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.

Increased cumulative doses and long-term BP treatment are the most important risk factors for BRONJ development. Type of BP, diabetes, hypothyroidism, smoking, and prior dental extractions may play a role in BRONJ development.

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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...

INTRODUCTIONHeparan sulfate proteoglycans are abundant molecules in the extracellular matrix and cell surface, consisting of a proteoglycan core protein attached to heparan sulfate chains 1 . Heparan sulfate chains are composed of alternating N-acetylglucosamine and glucuronic acid moieties. The fine structure of the heparan sulfate chains are modified through a series of enzymatic reactions 1 . Heparan sulfate serves as a docking site for multiple chemokines, lipids, and growth factors 2,3 . We hypothesized that the fine structure of the heparan sulfate chain influenced proliferative properties of the VSMC. This hypothesis was based on previous findings in transgenic models in which heparan sulfate modifying enzymes had been deleted resulting in altered proliferation of different cell types [4][5][6][7][8][9][10][11] .Expression of one of the many heparan sulfate modifying enzymes, Ndst1, is up-regulated 40 fold in response to vascular injury 12 . Ndst1 catalyzes an initial step in heparan sulfate modification -replacing N-acetyl groups with sulfate on the N-acetylglucoasamine residues 1, 13 . We utilized two cre recombinase mouse models to delete Ndst1 in smooth muscle to test the role of Ndst1 in vascular remodeling. The two promoters used to express cre recombinase Corresponding Author: Jennifer L. Hall, Ph.D. Lillehei Heart Institute, 312 Church St SE, Minneapolis, MN 55455, Voice: 612.626.4566, Fax: 612.624.8118, jlhall@umn.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. in smooth muscle were SM22α and SMMHC. The SM22α-cre + Ndst1 −/− mouse exhibited efficient and specific loss of Ndst1 in smooth muscle. As previously described in the literature 14 , the SMMHC-cre + Ndst1 −/− mouse exhibited ectopic recombination of floxed alleles in the germline and less efficient expression of cre-recombinase in smooth muscle compared to the SM22α promoter. Although loss of a single allele in the germline and less efficient deletion in smooth muscle in the SMMHC-cre + Ndst1 −/− was not a fatal flaw, this model was limited to initial studies. Compromised Ndst1 expression led to significantly reduced lesion formation in response to injury in both models. Additional studies in the targeted SM22α-cre + Ndst1 −/− lines showed a 50% loss of VSMC and decreased size of the femoral artery that was evident at day 7 which persisted through till adulthood. These findings may have important implications for the role of heparan sulfate in vascular structure and remodeling. NIH Public Access MATERIALS AND METHODS Generation of Ndst1 deficient mouse modelsNdst1 deficient mice Ndst1 flox/flo...

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Purpose To analyze serum markers of bone turnover, angiogenesis, endocrine function, and inflammation in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients who discontinued long-term intravenous bisphosphonate (BP) therapy. Patients and Methods Serum samples were obtained from 25 BRONJ patients who had discontinued long-term intravenous BP therapy for an average of 11.4±8.7 months and 48 non-BRONJ controls who continued receiving intravenous BP therapy. Samples were analyzed for total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OCN), C-telopeptide (CTX), vascular-endothelial growth factor (VEGF), triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), 25-hydroxyvitamin D, and C-reactive protein (CRP). Results The mean number of BP infusions was significantly higher in BRONJ subjects compared with controls (38.4±26.3 infusions vs 18.8±7.2, p<0.0001); however, the duration of BP therapy was not significantly different between the groups (p=0.23). Overall, there were no significant differences in any of the markers between BRONJ subjects and controls (all p values ≥ 0.16). In a subgroup analysis that matched BRONJ subjects and controls according to mean age and BP infusions (BRONJ, n=10 and controls, n=48), log10VEGF (2.9±0.4 vs 2.4±0.4, p=<0.001) and CRP (34±26 vs 13±8, p=<0.01) levels were significantly higher in BRONJ subjects compared with controls. Within BRONJ subjects, none of the serum markers were correlated with duration of BP discontinuation. Conclusions Bone turnover and endocrine markers in BRONJ subjects who discontinue long-term intravenous BP therapy are similar to non-BRONJ controls receiving intravenous BP therapy. However, angiogenesis and inflammation markers are higher in BRONJ subjects who discontinue long-term intravenous BP therapy. The prolonged skeletal half-life of BPs may suppress bone turnover markers in BRONJ subjects for several years following discontinuation of intravenous BP therapy, suggesting an extended effect on bone homeostasis.