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Biomedical subjects

Andrew C Newby

Publications and source records attributed to Andrew C Newby.

At least 19 recordsLinked to original sources
PubMed2006

Genomics of foam cells and nonfoamy macrophages from rabbits identifies arginase-I as a differential regulator of nitric oxide production.

OBJECTIVE: Conversion of macrophages to foam cells is a critical step in the initiation and progression of atherosclerosis. We sought to identify genes differentially regulated in foam cells, since these are likely to include new targets for intervention. METHODS AND RESULTS: We used suppression subtraction hybridization to compare foam cells and nonfoamy macrophages isolated from subcutaneous granulomas of rabbits fed a cholesterol-rich or normal chow diet and confirmed upregulation of 3 genes, including matrix metalloproteinase-12 (mRNA 2.0-fold, P<0.005; protein 3.9-fold, P<0.03). Arginase-I mRNA showed the biggest decrease among 11 downregulated genes in foam cells (2.7-fold, P<0.001) and was accompanied by significantly reduced arginase enzymatic activity (60-fold, P<0.01). Arginase-I competes for substrate L-arginine with nitric oxide synthase and consequently nitric oxide production was significantly increased (3-fold, P<0.02) in foam cells compared with nonfoamy macrophages despite no difference in nitric oxide synthase isoenzyme expression. We validated upregulation of matrix metalloproteinase-12 and downregulation of arginase-1 in foam cells of rabbit and human atherosclerotic plaques. CONCLUSIONS: Our study identified several differentially expressed genes in foam cells and nonfoamy macrophages derived from live rabbits. The altered pattern of gene expression in foam cells is likely to influence atherosclerosis formation and stability.

Animals↗
PubMed2006

On the biology of saphenous vein grafts fitted with external synthetic sheaths and stents.

Autologous saphenous vein is used as a conduit to bypass atherosclerotic lesions in both the coronary artery (coronary artery bypass graft surgery [CABG]) and in femoral arteries (infrainguinal bypass graft surgery [IIBS]). Despite the undoubted success and benefits of the procedures, graft failure occurs in 50% of cases within 10 years after surgery. A principal cause of late vein graft failure is intimal and medial hyperplasia and superimposed atherogenesis. Apart from lipid lowering therapy, no intervention has hitherto proved clinically effective in preventing late vein graft failure which clearly constitutes a major clinical and economic problem that needs to be urgently resolved. However, we have studied the effect of external synthetic stents and sheaths in pig models of vein into artery interposition grafting and found them to have a profound effect on vein graft remodelling and thickening. In this review, therefore, we will summarise the mechanisms underlying vein graft failure and how these stents influence these processes and the possible mechanisms involved as well as the application of these devices in preventing vein graft failure clinically.

Animals↗
PubMed2006

Short- and long-term effects of cytochalasin D, paclitaxel and rapamycin on wall thickening in experimental porcine vein grafts.

OBJECTIVES: Neointima formation and wall thickening caused by smooth muscle cell proliferation compromise long-term patency of human aorto-coronary vein-grafts. We investigated short- and long-term effects of anti-proliferative pharmacological agents on experimental pig vein-grafts with similar dimensions and kinetics to human coronary grafts. METHODS AND RESULTS: Saphenous veins were treated for 1 h ex vivo with vehicle or concentrations of cytochalasin D, paclitaxel or rapamycin found to be anti-proliferative in preliminary studies. Vehicle and treated veins were implanted contralaterally, end-to-end into the carotid arteries of pigs. Cytochalasin D 2.5 mug/ml non-significantly reduced neointima formation in 4-week vein-grafts (mean+/-standard error, 2.5+/-0.6 vs. 3.3+/-0.6 mm2, n = 10, p = NS), whilst paclitaxel 10 microM produced significant inhibition (1.7+/-0.2 vs. 3.0+/-0.3 mm2, n = 8, p < 0.01) as did rapamycin 0.1 mg/ml (0.6+/-0.3 vs. 1.7+/-0.5 mm(2), n = 8, p < 0.02). Similar effects were found on total wall cross-sectional area but medial area was unaffected. PCNA staining of 1-week vein grafts confirmed in vivo anti-proliferative effects of paclitaxel (21+/-2 vs. 36+/-3%, n = 5, p < 0.01) and rapamycin (32+/-1 vs. 57+/-6%, n = 6, p < 0.005); neither agent stimulated loss of endothelium at these concentrations. Neointima and total wall area increased significantly between 4- and 12-weeks in all vein-grafts such that there was no longer a significant effect on neointima formation of either paclitaxel (7.5+/-1.3 vs. 8.9+/-1.9 mm2 in control, n = 5, p = NS) or rapamycin (6.0+/-0.9 vs. 7.9+/-1.1 mm2 in control, n = 9, p = NS) or on total wall area in 12-week grafts. CONCLUSIONS: Pre-treatment of saphenous vein with anti-proliferative agents paclitaxel or rapamycin reduced neointima and total wall area after 4 weeks but continued growth abolished differences by 12 weeks. These results may help to understand the failure of clinical studies using anti-proliferative treatments in vein-grafts.

