MicroRNAs (miRNAs) are being developed to enhance tissue regeneration. Here we show that a hyperbranched polymer with high miRNA-binding affinity and negligible cytotoxicity can self-assemble into nano-sized polyplexes with a 'double-shell' miRNA distribution and high transfection efficiency. These polyplexes are encapsulated in biodegradable microspheres to enable controllable two-stage (polyplexes and miRNA) delivery. The microspheres are attached to cell-free nanofibrous polymer scaffolds that spatially control the release of miR-26a. This technology is used to regenerate critical-sized bone defects in osteoporotic mice by targeting Gsk-3β to activate the osteoblastic activity of endogenous stem cells, thus addressing a critical challenge in regenerative medicine of achieving cell-free scaffold-based miRNA therapy for tissue engineering.

Alternative splicing of VEGF-A gives rise to two families - the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family that differ by only six amino acids at their C-terminal end. The first verified and widely reported VEGFxxxb family member is VEGF165b, and here VEGF165b is a positive control.

Phagocyte-derived myeloperoxidase (MPO) and proinflammatory HDL are associated with metabolic syndrome (MetS) and increased cardiovascular disease risk. Therapeutic lifestyle changes (TLCs), such as a Mediterranean diet and exercise, decrease this risk. However, the link among TLCs, HDL, and MPO-mediated oxidative stress remains unclear.

High density lipoprotein (HDL) cholesterol levels and cholesterol efflux capacity (CEC) are inversely correlated with coronary artery disease (CAD) risk. Myeloperoxidase (MPO) derived oxidants and HDL proteome changes are implicated in HDL dysfunction in subjects with CAD in the United States; however, the effect of MPO on HDL function and HDL proteome in ethnic Chinese population is unknown. We recruited four matched ethnic Chinese groups (20 patients each): subjects with 1) low HDL levels (HDL levels in men <40mg/dL and women <50mg/dL) and non-CAD (identified by coronary angiography or cardiac CT angiography); 2) low HDL and CAD; 3) high HDL (men >50mg/dL; women >60mg/dL) with no CAD; and 4) high HDL with CAD. Serum cytokines, serum MPO levels, serum CEC, MPO-oxidized HDL tyrosine moieties, and HDL proteome were assessed by mass spectrometry individually in the four groups. The cytokines, MPO levels, and HDL proteome profiles were not significantly different between the four groups. As expected, CEC was depressed in the entire CAD group but more specifically in the CAD low-HDL group. HDL of CAD subjects had significantly higher 3-nitrotyrosine than non-CAD subjects, but the MPO-specific 3-chlorotyrosine was unchanged; CEC in the CAD low-HDL group did not correlate with either HDL 3-chlorotyrosine or 3-nitrotyrosine levels. Neither 3-chlorotyrosine, which is MPO-specific, nor 3-nitrotyrosine generated from MPO or other reactive nitrogen species was associated with CEC. MPO mediated oxidative stress and HDL proteome composition changes are not the primary cause HDL dysfunction in Chinese subjects with CAD. These studies highlight ethnic differences in HDL dysfunction between United States and Chinese cohorts raising possibility of unique pathways of HDL dysfunction in this cohort.

The mechanism regarding rapid progression of residual hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA) has been preliminarily discussed. However, most studies have mainly focused on RFA-induced changes in the tumor cells. The present study was designed to determine whether tumor-associated endothelial cells (TAECs) could contribute to the invasiveness of HCC after insufficient RFA.

Surfactant protein A2 (SP-A2) plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA), a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising 15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.

The prevalence of type 2 diabetes (T2D) is rapidly increasing worldwide. Effective therapies, such as insulin and Glucagon-like peptide-1 (GLP-1), require injections, which are costly and result in less patient compliance. Here, we report the identification of a tripeptide with significant potential to treat T2D. The peptide, referred to as Diapin, is comprised of three natural L-amino acids, GlyGlyLeu. Glucose tolerance tests showed that oral administration of Diapin effectively lowered blood glucose after oral glucose loading in both normal C57BL/6J mice and T2D mouse models, including KKay, db/db, ob/ob mice, and high fat diet-induced obesity/T2D mice. In addition, Diapin treatment significantly reduced casual blood glucose in KKay diabetic mice in a time-dependent manner without causing hypoglycemia. Furthermore, we found that plasma GLP-1 and insulin levels in diabetic models were significantly increased with Diapin treatment compared to that in the controls. In summary, our findings establish that a peptide with minimum of three amino acids can improve glucose homeostasis and Diapin shows promise as a novel pharmaceutical agent to treat patients with T2D through its dual effects on GLP-1 and insulin secretion.

