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Biomedical subjects

A C Newby

Publications and source records attributed to A C Newby.

At least 37 records · Page 2Linked to original sources
PubMed1999

Targets for gene therapy of vein grafts.

Poor long-term patency and a lack of suitable systemic pharmacologic therapy for the prevention of vein graft failure have prompted the search for effective local gene therapy. Vein grafts are particularly well suited for gene transfer in the clinic because direct access to vein is available during surgical preparation for grafting. In this review, the available animal models are discussed and a new mouse model is highlighted. Recent advances in gene transfer technology are reviewed, including the use of adeno-associated virus and modified adenoviruses that can prolong in vivo transgene expression for months. Gene therapy is intended to reduce early thrombosis, reduce neointima formation, and prevent atherosclerosis in vein grafts. Promising antithrombotic targets include tissue plasminogen activator and thrombomodulin. Nitric oxide synthase, prostacyclin synthase, and tissue inhibitors of metalloproteinases have been used to reduce neointima formation, and vein graft atheroma remains a challenge for the future.

Anastomosis, Surgical↗
PubMed1999

Expression of tissue inhibitor of metalloproteinase-1, -2, and -3 during neointima formation in organ cultures of human saphenous vein.

Degradation of the extracellular basement membrane is implicated in atherosclerosis, restenosis after angioplasty, and intimal thickening of vein grafts. Upregulation of metalloproteinase (MMP)-2 and MMP-9 accompanies neointima formation in cholesterol-fed rabbits, in rat and pig models of angioplasty, and in organ cultures of human saphenous veins. MMPs are inhibited by binding to tissue inhibitors of MMPs (TIMPs). Relatively little is known about their regulation in relationship to neointima formation; thus, we investigated TIMP expression in the organ culture model. Qualitative reverse transcriptase-polymerase chain reaction of mRNA extracted from veins showed that TIMP-1, TIMP-2, and TIMP-3 are each expressed before and after culture. Zymography revealed that TIMP-1 was the most abundant TIMP secreted and that its secretion increased dramatically between 0 to 2 and 12 to 14 days of culture. An enzyme-linked immunosorbent assay showed that TIMP-1 secretion increased from 3.2+/-1.5 (mean+/-SE) to 32+/-6 ng/mg wet weight per day (n=5, P<0.01). Immunocytochemical testing localized the increased expression of TIMP-1 to neointimal smooth muscle cells. Although less abundant, TIMP-2 secretion also increased from 0.8+/-0. 3 to 4.7+/-0.2 ng/mg wet weight per day (n=5, P<0.001), and tissue levels increased from 33+/-7 to 150+/-70 ng/mg wet weight (P<0.05). TIMP-2 was also immunolocalized to neointimal smooth muscle cells and their surrounding matrix. TIMP-3 was not secreted but was detected variably and constitutively in tissue extracts (160+/-120 and 170+/-100 ng/mg wet weight [n=9] on days 2 and 14, respectively). TIMP-3 was found in the cells and extracellular matrix of the media and adventitia before culture and to a lesser extent in the neointima after 14 days of culture. Rates of total TIMP secretion on day 14 exceeded those of MMP-2 and MMP-9 (10.6+/-1.9 and 15.6+/-2.3 ng/mg wet weight per day, respectively). Consistent with this, in situ zymography showed that MMP gelatinase activity was highly localized to cell bodies in the media and neointima. Secretion of TIMP-1 and TIMP-2 is greatly increased during neointima formation in human saphenous veins. TIMP-1 is readily released, whereas TIMP-2 remains partially attached and TIMP-3 exclusively attached to the extracellular matrix. Regulation of TIMP expression is therefore an important determinant of net MMP activity during neointima formation, restricting it to the pericellular environment.

Collagenases↗
PubMed1999

Increased secretion of basement membrane-degrading metalloproteinases in pig saphenous vein into carotid artery interposition grafts.

