- Email: [email protected]
Caveolae, Lipid Rafts, and Vascular Disease
Wolf BB, Goldstein JC, Stennicke HR, et al.: 1999. Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. Blood 94:1683 – 1692. Wu YP, Vink T, Schiphorst M, et al.: 2000. Platelet thrombus formation on collagen at high shear rates is mediated by von Willebrand factor– glycoprotein Ib interaction and inhibited by von Willebrand factor– glycoprotein IIb/IIIa interaction. Arterioscler Thromb Vasc Biol 20:1661 – 1667. Zaman GJR, Conway EM: 2000. The elusive factor Xa receptor: failure to detect transcripts that correspond to
the published sequence of EPR1. Blood 96:145 – 148. Zwaal RFA, Comfurius P, Smeets E, et al.: 1996. Platelet procoagulant activity and microvesicle formation. In Seghatchian MJ, Samama MM , Hecker SP, eds. Hypercoagulable states. CRC Press, Boca Raton, FL, pp. 29 – 36. Zwaal RFA, Schroit AJ: Pathophysiological implications of membrane phospholipid asymmetry in blood cells. Blood 89:1997. 1121 – 1132.
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Caveolae, Lipid Rafts, and Vascular Disease Xiang-An Li, William V. Everson, and Eric J. Smart*
Caveolae and lipid rafts are discrete regions within the plasma membrane that coordinate and regulate a variety of signaling processes. The exact relationship between caveolae and lipid rafts is unclear. However, caveolae contain a protein called caveolin that serves as a biochemical marker for caveolae. In addition, caveolin plays a role in maintaining the lipid composition of caveolae, the morphology of caveolae, and the signals that emanate from caveolae. The physiologic importance of caveolae is evidenced by recent studies using caveolin knockout mice that show dramatic abnormalities in the cardiovascular system, such as pulmonary hypertension and cardiac hypertrophy. In this review, we will focus on the role of caveolae in the cardiovascular system. (Trends Cardiovasc Med 2005;15:92–96) D 2005, Elsevier Inc.
The plasma membrane is composed of phospholipids, cholesterol, and other lipids, along with a wide variety of
Xiang An-Li, William V. Everson, and Eric J. Smart are at the University of Kentucky Medical School, Department of Pediatrics, 423 Sanders-Brown, Lexington, KY 405360230, USA. * Address correspondence to: Eric J. Smart, PhD, Department of Pediatrics, University of Kentucky, 423 Sanders-Brown, 800 Limestone St, Lexington, KY 40536-0230, USA. Tel.: (+1) 859-323-6412; fax: (+1) 859-2572120; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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proteins. However, the plasma membrane components are not evenly distributed, but rather are organized into discrete regions or domains. Examples of well-established domains are clathrincoated pits and cell-to-cell junctions, each of which is specialized for a specific function, such as endocytosis or intercellular communication. Caveolae and lipid rafts are also specialized domains that are rich in cholesterol, sphingolipids, saturated fatty acids, and signaling proteins (Anderson 1998, Smart et al. 1999). In this review, we will focus on recent progress concerning the role of caveolae in the cardiovascular system.
Caveolae Versus Lipid Rafts
Caveolae were first described by Yamada (1955) as small flask-shaped noncoated plasma membrane invaginations as observed by electron microscopy. The importance of caveolae was underappreciated until the 1990s when caveolin (VIP21) was cloned and identified as a component of caveolae (Glenney 1992, Glenney and Soppet 1992, Kurzchalia et al. 1992, Rothberg et al. 1992). The establishment of biochemical isolation methods for obtaining caveolae, using caveolin as a biochemical marker, made it possible to study the components and functions of caveolae (Anderson 1998). Detailed studies provided for an additional broader definition of caveolae, that is, not only existing as morphologically identifiable flask-shaped plasma membrane invaginations but also as flattened caveolin-rich membrane microdomains morphologically indistinguishable from the rest of the plasma membrane (Chang et al. 1992, Rothberg et al. 1990). The literature is confusing because some researchers use solely morphologic criteria to define caveolae, and others use both morphologic and biochemical criteria to define them. However, even the original definition of caveolae is not completely accurate because rapid-freeze deep-etch electron microscopy has been used to demonstrate that bclassicQ noncoated caveolae do indeed have a striated coat structure. In addition, the morphology of caveolae appears to be dynamic and depends on the cell type and experimental conditions. For example, in fibroblasts and endothelial cells, caveolae are able to aggregate to form patches of caveolae containing hundreds of units that occupy several square micrometers of the cell surface (Gratton et al. 2004). Lipid rafts are defined operationally by the basis used to isolate a population of membranes from cells (Anderson 1998, Simons and Ikonen 1997), which is detergent insolubility, a light buoyant density, and rich in cholesterol, sphingomyelin, and signaling proteins. Lipid rafts and caveolae have similar lipid and protein components, with two notable exceptions. Lipid rafts do not contain caveolin, and caveolae do not contain glycosyl–phosphatidylinositol-anchored proteins (Brown and Waneck 1992, Smart et al. 1999). Lipid rafts can be isolated from all cells, including cells TCM Vol. 15, No. 3, 2005
that express caveolin and cells that do not express caveolin. Some researchers consider caveolae as a type of lipid raft that contains caveolin, whereas other researchers consider the two microdomains as completely separate entities. Considering the reported functions of caveolin, the absence and presence of caveolin most likely affect the function of the microdomains, and thus the two domains can be considered functionally distinct from each other.
