Atherosclerosis in Watanabe heritable hyperlipidemic rabbit arteries

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Angiogenesis-dependent and independent phases of intimal hyperplasia.

BACKGROUND: Neointimal vascular smooth muscle cell (VSMC) proliferation is a primary cause of occlusive vascular disease, including atherosclerosis, restenosis after percutaneous interventions, and bypass graft stenosis. Angiogenesis is implicated in the progression of early atheromatous lesions in animal models, but its role in neointimal VSMC proliferation is undefined. Because percutaneous coronary interventions result in induction of periadventitial angiogenesis, we analyzed the role of this process in neointima formation. METHODS AND RESULTS: Local injury to the arterial wall in 2 different animal models induced periadventitial angiogenesis and neointima formation. Application of angiogenesis stimulators vascular endothelial growth factor (VEGF-A165) or a proline/arginine-rich peptide (PR39) to the adventitia of the injured artery induced a marked increase in neointimal thickening beyond that seen with injury alone in both in vivo models. Inhibition of either VEGF (with soluble VEGF receptor 1 [sFlt1]) or fibroblast growth factor (FGF) (with a dominant=negative form of FGF receptor 1 [FGF-R1DN]), respectively, signaling reduced adventitial thickening induced by VEGF and PR39 to the level seen with mechanical arterial injury alone. However, neither inhibitor was effective in preventing neointimal thickening after mechanical injury when administered in the absence of angiogenic growth factor. CONCLUSIONS: Our findings indicate that adventitial angiogenesis stimulates intimal thickening but does not initiate it

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

Vascular endothelial growth factor B (VEGF-B) is a potentmediator 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 knownmodulatory 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.Peer reviewe

Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.

Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells

AAV2 and AAV9 tropism and transgene expression in the mouse eye and major tissues after intravitreal and subretinal delivery

Introduction: The eye is an excellent target for gene therapy because of its anatomical features. Gene therapy to treat ocular disorders relies on efficient gene delivery and transgene expression in the target cells. The aim of this study was to compare the biodistribution and safety of two different AAV serotypes after intravitreal (IVT) and subretinal injections.Methods: AAV2 (1 × 1012 vg/mL) and AAV9 (5 × 1012 vg/mL) vectors expressing an enhanced green fluorescent protein (EGFP) and an AAV9-empty (6 × 1011 vg/mL) vector were injected intravitreally or subretinally into both eyes of adult C57Bl/OlaHsd mice. The biodistribution of the viral vectors in the eye and off-target tissues was studied using qPCR. GFP expression was studied from cryosections, and GFP transduction efficacy was verified using immunohistostaining for GFP. In addition, electroretinography (ERG) was used to assess the effect of vectors on retinal function.Results: In addition to the eyes, viral vector copies were found in distant off-target tissues such as the liver, especially after AAV9-EGFP IVT and subretinal injections. AAV9-EGFP injections showed more GFP expression throughout the retina compared to AAV2-EGFP. AAV2-EGFP IVT showed transgene expression mainly in the ganglion cell layer, whereas subretinal injection showed GFP expression in the retinal pigment epithelium. In addition, GFP was expressed at a moderate level in the liver after both injection routes of AAV9 and in parts of the brain after all injection groups except AAV9-empty. Lowered a- and b-amplitude values were seen in ERG in both scotopic and photopic experiments after AAV9-EGFP subretinal injection compared to all other groups.Discussion: This study shows that intraocular injection of AAV2 and AAV9 transduces retinal cells. Although the more efficient transduction of the retina, negative effect on the retinal function, and off-target transgene expression of AAV9 makes AAV2 a more suitable gene delivery vector to treat ocular disorders

Molecular Imaging to Monitor Left Ventricular Remodeling in Heart Failure

Purpose of Review: Cardiovascular diseases are the leading cause of deaths worldwide. Many complex cellular and molecular pathways lead to myocardial remodeling after ischemic insults. Anatomy, function, and viability of the myocardium can be assessed by modern medical imaging techniques by both visualizing and quantifying damages. Novel imaging techniques aim for a precise and accurate visualization of the myocardium and for the detection of alternations at the molecular level.Recent Findings: Magnetic resonance imaging assesses anatomy, function, and tissue characterization of the myocardium non-invasively with high spatial resolution, sensitivity, and specificity. Using hyperpolarized magnetic resonance imaging, molecular and metabolic conditions can be assessed non-invasively. Single photon-emission tomography and positron-emission tomography are the most sensitive techniques to detect biological processes in the myocardium. Cardiac perfusion, metabolism, and viability are the most common clinical targets. In addition, molecular-targeted imaging of biological processes involved in heart failure, such as myocardial innervation, inflammation, and extracellular matrix remodeling, is feasible.Summary: Novel imaging techniques can provide a precise and accurate visualization of the myocardium and for the detection of alternations at molecular level.</div