Blood lipid levels are heritable, treatable risk factors for cardiovascular disease. We systematically assessed genome-wide coding variation to identify new genes influencing lipid traits, fine map known lipid loci and evaluate whether low-frequency variants with large effects exist for these traits. Using an exome array, we genotyped 80,137 coding variants in 5,643 Norwegians. We followed up 18 variants in 4,666 Norwegians and identified ten loci with coding variants associated with a lipid trait (P < 5 × 10(-8)). One variant in TM6SF2 (encoding p.Glu167Lys), residing in a known genome-wide association study locus for lipid traits, influences total cholesterol levels and is associated with myocardial infarction. Transient TM6SF2 overexpression or knockdown of Tm6sf2 in mice alters serum lipid profiles, consistent with the association observed in humans, identifying TM6SF2 as a functional gene within a locus previously known as NCAN-CILP2-PBX4 or 19p13. This study demonstrates that systematic assessment of coding variation can quickly point to a candidate causal gene.

The mechanism of rapid growth of the residual tumor after radiofrequency (RF) ablation is poorly understood. In this study, we investigated the effect of hyperthermia on HepG2 cells and generated a subline with enhanced viability and dys-regulated angiogenesis in vivo, which was used as a model to further determine the molecular mechanism of the rapid growth of residual HCC after RF ablation.

Dysfunctional high-density lipoprotein (HDL) may have pro-inflammatory effects on the endothelial cells,which causes atherosclerosis in type 2 diabetes mellitus (T2DM). HDL is a major carrier of sphingosine-1-phosphate (S1P) in plasma while S1P exhibits multiple biological activities. However, potential role of HDL and S1P in T2DM remains unexplored. We hypothesized that diabetic HDL with higher contents of S1P exerts beneficial effects on the vascular system.

High density lipoprotein (HDL) has been proved to be a protective factor for coronary heart disease. Notably, HDL in atherosclerotic plaques can be nitrated (NO2-oxHDL) and chlorinated (Cl-oxHDL) by myeloperoxidase (MPO), likely compromising its cardiovascular protective effects.

Epithelial-mesenchymal transition (EMT) and angiogenesis is involved in tumor progression after radiofrequency ablation (RFA). ATPase inhibitory factor 1 (IF1) is a bad predictor of prognosis. Sorafenib inhibited EMT of hepatocellular carcinoma (HCC) after RFA. Whether IF1 promotes the EMT and angiogenesis of HCC and attenuates the effect of sorafenib after insufficient RFA is investigated. In this study, higher expression of IF1 was found in residual tumor after insufficient RFA. Hep3B or Huh7 cells after insufficient RFA were designated as Hep3B-H or Huh7-H cells in vitro. Hep3B-H or Huh7-H cells exhibited enhanced capacities of colony formation, migration, and increased expression of EMT associated markers and IF1 compared with Hep3B or Huh7 cells. IF1 knockdown in Hep3B-H or Huh7-H cells decreased the colony formation and migratory capacity, and IF1 overexpression in Hep3B or Huh7 cells increased these capacities. IF1 in HCC cells directly and indirectly affected angiogenesis of TAECs after insufficient RFA. IF1 promoted HCC cells growth and metastasis after insufficient RFA. IF1 increased HCC cells resistance after insufficient RFA to sorafenib. Higher IF1 expression indicated poor disease survival in HCC patients after sorafenib therapy. NF-κB activation induced by IF1 attenuated the effect of sorafenib on HCC cells after insufficient RFA. Our results demonstrated that IF1 promotes the EMT and angiogenesis, and attenuates HCC cell sensitivity to sorafenib after insufficient RFA through NF-κB signal pathway.