Caveolae and Cholesterol Homeostasis
Caveolae are enriched in cholesterol compared with the rest of the plasma membrane (Pike et al. 2002). In fact, caveolae formation/maintenance depends on cholesterol. Anderson and Jacobson (2002) proposed the blipid shellsQ hypothesis that proteins are not simply inserted into the caveolae but are encased in shells of cholesterol and other lipids. Murata et al. (1995) demonstrated that caveolin directly binds cholesterol with high affinity in a 1:1 ratio. It is speculated that the high concentration of cholesterol associated with caveolae is because of the large oligomeric caveolin complexes in caveolae. Cholesterol is a key component of caveolae structure and helps to regulate caveolae function. Interestingly, several observations support a role for caveolae in helping to maintain cellular cholesterol balance. One of the functions of caveolae is to serve as a site of cholesterol uptake from the extracellular environment. The classic pathway of cholesterol uptake is initiated in clathrin-coated pits via receptor-mediated endocytosis of low-density lipoprotein (LDL) (Goldstein et al. 1985). Alternative pathways for cholesterol uptake that do not require receptor-mediated endocytosis have also been described. These selective uptake mechanisms enable the internalization of cholesteryl esters from lipoproteins on the cell surface, without endocytic uptake of the particle. Scavenger receptor BI (SR-BI) functions as a receptor for high-density lipoprotein (HDL) and mediates selective uptake of cholesteryl esters via caveolae in nonhepatic cells (Krieger 1999, Trigatti et al. 2000). Cholesteryl esters are transferred by an unknown mechanism from the HDL particle to caveolae. The cholesTCM Vol. 15, No. 3, 2005
teryl esters are then internalized to the endoplasmic reticulum by a chaperone complex that consists of caveolin, annexin II, cyclophilin 40, and cyclophilin A. Caveolin association with cholesterol requires that caveolin by palmitoylated at cysteine 133 (Uittenbogaard et al. 2002). Once the cholesteryl esters are delivered to the endoplasmic reticulum, the complex recycles to the plasma membrane for another round of uptake. In addition to being sites for cholesterol uptake, caveolae also function as sites of cholesterol efflux. Fielding and Fielding (1995) have demonstrated that free cholesterol is effluxed directly from caveolae to pre–beta-HDL particles. More recently, Fu et al. (2004) demonstrated that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to caveolae. A mechanism for trafficking and consequently enriching caveolae in cholesterol was described (Uittenbogaard and Smart 2000). These researchers demonstrated that newly synthesized cholesterol can be trafficked directly to caveolae, independent of vesicles, by a process that takes 10 to 20 min. The cholesterol associates with a caveolin chaperone complex that consists of caveolin, HSP56, cyclophilin 40, and cyclophilin A. The cholesterol associates with caveolin that is palmitoylated on cysteine 143 and 156, and the protein–lipid complex moves to caveolae by an unknown mechanism. Interestingly, the complex that moves cholesterol to caveolae is very similar to the complex that internalizes cholesteryl esters from caveolae. The major difference is that annexin II is part of the complex for internalization, and HSP56 is part of the complex for efflux. Finally, mutational analysis demonstrated that differential acylation of caveolin is critical in the formation of each of the complexes. However, the mechanism controlling the acylation is unknown.
