Factors that influence the suitability of human organ-cultured corneas.

PURPOSE: The purpose was to assess the influence of donor and storage factors on the suitability of organ-cultured corneas for transplantation. METHODS: Data from 1340 donor corneas stored between 2009 and 2015 were analyzed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the suitability of grafts for transplantation. RESULTS: Forty-one percent (553/1340) of corneas were discarded. The leading causes were medical contraindication (20.2 %) and poor endothelial quality (19.3 %). Donor age influenced suitability for transplantation significantly. Corneas from donors aged 80 years and older were more likely to be discarded because of endothelial insufficiency (P < 0.0001). The cause of donor death including infection and multiple organ dsyfunction syndrom (MODS) increased the risk of bacterial or fungal contamination during organ culture (P = 0.007 and P = 0.014, respectively). Prolonged time between death and enucleation was associated with an increased risk of unsuitability for transplantation (P < 0.0001). The amount of time between death and corneoscleral disc excision and duration of storage influenced the suitability for transplantation (P = 0.0007 and P < 0.0001, respectively). CONCLUSION: Donor age, cause of death, storage time, death to enucleation and death to disc excision times influenced transplantation suitability. The percentage of discarded corneas may be reduced by shortening storage time, death to enucleation, and death to corneoscleral disc excision times. Setting a maximum donor age could reduce the percentage of discarded corneas. However, as long as there is a lack of donor corneas, we do not recommend any donor age limit.

In vivo biocompatibility of a new cyanine dye for ILM peeling.

PURPOSE: To investigate the biocompatibility of the new cyanine dye: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS) as a vital dye for intraocular application in an in vivo rat model and to evaluate the effects of this dye on retinal structure and function. METHODS: DSS at a concentration of 0.5% was applied via intravitreal injections to adult Brown Norway rats with BSS serving as a control. Retinal toxicity was assessed 7 days later by means of retinal ganglion cell (RGC) counts, light microscopy, optical coherence tomography (OCT), and electroretinography (ERG). RESULTS: No significant decrease in RGC numbers was observed. No structural changes of the central retina were observed either in vivo (OCT) or under light microscopy. ERGs detected a temporary reduction of retinal function 7 days after injection; this was no longer evident 14 days after injection. CONCLUSIONS: DSS showed good biocompatibility in a well-established experimental in vivo setting and may be usable for intraocular surgery as an alternative to other cyanine dyes. In contrast to indocyanine green, it additionally offers fluorescence in the visual spectrum. Further studies with other animal models are needed before translation into clinical application.

[Using intracameral cefuroxime reduces postoperative endophthalmitis rate: 5 years experience at the University Eye Hospital Tübingen]. / Reduktion der postoperativen Endophthalmitisrate durch intrakamerale Cerfuroximgabe: Ergebnisse aus 5 Jahren Erfahrungen an der Universitäts-Augenklinik Tübingen.

BACKGROUND: Cataract surgery is the most commonly performed surgical procedure in developed countries. The annual number of cataract surgeries in Germany is about 600,000. Acute postoperative endophthalmitis is a very severe and the most dreaded complication of cataract surgery. Various operative and non-operative measures have been suggested to prevent this serious complication. The European Society of Cataract & Refractive Surgeons (ESCRS) study of intracameral cefuroxime was the first prospective, randomised and partially placebo-controlled clinical trial showing the efficacy of antibiotic prophylaxis to prevent endophthalmitis in 2007. The aim of this retrospective study is to investigate a possible reduction of intracameral cefuroxime to prevent postoperative endophthalmitis at the University Eye Hospital Tübingen. PATIENTS AND METHODS: During the period from January 2002 to August 2013, 2 time periods were determined based on the adoption of intracameral cefuroxime injections after cataract surgery. From January 2002 to May 2009 patients received at the end of cataract surgery a subconjunctival administration of 50 mg of mezlocillin and postoperative antibiotic eye drops (gentamicin) without intracameral injection. From June 2009 to August 2013, patients received an intracameral injection of cefuroxime while antibiotic drops (moxifloxacin) were used too. The rates of postoperative infectious endophthalmitis during these 2 periods were calculated. RESULTS: 31 cases of endophthalmitis occurred in 31,386 cataract surgeries. The overall cumulative incidence was 0.99 per 1000 patients. The incidence in the first period without intracameral cefuroxime injection was 1.38 (95 % confidence interval [CI]: 1.03-1.72) per 1000 patients and in the second period 0.44 (95 % CI: 0.34-0.54) per 1000 patients (p < 0.001). CONCLUSION: Intracameral injection of cefuroxime reduces the rate of postoperative infectious endophthalmitis in cataract surgery significantly.

