Acetylated LDL Research Articles

Single-cell transcriptomics analysis reveals Cilp+Cd200+ fibroblasts promote mesenchymal endothelial transition (MEndoT) in cardiac injury

Abstract Background Cardiac fibroblasts are considered to be highly heterogeneous mesenchymal cells after a cardiac injury. Understanding the heterogeneity of cardiac fibroblasts is of fundamental importance for fibroblast-targeted strategies to treat patients with heart disease. Advances in the single-cell RNA-sequencing (scRNA-Seq) technology have provided a possibility to elaborate the heterogeneity of cells in more depth. However, currently, most studies on scRNA-Seq of cardiac fibroblasts are performed using non-myocyte cells isolated at a certain point in time, which include a very complex, interdependent community of interstitial cells. scRNA-Seq based on lineage-tracing of cardiac fibroblasts is essential for a deeper understanding of cellular heterogeneity of specific cell type populations, which can reveal how a pre-existing cardiac fibroblast population responds to cardiac injury. Our previous studies demonstrated that a subset of cardiac fibroblasts can adopt the endothelial cell phenotype by undergoing mesenchymal–endothelial transition (MEndoT) and contributes to cardiac repair by revascularizing the damaged myocardium. Purpose To decipher which subgroup of the preexisting cardiac fibroblasts involved in MEndoT respond to cardiac injury. Methods and Results For lineage-tracing-based studies, Col1a2 Cre ERT: R26R td-Tomato transgenic mice were generated for reliable cardiac fibroblast tracking. 10X scRNA-Seq was performed to analyze the diversity of prelabeled cardiac fibroblasts after sham or transverse aortic constriction injury. Using the Seurat R package, the td-Tomato labeled cells were further clustered into 11 distinct populations. In addition, the cell trajectory assay shows both Cilp and Cd200 positive (Cilp+/Cd200+) cluster located in the early state and showing the phenotype of mesenchymal stem cells, indicating that this clusters have high diversity with regard to differentiation and directly contribute to angiogenesis. Besides, we isolated Cilp+/Cd200+ cardiac fibroblasts from wild-type mice by flow sorting and performed matrigel tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake to measure the potential of MEndoT. Interestingly, compared with Cilp-/Cd200- cardiac fibroblasts, both matrigel tube formation and Ac-LDL uptake of the Cilp+/Cd200+ group were significantly increased (∼1.3 fold higher in matrigel tube formation, P<0.05;∼3.3 fold higher in Ac-LDL uptake, P<0.05). Conclusion Our findings indicated that Cilp+/Cd200+ is the main group of cardiac fibroblasts involved in MEndoT and plays a major role in the organization of the extracellular matrix and cell differentiation. This study provided new therapeutic strategies for cardiac hypertrophy by accurately regulating the cardiac fibroblasts.

Long-term glucocorticoid treatment increases CD204 expression by activating the MAPK pathway and enhances modified LDL uptake in murine macrophages

Atherosclerotic plaques develop from the accumulation of macrophage-derived foam cells via the uptake of modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Although glucocorticoids are suspected to exacerbate atherosclerosis, the precise mechanisms have not been fully elucidated. We investigated the effects of long-term treatment (2 weeks) with both a natural glucocorticoid (hydrocortisone, HC, 1 μM) and a synthetic glucocorticoid (dexamethasone, Dex, 100 nM) on murine bone marrow-derived macrophages using flow cytometry and western blotting. Treatment with HC and Dex enhanced CD204 expression but not CD36 expression and acetylated LDL (Ac-LDL) uptake. Treatment with HC and Dex also induced the phosphorylation of extracellular signal-regulated kinase (ERK). The Dex-induced enhancement in CD204 expression and Ac-LDL uptake were suppressed by an inhibitor of the mitogen-activated protein kinase (MAPK)/ERK kinase. These results suggest that glucocorticoids activate the MAPK/ERK pathway, which enhances CD204 expression and results in increased uptake of Ac-LDL in macrophages. The MAPK/ERK pathway in macrophages might be a key target to prevent atherosclerosis that is worsened by glucocorticoids.

Efficient generation of endothelial cells from induced pluripotent stem cells derived from a patient with peripheral arterial disease.

