Nonsteroidal Anti-Inflammatory Drugs and Opioids in Postsurgical Dental Pain.

Postsurgical dental pain is mainly driven by inflammation, particularly through the generation of prostaglandins via the cyclooxygenase system. Thus, it is no surprise that numerous randomized placebo-controlled trials studying acute pain following the surgical extraction of impacted third molars have demonstrated the remarkable efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen sodium, etodolac, diclofenac, and ketorolac in this prototypic condition of acute inflammatory pain. Combining an optimal dose of an NSAID with an appropriate dose of acetaminophen appears to further enhance analgesic efficacy and potentially reduce the need for opioids. In addition to being on average inferior to NSAIDs as analgesics in postsurgical dental pain, opioids produce a higher incidence of side effects in dental outpatients, including dizziness, drowsiness, psychomotor impairment, nausea/vomiting, and constipation. Unused opioids are also subject to misuse and diversion, and they may cause addiction. Despite these risks, some dental surgical outpatients may benefit from a 1- or 2-d course of opioids added to their NSAID regimen. NSAID use may carry significant risks in certain patient populations, in which a short course of an acetaminophen/opioid combination may provide a more favorable benefit versus risk ratio than an NSAID regimen.

Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells.

OBJECTIVE: The efficient introduction of regulatory genes into vascular smooth muscle cells (SMCs) is one of the most promising options for gene therapy of cardiovascular diseases. Cationic liposome-mediated gene transfer may become a favorable transfection technique with regard to patient's safety for in vivo administration. However, this method until now has its limitation in a low transfection efficiency. Therefore, the present study was designed to improve cationic liposome-mediated transfection of rabbit vascular SMCs in vitro and in vivo, in order to enhance transfection efficiency and present an optimized system which may offer a potential therapeutic benefit for in vivo application. METHODS AND RESULTS: Optimized lipofection of rabbit SMCs with the mammalian expression vector pE-N1 and the reporter gene green fluorescent protein resulted in a mean transfection efficiency of about 50%. The unique transfection of rabbit SMCs in vitro and in vivo with the inducible isoform of human nitric oxide synthase (NOSII), using the same vector, resulted in a successful transient transcription and translation of a functionally active human NOSII in rabbit SMC, persisting 5-6 days. We could further demonstrate that the transfection procedure and the transgene product did neither induce necrosis nor apoptosis under the conditions chosen and did not result in the induction of endogenous NOSII of transfected SMCs. CONCLUSION(S): These findings indicate potential therapeutic relevance for this nonviral gene transfer system for in vivo gene therapy for cardiovascular diseases.

Effects of local delivery of trapidil on neointima formation in a rabbit angioplasty model.

1. Smooth muscle cell (SMC) proliferation can result in luminal reduction of a vessel following balloon angioplasty. This study was designed (i) to determine if local administration of trapidil (triazolopyrimidine) into a vessel wall reduces neointima formation, and (ii) to explore the mechanism involved in the subsequent reduction in cell proliferation. 2. Following balloon angioplasty in 40 anaesthetized New Zealand White rabbits, trapidil (50-200 mg) or its vehicle (saline) was injected into the dilated vessel wall of the right femoral artery. Experimental groups and time of investigation: (I) vehicle (2 weeks, n = 3), (II) trapidil-100 mg (2 weeks, n = 3), (III) vehicle (3 weeks, n = 8), (IV) trapidil-50 mg (3 weeks, n = 5); (V) trapidil-100 mg (3 weeks, n = 9) or (V) trapidil-200 mg (3 weeks, n = 7). 3. After 2 weeks, there was a significant reduction of intimal hyperplasia (expressed as intima to media area ratio) in the trapidil group compared with vehicle (0.44 +/- 0.04 vs 0.93 +/- 0.04, *P < 0.05) and also a significant reduction in cell proliferation (% ratio of BrdU-positive cells to total cell number: vehicle 14 +/- 2% vs trapidil 6 +/- 1%, *P < 0.05). 4. After 3 weeks, there was a dose-dependent reduction of intimal hyperplasia in the trapidil groups compared with vehicle (trapidil 50 mg 1.14 +/- 0.04; trapidil 100 mg 0.91 +/- 0.09*; trapidil 200 mg 0.77 +/- 0.09* vs vehicle 1.67 +/- 0.23, *P < 0.05). 5. Thus, the local administration of trapidil to the rabbit femoral artery reduces the neointima formation, which occurs 2 or 3 weeks after balloon angioplasty via a mechanism, which is dependent on inhibition of cell proliferation.

