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ABSTRACT: Anchorage of cells to the extracellular matrix and integrin-mediated signals play crucial roles in cell survival. We have previously shown that during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (HUVECs), key molecules in focal adhesions and adherens junctions are cleaved by caspases. In this study we provide evidence for a selective upregulation of cell-associated matrix metalloproteinases (MMPs). We observe a physical association of MMP2 with beta1 and alphav integrins, which increased three- to fourfold during apoptosis and is dependent upon integrin beta1 levels and activation state. Both enforced activation of beta1 integrin by a specific antibody and inhibition of MMPs protect HUVECs from apoptosis. We hypothesize that, prior to the commitment to apoptosis, 'inside-out' signals initiated by the apoptotic stimulus alter cell shape together with the activation states and/or the availability of integrins, which promote matrix-degrading activity around dying cells. This 'auxiliary' apoptotic pathway may interrupt ECM-mediated survival signaling, and thus accelerate the efficient execution of the cell death program.
Cell Death and Differentiation 01/2003; 9(12):1360-7. · 8.85 Impact Factor
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ABSTRACT: The production of matrix metalloproteinases (MMPs), such as MMP9, by macrophages may be a critical factor in the rupture of unstable atherosclerotic plaques and aortic aneurysms. Therefore, we studied the role of matrix and soluble cytokines in the regulation of monocyte/macrophage expression of MMP9. Although freshly isolated monocytes synthesize little MMP9, cells cultured on tissue-culture plastic differentiate into macrophages and synthesize maximal amounts of MMP9. Differentiated macrophages cultured on plastic are unresponsive to further stimulation by interleukin 1beta, tumor necrosis factor alpha, or platelet-derived growth factor BB. In contrast, monocytes cultured on polymerized collagen synthesize much less MMP9 than cells cultured on plastic and demonstrate a more than three-fold increase in MMP9 synthesis in response to interleukin 1beta, tumor necrosis factor alpha, and platelet-derived growth factor BB. To determine whether the physical state of the collagen was critical for the decrease in basal synthesis of MMP9, monocytes were cultured in suspension for 5 days to allow differentiation and then seeded onto monomer or polymerized collagen. Synthesis of MMP9 was significantly decreased in cells on polymerized collagen and modestly increased in macrophages seeded on monomer collagen. These results suggest that MMP9 synthesis by macrophages in the vessel wall may be under negative control by native, polymerized collagen and that disruption of this native conformation could increase MMP9 production. In addition, cells in contact with the collagen matrix are potentially more responsive to soluble mediators such as platelet-derived growth factor, interleukin 1beta, and tumor necrosis factor alpha.
Journal of Vascular Surgery 01/2002; 34(6):1111-8. · 3.21 Impact Factor
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ABSTRACT: ADAM15 is a member of the family of metalloprotease-disintegrins that have been shown to interact with integrins in an RGD- and non-RGD-dependent manner. In the present study, we examined the effects of ADAM15 overexpression on cell-matrix and cell-cell interactions in NIH3T3 cells. Tetracycline-regulated ADAM15 overexpression in NIH3T3 cells leads to an inhibition of migration on a fibronectin-coated filter in a Boyden chamber assay and in a scratch wound model. The effects of ADAM15 overexpression on cell migration are not due to changes in matrix attachment or to the lack of extracellular signal-regulated kinase signaling response to PDGF or fibronectin. However, a decrease in monolayer permeability with ADAM15 overexpression and altered cell morphology suggest a possible increase in cell-cell interaction. Analysis of adhesion of NIH3T3 cells to a polyclonal population of cells retrovirally transduced to overexpress ADAM15 demonstrates a 45% increase in cell adhesion, compared with enhanced green fluorescent protein-expressing control cells. In addition, we demonstrate localization of HA-epitope-tagged ADAM15 to cell-cell contacts in an epithelial cell line that forms extensive cell-cell contact structures. Thus, overexpression of ADAM15 in NIH3T3 cells appears to enhance cell-cell interactions, as suggested by decreased cell migration, altered cell morphology at the wound edge, decreased monolayer permeability, and increased cell adhesion to monolayers of cells expressing ADAM15 by retroviral transduction.
