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Biomedical subjects

S J George

Publications and source records attributed to S J George.

At least 19 recordsLinked to original sources
PubMed2001

Macro-porosity is necessary for the reduction of neointimal and medial thickening by external stenting of porcine saphenous vein bypass grafts.

BACKGROUND: placing external non-restrictive macro-porous stents around porcine vein grafts prevents neointima formation and medial thickening in both the short and long term. Whether the porosity of the stent material influences this effect, however, has not been determined. Therefore, the effect on neointimal and medial thickening of external macro-porous (polyester) and micro-porous (polytetrafluorethylene) stents of equal diameter were compared. The effect on expression of platelet-derived growth factor (PDGF), a potent mediator of vascular smooth muscle cell migration and proliferation and its receptors was also investigated. METHODS AND RESULTS: saphenous vein-carotid artery interposition grafting was performed in Landrace pigs with external placement of 8 mm diameter macro- and micro-porous stents contralaterally. One month after surgery, graft wall dimensions, PDGF and PDGF receptor expression and cell proliferation using proliferating cell nuclear antigen (PCNA) were measured on histological sections. Macro-porous stents significantly reduced neointimal and medial thickening compared with micro-porous stents (0.1+/-0.02 vs. 0.25+/-0.03 mm, P<0.002, and 0.10+/-0.02 vs. 0.17+/-0.02 mm, P<0.014, respectively). Macro-porous stents significantly reduced the percentage of cells expressing PDGF and PCNA, compared with micro-porous stents (36+/-9 vs. 80+/-7, P < 0.002, and 11+/-3 vs. 21+/-2, P < 0.02, respectively). The percentage of cells expressing PDGF receptors was similar with both the stent types. Adventitial microvessel formation occurred across macro-porous stents but was markedly suppressed by micro-porous stents. CONCLUSIONS: porosity is crucial to the efficacy of external stents in reducing neointima formation in porcine vein grafts. Decreases in PDGF expression and cell proliferation accompany the reduction in neointima formation. In addition, macro-porous stents allow adventitial microvessels to connect with the vasculature outside the stent, thereby potentially improving oxygenation. Although external stenting is highly effective in reducing neointima formation after vein grafting, the properties of the stent material necessary for this effect have not been defined. This study establishes that macro-porosity is one essential feature required to reduce PDGF expression cell proliferation and neointima formation.

Animals↗
PubMed2001

Wild-type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis.

Patency of autologous human saphenous vein coronary artery bypass grafts (CABG) is compromised by intimal thickening and superimposed atherosclerosis, caused by migration of vascular smooth muscle cells (SMC) to the intima where they proliferate. Here, using adenoviral transfer, we have targeted SMCs using wild-type p53 (wt p53) overexpression. Initial in vitro analyses demonstrated that wt p53 overexpression had no effect on SMC proliferation but promoted apoptosis, which was inhibited by co-expression of bcl2 or crmA. Wt p53 inhibited SMC invasion through reconstituted matrices, a phenotype not affected by bcl2 or crmA. Overexpression of wt p53 in human saphenous vein before organ culture significantly induced apoptosis (P < 0.01, Student's t test) without affecting proliferation rates either in the media or in the intima. SMC migration was, however, significantly reduced by wt p53 (P < 0.01, Student's t test). Intimal thickening and the number of neointimal cells were reduced by 89% and 73%, respectively, after 14 days (P < 0.01 and P < 0.001, respectively, Student's t test). This study demonstrates that overexpression of wt p53 promotes apoptosis and inhibits migration of SMC leading to reduced intimal thickening. This maybe a useful approach for increasing patency rates in CABG procedures in the clinic.

Adenoviridae↗
PubMed2001

Injury induces dedifferentiation of smooth muscle cells and increased matrix-degrading metalloproteinase activity in human saphenous vein.

Long-term patency of human saphenous vein bypass grafts is low because of intimal thickening and superimposed atherosclerosis. Matrix-degrading metalloproteinases (MMPs) and changes in vascular smooth muscle cell (VSMC) phenotype are thought to be essential for the VSMC migration that contributes to intimal thickening. We examined VSMC phenotype and MMP activity in saphenous veins obtained before and after surgical manipulation. Surgical preparation of the veins significantly increased pro-MMP-1 expression by 2-fold and significantly reduced tissue inhibitor of MMPs (TIMP)-2 expression, whereas MMP-3 and TIMP-1 were unaffected. Furthermore, caseinolytic and gelatinolytic activities measured by in situ zymography were dramatically elevated by injury. The expression of desmin and smoothelin was significantly decreased by injury, whereas vimentin expression was significantly increased. In addition, these changes in phenotype and MMP activity were localized to a subpopulation of VSMCs, the circumferential medial VSMCs. Our data show that surgical preparative injury induces phenotypic modulation of a subpopulation of medial VSMCs to a synthetic phenotype and increases MMP activity. This may favor matrix degradation, VSMC migration, and the subsequent intimal thickening that leads to graft failure.