[Neuroprotective approaches]. / Neuroprotektive Ansätze.

After introduction of vitreoretinal surgery more than 40 years ago, further development of the procedure involved a continuous reduction of potential toxic effects by irrigating solutions, endoillumination or mechanical manipulation. Recently, additional efforts were made to prevent neurodegeneration via pharmacological intervention. Taurine as additive for irrigating solutions can be considered as an example for neuroprotectants in vitreoretinal surgery. Approval of neuroprotective agents demands an increased effort for preclinical and clinical evaluation. To date, only few neuroprotective substances are used in clinical routine in the context of vitreoretinal surgery, however, experimental data suggest a high potential of various neuroprotective agents. The following article gives an overview of current neuroprotective approaches feasible for vitreoretinal surgery and a critical analysis of their clinical relevance.

[Drug-induced malignant arrhythmias. IT prevents lethal drug mixtures]. / Medikamentös induzierte maligne Arrhythmien. EDV-Prävention verhindert tödliche Mixturen.

We report a case of nearly fatal ventricular tachyarrhythmia type Torsade de pointes caused by medication-induced prolongation of QTc duration (methadone, ondansetron, escitalopram). The etiology, pathophysiology, and trigger mechanisms of such malignant arrhythmias are discussed. In order to prevent similar iatrogenic complications in the future, we networked the qtdrug database with our medication interaction control program and installed an automatic electronic warning system for the physicians in charge in case of a digitally recorded prolonged QTc duration.

RASSF1A inhibits estrogen receptor alpha expression and estrogen-independent signalling: implications for breast cancer development.

The Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor whose inactivation is implicated in the development of many human cancers, including breast carcinomas. Little is known about the tumor-suppressive function of RASSF1A in breast tissue and whether its inactivation is mechanistically involved in the initiation and progression of breast tumors. Here, we show that RASSF1A inhibits breast cancer growth in vivo, and suppresses estrogen receptor (ERα) expression and function. Reconstitution of RASSF1A in MCF7 cells led to decreased ERα levels and reduced sensitivity to estrogen (E2). Concomitantly, we observed decreased expression of Id1 as well as the E2-responsive genes Bcl-2 and c-Myc that cooperatively contribute to the immortalization and transformation of breast epithelial cells. This downregulation was associated with induction of cell-cycle arrest and senescence that constitute early barriers to cancer initiation and progression. Knockdown of ERα showed that downregulation of ERα suffices to increase senescence and inhibit expression of Bcl-2, c-Myc and Id1. However, enforced expression of ERα only partially rescued RASSF1A-mediated growth inhibition and senescence, suggesting that suppression of ERα expression and activity is not the only mechanism by which RASSF1A inhibits growth and survival of breast cancer cells. Ectopic expression of Bcl-2, c-Myc and Id1 had little or no effect on RASSF1A-mediated growth arrest, indicating that RASSF1A acts dominantly over these oncogenes. Mechanistically, RASSF1A was found to suppress ERα expression through Akt1. It also transiently inhibited ERα-induced Ras-MAPK activity after exposure of cells to E2. Together, our data show that RASSF1A acts as a tumor suppressor in ERα+ mammary epithelial cells, in part through inhibiting ERα expression and activity. These findings suggest that RASSF1A has a key role in suppressing the transformation of human breast epithelial cells and ERα+ breast cancer initiation.

[Clinical results after cataract surgery in patients with Behçet's disease]. / Ergebnisse nach Kataraktchirurgie bei Patienten mit Behçet-Erkrankung.

BACKGROUND: Behçet's disease is a systemic vasculitis disorder of unknown aetiology. Ocular involvement, especially with vasculitis, is detected in up to 80 % of the cases. Anterior segment involvement such as cataract is also seen in the follow-up of patients who are then treated surgically. In this study, we aimed to analyze the outcomes of cataract surgery in patients with Behçet's disease retrospectively. PATIENTS AND METHODS: The records of 9 patients (12 eyes) with Behçet's disease who underwent phacoemulsification with IOL implantation in 11 eyes and extracapsular cataract extraction (ECCE) with IOL implantation in one eye between June 2001 and September 2009 were evaluated retrospectively. The visual outcome and complications were analysed. RESULTS: The mean follow-up was 33.8 months (range 3 to 88 months). The mean preoperative LogMAR BCVA was 1.15 ± 0.53 (95 % CI: 0.81 - 1.49) and increased to 0.36 ± 0.32 (95 % CI: 0.15 - 0.56) at last medical visit (p < 0.001). The most frequent postoperative complication was posterior capsular opacification, which developed in 2 eyes (17 %). Other complications were mild fibrinous reaction in 1 eye (8 %). CONCLUSIONS: The outcomes of cataract surgery in patients with Behçet's disease were satisfactory. The great majority of the patients regained and retained a good visual outcome and had fewer postoperative complications.