A major challenge in genetic association studies is that most associated variants fall in the non-coding part of the human genome. We searched for variants associated with bone mineral density (BMD) after enriching the discovery cohort for loss-of-function (LoF) mutations by sequencing a subset of the Nord-Trøndelag Health Study, followed by imputation in the remaining sample (N = 19,705), and identified ten known BMD loci. However, one previously unreported variant, LoF mutation in MEPE, p.(Lys70IlefsTer26, minor allele frequency [MAF] = 0.8%), was associated with decreased ultradistal forearm BMD (P-value = 2.1 × 10-18), and increased osteoporosis (P-value = 4.2 × 10-5) and fracture risk (P-value = 1.6 × 10-5). The MEPE LoF association with BMD and fractures was further evaluated in 279,435 UK (MAF = 0.05%, heel bone estimated BMD P-value = 1.2 × 10-16, any fracture P-value = 0.05) and 375,984 Icelandic samples (MAF = 0.03%, arm BMD P-value = 0.12, forearm fracture P-value = 0.005). Screening for the MEPE LoF mutations before adulthood could potentially prevent osteoporosis and fractures due to the lifelong effect on BMD observed in the study. A key implication for precision medicine is that high-impact functional variants missing from the publicly available cosmopolitan panels could be clinically more relevant than polygenic risk scores.

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe-/-) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe-/- mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe-/- mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.

Our previous study has revealed that exosomes from adipose-derived stem cells (ASCs) promote angiogenesis in subcutaneously transplanted gels by delivery of microRNA-31 (miR-31) which targets factor inhibiting hypoxia-inducible factor-1 (FIH1) in recipient cells. Here we hypothesized that ASC exosomes alleviate ischemic diseases through miR-31/FIH1/hypoxia-inducible factor-1α (HIF-1α) signaling pathway. Exosomes from ASCs were characterized with nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting analysis for exosomal markers. Results from immunoblotting and laser imaging of ischemic mouse hindlimb revealed that miR-31 enriched ASC exosomes inhibited FIH1 expression and enhanced the blood perfusion, respectively. These effects were impaired when using miR-31-depleted exosomes. Immunohistochemistry analysis showed that administration of exosomes resulted in a higher arteriole density and larger CD31+ area in ischemic hindlimb than miR-31-delpleted exosomes. Similarly, knockdown of miR-31 in exosomes reduced the effects of the exosomes on increasing ventricular fraction shortening and CD31+ area, and on decreasing infarct size. Exosomes promoted endothelial cell migration and tube formation. These changes were attenuated when miR-31 was depleted in the exosomes or when FIH1 was overexpressed in the endothelial cells. Furthermore, the results from immunocytochemistry, co-immunoprecipitation, and luciferase reporter assay demonstrated that the effects of exosomes on nuclear translocation, binding with co-activator p300, and activation of HIF-1α were decreased when miR-31 was depleted in the exosomes or FIH1 was overexpressed. Our findings provide evidence that exosomes from ASCs promote angiogenesis in both mouse ischemic hindlimb and heart through transport of miR-31 which targets FIH1 and therefore triggers HIF-1α transcriptional activation.

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe-/- mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm-/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

Atherosclerosis is the leading cause of cardiovascular diseases, which is also the primary cause of mortality among diabetic patients. Endothelial cell (EC) dysfunction is a critical early step in the development of atherosclerosis and aggravated in the presence of concurrent diabetes. Although the heterogeneity of the organ-specific ECs has been systematically analyzed at the single-cell level in healthy conditions, their transcriptomic changes in diabetic atherosclerosis remain largely unexplored. Here, we carried out a single-cell RNA sequencing (scRNA-seq) study using EC-enriched single cells from mouse heart and aorta after 12 weeks feeding of a standard chow or a diabetogenic high-fat diet with cholesterol. We identified eight EC clusters, three of which expressed mesenchymal markers, indicative of an endothelial-to-mesenchymal transition (EndMT). Analyses of the marker genes, pathways, and biological functions revealed that ECs are highly heterogeneous and plastic both in normal and atherosclerotic conditions. The metabolic transcriptomic analysis further confirmed that EndMT-derived fibroblast-like cells are prominent in atherosclerosis, with diminished fatty acid oxidation and enhanced biological functions, including regulation of extracellular-matrix organization and apoptosis. In summary, our data characterized the phenotypic and metabolic heterogeneity of ECs in diabetes-associated atherogenesis at the single-cell level and paves the way for a deeper understanding of endothelial cell biology and EC-related cardiovascular diseases.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.