Peripheral arterial disease (PAD) is caused by atherosclerotic plaque accumulation, which results in ischemia in lower extremity ischemia. Cell-based therapy using endothelial progenitor cells (EPCs) or endothelial cells (ECs) has been challenging due to an insufficient number and replicative senescence of primary cells isolated from patients. To overcome this limitation, we generated induced pluripotent stem cells (iPSCs) from a patient with PAD for the first time. The patient-specific iPSCs have unlimited proliferation and can be used to generate a clinically relevant number of functional ECs. Here we developed a strategy to efficiently generate high EC yields within 5days of differentiation. The generated iPSC-derived ECs from a PAD patient were phenotypically and functionally similar to the primary blood outgrowth endothelial cells (BOECs) and iPSC-ECs derived from healthy donors as evidenced by expression of EC-specific markers, capillary-like tube-forming potential, and the ability to uptake acetylated low-density lipoprotein (Ac-LDL). Our approach may provide an alternative renewable source of large-scale ECs for regenerative therapy. This study represents the first step toward the development of an autologous cell-based strategy for the treatment of PAD in the future.

Mechanism of isosorbide dinitrate combined with exercise training rehabilitation to mobilize endothelial progenitor cells in patients with coronary heart disease

ABSTRACT it was to explore effect of isosorbide dinitrate combined with exercise training and rehabilitation on endothelial progenitor cells (EPCs) in coronary heart disease. EPCs were isolated and cultured from peripheral blood of coronary heart disease patients, and morphology and surface markers were detected. Then, 116 patients were rolled into treatment group (isosorbide dinitrate + exercise rehabilitation training) and control group (isosorbide dinitrate). Characteristics of EPCs cells after treatment were compared. The mononuclear cells were round and small in size and were not evenly distributed in the culture plate. EPCs cells grew as colonies after 8d-culture, and the surrounding cells grew outward in a germinating manner with colonies as the center, forming multiple cell populations. Positive rates of EPCs surface markers CD133, CD34, and vascular endothelial growth factor receptor (KDR) were 11.25 ± 3.07%, 48.18 ± 9.13%, and 76.36 ± 8.27%, respectively. Proliferation activity of EPCs in the treatment group was dramatically higher versus controls at day three, five, and seven (P < 0.05). Adhesion ability of EPCs in treatment group was dramatically higher than controls at day three, five, and seven (P < 0.05). Migration ability of EPCs in treatment group was dramatically higher versus control group at day three, five, and seven (P < 0.05). In short, isosorbide dinitrate plus exercise rehabilitation greatly enhanced the proliferation activity, adhesion ability, and migration ability of EPCs cells, which also played a beneficial role in the repair of endothelial injury, with notable effects. Abbreviation listAbbreviation: Full name; EPCs: endothelial progenitor cells; RPMI: roswell Park Memorial Institute;VEGF: vascular endothelial growth factor; BFGF: basic fibroblast growth factor; DiL-acLDL: DiL-labeled acetyl low density lipoprotein; PBS: phosphate buffer; FITC-UEA-I: FITC-labeled Vitex agglutinin I; EDTA: ethylenediamine tetra-acetic acid; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium.

Insights into the ligand binding specificity of SREC-II (scavenger receptor expressed by endothelial cells).

SREC‐II (scavenger receptor expressed by endothelial cells II) is a membrane protein encoded by the SCARF2 gene, with high homology to class F scavenger receptor SR‐F1, but no known scavenging function. We produced the extracellular domain of SREC‐II in a recombinant form and investigated its capacity to interact with common scavenger receptor ligands, including acetylated low‐density lipoprotein (AcLDL) and maleylated or acetylated BSA (MalBSA or AcBSA). Whereas no binding was observed for AcLDL, SREC‐II ectodomain interacted strongly with MalBSA and bound with high affinity to AcBSA, a property shared with the SR‐F1 ectodomain. SREC‐II ectodomain also interacted with two SR‐F1‐specific ligands, complement C1q and calreticulin, with affinities in the 100 nm range. We proceeded to generate a stable CHO cell line overexpressing full‐length SREC‐II; binding of MalBSA to these cells was significantly increased compared with nontransfected CHO cells. In contrast, no increase in binding could be detected for C1q and calreticulin. We show for the first time that SREC‐II has the capacity to interact with the common scavenger receptor ligand MalBSA. In addition, our data highlight similarities and differences in the ligand binding properties of SREC‐II in soluble form and at the cell surface, and show that endogenous protein ligands of the ectodomain of SREC‐II, such as C1q and calreticulin, are shared with the corresponding domain of SR‐F1.

