Percutaneous minimally invasive fetoscopic surgery for spina bifida aperta. Part III: neurosurgical intervention in the first postnatal year.

OBJECTIVE: To evaluate the need for postnatal neurosurgical intervention after fetoscopic patch coverage of spina bifida aperta (SBA). METHODS: This was a retrospective analysis of a cohort of 71 fetuses which underwent minimally invasive fetoscopic patch coverage of SBA between 21 + 0 and 29 + 1 weeks of gestation. Postnatal neurosurgical procedures were classified into two types: re-coverage of the SBA within the first 3 months following birth, and shunt placement as treatment of associated hydrocephalus within the first year. RESULTS: Location of the SBA was lumbosacral in 59 cases, lumbar in seven, thoracic in three and sacral in two. In total, 20/71 (28%) patients underwent early postnatal neurosurgical intervention by means of re-coverage of the SBA. This was performed because of cerebrospinal fluid leakage in seven (35%), adhesions with functional deterioration in three (15%), incomplete coverage in five (25%) and skin defect in five (25%) cases. Ventriculoperitoneal shunt placement within 1 year was required in 32 (45%) cases and was preceded by ventriculostomy in two. Three (4%) infants needed Chiari decompression surgery in the first 12 months following birth, because of syringomyelia or gait disturbance. CONCLUSIONS: Fetoscopic patch coverage of SBA may require postnatal re-coverage in some cases. In most cases, conservative wound treatment shows good results, without requiring neurosurgical intervention. The low 1-year-shunt rate is comparable to data of the Management of Myelomeningocele Study and lower compared with published data of patients with postnatal only coverage of SBA.

Bacterial community dynamics during ensiling of perennial ryegrass at two compaction levels monitored by terminal restriction fragment length polymorphism.

AIMS: The purpose of this study was to investigate the effects of compaction and air infiltration on the bacterial community structure during the fermentation process and the aerobic exposure phase of grass silage. METHODS AND RESULTS: Perennial ryegrass was ensiled at laboratory scale in a high-density (HD) and low-density (LD) compaction variant. Silages were exposed to air, and degradation was monitored by analysing temperature changes within the silage. Fermentation dynamics were examined using chemical analysis and terminal restriction fragment length polymorphism (TRFLP) analysis of the 16S rRNA gene supported by cloning and sequencing of representative samples. Dominant Lactobacillus species in HD silages remained largely unchanged during aerobic exposure. LD silages revealed fundamental changes in the bacterial community structure when exposed to air. After 4 days aerobic storage, only 23% of the primary silage community remained and mainly opportunistic Bacillus species proliferated. CONCLUSION: The ensiling of ryegrass is a very dynamic microbial process. Aerobic spoilage was limited to the LD silages, marked by a complete change towards a Bacillus-dominated community. SIGNIFICANCE AND IMPACT OF THE STUDY: The TRFLP analysis supported by the identification of terminal restriction fragments, revealed novel insights into the dynamics of the bacterial community during ensiling, and at aerobic spoilage conditions.

Differences in the implications of maternal lipids on fetal metabolism and growth between gestational diabetes mellitus and control pregnancies.

