RESUMO
Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr -/-, Apobec1 -/- double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.
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Epidemiological studies support a strong association between non-high-density lipoprotein cholesterol levels and heart failure incidence. The objective of the current study was to evaluate the effect of selective cholesterol lowering adeno-associated viral serotype 8 (AAV8)-mediated low-density lipoprotein receptor (LDLr) gene transfer on cardiac remodelling and myocardial oxidative stress following transverse aortic constriction (TAC) in female C57BL/6 LDLr-/- mice with mild hypercholesterolemia. Cholesterol lowering gene transfer resulted in a 65.9% (p<0.0001) reduction of plasma cholesterol levels (51.2 ± 2.2 mg/dl) compared to controls (150 ± 7 mg/dl). Left ventricular wall area was 11.2% (p<0.05) lower in AAV8-LDLr TAC mice than in control TAC mice. In agreement, pro-hypertrophic myocardial proteins were potently decreased in AAV8-LDLr TAC mice. The degree of interstitial fibrosis and perivascular fibrosis was 31.0% (p<0.001) and 29.8% (p<0.001) lower, respectively, in AAV8-LDLr TAC mice compared to control TAC mice. These structural differences were associated with improved systolic and diastolic function and decreased lung congestion in AAV8-LDLr TAC mice compared to control TAC mice. Cholesterol lowering gene therapy counteracted myocardial oxidative stress and preserved the potential for myocardial fatty acid oxidation in TAC mice. In conclusion, cholesterol lowering gene therapy attenuates pressure overload-induced heart failure in mice with mild hypercholesterolemia.
Assuntos
Colesterol , Insuficiência Cardíaca , Hipercolesterolemia , Animais , Dependovirus , Terapia Genética , Vetores Genéticos , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/fisiopatologia , Hipercolesterolemia/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Estresse Oxidativo/fisiologia , Receptores de LDL/genética , Remodelação Ventricular/fisiologiaRESUMO
Hypercholesterolemia may be causally related to heart failure with preserved ejection fraction (HFpEF). We aimed to establish a HFpEF model associated with hypercholesterolemia and type 2 diabetes mellitus by feeding a high-sucrose/high-fat (HSHF) diet to C57BL/6J low-density lipoprotein receptor (LDLr)-/- mice. Secondly, we evaluated whether cholesterol-lowering adeno-associated viral serotype 8 (AAV8)-mediated LDLr gene transfer prevents HFpEF. AAV8-LDLr gene transfer strongly (p < 0.001) decreased plasma cholesterol in standard chow (SC) mice (66.8 ± 2.5 mg/dl versus 213 ± 12 mg/dl) and in HSHF mice (84.6 ± 4.4 mg/dl versus 464 ± 25 mg/dl). The HSHF diet induced cardiac hypertrophy and pathological remodeling, which were potently counteracted by AAV8-LDLr gene transfer. Wet lung weight was 19.0% (p < 0.001) higher in AAV8-null HSHF mice than in AAV8-null SC mice, whereas lung weight was normal in AAV8-LDLr HSHF mice. Pressure-volume loop analysis was consistent with HFpEF in AAV8-null HSHF mice and showed a completely normal cardiac function in AAV8-LDLr HSHF mice. Treadmill exercise testing demonstrated reduced exercise capacity in AAV8-null HSHF mice but a normal capacity in AAV8-LDLr HSHF mice. Reduced oxidative stress and decreased levels of tumor necrosis factor-α may mediate the beneficial effects of cholesterol lowering. In conclusion, AAV8-LDLr gene therapy prevents HFpEF.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/prevenção & controle , Terapia Genética/métodos , Insuficiência Cardíaca/prevenção & controle , Hipercolesterolemia/terapia , Receptores de LDL/genética , Animais , Colesterol/sangue , Dependovirus/genética , Diabetes Mellitus Tipo 2/etiologia , Cardiomiopatias Diabéticas/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Feminino , Insuficiência Cardíaca/fisiopatologia , Hipercolesterolemia/complicações , Hipercolesterolemia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Receptores de LDL/metabolismo , Volume Sistólico , Fator de Necrose Tumoral alfa/sangueRESUMO
