Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

3D vascularised proximal tubules-on-a-multiplexed chip model for enhanced cell phenotypes.

Modelling proximal tubule physiology and pharmacology is essential to understand tubular biology and guide drug discovery. To date, multiple models have been developed; however, their relevance to human disease has yet to be evaluated. Here, we report a 3D vascularized proximal tubule-on-a-multiplexed chip (3DvasPT-MC) device composed of co-localized cylindrical conduits lined with confluent epithelium and endothelium, embedded within a permeable matrix, and independently addressed by a closed-loop perfusion system. Each multiplexed chip contains six 3DvasPT models. We performed RNA-seq and compared the transcriptomic profile of proximal tubule epithelial cells (PTECs) and human glomerular endothelial cells (HGECs) seeded in our 3D vasPT-MCs and on 2D transwell controls with and without a gelatin-fibrin coating. Our results reveal that the transcriptional profile of PTECs is highly dependent on both the matrix and flow, while HGECs exhibit greater phenotypic plasticity and are affected by the matrix, PTECs, and flow. PTECs grown on non-coated Transwells display an enrichment of inflammatory markers, including TNF-a, IL-6, and CXCL6, resembling damaged tubules. However, this inflammatory response is not observed for 3D proximal tubules, which exhibit expression of kidney signature genes, including drug and solute transporters, akin to native tubular tissue. Likewise, the transcriptome of HGEC vessels resembled that of sc-RNAseq from glomerular endothelium when seeded on this matrix and subjected to flow. Our 3D vascularized tubule on chip model has utility for both renal physiology and pharmacology.

The tyrosine kinase inhibitor nilotinib targets the discoidin domain receptor DDR2 in calcific aortic valve stenosis.

BACKGROUND AND PURPOSE: Tyrosine kinase inhibitors (TKI) used to treat chronic myeloid leukaemia (CML) have been associated with cardiovascular side effects, including reports of calcific aortic valve stenosis. The aim of this study was to establish the effects of first and second generation TKIs in aortic valve stenosis and to determine the associated molecular mechanisms. EXPERIMENTAL APPROACH: Hyperlipidemic APOE*3Leiden.CETP transgenic mice were treated with nilotinib, imatinib or vehicle. Human valvular interstitial cells (VICs) were isolated and studied in vitro. Gene expression analysis was perfromed in aortic valves from 64 patients undergoing aortic valve replacement surgery. KEY RESULTS: Nilotinib increased murine aortic valve thickness. Nilotinib, but not imatinib, promoted calcification and osteogenic activation and decreased autophagy in human VICs. Differential tyrosine kinase expression was detected between healthy and calcified valve tissue. Transcriptomic target identification revealed that the discoidin domain receptor DDR2, which is preferentially inhibited by nilotinib, was predominantly expressed in human aortic valves but markedly downregulated in calcified valve tissue. Nilotinib and selective DDR2 targeting in VICs induced a similar osteogenic activation, which was blunted by increasing the DDR2 ligand, collagen. CONCLUSIONS AND IMPLICATIONS: These findings suggest that inhibition of DDR2 by nilotinib promoted aortic valve thickening and VIC calcification, with possible translational implications for cardiovascular surveillance and possible personalized medicine in CML patients.

The resolvin D1 receptor GPR32 transduces inflammation resolution and atheroprotection.

Chronic inflammation is a hallmark of atherosclerosis and results from an imbalance between proinflammatory and proresolving signaling. The human GPR32 receptor, together with the ALX/FPR2 receptor, transduces biological actions of several proresolving mediators that stimulate resolution of inflammation. However, since no murine homologs of the human GPR32 receptor exist, comprehensive in vivo studies are lacking. Using human atherosclerotic lesions from carotid endarterectomies and creating a transgenic mouse model expressing human GPR32 on a Fpr2×ApoE double-KO background (hGPR32myc×Fpr2-/-×Apoe-/-), we investigated the role of GPR32 in atherosclerosis and self-limiting acute inflammation. GPR32 mRNA was reduced in human atherosclerotic lesions and correlated with the immune cell markers ARG1, NOS2, and FOXP3. Atherosclerotic lesions, necrotic core, and aortic inflammation were reduced in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice as compared with Fpr2-/-×Apoe-/- nontransgenic littermates. In a zymosan-induced peritonitis model, the hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice had reduced inflammation at 4 hours and enhanced proresolving macrophage responses at 24 hours compared with nontransgenic littermates. The GPR32 agonist aspirin-triggered resolvin D1 (AT-RvD1) regulated leukocyte responses, including enhancing macrophage phagocytosis and intracellular signaling in hGPR32mycTg×Fpr2-/-×Apoe-/- transgenic mice, but not in Fpr2-/-×Apoe-/- nontransgenic littermates. Together, these results provide evidence that GPR32 regulates resolution of inflammation and is atheroprotective in vivo.

TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is a common age-related disease characterized by active calcification of the leaflets of the aortic valve. How innate immune cells are involved in disease pathogenesis is not clear. In this study we investigate the role of the pattern recognition receptor Toll-like receptor 7 (TLR7) in CAVS, especially in relation to macrophage subtype. Human aortic valves were used for mRNA expression analysis, immunofluorescence staining, or ex vivo tissue assays. Response to TLR7 agonist in primary macrophages and valvular interstitial cells (VICs) were investigated in vitro. In the aortic valve, TLR7 correlated with M2 macrophage markers on mRNA levels. Expression was higher in the calcified part compared with the intermediate and healthy parts. TLR7+ cells were co-stained with M2-type macrophage receptors CD163 and CD206. Ex vivo stimulation of valve tissue with the TLR7 ligand imiquimod significantly increased secretion of IL-10, TNF-α, and GM-CSF. Primary macrophages responded to imiquimod with increased secretion of IL-10 while isolated VICs did not respond. In summary, in human aortic valves TLR7 expression is associated with M2 macrophages markers. Ex vivo tissue challenge with TLR7 ligand led to secretion of immunomodulatory cytokine IL-10. These results connect TLR7 activation in CAVS to reduced inflammation and improved clearance.

FADS1 (Fatty Acid Desaturase 1) Genotype Associates With Aortic Valve FADS mRNA Expression, Fatty Acid Content and Calcification.

BACKGROUND: Aortic stenosis (AS) contributes to cardiovascular mortality and morbidity but disease mechanisms remain largely unknown. Recent evidence associates a single nucleotide polymorphism rs174547 within the FADS1 gene, encoding FADS1 (fatty acid desaturase 1), with risk of several cardiovascular outcomes, including AS. FADS1 encodes a rate-limiting enzyme for ω-3 and ω-6 fatty acid metabolism. The aim of this study was to decipher the local transcriptomic and lipidomic consequences of rs174547 in tricuspid aortic valves from patients with AS. METHODS: Expression quantitative trait loci study was performed using data from Illumina Human610-Quad BeadChip, Infinium Global Screening Arrays, and Affymetrix Human Transcriptome 2.0 arrays in calcified and noncalcified aortic valve tissue from 58 patients with AS (mean age, 74.2; SD, 5.9). Fatty acid content was assessed in aortic valves from 25 patients with AS using gas chromatography. Δ5 and Δ6 desaturase activity was assessed by the product-to-precursor ratio. RESULTS: The minor C-allele of rs174547, corresponding to the protective genotype for AS, was associated with higher FADS2 mRNA levels in calcified valve tissue, whereas FADS1 mRNA and other transcripts in proximity of the single nucleotide polymorphism were unaltered. In contrast, the FADS1 Δ5-desaturase activity and the FADS2 Δ6-desaturase activity were decreased. Finally, docosahexaenoic acid was decreased in calcified tissue compared with non-calcified tissue and C-allele carriers exhibited increased docosahexaenoic acid levels. Overall desaturase activity measured with ω-3 fatty acids was higher in C-allele carriers. CONCLUSIONS: The association between the FADS1 genotype and AS may implicate effects on valvular fatty acids.

Semicarbazide-Sensitive Amine Oxidase Increases in Calcific Aortic Valve Stenosis and Contributes to Valvular Interstitial Cell Calcification.