AIMS: To evaluate the potential contribution of maternal glucose and lipids to fetal metabolic variables and growth in pregnancies with normal glucose tolerance in comparison with pregnancies with well-controlled gestational diabetes previously reported by us. METHODS: In 190 pregnancies with normal oral glucose tolerance tests (controls), insulin, glucose and lipid components were determined in maternal and arterial cord blood serum. Birthweight and neonatal fat mass were obtained after delivery. Values were adjusted for maternal pre-pregnancy BMI, Caesarean section and gestational age. Measurements were compared with those of gestational diabetes previously reported. RESULTS: Maternal serum glucose, triacylglycerol, free fatty acid and cholesterol levels did not differ between control pregnancies and those with gestational diabetes, whereas insulin, homeostasis model assessment and glycerol values were significantly lower in the former (2.6 vs. 5.6 µmol/l and 176 vs. 193 µmol/l, respectively). In contrast, cord blood glucose and free fatty acids were significantly lower in control pregnancies than in those with gestational diabetes (3.9 vs. 4.4 mmol/l and 80.7 vs. 137 µmol/l, respectively); the same was valid for insulin (0.03 vs. 0.05 nmol/l) and homeostasis model assessment (1.0 vs. 1.87). In control pregnancies, maternal serum glucose, free fatty acids and glycerol correlated with those in cord blood, but not with neonatal weight and fat mass, as seen for free fatty acids in those with gestational diabetes. The negative correlation between cord blood triacylglycerols and neonatal weight or fat mass previously reported in gestational diabetes could not be confirmed in control pregnancies, where all fetal lipids showed a positive correlation to neonatal anthropometrics. CONCLUSION: In normal pregnancies, in contrast to those with gestational diabetes, maternal lipids do not influence neonatal weight. Similar levels of maternal lipids in pregnancies with gestational diabetes and control pregnancies, but higher free fatty acids in the cord blood of those with gestational diabetes, indicate their enhanced placental transport and/or enhanced lipolysis as a result of decreased fetal insulin responsiveness.

[Strategies to prevent surgical site infections]. / Strategien zur Prävention von postoperativen Wundinfektionen.

Surgical site infections (SSI) are a severe complication following surgical or orthopaedic procedures and are associated with significant increases in hospital length of stay (LOS), additional costs, morbidity and mortality. Hence, the prevention of SSI is essential and poses a major challenge in the healthcare system. Strategies and key points are presented and discussed. Infection control measures such as active surveillance of SSI, implementation of a checklist, compliance observations and instruction/training of healthcare workers as well as Staphylococcus aureus/MRSA screening, clipping instead of shaving, adherence to perioperative antibiotic prophylaxis, maintaining intraoperative normothermia and blood glucose control are essential for a comprehensive bundle in order to prevent SSI.

[Frequency of polyphasic health-care-associated outbreaks is highest in surgical departments - results of a systematic literature search]. / Chirurgen sind am häufigsten von mehrphasischen nosokomialen Ausbrüchen betroffen - Ergebnisse einer systematischen Literaturrecherche.

BACKGROUND: So-called polyphasic nosocomial outbreaks describe a situation in which additional infections occur after a certain case-free interval - despite the detection of the outbreak's source. This article summarises the results of a systematic search of the medical literature on polyphasic outbreaks. MATERIALS AND METHODS: For this purpose, the Outbreak Worldwide-Database, PubMed and reference lists of relevant articles were screened. RESULTS: A total of 124 polyphasic outbreaks (median duration of 50 weeks) was included in the analysis and then compared to 2089 monophasic nosocomial outbreaks. Surgical departments were significantly more often involved in polyphasic outbreaks than they were in monophasic events (33.9 % vs. 24.5 %; p < 0.05). Hepatitis B virus outbreaks were significantly more often seen as poly-phasic events. Either there had been more than one source initially, or a new source developed during the first phase of the outbreak and led to additional cases thereafter. CONCLUSIONS: Up to now, only little is known about polyphasic nosocomial outbreaks. Thus, there is a further need to close this gap of information in the future. Personnel on the ward as well as -infection control staff should always consider the possibility of the existence of more than one -source when investigating a nosocomial outbreak.

[Hand hygiene and campaigns]. / Händehygiene und Kampagnen.