Late saphenous vein bypass graft failure in humans involves medial and neointimal thickening as the result of migration and proliferation of vascular smooth muscle cells (SMCs). Recent work on angioplasty indicates that basement membrane-degrading metalloproteinases (MMPs) cooperate with growth factors to mediate SMC migration and proliferation. We sought evidence here for a similar role in experimental vein grafts in pigs. Tissue levels and secretion of MMP-2 and MMP-9 were compared by quantitative zymography in veins and grafts removed 2 to 168 days after implantation. Pro and active forms of MMP-2 were present in veins, but levels were increased in vein grafts after 7 days (4- and 6-fold, respectively) and 28 days (3-fold for both), returning to values in veins after 168 days. MMP-9 was not detected in veins, was increased in grafts after 2 days, further increased after 7 days (6-fold) and 28 days (15-fold), and declined to undetectable levels by 168 days. Immunocytochemistry detected increased expression of MMP-2 and MMP-9 with the same time course. MMP-2 was widely distributed, whereas MMP-9 was concentrated in highly proliferative SMCs at the superficial layers of the thickened neointima. We conclude that increased production of the basement membrane-degrading MMP-2 and MMP-9 spanned the period of neointima formation and SMC proliferation in experimental vein grafts. MMPs therefore constitute new therapeutic targets for reducing late vein graft failure.

Animals↗
PubMed1999

Increased secretion of tissue inhibitors of metalloproteinases 1 and 2 from the aortas of cholesterol fed rabbits partially counterbalances increased metalloproteinase activity.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) plays an important role in extracellular matrix turnover and thereby modulates atherosclerotic plaque development. MMP-1, -2, -3, and -9 activity is increased by atherosclerosis, but the status of TIMPs is less clear. We therefore compared secretion of TIMPs-1 and -2 from cultured aortic explants derived from arch, middle, and distal portions of thoracic aortas of normal rabbits and rabbits fed a 1% cholesterol diet for 8 weeks, using reverse zymography of conditioned media. Cholesterol feeding significantly increased secretion of TIMP-1 from arch and middle portions (both 2.6-fold), accompanied by 2.0- and 2.7-fold increases in TIMP-2, respectively. Atherosclerotic aortas exhibited increased immunoreactive TIMP-1 and TIMP-2 in endothelial cells, smooth muscle cells, and macrophages. Staining of extracellular matrix was also prominent within the noncellular boundary region between fibrous cap and the lipid core, and within the lipid core. Increased TIMP-2 staining was also found in the media subjacent to the lipid core. In situ gelatin zymography demonstrated excess MMP activity within the plaque with partial inhibition in the lipid core base and subjacent media, consistent with the distribution of TIMPs. Casein zymography and in situ zymography demonstrated that increased caseinolytic activity was confined to the pericellular zones of macrophages within the lipid core, again consistent with its restriction by TIMPs. In summary, atherosclerosis increases TIMP expression, which counterbalances, in part, increased MMP activity.

Animals↗
PubMed1999

Fibrous cap formation or destruction--the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation.

Endothelial activation and infiltration of monocyte macrophages are essential prerequisites for fibrous cap formation, which comprises proliferation and migration of smooth muscle cells and net matrix deposition. Macrophage foam cells and endothelium act as a source of growth factors and chemoattractants for smooth muscle cells. However, growth factors alone do not stimulate smooth muscle cell proliferation or migration. This requires, in addition, the remodelling of the extracellular matrix, at least partly mediated by metalloproteinases. In particular, loss of basement membrane components and contact with the interstitial matrix appears to be required to release a brake on proliferation and migration exerted by the basement membrane. Unless there is a change in the phenotype of macrophages in advanced lesions, it is not clear why fibrous cap destruction rather than formation should take place in macrophage-rich shoulder regions of plaques. Impaired cap formation caused by smooth muscle senescence, mummification and propensity to apoptosis may be as important as increased cap destruction in promoting plaque rupture.

Animals↗
PubMed1998

Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B.