Intravitreal Anti-VEGF Drugs and Signals of Dementia and Parkinson-Like Events: Analysis of the VigiBase Database of Spontaneous Reports

Introduction: Since vascular endothelial growth factor (VEGF) regulates several aspects of the central nervous system, particularly in dopaminergic neurons, VEGF inhibitors may be linked to Parkinson-like events and dementia, or variants of these diseases. Two recent case reports have found a potential link between intravitreal anti-VEGF use and Parkinson’s disease (PD) and dementia. Aim: To evaluate disproportionality in a large spontaneous reporting database concerning intravitreal anti-VEGF drugs and PD or dementia, and related conditions. Methods: Using VigiBase, individual case safety reports (ICSRs) attributed to intravitreal ranibizumab, aflibercept, pegaptanib, and bevacizumab were identified from 2010 to 2016. Within Standardised Narrow Medical Dictionary for Regulatory Activities (MedDRA®) Queries (SMQs) for “Parkinson-like events” and “Dementia,” suspected events were identified using preferred terms (PTs). The Proportional Reporting Ratio (PRR) was estimated with the lower 95% confidence intervals (CIs) for all drug-event pairs with ≥3 suspected events. The vigiGrade completeness score was reported for the ICSRs. The analyses were repeated, including only persons aged 65 and over. Results: Out of 18.9 million ICSRs, 7,945 (0.004%) concerned intravitreal anti-VEGF drugs. Of these, 27 (0.34%) were identified concerning the SMQs “Dementia” (N = 17, 62.96%) and “Parkinson-like events” (N = 10, 37.94%) in persons of all ages. Among persons age 65 and over, 4,758 (59.88% of relevant ICSRs) ICSRs were identified for anti-VEGF drugs. When restricting disproportionality analysis to persons aged 65 and over, no disproportionality was seen for any of the drug-event pairs at the level of SMQ. However, on analysing disproportionality by PT, a potential signal emerged for intravitreal ranibizumab and Parkinson’s disease [N = 6 ICSRs; PRR: 3.05 (95% CI: 1.36-6.81)]. In general, the vigiGrade completeness score was low for all the ICSRs of interest, as no ICSR had a score >0.8. Conclusion: Present findings suggest a potential signal for Parkinson’s disease related to intravitreal ranibizumab. This is supported by several biologically plausible mechanisms but requires confirmation through pharmacoepidemiological studies, especially because of the low number of cases

Human serum albumin nanoparticles loaded with phthalocyanine dyes for potential use in photodynamic therapy of atherosclerotic plaques

Diseases caused by obstruction or rupture of vulnerable plaques in the arterial walls such as cardiovascular infarction or stroke are the leading cause of death in the world. In the present work, we developed human serum albuminnanoparticles loaded by physisorption with zinc phthalocyanine, TT1, mainly used for industrial application as near-infrared photosensitizer and compared these to HSA NPsloaded with the well-known silicone phthalocyanine (Pc4). The use of NIR light allows for better tissue penetration, while the use of nanoparticles permitshigh local concentrations. The particles were characterized and tested for toxicity and stability as well as for their potential use as a contrast agent and NIR photosensitizer for photodynamic therapy in cardiovascular disease. We focused on the distribution of the nanoparticles in RAW264.7macrophage cells and atherosclerotic mice. The nanoparticles had an average size of 120 nm according todynamic light scattering, good loading capacity for zinc phthalocyanine,and satisfying stability in 50% (v/v) fetal bovine serum for 8 hours and in an aqueous environment at 4°C for 4–6 weeks. Under light irradiation we found a high production of singlet oxygen and the products showed no dark toxicity in vitro with macrophages(the target cells in vulnerable plaques),but at a low μg/mL nanoparticleconcentration killed efficiently the macrophagesupon LED illumination. Injection of the contrast agentin atherosclerotic mice led to a visible fluorescence signal of zinc phthalocyaninein the atherosclerotic plaque at 30 minutes and in the lungs with afast clearance of the nanoparticles. Zinc phthalocyanine loaded human serum albumin nanoparticles present an interesting candidate for the visualization and potentially photodynamictreatment of macrophages in atherosclerotic plaquesThe research leading to these results has received funding from FP7-NMP CosmoPHOS-Nano under grant agreement No. 310337. Additional funding was received by the Spanish groups from MINECO (CTQ2017-85393-P) and ERA-NET/MINECO EuroNanoMed2017-191 / PCIN-2017-04