Caseins↗
PubMed2000

Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite.

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.

Cytochrome c Group↗
PubMed2000

Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3.

BACKGROUND: Autologous saphenous vein coronary artery bypass graft surgery is complicated by late graft failure due to neointima formation and subsequent atherosclerosis. Growth factors and metalloproteinases (MMPs) act in concert to promote neointima formation. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an extracellular matrix-associated MMP inhibitor, uniquely promotes apoptosis of isolated vascular smooth muscle cells. Here, we overexpressed TIMP-3 at the luminal surface of human saphenous veins before organ culture and in pig saphenous veins before interposition grafting into carotid arteries in vivo to assess neointima formation. METHODS AND RESULTS: In both models, high TIMP-3 immunoreactivity occurred in the luminal and upper medial extracellular matrix after adenovirus delivery. MMP activity measured by in situ zymography was reduced throughout the veins, confirming a bystander effect. By use of 3 independent techniques, apoptosis levels in the neointima and medial layer were significantly elevated by TIMP-3 overexpression. Neointima formation was reduced by 84% in 14-day human organ cultures and by 58% in 28-day pig vein grafts (both P<0.05). In contrast, TIMP-2 overexpression had no effect on neointima formation in vivo. CONCLUSIONS: Our results highlight the potential therapeutic benefit for TIMP-3 overexpression to reduce neointima formation associated with late vein graft failure.

Adenoviridae↗
PubMed2000

The association of platelet-derived growth factor receptor expression, plaque morphology and histological features with symptoms in carotid atherosclerosis.

Platelet-derived growth factor may influence smooth muscle cell migration and proliferation and, therefore, carotid plaque composition and stenosis. Platelet-derived growth factor receptor expression and histological features were compared in carotid plaques from symptomatic and asymptomatic patients. Immunocytochemistry and histology determined platelet-derived growth factor-alpha and -beta receptor expression, white blood cell infiltration, smooth muscle cell, elastin, cholesterol, collagen and intraplaque haemorrhage in carotid artery plaques removed at surgery or the post-mortem. Plaques with > 70% stenosis from asymptomatic (n = 10) and symptomatic patients (n = 27) had higher expression of platelet-derived growth factor and beta receptors and higher scores for macrophages and intraplaque haemorrhage than plaques with < 70% stenosis from asymptomatic patients (n = 33). Plaques with > 70% stenosis from symptomatic patients had significantly lower alpha receptor expression than plaques with > 70% stenosis from asymptomatic patients. The reduction of alpha receptor expression, which may inhibit smooth muscle cell migration, suggests that differential expression of platelet-derived growth factor receptor subunits in plaques may be related to symptoms.

Aged↗
PubMed2000

A simplified technique for the cryopreservation of vein allografts.

OBJECTIVES: we assessed the effects of cryopreservation on smooth-muscle cell injury in human vein. MATERIALS AND METHODS: long saphenous vein was collected during surgery and cryopreserved. Smooth-muscle cell damage was assessed after thawing by in situ detection of fragmented DNA. The presence of cryoprotectant (10% dimethyl sulphoxide, DMSO), cooling and warming rates, and the rate of cryoprotectant removal after thawing were examined. RESULTS: control veins exhibited damage in 8.5% (95% confidence interval (CI) 4.7 to 13.4%,n=13) of smooth-muscle cells compared with 27.7% (95% CI 23.2 to 32.4%, n=115) in vein frozen in 10% DMSO (p=0.001). In the presence of DMSO, damage to smooth-muscle cells was independent of the rates of cooling (p=0.72) and warming (p=0.45). The rate of dilution to remove the cryoprotectant after thawing also had no effect on cell damage (p=0.64). In the absence of cryoprotectant, cell damage was doubled to approximately 50% by slow rather than rapid warming (p=0.01). CONCLUSION: cooling rate, and the presence of a cryoprotectant, has little effect on smooth-muscle damage, provided that the tissue is warmed rapidly. Slow warming, in the absence of DMSO, causes substantial damage. These results suggest that simplified methods of vein cryopreservation are feasible.

Confidence Intervals↗
PubMed2000

Lipid risk factor correlates of ischemic heart disease as diagnosed by myocardial perfusion scintigraphy.

