The Effect of Different Glucose Concentrations on the Antiproliferative Activity of Metformin in MCF-7 Breast Cancer Cells.

The glucose-lowering drug metformin has been reported to have anticancer properties through unknown mechanisms. Other unknown factors that may influence its anticancer potential include the glycemic status of the patient. Therefore, the objective of this study is to determine the effect of different glucose environments on the antiproliferative potency and the cellular mechanism of action of metformin. Human breast cancer cells, MCF-7, were incubated in low, normal, elevated, and high glucose environments and treated with metformin. The antiproliferative potential of metformin and its effect on protein expression as well as its ability to induce cellular apoptosis and autophagy under different glucose environments, were determined using different molecular techniques. Metformin significantly inhibited cellular proliferation in a time- and glucose-concentration-dependent manner. In comparison to elevated glucose, low normal glucose alone induced a significant level of autophagy that was further increased in the presence of metformin. While glucose concentration did not appear to have an effect on the antiproliferative potency of metformin, the cellular basis of action was shown to be glucose-dependent. The antiproliferative mechanism of action of metformin in elevated and low normal glucose environments is mTOR-dependent, whereas, in the high glucose environment, the antiproliferative mechanism is independent of mTOR. This is the first study to report that both the antiproliferative potency and the cellular mechanism of action aredependent on the concentration of glucose.

Angiogenic content of microparticles in patients with diabetes and coronary artery disease predicts networks of endothelial dysfunction.

BACKGROUND: Elevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD). METHODS: A total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs' apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array. RESULTS: Levels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-ß1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-ß1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes. CONCLUSION: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction.

Hyperglycaemia disrupts conducted vasodilation in the resistance vasculature of db/db mice.

Vascular dysfunction in small resistance arteries is observed during chronic elevations in blood glucose. Hyperglycaemia-associated effects on endothelium-dependent vasodilation have been well characterized, but effects on conducted vasodilation in the resistance vasculature are not known. Small mesenteric arteries were isolated from healthy and diabetic db/db mice, which were used as a model of chronic hyperglycaemia. Endothelium-dependent vasodilation via the Gq/11-coupled proteinase activated receptor 2 (PAR2) was stimulated with the selective agonist SLIGRL. The Ca2+-sensitive fluorescent indicator fluo-8 reported changes in endothelial cell (EC) [Ca2+]i, and triple cannulated bifurcating mesenteric arteries were used to study conducted vasodilation. Chronic hyperglycaemia did not affect either EC Ca2+ or local vasodilation to SLIGRL. However, both acute and chronic exposure to high glucose or the mannitol osmotic control attenuated conducted vasodilation to 10µM SLIGRL. This investigation demonstrates for the first time that a hypertonic solution containing glucose or mannitol can interfere with the spread of a hyperpolarizing current along the endothelium in a physiological setting. Our findings reiterate the importance of studying the effects of hyperglycaemia in the vasculature, and provide the basis for further studies regarding the modulation of junctional proteins involved in cell to cell communication by diseases such as diabetes.

Challenges in the Biomedical Research Enterprise in the 21st century: Antecedents in the writings of David Triggle.

David Triggle's career as an educator, researcher, essayist and ethicist in many ways has paralleled the post WWII emergence of the "biomedical research ecosystem" that originated in the concept of the "Endless Frontier". In the ensuing 70 years biomedical research has irreparably changed the nature of society with vaccines, the birth control pill and new generations of drugs and biologics to treat infections, sexually transmitted diseases, psychiatric disorders and cardiovascular diseases. These have led to a shift in the population demographic to the elderly and the chronically sick leading to major issues in the provision of effective and affordable healthcare for much of the world's population. In the present article, in addition to providing a brief biography and an appreciation of David Triggle's contributions to science, the authors expand on three topics in which he has had a major interest: improving the quality, reproducibility and relevance of basic biomedical research; declining career opportunities for graduates in an era of decreased funding-the end of the "Endless Frontier"; and the dynamic between the provision for universal healthcare and the dysfunctional, wasteful and politicized systems of the current medical-industrial complex in delivering healthcare.