Caveolae-Mediated Regulation of Endothelial Nitric Oxide Synthase
In addition to the role of caveolae in cholesterol homeostasis, caveolae are also important sites for signal transduction (Smart et al. 1999). One of the best documented functions of caveolae related to the cardiovascular system is
the regulation of endothelial nitric oxide synthase (eNOS) (Shaul 2003). eNOS is involved in the control of several important processes, such as angiogenesis, vasorelaxation, and permeability (Shaul 2002). Mice deficient in eNOS lose vascular tone and develop atherosclerosis (Knowles et al. 2000, Kuhlencordt et al. 2001). eNOS is normally myristoylated and palmitoylated, which targets the protein to caveolae, where it resides in an inactive signaling complex awaiting activation (Shaul 2002). Mutations of the palmitoylation or myristoylation sites in eNOS prevent caveolae targeting and result in a reduction in agoniststimulated nitric oxide release (GarciaCardena et al. 1996, Liu et al. 1996), suggesting that caveolae are important for temporal and spatial activation of the enzyme. Caveolin binds to eNOS through two scaffolding domains. Caveolin–eNOS association interferes with electron flux from the reductase to the oxygenase domain of eNOS, resulting in the lack of nitric oxide production (Garcia-Cardena et al. 1997, Ghosh et al. 1998). Interestingly, caveolin-mediated inhibition of eNOS can be antagonized by an increase in intracellular calcium resulting in a calmodulin– calcium complex displacing caveolin from eNOS (Ju et al. 1997, Michel et al. 1997a, Michel et al. 1997b). This discovery has led to the proposal of a caveolin– calmodulin regulatory cycle of eNOS inactivation and activation. In support of this hypothesis, a peptide from the scaffolding domain of caveolin-1 greatly inhibits eNOS activity in vivo, and the absence of caveolae in caveolin-deficient mice causes constitutive activation of eNOS (Bucci et al. 2000). These findings confirm the physiologic importance of caveolae in regulating eNOS activity. In addition to direct regulation of eNOS by caveolin, alterations in the lipid content of caveolae can regulate eNOS. Uittenbogaard et al. (2000) have demonstrated that oxidized LDL (oxLDL) binding to CD36, which is localized to caveolae in endothelial cells, results in the efflux of caveolae cholesterol to the lipoprotein particle. The decrease in caveolae cholesterol caused eNOS to move to an unidentified intracellular localization where it was nonresponsive to physiologic agonists. Surprisingly, the concomitant addition of HDL with the oxLDL prevented the movement of eNOS out of
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caveolae. The effect of HDL depends on binding to SR-BI. HDL donated cholesterol to caveolae and in effect compensated for the loss of cholesterol to oxLDL. Thus, the lipid milieu of caveolae was maintained, and eNOS remained associated with caveolae. Recent studies have demonstrated that eNOS can be stimulated by HDL binding to caveolae-localized SR-BI in endothelial cells (Li et al. 2002, Mineo et al. 2003, Yuhanna et al. 2001). Several mechanisms have been proposed for how HDL stimulates eNOS activity. Using an in vitro transient expression cell line and aortic rings from SR-BI knockout mice, Yuhanna et al. (2001) showed that HDL significantly stimulates eNOS activity in an SR-BI–dependent manner. Using a CHO cell line that stably expressed both SR-BI and eNOS, Li et al. (2002) demonstrated that the binding of HDL to SR-BI greatly increases the intracellular ceramide levels, which enhances eNOS activity. In contrast, Mineo et al. (2003) reported that HDL stimulates eNOS by activating the Akt kinase pathway. The reason for the reported differences is unclear but may relate to the type of cells used and the confluency of the cells. More recently, Gong et al. (2003) reported that estrogen associated with HDL is capable of stimulating eNOS activity in an SR-BI–dependent manner. Estrogeninduced stimulation of eNOS appears to be estrogen receptor dependent but independent of gene transcription. The regulatory mechanisms between HDL, estrogen, and eNOS activity are not known, but it is likely that these processes are directly involved in normal and abnormal cardiovascular functions.
Caveolin Knockout Mice and the Cardiovascular System
The recent generation of caveolinknockout mice has provided a useful tool for investigating the role of caveolae in physiologic processes. Drab et al. (2001) generated mice by the targeted deletion of exon 3 of the caveolin-1 gene. The deletion of caveolin-1 eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic
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tone. In addition, the lungs of caveolin-1 knockout mice displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1–disrupted mice. Razani et al. (2001) also created caveolin-1 null mice, and in tissues and cultured embryonic fibroblasts from these mice, they observed a lack of caveolae formation, degradation, and redistribution of caveolin-2, defects in the endocytosis of albumin (a caveolae ligand), and a hyperproliferative phenotype. In the lungs, the authors observed thickened alveolar septa and hypercellularity and an increase in the number of vascular endothelial growth factor receptor-positive endothelial cells. By measuring the physiologic response of aortic rings to various stimuli, they found that caveolin-1–deficient mice showed abnormal vasoconstriction and vasorelaxation responses. They observed that eNOS activity was upregulated in caveolin-1 null animals, and this activity could be blunted by a specific NOS inhibitor. Zhao et al. (2002) generated a third caveolin-1 knockout model and identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the caveolin1–deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility. These animals also had marked right ventricular hypertrophy, suggesting a chronic increase in pulmonary artery pressure that was later confirmed by direct measurement. However, not all of the effects caused by a lack of caveolin-1 are negative. A recent study using caveolin-1/apolipoprotein E double-knockout mice showed that the lack of caveolin-1 in apolipoprotein E knockout mice significantly reduces the development of atherosclerosis (Frank et al. 2004, Sessa 2004). However, the mice showed significant increases in plasma cholesterol and triglycerides levels. In addition to the caveolin-1 knockout mice, a caveolin-1/ caveolin-3 double-knockout mouse has been generated (Park et al. 2002). The caveolin-1/caveolin-3 double-knockout phenotype is a combination of the phenotypes seen for the single knockouts. However, there seems to be a more severe phenotype in the heart of the
double knockout mouse than might be expected from the studies of the single caveolin-1 or caveolin-3 knockout mice. The double knockouts show severe cardiomyopathy, with left ventricular hypertrophy and dilation. The mechanistic basis for this extreme phenotype is unknown. Clearly, caveolin has multiple functions in the cardiovascular system, and considerably more research is required to elucidate the molecular mechanisms involved.
