Neeta Adhikari

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Increase in GLUT1 in Smooth Muscle Alters Vascular Contractility and Increases Inflammation in Response to Vascular Injury

Objective—The goal of this study was to test the contributing role of increasing glucose uptake in vascular smooth muscle cells (VSMCs) in vascular complications and disease. Methods and Results—A murine genetic model was established in which glucose trasporter 1 (GLUT1), the non–insulin-dependent glucose transporter protein, was overexpressed in smooth muscle using the sm22&agr; promoter. Overexpression of GLUT1 in smooth muscle led to significant increases in glucose uptake (n=3, P<0.0001) as measured using radiolabeled 2-deoxyglucose. Fasting blood glucose, insulin, and nonesterified fatty acids were unchanged. Contractility in aortic ring segments was decreased in sm22&agr;-GLUT1 mice (n=10, P<0.04). In response to vascular injury, sm22&agr;-GLUT1 mice exhibited a proinflammatory phenotype, including a significant increase in the percentage of neutrophils in the lesion (n=4, P<0.04) and an increase in monocyte chemoattractant protein-1 (MCP-1) immunofluorescence. Circulating haptoglobin and glutathione/total glutathione were significantly higher in the sm22&agr;-GLUT1 mice postinjury compared with controls (n=4, P<0.05), suggesting increased flux through the pentose phosphate pathway. sm22&agr;-GLUT1 mice exhibited significant medial hypertrophy following injury that was associated with a significant increase in the percentage of VSMCs in the media staining positive for nuclear phosphoSMAD2/3 (n=4, P<0.003). Conclusion—In summary, these findings suggest that increased glucose uptake in VSMCs impairs vascular contractility and accelerates a proinflammatory, neutrophil-rich lesion in response to injury, as well as medial hypertrophy, which is associated with enhanced transforming growth factor-&bgr; activity.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

The Role of β-Transducin Repeat-Containing Protein (β-TrCP) in the Regulation of NF-κB in Vascular Smooth Muscle Cells

Objective—Degradation of I&kgr;B is an essential step in nuclear factor (NF)-&kgr;B activation. However, the determinants regulating this process have not been defined in vascular smooth muscle cells (VSMCs). We hypothesized that the E3-ligase, &bgr;-transducin repeat-containing protein 1 (&bgr;-TrCP1), was a rate-determining mediator that regulates the ubiquitin-mediated degradation of I&kgr;B&agr; (in VSMC). Methods and Results—Upregulation of &bgr;-TrCP1 accelerated the rate of I&kgr;B&agr; degradation, leading to increased NF-&kgr;B activity. In contrast, VSMCs harboring a dominant-negative &bgr;-TrCP1 transgene lacking the F-box domain exhibited a reduction in serum-stimulated NF-kB activity but no alteration in response to tumor necrosis factor (TNF). These findings suggest that &bgr;-TrCP1 increases the rate of NF-&kgr;B activation but is not rate-limiting in response to TNF in VSMCs. Endogenous &bgr;-TrCP1 expression was regulated through the conserved Wnt cascade. Upregulation of Wnt1 resulted in &bgr;-catenin–mediated activation of Tcf-4, leading to increased &bgr;-TrCP1 expression and NF-&kgr;B activity. Furthermore, VSMCs harboring a Tcf-4 mutant lacking a &bgr;-catenin binding domain exhibited a significant reduction in &bgr;-TrCP1 expression along with abolishment of NF-&kgr;B activity. Conclusions—We provide the first evidence of crosstalk between the Wnt cascade and NF-&kgr;B signaling in VSMCs. This crosstalk is mediated through the E3-ligase, &bgr;-TrCP1.

Heparan sulfate Ndst1 regulates vascular smooth muscle cell proliferation, vessel size and vascular remodeling

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.

RIP140 contributes to foam cell formation and atherosclerosis by regulating cholesterol homeostasis in macrophages

Atherosclerosis, a syndrome with abnormal arterial walls, is one of the major causes that lead to the development of various cardiovascular diseases. The key initiator of atherosclerosis is cholesterol accumulation. The uncontrolled cholesterol deposition, mainly involving low-density lipoprotein (LDL), causes atheroma plaque formation, which initiates chronic inflammation due to the recruitment of inflammatory cells such as macrophages. Macrophages scavenge excess peripheral cholesterol and transport intracellular cholesterol to high-density lipoprotein (HDL) for excretion or storage. Cholesterol-laden macrophage-derived foam cell formation is the main cause of atherogenesis. It is critical to understand the regulatory mechanism of cholesterol homeostasis in the macrophage in order to prevent foam cells formation and further develop novel therapeutic strategies against atherosclerosis. Here we identified a protein, RIP140 (receptor interacting protein 140), which enhances macrophage-derived foam cell formation by reducing expression of reverse cholesterol transport genes, A TP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1). In animal models, we found that reducing RIP140 levels by crossing macrophage-specific RIP140 knockdown (MϕRIP140KD) mice with ApoE null mice effectively ameliorates high-cholesterol diet-induced atherosclerosis. Our data suggest that reducing RIP140 levels in macrophages significantly inhibits atherosclerosis, along with markers of inflammation and the number of macrophages in a western diet fed ApoE null mouse. This study provides a proof-of-concept for RIP140 as a risk biomarker of, and a therapeutic target for, atherosclerosis.