INTRODUCTION: Calcific aortic valve stenosis (CAVS) is a common disease associated with aging. Oxidative stress participates in the valve calcification process in CAVS. Semicarbazide-sensitive amine oxidase (SSAO), also referred to as vascular adhesion protein 1 (VAP-1), transforms primary amines into aldehydes, generating hydrogen peroxide and ammonia. SSAO is expressed in calcified aortic valves, but its role in valve calcification has remained largely unexplored. The aims of this study were to characterize the expression and the activity of SSAO during aortic valve calcification and to establish the effects of SSAO inhibition on human valvular interstitial cell (VIC) calcification. METHODS: Human aortic valves from n = 80 patients were used for mRNA extraction and expression analysis, Western blot, SSAO activity determination, immunohistochemistry, and the isolation of primary VIC cultures. RESULTS: SSAO mRNA, protein, and activity were increased with increasing calcification within human aortic valves and localized in the vicinity of the calcified zones. The valvular SSAO upregulation was consistent after stratification of the subjects according to cardiovascular and CAVS risk factors associated with increased oxidative stress: body mass index, diabetes, and smoking. SSAO mRNA levels were significantly associated with poly(ADP-ribose) polymerase 1 (PARP1) in calcified tissue. Calcification of VIC was inhibited in the presence of the specific SSAO inhibitor LJP1586. CONCLUSION: The association of SSAO expression and activity with valvular calcification and oxidative stress as well as the decreased VIC calcification by SSAO inhibition points to SSAO as a possible marker and therapeutic target to be further explored in CAVS.

Proteoglycan 4 is Increased in Human Calcified Aortic Valves and Enhances Valvular Interstitial Cell Calcification.

Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.

Upregulated Autophagy in Calcific Aortic Valve Stenosis Confers Protection of Valvular Interstitial Cells.

Autophagy serves as a cell survival mechanism which becomes dysregulated under pathological conditions and aging. Aortic valve thickening and calcification causing left ventricular outflow obstruction is known as calcific aortic valve stenosis (CAVS). CAVS is a chronic and progressive disease which increases in incidence and severity with age. Currently, no medical treatment exists for CAVS, and the role of autophagy in the disease remains largely unexplored. To further understand the role of autophagy in the progression of CAVS, we analyzed expression of key autophagy genes in healthy, thickened, and calcified valve tissue from 55 patients, and compared them with nine patients without significant CAVS, undergoing surgery for aortic regurgitation (AR). This revealed a upregulation in autophagy exclusively in the calcified tissue of CAVS patients. This difference in autophagy between CAVS and AR was explored by LC3 lipidation in valvular interstitial cells (VICs), revealing an upregulation in autophagic flux in CAVS patients. Inhibition of autophagy by bafilomycin-A1 led to a decrease in VIC survival. Finally, treatment of VICs with high phosphate led to an increase in autophagic activity. In conclusion, our data suggests that autophagy is upregulated in the calcified tissue of CAVS, serving as a compensatory and pro-survival mechanism.

ERV1/ChemR23 Signaling Protects Against Atherosclerosis by Modifying Oxidized Low-Density Lipoprotein Uptake and Phagocytosis in Macrophages.

BACKGROUND: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. METHODS: Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. RESULTS: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. CONCLUSIONS: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5.

Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated ß-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis.

Iron alters valvular interstitial cell function and is associated with calcification in aortic stenosis.

AIMS: Aortic valve stenosis (AS) is the most common valvulopathy and is characterized by inflammation, extracellular matrix (ECM) remodelling and calcification, causing a narrowing of the valve and the consequential obstruction of the cardiac outflow. Although intraleaflet haemorrhage is associated with AS progression, the mechanisms involved are not known. The aims of this study were to identify valvular iron in relation to pathological changes associated with AS and the effects on valvular interstitial cells (VIC) in terms of iron uptake and iron-induced responses. METHODS AND RESULTS: Valvular iron accumulation was detected by Perls' staining on aortic valve sections and shown to increase with the extent of calcification. Furthermore, qRT-PCR analysis revealed that iron-containing valve regions exhibited increased expression of genes involved in ECM remodelling and calcification. In addition, we demonstrate that iron transporters are regulated by pathways with major impact on AS and that VIC can take up and accumulate iron, which resulted in increased proliferation and decreased elastin production. CONCLUSION: Iron, which may accumulate in the aortic valve by means of intraleaflet haemorrhages, can be taken up by VIC in a pro-inflammatory environment and actively contribute to VIC proliferation, ECM remodelling and calcification. These findings suggest a possible mechanism through which iron uptake by VIC may favour AS progression.