Future Studies
Given the range of putative functions of caveolae, one would expect caveolin knockout mice to be nonviable. However, caveolin-1, caveolin-2, and caveolin-3 knockout mice are all viable and fertile, suggesting that some of the caveolae functions are being performed by lipid rafts in the caveolin-1 knockout mice (Drab et al. 2001; Galbiati et al. 2001; Hagiwara et al. 2000; Razani et al. 2001; Razani et al. 2002; Zhao et al. 2002). Obviously, a great deal of additional research is required to understand the interactions between caveolae and lipid rafts. One of the most exciting developments has been the generation of bfloxedQ caveolin-1 mice, which can be used to study tissue-specific effects of caveolin-1/caveolae in mouse models (Cao et al. 2003). Finally, the recent development of new fluorescent dyes will make it possible to study the movement of protein into and out of caveolae in live cells in real time. Future studies will bring exciting discoveries concerning caveolae and the cardiovascular system.
Acknowledgments
The authors thank the Cardiovascular Research Center and Pediatric Research Center for invaluable advice and assistance. This work was supported, in part, by a COBRE grant (P20 RR15592) from the National Center for Research Resources at the National Institutes of Health and by the National Heart, Lung, and Blood Institute (HL62844, HL64056: EJS).
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A Role for Msx2 and Necdin in Smooth Muscle Differentiation of Mesoangioblasts and Other Mesoderm Progenitor Cells Silvia Brunelli and Giulio Cossu* The molecular regulation of smooth muscle differentiation is currently far less well understood than that of striated muscle, in part because in this cell type, the differentiated state is plastic and reversible. In recent years, however, several molecules, the best characterized of which is myocardin, have been shown to be necessary and sufficient to promote at least partial smooth muscle differentiation. Indeed, mice deficient in myocardin have a severe reduction of smooth muscle tissue. However, possibly because of multiple embryological origins, which include mesenchyme, neural crest, and even endothelium, different types of smooth muscle cells differ in their expression of myocardin and of other potential regulatory molecules. Here, we will review recent work on the topic, focusing on the mesoangioblast, a recently described vesselassociated stem cell, whose differentiation into smooth muscle is dependent upon expression of msx2 and necdin, but not of myocardin. (Trends Cardiovasc Med 2005;15:96–100) D 2005, Elsevier Inc. Silvia Brunelli and Giulio Cossu are at the Stem Cell Research Institute, Dibit, H. San Raffaele, Milan, Italy. Silvia Brunelli is also at the Department of Experimental, Environmental Medicine and Medical Biotechnology, University of Milano-Bicocca, Milan, Italy. Giulio Cossu is also at the Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park of Rome, IIo Medical School, University of Rome ‘‘La Sapienza’’, Rome, Italy. * Address correspondence to: Giulio Cossu, Stem Cell Research Institute, DIBIT-H San Raffaele, via Olgettina 58, 20132 Milan, Italy. Tel.: (+39) 0226234954, fax: (+39) 0226434621; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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Mesoangioblasts Are Novel Stem Cells That Can Differentiate Into Smooth Muscle
Mesoangioblasts are a recently characterized class of stem cells able to selfrenew and to give rise to different mesoderm cell types in vitro and in vivo (Minasi et al. 2002). They originate in the dorsal aorta of the early embryo (10-12 somites) from a hypothetical primitive ancestor common to hemoangioblasts, and they remain physically associated with the vascular compartment during development and throughout adult life (De Angelis et al. 1999, Cossu and Bianco 2003).
Mesoangioblasts can differentiate into skeletal, cardiac, and smooth muscle and thus appear as a possible source of cells for the therapy of genetic or acquired muscle diseases, in particular of muscular dystrophy. It has been recently shown that intra-arterial delivery of wild-type mesoangioblasts corrects morphologically and functionally the dystrophic phenotype of downstream muscles in a-sarcoglycan null mice, a model organism for limb-girdle muscular dystrophy (Sampaolesi et al. 2003). More recently, mesoangioblasts have also been found to be effective in ameliorating cardiac function after experimental infarction, although their differentiation into cardiomyocytes is a rare event (Galli et al. 2005). We have also shown that mesoangioblasts differentiate very efficiently to smooth muscle cells (SMCs) in vivo and vitro, in response to transforming growth factor h (TGFh) (Figure 1A and B) (Minasi et al. 2002, Tagliafico et al. 2004), a molecule known to induce smooth muscle (SM) differentiation in different types of cells (Grainger et al. 1998, Sinha et al. 2004). Accordingly, mesoangioblasts express at high level many members of the TGFh pathway, including receptors and several of the signaling proteins Smads (Tagliafico et al. 2004). Understanding the molecular mechanisms underlying specific cell type differentiation of mesoangioblasts is a necessary prerequisite to optimizing stem cell-based therapeutic strategies. In this brief review, we will focus on mesoangioblast differentiation into a SMC phenotype and on recently discovered molecules that may play an important role in this process.
