Current methods for purifying caveolae from tissue culture cells take advantage of the Triton X-100 insolubility of this membrane domain. To circumvent the use of detergents, we have developed a method that depends upon the unique buoyant density of caveolae membrane. The caveolae fractions that we obtain are highly enriched in caveolin. As a consequence we are able to identify caveolae-associated proteins that had previously gone undetected. Moreover, resident caveolae proteins that are soluble in Triton X-100 are retained during the isolation.

Abstract-It is well recognized that high-density lipoprotein (HDL)-cholesterol is antiatherogenic and serves a role in mediating cholesterol efflux from cells. However, HDL has multiple additional endothelial and antithrombotic actions that may also afford cardiovascular protection. HDL promotes the production of the atheroprotective signaling molecule nitric oxide (NO) by upregulating endothelial NO synthase (eNOS) expression, by maintaining the lipid environment in caveolae where eNOS is colocalized with partner signaling molecules, and by stimulating eNOS as a result of kinase cascade activation by the high-affinity HDL receptor scavenger receptor class B type I (SR-BI). HDL also protects endothelial cells from apoptosis and promotes their growth and their migration via SR-BI-initiated signaling. As importantly, there is evidence of a variety of mechanisms by which HDL is antithrombotic and thereby protective against arterial and venous thrombosis, including through the activation of prostacyclin synthesis. The antithrombotic properties may also be related to the abilities of HDL to attenuate the expression of tissue factor and selectins, to downregulate thrombin generation via the protein C pathway, and to directly and indirectly blunt platelet activation. Thus, in addition to its cholesterol-transporting properties, HDL favorably regulates endothelial cell phenotype and reduces the risk of thrombosis. With further investigation and resulting greater depth of understanding, these mechanisms may be harnessed to provide new prophylactic and therapeutic strategies to combat atherosclerosis and thrombotic disorders. Key Words: atherosclerosis Ⅲ endothelial nitric oxide synthase Ⅲ endothelium Ⅲ HDL Ⅲ nitric oxide Ⅲ prostacyclin Ⅲ protein C Ⅲ thrombin Ⅲ thrombosis T he risk of atherosclerosis is inversely related to circulating levels of high-density lipoprotein cholesterol (HDL-C), 1,2 and the Framingham Heart Study demonstrated that the association is independent of low-density lipoprotein (LDL) cholesterol. 3,4 In addition, clinical trials with agents that increase HDL show that elevations in the lipoprotein decrease the incidence of cardiovascular events. 5-7 Furthermore, there is evidence that the risk for restenosis following a vascular intervention is inversely related to HDL. 8,9 HDL classically functions in reverse cholesterol transport (RCT), removing cholesterol from peripheral tissues and delivering it to the liver and to steroidogenic organs by binding of the major HDL apolipoprotein apolipoprotein A-I (apoA-I) to the highaffinity HDL receptor scavenger receptor B type I (SR-BI). 10,11 In mouse models of atherosclerosis, both apoA-I and SR-BI provide atheroprotection, 12,13 and the provision of apoA-I or HDL also attenuates neointima formation after artery injury in the context of experimental hypercholesterolemia. 14,15 The protective nature of HDL has been previously attributed to its role in RCT. However, the basis for Original

An essential step in the epidermal growth factor (EGF)-dependent activation of MAP kinase is the recruitment of Raf-1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf-1 began to appear in caveolae but not in non-caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf-1 in this fraction was able to phosphorylate MAP kinase kinase, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overexpression of HaRas V12 caused the recruitment of Raf-1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA-5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.

Summary To date estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells and it stimulates cell-autonomous, ER-dependent and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally-modulated, non-aromatized ER ligand that promotes ER+ breast tumor growth.

Abstract-Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) ␣-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ER␣ and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E 2 , 10 -8 mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ER␣ antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ER␣ displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER␣ plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ER␣ protein in caveolae, and E 2 caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E 2 on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ER␣ is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2000;87:e44-e52.) Key Words: acetylcholine Ⅲ bradykinin Ⅲ caveolin Ⅲ cell membrane Ⅲ endothelium Ⅲ estrogens T he hormone estrogen classically exerts its effects by modifying gene expression through the activation of estrogen receptors (ERs), which serve as transcription factors. 1-3 However, there are also rapid, presumably nongenomic effects of estrogen in a variety of tissues including the vasculature. 4 -6 Estrogen has important atheroprotective properties that are at least partially related to its capacity to enhance the bioavailability of nitric oxide (NO). [5][6][7] NO is a potent regulator of blood pressure, platelet aggregation, leukocyte adhesion, and vascular smooth muscle mitogenesis that is produced in the vascular wall primarily by the endothelial isoform of NO synthase (eNOS) on the conversion of the substrate L-arginine to L-citrulline. 8 The function of the L-arginine/eNOS system is altered in a variety of vascular disorders. 9 We have previously shown that estrogen rapidly stimulates eNOS activity in endothelial cells, that the response is attenuated by ER antagonism but not by inhibiting gene transcription, and that ER␣ is expressed in endothelium. 10,11 We have also shown that the overexpression of ER␣ in endothelial cells causes enhancement of the acute response to estradiol (E 2 ) that is blocked by ER antagonism, specific to E 2 versus other agonists, and dependent on the ER␣ hormone binding domain. In addition, the acute stimulation of eNOS by E 2 can be reconstituted in COS-7 cells cotransfected with wild-type ER␣ and eNOS. 11 Thus, the short-term ef...