Bioactivation of nitroglycerin in vascular smooth muscle cells is different from that in non-vascular tissue.

The mechanism of biotransformation of nitroglycerin into the pharmacologically active radical nitric oxide (NO) or a related compound is still unclear. Different enzymes have been discussed to be involved in the bioactivation process. The effects of inhibition of glutathione-S-transferase and cytochrome P-450 enzymes were investigated on nitroglycerin-induced relaxation of bovine and porcine coronary arteries and on nitroglycerin-induced activation of guanylyl cyclase in cultivated porcine aortic smooth muscle cells. The glutathione-S-transferase inhibitor sulfobromophthalein had no effect on nitroglycerin-induced vascular relaxation, nor on nitroglycerin-induced elevation of cGMP levels in porcine coronary artery smooth muscle cells. The modulation of cytochrome P-450 activity by selective inhibitors as well as inducers did not alter the bioactivity of nitroglycerin in both systems. The data demonstrate that the isoenzymes of both enzyme families, which have been shown to be involved in the metabolism of nitroglycerin in different non-vascular tissues, do not play a role in bioactivation of nitroglycerin in the vascular system.

Tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 in coronary artery smooth muscle cells.

This study compares the antimitogenic effects of iloprost and prostaglandin E1 on platelet-derived growth factor-BB stimulated DNA synthesis ([3H]thymidine incorporation) in bovine coronary artery smooth muscle cells. When added 20-24 h after stimulation with platelet-derived growth factor-BB (20 ng/ml), both iloprost and prostaglandin E1, concentration-dependently (IC50 3-5 nM) inhibited DNA synthesis. However, when added together with the growth factor (0-24 h), the inhibition of DNA synthesis by iloprost was markedly attenuated, indicating tolerance development. In contrast, no tolerance to antimitogenic effects of prostaglandin E1 or forskolin were observed. When added to iloprost-tolerant cells, both prostaglandin E1 and forskolin, still inhibited DNA synthesis. There was no evidence for transcriptional down-regulation of prostacyclin receptor gene by iloprost. The data demonstrate a tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 and suggest that the receptors, mediating the antiproliferative actions of these prostaglandins, may be different.

Thromboxane A2 induces cell signaling but requires platelet-derived growth factor to act as a mitogen.

This study investigates thromboxane A2-induced cell signaling and mitogenesis of bovine coronary artery smooth muscle cells. The thromboxane mimetic U 46619 [(15S)-hydroxy-11,9-(epoxymethano) prosta-5Z,13E-dienoic acid] (10 microM) stimulated [Ca2+]i signals, phosphorylation of MAP kinase (mitogen-activated protein kinase), and expression of c-fos mRNA in smooth muscle cells. In contrast, no stimulation of DNA synthesis or cell proliferation by U 46619 was observed. However, platelet-derived growth factor-BB (20 ng/ml)-induced mitogenesis was potentiated by U 46619. Similar results were obtained with I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1] heptan-2-yl]-5-heptenoic acid]. These potentiating effects were abrogated by a specific thromboxane receptor antagonist, suggesting that the potentiation of platelet-derived growth factor-BB-induced smooth muscle cell mitogenesis by U 46619 and I-BOP was mediated by thromboxane receptors. It is concluded that thromboxane A2 generated by blood platelets at the site of vessel injury induces cell signaling in smooth muscle cells but acts as a mitogen only in the presence of growth factor(s).