SMCs: Origin, Function, and Differentiation
SMCs are multifunctional cells that exhibit spontaneous and agonistinduced contractile properties, secrete and assemble a wide variety of extracellular matrix proteins, display migratory and proliferative responses to tissue injury, and both produce and respond to a variety of paracrine-acting growth factors. SMCs regulate a variety of functions, such as arterial tone, airway resistance, and gastrointestinal and genitourinary tract contractility. TCM Vol. 15, No. 3, 2005
the published sequence of EPR1. Blood 96:145 – 148. Zwaal RFA, Comfurius P, Smeets E, et al.: 1996. Platelet procoagulant activity and microvesicle formation. In Seghatchian MJ, Samama MM , Hecker SP, eds. Hypercoagulable states. CRC Press, Boca Raton, FL, pp. 29 – 36. Zwaal RFA, Schroit AJ: Pathophysiological implications of membrane phospholipid asymmetry in blood cells. Blood 89:1997. 1121 – 1132.
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Caveolae, Lipid Rafts, and Vascular Disease Xiang-An Li, William V. Everson, and Eric J. Smart*
Caveolae and lipid rafts are discrete regions within the plasma membrane that coordinate and regulate a variety of signaling processes. The exact relationship between caveolae and lipid rafts is unclear. However, caveolae contain a protein called caveolin that serves as a biochemical marker for caveolae. In addition, caveolin plays a role in maintaining the lipid composition of caveolae, the morphology of caveolae, and the signals that emanate from caveolae. The physiologic importance of caveolae is evidenced by recent studies using caveolin knockout mice that show dramatic abnormalities in the cardiovascular system, such as pulmonary hypertension and cardiac hypertrophy. In this review, we will focus on the role of caveolae in the cardiovascular system. (Trends Cardiovasc Med 2005;15:92–96) D 2005, Elsevier Inc.
The plasma membrane is composed of phospholipids, cholesterol, and other lipids, along with a wide variety of
Xiang An-Li, William V. Everson, and Eric J. Smart are at the University of Kentucky Medical School, Department of Pediatrics, 423 Sanders-Brown, Lexington, KY 405360230, USA. * Address correspondence to: Eric J. Smart, PhD, Department of Pediatrics, University of Kentucky, 423 Sanders-Brown, 800 Limestone St, Lexington, KY 40536-0230, USA. Tel.: (+1) 859-323-6412; fax: (+1) 859-2572120; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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proteins. However, the plasma membrane components are not evenly distributed, but rather are organized into discrete regions or domains. Examples of well-established domains are clathrincoated pits and cell-to-cell junctions, each of which is specialized for a specific function, such as endocytosis or intercellular communication. Caveolae and lipid rafts are also specialized domains that are rich in cholesterol, sphingolipids, saturated fatty acids, and signaling proteins (Anderson 1998, Smart et al. 1999). In this review, we will focus on recent progress concerning the role of caveolae in the cardiovascular system.
Caveolae Versus Lipid Rafts
Caveolae were first described by Yamada (1955) as small flask-shaped noncoated plasma membrane invaginations as observed by electron microscopy. The importance of caveolae was underappreciated until the 1990s when caveolin (VIP21) was cloned and identified as a component of caveolae (Glenney 1992, Glenney and Soppet 1992, Kurzchalia et al. 1992, Rothberg et al. 1992). The establishment of biochemical isolation methods for obtaining caveolae, using caveolin as a biochemical marker, made it possible to study the components and functions of caveolae (Anderson 1998). Detailed studies provided for an additional broader definition of caveolae, that is, not only existing as morphologically identifiable flask-shaped plasma membrane invaginations but also as flattened caveolin-rich membrane microdomains morphologically indistinguishable from the rest of the plasma membrane (Chang et al. 1992, Rothberg et al. 1990). The literature is confusing because some researchers use solely morphologic criteria to define caveolae, and others use both morphologic and biochemical criteria to define them. However, even the original definition of caveolae is not completely accurate because rapid-freeze deep-etch electron microscopy has been used to demonstrate that bclassicQ noncoated caveolae do indeed have a striated coat structure. In addition, the morphology of caveolae appears to be dynamic and depends on the cell type and experimental conditions. For example, in fibroblasts and endothelial cells, caveolae are able to aggregate to form patches of caveolae containing hundreds of units that occupy several square micrometers of the cell surface (Gratton et al. 2004). Lipid rafts are defined operationally by the basis used to isolate a population of membranes from cells (Anderson 1998, Simons and Ikonen 1997), which is detergent insolubility, a light buoyant density, and rich in cholesterol, sphingomyelin, and signaling proteins. Lipid rafts and caveolae have similar lipid and protein components, with two notable exceptions. Lipid rafts do not contain caveolin, and caveolae do not contain glycosyl–phosphatidylinositol-anchored proteins (Brown and Waneck 1992, Smart et al. 1999). Lipid rafts can be isolated from all cells, including cells TCM Vol. 15, No. 3, 2005
that express caveolin and cells that do not express caveolin. Some researchers consider caveolae as a type of lipid raft that contains caveolin, whereas other researchers consider the two microdomains as completely separate entities. Considering the reported functions of caveolin, the absence and presence of caveolin most likely affect the function of the microdomains, and thus the two domains can be considered functionally distinct from each other.