BACKGROUND: Hand disinfection is a well-known and appropriate practise for infection prevention. Hence, it is logical to encourage its compliance and to provide its sustainability in the daily routine of a hospital. Several campaigns address an improvement of this important prevention measure. METHODS: In the Hannover Medical School the health staffs on the intensive care units and bone marrow transplantation wards were examined for this topic by a standardised questionnaire. The aim was to detect deficiencies and the level of knowledge. RESULTS: The forms were handed out to 838 health-care workers on 12 wards. 346 (41.2 %) were analysed. Inadequate hand disinfection due to a lack of time was the most common answer (43.1 %), followed by "there is no reason" (37.3 %). The alcoholic hand rub should be better available (50.3 %) and a continuing education programme should be provided (42.8 %) for improving hand hygiene practise. CONCLUSION: The survey revealed the known risk factors for non-compliance. At this point, the national hand campaign "Aktion Saubere Hände" supports training courses by providing instruction materials for all participants. These materials are used for training health-care workers individually.

Role of endotoxin in the pathogenesis of critical illness polyneuropathy.

Critical illness polyneuropathy (CIP) occurs in association with sepsis and multiple organ failure; however, little is known about the pathomechanisms of CIP and its therapy. In order to determine the parameters which interfere with development of CIP, electrophysiological investigations of peripheral nerves and biochemical measures were correlated to each other. The present study includes 20 consecutive patients in an intensive care unit developing severe sepsis or septic shock. Nerve conduction studies and electromyography were performed with occurring sepsis (day 1, 7, 14) and neurophysiological parameters were correlated with biochemical measures, especially indicators of infection and inflammation. It was found that all patients developed neurophysiological signs of axonal motor polyneuropathy. There was a significant correlation between serum concentrations of endotoxin and interleukin-2 receptors (IL2-R) and reduction of the amplitude of the compound motor action potentials. Other clinical and biochemical parameters showed no significant correlations with neurophysiological data. This finding apparently indicates that endotoxin damages nerve axons directly or indirectly, e.g. by activation of inflammatory cascades (IL2-R). Endotoxin appears to be an essential factor in the pathogenesis of CIP in sepsis, and therapeutic options neutralizing endotoxin may prevent development of CIP.

Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia.

Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis.

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

Local mechanical properties of plasma treated polystyrene surfaces.

We study how a local air plasma treatment affects the mechanical properties of polystyrene by performing indentation measurements on the polymer in the elastic and plastic regime. The local exposure to plasma was obtained by placing a shadow-mask with quadratic holes of 45 x 45 microm(2) on top of the polymer substrate, providing uncovered (exposed to the plasma) and covered (protected from the plasma) areas. We have analyzed quantitatively the topography and the elastic-plastic properties of such a sample with atomic force microscopy (AFM) measurements, both before and after plasma treatment. To enhance the differences between covered and uncovered areas, the sample has been exposed to solvent vapor. This generates regions which are differently swollen. The quantitative investigation of the mechanical properties of the swollen sample for different solvent exposure times gives further insight into the changes of polystyrene mechanical properties caused by the plasma.

Psychopathological and behavioral correlates of dopaminergic sensitivity in alcohol-dependent patients.

OBJECTIVES: To explore 2 facets of dopamine receptor sensitivity in alcoholics: (1) whether reduced sensitivity of central dopamine receptors is correlated with anxiety, depression, or novelty seeking and (2) whether this reduction is associated with poor treatment outcome. METHOD: Sixty-four alcohol-dependent patients were assessed according to their clinical outcome, sensitivity of central dopamine receptors (apomorphine-induced growth hormone secretion), mood states, and personality traits before and after detoxification. RESULTS: Patients with poor treatment outcome displayed a blunted growth hormone response before, but not after, detoxification. Growth hormone response was not significantly correlated with novelty seeking. Relapsing patients tended to be less depressed than patients who remained abstinent during observation. CONCLUSION: This study did not support the hypothesis that reduced sensitivity of dopamine receptors is associated with anxiety, depressed mood, or high novelty seeking in alcoholism.

ACE inhibitors are endothelium dependent vasodilators of coronary arteries during submaximal stimulation with bradykinin.

