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Background and aims Dipeptidyl peptidase 4 (DPP-4) inhibitors have anti-inflammatory and atheroprotective effects. We evaluated the effects of the DPP-4 inhibitor linagliptin on atherosclerotic plaque and hepatic inflammation using histology and 2-deoxy-2-[18F]-fluoro- d -glucose (18F-FDG), a positron emission tomography tracer of inflammation, in a mouse model of hypercholesterolemia and type 2 diabetes. Methods Igf2/Ldlr−/−Apob100/100 mice were fed a high-fat diet (HFD) for 8 weeks and then randomly allocated to receive HFD (n = 14), or HFD with added linagliptin (n = 15) for additional 12 weeks. Five mice fed a chow diet were studied as an additional control. At the end of the study, glucose tolerance, aortic and liver uptake of 18F-FDG, and histology were studied. Results Mice in linagliptin and HFD groups had similar fasting glucose concentrations, but linagliptin improved glucose tolerance. Aortas of linagliptin and HFD groups showed advanced atherosclerotic plaques with no difference in the mean intima-to-media ratio or number of macrophages in the plaques. Autoradiography showed similar 18F-FDG uptake by atherosclerotic plaques in linagliptin and HFD groups (plaque-to-wall ratio: 1.7 ± 0.25 vs. 1.6 ± 0.21; p = 0.24). In the liver, linagliptin reduced the histologic inflammation score but had no effect on 18F-FDG uptake. Compared with chow diet, uptake of 18F-FDG was similar in the aorta, but higher in the liver after HFD. Conclusions Linagliptin therapy improved glucose tolerance and reduced hepatic inflammation but had no effect on plaque burden or atherosclerotic inflammation, as determined by histology and 18F-FDG uptake, in atherosclerotic mice with type 2 diabetes.

Statins are effective drugs used to prevent and treat cardiovascular diseases but their effects in the absence of low density lipoprotein receptor (LDLR) and on the risk of diabetes are not yet well characterized. The aim of this study was to clarify systemic and pleiotropic effects of rosuvastatin on cardiovascular and diabetic phenotypes. IGF-II/LDLR-/-ApoB100/100 hypercholesterolemic prediabetic mice were used to test the effects of rosuvastatin on plasma glucose, insulin, lipids, atherosclerosis and liver steatosis. To get a more comprehensive view about changes in gene expression RNA-sequencing was done from the liver. Rosuvastatin significantly reduced plasma cholesterol in hypercholesterolemic mice in the absence of LDLR but had no effects on atherosclerosis at aortic sinus level or in coronary arteries. Rosuvastatin also significantly reduced liver steatosis without any harmful effects on glucose or insulin metabolism. RNA-sequencing showed relatively specific effects of rosuvastatin on genes involved in cholesterol metabolism together with a significant anti-inflammatory gene expression profile in the liver. In addition, significant changes were found in the expression of Perilipin 4 and 5 which are involved in lipid droplet formation in the liver. For the first time it could be shown that Tribbles proteins are affected by rosuvastatin treatment in the hyperlipidemic mice. Rosuvastatin had several positive effects on hypercholesterolemic mice showing early signs of diabetes, many of which are unrelated to cholesterol and lipoprotein metabolism. These results increase our understanding about the systemic and pleiotropic effects of rosuvastatin in the absence of LDLR expression.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Dipeptidyl peptidase 4 (DPP-4) inhibitors have anti-inflammatory and atheroprotective effects. We evaluated the effects of the DPP-4 inhibitor linagliptin on atherosclerotic plaque and hepatic inflammation using histology and 2-deoxy-2-[Igf2/LdlrMice in linagliptin and HFD groups had similar fasting glucose concentrations, but linagliptin improved glucose tolerance. Aortas of linagliptin and HFD groups showed advanced atherosclerotic plaques with no difference in the mean intima-to-media ratio or number of macrophages in the plaques. Autoradiography showed similarLinagliptin therapy improved glucose tolerance and reduced hepatic inflammation but had no effect on plaque burden or atherosclerotic inflammation, as determined by histology and