NO reduces PMN adhesion to human vascular endothelial cells due to downregulation of ICAM-1 mRNA and surface expression.

Reperfusion damage is largely due to the adherence of polymorphonuclear leukocytes to the endothelium initiated by adhesion molecule upregulation. The reduced endothelial nitric oxide release during ischemia may be involved in the upregulation of intercellular adhesion molecule 1. In this study, we tested if nitric oxide donors suppress polymorphonuclear leukocyte adherence to activated endothelial cells by inhibition of the intercellular adhesion molecule 1 surface expression. Confluent human umbilical vein endothelial cells were stimulated with tumor necrosis factor alpha (300 U/mL) after preincubation with increasing concentrations of the nitric oxide donors CAS 1609 (0.005-5 mM/L) and 3-(4-morpholinyl)-sydnonimine (0.01-1 mM/L). Intercellular adhesion molecule 1 surface expression was measured in a cell surface enzyme-linked immunosorbent assay, intercellular adhesion molecule 1 mRNA by Northern analysis. Human saphenous vein endothelial cells were transfected with the inducible nitric oxide synthase gene and stimulated with tumor necrosis factor alpha (300 U/mL). Fluorescein green-labeled polymorphonuclear leukocytes adhering to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells were quantified by epifluorescent microscopy. The intercellular adhesion molecule 1 surface expression of activated human umbilical vein endothelial cells/human saphenous vein endothelial cells was significantly diminished to 40 to 60% of the maximum after treatment with CAS 1609, 3-(4-morpholinyl)-sydnonimine, or transfection with the inducible nitric oxide synthase gene. Intercellular adhesion molecule 1 mRNA was diminished by CAS 1609 and 3-(4-morpholinyl)-sydnonimine in the same manner. The functional relevance of our data was shown by reduction of polymorphonuclear leukocyte adherence to activated human umbilical vein endothelial cells/human saphenous vein endothelial cells following treatment with CAS 1609 and 3-(4-morpholinyl)-sydnonimine or transfection with inducible nitric oxide synthase. Tumor necrosis factor-induced polymorphonuclear leukocyte adherence was abolished by blocking antibody against intercellular adhesion molecule 1. Thus, exogenous or endogenous substitution of nitric oxide diminishes the expression of endothelial intercellular adhesion molecule 1 and its mRNA following tumor necrosis factor alpha stimulation. This results in a reduced polymorphonuclear leukocyte adherence to activated endothelium.

Exogenous progesterone and embryo survival in Booroola-cross ewes.

To investigate if exogenous progesterone improves embryo survival, 209 multiparous Booroola Merino x South Australian Merino ewes, heterozygous for the F gene (F+) were allocated to seven treatment groups and inseminated at a synchronized oestrus. Six groups received progesterone from controlled internal drug release G dispensers on the following days after ovulation: 4-7, 4-11, 4-14, 7-11, 7-14 and 11-14. Concentration of peripheral progesterone increased (P less than 0.05) in most supplemented groups, but there were no significant differences in pregnancy rates between treatments. However, the number of fetuses per pregnancy was increased for progesterone treatments starting on Day 4 (Days 4-7, 4-11 and 4-14 combined v. control; P less than 0.05) and for all supplemented treatments compared with the control (P less than 0.05).

Evaluation of a nutritional strategy to increase ovulation rate in merino ewes mated in late spring-early summer.

A nutritional strategy for increasing ovulation rate in Merino ewes mated in late spring-early summer was evaluated on two commercial farms. The strategy used the 'ram effect' to induce oestrus in seasonally anoestrus ewes and supplementary feeding of lupin grain six days prior to oestrus to increase ovulation rate. Ewes that had been isolated from rams for 6 weeks were exposed to vasectomised rams for 2 weeks and then mated to fertile rams for 6 weeks. Feeding 500 g lupins/head/day for 14 days commencing 12 days after the introduction of vasectomised rams, increased the number of ovulations from 126 to 146 per 100 ewes exposed to rams (P < 0.05). This increase was reflected in an improvement in fecundity (lambs born per ewe lambing; P < 0.05) but not fertility (ewes lambing per ewe mated to rams). Net reproductive performance (the product of fertility, fecundity and lamb survival) was increased by 11 lambs weaned per 100 ewes exposed to rams due to lupin supplementation at mating.

