Short-term effects of sacubitril/valsartan therapy on myocardial oxygen consumption and energetic efficiency of cardiac work in heart failure with reduced ejection fraction: A randomized controlled study.

AIMS: We sought to evaluate the mechanism of angiotensin receptor-neprilysin inhibitor (ARNI) sacubitril/valsartan therapy and compare it with a valsartan-only control group in patients with heart failure with reduced ejection fraction (HFrEF). METHODS AND RESULTS: The study was a phase IV, prospective, randomized, double-blind, parallel-group study in patients with New York Heart Association class II-III heart failure and left ventricular ejection fraction (LVEF) ≤35%. During a 6-week run-in period, all patients received valsartan therapy, which was up-titrated to the highest tolerated dose level (80 mg bid or 160 mg bid) and then randomized to either valsartan or sacubitril/valsartan. Myocardial oxygen consumption, energetic efficiency of cardiac work, cardiac and systemic haemodynamics were quantified using echocardiography and 11 C-acetate positron emission tomography before and after 6 weeks of therapy (on stable dose) in 55 patients (ARNI group: n = 27, mean age 63 ± 10 years, LVEF 29.2 ± 10.4%; and valsartan-only control group: n = 28, mean age 64 ± 8 years, LVEF 29.0 ± 7.3%; all p = NS). The energetic efficiency of cardiac work remained unchanged in both treatment arms. However, both diastolic (-4.5 mmHg; p = 0.026) and systolic blood pressure (-9.8 mmHg; p = 0.0007), myocardial perfusion (-0.054 ml/g/min; p = 0.045), and left ventricular mechanical work (-296; p = 0.038) decreased significantly in the ARNI group compared to the control group. Although myocardial oxygen consumption decreased in the ARNI group (-5.4%) compared with the run-in period and remained unchanged in the control group (+0.5%), the between-treatment group difference was not significant (p = 0.088). CONCLUSIONS: We found no differences in the energetic efficiency of cardiac work between ARNI and valsartan-only groups in HFrEF patients. However, ARNI appears to have haemodynamic and cardiac mechanical effects over valsartan in heart failure patients.

The Ablation of VEGFR-1 Signaling Promotes Pressure Overload-Induced Cardiac Dysfunction and Sudden Death.

Molecular mechanisms involved in cardiac remodelling are not fully understood. To study the role of vascular endothelial growth factor receptor 1 (VEGFR-1) signaling in left ventricular hypertrophy (LVH) and heart failure, we used a mouse model lacking the intracellular VEGFR-1 tyrosine kinase domain (VEGFR-1 TK-/-) and induced pressure overload with angiotensin II infusion. Using echocardiography (ECG) and immunohistochemistry, we evaluated pathological changes in the heart during pressure overload and measured the corresponding alterations in expression level and phosphorylation of interesting targets by deep RNA sequencing and Western blot, respectively. By day 6 of pressure overload, control mice developed significant LVH whereas VEGFR-1 TK-/- mice displayed a complete absence of LVH, which correlated with significantly increased mortality. At a later time point, the cardiac dysfunction led to increased ANP and BNP levels, atrial dilatation and prolongation of the QRSp duration as well as increased cardiomyocyte area. Immunohistochemical analyses showed no alterations in fibrosis or angiogenesis in VEGFR-1 TK-/- mice. Mechanistically, the ablation of VEGFR-1 signaling led to significantly upregulated mTOR and downregulated PKCα phosphorylation in the myocardium. Our results show that VEGFR-1 signaling regulates the early cardiac remodelling during the compensatory phase of pressure overload and increases the risk of sudden death.

Recurrent hospitalizations are associated with increased mortality across the ejection fraction range in heart failure.

AIMS: The proportion of patients hospitalized for heart failure (HF) with preserved left ventricular ejection fraction (LVEF) is rising, but no approved treatment exists, in part owing to incomplete characterization of this particular HF phenotype. In order to better define the characteristics of HF phenotypes in Finland, a large cohort with 12 years' follow-up time was analysed. METHODS AND RESULTS: Patients diagnosed between 2005 and 2017 at the Hospital District of Southwest Finland were stratified according to LVEF measure and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels. For this retrospective registry study, previously diagnosed HF patients were defined as follows: patients with reduced ejection fraction (HFrEF; LVEF ≤ 40%; n = 4042), mid-range ejection fraction (HFmrEF; LVEF > 40-50% and NT-proBNP ≥ 125 pg/mL; n = 1468), and preserved ejection fraction (HFpEF; LVEF > 50% and NT-proBNP ≥ 125 pg/mL; n = 3122) and followed up for 15 022, 4962, and 10 097 patient-years, respectively. Cardiovascular (CV) hospitalization and mortality, influence of pre-selected covariates on hospitalization and mortality, and the proportion of HFpEF and HFmrEF patients with a drop in LVEF to HFrEF phenotype were analysed. All data were extracted from the electronic patient register. HFrEF patients were rehospitalized slightly earlier than HFpEF/HFmrEF patients, but the second, third, and fourth rehospitalization rates did not differ between the subgroups. Female gender and better kidney function were associated with reduced rehospitalizations in HFmrEF and HFrEF, with a non-significant trend in HFpEF. Each additional hospitalization was associated with a two-fold increased risk of death and 2.2- to 2.3-fold increased risk of CV death. All-cause mortality was higher in patients with HFpEF. Although CV mortality was less frequent in HFpEF patients, it was associated with increased NT-proBNP concentrations at index in all patient groups. During the 10 years following the index date, 26% of HFmrEF patients and 10% of HFpEF patients progressed to an HFrEF phenotype. CONCLUSIONS: These findings suggest that disease progression, in terms of increased frequency of hospitalizations, and the relationship between increased number of hospitalizations and mortality are similar by LVEF phenotypes. These data highlight the importance of effective treatments that can reduce hospitalizations and suggest a role for monitoring NT-proBNP levels in the management of HFpEF patients in particular.

