Nihar R. Pandey

Distinct Roles of Ca2+, Calmodulin, and Protein Kinase C in H2O2-Induced Activation of ERK1/2, p38 MAPK, and Protein Kinase B Signaling in Vascular Smooth Muscle Cells

We have shown earlier that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), two key mediators of growth-promoting and proliferative responses, are activated by hydrogen peroxide (H(2)O(2)) in A10 vascular smooth muscle cells (VSMC). In the present studies, using a series of pharmacological inhibitors, we explored the upstream mechanisms responsible for their activation in response to H(2)O(2). H(2)O(2) treatment of VSMC stimulated ERK1/2, p38 mitogen-activated protein kinase (MAPK), and PKB phosphorylation in a dose- and time-dependent fashion. BAPTA-AM and EGTA, chelators of intracellular and extracellular Ca(2+), respectively, inhibited H(2)O(2)-stimulated ERK1/2, p38 MAPK, and PKB phosphorylation. Fluphenazine, an antagonist of the Ca(2+)-binding protein calmodulin, also suppressed the enhanced phosphorylation of ERK1/2, p38 MAPK, and PKB. In contrast, the protein kinase C (PKC) inhibitors Gö 6983 and Rö 31-8220 attenuated H(2)O(2)-induced ERK1/2 phosphorylation, but had no effect on p38 MAPK and PKB phosphorylation. Taken together, these data demonstrate that the activation of Ca(2+)/calmodulin-dependent pathways represents a key component mediating the stimulatory action of H(2)O(2) on ERK1/2, p38 MAPK, and PKB phosphorylation. On the other hand, PKC appears to be an upstream modulator of the increased ERK1/2 phosphorylation, but not of p38 MAPK and PKB in response to H(2)O(2) in VSMC.

Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.

A uniquely formulated soy phospholipid, phosphatidylinositol (PI), is under development as a therapeutic agent for increasing plasma high-density lipoprotein (HDL) levels. Soy PI has been shown to increase plasma HDL and apolipoprotein A-I (apoA-I) levels in phase I human trials. Low micromolar concentrations of PI increase the secretion of apoA-I in model human hepatoma cell lines, through activation of G-protein and mitogen-activated protein (MAP) kinase pathways. Experiments were undertaken to determine the importance of the PI head group and acyl chain composition on hepatic apoA-I secretion. Phospholipids with choline and inositol head groups and one or more linoleic acid (LA) acyl chains were shown to stimulate apoA-I secretion by HepG2 cells and primary human hepatocytes. Phospholipids containing two LA groups (dilinoleoylphosphatidylcholine, DLPC) were twice as active as those with only one LA group and promoted a 4-fold stimulation in apoA-I secretion. Inhibition of cytosolic phospholipase A2 with pyrrolidine 1 (10 microM) resulted in complete attenuation of PI- and DLPC-induced apoA-I secretion. Pretreatment with the peroxisome proliferator-activated receptor alpha (PPARalpha) inhibitor MK886 (10 microM) also completely blocked PI- and DLPC-induced apoA-I secretion. Hepatic PPARalpha expression was significantly increased by both PI and DLPC. However, in contrast to that seen with the fibrate drugs, PI caused minimal inhibition of catalytic activities of cytochrome P450 and UGT1A1 enzymes. These data suggest that LA-enriched phospholipids stimulate hepatic apoA-I secretion through a MAP kinase stimulation of PPARalpha. LA-enriched phospholipids have a greater apoA-I secretory activity than the fibrate drugs and a reduced likelihood to interfere with concomitant drug therapies.

Phospholipids block nuclear factor-kappa B and tau phosphorylation and inhibit amyloid-beta secretion in human neuroblastoma cells.

