PubMed Health⌕ Search

Biomedical subjects

C M Holt

Publications and source records attributed to C M Holt.

At least 19 recordsLinked to original sources
PubMed2007

Vascular delivery of c-myc antisense from cationically modified phosphorylcholine coated stents.

c-Myc is involved in the formation of neointimal hyperplasia. We investigated in vitro, ex vivo and in vivo release of antisense c-myc from cationically modified phosphorylcholine-coated stents, as well as the effects on c-Myc expression and neointima formation in a porcine coronary stent model. In vitro experiments were performed to determine optimal loading of stents with antisense. Stents loaded with labelled antisense were deployed in porcine arteries ex vivo and in vivo. Antisense was detected in the vessel wall directly surrounding the stent of pig carotid and coronary artery up to 48 h after stent deployment. Nuclear uptake was observed in endothelial and vascular smooth muscle cells. Labelled antisense within peripheral tissues in vivo was <1.0% of that within stented arterial segments. Control and antisense loaded stents implanted into 10 pig coronary arteries and analysed at 28 days post-stenting showed that lumen area within the antisense stents was significantly increased (i.e. 30.5% greater, P<0.01), whilst both neointimal area and neointimal thickness were significantly reduced (17.5% and 19.5%, respectively, P<0.01) compared to control stents. Cationically modified phosphorylcholine coated stent-based delivery of c-myc antisense is feasible with minimal systemic delivery and is associated with a reduction of in-stent neointimal hyperplasia in pig coronary arteries.

Animals↗
PubMed2001

TGFbeta is active, and correlates with activators of TGFbeta, following porcine coronary angioplasty.

OBJECTIVE: Restenosis following angioplasty involves processes that may be influenced by local production of cytokines. We investigated the expression of active and total transforming growth factor beta (TGFbeta) following porcine coronary angioplasty (PTCA), and have correlated this with the expression of potential in vivo activators of TGFbeta: mannose-6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor and thrombospondin-1. METHODS: Oversized porcine PTCA was performed and the arteries excised after selected intervals. Levels of in situ active and total (active plus latent) TGFbeta were determined using a modified plasminogen activator-inhibitor/luciferase bioassay. RESULTS: Levels of active TGFbeta significantly increased 2 h to 7 days after angioplasty, compared to non-injured controls. Levels returned to baseline by 28 days. Active TGFbeta in tissues adjacent to the injured artery did not change. Total TGFbeta was significantly higher than controls 2-6 h after injury. M6P/IGF-II receptor mRNA was upregulated between 6 h and 3 days after injury, with protein detectable at 3-28 days. Thrombospondin-1 was detected between 1 h and 14 days. CONCLUSIONS: We conclude that balloon injury causes an early rapid increase in levels of active TGFbeta, that correlates with the expression of TGFbeta activators. Thus, TGFbeta is a good potential target for anti-restenotic therapies.

Angioplasty, Balloon, Coronary↗
PubMed2001

Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries.

Previous studies have shown that inhibition of the proto-oncogene c-myb inhibits neointimal formation in various animal models. However, the temporal and spatial expression of c-Myb in the vessel wall after injury is not known, and the mechanism of action of antisense oligonucleotide (AS-ODN-c-myb) inhibition remains unclear. One potential effect of cell cycle dysregulation by inhibition of c-myb is an increase in the rates of apoptosis. In this study, c-Myb expression after percutaneous transluminal coronary angioplasty (PTCA) injury and induction of apoptosis after AS-ODN-c-myb treatment were determined. Immunohistochemistry and cellular phenotyping were used to localize c-Myb expression in porcine coronary arteries at various time intervals after PTCA. In vitro, the effects of AS-ODN-c-myb on the apoptosis of porcine vascular smooth muscle cells (PVSMCs) and endothelial cells were determined by using a cell-death ELISA and time-lapse video microscopy. In vivo, local delivery of AS-ODN-c-myb was performed after PTCA of pig coronary arteries, and apoptosis was quantified at 6 hours. c-Myb is induced in pig coronary arteries after angioplasty, with maximal expression in inflammatory cells at 18 hours and in vascular smooth muscle cells at 3 to 7 days. In vitro, AS-ODN-c-myb enhanced PVSMCs (6.8+/-0.8% [P=<0.001] versus 0.5% serum) but not endothelial cell apoptosis (1.4+/-0.5% [P=NS] versus 0.5% serum). In vivo, 6 hours after porcine coronary angioplasty and delivery of AS-ODN-c-myb, the proportion of apoptotic cells within the media was 4.2+/-0.8% (PTCA alone), 2.3+/-0.2% (PTCA+vehicle), and 9.0+/-1.1% (PTCA+AS-ODN-c-myb; P<0.05 versus PTCA alone and P<0.01 versus PTCA+saline). c-Myb is expressed after PTCA of pig coronary arteries, and AS-ODN-c-myb induces apoptosis of PVSMCs in vitro and medial cells in vivo.