Matrix metalloproteinase (MMPs) enzymes are implicated in matrix remodelling during proliferative inflammatory processes including wound healing. We report here synergistic upregulation of MMP-9 protein and mRNA by platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF) in combination with interleukin-1alpha (IL-1alpha) or tumour necrosis factor-alpha (TNF-alpha) in primary rabbit and human dermal fibroblasts. The synergistic interaction between growth factors and cytokines implies that basement membrane remodelling is maximal physiologically when both are present together. The signalling pathways mediating this synergistic regulation are not understood, although analysis of the MMP-9 promoter has identified an essential proximal AP-1 element and an upstream nuclear factor kappa-B (NF-kappaB) site. Using electromobility shift assays, binding to the AP-1 site was only slightly increased by growth factors and cytokines. NF-kappaB binding was rapidly induced by IL-1alpha or TNF-alpha but was neither induced nor potentiated by bFGF or PDGF. Neither AP-1 nor NF-kappaB was therefore sufficient on its own for synergistic regulation. Using a recently developed adenovirus that overexpresses the inhibitory subunit, IkappaB alpha, we demonstrated an absolute requirement for NF-kappaB in upregulation of MMP-9. Activation of NF-kappaB binding by inflammatory cytokines was therefore necessary but not sufficient for synergistic upregulation of MMP-9.

Animals↗
PubMed1998

Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein.

Neointimal formation involving smooth muscle cell (SMC) migration and proliferation is a common feature of atherosclerosis, restenosis after angioplasty, and vein graft intimal thickening. Extracellular matrix remodeling by metalloproteinase (MMP) enzymes is an essential component of neointimal formation and therefore MMPs are a potential target for localized gene therapy. To evaluate this concept using human tissue, we used the highly reproducible organ culture model of neointimal formation in human saphenous vein to investigate the effect of adenovirus-mediated gene transfer of tissue inhibitor of metalloproteinase 1 (TIMP-1) and the bacterial LacZ gene (RAd35) as a control. Incubating veins with 100 microl of RAd35 (1.2 x 10(10) pfu/ml) led to expression of LacZ in 39 +/- 7% of surface cells but had no effect on SMC proliferation, migration, or neointimal formation. Similar infection with RAdTIMP-1 increased explanation of TIMP-1 in surface cells and significantly inhibited neointimal formation and SMC migration after 14 days by 54% and 78%, respectively (n = 6, p < 0.05 Student's paired t test). No effect on SMC proliferation or deleterious effect on cell viability was observed. A specific MMP inhibitory effect was detected using in situ zymography. These data confirm the importance of MMPs in neointimal formation and highlight the potential for application of TIMP gene therapy.

Adenoviridae↗
PubMed1998

Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis.

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related secreted proteins that limit matrix metalloproteinase (MMP) activity and also have direct effects on cell growth. We used the highly efficient adenoviral delivery system to overexpress individual TIMPs from the cytomegalovirus immediate early promoter in rat aortic smooth muscle cells. Overexpression of TIMP-1, -2, or -3, or a synthetic MMP inhibitor similarly inhibited SMC chemotaxis and invasion through reconstituted basement membrane. TIMP-1 overexpression did not effect cell proliferation. By contrast, TIMP-2 caused a dose-dependent reduction in proliferation, an effect not mimicked by a synthetic MMP inhibitor. TIMP-3 overexpression induced DNA synthesis, and promoted SMC death by apoptosis, a phenotype reproduced by adding TIMP-3 to uninfected cells, but not by a synthetic MMP inhibitor. Our study is the first to compare systematically the effect of overexpression of three TIMPs in any cell. We found similar effects on invasion mediated by inhibition of MMP activity, but widely divergent effects on proliferation and death through actions of TIMP-2 and -3 independent of MMP inhibition. These findings have important implications for the physiological roles of TIMPs and their use in gene therapy.

Adenoviridae↗
PubMed1998

External stenting reduces long-term medial and neointimal thickening and platelet derived growth factor expression in a pig model of arteriovenous bypass grafting.