Proteinase-activated receptors 1 and 2 and the regulation of porcine coronary artery contractility: a role for distinct tyrosine kinase pathways.

BACKGROUND AND PURPOSE: Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. EXPERIMENTAL APPROACH: Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1 /PAR2 -activating peptides, angiotensin-II, PGF2α , EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). KEY RESULTS: AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1 /PAR2 agonists, EGF and angiotensin-II, but not by PGF2α , the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1 /PAR2 -activating peptides and angiotensin-II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 >> PAR1 ) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α . CONCLUSION AND IMPLICATIONS: PAR1/2 -mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm.

Cytochrome P450 products and arachidonic acid-induced, non-store-operated, Ca2+ entry in cultured bovine endothelial cells.

Endothelial cells possess multiple mechanisms for the control of Ca2+ influx during agonist and mechanical stimulation. Increased intracellular Ca2+ during such events is important in the production of vasoactive substances including NO, prostacyclin, and, possibly, endothelium-derived hyperpolarizing factor(s). The present studies examined the effect of arachidonic acid on cellular Ca2+ entry and the underlying mechanisms by which this fatty acid regulates entry. Studies were conducted in cultured bovine aortic endothelial cells (passages 3 to 6) with changes in intracellular Ca2+ determined using the fluorescent Ca2+-sensitive indicator fura 2. Arachidonic acid (1 to 50 microM) stimulated Ca2+ entry from the superfusate without affecting Ca2+ release from intracellular stores. 2-aminoethoxydiphenyl borate (2APB) (100 microM) added at the peak of Ca2+ entry did not inhibit arachidonic acid-induced Ca2+ entry but, in contrast, significantly inhibited entry stimulated by ATP (1 microM). Arachidonic acid-induced Ca2+ entry was inhibited by econazole (1 microM), but not indomethacin (10 microM) or nordihydroguairetic acid (10 microM), suggesting the involvement of cytochrome P450 monooxygenase metabolite of arachidonic acid. Oleic acid (10 microM) was ineffective in inducing Ca2+ entry, whereas linoleic acid (10 microM) stimulated Ca2+ entry but by a mechanism insensitive to econazole. Collectively the data demonstrate that primary cultured aortic endothelial cells possess a Ca2+ entry mechanism modulated by arachidonic acid. This mode of Ca2+ entry appears to operate independently of store depletion-mediated mechanisms.

Endothelin blockade potentiates endothelial protective effects of ACE inhibitors in saphenous veins.

BACKGROUND: Angiotensin II and endothelin-1 are potent endothelium-derived contracting factors. The effects of acute endothelin antagonism on endothelial function in saphenous vein from patients treated with and without angiotensin-converting enzyme inhibitors were compared. METHODS: Vascular segments of saphenous vein were obtained perioperatively from 14 patients on angiotensin-converting enzyme inhibitors and 29 controls. In vitro endothelium-dependent and -independent responses to acetylcholine and sodium nitroprusside were assessed by constructing isometric dose-response curves in precontracted rings in the presence and absence of bosentan (endothelinA/B receptor antagonist) and BQ-123 (endothelinA antagonist) using isolated organ baths. Percent maximum relaxation and sensitivity were compared between interventions. RESULTS: Endothelium-dependent relaxation to acetylcholine was augmented in the angiotensin-converting enzyme inhibitor-treated group (p < 0.005). Both specific and mixed endothelin receptor blockade improved acetylcholine-mediated relaxation in the angiotensin-converting enzyme inhibitor-treated and untreated groups (p < 0.02). The effects of these antagonists were endothelium specific as endothelium-independent responses to sodium nitroprusside remain unaltered. CONCLUSIONS: These data demonstrate that (1) chronic angiotensin-converting enzyme inhibition improves endothelial function in saphenous veins, and (2) this effect can be further augmented by acute endothelin blockade. These data suggest that antagonism of both angiotensin II and endothelin may be important in attenuating saphenous vein arteriosclerosis.