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Biomedical subjects

Elaine W Raines

Publications and source records attributed to Elaine W Raines.

At least 19 recordsLinked to original sources
PubMed2006

Emerging roles for ectodomain shedding in the regulation of inflammatory responses.

The multistep model of leukocyte recruitment to sites of inflammation has helped elucidate specific molecular cues for each of the individual steps. However, it is less clear how cells transition between the different steps and how the complex interactions are coordinately regulated. Once a leukocyte sticks to the endothelium, it only takes a few minutes to reach the subendothelial basement membrane, so the transitions and regulatory mechanisms must be rapid. We put forward the hypothesis that proteolytic shedding of cell surface proteins provides a mechanism to aid in the rapid transition of cells and coordinate the complex, multistep process of leukocyte recruitment in response to inflammatory stimuli. Support for this hypothesis is provided from analyses of disease states and from studies with protease inhibitors and genetically engineered mutations that prevent "ectodomain shedding" of cell surface proteins and consequently perturb the inflammatory response.

ADAM Proteins↗
PubMed2005

Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice.

The majority of acute clinical manifestations of atherosclerosis are due to the physical rupture of advanced atherosclerotic plaques. It has been hypothesized that macrophages play a key role in inducing plaque rupture by secreting proteases that destroy the extracellular matrix that provides physical strength to the fibrous cap. Despite reports detailing the expression of multiple proteases by macrophages in rupture-prone regions, there is no direct proof that macrophage-mediated matrix degradation can induce plaque rupture. We aimed to test this hypothesis by retrovirally overexpressing the candidate enzyme MMP-9 in macrophages of advanced atherosclerotic lesions of apoE-/- mice. Despite a greater than 10-fold increase in the expression of MMP-9 by macrophages, there was only a minor increase in the incidence of plaque fissuring. Subsequent analysis revealed that macrophages secrete MMP-9 predominantly as a proform, and this form is unable to degrade the matrix component elastin. Expression of an autoactivating form of MMP-9 in macrophages in vitro greatly enhances elastin degradation and induces significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apoE-/- mice in vivo. These data show that enhanced macrophage proteolytic activity can induce acute plaque disruption and highlight MMP-9 as a potential therapeutic target for stabilizing rupture-prone plaques.

Animals↗
PubMed2005

Thematic review series: The immune system and atherogenesis. Cytokines affecting endothelial and smooth muscle cells in vascular disease.

The cellular and extracellular matrix accumulations that comprise the lesions of atherosclerosis are driven by local release of cytokines at sites of predilection for lesion formation, and by the specific attraction and activation of cells expressing receptors for these cytokines. Although cytokines were originally characterized for their potent effects on immune and inflammatory cells, they also promote endothelial cell dysfunction and alter smooth muscle cell (SMC) phenotype and function, which can contribute to or retard vascular pathologies. This review summarizes in vivo studies that have characterized endothelial- and smooth muscle-specific effects of altering cytokine signaling in vascular disease. Although multiple reports have identified cytokines as pivotal players in endothelial and SMC responses in vascular disease, they also have highlighted the need to delineate the critical genes and specific cellular functions regulated by individual cytokine signaling pathways.

Animals↗
PubMed2005

Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta.

OBJECTIVE: Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. METHODS AND RESULTS: We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 micromol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKKbeta activity, which regulates activation of NF- kappaB, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKKbeta activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKKbeta blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKKbeta recapitulates the effect of FFA on insulin-dependent NO production. CONCLUSIONS: Elevated levels of common FFA found in human serum activate IKKbeta in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction.

Animals↗
PubMed2005

Are suppressors of cytokine signaling proteins recently identified in atherosclerosis possible therapeutic targets?

Atherosclerosis is a slowly progressing chronic inflammatory disease characterized by focal arterial lesions that can ultimately occlude the entire blood vessel and lead to sudden death. Lesions associated with cardiovascular events are those enriched in macrophages and other inflammatory cells. Activation of inflammatory cells within lesions induces the release of cytokines which promotes more inflammation and associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. This review focuses on suppressors of cytokine signaling proteins recently identified in the atherosclerosis-prone ApoE(-/-) mouse and provides perspectives of their potential for intervention in atherosclerotic lesion progression.

Animals↗
PubMed2005

Activation of IKKbeta by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells.

Hyperglycemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk, but the molecular basis for this effect is unknown. In skeletal muscle, diabetes induces activation of inhibitor kappaB kinase (IKKbeta), a key cellular mediator of the response to inflammatory stimuli, and this impairs insulin signal transduction via the insulin receptor substrate-phosphatidylinositol 3-OH kinase (IRS-1/PI3-kinase) pathway. Since activation of endothelial nitric oxide synthase (eNOS) is dependent on IRS-1/PI3-kinase signaling, we hypothesized that activation of IKKbeta may contribute to the effect of glucose to impair NO production. Here, we show that exposure of bovine aortic endothelial cells to high glucose (25 mM) for 24 h impaired insulin-mediated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt, activation of eNOS, and production of NO. High glucose treatment also activated IKKbeta, and pretreatment with aspirin, a pharmacological inhibitor of IKKbeta, prevented both glucose-induced IKKbeta activation and the effect of high glucose to impair insulin-mediated NO production. These adverse responses to glucose were also blocked by selective inhibition of IKKbeta signaling via overexpression of a kinase-inactive form of the enzyme. Conversely, overexpression of wild-type IKKbeta recapitulated the deleterious effect of high glucose on insulin-mediated activation of eNOS. These data demonstrate that activation of IKKbeta plays a critical and novel role to mediate the deleterious effects of high glucose on endothelial cell function.

Animals↗
PubMed2005

The absence of platelet-derived growth factor-B in circulating cells promotes immune and inflammatory responses in atherosclerosis-prone ApoE-/- mice.

Both innate and adaptive immunity contribute to the progression of inflammatory-fibrotic lesions of atherosclerosis. Although platelet-derived growth factor (PDGF)-B has been investigated as a stimulant of smooth muscle cells in vascular diseases, its effects on the immune response during disease have not been evaluated in vivo. We used hematopoietic chimeras generated after lethal irradiation of ApoE-/- recipients to test the role of PDGF in atherosclerosis. Monocyte accumulation in early atherosclerotic lesions increased 1.9-fold in ApoE-/-/PDGF-B-/- chimeras. Lymphocytes from null chimeras showed a 1.6- to 2.0-fold increase in the number of activated CD4(+) T cells and a 2.5-fold elevation of interferon-gamma-secreting CD4(+) T cells on ex vivo challenge with modified low-density lipoprotein. Splenocyte transcript levels were also altered with a twofold decrease in interleukin-10 and 1.7- and 3.0-fold increases in interleukin-18 and CCR 5, respectively. These cellular and molecular changes were consistent with a shift to a proinflammatory phenotype in null chimeras. Our data also demonstrated for the first time the presence of a recently discovered family of negative regulators of innate and adaptive immunity, the suppressors of cytokine signaling (SOCS), in developing atherosclerotic lesions. Thus, our studies identify two independent negative immune regulatory pathways-PDGF-B and SOCS-that may help limit lesion expansion.

Animals↗
PubMed2004

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx.

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

ADAM Proteins↗
PubMed2004

Cleavage of focal adhesion kinase in vascular smooth muscle cells overexpressing membrane-type matrix metalloproteinases.

BACKGROUND: Membrane-type matrix metalloproteinases (MT-MMPs) were initially identified as cell surface activators of MMP-2 (gelatinase A). We have reported that MT1-MMPs and MT3-MMPs are expressed by activated vascular smooth muscle cells (SMCs) and play a role in the regulation of cell function. Overexpression of MT-MMPs results in cell rounding, decreased adherence, and increased migration. Because integrin-mediated cell adhesion regulates these events, we have investigated the functional relationship between MT-MMPs and focal adhesion assembly. METHODS AND RESULTS: Using adenoviral vectors we show that overexpression of MT-MMPs reduces the number of focal contacts, whereas the cell surface expression of integrin subunits remains unchanged. The 125-kDa focal adhesion kinase (FAK) is cleaved resulting in a 90-kDa fragment under these conditions, and paxillin is partially dissociated from FAK after its cleavage. Pretreatment of cells with BB94, a synthetic MMP inhibitor, rescues cell adhesion and prevents changes in focal adhesions, supporting a potential role for MT-MMP enzymatic activities. CONCLUSIONS: This study provides the first evidence that MT-MMPs are not only important in matrix degradation but also may affect the function of focal adhesions through FAK cleavage.

Actin Cytoskeleton↗
PubMed2004

A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16.

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.

ADAM Proteins↗
PubMed2004

PDGF and cardiovascular disease.

Platelet-derived growth factor (PDGF) was identified in a search for serum factors that stimulate smooth muscle cell (SMC) proliferation. During the development of lesions of atherosclerosis that can ultimately lead to vessel occlusion, SMC are stimulated by inflammatory factors to migrate from their normal location in the media. They accumulate within the forming lesion where they contribute to lesion expansion by proliferation and deposition of extracellular matrix. Different genetic manipulations in vascular cells combined with various inhibitory strategies have provided strong evidence for PDGF playing a prominent role in the migration of SMC into the neointima following acute injury and in atherosclerosis. Other activities of PDGF identified in vivo suggest additional functions for PDGF in the pathogenesis of cardiovascular disease.

Animals↗
PubMed2004

Role of discoidin domain receptors 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in atherosclerosis and lymphangioleiomyomatosis.

