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.

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 19F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of 19F 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 19F NMR experiments in a multi-protein format, offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r2 = 0.84–0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164–FJ010174.

This data set represents the first description of signalling pathways associated with the functional recovery of end-stage human heart failure and the identification of new targets in the human heart that are modified by this combination therapy.

receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/␤-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the ␤-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6⌬C) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the ␤-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a ␤-catenin-dependent signal. Expression of the dominant interfering LRP6⌬C transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D 1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptorrelated protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.␤-catenin; apoptosis; cyclin D 1; T cell factor-4 REGULATION OF VASCULAR SMOOTH MUSCLE CELL (VSMC) proliferation and apoptosis plays a critical role in the pathogenesis of vascular disease and remodeling (4,11,12,45). Recent work from our lab has identified a role for the evolutionary conserved Wnt signaling pathway in governing VSMC proliferation and survival in the context of vascular injury (54).Wnts are a family of secreted glycoproteins that bind to a class of Frizzled receptors (3, 9, 21, 40). The conserved Wnt cascade is known to govern cell fate, proliferation, differentiation, and polarity (3,9,21,40). Wnt binding to the Frizzled family of receptors stabilizes and translocates ␤-catenin to the nucleus and activates a family of T cell factor/lymphoid enhancing factors (Tcf/Lef) (3, 40). We demonstrated a robust temporal upregulation of ␤-catenin in intimal VSMCs that led to activation of the transcription factor Tcf-4 (54). We further demonstrated that activation of the Wnt cascade led to transactivation of the Tcf-responsive gene cyclin D 1 , an increase in VSMC proliferation, and inhibition of apoptosis (54). Loss of Tcf-4 function abolished both proliferative and survival promoting effects (54). The objective of this study was to extend these findings and assess the previously undefined role of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in the regulation of Wnt signaling in the vasculature.LRP6 and LRP5 are members of the LRP superfamily that have recently been found to be required for activation of the canonical Wnt signaling pathway (35,37,42,50,56). LRP5 and LRP6 are single transmembrane proteins that contain four epidermal growth factor-like repeats and three LDL receptor type A repeat...

Lead is a well-documented testicular toxicant. The present work was planned to study the occurrence of germ cell death after lead administration. Young growing rats were treated with 5, 10 and 20 mg kg(-1) body weight of lead for 2 weeks. Cell death was assessed by employing in situ TUNEL staining, DNA electrophoresis and morphological examination of the tubules. The results showed that Pb induced significant numbers of germ cells to undergo apoptosis in the seminiferous tubules of rats treated with 20 mg kg(-1) body weight. However, DNA fragmentation was not detected at any of the doses. The level of lead accumulation in the testis increased in a dose-dependent manner.

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-insulindependent glucose transporter protein, was overexpressed in smooth muscle using the sm22␣ promoter. Overexpression of GLUT1 in smooth muscle led to significant increases in glucose uptake (nϭ3, PϽ0.0001) 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␣-GLUT1 mice (nϭ10, PϽ0.04). In response to vascular injury, sm22␣-GLUT1 mice exhibited a proinflammatory phenotype, including a significant increase in the percentage of neutrophils in the lesion (nϭ4, PϽ0.04) and an increase in monocyte chemoattractant protein-1 (MCP-1) immunofluorescence. Circulating haptoglobin and glutathione/total glutathione were significantly higher in the sm22␣-GLUT1 mice postinjury compared with controls (nϭ4, PϽ0.05), suggesting increased flux through the pentose phosphate pathway. sm22␣-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 (nϭ4, PϽ0.003). 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-␤ activity. Key Words: Glut1 Ⅲ hypertrophy Ⅲ vascular smooth muscle Ⅲ haptoglobin Ⅲ phosphoSMAD2/3 Ⅲ glucose Ⅲ neutrophil Ⅲ macrophage C ardiovascular complications remain the number one cause of death from individuals with diabetes. Epidemiological studies to date have reported conflicting results over the role of glucose as a contributing risk factor to coronary artery disease in individuals with type 1 diabetes. 1-9 A recent report found that glycohemoglobin in nondiabetic adults was strongly associated with coronary artery disease, further suggesting that increasing cellular glucose uptake promotes vascular complications and coronary artery disease. 10 However, the mechanisms through which glucose increases the risk of coronary artery disease are not well understood. Specifically, the role of increasing glucose uptake in different cell types in contributing to coronary artery disease is not well understood.We tested the hypothesis that increasing glucose uptake in vascular smooth muscle cells would alter the contractility properties of the vessel. In addition, we tested the hypothesis that in response to vascular injury, increased glucose uptake would exacerbate vascular intima formation.We used a genetic approach to increase expression of the non-insulin-dependent glucose transporter protein GLUT1 in VSMCs using the sm22␣ promoter. This...

We screened a compendium of gene profiles from 19 paired human heart samples harvested at the time of implant and explant of a left ventricular assist device (LVAD) for novel genes regulating the Ras/MEK/ERK cascade. From this analysis we identified Sprouty1, an evolutionally conserved gene that acts as an intrinsic inhibitor of the Ras/MEK/ERK pathway. Sprouty1 mRNA and protein were significantly upregulated in the heart in response to mechanical unloading with a LVAD. The upregulation of Sprouty1 in the heart following mechanical unloading was accompanied by a significant decrease in phosphorylated ERK1/2. Gain of function experiments demonstrated that upregulation of Sprouty1 in isolated cardiac myocytes led to a significant decrease and altered kinetics of ERK1/2 phosphorylation. Immunohistochemistry of human hearts revealed that Sprouty1 was also expressed in the microvasculature. Upregulation of Sprouty1 in endothelial cells led to a significant decrease in VEGF-induced endothelial cell proliferation. To our knowledge, these findings are the first to define Sprouty expression in the heart and suggest that Sprouty1 may serve as an intrinsic mediator governing ventricular remodeling through a coordinated coupling of both myocyte and vascular alterations in response to mechanical load.