Caveolae and Cholesterol Homeostasis
Caveolae are enriched in cholesterol compared with the rest of the plasma membrane (Pike et al. 2002). In fact, caveolae formation/maintenance depends on cholesterol. Anderson and Jacobson (2002) proposed the blipid shellsQ hypothesis that proteins are not simply inserted into the caveolae but are encased in shells of cholesterol and other lipids. Murata et al. (1995) demonstrated that caveolin directly binds cholesterol with high affinity in a 1:1 ratio. It is speculated that the high concentration of cholesterol associated with caveolae is because of the large oligomeric caveolin complexes in caveolae. Cholesterol is a key component of caveolae structure and helps to regulate caveolae function. Interestingly, several observations support a role for caveolae in helping to maintain cellular cholesterol balance. One of the functions of caveolae is to serve as a site of cholesterol uptake from the extracellular environment. The classic pathway of cholesterol uptake is initiated in clathrin-coated pits via receptor-mediated endocytosis of low-density lipoprotein (LDL) (Goldstein et al. 1985). Alternative pathways for cholesterol uptake that do not require receptor-mediated endocytosis have also been described. These selective uptake mechanisms enable the internalization of cholesteryl esters from lipoproteins on the cell surface, without endocytic uptake of the particle. Scavenger receptor BI (SR-BI) functions as a receptor for high-density lipoprotein (HDL) and mediates selective uptake of cholesteryl esters via caveolae in nonhepatic cells (Krieger 1999, Trigatti et al. 2000). Cholesteryl esters are transferred by an unknown mechanism from the HDL particle to caveolae. The cholesTCM Vol. 15, No. 3, 2005
teryl esters are then internalized to the endoplasmic reticulum by a chaperone complex that consists of caveolin, annexin II, cyclophilin 40, and cyclophilin A. Caveolin association with cholesterol requires that caveolin by palmitoylated at cysteine 133 (Uittenbogaard et al. 2002). Once the cholesteryl esters are delivered to the endoplasmic reticulum, the complex recycles to the plasma membrane for another round of uptake. In addition to being sites for cholesterol uptake, caveolae also function as sites of cholesterol efflux. Fielding and Fielding (1995) have demonstrated that free cholesterol is effluxed directly from caveolae to pre–beta-HDL particles. More recently, Fu et al. (2004) demonstrated that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to caveolae. A mechanism for trafficking and consequently enriching caveolae in cholesterol was described (Uittenbogaard and Smart 2000). These researchers demonstrated that newly synthesized cholesterol can be trafficked directly to caveolae, independent of vesicles, by a process that takes 10 to 20 min. The cholesterol associates with a caveolin chaperone complex that consists of caveolin, HSP56, cyclophilin 40, and cyclophilin A. The cholesterol associates with caveolin that is palmitoylated on cysteine 143 and 156, and the protein–lipid complex moves to caveolae by an unknown mechanism. Interestingly, the complex that moves cholesterol to caveolae is very similar to the complex that internalizes cholesteryl esters from caveolae. The major difference is that annexin II is part of the complex for internalization, and HSP56 is part of the complex for efflux. Finally, mutational analysis demonstrated that differential acylation of caveolin is critical in the formation of each of the complexes. However, the mechanism controlling the acylation is unknown.
Caveolae-Mediated Regulation of Endothelial Nitric Oxide Synthase
In addition to the role of caveolae in cholesterol homeostasis, caveolae are also important sites for signal transduction (Smart et al. 1999). One of the best documented functions of caveolae related to the cardiovascular system is
the regulation of endothelial nitric oxide synthase (eNOS) (Shaul 2003). eNOS is involved in the control of several important processes, such as angiogenesis, vasorelaxation, and permeability (Shaul 2002). Mice deficient in eNOS lose vascular tone and develop atherosclerosis (Knowles et al. 2000, Kuhlencordt et al. 2001). eNOS is normally myristoylated and palmitoylated, which targets the protein to caveolae, where it resides in an inactive signaling complex awaiting activation (Shaul 2002). Mutations of the palmitoylation or myristoylation sites in eNOS prevent caveolae targeting and result in a reduction in agoniststimulated nitric oxide release (GarciaCardena et al. 1996, Liu et al. 1996), suggesting that caveolae are important for temporal and spatial activation of the enzyme. Caveolin binds to eNOS through two scaffolding domains. Caveolin–eNOS association interferes with electron flux from the reductase to the oxygenase domain of eNOS, resulting in the lack of nitric oxide production (Garcia-Cardena et al. 1997, Ghosh et al. 1998). Interestingly, caveolin-mediated inhibition of eNOS can be antagonized by an increase in intracellular calcium resulting in a calmodulin– calcium complex displacing caveolin from eNOS (Ju et al. 1997, Michel et al. 1997a, Michel et al. 1997b). This discovery has led to the proposal of a caveolin– calmodulin regulatory cycle of eNOS inactivation and activation. In support of this hypothesis, a peptide from the scaffolding domain of