The risk for cardiovascular disease from atherosclerosis is inversely proportional to serum levels of high density lipoprotein (HDL) 1 (1, 2). HDL classically serves to remove cholesterol from peripheral tissues in a process known as reverse cholesterol transport. However, the mechanisms by which HDL is atheroprotective are complex and not fully understood, since circulating levels of HDL and the major HDL apolipoprotein, apolipoprotein A-I, do not regulate reverse cholesterol transport (3). We previously reported that HDL stimulates endothelial nitric-oxide synthase (eNOS) activity in endothelial cells (EC) through apolipoprotein A-I binding to scavenger receptor type I (SR-BI), the high affinity HDL receptor (4). Similarly, HDL enhances endothelium-and NO-dependent relaxation in aortas from wild-type but not SR-BI knock-out mice. Recently, Li et al. (5) also reported that HDL binding to SR-BI activates eNOS. The HDL-induced increase in NO production may be critical to the atheroprotective features of HDL, since diminished bioavailablity of endothelium-derived NO has a key role in the early pathogenesis of hypercholesterolemia-induced vascular disease and atherosclerosis (6 -8). However, the mechanisms by which HDL activates eNOS are yet to be clarified. eNOS is one of three isoenzymes that convert L-arginine to L-citrulline plus NO. The activity of eNOS is regulated by complex signal transduction pathways that involve various phosphorylation events and protein-protein interactions. Many stimuli modulate eNOS activity by activating kinases that alter the phosphorylation of the enzyme (9 -15). Akt kinase (also known as PKB) activates eNOS by directly phosphorylating the enzyme at Ser-1179 (16 -19). Akt itself is phosphorylated and activated by phosphoinositide 3-kinase (PI3 kinase), which in turn is activated by a tyrosine kinase (TK). Both receptor TK and nonreceptor TK are involved in PI3 kinase-Akt mediated eNOS activation by various agonists (19 -22). In contrast to Ser-1179, phosphorylation of Thr-497 of eNOS attenuates enzyme activity (12,14,15). eNOS is also modulated by MAP kinases (23, 24), and unlike Akt, the effect of MAP kinases on eNOS activity can be either positive or negative (9,(25)(26)(27). The role of kinase cascades in signaling by HDL from SR-BI to eNOS is entirely unknown. To better understand the basis of HDL action in endothelium, the present investigation was designed to test the hypothesis that HDL activation of eNOS entails the phosphorylation of the enzyme. We also studied the potential roles of specific kinase cascades in HDL-mediated eNOS stimulation. Using pharmacological inhibition or dominant negative mutant forms of selective kinases in EC or COS M6 cells transfected with eNOS and the HDL receptor, SR-BI, we investigated the involvement of tyrosine kinases, PI3 kinase, Akt, and MAP kinases in HDL-mediated eNOS activation. In addition to improving our specific understanding of eNOS modulation, the elucidation of the signaling cascade(s) coupling SR-BI to the enzyme provides impor...

Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-I(-/-) mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI(-/-) mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.

In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF. To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR. All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae. By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor. Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrincoated pits. Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.The most common mutant EGF 1 receptor (EGFR) found in human tumor cells has a truncated extracellular domain, is constitutively active at the cell surface, and is not down regulated by either EGF or anti-EGFR IgG (1). Other oncogenic EGFR, those missing the cytoplasmic regulatory domain but having an active kinase domain, also fail to down regulate (2). Down-regulation is the term used to describe the attenuation of signal transduction by receptor-mediated endocytosis. Detailed studies of the normal EGFR have found that endocytosis under these conditions is a high affinity, saturable process that involves the interaction of endocytic codes in the receptor cytoplasmic tail with an unidentified set of molecules present in clathrin-coated pits (3, 4). Receptors that can not down regulate, therefore, are either missing the information required for capture or are incapable of accessing coated pits. None of the studies carried out so far have distinguished between these two mechanisms.To determine which of these two mechanisms accounts for the behavior of mutant, oncogenic EGFR that fail to down regulate, the location of the unstimulated receptors must first be determined. EGFR might be randomly distributed across the surface or confined to specialized membrane domains. Membrane fractionation and immunocytochemistry has been used to show that in quiescent fibroblasts wild-type EGFR (5, 6), as well as other receptor (7) and non-receptor (8) tyrosine kinases, are highly enriched in caveolae membrane fractions. Moreover, the first phases of signal transduction initiated by EGF binding, such as activation of tyrosine kinase activity (6, 7), phosphorylation of protein substrates (7), recruitment of adaptors (6, 7, 9) and essential kinases (7), and activation of MAP kinase (7, 10), all appear to take place in caveolae membranes. These and other studies (reviewed in Ref. 8) indicate that entire signaling pathways are pre-organized in caveolae. Rapid signal...