Angioplasty, Balloon, Coronary↗
PubMed2001

Utility of heel ultrasound bone density in men.

In women, heel ultrasound (US) bone mineral density (BMD) has been shown to predict fracture risk, but the usefulness of this screening tool in men is not known. We measured the heel quantitative ultrasound index (QUI( in a convenience sample 185 of men (136 Caucasian, 1 Asian, and 48 African-American) with an average age of 63 yr (range of 25-85) undergoing BMD of the spine and hip by dual X-ray absorptiometry (DXA) to determine whether the heel measurement could predict central BMD. The average DXA T-score was -0.97, -1.20, and -1.61 for the spine, total hip, and femoral neck, respectively. The mean heel US BMD T-score (using the only available T-score, which was defined for Caucasian postmenopausal women) was -0.92. There were significant correlations among the various DXA measurements and the heel US BMD T-score (r = 0.373-0.483, p < 0.001). We defined arbitrarily osteopenia as a spine, total hip, or femoral neck T-score by DXA of < -1.5. We also made two different arbitrary definitions of osteoporosis by DXA: < -2.0 and < -2.5. Using these numbers as disease definitions, we determined the specificity, sensitivity, as well as positive and negative predictive values of using the heel US T-score to predict osteopenia or osteoporosis. Using various cutoffs for the heel T-score, we found that increasing the cutoff toward 0 increased the sensitivity but lowered the specificity. No cutoff was found that provided both good sensitivity and specificity. By analyzing the men by ethnic and age groups, we found that the best set of receiver operating characteristic (ROC) curves was derived from data using heel US to predict osteopenia and osteoporosis in men younger than age 65, although the areas under the ROC curve were approx 0.8. In conclusion, despite a strong correlation between the heel QUI and the spine and hip BMD by DXA, no heel T-score could predict osteopenia or osteoporosis with satisfactory sensitivity and specificity. It is possible that the use of risk factor assessment plus heel QUI might have better predictive value, and further studies are needed to determine whether heel QUI or other US determination is an independent risk factor for fracture in men.

Absorptiometry, Photon↗
PubMed2001

Phosphorylcholine-coated stents in porcine coronary arteries: in vivo assessment of biocompatibility.

AIMS: To examine the angiographic (quantitative coronary angiography), morphometric, light microscopic (LM) (i.e., histology and immunohistochemical staining) and electron microscopic (EM) findings after implantation of phosphorylcholine (PC)-coated compared to uncoated stents in porcine coronary arteries. METHODS: Forty (25 PC-coated, 15 uncoated) divYsio stents were implanted into the coronary arteries of 20 pigs. Quantitative coronary angiography (QCA) was performed pre-stent and post-implantation in fifteen pigs, at 28 days. Two pigs were killed at 5 days (LM and scanning EM), one pig at 14 days (scanning EM) and 17 pigs at 28 days (LM, scanning EM, transmission EM). At 28 days, thirty-two of 34 stented segments excised were formalin-fixed, of which 30 were embedded in resin and sectioned for morphometry and LM. Remaining stents were examined by TEM and SEM. RESULTS: No angiographically occlusive thrombosis occurred in any of the stents. LM at 5 days showed endothelialization of PC-coated and uncoated stents, which was also confirmed by scanning EM at 14 days. At 28 days, QCA and morphometry showed no significant differences between PC-coated and uncoated stents. A few inflammatory cells were seen in both stent types at 5 days but there was no inflammatory or additional tissue reaction to PC-coated compared to uncoated stents at 28 days. CONCLUSIONS: The divYsio stents, with or without PC coating, performed equally well in terms of acute patency, 28-day QCA and morphometry. The PC coating allows a stent to endothelialize normally and is not associated with specific histological changes. The PC coating on the divYsio stent appears biocompatible.