Bypass of stenotic coronary arteries with autologous saphenous vein is an established treatment for ischemic heart disease. However, its long-term clinical success is limited. Late vein graft failure is the result of medial and intimal thickening consequent upon medial vascular smooth muscle cell migration, proliferation and extracellular matrix deposition, followed later by superimposed atherosclerosis. These changes directly compromise graft blood flow and provoke thrombosis. Vein graft wall thickening may represent an adaptation imposed by arterial hemodynamic factors, and these factors have been shown to promote vascular smooth muscle cell migration and proliferation through activation of key mediators including platelet-derived growth factor (PDGF). Many pharmacological interventions aimed at preventing these long-term changes have proven unsuccessful in clinical evaluation. We recently demonstrated in a pig saphenous vein graft model that application of an external polyester stent to the outside of carotid interposition vein grafts reduced intimal hyperplasia and total wall thickness 1 month after implantation. However, it is not known whether the benefits of the stent are maintained in the longer term or what mechanisms underlie its effect. The present study therefore compared morphological changes and PDGF expression in stented grafts and contralateral unstented grafts in the same pigs, 6 months after graft implantation. Reduced medial thickening, neointima formation, and cell proliferation were sustained in externally stented grafts, and these effects were associated with a significant reduction in PDGF expression.

Animals↗
PubMed1998

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins.

Metalloproteinases (MMPs) are implicated in neointima formation and hence vein graft failure. Gene transfer to elevate local levels of tissue inhibitor of metalloproteinases (TIMPs) is therefore a potential treatment. In this study, we have used lumenal application of a replication-defective recombinant adenovirus to overexpress TIMP-2 and observe the effects on neointimal thickening in a well characterised human saphenous vein organ culture model. Increased TIMP-2 expression was localised to lumenal surface cells but nevertheless increased total functional TIMP-2 secretion after 14 days culture from 4.0 +/- 2.0 to 21.8 +/- 2.9 ng/mg wet weight/day (P < 0.05, n = 3). In situ zymography revealed a marked inhibition of gelatinolytic activity by TIMP-2 gene transfer throughout the vein segments. Neointima formation and neointimal cell numbers were reduced 79% and 71%, respectively (P < 0.05; n = 8). TIMP-2 overexpression had no effect on smooth muscle cell proliferation, secretion of pro-MMP-2 or -9 and did not inhibit the processing of pro-MMP-2 to its active form. Our data indicate that TIMP-2 overexpression reduces neointimal thickening, primarily by inhibiting MMP activity and hence smooth muscle cell migration.

Adenoviridae↗
PubMed1997

Surgical preparative injury and neointima formation increase MMP-9 expression and MMP-2 activation in human saphenous vein.

OBJECTIVES: Injury stimulates smooth muscle cell (SMC) migration and proliferation by mechanisms that are incompletely understood. Surgical preparative injury is an important determinant of neointimal thickening in human saphenous vein bypass grafts. We investigate here whether basement-membrane-degrading metalloproteinases (MMPs) are stimulated by surgical preparation and culturing of human saphenous veins in organ culture. METHODS: Secretion of MMP-2 and MMP-9 was measured by zymography and Western blotting. Sites of MMP secretion were localised by immunocytochemistry and in situ hybridisation. RESULTS: Freshly isolated veins secreted pro-MMP-2 and much lower amounts of active MMP-2 and pro-MMP-9. MMP-2 was expressed in all cells whereas MMP-9 expression was confined to endothelial cells and at low levels to 55 +/- 10% (mean +/- s.e.m., n = 6) of medial SMC. Surgical preparative injury increased pro-MMP-2, active MMP-2 and pro-MMP-9 secretion. MMP-9 expression 3 h after surgical preparation occurred at high levels in 59 +/- 5% of medial SMC (P < 0.05 vs. freshly isolated, n = 6). Culturing in serum for 12 days increased pro-MMP-2, active MMP-2 and pro-MMP-9 secretion to equal levels in freshly isolated and surgically prepared veins. MMP-9 expression was greatest in the highly proliferative neointimal SMC and was more widespread in medial SMC of surgically prepared than freshly isolated veins (89 +/- 3 vs. 67 +/- 11%, n = 6, P < 0.05), paralleling the differences in proliferative index (18 +/- 3 vs. 8 +/- 4 cell/mm2, P < 0.05). CONCLUSIONS: The data provide new insights into the mechanisms underlying human SMC proliferation. Activation of MMP-2 and increased MMP-9 expression are shown to be important components of the response to injury in this model. Furthermore, MMP-9 expression is closely associated with medial and neointimal SMC proliferation.

