All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells.

OBJECTIVE: The vitamin-A derivative all-trans retinoic acid (atRA) is a potent regulator of cell growth, differentiation, and matrix formation of various cell types and plays an important role in embryogenesis. However, sparse data are available about its effects on human vessel diseases. Thus, we studied the effects of atRA on human arterial smooth muscle cell (haSMC) and endothelial cell (haEC) proliferation, migration, differentiation and extracellular matrix (ECM) turnover in mono- and transfilter cocultures. METHODS: Effects of atRA on human arterial cells in monocultures were determined using cell counting assays, BrdU-ELISA and MTT-tests. In transfilter cocultures haSMC-growth was studied under the stimulatory effect of proliferating haEC. Using Northern blot analysis, effects of atRA on mRNA expression of ECM-proteins were examined while protein expression and activity of matrix metalloproteinases were determined by Western blotting and zymography. RESULTS: atRA caused a dose dependent inhibition of haSMC-growth in monocultures (IC(50) at 0.022 microM) whereas haEC-growth was inhibited less potently (IC(50) at 97 microM). In addition, proliferation and migration of haSMC through a porous membrane were inhibited dose dependently by micromolar atRA-doses after non-stop and single dose application of atRA on the endothelial side of the complex transfilter coculture system. Immunostainings and Northern blotting demonstrated an enhanced alpha-smooth muscle actin and heavy chain myosin expression in haSMC after atRA-treatment. Whereas mRNA-expression of the glycoproteins thrombospondin-1 and fibronectin were decreased, collagen-1 mRNA expression was even slightly stimulated. Transcription of biglycan and TGF-beta1 were not influenced in a specific manner. Finally, protein expression and activity of the matrix metalloproteinases MMP-2 and MMP-9 were inhibited significantly by atRA. CONCLUSIONS: atRA was found to be a potent inhibitor of both haSMC-proliferation and -migration, even in coculture with haEC releasing growth factors. In addition, redifferentiation, ECM synthesis and ECM degradation were regulated by atRA which also influence haSMC migration and intima formation. Thus, atRA-treatment seems to be a promising strategy for the inhibition of processes involved both in atherosclerosis and restenosis.

Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake.

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-beta(1) (TGF-beta(1)) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 microg/ml of the liposome Cytofectin GSV((R)) (CF) and the dendrimer SuperFect (SF) reduced mitochondrial activity of haEC after > or =4 h transfection whereas viability of haSMC was not influenced. DAC-30((R)) showed significant toxic effects on haEC and haSMC at each dose after > or =4 h and Lipofectin((R)) (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that 'naked' ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2-5 h. With DAC-30, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-beta(1) mRNA expression and synthesis after transfection for 3-5 h using 0.1-1.0 microM ASO versus control oligonucleotides. We conclude that Cytofectin GSV is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin. Cytofectin GSV might offer a promising tool for antisense strategies in the treatment of vascular disorders.

Effects of cerivastatin on human arterial smooth muscle cell proliferation and migration in transfilter cocultures.

Statins competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity reducing mevalonate synthesis. In this study, antiproliferative and antimigratory effects of the new compound cerivastatin were analyzed and compared with classic statins of the first and second generation using mono- and cocultures of human arterial smooth muscle (haSMC) and endothelial (haEC) cells. Effects on the mitotic index and mitochondrial activity of haEC and haSMC monocultures were tested using BrdU enzyme-linked immunosorbent assay (ELISA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) tests, respectively. In lactate dehydrogenase (LDH) assays, cytotoxicity of statins was studied. Transfilter cocultures were performed for 14 days to evaluate haSMC growth under the stimulatory effect of proliferating haEC, which release growth factors [e.g., platelet-derived growth factor (PDGF)]. The hydrophobic statins simvastatin, lovastatin, and atorvastatin significantly inhibited haSMC and haEC growth in monocultures at 0.5-50 microM. However, most potent effects were exerted by cerivastatin in 10- to 30-fold lower doses without any significant cytotoxicity. More important, cerivastatin showed also significant effects on haSMC proliferation and migration in transfilter cocultures at extremely low doses (IC50, 0.04-0.06 microM), even when applied exclusively to the endothelial side and in the presence of low-density lipoprotein (LDL). Addition of mevalonate abolished the effects of cerivastatin completely. Even in the presence of growth-stimulating haEC and LDL, cerivastatin was found to be the most potent inhibitor of haSMC proliferation and migration in doses that also can be reached in human serum after oral drug administration. The results support the concept that statins seems to influence additional cellular mechanisms beyond cholesterol reduction, which might also have a relevance for the prevention of restenosis.