Animals↗
PubMed2000

In vitro evaluation of c7E3-Fab (ReoPro) eluting polymer-coated coronary stents.

OBJECTIVE: Stent thrombosis and in-stent restenosis remain problematic in certain patient sub-groups. c7E3-Fab (ReoPro, abciximab) inhibits the platelet glycoprotein IIb/IIIa receptor as well as the smooth muscle cell alpha(v)beta3 receptor, and thus may influence both processes, especially if high local concentrations could be achieved. We have studied the adsorption and elution characteristics of c7E3-Fab on commercially available polymer-coated stents. We have also investigated the effect of such antibody binding on platelet deposition in vitro, and on antibody deposition into ex vivo human saphenous vein wall to assess whether such stents may influence stent thrombosis and restenosis. METHODS AND RESULTS: Adsorption was measured using a radioisotope technique after immersing segments of polymer-coated stents in c7E3-Fab solutions. Uptake was dependent on antibody concentration and duration of immersion of wire in the solution. After 22 h (at 5 mg ml(-1)), 1146+/-101 ng cm(-1) wire was adsorbed. In an in vitro perfusion circuit, the antibody eluted slowly, with 53% remaining after 12 days washing. To determine the value that such stents might have in clinical practise, adsorption to balloon-mounted stents was assessed at room temperature, using commercially available c7E3-Fab (2 mg ml(-1)). Efficacy of eluting c7E3-Fab was determined by measuring deposition of 111-Indium platelets. Immersing stents in c7E3-Fab for 20 min inhibited platelet deposition by 82.3% compared to controls (P=0.018). Deployment of treated stents in ex vivo saphenous vein resulted in the deposition of c7E3-Fab in the intima and media. CONCLUSIONS: c7E3-Fab can be passively adsorbed onto polymer-coated stents. It elutes slowly and in a predictable manner, significantly inhibiting platelet deposition in vitro. These studies pave the way to developing stent-based delivery of a potent anti-platelet agent that may additionally affect smooth muscle cell activity.

Abciximab↗
PubMed2000

Effects of radiographic contrast media on proliferation and apoptosis of human vascular endothelial cells.

The aim of the study was to determine the effects of radiographic contrast media (RCM) on proliferation and apoptosis of human vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed for either 1 min or 15 min to RCM (diatrizoate, ioxaglate, iopromide, iotrolan) at an iodine concentration of 250 mgl ml-1. Controls were complete growth medium (CGM) and saturated mannitol (osmotic control). [3H]thymidine incorporation was used to determine cell proliferation 24 h after exposure. Apoptosis was determined at 1 h and 6 h by terminal uridine nick end labelling (TUNEL), time lapse video microscopy (TLVM) and DNA electrophoresis. Mean proliferation rates (%) (+/- SEM) (p-values compared with the CGM control) at 1 min and 15 min, respectively, were: diatrizoate: 31.9 (10.6), 5.8 (1.5) (p < 0.001); ioxaglate: 48.4 (10.9), 20.4 (4.5) (p < 0.001); iopromide: 63.4 (8.7), 58.2 (10.2) (p < 0.05); iotrolan: 84.7 (7.3), 72.8 (12.4) (p = ns); saturated mannitol 50.5 (9.6), 45.9 (10.0) (p < 0.001). Mean apoptotic indices (%) (+/- SEM) at 1 h and 6 h following 1 min exposure, respectively, were: CGM: 0.25 (0.13), 0.23 (0.08); diatrizoate: 2.18 (0.19), 2.69 (0.34) (p < 0.001); ioxaglate: 1.90 (0.23), 1.69 (0.02) (p < 0.05); iopromide: 0.59 (0.04), 0.33 (0.02) (p = ns); iotrolan: 0.30 (0.07), 0.27 (0.1) (p = ns); saturated mannitol 2.11 (0.24), 1.4 (0.1) (p < 0.05). After 15 min exposure, apoptosis rates at both 1 h and 6 h, respectively, were: iotrolan: 0.29 (0.17), 0.51 (0.16) (p = ns); diatrizoate: 3.19 (0.81), 11.66 (1.75) (p < 0.001); ioxaglate: 1.88 (0.14), 2.87 (0.20) (p < 0.05); iopromide: 1.06 (0.11), 1.52 (0.15) (p < 0.05); saturated mannitol 1.62 (0.09), 4.63 (0.74) (p < 0.05). TLVM and DNA electrophoresis confirmed the occurence of apoptosis after exposure to RCM. In conclusion, saturated mannitol and all tested RCM, with the exception of iotrolan, (diatrizoate > ioxaglate > iopromide) reduced proliferation and increased apoptosis of HUVECs. The effects were more pronounced with ionic RCM and seem to depend on osmolality as well as the chemical structure of these agents. Endothelial injury and apoptosis may be responsible for some of the side effects associated with intravascular use of RCM.