OBJECTIVE: The effect of angiotensin converting enzyme (ACE) inhibitors on vascular tone of isolated coronary arteries was determined in the presence of bradykinin and other vasodilators to elucidate the mechanisms leading to augmented bradykinin effects during ACE inhibition. METHODS: Rings of isolated bovine and human coronary arteries were mounted in organ chambers for measurement of isometric force. The effects of lisinopril, enalaprilat, and captopril were investigated in the presence of submaximal concentrations of bradykinin or other vasodilators. RESULTS: ACE inhibitors alone did not affect vascular tone. Threshold concentrations of bradykinin (10(-10) M), kallidin (10(-9.5) M), and the slowly degraded bradykinin agonists D-Arg(Hyp3)-bradykinin (10(-9.5) M) and [Hyp3-Tyr(Me)8]-bradykinin (10(-10.5) M) caused partial relaxation of bovine rings with endothelium. Subsequent addition of ACE inhibitors markedly potentiated the relaxations to the kinins. Bradykinin concentrations in the organ bath measured by a specific bradykinin radioimmunoassay remained stable during the addition of lisinopril. Variation of the exposure time to bradykinin (10 to 60 min) did not affect the relaxations to the ACE inhibitor. The relaxations to lisinopril were not observed after either removal of the endothelium or incubation with nitro-l-arginine or the bradykinin-2 receptor antagonist Hoe 140. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium dependent relaxation to lisinopril and captopril was also observed in human coronary arteries treated with bradykinin (> or = 10(-7) M), but not in those treated with substance P (10(-8) M). CONCLUSIONS: ACE inhibitors selectively potentiate endothelium dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries by a local mechanism. This effect on endothelial cells might occur in addition to augmented bradykinin concentrations in the blood and reduced angiotensin II generation.

Angiotensin II directly increases transforming growth factor beta1 and osteopontin and indirectly affects collagen mRNA expression in the human heart.

OBJECTIVES: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFbeta-1 and osteopontin are involved in this process. METHODS AND RESULTS I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFbeta-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFbeta-1 (183+/-21% of control, p<0.05) and osteopontin mRNA (275+/-58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95+/-8%). Stimulation with TGFbeta-1 led to an increase in collagen I and III mRNA (127+/-10%, p<0.05; 140+/-15%, p<0.02). METHODS AND RESULTS II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFbeta-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, II or fibronectin mRNA content after 24 h. In contrast, TGFbeta-1 stimulation significantly increased collagen I and III mRNA expression (124+/-5% and 128+/-5%, p<0.002). CONCLUSIONS: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFbeta-1 and osteopontin mRNA expression. Since TGFbeta-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.

Psychosocial determinants of self-reported hand hygiene behaviour: a survey comparing physicians and nurses in intensive care units.

BACKGROUND: Research applying psychological behaviour change theories to hand hygiene compliance is scarce, especially for physicians. AIM: To identify psychosocial determinants of self-reported hand hygiene behaviour (HHB) of physicians and nurses in intensive care units (ICUs). METHODS: A cross-sectional survey using a self-administered questionnaire that applied concepts from the Health Action Process Approach on hygienic hand disinfection was conducted in 10 ICUs and two haematopoietic stem cell transplantation units at Hannover Medical School, Germany. Self-reported compliance was operationalized as always disinfecting one's hands when given tasks associated with risk of infection. Using seven-point Likert scales, behavioural planning, maintenance self-efficacy and action control were assessed as psychological factors, and personnel and material resources, organizational problems and cooperation on the ward were assessed as perceived environmental factors. Multiple logistic regression analysis was employed. FINDINGS: In total, 307 physicians and 348 nurses participated in this study (response rates 70.9% and 63.4%, respectively). Self-reported compliance did not differ between the groups (72.4% vs 69.4%, P = 0.405). While nurses reported stronger planning, self-efficacy and action control, physicians indicated better personnel resources and cooperation on the ward (P < 0.02). Self-efficacy [odds ratio (OR) 1.4, P = 0.041], action control (OR 1.8, P < 0.001) and cooperation on the ward (OR 1.5, P = 0.036) were positively associated with HHB among physicians, but only action control was positively associated with HHB among nurses (OR 1.6, P < 0.001). CONCLUSION: The associations between action control (self-regulatory strategies where behaviour is evaluated continuously and automatically against guidelines) and compliance indicate that HHB is a habit in need of self-monitoring. The fact that perceived cooperation on the ward was the only environmental correlate of HHB among physicians stresses the importance of team-directed interventions.

