Platelet receptor desensitization induced by elevated prostacyclin levels causes platelet-endothelial cell adhesion.

OBJECTIVES: The purpose of this study was to investigate the role of platelet prostacyclin receptor desensitization in platelet-endothelial cell adhesion. BACKGROUND: Platelet-endothelial cell adhesion is regulated by endothelial cell-derived mediators, such as prostacyclin and endothelium-derived relaxing factor. Prostacyclin activates platelet adenylate cyclase and augments cyclic adenosine monophosphate formation by way of specific membrane receptors. Platelet exposure to prostacyclin or chemically stable analogs results in a time- and dose-dependent prostacyclin receptor desensitization as it occurs during infusion therapy with prostacyclin analogs or in pathophysiologic situations such as acute myocardial infarction. METHODS: Adhesion of washed and radiolabeled human platelets stimulated with thrombin to cultured umbilical vein endothelial cells was measured under control conditions and under conditions of platelet prostacyclin receptor desensitization induced by incubation with the prostacyclin analog iloprost (10 to 100 nmol/liter) for 3 h. RESULTS: Thrombin (0.08 to 0.2 U/ml) increased platelet adhesion in a dose-dependent manner from 2.7 +/- 0.3% to 6.4 +/- 0.6% (mean value +/- SEM). Preincubation of platelets resulted in a dose-dependent down-regulation of 3H-iloprost binding up to 58.8 +/- 6.7% of control platelets with 100 nmol/liter of iloprost. Co-incubation of prostacyclin receptor-desensitized platelets with endothelial cells resulted in a marked augmentation of thrombin-induced adhesion up to 28.6 +/- 4.5%. Approximately the same increase in platelet adhesion was seen after complete abrogation of endothelial cell prostacyclin synthesis by pretreatment with aspirin. Comparison of iloprost-induced receptor desensitization and increased platelet-endothelial cell adhesion indicated a positive correlation. CONCLUSIONS: Platelet prostacyclin receptor desensitization was observed in humans in vivo during acute myocardial infarction or during therapeutic administration of prostacyclin analogs. In vitro platelet prostacyclin receptor desensitization caused a marked augmentation of platelet-endothelial cell adhesion. This increase in adhesion might result in an enhanced tendency toward thrombus formation in humans.

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.

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.

Innovative approaches to educating medical students for practice in a changing health care environment: the National UME-21 Project.

In today's continually changing health care environment, there is serious concern that medical students are not being adequately prepared to provide optimal health care in the system where they will eventually practice. To address this problem, the Health Resources and Services Administration (HRSA) developed a $7.6 million national demonstration project, Undergraduate Medical Education for the 21st Century (UME-21). This project funded 18 U.S. medical schools, both public and private, for a three-year period (1998-2001) to implement innovative educational strategies. To accomplish their goals, the 18 UME-21 schools worked with more than 50 organizations external to the medical school (e.g., managed care organizations, integrated health systems, Area Health Education Centers, community health centers). The authors describe the major curricular changes that have been implemented through the UME-21 project, discuss the challenges that occurred in carrying out those changes, and outline the strategies for evaluating the project. The participating schools have developed curricular changes that focus on the core primary care clinical clerkships, take place in ambulatory settings, include learning objectives and competencies identified as important to providing care in the future health care system, and have faculty development and internal evaluation components. Curricular changes implemented at the 18 schools include having students work directly with managed care organizations, as well as special demonstration projects to teach students the knowledge, skills, and attitudes necessary for successfully managing care. It is already clear that the UME-21 project has catalyzed important curricular changes within 12.5% of U.S. medical schools. The ongoing national evaluation of this project, which will be completed in 2002, will provide further information about the project's impact and effectiveness.

The reluctant entrepreneur.

Unlike a lot of corporate executives, Ken Veit never longed to be his own boss. But after 30 years on the fast track, he lost his high-powered job at one of the world's largest insurance companies and was forced to take an entrepreneurial leap of faith. In 1989, Veit signed a franchise agreement to own and operate a Cartoon Corner store in a mall in Scottsdale, Arizona. Cartoon Corner was based on the Disney store idea, but it carried hundreds of products featuring cartoon characters from every movie studio. Most important, Cartoon Corner offered extensive training and an elaborate management support system for its franchisees. The company planned to franchise 100 stores over the next few years, then go public. If all went well, its young executives claimed, the Cartoon Corner chain would build a market valuation of up to $100 million by the mid-1990s. In addition, the mall, which was in the planning stages when Veit signed on, was supposed to become a new kind of entertainment mall, with seven movie theaters, a space-flight simulator, and a shark-filled aquarium. It had all sounded too good to be true--and it was. Despite Veit's careful forecasting, he suffered a series of unexpected catastrophes. The mall failed to keep its promises. The franchisor lost its venture capital. The Gulf War dried up retail traffic. But it was too late to back out. Veit went forward on his own, truly alone for the first time in his life. When the mall and his store finally opened in May 1991, they did so in the midst of a recession. Despite the inspirational stories of other former executives, Veit has learned that the life of an entrepreneur is not all it's cracked up to be. As he notes, "I began with well-above-average experience, a proven concept, and excellent capitalization, yet in my case, personal bankruptcy remains a distinct possibility."

Three-dimensional body scanning: a new method to estimate body surface area in neonates.