Apoptosis↗
PubMed1999

Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein.

OBJECTIVES: Saphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis. METHODS: Surgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37 degrees C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferation cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel was thicknesses. RESULTS: Control, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/microgram DNA, P < 0.05, n = 6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P < 0.05, n = 5. CONCLUSIONS: These findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

Adult↗
PubMed1998

Apoptosis and cell proliferation after porcine coronary angioplasty.

BACKGROUND: Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS: Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS: These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.

Actins↗
PubMed1998

Domain mapping of human apurinic/apyrimidinic endonuclease. Structural and functional evidence for a disordered amino terminus and a tight globular carboxyl domain.

We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity.

Binding Sites↗
PubMed1998

Interleukin-1 in myocardium and coronary arteries of patients with dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (DCM) is characterised by a severe dysfunction of the heart muscle resulting in terminal heart failure. Its pathogenesis is believed to be multifactorial involving genetic predisposition, viral infection and autoimmunity, but little is known in detail, and there is no curative treatment except transplantation. Interleukin-1 (IL-1) mediates inflammatory responses to infection and injury. It can be produced by several widely-distributed cell types, including macrophages, and is thought to depress myocyte contractility by stimulating nitric oxide synthase. To investigate whether this pro-inflammatory cytokine may be a pathogenic mediator in DCM, IL-1beta mRNA and protein were evaluated in coronary arteries and myocardium from patients undergoing cardiac transplantation for DCM.IL-1beta mRNA was detected by PCR of cDNA and northern blots of mRNA in coronary arteries and myocardium from patients with DCM. By comparison, samples from patients with ischaemic heart disease (IHD) contained much less IL-1beta mRNA. In contrast, mRNA for other cytokines (TNFalpha, IL-6, IL-10, PDGFA) were similar in both pathologies. In DCM, IL-1beta mRNA and protein were localised to infiltrating macrophages in interstitial regions between myocytes, some of the myocytes themselves, and endothelial cells of vessels in the wall of the arteries. These results suggest that local production of the pro-inflammatory cytokine, IL-1beta may play a part in the pathogenesis of DCM.

Adolescent↗
PubMed1998

Intravascular stents: a new technique for tissue processing for histology, immunohistochemistry, and transmission electron microscopy.