Comparative Evaluation on Impacts of Fibronectin, Heparin-Chitosan, and Albumin Coating of Bacterial Nanocellulose Small-Diameter Vascular Grafts on Endothelialization In Vitro.

In this study, we contrast the impacts of surface coating bacterial nanocellulose small-diameter vascular grafts (BNC-SDVGs) with human albumin, fibronectin, or heparin–chitosan upon endothelialization with human saphenous vein endothelial cells (VEC) or endothelial progenitor cells (EPC) in vitro. In one scenario, coated grafts were cut into 2D circular patches for static colonization of a defined inner surface area; in another scenario, they were mounted on a customized bioreactor and subsequently perfused for cell seeding. We evaluated the colonization by emerging metabolic activity and the preservation of endothelial functionality by water soluble tetrazolium salts (WST-1), acetylated low-density lipoprotein (AcLDL) uptake assays, and immune fluorescence staining. Uncoated BNC scaffolds served as controls. The fibronectin coating significantly promoted adhesion and growth of VECs and EPCs, while albumin only promoted adhesion of VECs, but here, the cells were functionally impaired as indicated by missing AcLDL uptake. The heparin–chitosan coating led to significantly improved adhesion of EPCs, but not VECs. In summary, both fibronectin and heparin–chitosan coatings could beneficially impact the endothelialization of BNC-SDVGs and might therefore represent promising approaches to help improve the longevity and reduce the thrombogenicity of BNC-SDVGs in the future.

A metal-organic-framework incorporated vascular graft for sustained nitric oxide generation and long-term vascular patency

Copper-MOFs (Cu-MOFs) have been reported to demonstrate great potential as cardiovascular biomaterials, due to enhanced catalytic ability of Cu2+ to generate nitric oxide (NO) from endogenous S-nitrosothiols (RSNOs). However, free Cu-MOFs usually show rapid degradation under physiological conditions, resulting in short catalytic half-life and risk of copper ion toxicity. Therefore, how to increase the stability of Cu-MOFs is of great importance in cardiovascular biomaterials research. Herein, we chose M199 MOF as an example and developed Cu-MOF-based scaffold, using the electrospinning method to embed Cu-MOF nanoparticles into polycaprolactone (PCL) fibers. Entrapment of Cu-MOF nanoparticles within PCL could simultaneously enhance Cu-MOF stability in serum and allow for long-term NO catalytic activity, as assessed by in vitro assays and using in situ implantation models. Additionally, the optimized concentration of Cu-MOFs loaded within the scaffolds significantly promoted endothelial cell (EC) migration and increased acetylated low-density lipoprotein (Ac-LDL) uptake. Moreover, Cu-MOF-based scaffolds dramatically inhibited platelet adhesion and activation, which markedly reduced acute thrombosis in arterio-venous shunt models. In situ implantation experiments revealed that the PCL/Cu-MOF scaffolds accelerated the formation of an intact endothelial monolayer. Together, these results suggest that the incorporation of Cu-MOFs into electrospun fibers could serve as a promising approach to achieve stable catalytic performance and long-term activity required for implant materials.

Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.

Aims: It was suggested that group V secretory phospholipase A2 (sPLA2-V) existed in the nucleus. This study examined whether nuclear sPLA2-V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells. Methods: RAW264.7 cells were transfected with shRNA vector targeting sPLA2-V (sPLA2-V-knockdown [KD] cells) or empty vector (sPLA2-V-wild-type [WT] cells). AcLDL endocytosis was assessed by incubation with125I-AcLDL or AcLDL conjugated with pHrodo. Actin polymerization was assessed by flow cytometry using Alexa Fluor 546-phalloidin. Results: In immunofluorescence microscopic studies, sPLA2-V was detected in the nucleus. ChIP-Seq and ChIP-qPCR analyses showed binding of sPLA2-V to the promoter region of thephosphoglycerate kinase 1 (Pgk1) gene. In the promoter assay, sPLA2-V-KD cells had lower promoter activity of thePgk1 gene than sPLA2-V-WT cells, and this decrease could be reversed by transfection with a vector encoding sPLA2-V-H48Q that lacks enzymatic activity. Compared with sPLA2-V-WT cells, sPLA2-V-KD cells had decreased PGK1 protein expression, beclin 1 (Beclin1) phosphorylation at S30, and class III PI3-kinase activity that could also be restored by transfection with sPLA2-V-H48Q. sPLA2-V-KD cells had impaired actin polymerization and endocytosis, which was reversed by introduction of sPLA2-V-H48Q or PGK1 overexpression. In sPLA2-V-WT cells, siRNA-mediated depletion of PGK1 suppressed Beclin1 phosphorylation and impaired actin polymerization and intracellular trafficking of pHrodo-conjugated AcLDL. Conclusions: Nuclear sPLA2-V binds to thePgk1 gene promoter region and increases its transcriptional activity. sPLA2-V regulates AcLDL endocytosis through PGK1-Beclin1 in a manner that is independent of its enzymatic activity in RAW264.7 cells.