Experimental Cell Research 12/2001; 271(1):152-60. · 3.58 Impact Factor
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W E Kaminski,
P Lindahl,
N L Lin,
V C Broudy,
J R Crosby,
M Hellström,
B Swolin,
D F Bowen-Pope,
P J Martin, R Ross,
C Betsholtz,
E W Raines
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ABSTRACT: Platelet-derived growth factor (PDGF)-B and PDGF beta-receptor (PDGFR beta) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B(-/-), PDGFR beta(-/-), or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFR beta expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses. (Blood. 2001;97:1990-1998)
Blood 05/2001; 97(7):1990-8. · 9.90 Impact Factor
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ABSTRACT: Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125(FAK)), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125(FAK) is a substrate for both calpain I- and II-mediated processing. Mapping of the proteolytic cleavage fragments of pp125(FAK) predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125(FAK) to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125(FAK) with the cytoskeletal fraction, while pp125(FAK) cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125(FAK) cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125(FAK), paxillin, and talin and dissolution of the focal adhesion complex.
The Journal of Cell Biology 12/1999; 147(3):619-30. · 10.26 Impact Factor
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ABSTRACT: The transcription factor NF-kappa B is an important regulator of gene expression during immune and inflammatory responses, and can also protect against apoptosis. Here we show that endothelial cells undergo apoptosis when deprived of growth factors. Surviving viable cells exhibit increased activity of NF-kappa B, whereas apoptotic cells show caspase-mediated cleavage of the NF-kappa B p65/ReIA subunit. This cleavage leads to loss of carboxy-terminal transactivation domains and a transcriptionally inactive p65 molecule. The truncated p65 acts as a dominant-negative inhibitor of NF-kappa B, promoting apoptosis, whereas an uncleavable, caspase-resistant p65 protects the cells from apoptosis. The generation of a dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and NF-kappa B inactivation.
Nature Cell Biology 09/1999; 1(4):227-33. · 19.49 Impact Factor
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ABSTRACT: Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell-matrix and cell-cell contacts. In this study, we show that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. beta-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding alpha-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved beta-catenin from lysates of apoptotic cells does not bind to endogenous alpha-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell-cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this "shedding" of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival.
Molecular Biology of the Cell 07/1998; 9(6):1589-601. · 4.94 Impact Factor
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ABSTRACT: Focal recruitment of monocytes and lymphocytes is one of the earliest detectable cellular responses in the formation of lesions of atherosclerosis. This localized accumulation of leukocytes is a multistep process in which the endothelium remains intact and may regulate leukocyte recruitment by expressing specific adhesion molecules. To examine the relationship of adhesion molecule expression to initiation factors and the sites of lesion formation, we analyzed the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet-endothelial cell adhesion molecule-1 (PECAM-1) en face on the aortic endothelium of control mice and homozygous apolipoprotein E-deficient (ApoE -/-) mice that develop complex lesions of atherosclerosis similar to those in humans. In control mice, VCAM-1 staining was weak and limited to sites of altered blood flow. In contrast, in the ApoE -/- mice, VCAM-1 appeared to be localized over the surface of groups of endothelial cells in lesion-prone sites. Expression of VCAM-1 preceded lesion formation, and increased expression above control levels appeared to be correlated with the extent of exposure to plasma cholesterol. Although ICAM-1 was the most prominent adhesion molecule in lesion-prone sites, its expression appeared to be independent of plasma cholesterol levels and was upregulated in both ApoE -/- and control mice. At lesion-prone sites associated with altered blood flow, ICAM-1 was located over the surface of each endothelial cell and on microvilli, whereas VCAM-1 was confined to the cell periphery in non-lesion-prone sites. PECAM-1 was localized at the cell periphery throughout the aorta, and its expression did not appear to be regulated. Thus, the levels, localization, and characteristics of expression of VCAM-1, ICAM-1, and PECAM-1 appear to be differentially regulated. Upregulation of VCAM-1 and ICAM-1 is associated with sites of lesion formation.
Arteriosclerosis Thrombosis and Vascular Biology 06/1998; 18(5):842-51. · 6.37 Impact Factor
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ABSTRACT: Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A-associated cyclin-dependent kinase 2(cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21Cip1/Waf1 and p27Kip1 results in a substantial reduction in their association with nuclear cyclin-cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.