Objective- We investigated the hypothesis that HDL (high-density lipoprotein) dysfunction in Scarb1-/- mice negatively affects cardiac function both in the absence and in the presence of pressure overload. Second, we evaluated whether normalization of HDL metabolism in Scarb1-/- mice by hepatocyte-specific SR-BI (scavenger receptor class B, type I) expression after E1E3E4-deleted adenoviral AdSR-BI (E1E3E4-deleted adenoviral vector expressing SR-BI protein in hepatocytes) transfer abrogates the effects of total body SR-BI deficiency on cardiac structure and function. Approach and Results- Transverse aortic constriction (TAC) or sham operation was performed at the age of 14 weeks, 2 weeks after saline injection or after gene transfer with AdSR-BI or with the control vector Adnull. Mortality rate in Scarb1-/- TAC mice was significantly increased compared with wild-type TAC mice during 8 weeks of follow-up (hazard ratio, 2.02; 95% CI, 1.14-3.61). Hepatocyte-specific SR-BI gene transfer performed 2 weeks before induction of pressure overload by TAC potently reduced mortality in Scarb1-/- mice (hazard ratio, 0.329; 95% CI, 0.180-0.600). Hepatocyte-specific SR-BI expression abrogated increased cardiac hypertrophy and lung congestion and counteracted increased myocardial apoptosis and interstitial and perivascular fibrosis in Scarb1-/- TAC mice. Scarb1-/- sham mice were, notwithstanding the absence of detectable structural heart disease, characterized by systolic and diastolic dysfunction and hypotension, which were completely counteracted by AdSR-BI transfer. Furthermore, AdSR-BI transfer abrogated increased end-diastolic pressure and diastolic dysfunction in Scarb1-/- TAC mice. Increased oxidative stress and reduced antioxidant defense systems in Scarb1-/- mice were rescued by AdSR-BI transfer. Conclusions- The detrimental effects of SR-BI deficiency on cardiac structure and function are nullified by hepatocyte-specific SR-BI transfer, which restores HDL metabolism.
Assuntos
Cardiomegalia/terapia , Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Receptores Depuradores Classe B/genética , Animais , Apoptose , Pressão Sanguínea , Cardiomegalia/sangue , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Células Cultivadas , HDL-Colesterol/sangue , Feminino , Fibrose , Expressão Gênica , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse OxidativoRESUMO
Epidemiological studies support an independent inverse association between high-density lipoprotein (HDL) cholesterol levels and heart failure incidence. The effect of selective HDL-raising adeno-associated viral serotype 8-human apolipoprotein (apo) A-I (AAV8-A-I) gene transfer on cardiac remodeling induced by transverse aortic constriction (TAC) was evaluated in C57BL/6 low-density lipoprotein receptor-deficient mice. Septal wall thickness and cardiomyocyte cross-sectional area were reduced by 16.5% (p < 0.001) and by 13.8% (p < 0.01), respectively, eight weeks after TAC in AAV8-A-I mice (n = 24) compared to control mice (n = 39). Myocardial capillary density was 1.11-fold (p < 0.05) higher and interstitial cardiac fibrosis was 45.3% (p < 0.001) lower in AAV8-A-I TAC mice than in control TAC mice. Lung weight and atrial weight were significantly increased in control TAC mice compared to control sham mice, but were not increased in AAV8-A-I TAC mice. The peak rate of isovolumetric contraction was 1.19-fold (p < 0.01) higher in AAV8-A-I TAC mice (n = 17) than in control TAC mice (n = 29). Diastolic function was also significantly enhanced in AAV8-A-I TAC mice compared to control TAC mice. Nitro-oxidative stress and apoptosis were significantly reduced in the myocardium of AAV8-A-I TAC mice compared to control TAC mice. In conclusion, selective HDL-raising human apo A-I gene transfer potently counteracts the development of pressure overload-induced cardiomyopathy.
