The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SMmicelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A 2 , or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn 2؉ -stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now show that Zn-SMase activity is secreted by human and murine macrophages, human skin fibroblasts, microglial cells, and several other cells in culture and is markedly up-regulated during differentiation of human monocytes to macrophages. Remarkably, peritoneal macrophages from mice in which the acid SMase gene had been disrupted by homologous recombination secreted no Zn-SMase activity, indicating that this enzyme and the intracellular lysosomal SMase, which is Zn-independent, arise from the same gene. Furthermore, skin fibroblasts from patients with types A and B NiemannPick disease, which are known to lack lysosomal SMase activity, also lack Zn-SMase activity in their conditioned media. Chinese hamster ovary cells stably transfected with a cDNA encoding lysosomal SMase massively overexpress both cellular lysosomal SMase and secreted ZnSMase activities. Thus, Zn-SMase arises independently of alternative splicing, suggesting a post-translational process. In summary, a wide variety of cell types secrete Zn-SMase activity, which arises from the same gene as lysosomal SMase. This secreted enzyme may play roles in physiological and pathophysiological processes involving extracellular sphingomyelin hydrolysis. Mammalian SMases1 have been implicated in many important physiological and pathophysiological processes, including lysosomal digestion of sphingomyelin, which is important for normal neuronal and vascular function (1); ceramide-mediated signal transduction, leading to cytokine-induced apoptosis, cellular differentiation, and various immune and inflammatory responses (2, 3); lipoprotein aggregation within the vessel wall, which is a key event in atherogenesis (4 -7); and intracellular cholesterol trafficking and metabolism (8 -10).2 Several different forms of mammalian SMases have been identified, including: (a) a lysosomal SMase that is present in all tissues, acts optimally at low pH, and shows no dependence on divalent cations (11). This enzyme, which is defective in types A and B Niemann-Pick disease (11), has been purified and cloned (12), and mice in which the gene encoding lysosomal SMase has been inactivated have recently been generated (13, 14); (b) a neutral, membrane-associated, Mg 2ϩ -stimulated SMase that is found predominantly in brain and kidney (15) and that is known to arise from a separate gene from lysosomal SMase (16) Although the molecular identity and functions are definitively known only for lysosomal SMase (15), two of the other forms, namely, Mg 2ϩ -stimulated SMase and cytosolic SMase, also have been studied. Both have been partially purified (17,19,20), and these two enzymes, in addition to lysosomal SMase, ...

Aggregation and retention of LDL in the arterial wall are key events in atherogenesis, but the mechanisms in vivo are not known. Previous work from our laboratories has shown that exposure of LDL to bacterial sphingomyelinase (SMase) in vitro leads to the formation of LDL aggregates that can be retained by extracellular matrix and that are able to stimulate macrophage foam cell formation. We now provide evidence that retained LDL is hydrolyzed by an arterial-wall SMase activity. First, we demonstrated that SMase-induced aggregation is caused by an increase in particle ceramide content, even in the presence of excess sphingomyelin (SM). This finding is compatible with previous data showing that lesional LDL is enriched in SM, though its ceramide content has not previously been reported. To address this critical compositional issue, the ceramide content of lesional LDL was assayed and, remarkably, found to be 10-50-fold enriched compared with plasma LDL ceramide. Furthermore, the ceramide was found exclusively in lesional LDL that was aggregated; unaggregated lesional LDL, which accounted for 20-25% of the lesional material, remained ceramide poor. When

We recently reported that macrophages and fibroblasts secrete a Zn 2؉ -dependent sphingomyelinase (SSMase), which, like lysosomal SMase, is a product of the acid SMase gene. S-SMase may cause subendothelial retention and aggregation of lipoproteins during atherogenesis, and the acid SMase gene has been implicated in ceramide-mediated cell signaling, especially involving apoptosis of endothelial cells. Because of the central importance of the endothelium in each of these processes, we now sought to examine the secretion and regulation of S-SMase by vascular endothelial cells. Herein we show that cultured human coronary artery and umbilical vein endothelial cells secrete massive amounts of S-SMase (up to 20-fold more than macrophages). Moreover, whereas S-SMase secreted by macrophages and fibroblasts is almost totally dependent on the addition of exogenous Zn 2؉ , endothelium-derived S-SMase was partially active even in the absence of added Zn 2؉. Secretion of S-SMase by endothelial cells occurred both apically and basolaterally, suggesting an endothelial contribution to both serum and arterial wall SMase. When endothelial cells were incubated with inflammatory cytokines, such as interleukin-1␤ and interferon-␥, S-SMase secretion by endothelial cells was increased 2-3-fold above the already high level of basal secretion, whereas lysosomal SMase activity was decreased. The mechanism of interleukin-1␤-stimulated secretion appears to be through increased routing of a SMase precursor protein through the secretory pathway. In summary, endothelial cells are a rich and regulatable source of enzymatically active S-SMase, suggesting physiologic and pathophysiologic roles for this enzyme.