Induction of Angiogenesis by a Type III Phosphodiesterase Inhibitor, Cilostazol, Through Activation of Peroxisome Proliferator-Activated Receptor-γ and cAMP Pathways in Vascular Cells.

OBJECTIVE: Peripheral arterial disease is highly prevalent in the elderly and in the subjects with cardiovascular risk factors such as diabetes. Approximately 2% to 4% of those affected with peripheral arterial disease commonly complain of intermittent claudication. Cilostazol, a type III phosphodiesterase inhibitor, is the only Food and Drug Administration-approved drug for the treatment of intermittent claudication. Cilostazol has been shown to be beneficial for the improvement of pain-free walking distance in patients with intermittent claudication in a series of randomized clinical trials. However, the underlying mechanism how cilostazol improved intermittent claudication symptoms is still unclear. APPROACH AND RESULTS: In this study, the effect of cilostazol on ischemic leg was investigated in mouse ischemic hindlimb model. Administration of cilostazol significantly increased the expression of hepatocyte growth factor (HGF), vascular endothelial growth factor, angiopoietin-1, and peroxisome proliferator-activated receptor-γ in vasculature. The capillary density in ischemic leg was also significantly increased in cilostazol treatment group when compared with control and aspirin treatment group. However, an increase in capillary density and the expression of growth factors was almost completely abolished by coadministration of HGF-neutralizing antibody, suggesting that cilostazol enhanced angiogenesis mainly through HGF. In vitro experiment revealed that cilostazol treatment increased HGF production in vascular smooth muscle cells via 2 major pathways: peroxisome proliferator-activated receptor-γ and cAMP pathways. CONCLUSIONS: Our data suggest that the favorable effects of cilostazol on ischemic leg might be through the angiogenesis through the induction of HGF via peroxisome proliferator-activated receptor-γ and cAMP pathways.

Gene therapy in peripheral artery disease.

INTRODUCTION: Despite the remarkable progress of medicine and endovascular procedures for revascularization, patients with critical limb ischemia (CLI) remain at high risk for amputation and often have a low quality of life due to pain and ulcers in the ischemic leg. Thus, a novel strategy for generating new blood vessels in CLI patients without treatment options is vital. Pre-clinical studies and Phase I clinical trials using VEGF and fibroblast growth factor (FGF) demonstrated promising results; however, more rigorous Phase II and III clinical trials failed to demonstrate benefits for CLI patients. Recently, two multicenter, double-blind, placebo-controlled clinical trials in Japan (Phase III) and the USA (Phase II) showed the benefits of hepatocyte growth factor (HGF) gene therapy for CLI patients. Although the number of patients included in these trials was relatively small, these results imply a distinct beneficial function for HGF over other angiogenic growth factors in a clinical setting. AREAS COVERED: In this review, data from Phase I-III clinical trials of gene therapy for patients with peripheral artery disease (PAD) are examined. In addition, the potential mechanisms behind the success or failure of clinical trials are discussed. EXPERT OPINION: Compared with VEGF and FGF, HGF has a unique molecular effect on inflammation, fibrosis and cell senescence under pathological conditions. These features may explain the clinical benefits of HGF in PAD patients.

MicroRNAs in biliary diseases.

Cholangiopathies are a group of diseases primarily or secondarily affecting bile duct cells, and result in cholangiocyte proliferation, regression, and/or transformation. Their etiopathogenesis may be associated with a broad variety of causes of different nature, which includes genetic, neoplastic, immune-associated, infectious, vascular, and drug-induced alterations, or being idiopathic. miRNAs, small non-coding endogenous RNAs that post-transcriptionally regulate gene expression, have been associated with pathophysiological processes in different organs and cell types, and are postulated as potential targets for diagnosis and therapy. In the current manuscript, knowledge regarding the role of miRNAs in the development and/or progression of cholangiopathies has been reviewed and the most relevant findings in this promising field of hepatology have been highlighted.