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PubMed2006

Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis.

BACKGROUND: Matrix metalloproteinase (MMP)-associated extracellular matrix degradation is thought to contribute to the progression and rupture of atherosclerotic plaques. However, direct evidence of this concept remains elusive. We hypothesized that overexpression of tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2 would attenuate atherosclerotic plaque development and instability in high fat-fed apolipoprotein E-knockout (apoE(-/-)) mice. METHODS AND RESULTS: Seventy male apoE(-/-) mice (n=10/group) fed a high-fat diet for 7 weeks were injected intravenously with first-generation adenoviruses expressing the gene for human TIMP-1 (RAdTIMP-1) or TIMP-2 (RAdTIMP-2) or a control adenovirus (RAd66) and were fed a high-fat diet for a further 4 weeks. Analysis of brachiocephalic artery plaques revealed that RAdTIMP-2 but not RAdTIMP-1 infection resulted in a marked reduction (48+/-13%, P<0.05) in lesion area compared with that in control animals. Markers associated with plaque instability, assessed by smooth muscle cell and macrophage content and the presence of buried fibrous caps, were significantly reduced by RAdTIMP-2. Effects on lesion size were not sustained with first-generation adenoviruses, but murine TIMP-2 overexpression mediated by helper-dependent adenoviral vectors exerted significant effects on plaques assessed 11 weeks after infection. In an attempt to determine the mechanism of action, we treated macrophages and macrophage-derived foam cells with exogenous TIMP-2 in vitro. TIMP-2 significantly inhibited migration and apoptosis of macrophages and foam cells, whereas TIMP-1 failed to exert similar effects. CONCLUSIONS: Overexpression of TIMP-2 but not TIMP-1 inhibits atherosclerotic plaque development and destabilisation, possibly through modulation of macrophage and foam cell behavior. Helper-dependent adenovirus technology is required for these effects to be maintained long term.

Adenoviridae↗
PubMed2006

Altered S-phase kinase-associated protein-2 levels are a major mediator of cyclic nucleotide-induced inhibition of vascular smooth muscle cell proliferation.

Cyclic nucleotides inhibit vascular smooth muscle cell (VSMC) proliferation but the underlying molecular mechanisms are incompletely understood. We studied the role of S-phase kinase-associated protein-2 (Skp2), an F-box protein of SCFSkp2 ubiquitin ligase responsible for polyubiquitylation of and subsequent proteolysis of p27Kip1, a key step leading to cell cycle progression. Skp2 mRNA and protein were upregulated in mitogen-stimulated VSMCs and after balloon injury in rat carotid arteries, where the time course and location of Skp2 expression closely paralleled that of proliferating cell nuclear antigen. Skp2 small interference RNA (siRNA) reduced Skp2 expression, increased p27Kip1 levels, and inhibited VSMC proliferation in vitro. cAMP-elevating agents prominently inhibited VSMC proliferation and Skp2 expression through inhibiting Skp2 transcription as well as decreasing Skp2 protein stability. Consistent with this, activation of protein kinase A, a downstream target of cAMP, was shown to negatively regulate focal adhesion kinase (FAK) phosphorylation and Skp2 expression. Adenovirus-mediated Skp2 expression reversed cAMP-induced p27Kip1 upregulation and rescued cAMP-related S-phase entry inhibition up to 50%. 8-bromo-cGMP also moderately reduced Skp2 and cell proliferation when VSMCs were incubated with low serum concentration. Interestingly, we showed that 8-bromo-cGMP inhibited Skp2 expression also through activation of protein kinase A, not protein kinase G, which conversely enhanced FAKY397 phosphorylation and Skp2 expression. After balloon injury of rat carotid arteries, local forskolin treatment significantly reduced FAKY397 phosphorylation, Skp2 expression, VSMC proliferation, and subsequent neointimal thickening. These data demonstrate for the first time that Skp2 is an important factor in VSMC proliferation and its inhibition by cyclic nucleotides.