Targeted ED-B fibronectin SPECT in vivo imaging in experimental atherosclerosis.

AIM: The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions. However, functional in vivo imaging of ED-B-containing plaques has not been explored. This study evaluated whether [(99m)Tc]-conjugated AP39 ([(99m)Tc]-AP39), a single-chain antibody specific to ED-B, can be used for in vivo detection of atherosclerotic plaques in Western diet (WD)-fed, apolipoprotein E-deficient (apoE-/-) mice as compared to wildtype (WT) control mice. METHODS: Using SPECT, 12-month-old WD-fed apoE-/- and WT mice were studied 4 hours after injecting [(99m)Tc]-AP39 (148 MBq). Subsequently, mice were sacrificed, thoracic aortas measured in a g-counter, and plaques analyzed using histology, immuno-histochemistry, autoradiography, and morphometry. RESULTS: In vivo [(99m)Tc]-AP39-SPECT imaging of apoE-/- mice demonstrated a significant signal activity in the plaque-ridden thoracic aorta (52.236 ± 40.646 cpm/cm³) that co-localized with the aortic arch and the supra-aortic arteries in MRI scans. Low signal activity (9.468 ± 4.976 cpm/cm³) was observed in WT mice. In apoE-/- mice, the strongest signals were detected in the aortic root, aortic arch and along the abdominal aorta. Autoradiography analysis of aortas from apoE-/- mice confirmed the in vivo observation by demonstrating signal localization in atherosclerotic plaques. The size of autoradiography-positive plaque areas correlated significantly with the size of ED-B-positive (r=0.645, P=0.044) or macrophage-infiltrated (r=0.84, P<0.002) plaques. A significant correlation was found between the sizes of ED-B-positive and macrophage-infiltrated plaque areas (r=0.93, P<0.01). CONCLUSION: [(99m)Tc]-AP39-SPECT in vivo imaging detects inflammatory plaque lesions in WD-fed apoE-/- mice.

Effect of the ergot derivative lisuride hydrogen maleate on serum prolactin concentrations in female rats.

The influence of a new synthetic ergot derivative, lisuride hydrogen maleate (LHM) on serum prolactin (PRL) concentrations was investigated in female rats using different test models: 1. in reserpine (R)-pretreated intact females, and 2. in ovariectomized (OVX) estradiol benzoate (E2)-primed animals with or without an additional pretreatment with R. In all the models used LHM was strongly effective in lowering serum PRL. Doses from 0.025 to 0.5 mg/kg LHM, given orally as well as subcutaneously, suppressed serum PRL. Depending on the dose used, the serum PRL was lowered to a different extent for up to 12 h. LHM was at least as effective as the well-known potent inhibitor of PRL secretion CB-154 in lowering serum PRL in OVX rats primed with E2. The effects of R, E2, and LHM are described in relation to their mode of action within the hypothalamic-hypophyseal system which regulates PRL secretion. While the increase in serum PRL induced by R seems to be directly relatable to its known catecholamine depletion, the circadian rhythm of PRL secretion induced by E2 seems to be influenced or mediated by central neural mechanisms. The effects of LHM on serum PRL in these test models can be related to its dopaminergic action and constitute further evidence for the central functions of dopaminergic mechanisms in the regulation of PRL secretion.

Regulation and differential expression of neutral endopeptidase 24.11 in human endothelial cells.