Introduction Concentric left ventricular hypertrophy (LVH) and diastolic dysfunction develops as an adaptive response to pressure overload. Eventually this may lead to decompensated hypertrophy characterised by interstitial fibrosis, contractile dysfunction as well as changes in metabolism and electrophysiology, consequentially triggering heart failure. The molecular mechanisms involved in cardiac remodelling are not fully understood but maladaptive angiogenesis could promote the transition from adaptive LVH to decompensated heart failure. Angiogenesis is mediated by vascular endothelial growth factors but their role in LVH has remained unresolved. Purpose In this study, we wanted to investigate whether vascular endothelial growth factor receptor 1 (VEGFR-1) signaling has a role in the progression of LVH and development of heart failure. Methods We used wild type littermate controls and domain specific knock out mouse lacking the intracellular VEGFR-1 tyrosine kinase domain (VEGFR-1 TK−/−) and induced pathological hypertrophy with subcutaneous angiotensin II infusion. We examined the cardiac function with echocardiography and acquired surface ECG signal during the development of LVH. Mice were followed up for 14 days before sacrification and sample collection. Cross-sectional cardiac samples were stained with Masson's trichrome to assess the level of fibrosis and immunostained for lectin to determine capillary area. Additionally, we performed a CD31 whole mount staining to visualise capillary 3D network. To analyse changes in gene expression levels, we performed RT qPCR measurements. Results VEGFR-1 TK deficiency led to increased mortality (33.3%) and lack of adaptive LVH. Whereas wild type mice responded to angiotensin II infusion with a significant increase in ejection fraction (55.5% to 69.9%) within the first 6 days, VEGFR-1 TK−/− mice displayed a 5.2% decrease and without adaptive thickening of the LV anterior wall. The most striking difference was seen in LV volume, where wild type mice displayed a 63.3% reduction but in VEGFR-1 TK−/− mice it remained unaltered after angiotensin II infusion. Histological analyses showed that VEGFR-1 TK−/− mice displayed significant cardiomyocyte hypertrophy combined with ventricular dilatation but without changes in fibrosis or angiogenesis. ECG analysis revealed that VEGFR-1 TK−/− mice exhibited widening of the QRS complex, similar to human LVH, and this was accompanied by increased ANP/BNP levels. Conclusions In this study, we show that the ablation of VEGFR-1 TK signaling has an unexpected role in pressure overload inducing mortality. VEGFR-1 TK−/− mice displayed dilated LVH and a protracted response to angiotensin II infusion, suggesting that VEGFR-1 signaling is required for the adaptive response and concentric hypertrophy of the myocardium.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4–5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Key points Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation–contraction coupling phenotype in the heart. Abstract The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation–contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.

Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

Despite considerable improvements in therapies, atherosclerotic cardiovascular diseases remain the leading cause of death worldwide. Therefore, in addition to current treatment options, new therapeutic approaches are still needed.In this review, novel gene and RNA interference-based therapy approaches and promising target genes for treating atherosclerosis are addressed. In addition, relevant animal models for the demonstration of the efficacy of different gene therapy applications, and current progress toward more efficient, targeted and safer gene transfer vectors are reviewed.Atherosclerosis represents a complex multifactorial disease that is dependent on the interplay between lipoprotein metabolism, cellular reactions and inflammation. Recent advances and novel targets, especially in the field of RNA interference-based therapies, are very promising. However, it should be noted that the modulation of a particular gene is not as clearly associated with a complex polygenic disease as it is in the case of monogenic diseases. A deeper understanding of molecular mechanisms of atherosclerosis, further progress in vector development and the demonstration of treatment efficacy in relevant animal models will be required before gene therapy of atherosclerosis meets its clinical reality.

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE−/− mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors., published version, peerReviewed

Cardiac‐specific overexpression of vascular endothelial growth factor (VEGF)‐B167 is known to induce left ventricular hypertrophy due to altered lipid metabolism, in which ceramides accumulate to the heart and cause mitochondrial damage. The aim of this study was to evaluate and compare different imaging methods to find the most sensitive way to diagnose at early stage the progressive left ventricular remodeling leading to heart failure. Echocardiography and cardiovascular magnetic resonance imaging were compared for imaging the hearts of transgenic mice with cardiac‐specific overexpression of VEGF‐B167 and wild‐type mice from 5 to 14 months of age at several time points. Disease progression was verified by molecular biology methods and histology. We showed that left ventricular remodeling is already ongoing at the age of 5 months in transgenic mice leading to heart failure by the age of 14 months. Measurements from echocardiography and cardiovascular magnetic resonance imaging revealed similar changes in cardiac structure and function in the transgenic mice. Changes in histology, gene expressions, and electrocardiography supported the progression of left ventricular hypertrophy. Longitudinal relaxation time in rotating frame (T1ρ) in cardiovascular magnetic resonance imaging could be suitable for detecting severe fibrosis in the heart. We conclude that cardiac‐specific overexpression of VEGF‐B167 leads to left ventricular remodeling at early age and is a suitable model to study heart failure development with different imaging methods.

Mouse models are extremely important in studying cardiac pathologies and related electrophysiology, but very few mouse ECG analysis programs are readily available. Therefore, a mouse ECG analysis algorithm was developed and validated. Surface ECG (lead II) was acquired during transthoracic echocardiography from C57Bl/6J mice under isoflurane anesthesia. The effect of aging was studied in young (2–3 months), middle‐aged (14 months) and old (20–24 months) mice. The ECG changes associated with pharmacological interventions and common cardiac pathologies, that is, acute myocardial infarction (AMI) and progressive left ventricular hypertrophy (LVH), were studied. The ECG raw data were analyzed with an in‐house ECG analysis program, modified specially for mouse ECG. Aging led to increases in P‐wave duration, atrioventricular conduction time (PQ interval), and intraventricular conduction time (QRS complex width), while the R‐wave amplitude decreased. In addition, the prevalence of arrhythmias increased during aging. Anticholinergic atropine shortened PQ time, and beta blocker metoprolol and calcium‐channel blocker verapamil increased PQ interval and decreased heart rate. The ECG changes after AMI included early JT elevation, development of Q waves, decreased R‐wave amplitude, and later changes in JT/T segment. In progressive LVH model, QRS complex width was increased at 2 and especially 4 weeks timepoint, and also repolarization abnormalities were seen. Aging, drugs, AMI, and LVH led to similar ECG changes in mice as seen in humans, which could be reliably detected with this new algorithm. The developed method will be very useful for studies on cardiovascular diseases in mice.