Development of a nutritional strategy for increasing lamb survival in Merino ewes mated in late spring/early summer.

A nutritional strategy for increasing lamb survival in Merino ewes mated in late spring/early summer was evaluated in a commercial flock over two consecutive years (Year 1, n = 680; Year 2, n = 325). The strategy combined the 'ram effect' to synchronise oestrus and hence parturition, plus supplementary feeding of lupin grain for 14 days in the expected early post-parturient period. Supplementary lupin feeding commenced 12 days after the expected start of lambing. Lambing was highly synchronised over a 14-day period commencing 17-19 days after the expected start of lambing, in both years. Supplementary feeding did not affect lamb birthweight in either year but subsequent increases in weight were observed at weaning in Year 1 (1.4 kg; P = 0.06) and tail docking in Year 2 (1.3 kg; P < 0.05). Lamb survival was increased by 7 lambs per 100 ewes exposed to rams in both years. (Year 1 at weaning, NS; Year 2 at tail docking, P < 0.001). It was concluded that the strategy improved both lamb survival and lamb performance possibly due to an effect of lupin supplementation on colostrum and subsequent milk production.

Real-time ultrasound imaging for predicting ovine fetal age.

Fetal head width and thoracic depth were observed via real-time ultrasound imaging in 12 single-, 12 twin- and 4 triplet-bearing BooroolaxSouth Australian Merino ewes at weekly intervals during mid pregnancy. Fetal age varied by a maximum of 24 h. Relationships between fetal age and the linear measures (head width, thoracic depth) were determined within litter size categories, using both linear and quadratic terms. All relationships tested for linearity were statistically significant (P<0.05). A curvilinear relationship (P<0.05) was observed for fetal age and thoracic depth of twin fetuses. Significant (P<0.05) curvilinear relationships were also observed for fetal age and head width, and fetal age and thoracic depth when litter size data were pooled. We conclude that head width and thoracic depth are acceptable linear measurements for estimating fetal age in the ovine species. Implications of these results for research and for the commercial field are discussed.

The translational therapeutics of prostaglandin inhibition in atherothrombosis.

Prostaglandins, products of the cyclo-oxygenase (COX) enzymes, can both promote and restrain atherothrombosis. While non-steroidal anti-inflammatory drugs (NSAIDs) selective for inhibition of COX-2 predispose to myocardial infarction, heart failure, hypertension and stroke, suppression of products of COX-1, such as thromboxane (Tx) A2, underlie cardioprotection from low-dose aspirin. Data from clinical pharmacology, rodent models, human genetics, observational studies and randomized trials provide insight into the implications of inhibiting COX product synthesis or function. Many lines of evidence afford a mechanistic explanation for the cardiovascular (CV) hazard from NSAIDs. Elucidation of the biology of this pathway using diversified approaches is also relevant to understanding the implications of substrate rediversion following inhibition of enzymes downstream of COXs, such as the microsomal PGE synthase (mPGES)-1 and of combining D prostanoid antagonism with niacin to attenuate facial flushing.

Iloprost-induced inhibition of proliferation of coronary artery smooth muscle cells is abolished by homologous desensitization.

In addition to inhibition of platelet function, prostacyclin and its stable analogues are reported to attenuate vascular smooth muscle cell proliferation. However, desensitization of prostacyclin responsiveness is a known phenomenon in platelets. In this study we investigated the time-dependent effects of the prostacyclin-mimetic iloprost and of PGE1, respectively, on PDGF-induced proliferation of cultured coronary artery smooth muscle cells. Proliferation, assessed by [3H]thymidine incorporation was markedly inhibited by coincubation with iloprost (100 nM) and PGE1 (100 nM) for 4 h. In contrast, addition of iloprost (100 nM) for 24 h did not decrease smooth muscle cell proliferation, whereas inhibition by PGE1 or by forskolin was not diminished. These results suggest a homologous desensitization of anti-mitogenic effects of iloprost in coronary artery smooth muscle cells, probably at receptor-level.