The acid sphingomyelinase (ASM) gene, which has been implicated in ceramide-mediated cell signaling and atherogenesis, gives rise to both lysosomal SMase (L-SMase), which is reportedly cation-independent, and secretory SMase (S-SMase), which is fully or partially dependent on Zn 2؉ for enzymatic activity. Herein we present evidence for a model to explain how a single mRNA gives rise to two forms of SMase with different cellular trafficking and apparent differences in Zn 2؉dependence. First, we show that both S-SMase and LSMase, which contain several highly conserved zincbinding motifs, are directly activated by zinc. In addition, SMase assayed from a lysosome-rich fraction of Chinese hamster ovary cells was found to be partially zinc-dependent, suggesting that intact lysosomes from these cells contain subsaturating levels of Zn 2؉. Analysis of Asn-linked oligosaccharides and of N-terminal amino acid sequence indicated that S-SMase arises by trafficking through the Golgi secretory pathway, not by cellular release of L-SMase during trafficking to lysosomes or after delivery to lysosomes. Most importantly, when Zn 2؉ -dependent S-SMase was incubated with SMasenegative cells, the enzyme was internalized, trafficked to lysosomes, and became zinc-independent. We conclude that L-SMase is exposed to cellular Zn 2؉ during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn 2؉ ; thus S-SMase requires exogeneous Zn 2؉ for full activity. This model provides important information for understanding the enzymology and regulation of L-and S-SMase and for exploring possible roles of ASM gene products in cell signaling and atherogenesis.

Apolipoprotein E knockout (apoE0) mice accumulate atherogenic remnant lipoproteins in plasma. We now provide evidence that these particles are enriched in sphingomyelin (SM), and explore the mechanisms and possible pathophysiological consequences of this finding. The phosphatidylcholine/sphingomyelin (PC/SM) ratio was reduced in all lipoproteins in apoE0 mice compared with wild-type (Wt) mice (2.0+/-0.2 vs. 4.7+/-0.5; 2.8+/-0.5 vs. 5.5+/-0.9; 1.9+/-0. 5 vs. 4.6+/-0.5 for VLDL, LDL, and HDL), reflecting 400 and 179% increases in plasma pools of SM and PC, respectively. Turnover studies using [14C]PC/[3H]SM VLDL or HDL showed that the fractional catabolic rate (FCR) of VLDL-SM and HDL-SM were markedly reduced in the apoE0 mice compared with Wt mice, while the FCRs of VLDL-PC and HDL-PC were similar. By contrast, the FCRs of [3H]PC ether and [14C]SM were identical in apoE0 and Wt mice. The production rates of VLDL-SM and HDL-SM in apoE0 mice were much higher than in Wt mice, while the production rates of lipoprotein PC were similar. To assess the underlying mechanisms, we also measured the PC/SM ratio in VLDL and LDL of LDL receptor knockout (LDLr0) and hepatic LDL receptor-related protein knockout/LDLr0 mice, but found no difference with Wt mice. Using S-sphingomyelinase, an enzyme secreted by macrophages and endothelial cells, we found that VLDL and LDL from apoE0, but not from Wt or LDLr0 mice, were significantly aggregated, and that aggregation was not prevented by adding back apoE. We then enriched the apoE0-VLDL and Wt-VLDL with different amounts of SM, and found that VLDL aggregation was enhanced. Thus, the increased SM content of lipoproteins in apoE0 mice is due to combined synthesis and clearance defects. Impaired SM clearance reflects resistance to intravascular enzymes and delayed removal by a non-LDLr, non-LDLr related protein pathway. The increased SM content in slowly cleared remnant lipoproteins may enhance their susceptibility to arterial wall SMase and increase their atherogenic potential.

Background-The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation. Methods and Results-To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. ␤-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). ␤-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Speg␣ and Speg␤ isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth. Conclusions-These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy. Key Words: cardiomyopathy Ⅲ hypertrophy Ⅲ myocytes Ⅲ genes M yosin light chain kinases (MLCK) are a family of proteins that are important for myocyte function, including structure and regulation of the actin-based cytoskeleton. 1,2 One member of this family, striated preferentially expressed gene (Speg), has isoforms in both striated and smooth muscle cells. 3 The Speg locus contains 2 transcriptional start sites, and through alternative promoter use and splicing in a tissue-specific manner, it generates 4 different isoforms. 3 Speg␣ and Speg␤ are expressed in striated muscle (cardiac and skeletal), aortic preferentially expressed gene (Apeg-1) is expressed in smooth muscle (predominantly vascular), and brain preferentially expressed gene (Bpeg) is expressed in the brain and aorta. [3][4][5] Speg␣ and Speg␤ share homology with MLCK family members. Specifically, the Speg isoforms, along with obscurin-MLCK, are unique members of the MLCK family, because they contain 2 tandemly arranged serine/threonine kinase (MLCK) domains. 2 Beyond the MLCK domains, Speg␣ and Speg␤ also contain immunoglobulin and fibronectin domains that are characteristic of the MLCK family of proteins. Prior investigations in our laboratory revealed that Speg␣ and Speg␤ are sensitive markers of striated muscle differentiation and that Speg colocalizes with desmin in the sarcomeric Z disc 3 ; however, the functional significance of Speg isoforms is yet to be elucidated. Editorial p 213 Clinical Perspective p 268In contrast to smooth muscle cells, striated muscle cells of both cardiac and skeletal origin undergo terminal differenti- Received June 15, 2008; accepted October 14, 2008. From Pulmona...

Background-Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. Methods and Results-To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2-deficient mice to a model of chronic normobaric hypoxia. COX-2-null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2-deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2-deficient mice had significant upregulation of the endothelin-1 receptor (ET A ) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET A receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2-deficient PASMCs with iloprost, a prostaglandin I 2 analog, and prostaglandin E 2 abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. Conclusions-Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET A receptor expression and increased PASMC hypertrophy. COX-2-deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2-derived prostaglandin I 2 or prostaglandin E 2 .