BACKGROUND: Body surface area (BSA) is usually estimated by calculation with mathematical formulae. Three-dimensional body scanning (3D scan) offers a suitable alternative. OBJECTIVES: We determined the BSA in healthy term and near-term neonates by 3D scanning. This system should be useful in the setting of intensive care medicine. METHODS: The measuring system consisted of a projector, two cameras, mirrors and a computer, and used the fringe projection technique with visible light. The infants were examined in a supine position; the hidden parts of the bodies were corrected for using a mathematical factor developed with a baby doll model. Results of the 3D scans were compared with those from five mathematical formulae for each subject. RESULTS: A total of 209 infants were studied by 3D scanning, of whom 53 had acceptable images and were selected for further analysis. The mean BSA was 2,139 cm(2) (SD 223.72). The minimal BSA was 1,587 cm(2), the maximal 2,670 cm(2), with a good correlation to body weight and length. One mathematical formula (Du Bois and Du Bois) showed a distinct underestimation of BSA compared to 3D scanning, the others an overestimation. Mean percentage similarity was from 96.8 to 100.9%. CONCLUSIONS: 3D scanning is an accurate and practical method to estimate BSA in newborns. Individual and repeated measurements from day to day are possible. Further studies are warranted in preterm and sick neonates.

Perioperative nursing data set for the adult patient having a cystoscopy.

This article gives a brief summary of a student project required in a perioperative internship program in a large medical center hospital. The Perioperative Nursing Data Set (PNDS) was used to determine activities necessary in developing a nursing care plan for patients undergoing cystoscopy. The project description and the total care plan is shared with the reader.

Diminished inhibition of adhesion molecule expression in prostacyclin receptor desensitized human platelets.

Long-term exposure of platelets to prostacyclin or iloprost (100nM, 3hr) results in receptor desensitization measured as decrease in 3H-iloprost binding sites by 47 +/- 14%. Desensitized platelets respond with an increased adhesion to endothelial cells. The mechanism of increased adhesiveness was studied by measuring the expression of the adhesion molecule CD62p (p-selectin; GMP140) on washed human platelets by flowcytometry. In thrombin stimulated platelets CD62p expression was dose-dependently reduced by iloprost. In receptor desensitized platelets IC50 for iloprost inhibition of thrombin-induced CD62p expression increased from 0.48 +/- 0.10 to 2.4 +/- 0.7 nM.

Prostacyclin receptor desensitization is a reversible phenomenon in human platelets.

BACKGROUND: Long-term exposure of platelets to endogenous or exogenous prostacyclin or its analogues might result in desensitization of the platelet prostacyclin receptor in vitro and in vivo accompanied by a loss in receptor density on the platelet surface and a reduced sensitivity toward the inhibitory effects of prostacyclins. However, the reversibility of this process in platelets has not yet been investigated. METHODS AND RESULTS: Human platelets desensitized by the chemically stable prostacyclin analogue iloprost showed a significant reduction in [3H]-iloprost binding sites that was reversed by saponin permeabilization. This indicates functionally active internalized prostacyclin receptors. To assess whether the internalized prostacyclin receptors recycle to the cell surface after withdrawal of the agonist, iloprost sensitivity and prostacyclin receptor binding properties of iloprost (30 nmol/L, 2 hours) desensitized platelets incubated in iloprost-free autologous plasma were investigated. While desensitized platelets showed a significant increase in IC50 for iloprost inhibition of thrombin-induced platelet aggregation, serotonin release, and p-selectin expression and a reduced iloprost-stimulated cAMP formation, platelet iloprost sensitivity was restored 3 hours after iloprost withdrawal. In addition, the significant reduction in Bmax and the increase in K(D) of prostacyclin receptors in desensitized platelets as revealed by [3H]-iloprost binding studies also returned to the initial values. CONCLUSIONS: These results indicate that prostacyclin receptors internalized during short-term desensitization are not degraded but can be recycled rapidly to the platelet surface in a functionally active form after withdrawal of the agonist.

[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.

Fusion of computed tomography data and optical 3D images of the dentition for streak artefact correction in the simulation of orthognathic surgery.

OBJECTIVE: To determine the limits of accuracy of fusion of optical three-dimensional (3D) imaging and computed tomography (CT) with and without metal artefacts in an experimental setting and to show the application of this hybrid system in 3D orthognathic surgery simulation. METHODS: Ten plaster casts of dental arches were subjected to a CT scan and optical 3D surface imaging. Subsequently, the first molars in the plaster casts were supplied with metal restorations, bilaterally, and new CT scans and optical surface images were assessed. The registration of the surface data of the two imaging modalities of the study models without and with metal restorations was carried out. The mean distance between the two data sets was calculated. From a patient a CT scan of the skull as well as optical 3D images of plaster casts of the dental arches were acquired. Again the two imaging modalities were registered and virtual orthognathic surgery simulation was carried out. RESULTS: The mean distance between the corresponding data points of CT and optical 3D surface images was 0.1262+/-0.0301 mm and 0.2671+/-0.0580 mm, respectively, for the plaster casts without and with metal restorations. The differences between these data were statistically significant (P<0.0005). For the patient case a mean difference of 0.66+/-0.49 mm and 0.56+/-0.48 mm for mandible and maxilla, respectively, was calculated between CT and optical surface data. CONCLUSION: The accuracy of the fusion of 3D CT surface data and optical 3D imaging is significantly reduced by metal artefacts. However, it seems appropriate for virtual orthognathic surgery simulation, as post-operative orthodontics are performed frequently.