Animals↗
PubMed2006

Do metalloproteinases destabilize vulnerable atherosclerotic plaques?

PURPOSE OF REVIEW: Atherosclerotic plaque rupture and thrombosis underlie most myocardial infarctions. Matrix metalloproteinases are a family of enzymes that remodel the extracellular matrix. Metalloproteinases could stabilize rupture-prone plaques by promoting smooth muscle cell migration and proliferation. Alternatively, metalloproteinases could destabilize vulnerable plaques by promoting matrix destruction, angiogenesis, leucocyte infiltration, and apoptosis. Evidence is reviewed from genetically modified mice and human biomarker and genetic studies that sheds light on this dual role of metalloproteinases. RECENT FINDINGS: Inhibition of metalloproteinases in mice using tissue inhibitors of metalloproteinases increases plaque stability; however, double knockouts of apolipoprotein E with matrix metalloproteinase 2, 3, 7, 9, 12, and 13 have more or less stable plaques, consistent with harmful or protective effects of individual metalloproteinases. Overexpression studies in mice or rabbits show that high activities of matrix metalloproteinase 9 and 12 decrease stability. Biomarker and human genetic studies demonstrate that increased metalloproteinase activity is associated with vascular repair or myocardial infarction. SUMMARY: Recent studies reinforce evidence for a dual role of matrix metalloproteinases in plaque stabilization and rupture, which probably depends on the stage, site, and severity of disease. Dysregulated metalloproteinase activity in end-stage coronary artery disease appears a valid target for therapy.

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PubMed2005

Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates.

Intimal thickening occurs in blood vessels in response to injury or atherosclerosis. The balance of migration and proliferation of vascular smooth muscle cells (VSMC) over death by apoptosis has an important impact on the final size of intimal thickening and may also affect atherosclerotic plaque stability. All aspects of VSMC behaviour are under coordinated control by growth factors, cell-matrix and cell-cell interactions. We review the evidence that matrix-degrading metalloproteinases (MMPs) regulate migration, proliferation and survival of VSMC. Moreover, we discuss critically the underlying mechanisms, which include changing growth factor availability and remodelling cell-matrix and cell-cell contacts. We conclude that MMPs influence VSMC behaviour by cleaving both matrix and non-matrix substrates.

Animals↗
PubMed2005

Regulation of vascular smooth muscle cell proliferation, migration and death by heparan sulfate 6-O-endosulfatase1.

Migration, proliferation and death of vascular smooth muscle cells (VSMC) are important events in vascular pathology regulated by heparan sulfate proteoglycans and hence potentially by cell surface HS 6-O-endosulfatase1 (sulf1). Sulf1 mRNA expression was increased in cultured VSMC compared to rat aorta. Furthermore, adenovirus mediated overexpression of quail sulf1 decreased adhesion, and increased proliferation and apoptosis of VSMC. Overexpression of a dominant negative variant also decreased adhesion of VSMC and increased proliferation, apoptosis, migration and chemotaxis of VSMC. Our results imply that only normal levels of 6-O-sulfation maintained by sulf1 are optimal for several functions of VSMC.

Adenoviridae↗
PubMed2005

Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries.