[Biocompatibility of dyes for vitreoretinal surgery]. / Biokompatibilität von Farbstoffen für die vitreoretinale Chirurgie.

For the last 8 years vital dyes have been used to visualize preretinal semitransparent structures in the eye during vitroretinal surgery. However, indocyanine green (ICG), which was the first commonly used dye, proved to be partly toxic to retinal cells and the retinal pigment epithelium in various in vitro and in vivo studies. Therefore, an intensive search for new and safer dyes was started. At present three dyes, trypan blue, patent blue and brilliant blue are predominantly used for vitreoretinal surgery, in addition to ICG. In this article an overview of preclinical biocompatibility studies for common vital dyes is presented. Additionally, a systematic approach for testing of new candidate dyes for vitreoretinal surgery will be proposed.

[Vital dyes in vitreoretinal surgery. Current application concepts]. / Farbstoffe in der vitreoretinalen Chirurgie. Aktuelle klinische Anwendung.

This article reviews the most relevant vital dyes and adjuncts currently available for use in vitreoretinal surgical procedures. The current concepts of intraocular application as well as the staining properties are described, and the issue of biocompatibility is discussed.

An in vivo evaluation of Brilliant Blue G in animals and humans.

BACKGROUND/AIMS: To evaluate the retinal toxicity of Brilliant Blue G (BBG) following intravitreal injection in rat eyes and examine the biocompatibility and the staining properties in humans. METHODS: BBG was injected into the 11 rat eyes to evaluate toxic effects with balanced salt solution (BSS) serving as control. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts and by light microscopy 7 days later. In addition, BBG was applied during vitrectomy for macular hole (MH) (n = 15) or epiretinal membranes (ERM) (n = 3) in a prospective, non-comparative consecutive series of patients. Before and after surgery, all patients underwent a complete clinical examination including measurement of best corrected visual acuity (VA) and intraocular pressure, perimetry, fundus photography and optical coherence tomography. Patients were seen 1 day before surgery and then in approximately four weeks intervals. RESULTS: No significant reduction in RGC numbers and no morphological alterations were noted. A sufficient staining of the internal limiting membrane (ILM) was seen in patients with MH, while the staining pattern in ERM cases was patchy, indicating that parts of the ILM were peeled off along with the ERM in a variable extent. All MHs could be closed successfully. VA improved in 10 eyes (56%; 8/15 MH patients, 2/3 ERM patients), was unchanged in four eyes (22%; all MH patients) and was reduced in four eyes (22%; 3/15 MH, 1/3 ERM). No toxic effects attributable to the dye were noted during patient follow-up. The ultrastructure of tissue harvested during surgery was unremarkable. CONCLUSION: Brilliant Blue provides a sufficient and selective staining of the ILM. No retinal toxicity or adverse effects related to the dye were observed in animal and human studies. The long-term safety of this novel dye will have to be evaluated in larger patient series and a longer follow-up.

Staining and peeling of the internal limiting membrane using a fluorescent dye (Rhodamine 6 G).

AIM: To assess whether low concentrations of a fluorescent dye such as Rhodamine 6G would help the unaided human eye visualise the vitreous and the internal limiting membrane (ILM) under standard halogen illumination. MATERIAL/METHODS: The UV/Vis absorption (E) and fluorescence (I) spectra of Rhodamine 6G in water were measured and compared with Indocyanine Green (ICG). Surgery was performed in two rhesus monkeys and consisted of standard pars plana vitrectomy with halogen light source used for illumination. Rhodamine 6G was diluted in balanced salt solution (BSS). A few drops of the dye in a concentration of 0.1% (307 mOsm) were applied over the posterior pole in the air-filled globe and washed out by irrigation after 1 min. Immediately after surgery, the globes were enucleated, fixated and prepared for histological evaluation. RESULTS: In contrast to ICG, both the maximum of the absorption and emission of Rhodamin 6G are very much within the spectral sensitivity of the human eye. The Rhodamine 6G-BSS itself appears red in colour. Using a dye concentration of 0.1%, there was no visible red-staining of the ILM as such. As the dye was irrigated out with BSS, a marked green fluorescence of the fluid within the vitreous cavity was noted. With halogen illumination through a standard 20-gauge light pipe, the dye provided a sufficient green fluorescence to identify and safely remove the ILM and to clearly differentiate areas of peeled from non-peeled ILM. During light microscopy, eyes revealed a peeled ILM demarcation with no signs of acute retinal toxicity. CONCLUSION: The findings indicate that a fluorescent dye can be used for ILM peeling. Assuming that the fluorophore provides a high enough fluorescence quantum yield after adsorption to the ILM, much lower dye concentrations could be used compared with absorbent dyes, thereby minimising toxic effects.