Blotting, Western↗
PubMed1997

Increased secretion of gelatinases A and B from the aortas of cholesterol fed rabbits: relationship to lesion severity.

Basement membrane degrading metalloproteinases (gelatinases) have been implicated in the regulation of vascular smooth muscle cell migration and proliferation in culture and during neointima formation in vivo. We compared the expression and activation of gelatinases A and B in explants derived from the arch, mid and distal portions of thoracic aortas of normal rabbits and those given a 1% cholesterol-containing diet for 8 weeks. Neointimal/medial ratio was less than 0.01 in normal rabbits but was significantly increased by cholesterol feeding in the arch (1.08 +/- 0.26), mid (0.75 +/- 0.28) and distal (0.32 +/- 0.12) portions of the aorta (mean +/- S.E.M., n = 6), and to a significantly (P < 0.05) greater extent in the arch and mid than distal portions. Secretion of gelatinase B measured by densitometric scanning of zymograms was undetectable from normal aortas, but was significantly increased by cholesterol feeding in the arch (0.16 +/- 0.06), mid (0.26 +/- 0.08) and distal (0.11 +/- 0.05) portions (optical density units, n = 6, each P < 0.05 versus normal diet). The increase in gelatinase B expression was localised by in situ hybridisation to neointimal vascular smooth muscle cells, macrophages and endothelial cells. Secretion of pro-gelatinase A was detected from normal aortas; it was increased by cholesterol feeding from the arch (4.0 versus 2.8, P < 0.05) and mid (3.6 versus 2.8, P < 0.05) but not distal portions of the aorta (1.8 versus 1.2, n.s.). Similar results were obtained for active gelatinase A secretion from the arch (0.50 versus 0.28, P < 0.05) and mid (0.47 versus 0.23, P < 0.05) but not distal portions (0.19 versus 0.20, n.s.). Increases in pro- and active gelatinase A secretion therefore paralleled the severity of atheroma formation. The results imply that increased basement membrane turnover mediated by gelatinases occurs during cholesterol induced atherosclerosis formation.

Animals↗
PubMed1996

Influence of external stent size on early medial and neointimal thickening in a pig model of saphenous vein bypass grafting.

BACKGROUND: Late saphenous vein graft failure results from intimal and medial thickening due to migration and proliferation of vascular smooth muscle cells and superimposed atheroma. These changes may represent an adaptation by the vein to its insertion into the arterial system. Using a porcine model of arteriovenous bypass grafting, we recently demonstrated that supporting the graft with a nonrestrictive external Dacron velour stent significantly reduced intimal hyperplasia and total wall thickness. In the present study, we investigated the influence of different external stent sizes on graft wall dimensions and cell proliferation. METHODS AND RESULTS: Three stent sizes were tested: mildly restrictive, nonrestrictive, and oversized (5, 6, and 8 mm in diameter, respectively). Four weeks after grafting, total wall thickness was decreased 40% by 5-mm stents (P = .02), 66% by 6-mm stents (P = .0004), and 81% by 8-mm stents (P = .02 versus unstented grafts). Neointimal thickness was reduced almost 62% by 6-mm and 72% by 8-mm stents (both P = .01) but not by 5-mm stents. As a result, the encroachment of the intima into the lumen was reduced approximately 70% by 6- or 8-mm stents (P = .02 and P = .01 versus unstented grafts, respectively). Both neointimal and medial cell proliferation were significantly reduced by all three stents compared with unstented grafts. CONCLUSIONS: External stenting of saphenous vein bypass grafts reduces early intimal and medial hyperplasia. Oversized stents give equally profound suppression of intimal thickening, obviating the need for precise size matching with the graft and greatly simplifying the surgical procedure.