Neutral endopeptidase 24.11, a membrane-bound metallopeptidase, cleaves, and degrades vasoactive peptides such as atrial natriuretic peptide, endothelin, angiotensin I, substance P, and bradykinin. Therefore, the presence of this metallopeptidase may contribute to the regulation of vascular tone and local inflammatory responses in the vascular endothelium and elsewhere. We determined neutral endopeptidase in cultured human endothelial cells from different vascular beds and studied its regulation by protein kinase C. Neutral endopeptidase was detected in all cultured endothelial cell types. Lowest concentrations were measured in human endothelial cells from umbilical veins (360 +/- 14 pg/mg protein), followed by pulmonary and coronary arteries; higher concentrations were found in endothelial cells from the cardiac microcirculation (1099 +/- 73 pg/mg protein). Neutral endopeptidase content increased during cell growth but was not affected by endothelial cell growth factor or modifications of the growth medium. Stimulation of protein kinase C with 1-oleoyl-2-acetyl-rac-glycerol (0.1 to 1 mumol/L) and phorbol 12-myristate 13-acetate (0.01 to 0.1 mumol/L) induced a time- and concentration-dependent increase of endothelial cells that was inhibited by cycloheximide (5 mumol/L), an inhibitor of protein synthesis. Incubation with phospholipase C (1 mumol/L) and thrombin (10 IU/mL) induced upregulation of neutral endopeptidase, resulting in 158 +/- 26% and 150 +/- 22% increases, respectively, compared with controls. The thrombin effect was inhibited by calphostin C (1 mumol/L), an inhibitor of protein kinase C. Endothelial neutral endopeptidase is constitutively expressed in endothelial cells from different origins and is inducible by thrombin via activation of the protein kinase C pathway.

Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells.

Migration of vascular smooth muscle cells (VSMCs) is a crucial response to vascular injury resulting in neointima formation and atherosclerosis. Platelet-derived growth factor (PDGF-BB) functions as a potent chemoattractant for VSMCs and enhances these pathologies in the vasculature. However, little is known about the intracellular pathways that mediate VSMC migration. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) activation in this function, since PDGF-BB as well as other growth factors activate this pathway. Using an in-gel kinase assay, we observed that PD 98059 an inhibitor of MEK that activates MAP kinase, inhibited PDGF-BB-induced activation of ERK-1 and ERK-2 in cultured rat aortic smooth muscle cells in a concentration-dependent manner. In contrast, PDGF-mediated activation of intracellular calcium release was not affected by PD 98059. The chemotactic response of both rat aortic smooth muscle cells (RASMCs) and human umbilical vein smooth muscle cells (HUSMCs) toward PDGF-BB (10 ng/mL) was significantly reduced by PD 98059 (10 mumol/L) to 41.7 +/- 7.1% in RASMCs (P < .01) and to 47.2 +/- 5.3% in HUSMCs (P < .01). Similar inhibition was seen at 30 mumol/L, less at 1 mumol/L. To further confirm the specificity of these results implicating the MAPK pathway, an antisense oligodeoxynucleotide (ODN) directed against the initiation translation site of rat ERK-1 and ERK-2 mRNA was used to suppress MAP kinase synthesis and function in rat VSMCs. Liposomal transfection with 0.4 mumol/L antisense ODN reduced ERK-1 and ERK-2 protein by 65% (P < .01) after 48 hours. The chemotactic response to PDGF-BB (10 ng/mL) was reduced by 75% (P < .01) in rat VSMCs transfected with the same antisense ODN concentration. Sense and scrambled control ODNs (0.4 mumol/L) did not affect ERK-1 and ERK-2 protein concentrations or chemotaxis of VSMCs induced by PDGF-BB. These experiments provide the first evidence that activation of MAPK is a critical event in PDGF-mediated signal transduction regulating VSMC migration.

Angiotensin II and alpha(v)beta(3) integrin expression in rat neonatal cardiac fibroblasts.

