Marie-Ann Ewart

Rosiglitazone Stimulates Nitric Oxide Synthesis in Human Aortic Endothelial Cells via AMP-activated Protein Kinase

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-γ inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.

The role of insulin and the adipocytokines in regulation of vascular endothelial function.

Vascular integrity in the healthy endothelium is maintained through the release of a variety of paracrine factors such as NO (nitric oxide). Endothelial dysfunction, characterized by reduced NO bioavailability, is associated with obesity, insulin resistance and Type II diabetes. Insulin has been demonstrated to have direct effects on the endothelium to increase NO bioavailability. Therefore altered insulin signalling in the endothelium represents a candidate mechanism underlying the association between insulin resistance and endothelial dysfunction. In recent years, it has become apparent that insulin sensitivity is regulated by the adipocytokines, a group of bioactive proteins secreted by adipose tissue. Secretion of adipocytokines is altered in obese individuals and there is increasing evidence that the adipocytokines have direct effects on the vascular endothelium. A number of current antidiabetic strategies have been demonstrated to have beneficial effects on endothelial function and to alter adipocytokine concentrations in addition to their effects on glucose homoeostasis. In this review we will explore the notion that the association between insulin resistance and endothelial dysfunction is accounted for by adipocytokine action on the endothelium. In addition, we examine the effects of weight loss, exercise and antidiabetic drugs on adipocytokine availability and endothelial function.

AMPK and vasculoprotection.

AMP-activated protein kinase (AMPK) is proposed to be a key regulator of cellular and organismal metabolism and has reported vasculoprotective effects. In addition, many therapeutic agents used in the treatment of diabetes and atherosclerosis such as metformin, thiazolidinediones and statins may exert their vasculoprotective effects through activation of AMPK. Activation of AMPK has a number of potentially beneficial anti-atherosclerotic effects including reducing adhesion of inflammatory cells to the blood vessel endothelium, reducing lipid accumulation and the proliferation of inflammatory cells caused by oxidised lipids, stimulation of gene expression responsible for cellular antioxidant defenses and stimulation of enzymes responsible for nitric oxide formation. In humans and animals the AMPK cascade triggers vascular protective mechanisms that have been shown to reduce myocardial ischaemic injury and mutations in AMPK can cause familial hypertrophic cardiomyopathy. Taken together, these data suggest that activation and function of AMPK contributes to cardiovascular health. In this review we propose to focus on the vasculoprotective effects of AMPK, the evidence for AMPK activation with currently used therapeutic agents and the potential for agents which specifically activate AMPK as a treatment for vascular disease.

Inhibition of Tumor Necrosis Factor α–Stimulated Monocyte Adhesion to Human Aortic Endothelial Cells by AMP-Activated Protein Kinase

Objective—Proatherosclerotic adhesion of leukocytes to the endothelium is attenuated by NO. As AMP-activated protein kinase (AMPK) regulates endothelial NO synthesis, we investigated the modulation of adhesion to cultured human aortic endothelial cells (HAECs) by AMPK. Methods and Results—HAECs incubated with the AMPK activator, AICAR, or expressing constitutively active AMPK demonstrated reduced TNF&agr;-stimulated adhesion of promonocytic U-937 cells. Rapid inhibition of TNF&agr;-stimulated U-937 cell adhesion by AICAR was NO-dependent, associated with unaltered cell surface adhesion molecule expression, and reduced MCP-1 secretion by HAECs. In contrast, inhibition of TNF&agr;-stimulated U-937 cell adhesion by prolonged AMPK activation was NO-independent and associated with reduced cell surface adhesion molecule expression. Conclusions—AMPK activation in HAECs inhibits TNF&agr;-stimulated leukocyte adhesion by a rapid NO-dependent mechanism associated with reduced MCP-1 secretion and a late NO-independent mechanism whereby adhesion molecule expression, in particular E-selectin, is suppressed.