caveolin-1 greatly inhibits eNOS activity in vivo, and the absence of caveolae in caveolin-deficient mice causes constitutive activation of eNOS (Bucci et al. 2000). These findings confirm the physiologic importance of caveolae in regulating eNOS activity. In addition to direct regulation of eNOS by caveolin, alterations in the lipid content of caveolae can regulate eNOS. Uittenbogaard et al. (2000) have demonstrated that oxidized LDL (oxLDL) binding to CD36, which is localized to caveolae in endothelial cells, results in the efflux of caveolae cholesterol to the lipoprotein particle. The decrease in caveolae cholesterol caused eNOS to move to an unidentified intracellular localization where it was nonresponsive to physiologic agonists. Surprisingly, the concomitant addition of HDL with the oxLDL prevented the movement of eNOS out of
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caveolae. The effect of HDL depends on binding to SR-BI. HDL donated cholesterol to caveolae and in effect compensated for the loss of cholesterol to oxLDL. Thus, the lipid milieu of caveolae was maintained, and eNOS remained associated with caveolae. Recent studies have demonstrated that eNOS can be stimulated by HDL binding to caveolae-localized SR-BI in endothelial cells (Li et al. 2002, Mineo et al. 2003, Yuhanna et al. 2001). Several mechanisms have been proposed for how HDL stimulates eNOS activity. Using an in vitro transient expression cell line and aortic rings from SR-BI knockout mice, Yuhanna et al. (2001) showed that HDL significantly stimulates eNOS activity in an SR-BI–dependent manner. Using a CHO cell line that stably expressed both SR-BI and eNOS, Li et al. (2002) demonstrated that the binding of HDL to SR-BI greatly increases the intracellular ceramide levels, which enhances eNOS activity. In contrast, Mineo et al. (2003) reported that HDL stimulates eNOS by activating the Akt kinase pathway. The reason for the reported differences is unclear but may relate to the type of cells used and the confluency of the cells. More recently, Gong et al. (2003) reported that estrogen associated with HDL is capable of stimulating eNOS activity in an SR-BI–dependent manner. Estrogeninduced stimulation of eNOS appears to be estrogen receptor dependent but independent of gene transcription. The regulatory mechanisms between HDL, estrogen, and eNOS activity are not known, but it is likely that these processes are directly involved in normal and abnormal cardiovascular functions.
Caveolin Knockout Mice and the Cardiovascular System
The recent generation of caveolinknockout mice has provided a useful tool for investigating the role of caveolae in physiologic processes. Drab et al. (2001) generated mice by the targeted deletion of exon 3 of the caveolin-1 gene. The deletion of caveolin-1 eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic
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tone. In addition, the lungs of caveolin-1 knockout mice displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1–disrupted mice. Razani et al. (2001) also created caveolin-1 null mice, and in tissues and cultured embryonic fibroblasts from these mice, they observed a lack of caveolae formation, degradation, and redistribution of caveolin-2, defects in the endocytosis of albumin (a caveolae ligand), and a hyperproliferative phenotype. In the lungs, the authors observed thickened alveolar septa and hypercellularity and an increase in the number of vascular endothelial growth factor receptor-positive endothelial cells. By measuring the physiologic response of aortic rings to various stimuli, they found that caveolin-1–deficient mice showed abnormal vasoconstriction and vasorelaxation responses. They observed that eNOS activity was upregulated in caveolin-1 null animals, and this activity could be blunted by a specific NOS inhibitor. Zhao et al. (2002) generated a third caveolin-1 knockout model and identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the caveolin1–deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility. These animals also had marked right ventricular hypertrophy, suggesting a chronic increase in pulmonary artery pressure that was later confirmed by direct measurement. However, not all of the effects caused by a lack of caveolin-1 are negative. A recent study using caveolin-1/apolipoprotein E double-knockout mice showed that the lack of caveolin-1 in apolipoprotein E knockout mice significantly reduces the development of atherosclerosis (Frank et al. 2004, Sessa 2004). However, the mice showed significant increases in plasma cholesterol and triglycerides levels. In addition to the caveolin-1 knockout mice, a caveolin-1/ caveolin-3 double-knockout mouse has been generated (Park et al. 2002). The caveolin-1/caveolin-3 double-knockout phenotype is a combination of the phenotypes seen for the single knockouts. However, there seems to be a more severe phenotype in the heart of the
double knockout mouse than might be expected from the studies of the single caveolin-1 or caveolin-3 knockout mice. The double knockouts show severe cardiomyopathy, with left ventricular hypertrophy and dilation. The mechanistic basis for this extreme phenotype is unknown. Clearly, caveolin has multiple functions in the cardiovascular system, and considerably more research is required to elucidate the molecular mechanisms involved.