Assuntos
Apolipoproteína A-I/metabolismo , Cardiomegalia/terapia , Cardiomiopatias/terapia , Terapia Genética/métodos , Lipoproteínas HDL/metabolismo , Animais , Apolipoproteína A-I/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Fibrose/genética , Fibrose/terapia , Coração/fisiopatologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
A causal role of hypercholesterolemia in non-ischemic heart failure has never been demonstrated. Adeno-associated viral serotype 8 (AAV8)-low-density lipoprotein receptor (AAV8-LDLr) gene transfer was performed in LDLr-deficient mice without and with pressure overload induced by transverse aortic constriction (TAC). AAV8-LDLr gene therapy resulted in an 82.8% (p < 0.0001) reduction of plasma cholesterol compared with controls. Mortality rate was lower (p < 0.05) in AAV8-LDLr TAC mice compared with control TAC mice (hazard ratio for mortality 0.457, 95% confidence interval [CI] 0.237-0.882) during 8 weeks of follow-up. AAV8-LDLr gene therapy attenuated cardiac hypertrophy, reduced interstitial and perivascular fibrosis, and decreased lung congestion in TAC mice. Cardiac function, quantified by invasive hemodynamic measurements and magnetic resonance imaging, was significantly improved 8 weeks after sham operation or after TAC in AAV8-LDLr mice compared with respective control groups. Myocardial protein levels of mammalian target of rapamycin and of acetyl-coenzyme A carboxylase were strikingly decreased following cholesterol lowering in mice without and with pressure overload. AAV8-LDLr therapy potently reduced cardiac glucose uptake and counteracted metabolic remodeling following pressure overload. Furthermore, oxidative stress and myocardial apoptosis were decreased following AAV8-LDLr therapy in mice with pressure overload. In conclusion, cholesterol-lowering gene therapy potently counteracts structural and metabolic remodeling, and enhances cardiac function.
Assuntos
Cardiomegalia/terapia , Cardiomiopatias/terapia , Colesterol/metabolismo , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Receptores de LDL/genética , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Aorta/cirurgia , Biomarcadores/metabolismo , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/mortalidade , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/mortalidade , Constrição Patológica/complicações , Constrição Patológica/metabolismo , Constrição Patológica/patologia , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Testes de Função Cardíaca , Hemodinâmica , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Receptores de LDL/deficiência , Análise de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Cardiomiopatias/patologia , Óleo de Coco/efeitos adversos , Resistência à Insulina , Miocárdio/patologia , Obesidade/patologia , Pressão , Animais , Aorta/patologia , Peso Corporal , Capilares/patologia , Cardiomiopatias/sangue , Cardiomiopatias/complicações , Cardiomiopatias/fisiopatologia , Colesterol , Constrição Patológica , Diástole , Dieta , Feminino , Fibrose , Glucose/metabolismo , Hemodinâmica , Hipertrofia Ventricular Direita/complicações , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Estimativa de Kaplan-Meier , Pulmão/patologia , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Transdução de Sinais , Sístole , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Obesity is associated with vascular inflammation, fibrosis and reduced high-density lipoproteins (HDL)-cholesterol. We aimed to investigate whether adenoviral gene transfer with human apolipoprotein (apo) A-I (Ad.A-I), the main apo of HDL, could exert immunomodulatory effects and counteract vascular inflammation and fibrosis in ob/ob mice. METHODS: Ad.A-I transfer was performed in 8 weeks (w) old ob/ob mice, which were sacrificed 7 w later. The aorta was excised for mRNA analysis and the spleen for splenocyte isolation for subsequent flow cytometry and co-culture with murine fibroblasts. HDL was added to mononuclear cells (MNC) and fibroblasts to assess their impact on adhesion capacity and collagen deposition, respectively. RESULTS: Ad.A-I led to a 1.8-fold (p < 0.05) increase in HDL-cholesterol versus control ob/ob mice at the day of sacrifice, which was paralleled by a decrease in aortic TNF-α and VCAM-1 mRNA expression. Pre-culture of MNC with HDL decreased their adhesion to TNF-α-activated HAEC. Ad.A-I exerted immunomodulatory effects as evidenced by a downregulation of aortic NOD2 and NLRP3 mRNA expression and by a 12 %, 6.9 %, and 15 % decrease of the induced proliferation/activity of total splenic MNC, CD4+, and CD8+ cells in ob/ob Ad.A-I versus control ob/ob mice, respectively (p < 0.05). Ad.A-I further reduced aortic collagen I and III mRNA expression by 62 % and 66 %, respectively (p < 0.0005), and abrogated the potential of ob/ob splenocytes to induce the collagen content in murine fibroblasts upon co-culture. Finally, HDL decreased the TGF-ß1-induced collagen deposition of murine fibroblasts in vitro. CONCLUSIONS: Apo A-I transfer counteracts vascular inflammation and fibrosis in ob/ob mice.