Matrix metalloproteinases (MMPs) are thought to be involved in the growth, destabilization, and eventual rupture of atherosclerotic lesions. Using the mouse brachiocephalic artery model of plaque instability, we compared apolipoprotein E (apoE)/MMP-3, apoE/MMP-7, apoE/MMP-9, and apoE/MMP-12 double knockouts with their age-, strain-, and sex-matched apoE single knockout controls. Brachiocephalic artery plaques were significantly larger in apoE/MMP-3 and apoE/MMP-9 double knockouts than in controls. The number of buried fibrous layers was also significantly higher in the double knockouts, and both knockouts exhibited cellular compositional changes indicative of an unstable plaque phenotype. Conversely, lesion size and buried fibrous layers were reduced in apoE/MMP-12 double knockouts compared with controls, and double knockouts had increased smooth muscle cell and reduced macrophage content in the plaque, indicative of a stable plaque phenotype. ApoE/MMP-7 double knockout plaques contained significantly more smooth muscle cells than controls, but neither lesion size nor features of stability were altered in these animals. Hence, MMP-3 and MMP-9 appear normally to play protective roles, limiting plaque growth and promoting a stable plaque phenotype. MMP-12 supports lesion expansion and destabilization. MMP-7 has no effect on plaque growth or stability, although it is associated with reduced smooth muscle cell content in plaques. These data demonstrate that MMPs are directly involved in atherosclerotic plaque destabilization and clearly show that members of the MMP family have widely differing effects on atherogenesis.

Animals↗
PubMed2005

Biphasic effect of p21Cip1 on smooth muscle cell proliferation: role of PI 3-kinase and Skp2-mediated degradation.

OBJECTIVE: Proliferation of vascular smooth muscle cells (VSMC) is an important event in atherogenesis, in-stent restenosis and late vein-graft failure. Cell-cycle progression is positively regulated by cyclin:cdk complexes and negatively regulated by cyclin-dependent kinase inhibitors, including p21Cip1. Here we investigate the mechanisms regulating p21Cip1 levels in VSMCs and its role in controlling VSMC proliferation. METHODS AND RESULTS: We studied the S-phase-associated kinase protein-2 (Skp2), an F-box protein implicated in the ubiquitination of p21Cip1. Overexpression of wild-type Skp2 or dominant-negative Skp2 decreased or increased p21Cip1 levels, respectively. Interestingly, levels of endogenous p21Cip1 and Skp2 were both increased in a phosphoinositide PI 3-kinase-dependent manner in late G1 phase. Increased expression of p21Cip1 occurred despite significantly increased Skp2-mediated proteasomal degradation. To determine the role of p21Cip1 in regulating VSMC proliferation, we used adenovirus-mediated overexpression and small-interfering RNA to elevate or silence p21Cip1 expression, respectively. Overexpression of p21Cip1 significantly inhibited VSCM proliferation. p21Cip1 silencing also inhibited proliferation and increased apoptotic cell death. CONCLUSIONS: Taken together, this data demonstrates that a balance between PI 3-kinase-driven upregulation and Skp2-mediated degradation controls the level of p21Cip1, which regulates VSMC proliferation in a biphasic manner. Low levels of p21Cip1 are also essential to counter apoptosis during cell-cycle progression.

Animals↗
PubMed2005

Stent-based delivery of tissue inhibitor of metalloproteinase-3 adenovirus inhibits neointimal formation in porcine coronary arteries.

BACKGROUND: Stent-based antiproliferative therapy appears to decrease in-stent restenosis. However, alternative approaches might produce equivalent efficacy with better long-term safety. In previous work, an adenovirus capable of expressing the tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) inhibited neointima formation in cell cultures and porcine saphenous vein grafts. RAdTIMP-3 decreased smooth muscle cell migration, stabilized the extracellular matrix, and uniquely promoted apoptosis. The current study developed eluting stent technology to deliver RAdTIMP-3 during stenting of pig coronary arteries. METHODS AND RESULTS: Binding of virus to and elution from stents and transduction of pig coronary arteries were confirmed using beta-galactosidase as a reporter gene in vitro and in vivo. Deployment of RAdTIMP-3-coated stents increased apoptosis and reduced neointimal cell density, but did not increase inflammation or proliferation compared with beta-galactosidase-expressing adenovirus (RAdlacZ). Neointimal area after 28 days was significantly reduced to 1.27+/-0.19 mm2 with RAdTIMP-3 versus 2.61+/-0.31 mm2 with RAdlacZ stents (P<0.001) and 2.12+/-0.20 mm2 with bare stents (P<0.005). CONCLUSIONS: Our results demonstrate for the first time to our knowledge the feasibility of adenovirus-coated stent technology and highlight the potential of TIMP-3 to produce significant inhibition of in-stent neointima formation.