Identification of Differentially Expressed Transcripts and Pathways in Blood One Week and Six Months Following Implant of Left Ventricular Assist Devices

Introduction Continuous-flow left ventricular assist devices (LVADs) are an established therapy for patients with end-stage heart failure. The short- and long-term impact of these devices on peripheral blood gene expression has not been characterized, and may provide insight into the molecular pathways mediated in response to left ventricular remodeling and an improvement in overall systemic circulation. We performed RNA sequencing to identify genes and pathways influenced by these devices. Methods RNA was extracted from blood of 9 heart failure patients (8 male) prior to LVAD implantation, and at 7 and 180 days postoperatively. Libraries were sequenced on an Illumina HiSeq2000 and sequences mapped to the human Ensembl GRCh37.67 genome assembly. Results A specific set of genes involved in regulating cellular immune response, antigen presentation, and T cell activation and survival were down-regulated 7 days after LVAD placement. 6 months following LVAD placement, the expression levels of these genes were significantly increased; yet importantly, remained significantly lower than age and sex-matched samples from healthy controls. Conclusions In summary, this genomic analysis identified a significant decrease in the expression of genes that promote a healthy immune response in patients with heart failure that was partially restored 6 months following LVAD implant.

SH2B3 Is a Genetic Determinant of Cardiac Inflammation and Fibrosis

Background—Genome-wide association studies are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes affect disease outcome. Such is the case for SH2B adaptor protein 3 (SH2B3), which is a negative regulator of multiple cytokine signaling pathways and is associated with increased risk of myocardial infarction (MI), but its role in post-MI inflammation and fibrosis is completely unknown. Methods and Results—Using an experimental model of MI (left anterior descending artery occlusion/reperfusion injury) in wild-type and Sh2b3 knockout rats (Sh2b3em2Mcwi), we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle contractility, and inflammatory gene expression. Compared with wild-type, Sh2b3em2Mcwi rats had significantly increased fibrosis (2.2-fold; P<0.05) and elevated leukocyte infiltration (>2-fold; P<0.05), which coincided with decreased left ventricle fractional shortening (−&Dgr;11%; P<0.05) at 7 days post left anterior descending artery occlusion/reperfusion injury. Despite an increased angiogenic potential in Sh2b3em2Mcwi rats (1.7-fold; P<0.05), we observed no significant differences in left ventricle capillary density between wild-type and Sh2b3em2Mcwi rats. In total, 12 genes were significantly elevated in the post left anterior descending artery occluded/reperfused hearts of Sh2b3em2Mcwi rats relative to wild-type, of which 3 (NLRP12, CCR2, and IFN&ggr;) were significantly elevated in the left ventricle of heart failure patients carrying the MI-associated rs3184504 [T] SH2B3 risk allele. Conclusions—These data demonstrate for the first time that SH2B3 is a crucial mediator of post-MI inflammation and fibrosis.

Identification of a new target of miR-16, Vacuolar Protein Sorting 4a.

Rationale The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF). Objective The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD). Methods and Results BEDTools v2.14.3 was used to discriminate SNPs within predicted 3′UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3′UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001). Conclusions We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.

Smooth Muscle Specific Deletion of Ndst1 Leads to Decreased Vessel Luminal Area and No Change in Blood Pressure in Conscious Mice

Heparan sulfate proteoglycans are abundant matrix and membrane molecules. Smooth muscle specific deletion of one heparan sulfate biosynthetic enzyme, N-deacetylase-N-sulfotransferase1 leads to decreased vascular smooth muscle cell proliferation, and vascular wall thickness. We hypothesized that this may lead to changes in blood pressure in conscious mice. Blood pressure was measured via telemetry in SM22αCre+Ndst1−/−(n = 4) and wild type (n = 8) mice. Aorta and thoracodorsal artery luminal area is significantly smaller in SM22αCre+Ndst1−/− (n = 4–8, P = 0.02, P = 0.0002) compared to wild type (n = 7) mice. Diurnal differences were observed in both cohorts for systolic, diastolic, mean arterial blood pressure, and heart rate (P < 0.001 from T test). No significant differences were found in the above parameters between the cohorts in either light or dark times using a linear mixed model. In conclusion, deletion of N-deacetylase-N-sulfotransferase1 in smooth muscle did not influence any of the blood pressure parameters measured despite significant decrease in aorta and thoracodorsal artery luminal area.