An advanced method for quantitative measurements of cholesterol crystallization

BackgroundCholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture – a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AimTo develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. Methods and resultsAn enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. ConclusionsThe in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells

BackgroundHuman induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages.MethodsFormation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system.ResultsOur protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development.ConclusionThis protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Ablation of Galectin-12 Inhibits Atherosclerosis through Enhancement of M2 Macrophage Polarization

The formation of foam cells, which are macrophages that have engulfed oxidized low-density lipoprotein (OxLDL), constitutes the first stage in the development of atherosclerosis. Previously, we found that knocking down galectin-12, a negative regulator of lipolysis, leads to reduced secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that plays an important role in atherosclerosis. This prompted us to study the role of galectin-12 in atherosclerosis. With that aim, we examined foam cell formation in Gal12‒/‒ murine macrophages exposed to OxLDL and acetylated LDL (AcLDL). Then, we generated an LDL receptor and galectin-12 double knockout (DKO) mice and studied the effect of galectin-12 on macrophage function and atherosclerosis. Lastly, we evaluated the role of galectin-12 in human THP-1 macrophages using a doxycycline-inducible conditional knockdown system. Galectin-12 knockout significantly inhibited foam cell formation in murine macrophages through the downregulation of cluster of differentiation 36 (CD36), and the upregulation of ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1), and scavenger receptor class B type 1 (SRB1). Consistent with this, galectin-12 knockdown inhibited foam cell formation in human macrophages. In addition, the ablation of galectin-12 promoted M2 macrophage polarization in human and murine macrophages as evidenced by the upregulation of the M2 marker genes, CD206 and CD163, and downregulation of the M1 cytokines, tumor necrosis factor α (TNF- α), interleukin-6 (IL-6), and MCP-1. Moreover, the ablation of galectin-12 decreased atherosclerosis formation in DKO mice. Based on these results, we propose galectin-12 as a potential therapeutic target for atherosclerosis.

Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells.

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs via its collagen-like region. We identified two different binding regions on SR-F1: the N-terminal moiety interacts with C1q and CRT whereas the C-terminal moiety binds AcLDL. The role of SR-F1 N-linked glycans was also tested by mutating each of the three glycosylated asparagines. The three mutants retained binding activities for both AcLDL and C1q. A stable THP-1 cell line overexpressing SR-F1 was generated and C1q was shown to bind more strongly to the surface of SR-F1 overexpressing macrophages, with C1q/SR-F1 colocalization observed in some membrane areas. We also observed a higher level of CRT internalization for THP-1 SR-F1 cells. Increasing SR-F1 negatively modulated the uptake of apoptotic cells. Indeed, THP-1 cells overexpressing SR-F1 displayed a lower phagocytic capacity as compared with mock-transfected cells, which could be partially restored by addition of C1q in the extracellular milieu. Our data shed some light on the role of SR-F1 in efferocytosis, through its capacity to bind C1q and CRT, two proteins involved in this process.

JAK-STAT-dependent regulation of scavenger receptors in LPS-activated murine macrophages

In atherosclerosis progression, atherosclerotic plaques develop upon accumulated foam cells derived from macrophages that take up modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Lipopolysaccharide (LPS) exacerbates atherosclerosis by enhancing the expression of scavenger receptors and thus increasing the uptake of modified LDL into macrophages. However, the signaling pathways that mediate LPS and scavenger receptor expression have not been fully elucidated.We used mouse bone marrow-derived macrophages and investigated the effects of LPS in vitro. LPS enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-1 (STAT-1). Inhibitors of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) pathway (U0126 and PD0325901) suppressed the uptake of acetylated-LDL (Ac-LDL) and the expression of CD204 but not CD36 in LPS-activated macrophages. Inhibitors of the Janus tyrosine kinase (JAK)-STAT pathway (ruxolitinib and tofacitinib) suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. We next injected LPS into the peritoneal cavity of mice and analyzed the effects of LPS. MEK inhibitor U0126 suppressed the uptake of Ac-LDL and the expression of CD204 but not CD36 in LPS-activated macrophages. JAK inhibitor ruxolitinib suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. These results suggest that scavenger receptors in LPS-activated mouse macrophages are regulated through a JAK-STAT-dependent pathway. Although further evaluation is necessary, JAK-STAT inhibition could be useful in atherosclerosis therapy, at least for atherosclerosis exacerbated by LPS.

Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1-M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE-/-) mouse model for early lesions, Acat1-M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1-M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1-M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1-M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE-/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -β/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Isolation of induced pluripotent stem cell-derived endothelial progenitor cells from sac-like structures

Transplanted endothelial progenitor cells (EPCs) repair blood vessels and exert regenerative effects on disorders such as lower limb ischemia. EPCs serve as a model for pathophysiological and pharmacokinetic studies, which is important for drug discovery. However, primary human EPCs are phenotypically unstable, which limits their clinical utility. Therefore, we employed human induced pluripotent stem (iPS) cells to circumvent this problem. Here we focused on human iPS cell-derived sac-like structures (iPS-sacs), which contain endothelial lineage cells and hematopoietic lineage cells. Previous studies isolated only hematopoietic lineage cells from iPS-sacs. Therefore, here we attempted to isolate EPCs. However, iPS-sacs generated by a published protocol did not contain sufficient EPCs. Therefore, to generate iPS-sacs highly enriched in EPCs, we added the glycogen synthase kinase 3 beta (GSK3β) inhibitor CHIR-99021 to the culture medium early during differentiation. The cells rapidly differentiated into mesoderm to yield abundant EPCs, and CHIR-99021 increased the proportion of EPCs contained in iPS-sacs. EPCs, which were purified using anti-platelet endothelial cell adhesion molecule (PECAM1) antibody-conjugated beads, expressed markers of immature endothelial cells. Purified EPCs formed tube-like structures and incorporated acetylated low density lipoprotein (Ac-LDL), reflecting endothelial phenotypes. The simple method described here will likely improve regenerative medicine and facilitate basic studies on the endothelial lineage.

Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica).

A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.

SUN-042 Loss of the 18-Kda Translocator Protein Tspo Destabilizes Cholesterol Homeostasis in a Hepatic Steatosis Cell Model

The 18-kDa translocator protein TSPO is a conserved ubiquitous high affinity cholesterol binding protein localized in the outer mitochondrial membrane. It plays a critical role in the segregation and transport of free cholesterol (FC) from the outer to the inner mitochondrial membrane in steroidogenic cells. Numerous studies have shown the presence of TSPO in liver. Imaging studies in humans, using specific TSPO ligands, showed that TSPO is highly expressed and accurately mirrors the histological picture of non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The function of TSPO in simple steatosis and NASH is unknown. Wild type Huh7 (WT) is a well-differentiated human hepatocyte-derived carcinoma cell line, which contains TSPO in mitochondria. In the present study, we generated a stable TSPO knockdown (TSPO KD) in the Huh7 cell line. To mimic hepatic steatosis in vitro, we treated WT and TSPO KD cells with either acetylated LDL (AcLDL), to deliver cholesterol into the cells, or AcLDL plus 58035, an Acetyl-CoA Acetyltransferase (ACAT) and thus cholesterol esterification inhibitor, to induce the accumulation of FC. AcLDL treatment significantly increased FC levels in WT cells; this effect was more pronounced in TSPO KD cells. In contrast, treatment of the cells with AcLDL and 58035 resulted in reduced FC levels likely due to the damage induced by FC accumulation that led to reduced cell proliferation. Considering that TSPO binds FC with high affinity, we investigated the underlying mechanism/link between TSPO KD and FC accumulation by examining the conversion between FC and cholesterol ester (CE) upon AcLDL treatment. The results obtained showed that knocking down TSPO expression reduced ACAT2 (transforming FC to CE) levels, but not cholesterol ester hydrolase (CEH) converting CE to FC, expression. TSPO was found to directly interact with ACAT2. Under these conditions, the accumulation of FC led to the appearance of lipid droplets (LD) of increased size in TSPO KD compared to WT cells, suggesting that TSPO plays a role in LD formation and/or dynamics in liver cells. GRP78, IRE1A, and PERK endoplasmic reticulum stress markers were also elevated in TSPO KD cells following AcLDL treatment while the mitochondrial membrane potential decreased compared to WT cells. Moreover, increased numbers of lysosomes and autophagosomes were observed in TSPO KD cells, an effect further potentiated by the treatment with AcLDL plus 58035. This finding was validated by the up regulation of the LAMP1 and LC3B, lysosome and autophagic structure markers, respectively. Taken together, these data suggest that under cholesterol treatment, TSPO loss would trigger ER stress, mitochondrial dysfunction, and lipid droplet accumulation via FC accumulation, and ultimately cause intracellular protein and organelle degradation, hallmarks of hepatic steatosis.