RESUMO
UNLABELLED: Plasma homocysteine levels predict heart failure incidence in prospective epidemiological studies. We evaluated whether selective homocysteine-lowering gene transfer beneficially affects cardiac remodeling and function in a model of pressure overload-induced cardiomyopathy induced by transverse aortic constriction (TAC). Female C57BL/6 low-density lipoprotein receptor (Ldlr (-/-)) cystathionine-ß-synthase (Cbs (+/-)) mice were fed standard chow (control mice) or a folate-depleted, methionine-enriched diet to induce hyperhomocysteinemia (diet mice). Three weeks after initiation of thisdiet, mice were intravenously injected with 5 × 10(10) viral particles of an E1E3E4-deleted hepatocyte-specific adenoviral vector expressing Cbs (AdCBS), with the same dose of control vector, or with saline buffer. TAC or sham operation was performed 2 weeks later. AdCBS gene transfer resulted in 86.4 % (p < 0.001) and 84.6 % (p < 0.001) lower homocysteine levels in diet sham mice and diet TAC mice, respectively. Mortality rate was significantly reduced in diet AdCBS TAC mice compared to diet TAC mice during a follow-up period of 8 weeks (hazard ratio for mortality 0.495, 95 % CI 0.249 to 0.985). Left ventricular hypertrophy (p < 0.01) and interstitial myocardial fibrosis (p < 0.001) were strikingly lower in control TAC mice and diet AdCBS TAC mice compared to diet TAC mice. Diastolic function in diet AdCBS TAC mice was similar to that of control TAC mice and was significantly improved compared to diet TACmice. AdCBS gene transfer potently reduced oxidative stress as evidenced by a reduction of plasma TBARS and a reduction of myocardial 3-nitrotyrosine-positive area (%). In conclusion, selective homocysteine lowering potently attenuates pressure overload-induced cardiomyopathy via reduced oxidative stress. KEY MESSAGE: Plasma homocysteine levels predict heart failure incidence in epidemiological studies. Transverse aortic constriction (TAC) induces pressure overload. Selective homocysteine-lowering gene therapy reduces mortality after TAC. Selective homocysteine lowering attenuates cardiac hypertrophy and fibrosis after TAC. Decreased homocysteine levels enhance diastolic function and lower oxidative stress.
Assuntos
Cardiomiopatias/genética , Cardiomiopatias/terapia , Cistationina beta-Sintase/genética , Homocisteína/sangue , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/terapia , Animais , Cardiomegalia/complicações , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/terapia , Cardiomiopatias/sangue , Cardiomiopatias/complicações , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Hemodinâmica , Homocisteína/genética , Hiper-Homocisteinemia/sangue , Hiper-Homocisteinemia/complicações , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Estresse OxidativoRESUMO
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.
RESUMO
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)â»/â» cystathionine-ß-synthase (Cbs)âº/â» 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 Ldlrâ»/â» Cbsâº/â» mice. The current study corroborates the view that hyperhomocysteinemia exerts direct effects on the myocardium and may potentiate the development of heart failure.
Assuntos
Cistationina beta-Sintase/genética , Insuficiência Cardíaca/terapia , Coração/fisiopatologia , Homocistinúria/terapia , Hipercolesterolemia/terapia , Hiper-Homocisteinemia/terapia , Infarto do Miocárdio/terapia , Adenoviridae/genética , Animais , Cistationina beta-Sintase/metabolismo , Diástole/fisiologia , Dieta Hiperlipídica , Feminino , Vetores Genéticos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Homocisteína/sangue , Homocistinúria/genética , Homocistinúria/patologia , Homocistinúria/fisiopatologia , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/patologia , Hiper-Homocisteinemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Transgenes , Remodelação Ventricular/fisiologiaRESUMO
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.
Assuntos
HDL-Colesterol/sangue , Vasos Coronários/metabolismo , Diástole/genética , Células Endoteliais/citologia , Técnicas de Transferência de Genes , Coração/fisiologia , Células-Tronco/citologia , Animais , Apolipoproteína A-I/genética , Capilares/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Receptores de LDL/genéticaRESUMO
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.
RESUMO
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.