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D C Crossman

Publications and source records attributed to D C Crossman.

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

Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro.

BACKGROUND: Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS: Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS: Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

Animals↗
PubMed1999

Interleukin-1 receptor antagonist gene polymorphism and coronary artery disease.

BACKGROUND: Cytokine gene variations are contributory factors in inflammatory pathology. Allele frequencies of interleukin (IL)-1 cluster genes [IL-1A(-889), IL-1B(-511), IL-1B(+3953), IL-1RN Intron 2 VNTR] and tissue necrosis factor (TNF)-alpha gene [TNFA(-308)] were measured in healthy blood donors (healthy control subjects), patients with angiographically normal coronary arteries (patient control subjects), single-vessel coronary disease (SVD), and those with multivessel coronary disease (MVD). METHODS AND RESULTS: Five hundred fifty-six patients attending for coronary angiography in Sheffield were studied: 130 patient control subjects, 98 SVD, and 328 MVD. Significant associations were tested in an independent population (London) of 350: 57 SVD, 191 MVD, and 102 control subjects. IL-1RN*2 frequency in Sheffield patient control subjects was the same as in 827 healthy control subjects. IL-1RN*2 was significantly overrepresented in Sheffield SVD patients (34% vs 23% in patient control subjects); IL-1RN*2 homozygotes in the SVD population (chi2 carriage=8.490, 1 df, P=0.0036). This effect was present though not quite significant in the London population (P=0. 0603). A summary trend test of the IL-1RN SVD genotype data for Sheffield and London showed a significant association with *2 (P=0. 0024). No significant effect of genotype at IL-1RN was observed in the Sheffield or London MVD populations. Genotype distribution analysis comparing the SVD and MVD populations at IL-1RN showed a highly significant trend (P=0.0007) with the use of pooled data. No significant associations were seen for the other polymorphisms. CONCLUSIONS: IL-1RN*2 was significantly associated with SVD. A difference in genetic association between SVD and MVD was also apparent.

Coronary Disease↗
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

Transforming growth factor-beta1 gene polymorphisms and coronary artery disease.

1. Transforming growth factor-beta1 is a cytokine with a very wide spectrum of biological activities. Previous studies have shown that it is involved in a number of physiological and pathological processes including heart disease. In our study we aimed to scan the transforming growth factor-beta1 locus for polymorphisms and to identify haplotypes significantly associated with a predisposition to coronary atherosclerosis.2. Two patient groups comprising 244 angiographically normal individuals and 655 patients with coronary artery disease were recruited from London and Sheffield. DNA samples from these subjects were screened for mutations in the transforming growth factor-beta1 locus and all subjects were genotyped by a coupled polymerase chain reaction-restriction enzyme digestion method.3. Five polymorphisms have been identified in the transforming growth factor-beta1 gene at positions G-800A, C-509T in the promoter region, Leu10-->Pro, Arg25-->Pro in exon 1 and Thr263-->Ile in exon 5. No significant difference in frequencies for any of the five polymorphisms was found between controls and patients with coronary artery disease. Similarly, there was no correlation between these polymorphisms and hypertension.4. The genotypes of all the individuals participating in the study were assigned to seven main haplotypes of the transforming growth factor-beta1 locus. Based on species comparison data we propose that GCCGC is the ancestral haplotype in humans.5. Our data suggest that these transforming growth factor-beta1 polymorphisms are not associated with coronary artery disease and therefore their presence alone would not be a genetic risk factor for predisposition to coronary artery disease.

Case-Control Studies↗
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

Methylenetetrahydrofolate reductase polymorphism (C-677T) and coronary artery disease.

1. Many studies have shown that hyperhomocysteinaemia is a risk factor for atherosclerotic vascular disease. A mutation (C-677T) in the gene coding for the methylenetetrahydrofolate reductase (MTHFR) enzyme has been shown to produce a thermolabile form of the enzyme. Homozygosity for this mutation has been correlated with an elevated plasma homocysteine concentration. The present study aimed to determine whether this mutation was a risk factor for coronary artery disease (CAD). This was achieved by comparing the frequency of the C-677T mutation in patients with angiographically proven CAD against angiographically normal patients in two separate U.K. samples. The analysis was repeated with CAD patients split into those with >=99% stenosis of arteries and those without, to establish whether the C-677T mutation could be correlated with severity of CAD.2. Two patient groups were selected from London and Sheffield. The London group comprised 174 cases and 148 controls. The Sheffield group comprised 93 cases and 85 controls. The DNA samples of the patients were genotyped by polymerase chain reaction and restriction enzyme digestion.3. For London the homozygous C-677T frequencies were: 0.07 (controls), 0.09 (CAD without >=99% stenosis) and 0.10 (CAD with >=99% stenosis). For Sheffield the homozygous C-677T frequencies were: 0.08 (controls), 0.10 (CAD without >=99% stenosis) and 0.11 (CAD with >=99% stenosis). No association was found between the C-677T mutation and CAD in our sample geographical groups. Statistical comparison by genotype distribution for 0 VD (no vessel disease, i.e. 0% diameter reduction in all epicardial arteries) versus CAD without >=99% stenosis: London, P=0.19; Sheffield, P=0.53; 0 VD versus CAD with >=99% stenosis: London, P=0. 23; Sheffield, P=0.55.