Perivascular fat, AMP-activated protein kinase and vascular diseases

Perivascular adipose tissue (PVAT) is an active endocrine and paracrine organ that modulates vascular function, with implications for the pathophysiology of cardiovascular disease (CVD). Adipocytes and stromal cells contained within PVAT produce mediators (adipokines, cytokines, reactive oxygen species and gaseous compounds) with a range of paracrine effects modulating vascular smooth muscle cell contraction, proliferation and migration. However, the modulatory effect of PVAT on the vascular system in diseases, such as obesity, hypertension and atherosclerosis, remains poorly characterized. AMP‐activated protein kinase (AMPK) regulates adipocyte metabolism, adipose biology and vascular function, and hence may be a potential therapeutic target for metabolic disorders such as type 2 diabetes mellitus (T2DM) and the vascular complications associated with obesity and T2DM. The role of AMPK in PVAT or the actions of PVAT have yet to be established, however. Activation of AMPK by pharmacological agents, such as metformin and thiazolidinediones, may modulate the activity of PVAT surrounding blood vessels and thereby contribute to their beneficial effect in cardiometabolic diseases. This review will provide a current perspective on how PVAT may influence vascular function via AMPK. We will also attempt to demonstrate how modulating AMPK activity using pharmacological agents could be exploited therapeutically to treat cardiometabolic diseases.

Sex affects bone morphogenetic protein type II receptor signaling in pulmonary artery smooth muscle cells

RATIONALE Major pulmonary arterial hypertension (PAH) registries report a greater incidence of PAH in women; mutations in the bone morphogenic protein type II receptor (BMPR-II) occur in approximately 80% of patients with heritable PAH (hPAH). OBJECTIVES We addressed the hypothesis that women may be predisposed to PAH due to normally reduced basal BMPR-II signaling in human pulmonary artery smooth muscle cells (hPASMCs). METHODS We examined the BMPR-II signaling pathway in hPASMCs derived from men and women with no underlying cardiovascular disease (non-PAH hPASMCs). We also determined the development of pulmonary hypertension in male and female mice deficient in Smad1. MEASUREMENTS AND MAIN RESULTS Platelet-derived growth factor, estrogen, and serotonin induced proliferation only in non-PAH female hPASMCs. Female non-PAH hPASMCs exhibited reduced messenger RNA and protein expression of BMPR-II, the signaling intermediary Smad1, and the downstream genes, inhibitors of DNA binding proteins, Id1 and Id3. Induction of phospho-Smad1/5/8 and Id protein by BMP4 was also reduced in female hPASMCs. BMP4 induced proliferation in female, but not male, hPASMCs. However, small interfering RNA silencing of Smad1 invoked proliferative responses to BMP4 in male hPASMCs. In male hPASMCs, estrogen decreased messenger RNA and protein expression of Id genes. The estrogen metabolite 4-hydroxyestradiol decreased phospho-Smad1/5/8 and Id expression in female hPASMCs while increasing these in males commensurate with a decreased proliferative effect in male hPASMCs. Female Smad1(+/-) mice developed pulmonary hypertension (reversed by ovariectomy). CONCLUSIONS We conclude that estrogen-driven suppression of BMPR-II signaling in non-PAH hPASMCs derived from women contributes to a pro-proliferative phenotype in hPASMCs that may predispose women to PAH.

The role of AMP-activated protein kinase in the functional effects of vascular endothelial growth factor-A and -B in human aortic endothelial cells

BackgroundVascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis.MethodsHuman aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK.ResultsIncubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.ConclusionsThese data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling

The energy-sensing pathway kinase AMPK inhibits proinflammatory JAK1 signaling. New uses for AMPK activators Inflammatory cytokines stimulate the JAK-STAT pathway and promote cell survival and migration in hematopoietic cells. Chronic or excessive inflammation also contributes to diseases. Drugs that activate the kinase AMPK, such as salicylate and the diabetes drug metformin, have anti-inflammatory effects. Rutherford et al. found that AMPK phosphorylates JAK, inhibiting JAK-mediated STAT activation in vascular endothelial cells exposed to IL-6. Pharmacological activation of AMPK suppressed JAK-STAT signaling in cells expressing a JAK1 mutant associated with acute lymphoblastic leukemia (ALL). JAK inhibitors have had limited success in patients. Thus, the findings not only identify JAK1 as a substrate for AMPK in its anti-inflammatory function but also suggest that metformin might be repurposed to inhibit JAK-STAT signaling in patients with ALL and, perhaps, other inflammatory diseases. Adenosine 5′-monophosphate–activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

Oestrogen receptor alpha in pulmonary hypertension

Aims Pulmonary arterial hypertension (PAH) occurs more frequently in women with mutations in bone morphogenetic protein receptor type 2 (BMPR2) and dysfunctional BMPR2 signalling underpinning heritable PAH. We have previously shown that serotonin can uncover a pulmonary hypertensive phenotype in BMPR2+/− mice and that oestrogen can increase serotinergic signalling in human pulmonary arterial smooth muscle cells (hPASMCs). Hence, here we wished to characterize the expression of oestrogen receptors (ERs) in male and female human pulmonary arteries and have examined the influence of oestrogen and serotonin on BMPR2 and ERα expression. Methods and results By immunohistochemistry, we showed that ERα, ERβ, and G-protein-coupled receptors are expressed in human pulmonary arteries localizing mainly to the smooth muscle layer which also expresses the serotonin transporter (SERT). Protein expression of ERα protein was higher in female PAH patient hPASMCs compared with male and serotonin also increased the expression of ERα. 17β-estradiol induced proliferation of hPASMCs via ERα activation and this engaged mitogen-activated protein kinase and Akt signalling. Female mice over-expressing SERT (SERT+ mice) develop PH and the ERα antagonist MPP attenuated the development of PH in normoxic and hypoxic female SERT+ mice. The therapeutic effects of MPP were accompanied by increased expression of BMPR2 in mouse lung. Conclusion ERα is highly expressed in female hPASMCs from PAH patients and mediates oestrogen-induced proliferation of hPASMCs via mitogen-activated protein kinase and Akt signalling. Serotonin can increase ERα expression in hPASMCs and antagonism of ERα reverses serotonin-dependent PH in the mouse and increases BMPR2 expression.

Soluble receptor for advanced glycation end products (sRAGE) attenuates haemodynamic changes to chronic hypoxia in the mouse.

The calgranulin-like protein MTS1/S100A4 and the receptor for advanced glycation end-products (RAGE) have recently been implicated in mediating pulmonary arterial smooth muscle cell proliferation and vascular remodelling in experimental pulmonary arterial hypertension (PH). Here, the effects of RAGE antagonism upon 2 weeks of hypobaric hypoxia (10% O2)-induced PH in mice were assessed. Treatment with sRAGE was protective against hypobaric hypoxia-induced increases in right ventricular pressure but distal pulmonary vascular remodelling was unaffected. Intralobar pulmonary arteries from hypobaric hypoxic mice treated with sRAGE showed protection against a hypoxia-induced reduction in compliance. However, a combination of sRAGE and hypoxia also dramatically increased the force of contractions to KCl and 5-HT observed in these vessels. The acute addition of sRAGE to the organ bath produced a small, sustained contraction in intralobar pulmonary vessels and produced a synergistic enhancement of the maximal force of contraction in subsequent concentration-response curves to 5-HT. sRAGE had no effect on 5-HT-induced proliferation of Chinese hamster lung fibroblasts (CCL39), used since they have a similar pharmacological profile to mouse pulmonary fibroblasts but, surprisingly, produced a marked increase in hypoxia-induced proliferation. These data implicate RAGE as a modulator of both vasoreactivity and of proliferative processes in the response of the pulmonary circulation to chronic-hypoxia.