Inhibitors of apoptosis confer resistance to tumour suppression by adoptively transplanted cytotoxic T-lymphocytes in vitro and in vivo.

Deregulation of apoptosis signalling is commonly found in cancer and results in resistance to cytotoxic therapies. Immunotherapy is a promising strategy to eliminate resistant cancer cells. The transfer of T-lymphocytes during allogeneic stem cell transplantation is clinically explored to induce a 'graft-versus-tumor' effect (GvT). Cytotoxic T-lymphocytes (CTL), which are major effectors of GvT, eliminate cancer cells by inducing apoptosis via multiple parallel pathways. Here, we study in vitro and in vivo the susceptibility of murine cancer cells engineered to express single antiapoptotic genes to CTL-mediated cytotoxicity. Interestingly, we find that single inhibitors of caspase activation, such as BCL-XL or dominant-negative mutants of FADD and caspase-9, protect cancer cells against antigen-specific CTL in vitro. Moreover, expression of BCL-XL impairs the growth suppression by adoptively transplanted CTL of established tumours in vivo. Hence, apoptosis defects that provide protection to cytotoxic cancer therapies can confer crossresistance to immunotherapy by tumour-reactive CTL.

A packaging cell line generating CD4-specific retroviral vectors for efficient gene transfer into primary human T-helper lymphocytes.

Murine leukemia virus (MuLV) can be pseudotyped with a variant of the human immunodeficency virus (HIV) envelope gene encoding the surface glycoprotein gp120-SU and a carboxy-terminally truncated transmembrane (TM) protein with only seven cytoplasmic amino acids. MuLV/HIV-1 pseudotyped retroviral vectors selectively target gene transfer to human cells expressing both CD4 and CXCR4. To apply this vector system to gene therapy of human diseases, we generated a stable packaging cell line, FLY-HIV-87, expressing the MuLV gag and pol genes and the C-terminally truncated variant of the HIV-1 envelope gene, but no retroviral vector genome. Production of infectious vector particles was tested after the introduction of different vector genomes and was in the range of 5x10(5) IU/ml. The vector particles could be concentrated up to 25-fold. Specific and efficient gene transfer into CD4/CXCR4 expressing cell lines and stimulated primary human CD4+ peripheral blood lymphocytes was achieved. Thus the packaging cell line FLY-HIV-87 is highly suitable for gene therapy of disorders of human T-helper cells.

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

[Comparison of two different protocols on change of medication in central venous catheterization in patients with bone marrow transplantation: results of a randomized multicenter study]. / Confronto di due differenti protocolli nell'intervallo del cambio di medicazione per cateteri venosi centrali in pazienti trapiantati di midollo osseo: risultati di uno studio multicentrico randomizzato. Gruppo Italiano di Infermieri Trapianto Midollo Osseo (GITMO).

Care of central venous catheter (CVC) in patients undergoing bone marrow transplantation (BMT) raises significant problems related to the high risk of local infections, to the immunodeficient status, which in itself is a predisposing factor for systematic blood stream infections. Although frequent changes of CVC dressing might theoretically reduce the incidence of infections, they are also accompanied by significant skin toxicity and patient discomfort. No study has yet addressed these points. The objective of this study was to compare two different time interval protocols for CVC dressing, in order to assess the effects on local infections and toxicity. In a multicentre study, 339 bone marrow transplant (BMT) patients with a tunnelled CVC (group A, 230 pts) or a non tunnelled one (Group B, 169 patients) were randomly allocated to receive CVC dressing changes every 5 or 10 days if belonging to group A or 2 or 5 days if in group B. Transparent impermeable polyurethane dressings were used for all patients. The rate of local infection at the site of CVC insertion was assessed by microbiological assay every 10 days, while severity of skin toxicity was measured according to the ECOG scale. Sixty-five per cent of enrolled patients were finally evaluable. Patients (in both groups) receiving CVC dressing changes at longer intervals did not show a significant increase in the rate of local infections, while those who received dressing every two days had a significant increase in local skin toxicity. Longer intervals were accompanied by a reduction in costs. The results of this study demonstrate that the increase in time interval between CVC dressing changes in BMT patients did not increment the risk of local infections, while significantly reducing patients discomfort and costs.