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↗
PubMed1994

Substance P potentiates the algogenic effects of intraarterial infusion of adenosine.

OBJECTIVES: This study investigated whether substance P potentiates the muscular and cardiac pain caused by the intraarterial infusion of adenosine, an autocoid known to induce muscular and cardiac ischemic-like pain in humans. BACKGROUND: Substance P is involved in the generation of neurogenic inflammation and causes cutaneous hyperalgesia. Because substance P is present in perivascular nerves it might also cause muscular and cardiac hyperalgesia. To test this hypothesis its effects on adenosine-induced muscular and cardiac pain were investigated in humans. METHODS: A randomized, crossover study of the algogenic effects of the intrailiac infusion of increasing scalar doses (from 125 to 2,000 micrograms/min) of adenosine or substance P (11.2 pmol/min) for 3 min, followed by the simultaneous infusion of substance P plus the same doses of adenosine, was carried out in nine patients with no evidence of peripheral vascular disease. A similar protocol was carried out by infusing increasing scalar doses of adenosine (from 50 to 800 micrograms/min) or substance P (11.2 pmol/min) for 3 min, followed by the simultaneous infusion of substance P plus the same doses of adenosine, into the left coronary artery of eight patients with angina. Pain severity, assessed by a visual analog scale, is presented as median. The remaining data are presented as mean value +/- 1 SD. RESULTS: All patients experienced pain during both adenosine and substance P plus adenosine infusion; no patient experienced pain during the infusion of substance P alone. During intrailiac infusion, all patients experienced pain in the right leg that occurred earlier (207 +/- 152 vs. 321 +/- 154 s, p < 0.05) and was greater (47 vs. 30 mm, p < 0.05) during the simultaneous infusion of substance P plus adenosine than during the infusion of adenosine. Similarly, during intracoronary infusion, all patients experienced chest pain that occurred earlier (409 +/- 242 vs. 596 +/- 210 s, p < 0.05) and was greater (51 vs. 33 mm, p < 0.05) during the simultaneous infusion of substance P plus adenosine than during infusion of adenosine. No patient exhibited electrocardiographic signs of ischemia. CONCLUSIONS: Substance P does not cause muscular or cardiac pain, but it provokes muscular and cardiac hyperalgesia.

Adenosine↗
PubMed1994

Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter.

Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.

Base Sequence↗
PubMed1993

Sodium cromoglycate: evidence of tachykinin antagonist activity in the human skin.

The mechanism of action of the antiasthmatic drug sodium cromoglycate (SCG) is unclear. One possibility is that SCG antagonizes the effects of the tachykinin substance P (SP), an agent known to cause airway edema. However, when SP is inhaled by humans, it has no demonstrable effect on airway function; therefore, the possibility that SCG prevents SP-induced changes in microvascular permeability was examined in human skin in vivo where potent edema-producing effects are seen. SCG (5-500 nmol) caused significant (P < 0.05) dose-dependent inhibition of SP-induced edema (wheal) formation when coadministered by intradermal injection. There was no effect on the nonreceptor-mediated flare response. SCG also significantly (P < 0.05) inhibited the wheal response to the related tachykinin neurokinin B but had no inhibitory effect on the cutaneous responses to histamine and prostaglandin E2. In addition, SCG (0.1-10 mM) caused dose-dependent inhibition of binding of SP labeled with 125I-labeled Bolton-Hunter to a number of tissues known to contain SP binding sites, as assessed by autoradiography. These concentrations were equivalent to the final concentrations of SCG found to inhibit the wheal response in the skin. The possibility that SCG interacted with SP was investigated both by gel filtration and high-performance liquid chromatography. No strong interaction was demonstrated with an 8,000 M excess of SCG under both hydrophobic and hydrophilic conditions. These results raise the possibility that SCG may have tachykinin antagonist properties.

Adult↗
PubMed1992

Endothelin-1 induces a histamine-dependent flare in vivo, but does not activate human skin mast cells in vitro.

The role of the mast cell in endothelin-1 induced flare has been investigated by in vivo and in vitro experiments. The intradermal injection of endothelin-1 (10 pmol) into human skin induced a pallor with surrounding axon-reflex flare which is similar to the flare response to histamine (1 nmol). At these doses, chosen to give identical flare areas, blood flow was increased in the area of the endothelin-induced flare over a longer period. A systemic H1-receptor antagonist significantly inhibited the area of both flares, although blood flow in the remaining portion of the endothelin-induced flare remained raised. Endothelin-1 at concentrations up to 3 x 10(-5) M failed to release histamine from human dispersed skin mast cells. We suggest from these results that a major component of endothelin-1 induced flare results from mast cell activation that is secondary to direct stimulation of sensory nerves by the peptide.

