Prog. Lipid Res. Vol. 29, pp. 45-63, 1990 Printed in Great Britain. All rights reserved

LIPID METABOLISM AND SIGNAL TRANSDUCTION IN ENDOTHELIAL CELLS RALPH

E.

WHATLEY,

GUY

and

A.

ZIMMERMAN,

STEPHEN

THOMAS

M. MCINTYRE

M. PRESCOTT

Nora Eccles Harrison Cardiovascular Research and Training Institute and the Departments of Medicine and Biochemistry, University o/Utah School of Medicine and the Veterans Affairs Medical Center, Salt Lake City, Utah 84112, U.S.A. CONTENTS I.

INTRODUCTION

II. LIPID METABOLISMBY ENDOTHELIALCELLS A. Overview of lipid metabolism by endothelial cells B. Arachidonic acid metabolism I. Uptake and incorporation of arachidonate by endothelial cells 2. Pathways of arachidonate metabolism in endothelial cells 3. Effect of other fatty acids on arachidonate metabolism in endothelial cells 4. Methods for studying arachidonate metabolism in endothelial cells C. Platelet-activating factor metabolism I. Synthesis of PAF by endothelial cells 2. Methods for studying PAF production in endothelial cells 3. Relationship of PAF production to arachidonate metabolism D. Production of other bioactive lipids by endothelial cells III. SIGNAL TRANSDUCTIONIN ENDOTHELIALCELL LIPID METABOLISM A. Overview B. Hormonal stimulation of endothelial cell lipid metabolism C. The role of calcium in endothelial cell signal transduction D. The role of calcium in lipid metabolism E. Protein kinases as regulators of lipid metabolism IV. SUMMARY ACKNOWLEDGEMENTS f&PERENCE.S

45 46 46 46 46 48 48 49 49 49

51 52 52 53 53 54 54 56 57 58 60 60

I. INTRODUCTION

There is abundant evidence that the endothelium is actively involved in processes that affect human health and disease. Its unique position, interposed between the intravascular and extravascular spaces, allows the endothelial cell to play a key role in regulating vascular homeostasis. The active processes of the endothelial cell include secretion,52**3 modulation of coagulation components,g”3~3*~‘2’interaction with neutrophils, platelets and other inflammatory cells7~10~2gJ7~s5*‘6’*‘63 and production of bioactive lipids such as arachidonic acid metabolites45*63*7’*‘4g and platelet-activating factor. 23*86~“6~‘53 In the basal physiologic state, these processes must provide a non-thrombogenic, non-inflammatory vascular lining so that uncontrolled inflammation and coagulation do not occur. In contrast, the endothelial cell must respond to injury by activation of procoagulants and production of inflammatory mediators.g*ss There is increasing evidence that lipids produced by the endothelial cell play a central role in both vascular homeostasis and the response to injury.37*73 These lipids include oxygenated arachidonate metabolites (eicosanoids) and platelet-activating factor (1-O -alkyl-2-acetyl-sn -glycero-3-phosphocholine; PAF). The biologic potential of these lipids is suggested by their potent vasoactive and pro-inflammatory effects.53*‘0s Although their precise role in vascular physiology remains undefined, their production in response to specific stimuli and their potent effects suggests an important role. In addition, the endothelial cell uses several lipid second messengers in the regulation of intracellular events that contribute to vascular homeostasis. These include inositol phosphates and diacylglycerol generated by phospholipase C-mediated hydrolysis of phosphoinositides.5’*78 Inositol phosphates affect calcium homeostasis, and diacylglycerol is a physiologic regulator of protein kinase C. Consequently, when appropriately stimulated, the endothelial 45

R. E. Whatley

46

et al.

cell produces lipids that likely have potent extracellular functions (eicosanoids, plateletactivating factor) as well as lipids that are important intracellular regulators. Ideally, this lipid metabolism contributes to vascular integrity in the steady-state and provides a salutory response to injury when necessary. Thus, a description of the pathways and regulation of lipid biosynthesis in the endothelial cell is critical to understanding vascular physiology and disease. II.

LIPID

METABOLISM

BY

ENDOTHELIAL

CELLS

A. Overview of Lipid Metabolism by Endothelial Cells The lipid composition

of cultured

endothelial

cells has been studied by several different from that of other types of mammalian cells in culture. In some cases, pathways of lipid uptake and metabolism have been elucidated in endothelial ce11s;‘9.82.9O.92,‘3?.134.I4I,I42,ISO however, specific details of many aspects of endothelial cell lipid metabolism are inferred by analogy to other cell types.13’ Endothelial cells take up fatty acids and other lipid precursors from culture medium and incorporate them into phospholipids, mono-, di- and triglycerides.5g.‘34 By means of specific receptors, endothelial cells also bind and incorporate lipoprotein particles.6g In addition, cultured endothelial cells are capable of modifying low density lipoprotein (LDL) particles in a form that binds to the LDL scavenger receptor on macrophages,s7 a process that may be relevant to atherogenesis. Thus, although endothelial cell lipid metabolism has clear similarities to other cell types, there are differentiated pathways of lipid metabolism that are physiologically and pathologically important. The ability to produce eicosanoids and other bioactive lipids in response to specific stimuli, while not a unique property of endothelial cells, is a differentiated function that is likely an important component of vascular homeostasis. investigators62390*1 19.134*150 and is not fundamentally

B. Arachidonic Acid Metabolism 1. Uptake and Incorporation

of Arachidonate by Endothelial Cells

Endothelial cells take up arachidonic acid, incorporate it into cellular lipids, and, when appropriately stimulated, release and metabolize it to a variety of oxygenated compounds termed eicosanoids. Arachidonate, an essential fatty acid for humans, is derived primarily from dietary sources and is incorporated intact into cellular lipids. Although not well studied in endothelial cells, arachidonate-specific acyl-CoA synthetases have been found in platelets’07~is7 and lymphocytes;i4’ presumably, similar mechanisms are present in endothelial cells to facilitate incorporation of arachidonate into cellular lipids. In addition, endothelial cells have the capacity to incorporate arachidonate contained in lipoprotein particles. Arachidonate esterified to cholesterol in high density lipoproteins (HDL) and LDL particles can be taken up by cultured endothelial cells, hydrolyzed and re-esterified to phospho- and neutral lipids and metabolized to eicosanoids upon stimulation of the cell~.“~*“~ The relative contribution of this source of arachidonate to the total pool is not known. Notably, human endothelial cells under usual culture conditions do not convert significant amounts of linoleate to arachidonate. 136Linoleate is converted to arachidonate by successive elongation and desaturation steps, a pathway that is present in some types of cultured cells. Although cultured endothelial cells can elongate linoleate (18:2) to docosadienoic acid (20:2), they lack the A6 desaturase needed to convert docosadienoic acid to eicosatrienoic acid (20:3), the immediate precursor of arachidonate (20:4).136 This presumably is the case in vivo as well, although studies have demonstrated that the A6 desaturase, which is limiting, can be induced in lipid-free medium.122 Arachidonate is primarily incorporated into endothelial cell phospholipids (Table 1) with a smaller percentage in mono-, di- and triglycerides and comprises ca. 10-20 mole percent of cellular

Lipid TABLE Phospholipid

1. Distribution

and signal

of Arachidonic

Acid

Percent of total Human1

class

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Others (sohinaomvelin.

metabolism

cardiolipin)

30 46 I9 5 NR

transduction

in Endothelial arachidonate* Porcine$ 33 46 I9 1.4 I.2

47 Cell Phospholipids Mole Human1 I 20 33 7 NR

percent

of FAT Porcine$ 6 I7 30 12