Inflammation and oxidative stress have been shown to play a critical role in the pathophysiology that leads to neurodegeneration. Omega-6 phospholipids, e.g. dilinoleoylphosphatidylcholine (DLPC), have been shown to have anti-inflammatory properties and therefore experiments were undertaken to determine whether DLPC can prevent inflammatory neurodegenerative events in the model neuronal cell line, SH-SY5Y. Tumor necrosis factor (TNF-alpha) and H(2)O(2) activate mitogen-activated protein kinase (MAPK) in SH-SY5Y cells within 5 min and this activation is completely blocked by DLPC (12 microM). DLPC blocks IkappaBalpha phosphorylation in the SH-SY5Y cells and prevents the phosphorylation and activation of nuclear factor-kappa B (NF-kappaB). The phospholipid inhibits induction of MAPK and NF-kappaB in similar fashion to the MEK1/2-inhibitor, U0126 (10 microM). DLPC completely abolishes TNF-alpha, H(2)O(2) and lipopolysaccaride (LPS)-induced neuronal tau phosphorylation. Cellular amyloid precursor protein levels are reduced by DLPC and LPS-induced amyloid-beta expression and secretion in SH-SY5Y cells are completely blocked by DLPC. Taken together, these data suggest that DLPC can act through MAPK to block neuronal inflammatory cascades and prevent potential pathological consequences in the neuronal metabolism of amyloid and tau proteins.

Lipoprotein charge and vascular lipid metabolism

Lipoproteins play a central role in transporting hydrophobic molecules through the bloodstream and between specific tissues. Lipoprotein molecules have a distinctive electrical charge and changes in electrostatic properties directly affect the metabolism of the lipoprotein. Lipoprotein charge controls interfacial interactions and determines the ability of the lipoprotein to interact with intravascular enzymes and cell surface proteins. Uniquely charged constituents of the lipoprotein thereby control the metabolism of lipoproteins by creating a regulatory system wherein the electrostatic properties of plasma lipoproteins determine the fate of intravascular lipids.

Hepatic High-Density Lipoprotein Secretion Regulates the Mobilization of Cell-Surface Hepatic Lipase †

HDL acts much like heparin to liberate hepatic lipase (HL) from cell surface proteoglycans and stimulate triglyceride clearance. Experiments were undertaken to evaluate the effects of factors that stimulate the secretion of HDL from the liver on the release of HL. Treatment of HepG2 cells with linoleic acid phospholipids (LAPL) (12 muM) promotes a similar increase in the accumulation of both HDL and HL in the cell media. LAPL also induce both apoA-I and HL release from primary human hepatocytes. Dilinoleoylphosphatidylcholine has a greater effect on both apoA-I secretion and HL release than palmitoyllinoleoylphosphatidylcholine. HL released from HepG2 cells is inactive and associated with a large HDL complex containing both apoA-I and apoA-II. Inclusion of the PPARalpha inhibitor, MK-886, or MAPK inhibitor, U0126, completely blocks the LAPL-induced apoA-I and HL accumulation in the media. LAPL-treated cell lysates, however, showed no change in HL protein expression nor HL mRNA. LAPL-induced HL release appears to be a consequence of the displacement ability of newly secreted HDL. Overexpression of pre-pro-apoA-I in HepG2 cells increased HL release, while siRNA inhibition of the apoA-I gene reduced HL in the media. The data show that factors that stimulate HDL secretion in hepatocytes act to also increase the release of HL. This may partly explain why HDL therapeutics often impact plasma triglyceride levels.

An Induction in Hepatic HDL Secretion Associated with Reduced ATPase Expression

Linoleic acid-phospholipids stimulate high-density lipoprotein (HDL) net secretion from liver cells by blocking the endocytic recycling of apoA-I. Experiments were undertaken to determine whether apoA-I accumulation in the cell media is associated with membrane ATPase expression. Treatment of HepG2 cells with dilinoeoylphosphatidylcholine (DLPC) increased apoA-I secretion fourfold. DLPC also significantly reduced cell surface F1-ATPase expression and reduced cellular ATP binding cassette (ABC)A1 and ABCG1 protein levels by approximately 50%. In addition, treatment of HepG2 cells with the ABC transporter inhibitor, glyburide, stimulated the apoA-I secretory effects of both DLPC and clofibrate. Pretreatment of HepG2 cells with compounds that increased ABC transport protein levels (TO901317, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, and resveratrol) blocked the DLPC-induced stimulation in apoA-I net secretion. Furthermore, whereas HepG2 cells normally secrete nascent prebeta-HDL, DLPC treatment promoted secretion of alpha-migrating HDL particles. These data show that an linoleic acid-phospholipid induced stimulation in hepatic HDL secretion is related to the expression and function of membrane ATP metabolizing proteins.

Phosphatidylinositol acts through mitogen-activated protein kinase to stimulate hepatic apolipoprotein A-I secretion.