Adenoviridae↗
PubMed2005

Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture.

Intimal thickening, the accumulation of cells and extracellular matrix within the inner vessel wall, is a physiological response to mechanical injury, increased wall stress, or chemical insult (e.g., atherosclerosis). If excessive, it can lead to the obstruction of blood flow and tissue ischemia. Together with expansive or constrictive remodeling, the extent of intimal expansion determines final lumen size and vessel wall thickness. Plaque rupture represents a failure of intimal remodeling, where the fibrous cap overlying an atheromatous core of lipid undergoes catastrophic mechanical breakdown. Plaque rupture promotes coronary thrombosis and myocardial infarction, the most prevalent cause of premature death in advanced societies. The matrix metalloproteinases (MMPs) can act together to degrade the major components of the vascular extracellular matrix. All cells present in the normal and diseased blood vessel wall upregulate and activate MMPs in a multistep fashion driven in part by soluble cytokines and cell-cell interactions. Activation of MMP proforms requires other MMPs or other classes of protease. MMP activation contributes to intimal growth and vessel wall remodeling in response to injury, most notably by promoting migration of vascular smooth muscle cells. A broader spectrum and/or higher level of MMP activation, especially associated with inflammation, could contribute to pathological matrix destruction and plaque rupture. Inhibiting the activity of specific MMPs or preventing their upregulation could ameliorate intimal thickening and prevent myocardial infarction.

Animals↗
PubMed2004

Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability. A novel mechanism regulating smooth muscle cell proliferation.

Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.

Animals↗
PubMed2004

R-cadherin:beta-catenin complex and its association with vascular smooth muscle cell proliferation.

OBJECTIVE: Vascular smooth muscle cell (VSMC) proliferation is an important component of atherosclerosis, restenosis after angioplasty and stent placement, and vein graft failure. Outside-in signaling from the cadherin:beta-catenin complex can increase transcription of the cell-cycle gene cyclin D1; however, its role in VSMC proliferation has only recently been considered. METHODS AND RESULTS: We examined the involvement of R-cadherin and beta-catenin in VSMC proliferation in balloon-injured carotid arteries in vivo and aortic rings in vitro. The number of medial VSMCs positive for R-cadherin was significantly reduced by 32%+/-5%, 52%+/-10%, and 23%+/-2% at 0.25, 24, and 48 hours after injury in vivo, respectively. These changes in cadherin expression coincided with the detection of nuclear beta-catenin and elevated cyclin D1 expression. Furthermore, loss of R-cadherin expression was associated with medial VSMC proliferation. Inhibition of classical cadherin function with a HAV peptide and R-cadherin neutralizing antibodies significantly increased proliferation by 4.3+/-1.0-fold and 4.1+/-0.98-fold, and increased the number of cells with beta-catenin in the nucleus and expressing cyclin D1 in aortic rings. CONCLUSIONS: These results suggest that R-cadherin expression and beta-catenin signaling may be associated with increased cyclin D1 expression and VSMC proliferation and may therefore play an important role in vascular disease.

Animals↗
PubMed2003

Regulation of matrix metalloproteinase (matrixin) genes in blood vessels: a multi-step recruitment model for pathological remodelling.

Matrix metalloproteinases (MMPs; matrixins) are a family of structurally related enzymes that collectively promote turnover of all components of the extracellular matrix. Matrix turnover is required for vascular repair, but, if excessive, leads to pathologies that include aneurysm formation and atherosclerotic plaque instability. We review the positive and negative regulation of metalloproteinase gene induction. We propose that multiple steps of gene induction recruit a wider spectrum of MMPs, which may ultimately lead to a transition from matrix turnover to matrix destruction. Studying the detailed mechanisms involved may suggest possibilities for intervening selectively against pathological MMP induction.

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