BACKGROUND: Study of the vascular response to stent implantation has been hampered by difficulties in sectioning metal and tissue without distortion of the tissue stent interface. The metal is often removed before histochemical processing, causing a loss of arterial architecture. Histological and immunohistochemical sections should be 5 microns with an intact tissue stent interface. OBJECTIVES: To identify the most suitable cutting and grinding equipment, embedding resin, and slides for producing thin sections of stented arteries with the stent wires in situ for histological, immunohistochemical, and transmission electron microscopic (TEM) analyses. METHODS: 20 balloon stainless steel stents were implanted in the coronary arteries of 10 pigs. Twenty eight days later the stented arterial segments were excised, formalin fixed, embedded in five different resins (Epon 812, LR white, T9100, T8100, and JB4), and sectioned with two different high speed saws and a grinder for histological, immunohistochemical, and TEM analyses. Five stented human arteries were obtained at necropsy and processed using the best of the reported methods. RESULTS: The Isomet precision saw and grinder/polisher unit reliably produced 5 microns sections with most embedding resins; minimum section thickness with the horizontal saw was 400 microns. Resin T8100, a glycol methacrylate, enabled satisfactory sectioning, grinding, and histological (toluidine blue, haematoxylin and eosin, and trichromatic and polychromatic stains) and immunohistochemical analyses (alpha smooth muscle actin, von Willebrand factor, vimentin, proliferating cell nuclear antigen, and CD68 (mac 387)). T9100 and T8100 embedded stented sections were suitable for ultrastructural examination with TEM. Stented human arterial sections showed preserved arterial architecture with the struts in situ. CONCLUSION: This study identified the optimal methods for embedding, sawing, grinding, and slide mounting of stented arteries to achieve 5 microns sections with an intact tissue metal interface, excellent surface qualities, histological and immunohistochemical staining properties, and suitability for TEM examination. The technique is applicable to experimental and clinical specimens.

Animals↗
PubMed1998

Interleukin-8 mRNA expression in circulating leucocytes during cardiopulmonary bypass.

Interleukin-8 (IL-8) is found in patients following cardiopulmonary bypass (CPB). It may contribute to microvascular injury by activating neutrophils. We examined IL-8 mRNA expression in leucocytes in bypass. IL-8 mRNA levels were measured by Northern analysis and densitometry of isolated mononuclear leucocytes and neutrophils from blood samples taken before, during, and 2 and 48 h after CPB. Plasma IL-8 was measured at each time-point by immunoassay. A strong signal for IL-8 mRNA was detected in neutrophils in five of five and, more weakly, in mononuclear leucocytes of three of five patients studied. The signal peaked consistently during, and fell following bypass, usually to undetectable levels by 48 h. There was always a detectable signal in neutrophils preoperatively. Plasma IL-8 increased from undetectable prebypass levels and peaked later (2 h postbypass) in four of five patients. In the other patient, the cytokine remained detectable throughout. These data demonstrate that IL-8 transcription occurs in leucocytes before and during CPB, but suggest that much of the IL-8 detectable in plasma following bypass may derive, not from these leucocytes, but from other cell types. The release of some IL-8 by neutrophils could lead to local positive feedback in neutrophil recruitment and associated endothelial injury.

Aged↗
PubMed1997

The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty.

Proto-oncogenes, including c-myb, are expressed early after vascular injury. The application of antisense oligodeoxynucleotides (AS-ODNs) against these genes inhibits cell proliferation and neointima formation in small animals and in peripheral arteries. The aim of this study was to investigate the specificity of action of AS-ODN-c-myb in vitro and to assess its effect, when delivered locally, on neointima formation after percutaneous transluminal coronary angioplasty (PTCA) in porcine coronary arteries. AS-ODN-c-myb inhibited the proliferation of vascular smooth muscle cells (VSMCs) in vitro in a dose-dependent manner. There was a corresponding reduction in steady state levels of c-myb mRNA and protein. Expression of another early gene, c-fos, was unaffected. S1 nuclease analysis demonstrated intact full-length AS-ODN-c-myb retrieved from VSMCs in culture after 12 hours. A range of ODNs, related and unrelated to c-myb, with and without a GGGG sequence, inhibited VSMC proliferation. Phosphorothioated AS-ODN-c-myb was 30 times less potent than unphosphorothioated AS-ODN-c-myb. PTCA induced porcine coronary artery neointima formation. c-myb mRNA was maximally induced 18 hours after injury. Unmodified AS-ODN-c-myb, sense-ODN-c-myb, saline, or nothing was delivered immediately after balloon dilatation via a double-skinned porous balloon (Transport, SciMed). Fluorescence-labeled AS-ODN-c-myb was deposited throughout the vessel wall. Mean maximum intima/media cross-sectional area 4 weeks after PTCA was reduced with AS-ODN-c-myb by 79% compared with saline (P < .05), 82% compared with sense-ODN-c-myb, and 63% compared with nothing (P < .10). Conclusions are as follows: (1) c-myb is expressed in VSMCs after vascular injury. (2) AS-ODN-c-myb is retained intact in VSMCs, reducing their proliferation in vitro in dose-dependent fashion, with reduction in c-myb mRNA and protein, whereas sense-ODN-c-myb is not. (3) A range of ODNs can reduce VSMC proliferation by a non-sequence-specific mechanism. (4) Phosphorothioate protection of antisense molecules may reduce their efficacy. (5) Local delivery of unmodified AS-ODN-c-myb via the Transport catheter reduces neointima formation after porcine PTCA. (6) Local delivery of fluid may exacerbate neointimal thickening.

