Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Targeted polyelectrolyte complex micelles treat vascular complications in vivo.

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG /Apoe-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.

Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics.

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.

Large-Scale Proteomic Identification of Targets of Cellular miR-197 Downregulated by Enterovirus A71.

MicroRNAs are noncoding RNA species comprising 18-23 nucleotides that regulate host-virus interaction networks. Here, we show that enterovirus A71 infection in human rhabdomyosarcoma (RD) is regulated by miR-197 expression. Transfection of miR-197 mimic into RD cells inhibited virus replication by interfering with the viral RNA synthesis. We employed a combination of mass-spectrometry-based quantitative proteomics with the stable isotope labeling with amino acids in cell culture (SILAC) approach for the identification of the miR-197 target genes in RD cells and to investigate the differential expression of the prospective target proteins. A total of 1822 proteins were repeatedly identified in miR-197-transfected RD cells, 106 of which were predicted to have seed sites by TargetScan. Notably, seven of eight selected genes potentially related to viral replication and immune response were validated as direct miR-197 targets, using a luciferase 3'-untranslated region (UTR) reporter assay. The expression levels of three selected endogenous molecules (ITGAV, ETF1, and MAP2K1/MEK1) were significantly reduced when RD cells were transfected with a miR-197 mimic. Our results provide a comprehensive database of miR-197 targets, which might provide better insights into the understanding of host-virus interaction.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

p-GaN/n-ZnO nanorods: the use of graphene nanosheets composites to increase charge separation in self-powered visible-blind UV photodetectors.

ZnO-based heterojunctions have found applications as self-powered ultraviolet photodetectors (PDs). However, high doping levels are not compatible with high mobility for metallic doped ZnO-based PDs so further development has been inhibited. This study demonstrates a method to increase the open-circuit voltage (V oc) that allows keeping a sufficiently high level of mobility of ZnO, using a ZnO nanorod/GaN heterojunction that incorporates graphene nanosheets as the active layer. These hybrid PDs have triple the value for V oc of PDs that have only pure ZnO and better exhibit photo-response characteristics. The results of surface Kelvin probe microscopy and x-ray photoelectron spectrometer show that the complex defects that occur because Zn interstitials form a shallow donor in ZnO are mainly responsible for the increase in the value of V oc. Using this functional nanostructure as an active layer represents a new method for the manufacture of high-performance self-powered PDs.

Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling.

RATIONALE: Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome-wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS-associated GWAS genes remains poorly understood. OBJECTIVES: To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2-mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury. METHODS: Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor-α, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS-associated GWAS genes, and lung pathophysiology. MEASUREMENTS AND MAIN RESULTS: KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras-related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS-induced lung injury in mice. CONCLUSIONS: Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein.

UNLABELLED: Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. IMPORTANCE: Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.

Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces.

RATIONALE: PhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis. OBJECTIVE: To establish the critical role of PPAP2B in endothelial responses to hemodynamics. METHODS AND RESULTS: Reduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells. CONCLUSIONS: Atherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Mechanosensitive super-enhancers regulate genes linked to atherosclerosis in endothelial cells.

Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms.

Targeting mechanosensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo.

Although atherosclerosis preferentially develops at arterial curvatures and bifurcations where disturbed flow (DF) activates endothelium, therapies targeting flow-dependent mechanosensing pathways in the vasculature are unavailable. Here, we provided experimental evidence demonstrating a previously unidentified causal role of DF-induced endothelial TXNDC5 (thioredoxin domain containing 5) in atherosclerosis. TXNDC5 was increased in human and mouse atherosclerotic lesions and induced in endothelium subjected to DF. Endothelium-specific Txndc5 deletion markedly reduced atherosclerosis in ApoE-/- mice. Mechanistically, DF-induced TXNDC5 increases proteasome-mediated degradation of heat shock factor 1, leading to reduced heat shock protein 90 and accelerated eNOS (endothelial nitric oxide synthase) protein degradation. Moreover, nanoparticles formulated to deliver Txndc5-targeting CRISPR-Cas9 plasmids driven by an endothelium-specific promoter (CDH5) significantly increase eNOS protein and reduce atherosclerosis in ApoE-/- mice. These results delineate a new molecular paradigm that DF-induced endothelial TXNDC5 promotes atherosclerosis and establish a proof of concept of targeting endothelial mechanosensitive pathways in vivo against atherosclerosis.

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells.

Single-cell motility is spatially heterogeneous and driven by metabolic energy. Directly linking cell motility to cell metabolism is technically challenging but biologically important. Here, we use single-cell metabolic imaging to measure glycolysis in individual endothelial cells with genetically encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. We show that cellular glycolysis fuels endothelial activation, migration and contraction and that sites of high lactate production colocalize with active cytoskeletal remodelling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Our data indicate that Rho-GTPase signalling coordinates energy metabolism with cytoskeleton remodelling to regulate endothelial cell motility.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

Development and comparison of forecast models of hand-foot-mouth disease with meteorological factors.

Hand-foot-mouth disease (HFMD) is an acute intestinal virus infectious disease which is one of major public health problems in mainland China. Previous studies indicated that HFMD was significantly influenced by climatic factors, but the associated factors were different in different areas and few study on HFMD forecast models was conducted. Here, we analyzed epidemiological characteristics of HFMD in Yiwu City, Zhejiang Province and constructed three forecast models. Overall, a total of 32554 HFMD cases were reported and 12 cases deceased in Yiwu City, Zhejiang Province. The incidence of HFMD peaked every other year and the curve of HFMD incidence had an approximately W-shape. The majority of HFMD cases were children and 95.76% cases aged ≤5 years old from 2008 to 2016. Furthermore, we constructed and compared three forecast models using autoregressive integrated moving average (ARIMA) model, negative binomial regression model (NBM), and quasi-Poisson generalized additive model (GAM). All the three models had high agreements between predicted values and observed values, while GAM fitted best. The exposure-response curve of monthly mean temperature and HFMD was approximately V-shaped. Our study explored epidemiological characteristics of HFMD in Yiwu City and provided accurate methods for early warning which would be great importance for the control and prevention of HFMD.

HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium.

Hemodynamic forces regulate vascular functions. Disturbed flow (DF) occurs in arterial bifurcations and curvatures, activates endothelial cells (ECs), and results in vascular inflammation and ultimately atherosclerosis. However, how DF alters EC metabolism, and whether resulting metabolic changes induce EC activation, is unknown. Using transcriptomics and bioenergetic analysis, we discovered that DF induces glycolysis and reduces mitochondrial respiratory capacity in human aortic ECs. DF-induced metabolic reprogramming required hypoxia inducible factor-1α (HIF-1α), downstream of NAD(P)H oxidase-4 (NOX4)-derived reactive oxygen species (ROS). HIF-1α increased glycolytic enzymes and pyruvate dehydrogenase kinase-1 (PDK-1), which reduces mitochondrial respiratory capacity. Swine aortic arch endothelia exhibited elevated ROS, NOX4, HIF-1α, and glycolytic enzyme and PDK1 expression, suggesting that DF leads to metabolic reprogramming in vivo. Inhibition of glycolysis reduced inflammation suggesting a causal relationship between flow-induced metabolic changes and EC activation. These findings highlight a previously uncharacterized role for flow-induced metabolic reprogramming and inflammation in ECs.

Inhibition of atherosclerosis-promoting microRNAs via targeted polyelectrolyte complex micelles.

Polyelectrolyte complex micelles have great potential as gene delivery vehicles because of their ability to encapsulate charged nucleic acids forming a core by neutralizing their charge, while simultaneously protecting the nucleic acids from non-specific interactions and enzymatic degradation. Furthermore, to enhance specificity and transfection efficiency, polyelectrolyte complex micelles can be modified to include targeting capabilities. Here, we describe the design of targeted polyelectrolyte complex micelles containing inhibitors against dys-regulated microRNAs (miRNAs) that promote atherosclerosis, a leading cause of human mortality and morbidity. Inhibition of dys-regulated miRNAs in diseased cells associated with atherosclerosis has resulted in therapeutic efficacy in animal models and has been proposed to treat human diseases. However, the non-specific targeting of microRNA inhibitors via systemic delivery has remained an issue that may cause unwanted side effects. For this reason, we incorporated two different peptide sequences to our miRNA inhibitor containing polyelectrolyte complex micelles. One of the peptides (Arginine-Glutamic Acid-Lysine-Alanine or REKA) was used in another micellar system that demonstrated lesion-specific targeting in a mouse model of atherosclerosis. The other peptide (Valine-Histidine-Proline-Lysine-Glutamine-Histidine-Arginine or VHPKQHR) was identified via phage display and targets vascular endothelial cells through the vascular cell adhesion molecule-1 (VCAM-1). In this study we have tested the in vitro efficacy and efficiency of lesion- and cell-specific delivery of microRNA inhibitors to the cells associated with atherosclerotic lesions via peptide-targeted polyelectrolyte complex micelles. Our results show that REKA-containing micelles (fibrin-targeting) and VHPKQHR-containing micelles (VCAM-1 targeting) can be used to carry and deliver microRNA inhibitors into macrophages and human endothelial cells, respectively. Additionally, the functionality of miRNA inhibitors in cells was demonstrated by analyzing miRNA expression as well as the expression or the biological function of its downstream target protein. Our study provides the first demonstration of targeting dys-regulated miRNAs in atherosclerosis using targeted polyelectrolyte complex micelles and holds promising potential for translational applications.

Proteolytic processing of porcine deoxyribonuclease II occurs in lysosomes but is not required for enzyme activation.

DNase II purified from porcine spleen (pDNase II) comprises alpha(1), alpha(2) and beta subunits. The three subunits are encoded by one cDNA, in the sequence alpha(1), beta, and alpha(2), and the peptides linking these subunits are presumably cleaved out post-translationally. To understand the relevance of post-translational cleavage to pDNase II, recombinant pDNase II (rpDNase II) was produced in human 293T cells by transfection with an expression plasmid containing pDNase II cDNA (pcDNaseII). An 11.5, a 35 and a 46.5 kDa protein were detected in the cell lysates, whereas only a 46.5 kDa protein was detected in the culture medium of the pcDNaseII-transfected cells. The 46.5 kDa rpDNase II secreted into the medium was purified to homogeneity and characterized. MALDI-TOF MS and N-terminal amino acid sequencing of the 46.5 kDa protein revealed a single contiguous polypeptide chain of pDNase II. Zymographic analysis showed that the 46.5 kDa protein digested DNA in acidic conditions and that the specific activity of this rpDNase II was about twice that of pDNase II purified from porcine spleen. Treatment with chloroquine, a lysosomal inhibitor, resulted in the accumulation of only the 46.5 kDa protein in the pcDNaseII-transfected cells. Treatments with cycloheximide 22 h after transfection led to accumulation of the processed enzyme and disappearance of the 46.5 kDa protein. These results suggest that the proteolytic processing of rpDNase II occurs in the lysosome, which is not involved in the activation of pDNase II.