Phosphatidylinositol (PI) has been shown to stimulate reverse cholesterol transport in animal models and to increase plasma apolipoprotein (apo) A-I levels and high-density lipoprotein cholesterol in human subjects. The objective of this study was to determine the molecular mechanism through which PI stimulates apo A-I secretion in hepatic cells. PI (12 mumol/L) significantly stimulates apo A-I secretion from HepG2 cells over 24 hours. The stimulation in apo A-I secretion is completely blocked by phospholipase C inhibitors (D609 and U73122) and the Ras inhibitor sulindac sulfide. Apolipoprotein A-I secretion is augmented with a protein kinase C agonist (dioctanoyl glycerol) and inhibited by a protein kinase C inhibitor (dioleoyl ethylene glycol). The PI-induced apo A-I secretion is unaffected by PI-3-kinase inhibitors but is sensitive to mitogen-activated protein kinase (MAPK) inhibitors. Whereas the p38MAPK inhibitor SB203580 has no effect on PI-induced apo A-I secretion, the MAPK kinase 1/2 inhibitor U0126 and the c-Jun-N-terminal kinase/stress-activated protein kinase inhibitor SP600125 block PI-induced apo A-I secretion. PI also increased extracellular-regulated protein kinase 1 and 2 phosphorylation in HepG2 cells in a time-dependent manner. PI does not appear to stimulate apo A-I gene transcription, as cellular apo A-I messenger RNA levels remained unchanged over the 24-hour incubation. However, PI significantly decreases apo A-I binding and degradation in HepG2 cells. Collectively, the data suggest that PI acts through MAPK pathways to increase plasma apo A-I levels by protecting it from reuptake and degradation.

Cardiovascular effects of tight versus usual blood-pressure control

We would like to draw the attention of readers to some important limitations of the study by Paolo Verdecchia and colleagues (Aug 15, p 525). First, the study is solely powered on left-ventricular hypertrophy (LVH) as the primary outcome of systolic hypertension. However, angiotensinreceptor blockers (ARBs) can prevent LVH independently of their ability to control blood pressure. In Verdecchia and colleagues’ study, the use of ARBs was a signifi cant 17% higher in the tight-control group than in the usual-control group, which could have reduced the incidence of LVH in itself. Second, Verdecchia and colleagues do not detail the diff erence in combination therapy in each group. Combination antihypertensive therapy, especially dual blockage of the renin-angiotensin system, regresses LVH more quickly and completely than do single agents. Further analysis of the results with a view to comparing various antihypertensive combinations used in both the groups is required before the lesser incidence of LVH is attributed solely to stricter blood pressure control. Third, Verdecchia and colleagues mention intensifi cation and downtitration of treatment. However, they do not mention the total incidence of such episodes during the whole period. In our opinion it is important to know how many such “events” took place. Finally, although Verdecchia and colleagues recognise the eff ect of lack of double-blinding on clinical decisions related to secondary outcome events, they do not acknowledge that this bias may have indirectly contributed to the higher incidence of LVH in the usual-control group. The bias in two treatment groups is refl ected by the fact that usual-control of blood pressure was achieved in 64% of patients at 1 year and in 67% at 2 years, whereas for the tight-control group the fi gures were 75% and 79%, respectively.

Day or night blood pressures for prognosis

114 www.thelancet.com Vol 371 January 12, 2008 high blood pressure now represents. New guidelines acknowledge global risk assessment rather than single factors, even one as central as blood pressure. Laurent and colleagues and ourselves pioneered the prognostic usefulness of non-invasive yet direct assessment of arterial structure and function or “stiff ness” by use of aortic pulse-wave velocity. This simple (if for now still relatively expensive) method is a powerful index of prognosis in all 12 studies reported so far, independent of blood pressure. All other classic risk factors, including age, smoking, blood lipids, diabetes, etc, also act on the large-vessel walls, as does peripheral resistance from small vessels, so aortic pulse-wave velocity probably represents an integrated estimate of risk, not just that from a single factor. Measures of arterial stiff ness taken at one time point with good reproducibility surely represent a potentially better use of resources than day or night pressures alone or together. That hypothesis holds even if aortic pulse-wave velocity has not yet been studied for eff ects of treatment.