Controversy over the atherogenicity of lipoprotein-X.

Lipoprotein-X (Lp-X) is an abnormal lipoprotein containing abundant free cholesterol and phospholipids, as well as some apolipoprotein E (apoE). Serum Lp-X increases in patients with cholestasis and lecithin-cholesterol acyltransferase deficiency, as well as in those receiving intravenous lipid emulsion. Lp-X is often associated with skin xanthomas in cholestatic patients. However, earlier studies showed that Lp-X is not taken up by murine macrophages, but exerts antiatherogenic actions. In this review, we discuss the heterogeneity of Lp-X and its potential atherogenicity. Mass spectrometry revealed that Lp-X of cholestatic patients is similar in lipid composition to low-density lipoprotein (LDL) and high-density lipoprotein, but not to bile acids, suggesting that Lp-X is synthesized in the liver. Palmar xanthomas appear in patients with cholestasis, but regress over months after improvement of hypercholesterolemia. Lp-X isolated from cholestatic patients is rich in apoE, and causes more lipid accumulation than oxidized LDL and acetyl LDL in human monocyte-derived macrophages. Lp-X is heterogeneous in apoE content. Lp-X is taken up in cholestatic patients by apoE-recognizing lipoprotein receptors. Further research is warranted to fully understand the atherogenicity of Lp-X and the clinical significance of elevated Lp-X concentrations, particularly in cholestatic patients.

Cycloxygenase-2 inhibition potentiates trans-differentiation of Wharton's jelly–mesenchymal stromal cells into endothelial cells: Transplantation enhances neovascularization-mediated wound repair

BackgroundNeo-vascularization, an indispensible phenomenon for tissue regeneration, facilitates repair and remodeling of wound tissues. This process is impaired in chronic wounds due to reduced number and recruitment of endothelial cells (ECs), thereby necessitating development of newer strategies to enhance the EC repertoire as a therapeutic approach. MethodsWe explored the ‘plasticity’ of Wharton's jelly derived–mesenchymal stromal cells (WJ-MSCs) using an anti-inflammatory drug-mediated enhanced trans-differentiation into ECs, based on our observation of temporal decrease in COX-2 expression during trans-differentiation of MSCs into ECs at day 7 and 14 along with mature ECs. ResultsAt a physiological level, an increased DiI-labeled acetylated-low density lipoprotein (DiI-Ac-LDL) uptake, proliferation, migration and chick chorio allantoic membrane (CAM)-vasculogenesis occurred while at a molecular level significant up-regulation in messenger RNA (mRNA) and protein expression of endothelial-specific markers, Vegfr2, Pecam, eNOS, VE-Cadh and Tie-2, along with an activated p-VEGFR2 and its downstream mediators were observed in celecoxib-preconditioned ECs as compared with WJ-MSCs. Green fluorescent protein (GFP)-expressing stable WJ-MSCs and trans-differentiated EC-D14 in the absence/presence of celecoxib were generated using antibiotic selection for intradermal transplantation at the wound site on a murine ‘excisional splinting wound’ model. Engraftment of transplanted human cells in immunosuppressant-treated mice was confirmed by a significant increase in the expression levels of human gene-specific endothelial markers at the regenerated wound sites. Morphometrically, increased vascularity and percent wound closure were observed in regenerated wounds of mice transplanted with celecoxib-preconditioned-EC-D14. ConclusionCox-2 inhibition led to an enhanced trans-differentiation of WJ-MSCs into ECs that, when transplanted, accelerated the skin regeneration by engraftment and neo-vascularization at the wound bed, suggesting a plausible new therapeutic role of celecoxib.