Polycarbophil-cysteine conjugates as platforms for oral polypeptide delivery systems.

The purpose of the present study was to evaluate the potential of polycarbophil-cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35-50 microM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil-cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4. 17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p < 0.05) improved. As the zinc binding affinity of polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21. 36), the inhibitory effect of the polymer-cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p < 0.05) higher than that of the unmodified polymer. Because of these inhibitory features, polycarbophil-cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine.

Comparison of two different time interval protocols for central venous catheter dressing in bone marrow transplant patients: results of a randomized, multicenter study. The Italian Nurse Bone Marrow Transplant Group (GITMO).

BACKGROUND AND OBJECTIVE: Care of central venous catheter (CVC) in patients undergoing bone marrow transplantation (BMT) raises significant problems related to the high risk of local infections due to the immunodeficient status, which in itself is a predisposing factor for systemic blood-stream infections. Although frequent changes of CVC dressing might theoretically reduce the incidence of infections, they are also accompanied by significant skin toxicity and patient discomfort. No study has yet addressed these points. The objective of this study was to compare two different time interval protocols for CVC dressing in order to assess the effects on local infections and toxicity. DESIGN AND METHODS: In a multicenter study, 399 bone marrow transplant (BMT) patients with a tunneled CVC (Group A, 230 pts) or a non-tunneled one (Group B, 169 pts) were randomly allocated to receive CVC dressing changes every 5 or 10 days, if belonging to Group A, or 2 or 5 days, if in Group B. Transparent, impermeable polyurethane dressings were used for all patients. The rate of local infections at the site of CVC insertion was assessed by microbiological assays every 10 days, while the severity of skin toxicity was measured according to the ECOG scale. RESULTS: Sixty-five per cent of enrolled patients were finally evaluable. Patients (in both Groups) receiving CVC dressing changes at longer intervals did not show a significant increase in the rate of local infections, while those who received dressing every 2 days had a significant increase in local skin toxicity. Longer intervals were accompanied by a reduction in costs. INTERPRETATION AND CONCLUSIONS: The results of this study demonstrate that the increase in time interval between CVC dressing changes in BMT patients did not raise the risk of local infections, while significantly reducing patient discomfort and costs.

Truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles.

The incorporation of human immunodeficiency virus-type-2 (HIV-2) envelope glycoprotein into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We observed that wild-type HIV-2 envelope protein or a frameshift mutant with 187 unrelated carboxyl-terminal residues did not allow the formation of infectious retroviral particles. In view of recent findings that an HIV-1 envelope protein variant with a shortened cytoplasmic domain was incorporated into MuLV particles, we constructed carboxyl-terminal truncations of the HIV-2 envelope protein. An envelope variant with 18 cytoplasmic amino acids formed only very few viral pseudotypes. The further removal of an additional 11 amino acids allowed the efficient pseudotyping of MuLV particles. As with the HIV-1 envelope protein, an HIV-2 envelope variant with 7 cytoplasmic amino acids was incorporated into functional MuLV particles. The pseudotyped vectors obtained are able to infect human CD4/CXCR4-expressing cells. Cell lines expressing human CD4 and other coreceptors could not be infected. This retroviral vector will prove useful for the study of HIV infection events mediated by the HIV-2 envelope glycoproteins, as well as for the targeting of CD4+ cells in the context of gene therapy of AIDS.

Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer.

Mutations in Ras family members that confer oncogenic potential are frequently observed in specific human cancers. We report that human non-small cell lung cancer (NSCLC) lines that harbor oncogenic mutations in Ki-Ras (H460, A549, H2122) synthesized high levels of prostaglandin E2 (PGE2) compared with NSCLC lacking Ras mutations or non-transformed lung epithelial cells (BEAS-2B). This increased PGE2 production was mediated by constitutively high expression of 85-kDa cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2). The increased expression of cPLA2 protein was mediated through elevated mRNA levels and activation of the cPLA2 promoter. Induction of cPLA2 promoter activity was blocked by expression of dominant-negative forms of Ras. Inhibition of Ras by the farnesyltransferase inhibitor BZA-5B inhibited prostaglandin synthesis in H2122 cells by decreasing expression of both cPLA2 and COX-2. Finally, inhibitors of eicosanoid synthesis blocked anchorage-independent growth of NSCLC lines exhibiting Ki-Ras mutations. These results identify cPLA2 as a novel Ras-inducible regulator of eicosanoid synthesis that participates in cellular transformation.

Suppression of smooth-muscle alpha-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2.

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.