Angioplasty, Balloon↗
PubMed1996

Interleukin-1 beta in coronary arteries of patients with ischemic heart disease.

Interleukin-1 beta (IL-1 beta) is known to have a number of effects on the different cell types present within coronary arteries. In this study we identified the location and phenotype of cells containing IL-1 beta in human coronary artery specimens from patients suffering from either coronary atherosclerosis or cardiomyopathy and correlated the presence of IL-1 beta with disease severity. Luminal endothelial cells, adventitial vessel wall cells, and macrophages were double labeled immunohistochemically for IL-1 beta protein and a cell type-specific monoclonal antibody for either endothelial cells or macrophages. In situ hybridization was performed to locate the presence of IL-1 beta mRNA within the coronary artery wall. In this study IL-1 beta protein was found to be increased in the adventitial vessel walls of atherosclerotic coronary arteries compared with coronary arteries from nonischemic cardiomyopathic hearts. This increase was directly proportional to the severity of coronary atherosclerosis. IL-1 beta protein was also detected in luminal endothelium and macrophages of atherosclerotic coronary arteries and coronary arteries from nonischemic cardiomyopathic hearts. IL-1 beta mRNA was found in luminal endothelial cells, adventitial vessel endothelial cells, and macrophages. We conclude that IL-1 beta is produced by endothelial cells and macrophages in coronary arteries from ischemic hearts and to a lesser extent from nonischemic cardiomyopathic hearts.

Adult↗
PubMed1994

Release of platelet derived growth factor in serum-free organ culture of human coronary artery.

OBJECTIVE: The aim was to test the hypothesis that platelet derived growth factor (PDGF) is synthesized in intact coronary arteries and that it is associated with cell proliferation in atherosclerotic plaques. METHODS: Segments of human coronary artery obtained from heart transplant recipients were cultured in serum-free media for 24 h. Tissue viability was assessed by ATP concentration. Cell proliferation was determined by incorporation of [3H] thymidine, autoradiography, and proliferating cell nuclear antigen (PCNA) immunostaining. Coronary artery conditioned media were tested for mitogenic activity using a fibroblast proliferation bioassay. Reverse transcription polymerase chain reaction (RT-PCR) for PDGF A and B was subsequently performed in order to confirm the endogenous nature and isoform of this mitogen. RESULTS: Tissue viability remained unchanged during culture, and cell proliferation was detected by incorporation of [3H] thymidine. Autoradiography and PCNA immunostaining showed proliferating cells within the intimal and medial layers. Coronary artery conditioned media produced significant stimulation of cell growth [127(SEM 29)%, n = 15] above that caused by culture medium alone. This mitogenic activity was inhibited by 42(8)% (n = 8) with a polyclonal neutralising antibody to PDGF. The endogenous nature of this mitogenic activity was confirmed by detection of PDGF-A and PDGF-B mRNA expression using RT-PCR and the identity of the amplified products confirmed by DNA sequencing. CONCLUSIONS: The data provide evidence for active PDGF production and gene expression within cells of the vessel wall. They also suggest that endogenously produced PDGF may play a role in controlling vascular smooth muscle cell proliferation in human coronary arteries.

Adult↗