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.

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.

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.

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.

Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10-72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10-4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10-5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids.

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.

Genes underneath signals from genome-wide association studies (GWAS) for kidney function are promising targets for functional studies, but prioritizing variants and genes is challenging. By GWAS meta-analysis for creatinine-based estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics Consortium and UK Biobank (n = 1,201,909), we expand the number of eGFRcrea loci (424 loci, 201 novel; 9.8% eGFRcrea variance explained by 634 independent signal variants). Our increased sample size in fine-mapping (n = 1,004,040, European) more than doubles the number of signals with resolved fine-mapping (99% credible sets down to 1 variant for 44 signals, ≤5 variants for 138 signals). Cystatin-based eGFR and/or blood urea nitrogen association support 348 loci (n = 460,826 and 852,678, respectively). Our customizable tool for Gene PrioritiSation reveals 23 compelling genes including mechanistic insights and enables navigation through genes and variants likely relevant for kidney function in human to help select targets for experimental follow-up.

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.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis, yet little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMCs), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both angiotensin II- (Ang II-) and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMCs resulted in loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its coactivator P300 and the SWI/SNF complex and suppressing VSMC inflammation by promoting a repressive chromatin state of NF-κB target genes as well as preventing VSMC apoptosis through transcriptional activation of KLF5-dependent B cell lymphoma 2 (BCL2) expression. Our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.

Increasing energy expenditure by promoting "browning" in adipose tissues is a promising strategy to prevent obesity and associated diabetes. To uncover potential targets of cold exposure, which induces energy expenditure, we performed phosphoproteomics profiling in brown adipose tissue of mice housed in mild cold environment at 16°C. We identified CDC2-like kinase 1 (CLK1) as one of the kinases that were significantly downregulated by mild cold exposure. In addition, genetic knockout of CLK1 or chemical inhibition in mice ameliorated diet-induced obesity and insulin resistance at 22°C. Through proteomics, we uncovered thyroid hormone receptor-associated protein 3 (THRAP3) as an interacting partner of CLK1, further confirmed by co-immunoprecipitation assays. We further demonstrated that CLK1 phosphorylates THRAP3 at Ser243, which is required for its regulatory interaction with phosphorylated peroxisome proliferator-activated receptor gamma (PPARγ), resulting in impaired adipose tissue browning and insulin sensitivity. These data suggest that CLK1 plays a critical role in controlling energy expenditure through the CLK1-THRAP3-PPARγ axis.

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a "plug and play" module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

In eukaryotic cells, DNA double strand breaks (DSB) are primarily repaired by canonical non-homologous end joining (c-NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ). Zinc finger and SCAN domain containing 4 (ZSCAN4), sporadically expressed in 1-5% mouse embryonic stem cells (mESCs), is known to regulate genome stability by promoting HR.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.