Potentiation of PDGF-induced growth responses in coronary artery smooth muscle cells by thromboxane.

Mitogenic effects of TXA2 in vascular smooth muscle cells are discussed to be dependent on the age of the donor organism. The present study investigates the contribution of TXA2 on PDGF-induced proliferation of bovine coronary artery smooth muscle cells (BCA-SMC) isolated from adult animals. Radioligand binding studies revealed high affinity TXA2 binding sites (Kd = 1.6 nM) in these cells. TXA2-mimetics alone showed no proliferative effect in BCA-SMC, assessed by [3H]thymidine incorporation. However, PDGF-stimulated proliferation was potentiated two-fold receptor-dependently by TXA2-mimetics. Thus, vasoconstrictory eicosanoids released from activated platelets might aggravate proliferation of vascular smooth muscle cells at sites of vessel injury in the adult organism.

[Local drug delivery and gene therapy]. / Lokale Medikamentengabe und Gentherapie.

One of the most important problems in clinical cardiology is still unresolved, i.e., the development of a restenosis following coronary balloon angioplasty. Our knowledge about the sequelae of pathophysiologic events occurring during neointima formation is still far from complete (Figure 1) and numerous therapeutic trials using systemic administration of drugs with different mechanisms of action have failed. Possible innovative strategies are the local administration of high doses of drugs into the coronary arteries and local gene therapeutic interventions to inhibit neointima formation by reducing the proliferation of vascular smooth muscle cells. Numerous catheter devices were developed (Figure 2) in order to enable the local application of high doses of a drug or DNA. Additionally, galenic techniques are being developed to guarantee a steady release of locally administered drugs, e.g. from drug containing liposomes or microcarriers (Figure 3). There are already several animal models in which the development of a neointima was reduced by injecting antisense oligonucleotides directed towards the RNA encoding cell cycle regulatory proteins or peptides. Alternatively, the transfer of cDNA encoding proteins or protein products which inhibit the cellular proliferation and migration are being tested in vitro and in vivo with the help of reporter genes (Figure 4). Although, gene transfer techniques are believed to offer great therapeutic options for the future, the clinical data available today regarding this method are very limited and are derived from studies in patients with peripheral arterial disease. Thus, it is still questionable if gene transfer techniques will ever be able to become an integral part of our standard treatment for patients with vascular diseases.

Involvement of PKC and NF-kappaB in nitric oxide induced apoptosis in human coronary artery smooth muscle cells.

Apoptosis of vascular smooth muscle cells is critically involved in progression of atherosclerosis and may prevent intimal hyperplasia in restenosis and vascular remodeling. Nitric oxide (NO) is known to induce apoptosis, but the signaling pathways still remain unclear. We investigated p53 accumulation, protein kinase C (PKC) activation and nuclear transcription factor (NF-kappaB) binding activity as possible signaling mechanisms of NO-induced apoptosis. Apoptosis was induced dose-dependently with the NO-donors sodiumnitroprusside (SNP: 232+/-48%) and SIN-1 (241+/-90% of actinomycin D induced apoptosis; means +/- SEM, *p< or =0.05 vs. control) in HSMC. Inhibition of PKC significantly attenuated NO-induced apoptosis. Staurosporine reduced SIN-1/SNP-mediated DNA fragmentation by 55.3+/-13.8% and 38.3+/-13.9% respectively. Comparable results were obtained for calphostin C. However, NO-mediated induction of apoptosis was not preceded by p53 accumulation. SNP decreased NF-kappaB binding activity in HSMC. These results suggest that induction of apoptosis by exogenous NO in HSMC is not dependent on p53 accumulation but involves protein kinase C signaling and regulation of NF-kappaB binding activity. This opens a new therapeutical approach in preventing restenosis after angioplasty.