Effects of dipeptidyl peptidase 4 inhibition on inflammation in atherosclerosis: A 18F-fluorodeoxyglucose study of a mouse model of atherosclerosis and type 2 diabetes.

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.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing 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.

Real-world clinical diagnostics of heart failure patients with reduced or preserved ejection fraction.

AIMS: The study aimed at investigating the use of guideline-recommended diagnostic tools and medication in patients with heart failure (HF) in specialty care in Southwest Finland. We also compared the characteristics of the diagnosed and undiagnosed patients as well as laboratory tests, procedures, and treatments in everyday clinical practice. METHODS AND RESULTS: Patients diagnosed with HF, cardiomyopathy, or hypertension-induced heart disease (n = 20 878, primary cohort) or not diagnosed with HF but having a record of elevated N-terminal pro-brain natriuretic peptide (NT-proBNP) (>125 ng/L, n = 24 321, secondary cohort) were included in the study from the specialty care patient register of the Hospital District of Southwest Finland during the years 2005-2017. Among patients with an International Classification of Diseases, Tenth Revision (ICD-10) code for HF, only 50% had ejection fraction (EF) data to be found by data mining from the electronic health records. Of these patients, 39% (n = 4042) had EF ≤ 40% [HF with reduced EF (HFrEF)] and 61% (n = 6347) had EF > 40%. Elevated NT-proBNP together with EF > 40% narrowed down the number to 4590 patients, a population defined as HF with preserved EF (HFpEF) patients. HFpEF patients were further stratified into HF with mildly reduced EF (HFmrEF; EF 41-50%, n = 1468) and EF > 50% patients (n = 3122) to compare clinical characteristics. NT-proBNP was higher within the HFrEF patients vs. HFpEF {4580 [inter-quartile range (IQR): 2065-9765] vs. 2900 [2065-9765] ng/L, P < 0.001}. Baseline co-morbidities differed between HFpEF and HFrEF groups. Further, HFpEF patients had more procedures and lab tests taken prior to diagnosis than had HFrEF patients. HFmrEF patients were found to resemble more HFrEF than EF > 50% patients. In 70% (n = 17 156) of patients in the secondary cohort, the NT-proBNP concentrations were >300 ng/L, median was 1090 (IQR 551-2558) ng/L and EF 58.4 ± 12.1% (n with EF available = 6845). Reduced EF was present in 6.8% of patients lacking HF diagnosis. CONCLUSIONS: Half of the patients with ICD-10 code for HF did not have EF data available after a visit at specialty care. In particular, the diagnosis of HFpEF seems challenging, reflected as an increase in procedures and laboratory test preceding diagnosis compared with those in HFrEF patients. Also, a large proportion of patients did not have HF diagnosis, yet they presented elevated NT-proBNP concentrations and clinical characteristics resembling those of HFpEF patients.

Heart failure in Finland: clinical characteristics, mortality, and healthcare resource use.

AIMS: The aims of this study were to describe patient characteristics of the adult chronic heart failure (HF) population and to estimate the prevalence, incidence, healthcare resource utilization (HCRU), and mortality associated with HF in Southwest Finland. METHODS AND RESULTS: This was a retrospective biobank and clinical registry study. Adult patients with an HF diagnosis (International Statistical Classification of Diseases and Related Health Problems (ICD) code I50) during 2004-2013 in secondary care were included in the study and compared with age-matched and gender-matched control patients without an I50 diagnosis. HF patients were stratified in groups by left ventricular ejection fraction (LVEF) as follows: LVEF < 40% [HF with reduced ejection fraction (HFrEF)]; LVEF ≥ 40% [HF with preserved ejection fraction (HFpEF)]; or unknown (LVEF unknown). HCRU was stratified by inpatient, outpatient, and emergency room visits. In 2013, the incidence of HF was 3.2/1000, and the prevalence was 13.9/1000 inhabitants (n = 15 594). In the stratified analysis of HF patients (n = 8833, average ± SD age 77.1 ± 11.2), 1115 (12.6%) patients had HFrEF (female 31.3%), 1449 (16.4%) had HFpEF (female 50.9%), and 6269 (71%) had unknown LVEF (female 52.1%). The most common co-morbidities were essential hypertension (58%), chronic elevated serum creatinine (57.3%), atrial fibrillation and flutter (55.1%), and chronic ischaemic heart disease (46.4%). Patients with HF diagnosis had higher HCRU compared with that of age-matched and gender-matched controls (3.7 more days per year at the hospital for HF patients compared with the controls). The total 5 year mortality was 62.6% for HF patients and 28.3% for controls, with higher age being the strongest predictor of mortality. Moreover, multivariable Cox regression analysis showed that patients with HFrEF had a 13% (95% confidence interval 2.7-25%) increased risk of mortality compared with HFpEF patients. CONCLUSIONS: The high mortality rate and HCRU among the studied HF patients highlight the severity of the disease and the economic and social burden on both patients and society. This calls for improved methods of care for this large patient population.