Patients with known coronary artery disease frequently change their lifestyles (e.g., diet, exercise, and smoking habit) after the diagnosis is made. Such changes can alter lipid risk factor levels and obscure etiologic risk factor associations with the presence of coronary artery disease. It is therefore preferable to determine the contribution of potential risk factors before the diagnosis of coronary artery disease has been established. In this trial, we used stress nuclear myocardial perfusion imaging to diagnose coronary artery disease in patients presenting for evaluation of chest pain. Two groups of age- and sex-matched patients were identified: a normal group (patients with no evidence of coronary artery disease), and an abnormal group (patients whose scans indicated the presence of significant coronary artery disease due to either fixed or reversible perfusion defects). Blood samples were drawn before scanning and analyzed for lipid risk factors. Compared to the normal group, the abnormal group had higher levels of triglycerides (189±91 vs. 135±51 mg/dL, p=0.003), lower levels of high-density lipoprotein cholesterol (39±9 vs. 45±14 mg/dL, p=0.037), and higher levels of small, dense low-density lipoprotein (LDL(3)) (42±18 vs. 32±13 mg/dL, p=0.007). Total cholesterol, low-density lipoprotein, and lipoprotein(a) levels were similar between groups. These findings suggest that ischemic heart disease, as assessed by myocardial perfusion scintigraphy, is more closely associated with the low high-density lipoprotein/high triglyceride syndrome than with increased low-density lipoprotein or total cholesterol levels. (c) 2000 by CHF, Inc.

Journal Article↗
PubMed2000

Therapeutic potential of matrix metalloproteinase inhibitors in atherosclerosis.

The activity of matrix-degrading metalloproteinases (MMPs) is essential for many of the processes involved in atherosclerotic plaque formation, for example, infiltration of inflammatory cells, smooth muscle cell migration and proliferation and angiogenesis. Furthermore, matrix degradation by MMPs may cause the plaque instability and rupture that leads to the clinical symptoms of atherosclerosis; unstable angina, myocardial infarction and stroke. Together, the family of MMPs can degrade all of the components of the blood vessel extracellular matrix and their activity therefore, is tightly regulated in normal blood vessels. The increased MMP activity during atherosclerotic plaque development and instability must therefore be caused by increased cytokine and growth factor-stimulated gene transcription, elevated zymogen activation and an imbalance in the MMP:TIMP ratio. It is therefore conceivable that inhibition of MMPs or re-establishing the MMP:TIMP balance may be useful in treating the symptoms of atherosclerosis. Recent studies using synthetic MMP inhibitors and gene therapy have highlighted the potential of such an approach.

Animals↗
PubMed1999

Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1.

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.

Binding Sites↗
PubMed1999

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem.

Phanerochaete chrysosporium lignin peroxidase isoenzyme H2 (LiP H2) exhibits a transition to a stable, inactive form at pH 9.0 with concomitant spectroscopic changes. The Söret peak intensity decreases some 55% with a red shift from 408 to 412 nm; the bands at 502 nm and 638 nm disappear and the peak at 536 nm increases. The EPR spectrum changes from a signal typical of high spin ferric haem to an exclusively low spin spectrum with g=2.92, 2.27, 1.50. These data indicate that the active pentaco-ordinated haem is converted into a hexaco-ordinated species at alkaline pH. Room temperature near-IR MCD data coupled with the EPR spectrum allow us to assign the haem co-ordination of alkali-inactivated enzyme as bishistidine. Re-acidification of the alkali-inactivated enzyme to pH 6 induces further spectroscopic changes and generates an irreversibly inactivated species. By contrast, a pH shift from 9.0 to 6.0 with simultaneous addition of 50 mM CaCl(2) results in the recovery of the initial activity together with the spectroscopic characteristics of the native ferric enzyme. Incubating with 50 mM CaCl(2) at a pH between 6.0 and 9.0 can also re-activate the enzyme. Divalent metals other than Ca(2+) do not result in restoration of activity. Experiments with (45)Ca indicate that two tightly bound calcium ions per enzyme monomer are lost during inactivation and reincorporated during subsequent re-activation, consistent with the presence of two structural Ca(2+) ions in LiP H2. It is concluded that both the structural Ca(2+) ions play key roles in the reversible alkaline inactivation of LiP H2.

Calcium↗
PubMed1999

Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3.

Dysregulation of matrix degrading metalloproteinase enzymes (MMPs) leads to increased extracellular matrix turnover, a key event in the local invasion and metastasis of many tumours. The tissue inhibitors of metalloproteinases (TIMPs) limit the activity of MMPs, which suggests their use in gene therapy. We have previously shown that overexpression of TIMP-1, -2 or -3 inhibits vascular smooth muscle and melanoma cell invasion, while TIMP-3 uniquely promotes apoptosis. We have therefore sought to determine whether TIMP-3 can inhibit invasion and promote apoptosis in other cancer cell types. Adenoviral-mediated overexpression of TIMP-3 inhibited invasion of HeLa and HT1080 cells through artificial basement membrane to similar levels as that achieved by TIMP-1 and -2. However, TIMP-3 uniquely promoted cell cycle entry and subsequent death by apoptosis. Apoptosis was confirmed by morphological analysis, terminal dUTP nick end labelling (TUNEL) and flow cytometry. The apoptotic phenotype was mimicked by addition of exogenous recombinant TIMP-3 to uninfected cultures demonstrating that the death signal is initiated extracellularly and that a bystander effect exists. These results show that TIMP-3 inhibits invasion in vitro and promotes apoptosis in cancer cell type of differing origin. This clearly identifies the potential of TIMP-3 for gene therapy of multiple cancer types.

Adenoviridae↗