Molecular Cell 04/1998; 1(4):553-63. · 14.18 Impact Factor
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ABSTRACT: The accumulation of proteoglycans (PGs) in atherosclerosis contributes to disease progression and stenosis and may partly depend on local regulation by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. In this study, the distribution of the major extracellular PGs is compared with that of PDGF and TGF-beta isoforms in developing lesions of atherosclerosis from hypercholesterolemic nonhuman primates. Strong immunostaining for decorin, biglycan, versican, and hyaluronan is observed in both intermediate and advanced lesions. Perlecan staining is weak in intermediate lesions but strong in advanced lesions in areas bordering the plaque core. Immunostaining for PDGF-B and TGF-beta1 is particularly prominent in macrophages in intermediate and advanced lesions. In contrast, TGF-beta2 and TGF-beta3 and PDGF-A are present in both macrophages and smooth muscle cells. Overall, PG deposits parallel areas of intense growth factor immunostaining, with trends in relative localization that suggest interrelationships among certain PGs and growth factors. Notably, decorin and TGF-beta1 are distributed similarly, predominantly in the macrophage-rich core, whereas biglycan is prominent in the smooth muscle cell matrix adjoining TGF-beta1-positive macrophages. Versican and hyaluronan are enriched in the extracellular matrix adjacent to both PDGF- and TGF-beta1-positive cells. These data demonstrate that PG accumulation varies with lesion severity, structural characteristics, and the proximity of PDGF and TGF-beta.
American Journal Of Pathology 03/1998; 152(2):533-46. · 4.89 Impact Factor
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ABSTRACT: Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation-induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3-like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130(Cas) (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.
Journal of Experimental Medicine 03/1998; 187(4):579-86. · 13.85 Impact Factor
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ABSTRACT: Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.
Journal of Clinical Investigation 09/1997; 100(4):875-85. · 15.39 Impact Factor
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Annals of the New York Academy of Sciences 05/1997; 811:245-52; discussion 252-4. · 3.15 Impact Factor
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Annals of the New York Academy of Sciences 05/1997; 811:76-85; discussion 85-7. · 3.15 Impact Factor
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Annals of the New York Academy of Sciences 05/1997; 811:498-505. · 3.15 Impact Factor
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ABSTRACT: Metalloproteinase-like, disintegrin-like, and cysteine-rich proteins (MDCs) are potential novel regulators of cell-cell and cell-matrix interactions, as well as of matrix degradation. We have asked whether MDCs are expressed in cultured diploid vascular cells, and have identified MDC 15 in human aortic smooth muscle (SMC) and umbilical vein endothelium (HUVEC). MDC 15 mRNA is expressed at higher levels in HUVECs than in SMCs. In cultured SMCs, MDC 15 mRNA levels are not regulated by PDGF or IGF-I or by adherence to different extracellular matrices. Nor is regulation of MDC 15 mRNA levels observed in HUVEC monolayers at different cell densities, after multi-scratch wounding, or after treatment with TNF-alpha, LPS, or thrombin. However, differences in proteolytic processing of MDC 15 are observed in different HUVEC strains. In contrast to cultured arterial cells, MDC 15 protein is not expressed in vivo in normal vessels, but is up-regulated in lesions of atherosclerosis. These findings suggest that MDC 15 may be a potential regulator of vascular cell function and may be involved in the development of lesions of atherosclerosis.
The FASEB Journal 03/1997; 11(2):173-80. · 5.71 Impact Factor
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ABSTRACT: Arterial smooth muscle cells (SMCs) are arrested in the G1 phase of the cell cycle on polymerized type I collagen fibrils, while monomer collagen supports SMC proliferation. Cyclin E-associated kinase and cyclin-dependent kinase 2 (cdk2) phosphorylation are inhibited on polymerized collagen, and levels of the cdk2 inhibitors p27Kip1 and p21Cip1/Waf1 are increased compared with SMCs on monomer collagen. p27Kip1 associates with the cyclin E-cdk2-p21Cip1/Waf1 complex in SMCs on polymerized collagen. Monovalent blocking antibodies to alpha2 integrins, integrins that mediate adhesion to both forms of collagen, mimic these effects on monomer collagen. Furthermore, polymerized collagen rapidly suppresses p70 S6 kinase, a possible regulator of p27Kip1. Thus, fibrillar collagen specifically regulates early integrin signaling that may lead to up-regulation of cdk2 inhibitors and inhibition of SMC proliferation.
