Regulation of Placental Growth Factor by Fluid Shear Stress in the Vessel Wall

Arteriogenesis is the process by which mature arteries form from collateral arterioles after upstream arterial stenosis or occlusion. During collateral remodeling, monocytes are recruited to the vessel wall. Placental growth factor (PLGF) is a potent stimulator of arteriogenesis via monocyte recruitment. Although fluid shear stress (FSS) is thought to be the primary signal for arteriogenesis, its role in regulating PLGF expression is unknown. However, PLGF is increased in collaterals by upstream arterial ligation. Therefore, we hypothesized that PLGF expression is regulated by FSS. To test this hypothesis, we created an in vitro model of coronary vessels, consisting of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) cocultured on porous Transwell inserts. We used a cone and plate apparatus to expose the HCAEC of the coculture to different FSS waveforms. We also employed an ex vivo model consisting of isolated mesenteric arterioles perfused at various flow rates. Flow or FSS significantly increased PLGF expression at both the mRNA and protein level, and this effect was dependent on the duration and magnitude of FSS, confirming our hypothesis. PLGF was predominantly produced by HCAEC, but HCASMC also played a role in modulating HCAEC PLGF expression and response to FSS. Next, we set out to identify the signaling pathways mediating the effects of FSS on PLGF. Hydrogen peroxide and NADPH oxidase 4 were required for FSS to upregulate PLGF. Similarly, heme oxygenase 1 activity was also required for FSS to affect PLGF expression. Heme oxygenase 1 also mediated the effects of hypoxia on PLGF, and our studies suggest the existence of a common pathway between these two stimuli (FSS and hypoxia). This study builds on several recent studies using various redox knockout mice to study vascular remodeling. It further emphasizes the importance of PLGF for arteriogenesis and presents a novel possible mechanism by which arteriogenesis progresses.Veterinary Biomedical Science

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice

Supraphysiological levels of the osteoblast‐enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase‐1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast‐specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6‐week‐old mice lacking osteoblast NPP1 expression (osteoblast‐specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast‐specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast‐specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin–sensitizing under‐carboxylated osteocalcin (195% increase; p < .05). However, following high‐fat‐diet challenge, osteoblast‐specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity

Glial Growth Factor 2 Regulates Glucose Transport in Healthy Cardiac Myocytes and During Myocardial Infarction via an Akt-Dependent Pathway

Neuregulin (NRG), a paracrine factor in myocytes, promotes cardiac development via the ErbB receptors. NRG-1β also improves cardiac function and cell survival after myocardial infarction (MI), although the mechanisms underlying these cardioprotective effects are not well elucidated. Increased glucose uptake has been shown to be cardio-protective during MI. We hypothesized that treatment with a recombinant version of NRG-1β, glial growth factor 2 (GGF2), will enhance glucose transport in the healthy myocardium and during MI. Cardiac myocytes were isolated from MI and healthy adult rats, and subsequently incubated with or without insulin or GGF2. Glucose uptake was measured using a fluorescent D-glucose analog. The translocation of glucose transporter (GLUT) 4 to the cell surface, the rate-limiting step in glucose uptake, was measured using a photolabeled biotinylation assay in isolated myocytes. Similar to insulin, acute in vitro GGF2 treatment increased glucose uptake in healthy cardiac myocytes (by 40 and 49%, respectively, P = 0.002). GGF2 treatment also increased GLUT4 translocation in healthy myocytes by 184% (P &lt; 0.01), while ErbB 2/4 receptor blockade (by afatinib) abolished these effects. In addition, GGF2 treatment enhanced Akt phosphorylation (at both threonine and serine sites, by 75 and 139%, respectively, P = 0.029 and P = 0.01), which was blunted by ErbB 2/4 receptor blockade. GGF2 treatment increased the phosphorylation of AS160 (an Akt effector) by 72% (P &lt; 0.05), as well as the phosphorylation of PDK-1 and PKC (by 118 and 92%, respectively, P &lt; 0.05). During MI, cardiac GLUT4 translocation was downregulated by 44% (P = 0.004) and was partially rescued by both in vitro insulin and GGF2 treatment. Our data demonstrate that acute GGF2 treatment increased glucose transport in cardiac myocytes by activating the ErbB 2/4 receptors and subsequent key downstream effectors (i.e., PDK-1, Akt, AS160, and PKC). These findings highlight novel mechanisms of action of GGF2, which warrant further investigation in patients with heart failure

A novel role for the mineralocorticoid receptor in glucocorticoid driven vascular calcification