Obstructive diseases of blood vessels and the lung are characterized by degradation and synthesis of new extracellular matrix (ECM) components. Regulated remodeling of the ECM in diseases such as atherosclerosis and lymphangioleiomyomatosis (LAM), both characterized by excessive accumulation of smooth muscle cells (SMCs), is thought to be controlled in part by cell surface receptors for specific ECM components. Discoidin domain receptors (DDR) 1 and 2 represent a family of tyrosine kinase collagen receptors that are activated by fibrillar collagens. To test the hypothesis that DDR may be involved in ECM remodeling by SMCs in vivo, we analyzed DDR expression by reverse transcriptase-polymerase chain reaction and immunohistochemistry and demonstrate that both DDR1 and DDR2 are up-regulated in nodules of LAM as compared to normal controls, and are expressed in lesions of atherosclerosis. In vitro, retroviral overexpression of DDR1 or DDR2 in human SMCs cultured on polymerized collagen gels leads to a reduction of collagen expression and induces matrix metalloproteinase (MMP) 1 at both mRNA and protein levels, but only DDR2 enhances MMP2 activation. Moreover, DDR2 overexpression increases SMC-mediated collagen and elastin degradation in vitro. Using laser microdissection, we extend our studies to the analysis of SMCs from LAM nodules where we observe higher MMP1 expression and MMP2 activation. Taken together, these data provide evidence for the potential roles of DDR1 and DDR2 in the regulation of collagen turnover mediated by SMCs in obstructive diseases of blood vessels and the lung.

Arteriosclerosis↗
PubMed2004

Bcl-2 overexpression leads to increases in suppressor of cytokine signaling-3 expression in B cells and de novo follicular lymphoma.

The t(14;18)(q32;q21), resulting in deregulated expression of B-cell-leukemia/lymphoma-2 (Bcl-2), represents the genetic hallmark in human follicular lymphomas. Substantial evidence supports the hypothesis that the t(14;18) and Bcl-2 overexpression are necessary but not solely responsible for neoplastic transformation and require cooperating genetic derangements for neoplastic transformation to occur. To investigate genes that cooperate with Bcl-2 to influence cellular signaling pathways important for neoplastic transformation, we used oligonucleotide microarrays to determine differential gene expression patterns in CD19+ B cells isolated from Emu-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by > or =2-fold in Emu-Bcl-2 transgenic mice (P < 0.05). The suppressor of cytokine signaling-3 (SOCS3) gene was found to be overexpressed 5-fold in B cells from Emu-Bcl-2 transgenic mice. Overexpression of Bcl-2 in both mouse embryo fibroblast-1 and hematopoietic cell lines resulted in induction of SOCS3 protein, suggesting a Bcl-2-associated mechanism underlying SOCS3 induction. Immunohistochemistry with SOCS3 antisera on tissue from a cohort of patients with de novo follicular lymphoma revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic follicular lymphoma cells and colocalized with Bcl-2 expression in 9 of 12 de novo follicular lymphoma cases examined. In contrast, SOCS3 protein expression was not detected in the follicular center cell region of benign hyperplastic tonsil tissue. These data suggest that Bcl-2 overexpression leads to the induction of activated signal transducer and activator of transcription 3 (STAT3) and to the induction of SOCS3, which may contribute to the pathogenesis of follicular lymphoma.

Animals↗
PubMed2003

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17).

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.

ADAM Proteins↗
PubMed2003

An NF-kappaB-dependent transcriptional program is required for collagen remodeling by human smooth muscle cells.

Although remodeling of vessels can dramatically alter lumen diameter and clinical sequelae, the molecular mechanisms regulating extracellular matrix turnover and remodeling are still not well understood. To investigate these processes in human smooth muscle, we have compared their culture on monomer and polymerized collagen gels, conditions that mimic some of the features of injured and normal vessels, respectively. We show that culture on polymerized, but not monomer, collagen leads to the activation of the transcription factor NF-kappaB through phosphorylation and degradation of its inhibitor, IkappaBalpha. Coincident with NF-kappaB activation, expression of MMP1, MMP2, and alpha2 integrin increases on polymerized collagen. Specific inhibition of NF-kappaB by retroviral overexpression of wild-type IkappaBalpha or phosphorylation-resistant, IkappaBalpha-stabilized mutant (IkappaBalphaSer32,36/Ala) reverses the increases in MMP1 and alpha2beta1 on polymerized collagen and decreases collagen gel contraction and degradation. However, forced overexpression of alpha2beta1 integrin or MMP1 in smooth muscle cells expressing IkappaBalphaSer32,36/Ala rescues their ability to contract collagen gels. Thus, polymerized collagen induces NF-kappaB-dependent expression of MMP1 and alpha2beta1 integrin, that are required for smooth muscle extracellular matrix remodeling.

Collagen↗