Animals↗
PubMed1996

Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells.

The migration and proliferation of vascular smooth muscle cells (SMCs) during neointima formation in atherosclerosis and angioplasty restenosis is mediated by certain growth factors and cytokines, one action of which may be to promote basement-membrane degradation. To test this hypothesis further, the effects of such growth factors and cytokines on the synthesis of two basement-membrane-degrading metalloproteinases, namely the 72 kDa gelatinase (MMP-2, gelatinase A) and the 95 kDa gelatinase (MMP-9, gelatinase B) and three tissue inhibitors of metalloproteinases (TIMPs) was studied in primary cultured rabbit aortic SMCs. Expression of the 95 kDa gelatinase was increased by phorbol myristate acetate, foetal calf serum, thrombin and interleukin-1alpha (IL-1alpha); platelet-derived growth factor (PDGF) BB alone had no effect but acted synergistically with IL-1alpha. A selective protein kinase C inhibitor, Ro 31-8220, abolished induction of the 95 kDa gelatinase. In contrast, none of the agents tested modulated the synthesis of the 72 kDa gelatinase. We conclude that maximal up-regulation of 95 kDa gelatinase expression requires the concerted action of growth factors and inflammatory cytokines mediated, in part, by a protein kinase C-dependent pathway. TIMP-1 and TIMP-2 were highly expressed, and their synthesis was not affected by growth factors or cytokines. Expression of TIMP-3 mRNAs was, however, increased by PDGF and transforming growth factor beta, especially in combination. Divergent regulation of gelatinase and TIMP expression implies that either net synthesis or net degradation of basement membrane can be mediated by appropriate combinations of growth factors and cytokines.

Animals↗
PubMed1996

An essential role for platelet-derived growth factor in neointima formation in human saphenous vein in vitro.

The role of platelet-derived growth factor (PDGF), a potent vascular smooth muscle cells (SMC) mitogen and chemoattractant, was investigated during neointima formation in human saphenous vein organ culture. PDGFA and B messenger ribonucleic acid (mRNA) expression was detected by RNase protection assay and in situ hybridisation and PDGF protein by immunocytochemistry. The expression of PDGFA and B mRNA was low in veins before culture while PDGF protein was detected in all cell types. A neointima consisting of densely packed SMC developed after 14 days of culture. The dense packing and high expression of PDGFA and B mRNA in neointimal SMC led to higher PDGF protein concentrations in the neointima, the role of which was examined by culturing with neutralising anti-(human PDGF) antibodies. The anti-PDGF antibodies significantly reduced neointimal thickness by approximately 66% and the number of neointimal cells by approximately 50%, without affecting neointimal or medial proliferation indices or cell viability. These results suggest that PDGF played an essential role in SMC migration into the neointima in human saphenous vein.

Adult↗
PubMed1996

External stenting reduces early medial and neointimal thickening in a pig model of arteriovenous bypass grafting.

The long-term clinical success of coronary artery bypass grafting with autologous saphenous vein is limited by progressive medial and neointimal thickening in the graft and superimposed atherosclerosis. We sought to reduce wall thickening by applying an external stent to experimental grafts in pigs. The diameter of the stent was designed to allow unrestricted initial expansion of the vein in response to arterial pressure and the stent material was highly porous so as to minimize adventitial [correction of advential] disruption. Four weeks after graft implantation, stented grafts had a larger lumen (11.2 +/- 6.2 [standard deviation] mm2 versus 7.6 +/- 3.4 mm2, p < 0.05, n = 9) and an almost fourfold thinner media (0.14 +/- 0.08 versus 0.49 +/- 0.22, p < 0.001) and neointima (0.10 +/- 0.07 versus 0.35 +/- 0.24, p < 0.001) than paired unstented grafts in the same animals. Cell proliferation was also greatly reduced by stenting in the neointimal and medial layers. The stenting procedure devised here is readily applicable to clinical coronary bypass grafts.

Animals↗