased phosphorylation of extracellular signal-regulated kinases, of the transcription factor STAT3, and of the pro-survival kinase Akt. Furthermore, it has previously been shown that HDL inhibits cardiomyocyte apoptosis underconditions of hyperglycemia. Finally, the development of diabetic cardiomyopathy is inhibited by human apo A-I gene transfer in rats. Taken together, epidemiological and experimental studies provide converging linesof evidence that HDL may exert direct protective effects on the myocardium.
In chapter 3.3, we examined the effect of lipid lowering gene transfer and selective HDL raising gene transfer on EPC number and EPC function and on cardiac structure and function in hypercholesterolemic C57BL/6 low density lipoprotein receptor deficient (LDLr-/-) mice. 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 circulatingnumber 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 AdLDLrtransfer. Similarly, HDL raising gene transfer increased EPC number andfunction 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. In conclusion, 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 animal models that are often characterized by a predominance of HDL.
In chapter 3.4 we assessed effects of selective HDL raising gene transfer in a murine model of ischemic cardiomyopathy. Specifically, the objective of this study was to examine whether human apo A-I gene transfer in hypercholesterolemic C57BL/6 LDLr-/- mice affects survival, infarct size, and cardiac function after permanent ligation of the left anterior descending coronary artery. In order to focus on the effect of HDL on myocardial biology, a model of permanent ligation was usedand not a model of ischemia-reperfusion injury. Gene transfer in C57BL/6 LDLr-/- mice was performed with the E1E3E4-deleted adenoviral vector AdA-I, inducing hepatocyte-specific expression of human apolipoprotein (apo A-I), or with the control vector Adnull. A ligation of the left anterior descending coronary artery was performed two weeks after transfer orsaline injection. HDL cholesterol levels were persistently 1.5-times (p<0.0001) higher in AdA-I mice compared to controls. Survival was increased (p<0.01) in AdA-I MI mice compared to control MI mice during the 28-day follow-up period (hazard ratio for mortality 0.42; 95% CI 0.24 to 0.76). Longitudinal morphometric analysis demonstrated attenuated infarct expansion and inhibition of left ventricular dilatation in AdA-I MI mice compared to 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. Inconclusion, selective HDL raising gene transfer exerts cardioprotectiveeffects following myocardial infarction.
Taken together, the data of this Ph.D. thesis provide a solid base for future pre-clinical development of new therapeutic modalities for HDL-targeted interventions. Topical HDL therapy may constitute a future innovative and clinically feasible therapeutic strategy to enhance healing of diabetic foot ulcers. Clinical translation of apo A-I gene transfer as a means to systemically increase HDL levels will depend on the development of potent and safe gene transfer vectors and on progress made in the adeno-associated viral gene therapy field.
Date:1 Oct 2009 → 30 Sep 2013
Keywords:Tissue repair, HDL therapy
Disciplines:Cardiac and vascular medicine
Project type:PhD project