Adult↗
PubMed1991

Substance P for evaluation of coronary endothelial function after cardiac transplantation.

The endothelium-dependent vasodilator substance P dilates normal and diseased coronary vessels in humans in vivo and produces a maximal response similar to that seen with intracoronary isosorbide dinitrate. Twelve cardiac transplant recipients underwent intracoronary infusion of substance P after routine annual investigations. All patients were well, with no evidence of rejection and with angiographically normal coronary arteries. Substance P was infused at 2 ml/min for 2 min into the coronary artery, starting at a dose of 1.4 pmol/min and increasing by doubling increments, and followed by isosorbide dinitrate (1 mg/min) infused over 2 min. Coronary artery diameter was measured in 23 vessel segments from 12 transplant recipients. The following doses were infused: saline solution (1 ml/min), substance P (0.7 [three patients], 1.4, 2.8, 5.6, 11.2, 22.4 pmol/min) and isosorbide dinitrate (1 mg/min). The mean percent increase in diameter (+/- SEM) in response to increasing doses of substance P was as follows: 0, 6.5 +/- 2.9%, 10.9 +/- 2.9%, 12.1 +/- 2.9%, 16.5 +/- 2.6%, 19.2 +/- 3.1% and 25.8 +/- 2.2%, respectively. Half maximal dilation was produced with 1.4 to 2.8 pmol/min of substance P; the maximal response (mean percent diameter change) was 22 +/- 2.5%. This was not significantly different from that achieved with isosorbide dinitrate. It is concluded that coronary endothelial function as assessed by response to substance P is preserved in cardiac transplant recipients with angiographically normal coronary arteries. Substance P may be a suitable agent for testing endothelial function in these patients.

Coronary Angiography↗
PubMed1991

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury.

Adult↗
PubMed1991

Responses of atherosclerotic human coronary arteries in vivo to the endothelium-dependent vasodilator substance P.

BACKGROUND: The effects of the endothelium-dependent vasodilator substance P (SP) on atherosclerotic human coronary arteries was studied. METHODS AND RESULTS: [125I]-SP binding to luminal cells was shown to be preserved in the atherosclerotic epicardial coronary arteries of four patients. No binding to medial smooth muscle cells was demonstrated. Intracoronary infusions of SP were undertaken in patients with coronary artery disease. SP was infused for 2-minute periods starting at a dose of 2.8 pmol/min rising by doubling increments to 22.4 pmol/min. Analysis of the epicardial coronary artery diameter, using a computerized analysis system (CAAS) of the angiograms, was performed at the end of each infusion. Analysis of seven smooth vessel segments from seven coronary vessels, which were stenosed at more proximal sites, was performed. Significant dose-dependent dilatation was seen (p = 0.04), which was maximal at 5.6 pmol/min SP. No additional dilatation was produced with 2 mg intracoronary isosorbide dinitrate (ISDN). Two of these seven patients showed no response to SP, and only one of these appeared to sustain dilatation with ISDN (2 mg intracoronary). In a second group of six patients with discrete coronary stenoses, analysis at the site of the stenosed segments appeared to reveal dilatation in response to SP in only one instance. One other stenotic segment dilated with isosorbide dinitrate but failed to dilate with SP; the remaining four were fixed. The segment immediately proximal to the stenosis preserved a dose-dependent vasodilator response. CONCLUSIONS: These findings demonstrate that the endothelium-dependent vasodilator substance P can still produce epicardial vasodilatation in vivo in the presence of coronary atherosclerosis.

Autoradiography↗
PubMed1990

The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester.

Tissue factor (TF) is the membrane-bound glycoprotein whose cofactor activity with factor VIIa causes activation of the extrinsic pathway of coagulation. The transition of endothelium to a procoagulant state by agents such as bacterial lipopolysaccharide (LPS) is the result of TF expression by these cells. The mechanism of TF induction in human umbilical vein endothelial cells (HUVEC) was investigated in response to LPS and phorbol 12-myristate 13-O-acetate (PMA). Northern blot analysis of total RNA from HUVEC showed a rapid rise in TF mRNA levels which was maximal at 2 h and had fallen to low levels by 6 h following both LPS (10 micrograms/ml) and PMA (10 ng/ml) stimulation. Nuclear-run on experiments showed at most a 2-fold increase in transcription of the TF gene following LPS stimulation but a 10-fold increase following PMA stimulation. In addition 24-h pre-incubation with PMA desensitized HUVEC to further PMA exposure, but caused no alteration in the response to LPS. Cycloheximide (10 micrograms/ml) alone caused induction of TF mRNA. Treatment of cells previously exposed to LPS for 1 or 4 h with actinomycin D indicated a 12-fold difference in the TF mRNA half-life. Therefore the rapid accumulation of TF mRNA in HUVEC stimulated by LPS is largely a result of an increase in mRNA stability rather than an increased rate of transcription of the gene.

Cell Nucleus↗