AbstractVascular calcification, which is common in the elderly and in patients with atherosclerosis, diabetes and chronic renal disease, increases the risk of cardiovascular morbidity and mortality. It is a complex, active and highly regulated cellular process that resembles physiological bone formation. It has previously been established that pharmacological doses of glucocorticoids facilitate arterial calcification. However, the consequences for vascular calcification of endogenous glucocorticoid elevation have yet to be established. Glucocorticoids (cortisol, corticosterone) are released from the adrenal gland, but can also be generated within cells from 11-keto metabolites of glucocorticoids (cortisone, 11-dehydrocorticosterone [11-DHC]) by the enzyme, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In the current study we hypothesized that endogenous glucocorticoids facilitate vascular smooth muscle cell (VSMC) calcification and investigated the receptor-mediated mechanism underpinning this process.In vitro studies revealed increased phosphate-induced calcification in mouse VSMCs following treatment for 7days with corticosterone (100nM; 7.98 fold; P<0.01), 11-DHC (100nM; 7.14 fold; P<0.05) and dexamethasone (10nM; 7.16 fold; P<0.05), a synthetic glucocorticoid used as a positive control. Inhibition of 11β-HSD isoenzymes by 10μM carbenoxolone reduced the calcification induced by 11-DHC (0.37 fold compared to treatment with 11-DHC alone; P<0.05). The glucocorticoid receptor (GR) antagonist mifepristone (10μM) had no effect on VSMC calcification in response to corticosterone or 11-DHC. In contrast, the mineralocorticoid receptor (MR) antagonist eplerenone (10μM) significantly decreased corticosterone- (0.81 fold compared to treatment with corticosterone alone; P<0.01) and 11-DHC-driven (0.64 fold compared to treatment with 11-DHC alone; P<0.01) VSMC calcification, suggesting this glucocorticoid effect is MR-driven and not GR-driven. Neither corticosterone nor 11-DHC altered the mRNA levels of the osteogenic markers PiT-1, Osx and Bmp2. However, DAPI staining of pyknotic nuclei and flow cytometry analysis of surface Annexin V expression showed that corticosterone induced apoptosis in VSMCs.This study suggests that in mouse VSMCs, corticosterone acts through the MR to induce pro-calcification effects, and identifies 11β-HSD-inhibition as a novel potential treatment for vascular calcification

New perspectives on rare connective tissue calcifying diseases

Connective tissue calcifying diseases (CTCs) are characterized by abnormal calcium deposition in connective tissues. CTCs are caused by multiple factors including chronic diseases (Type II diabetes mellitus, chronic kidney disease), the use of pharmaceuticals (e.g. warfarin, glucocorticoids) and inherited rare genetic diseases such as pseudoxanthoma elasticum (PXE), generalized arterial calcification in infancy (GACI) and Keutel syndrome (KTLS). This review explores our current knowledge of these rare inherited CTCs, and highlights the most promising avenues for pharmaceutical intervention. Advancing our understanding of rare inherited forms of CTC is not only essential for the development of therapeutic strategies for patients suffering from these diseases, but also fundamental to delineating the mechanisms underpinning acquired chronic forms of CTC

The effects of prenatal hypoxia on the levels of the α-subunits of G proteins in the heart of the Broiler chicken (Gallus gallus)

Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit

Synthesis and Biological Evaluation of Some New Pyridines, Isoxazoles and Isoxazolopyridazines Bearing 1,2,3-Triazole Moiety

Some new isoxazole derivatives 3a-d were synthesized via the reaction of 3-(dimethylamino)-1-(5-methyl-1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)prop-2-en-1-one (1) with different hydroximoyl chlorides derivatives 2a-d. From these new isoxazoles 3a-d a new series of isoxazolopyridazines 4a-d was derived using hydrazine hydrate. In addition, enaminone 1 was reacted with ethyl acetoacetate to afford the corresponding ester derivative 6, the latter was submitted to react with different chemical reagents to obtain a variety of bioactive substituted pyridine derivatives. The azido derivative 14, was used as the key molecule for the synthesis of new urea and aryl carbamate derivatives upon its reaction with different amines and phenol through Curtius rearrangement. The chemical structures of all new compounds were investigated from their spectral and microanalytical data. The synthesized compounds were tested for their pharmacological potency as, anti-hepatic cancer and anti-microbial agents. Most of the tested compounds showed good anti-hepatic cancer results comparing with the standard drug doxorubicin especially when their toxic effects on the normal cell lines were studied. Referring to the anti-microbial test most of the compounds showed strong effects