Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal-transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial cell like phenotype after acute ischemic cardiac injury. Fibroblast derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast derived endothelial cells, reduces post infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal to endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial-transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

Evidence accumulated over the past several years indicates that the AMP-activated protein kinase (AMPK) 2 may be a therapeutic target for treating insulin resistance and type 2 diabetes (1). AMPK is a heterotrimeric protein formed by an ␣ subunit, which contains the catalytic activity, and by the ␤ and ␥ regulatory subunits important in maintaining stability of the heterotrimer complex (2). AMPK belongs to a family of energy-sensing enzymes functioning as a "fuel gauge" that monitors changes in the energy status of a cell (3, 4). When activated, AMPK shuts down anabolic pathways and promotes catabolism in response to an elevated AMP/ATP ratio by down-regulating the activity of several key enzymes of intermediary metabolism (4). Two primary acute consequences of AMPK activation are 1) an increase in glucose uptake by induction of glucose 4 transporter microvesicle cytoplasm to membrane translocation and fusion and 2) an increase in fatty acid oxidation by phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis (5). Therefore, the AMPK signal pathways are thought to play a central role in the regulation of cellular glucose and lipid homeostasis. The control of AMPK activity is complex, and the classic view is that AMPK is activated allosterically by an increase in the intracellular AMP/ATP ratios and/or by the phosphorylation of threonine 172 within the ␣ subunit. Several protein kinases responsible for this phosphorylation have been identified. They include Peutz-Jeghers syndrome kinase LKB1 (LKB1) (6), and the Ca 2ϩ /calmodulin-dependent protein kinase kinase (7). Protein phosphorylation signal transduction systems are balanced and regulated delicately by both phosphatase and kinase. Since AMPK is activated by (a) protein kinase(s) at the threonine 172 residue, one can easily assume that AMPK can be regulated negatively by (a) serine/threonine phosphatase(s). To date, a wide range of physiological stressors, pharmacological agents, and hormones associated with increase in the intracellular AMP/ATP ratios have been demonstrated to activate AMPK (8). AMPK is also thought to be regulated by glycogen (9), which is the major cellular storage form of carbohydrates and thus, an additional indicator of cellular energy status. Lipids are the other major energy source for cellular metabolism. Recent studies (10, 11) in heart and liver have revealed that AMPK may be sensitive to the "lipid status" of a cell, and activation may be influenced by intracellular fatty BSA, bovine serum albumin; eNOS, endothelial nitric-oxide synthase; LKB1, Peutz-Jeghers syndrome kinase LKB1; OA, okadaic acid; ONOO Ϫ , peroxynitrite; VSMC, vascular smooth muscle cell; siRNA, short interference RNA; FFA, free fatty acid; EBM, endothelial basal medium; 2-BrP, 2-bromopalmitate; HFD, high fat diet; PP2C, protein phosphatase 2C.

Background-Tetrahydrobiopterin (BH4) deficiency is reported to uncouple the enzymatic activity of endothelial nitric oxide synthase in diabetes mellitus. The mechanism by which diabetes actually leads to BH4 deficiency remains elusive. Here, we demonstrate that diabetes reduced BH4 by increasing 26S proteasome-dependent degradation of guanosine 5Ј-triphosphate cyclohydrolase I (GTPCH), a rate-limiting enzyme in the synthesis of BH4, in parallel with increased formation of both superoxide and peroxynitrite (ONOO Ϫ ). Methods and Results-Exposure

Background-Oxidative stress plays a causal role in vascular injury in diabetes mellitus, but the mechanisms and targets remain poorly understood. Methods and Results-Exposure of cultured human umbilical vein endothelial cells to either peroxynitrite (ONOO

SUMMARY1. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase involved in the regulation of cellular and organismal metabolism. AMPK has a heterotrimeric structure, consisting of a catalytic a-subunit and regulatory b-and g-subunits, each of which has two or more isoforms that are differentially expressed in various tissues and that arise from distinct genes. The AMPK system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. In addition, AMPK is activated by physiological stimuli and oxidants.2. The importance of AMPK in cardiovascular functions is best demonstrated by recent studies showing that widely used drugs, including statins, metformin and rosiglitazone, execute cardiovascular protective effects at least partly through the activation of AMPK. As a consequence, AMPK has been proposed as a candidate target for therapeutic intervention in the treatment of both Type 2 diabetes and metabolic syndrome owing to its central role in the regulation of energy balance; it may also have a role in weight control.3. In the present brief review, we summarize the recent progress of AMPK signalling and regulation focusing on vascular endothelial cells. We further hypothesize that AMPK is a dual sensor for energy and redox status within a cell and AMPK may be a therapeutic target for protecting vascular endothelial function.Key words: AMP-activated kinase, atherosclerosis, diabetes mellitus, endothelium, energy metabolism, hypertension, nitric oxide, oxidative stress, peroxynitrite, superoxide anions. OVERALL REVIEW OF AMP-ACTIVATED KINASEThe AMP-activated protein kinase (AMPK) is a serine/threonine kinase and a member of the Snf1/AMPK protein kinase family that is found in all eukaryotes. 1,2 AMPK has been proposed to act as a cellular energy sensor, which switches on catabolic pathways that produce ATP and switches off anabolic pathways that consume ATP. AMPK is a heterotrimer, containing a-, b-and g-subunits, each of which has at least two isoforms. The a-subunit contains the catalytic site; however, all subunits are necessary for full activity. Increases in the ratio of AMP to ATP activate AMPK by a number of mechanisms, including direct allosteric activation and covalent modification due to activation by an AMP-dependent AMPK kinase (AMPKK), which phosphorylates the a-subunit on Thr 172 . Until recently, it was believed that this occurred as a result of activation of AMPKK by an increase in the AMP/ATP ratio. However, recent studies from Woods et al. 3 and Carlson et al. 4 failed to demonstrate such an increase in AMPKK activity in response to various AMPK activators. Rather, their results indicate that AMP binds to AMPK and that this makes it more susceptible to phosphorylation by AMPKK. Interestingly, the first AMPKK that has been identified is LKB1, a tumour suppressor that is mutated in humans with Peutz-Jegher syndrome, 5 a disorder associated with an increased risk of developing carcinomas of the colon, stomach and pancreas. A second AMPKK has been identif...