Therapeutic effects of rosuvastatin in hypercholesterolemic prediabetic mice in the absence of low density lipoprotein receptor.

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.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

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.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60-70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.

Left ventricular remodeling leads to heart failure in mice with cardiac-specific overexpression of VEGF-B167: echocardiography and magnetic resonance imaging study.

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.

Peroxisome proliferator-activated receptor-γ coactivator 1 α1 induces a cardiac excitation-contraction coupling phenotype without metabolic remodelling.

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.

Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart.

Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRß) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRß agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses.

Expression of vascular endothelial growth factor (VEGF)-B and its receptor (VEGFR1) in murine heart, lung and kidney.

Metabolic diseases, such as obesity and diabetes, are a serious burden for the health system. Vascular endothelial growth factor (VEGF)-B has been shown to regulate tissue uptake and accumulation of fatty acids and is thus involved in these metabolic diseases. However, the cell-type-specific expression pattern of Vegfb and its receptor (VEGFR1, gene Flt1) remains unclear. We explore the expression of Vegfb and Flt1 in the murine heart, lung and kidney by utilizing ß-galactosidase knock-in mouse models and combining the analysis of reporter gene expression and immunofluorescence microscopy. Furthermore, Flt1 heterozygous mice were analyzed with regard to muscular fatty acid accumulation and peripheral insulin sensitivity. Throughout the heart, Vegfb expression was found in cardiomyocytes with a postnatal ventricular shift corresponding to known changes in energy requirements. Vegfb expression was also found in the pulmonary myocardium of the lung and in renal epithelial cells of the thick ascending limb of Henle's loop, the connecting tubule and the collecting duct. In all analyzed organs, VEGFR1 expression was restricted to endothelial cells. We also show that reduced expression of VEGFR1 resulted in decreased cardiac fatty acid accumulation and increased peripheral insulin sensitivity, possibly as a result of attenuated VEGF-B/VEGFR1 signaling. Our data therefore support a tightly controlled, paracrine signaling mechanism of VEGF-B to VEGFR1. The identified cell-specific expression pattern of Vegfb and Flt1 might form the basis for the development of cell-type-targeted research models and contributes to the understanding of the physiological and pathological role of VEGF-B/VEGFR1 signaling.

Mouse ECG findings in aging, with conduction system affecting drugs and in cardiac pathologies: Development and validation of ECG analysis algorithm in mice.

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.

Prospect and progress of gene therapy in treating atherosclerosis.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation.

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.

HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery.

BACKGROUND: Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors (HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. METHODS: Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and (31)P-magnetic resonance spectroscopy ((31)P-MRS), respectively. RESULTS: HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, AdHIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. CONCLUSIONS: We conclude that both AdHIF-1α and AdHIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy.

Hypoxia-inducible factor 1-induced G protein-coupled receptor 35 expression is an early marker of progressive cardiac remodelling.

AIMS: G protein-coupled receptor 35 (GPR35) has been characterized to be one of the genes that are up-regulated in human heart failure. Since mechanisms controlling GPR35 expression are not known, we investigated the regulation of GPR35 gene and protein expression in cardiac myocytes and in the mouse models of cardiac failure. METHODS AND RESULTS: In cardiac myocytes, GPR35 gene expression was found to be exceptionally sensitive to hypoxia and induced by hypoxia-inducible factor-1 (HIF-1) activation. HIF-1-dependent regulation was established by genetic (HIF-1/VP16, Inhibitory Per/Arnt/Sim domain protein) and chemical [desferrioxamine (DFO)] modulation of the HIF-1 pathway and further confirmed by mutation analysis of the GPR35 promoter and by demonstrating direct binding of endogenous HIF-1 to the gene promoter. Hypoxia increased the number and density of GPR35 receptors on the cardiomyocyte cell membranes. Chemical GPR35 agonist Zaprinast caused GPR35 activation and receptor internalization in cardiac myocytes. In addition, overexpressed GPR35 disrupted actin cytoskeleton arrangement and caused morphological changes in cultured cardiomyocytes. GPR35 gene and protein expressions were also induced in mouse models of cardiac failure; the acute phase of myocardial infarction and during the compensatory and decompensatory phase of pressure-load induced cardiac hypertrophy. CONCLUSIONS: Cardiac expression of GPR35 is regulated by hypoxia through activation of HIF-1. The expression of GPR35 in mouse models of cardiac infarction and pressure load suggests that GPR35 could be used as an early marker of progressive cardiac failure.