Zusammenfassung
Die Herzinsuffizienz mit erhaltener Ejektionsfraktion („heart failure with preserved ejection fraction“ HFpEF) stellt ein heterogenes Syndrom mit unterschiedlichen Ätiologien und ...pathophysiologischen Faktoren dar. Adipositas und Diabetes mellitus Typ 2 (T2DM), die überzufällig häufig koexistieren, induzieren eine Vielfalt von metabolischen und nichtmetabolischen Signalstörungen, die Entzündung, Fibrose und Myozytensteifigkeit – alles Kennzeichen der HFpEF – begünstigen. Dabei sind im Gegensatz zu anderen HFpEF-Risikofaktoren die Adipositas und der T2DM häufig mit der vermehrten Bildung von viszeralem Fettgewebe assoziiert, das einen hochaktiven endokrinen Gewebeverbund darstellt, der Inflammation und Fibrose über verschiedene para- und vasokrine Signale verstärken und so die Funktion von Herz, Niere und pulmonalem Gefäßbett nachhaltig beeinflussen kann. Ein abnorm ausgedehntes epikardiales Fettgewebe („epicardial adipose tissue“ EAT) bedingt hierbei nicht nur eine mechanische Einengung des diastolischen Füllungsvorgangs des Herzens, sondern ist aufgrund der hohen Konzentrationen freigesetzter proinflammatorischer Adipokine insbesondere auch mit einer erhöhten Inzidenz von Vorhofflimmern und eingeschränkten linksventrikulären Kontraktionsparametern vergesellschaftet. Adipöse Patienten mit HFpEF gehören daher zu einem HFpEF-Phänotyp mit besonders schlechter Prognose und könnten von einer EAT-ausgerichteten phänotypspezifischen Intervention profitieren. Dabei könnte neben Statinen und Antidiabetika wie Metformin gerade Glucagon-like-peptide-1(GLP-1)-Rezeptor-Agonisten oder Natrium-Glukose-Kotransporter-2(SGLT-2)-Inhibitoren eine besondere Rolle zukommen.
The kallikrein-kinin system participates in the maintenance of the cardiovascular phenotype. We previously demonstrated that human tissue kallikrein gene (hTK) transfer promotes the healing of ...ischemic limbs. The present investigation aimed to test the original hypothesis that hTK delivery to the peri-infarct myocardium would prevent post-ischemic heart failure.
Myocardial infarction (MI) was induced in anesthetized mice by permanently occluding the left coronary descendant. hTK was delivered to the peri-infarct myocardium via an adenoviral vector (Ad.hTK). Controls received Ad.Null or saline. Survival rate was similar among groups. Ad.hTK increased the number of circulating endothelial progenitor cells and promoted the growth of capillaries and arterioles in the peri-infarct myocardium. In addition, Ad.hTK increased the abundance of cardiac progenitor cells (CPCs) in the peri-infarct and suppressed the apoptotic death of peri-infarct cardiomyocytes in vivo and ex vivo. As a consequence of these beneficial effects, at 5 weeks from MI, hTK-transduced hearts were protected from post-MI ventricular dilatation and showed better systolic and diastolic functions.
Ad.hTK benefits the neovascularization and viability of peri-infarct myocardium and increases CPC abundance, thereby decreasing ventricular dysfunction. Our study significantly adds to the knowledge of the protective effects of TK gene transfer on ischemic diseases and opens new avenues for the treatment of post-MI cardiac failure.
Background: Long term expression after gene transfer is dependent on the absence of an adaptive immune response against the transgene product. In contrast, genetic vaccination requires the induction ...of a vigorous immune response. The effect of promoter, transgene product, vector dose, murine strain and animal species on humoral immune responses against the transgene product after adenoviral transfer was evaluated.Methods: Gene transfer was performed with E1E3E4-deleted vectors containing the 1.5 kb human α 1 -antitrypsin promoter in front of the genomic human apo A-I sequence and 4 copies of the human apo E enhancer (AdAT4 .apo A-I). Alternatively, human plasminogen (92 kDa) (AdAT4 .plasm) or human microplasminogen (28.6 kDa) (AdAT4 .μplasm) was expressed under control of the same promoter and enhancers. To determine the effect of promoter, human apo A-I was expressed under control of the cytomegalovirus promoter (AdCMV.apoA-I) or the murine MHC II Eβ promoter (AdEβ.apoA-I).Results: Gene transfer with 5 × 10 10 particles of AdAT4 .apo A-I was not associated with an immune response against human apo A-I in C57BL/6, Balb/c, C3H/HeJ or Swiss Webster mice. Classical immunization with human apo A-I protein formulated in complete Freund adjuvant followed by rechallenge with human apo A-I protein formulated in incomplete Freund adjuvant induced a vigorous immune response in naive Balb/c mice, but not so in Balb/c mice injected with 5 × 10 10 particles of AdAT4 .apo A-I, indicating that transfer with this dose of AdAT4 .apo A-I induced immunological tolerance for human apo A-I. In contrast, gene transfer with 1 x 1010 particles of AdAT4 .apo A-I in Balb/c mice did not induce antibodies but classical immunization with human apo A-I protein induced a vigorous immune response in these mice, indicating that the absence of an immune response after transfer with this dose is solely due to immunological ignorance. Gene transfer with AdCMV.apoA-I induced high titers of antibodies against human apo A-I in Balb/c mice, but not in the other three strains. In contrast, transfer with AdEβ.apoA-I induced a strong immune response in Balb/c, C3H/HeJ and Swiss Webster mice and moderate antibody titers in C57BL/6 mice. Antibodies against human plasminogen were observed in C57BL/6 mice after transfer with AdAT 4 .plasm, but there were no antibodies against microplasminogen after transfer with AdAT4 .μplasm, indicating that tolerance and/or ignorance for the transgene product in mice is not only determined by the expression cassette, but also by the transgene product. In contrast to mice, neutralising antibodies against human apo A-I were present after transfer with 4 × 10 12 particles/kg of AdAT4 .apo A-I in three rabbit strains. In Sprague Dawley rats, a large interindividual variability in immune response after transfer with AdAT4 .apo A-I was observed.Conclusion: The effect of expression cassette on the immune response against the transgene product in mice should be extended with caution to other species. The induction of immunological tolerance against the transgene product in mice is dependent on the antigen levels. The murine MHC II Eβ promoter appears to be more efficient than the CMV promoter for inducing antibodies against the transgene product in mice.