Cell 01/1997; 87(6):1069-78. · 32.40 Impact Factor
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ABSTRACT: Within the last five years, a number of specific growth factors have been localized in developing lesions of atherosclerosis. This localization of growth factors that is not observed in normal vessels, together with the pleotrophic activities of growth factors, have suggested a role for growth factors in atherosclerotic lesion progression. However, based on in vitro studies, many of the growth factors identified in lesions have overlapping target cells and are derived from the same cellular sources. What is the relative role of the specific growth factors identified? How is the their activity altered by the local conditions in the vessel wall? How do different risk factors for atherosclerosis alter the balance between growth factors and their natural regulators? Evidence for the involvement of specific growth factors in the progression of lesions of atherosclerosis is discussed, as well as the multiple levels at which the activities of these growth factors may be regulated by the vessel wall.
BioEssays 05/1996; 18(4):271-82. · 4.95 Impact Factor
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ABSTRACT: We have developed a high resolution magnetic resonance (MR) imaging technique to serially assess lesions of atherosclerosis in a rabbit model. A volume phased array coil was designed and used to image the abdominal aortas of six atherosclerotic rabbits and two age-, sex-, and weight-matched controls. Lesions of atherosclerosis were induced by a combination of repeat balloon injury and a hyperlipidemic diet. All animals were imaged on at least two occasions 9-16 months after initiation of atherosclerosis. In addition, animals were imaged immediately after sacrifice. Anatomic dissection and histology were performed to verify the MR findings. The volume phased array coil improves the image signal-to-noise ratio over existing extremity coils and resulted in higher resolution images of the abdominal aorta. Proton density-weighted images acquired with 2D/3D fast spin-echo are the most useful sequence to outline the vessel wall and to differentiate wall from lumen and background. Progressive wall thickening and lumen stenosis were observed in the serial images of the diseased rabbits. Wall thickness and lumen area derived noninvasively from the in vivo MR images correlate with postmortem MR images and sections of aorta examined by dissection microscopy and histology. Spin-echo and fast spin-echo imaging with a phased array body coil can be used to accurately assess plaque dimensions, and potentially can be used to image intraplaque features and to monitor lesion progression or regression. It should also be possible to adapt these techniques to assess human disease, especially for peripheral vascular problems.
Magnetic Resonance Imaging 02/1996; 14(1):93-102. · 1.99 Impact Factor
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ABSTRACT: The abilities of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) to regulate cAMP metabolism and mitogen-activated protein kinase (MAP kinase) activity were compared in human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP accumulation up to 150-fold in a concentration-dependent manner (EC50 approximately 0.7 nM). The peak of cAMP formation and cAMP-dependent protein kinase (PKA) activity occurred approximately 5 min after the addition of PDGF and rapidly declined thereafter. Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA activity by an additional 2.5-3-fold, whereas IBMX alone was essentially without effect. The PDGF-stimulated increase in cAMP was prevented by addition of the cyclooxygenase inhibitor indomethacin, consistent with release of prostaglandins stimulating cAMP. PDGF, but not IGF-I, stimulated MAPK activity, cytosolic phospholipase A2 (cPLA2) phosphorylation, and cAMP synthesis which indicated a key role for MAP kinase in the activation of cPLA2. Further, PDGF stimulated the rapid release of arachidonic acid and synthesis of prostaglandin E2 (PGE2) which could be inhibited by a cPLA2 inhibitor (AACOCF3). Calcium mobilization was required for PDGF-induced arachidonic acid release and PGE2 synthesis but not for MAPK activation, whereas PKC was required for PGE2-mediated activation of PKA. In summary, these results demonstrated that PDGF increases cAMP formation and PKA activity through a MAP kinase-mediated activation of cPLA2, arachidonic acid release, and PGE2 synthesis in human arterial smooth muscle cells.
Journal of Biological Chemistry 02/1996; 271(1):505-11. · 4.77 Impact Factor