Future Studies
Given the range of putative functions of caveolae, one would expect caveolin knockout mice to be nonviable. However, caveolin-1, caveolin-2, and caveolin-3 knockout mice are all viable and fertile, suggesting that some of the caveolae functions are being performed by lipid rafts in the caveolin-1 knockout mice (Drab et al. 2001; Galbiati et al. 2001; Hagiwara et al. 2000; Razani et al. 2001; Razani et al. 2002; Zhao et al. 2002). Obviously, a great deal of additional research is required to understand the interactions between caveolae and lipid rafts. One of the most exciting developments has been the generation of bfloxedQ caveolin-1 mice, which can be used to study tissue-specific effects of caveolin-1/caveolae in mouse models (Cao et al. 2003). Finally, the recent development of new fluorescent dyes will make it possible to study the movement of protein into and out of caveolae in live cells in real time. Future studies will bring exciting discoveries concerning caveolae and the cardiovascular system.
Acknowledgments
The authors thank the Cardiovascular Research Center and Pediatric Research Center for invaluable advice and assistance. This work was supported, in part, by a COBRE grant (P20 RR15592) from the National Center for Research Resources at the National Institutes of Health and by the National Heart, Lung, and Blood Institute (HL62844, HL64056: EJS).
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PII S1050-1738(05)00028-9
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A Role for Msx2 and Necdin in Smooth Muscle Differentiation of Mesoangioblasts and Other Mesoderm Progenitor Cells Silvia Brunelli and Giulio Cossu* The molecular regulation of smooth muscle differentiation is currently far less well understood than that of striated muscle, in part because in this cell type, the differentiated state is plastic and reversible. In recent years, however, several molecules, the best characterized of which is myocardin, have been shown to be necessary and sufficient to promote at least partial smooth muscle differentiation. Indeed, mice deficient in myocardin have a severe reduction of smooth muscle tissue. However, possibly because of multiple embryological origins, which include mesenchyme, neural crest, and even endothelium, different types of smooth muscle cells differ in their expression of myocardin and of other potential regulatory molecules. Here, we will review recent work on the topic, focusing on the mesoangioblast, a recently described vesselassociated stem cell, whose differentiation into smooth muscle is dependent upon expression of msx2 and necdin, but not of myocardin. (Trends Cardiovasc Med 2005;15:96–100) D 2005, Elsevier Inc. Silvia Brunelli and Giulio Cossu are at the Stem Cell Research Institute, Dibit, H. San Raffaele, Milan, Italy. Silvia Brunelli is also at the Department of Experimental, Environmental Medicine and Medical Biotechnology, University of Milano-Bicocca, Milan, Italy. Giulio Cossu is also at the Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park of Rome, IIo Medical School, University of Rome ‘‘La Sapienza’’, Rome, Italy. * Address correspondence to: Giulio Cossu, Stem Cell Research Institute, DIBIT-H San Raffaele, via Olgettina 58, 20132 Milan, Italy. Tel.: (+39) 0226234954, fax: (+39) 0226434621; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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Mesoangioblasts Are Novel Stem Cells That Can Differentiate Into Smooth Muscle
Mesoangioblasts are a recently characterized class of stem cells able to selfrenew and to give rise to different mesoderm cell types in vitro and in vivo (Minasi et al. 2002). They originate in the dorsal aorta of the early embryo (10-12 somites) from a hypothetical primitive ancestor common to hemoangioblasts, and they remain physically associated with the vascular compartment during development and throughout adult life (De Angelis et al. 1999, Cossu and Bianco 2003).
Mesoangioblasts can differentiate into skeletal, cardiac, and smooth muscle and thus appear as a possible source of cells for the therapy of genetic or acquired muscle diseases, in particular of muscular dystrophy. It has been recently shown that intra-arterial delivery of wild-type mesoangioblasts corrects morphologically and functionally the dystrophic phenotype of downstream muscles in a-sarcoglycan null mice, a model organism for limb-girdle muscular dystrophy (Sampaolesi et al. 2003). More recently, mesoangioblasts have also been found to be effective in ameliorating cardiac function after experimental infarction, although their differentiation into cardiomyocytes is a rare event (Galli et al. 2005). We have also shown that mesoangioblasts differentiate very efficiently to smooth muscle cells (SMCs) in vivo and vitro, in response to transforming growth factor h (TGFh) (Figure 1A and B) (Minasi et al. 2002, Tagliafico et al. 2004), a molecule known to induce smooth muscle (SM) differentiation in different types of cells (Grainger et al. 1998, Sinha et al. 2004). Accordingly, mesoangioblasts express at high level many members of the TGFh pathway, including receptors and several of the signaling proteins Smads (Tagliafico et al. 2004). Understanding the molecular mechanisms underlying specific cell type differentiation of mesoangioblasts is a necessary prerequisite to optimizing stem cell-based therapeutic strategies. In this brief review, we will focus on mesoangioblast differentiation into a SMC phenotype and on recently discovered molecules that may play an important role in this process.
SMCs: Origin, Function, and Differentiation
SMCs are multifunctional cells that exhibit spontaneous and agonistinduced contractile properties, secrete and assemble a wide variety of extracellular matrix proteins, display migratory and proliferative responses to tissue injury, and both produce and respond to a variety of paracrine-acting growth factors. SMCs regulate a variety of functions, such as arterial tone, airway resistance, and gastrointestinal and genitourinary tract contractility. TCM Vol. 15, No. 3, 2005