The hepatotropism and intrahepatic distribution of adenoviral vectors may be species dependent. Hepatocyte transduction was evaluated in three rabbit strains after transfer with E1E3E4-deleted ...adenoviral vectors containing a hepatocyte specific α1 -antitrypsin promoter driven expression cassette (AdAT4 ). Intravenous administration of 4 × 1012 particles/kg of AdAT4 induced human apo A-I levels above 40 mg/dl in Dutch Belt, but below 1 mg/dl in New Zealand White and Fauve de Bourgogne rabbits. Human apo A-I levels did not correlate with transgene DNA levels, but with human apo A-I mRNA/DNA ratio in the liver. This human apo A-I mRNA/DNA ratio in the liver was 1.6 ± 0.37 at day 14 in Dutch Belt rabbits and was 77-fold (p<0.01) and 69-fold (p<0.01) higher than in New Zealand White and Fauve de Bourgogne rabbits, respectively, indicating strain differences of intrahepatic transgene DNA distribution. Diameters of sinusoidal fenestrae determined by transmission electron microscopy were significantly (p=0.0014) larger in Dutch Belt (124 ± 3.4 nm) than in New Zealand White (108 ± 1.3 nm) and Fauve de Bourgogne (105 ± 2.6 nm) rabbits. Since the diameter of adenoviral particles determined by cryo-electon microscopy is 110 nm, the observed differences in diameters of sinusoidal fenestrae are a critical determinant of hepatocyte transduction by adenoviral vectors.The number of fenestrae per μm 2 determined by scanning electron microscopy was not significantly different between New Zealand White rabbits (19 ± 0.66) and Dutch Belt rabbits (19 ± 0.60) and was not determined in Fauve de Bourgogne rabbits. Consitent with the presence of an anatomical barrier for the passage of adenoviral vectors at the level of the sinusoidal endothelium, intraportal transfer preceded by injection of sodium decanoate, which enhances endothelial permeability, increased human apo A-I levels 32- and 120-fold in New Zealand White and Fauve de Bourgogne rabbits, respectively, but did not affect expression in Dutch Belt rabbits. In conclusion, size of sinusoidal fenestrae appears to be a critical determinant of hepatocyte transduction after adenoviral transfer. The existence of several pathophysiological conditions in humans in which the anatomy of the sinusoidal endothelium is altered, underscores the relevance of these observations for the development of hepatocyte directed gene transfer. Since the diameter of lentiviral vectors is higher than adenoviral vectors and since genetic variation in the diameters of sinusoidal fenestrae in humans with a healthy liver is unknown, this determinant of parenchymal liver cell transduction should be taken into account for the development of cllinical gene transfer.
High density lipoprotein (HDL) metabolism and lecithin:cholesterol acyltransferase (LCAT)-induced HDL remodeling were investigated in transgenic mice expressing human apolipoprotein (apo) AI or an ...apoAI/apoAII chimera in which the Arg123-Tyr166 domain of apoAI was substituted with the Ser12-Ala75 domain of apoAII. Expression of apoAI and of the apoAI/apoAII chimera resulted in a respective 3. 5-fold and 2.9-fold increase of HDL cholesterol. Human LCAT gene transfer into apoAI-transgenic mice resulted in a 5.1-fold increase of endogenous LCAT activity. This increase was associated with a 2. 4-fold increase of the cholesterol ester-to-free cholesterol ratio of HDL, a shift from HDL(3) to HDL(2), and a 2.4-fold increase of HDL cholesterol levels. Agarose gel electrophoresis revealed that human LCAT gene transfer into human apoAI-transgenic mice resulted in an increase of pre-beta-HDL and of pre-alpha-HDL. In contrast, human LCAT gene transfer did not affect cholesterol levels and HDL distribution profile in mice expressing the apoAI/apoAII chimera. Mouse LCAT did not "see" a difference between wild-type and mutant human apoAI, whereas human LCAT did, thus localizing the species-specific interaction in the central domain of apoAI. In conclusion, the Arg123-Tyr166 central domain of apoAI is not critical for in vivo lipoprotein association. It is, however, critical for LCAT-induced hyperalphalipoproteinemia and HDL remodeling independent of the lipid-binding properties of apoAI.
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