We recently demonstrated that alpha(v)beta(3) integrins are involved in the mechanisms of angiotensin II (Ang II)-induced DNA synthesis and collagen gel contractions in rat cardiac fibroblasts (CFBs), cellular mechanisms that are relevant for cardiac remodeling. The aim of the present study was to elucidate the effect of Ang II and other growth factors on the regulation of the alpha(v)beta(3) integrins in fibroblasts from neonatal rat hearts. The alpha(v)beta(3) integrin receptor expression was significantly increased (P<0.05) at the mRNA level after treatment with Ang II, transforming growth factor-beta(1) (TGF-beta(1)), and platelet-derived growth factor (PDGF) for 8 and 16 hours. The surface expression of the alpha(v) and beta(3) integrin subunits was elevated after 32 and 48 hours (P<0.05) as determined with flow cytometry. To investigate fibroblast motility, we performed chemotaxis experiments with transwell chambers. Ang II was chemotactic for CFBs, as tested with checkerboard experiments. The chemotactic effect was concentration dependent and was completely blocked by Ang II type 1 receptor blockers but not by Ang II type 2 receptor blocker PD 123319. Ang II- and PDGF-BB-mediated chemotaxis could be significantly inhibited by RGD peptides and the blocking antibodies against alpha(v)beta(3) integrin (both P<0.01). Adhesion of CFBs to vitronectin was partially inhibited by an antibody to alpha(v)beta(3) integrin but was mainly mediated by an alpha(v)beta(5) integrin. Relevant in vivo expression of alpha(v)beta(3) integrin by CFBs was confirmed with in situ hybridization with probes for alpha(v) and beta(3) mRNA in rat hearts. The present study demonstrates that the expression of alpha(v)beta(3) integrin is augmented by Ang II, PDGF, and TGF-beta(1) in neonatal CFBs. Furthermore, this integrin is involved in the chemotaxis, motility, and adhesion of CFBs. The present findings support the current concept that integrins participate in the control of fibroblast behavior during cardiac remodeling mechanisms.

Angiotensin II and PDGF-BB stimulate beta(1)-integrin-mediated adhesion and spreading in human VSMCs.

beta(1)-Integrins play an important role for adhesion and spreading of human smooth muscle cells. In the present study we examined the influence of angiotensin II and platelet-derived growth factor (PDGF)-BB on beta(1)-integrin-dependent functions of human smooth muscle cells obtained from iliac arteries. Treatment of these cells with PDGF-BB (20 ng/mL) and Angiotensin II (1 micromol/L) did not change beta(1)-integrin expression up to 48 hours as analyzed by flow cytometry and reverse transcription polymerase chain reaction. beta(1)-integrins predominantly mediated adhesion of human smooth muscle cells to collagen I (79.7+/-4.4%, P<0.01) and fibronectin (66. 6+/-2.4%, P<0.01). Treatment of smooth muscle cells with Angiotensin II (1 micromol/L) and PDGF-BB (20 ng/mL) significantly increased the adhesion to collagen I by 56.5% and 44.3%, respectively, and to fibronectin by 49.6% and 36.4%, respectively (all P<0.05). Angiotensin II-induced effects were mediated by the AT(1) receptor. The PDGF-BB mediated increase of adhesion was inhibited in the presence of genestein, a tyrosine-kinase inhibitor and by protein kinase C downregulation with phorbol 12-myristate 13-acetate. Spreading of smooth muscle cells also was beta(1)-integrin dependent on collagen I and alpha(5)beta(1)-integrin dependent on fibronectin. Angiotensin II and PDGF-BB increased cell spreading on fibronectin up to 276% and 318%, respectively, and on collagen I up to 133% and 138% (all P<0.05). These increases were significantly inhibited by blocking antibodies against beta(1)-integrin, alpha(5)-integrin on fibronectin, the AT(1) receptor blocker irbesartan, and genestein. The present data demonstrate that angiotensin II and as well PDGF-BB enhance beta(1)-integrin-dependent adhesion and spreading of human vascular smooth muscle cells. Furthermore, the experiments with PDGF suggest an involvement of protein kinase C activation leading to these enhanced effects.