Growth factor expression of human arterial smooth muscle cells and endothelial cells in a transfilter coculture system.

By releasing growth factors, vascular cells can modulate proliferation and migration of neighboring cells in the arterial wall. Previous histological studies in transfilter cocultures, a culture model aimed to simulate vessel wall architecture, indicated that human arterial endothelial cells (haEC) can influence human arterial smooth muscle cell (haSMC) growth significantly. The aim of this study was to investigate the expression and secretion of various growth factors in order to better define the functional interactions between haEC and haSMC. Protein levels of platelet-derived-growth factor-AB (PDGF-AB), transforming-growth factor-beta1 (TGF-beta1), and tumor-necrosis factor-alpha (TNF-alpha) in mono- and cocultures were determined by ELISA 6, 12, 24, 48, 72 h after serum reduction. Highest PDGF-AB levels were found in monocultures with proliferative haEC, showing a peak after 24 h. In cocultures of haEC and haSMC, PDGF-AB levels were significantly lower. In contrast, neither proliferative, nor confluent haSMC released PDGF-AB significantly. Highest TGF-beta1 concentrations were detected in cocultures, followed by monocultures of haSMC and monocultures of haEC. In all cultures, TGF-beta1 levels increased in parallel with cultivation time and cell numbers, showing a maximum after 72 h. TNF-alpha could not be detected in any culture. Northern blots demonstrated a strong expression of PDGF-B chain-mRNA in haEC, but not in haSMC. PDGF-A chain and TGF-beta1-mRNA were expressed by haSMC and haEC. Addition of PDGF-AB to haSMC resulted in a potent growth stimulation, whereas TGF-beta1 and TNF-alpha exerted only moderate, divergent effects on haSMC. Histological observations in transfilter cocultures demonstrated that proliferative haEC induce the formation of fibromuscular plaques. These results suggest that proliferative haEC act as potent growth stimulators for haSMC, predominantly by PDGF-AB or -BB release.

[Primary pulmonary hypertension and pregnancy]. / Primäre pulmonale Hypertonie und Schwangerschaft.

Primary pulmonary hypertension (PPH) is an uncommon disease with a poor prognosis. Most patients with PPH are young women of child-bearing age. We report on such a case of PPH during pregnancy, discuss the aetiology, pathophysiology, morphological characteristics and the management in pregnancy and delivery.

[Cesarean section in multiple pregnancy especially with triplets and quadruplets--preparation and execution]. / "Die Schnittentbindung bei Mehrlingen vor allem bei Drillingen und Vierlingen"--Vorbereitung und Durchführung.

The frequency of multiple pregnancies has increased in perinatal centers during the last years. The result is a rise in perinatal risk. Because of prematurity and abnormal presentations and positions, cesarean section rate is high in this group. This paper discusses the intra- and perioperative problems that may occur in cesarean section of multiple pregnancies, advices are given for perioperative management. In the discussion of surgical techniques, the amnion preserving incision according to Hillemanns is described as a reliable procedure, especially in multiple pregnancies combined with prematurity.

[Systemic lupus erythematosus and pregnancy. Clinical aspects, serology and management]. / Systemischer Lupus Erythematodes und Schwangerschaft. Klinik, Serologie und Management.

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting the connective tissue of the skin and the vascular system. In about 90% of the cases, the first diagnosis is made in women of child-bearing age. We report on 11 pregnancies in 5 patients with SLE. The incidence of SLE was found to be 1:2966 in relation to obstetric cases in our hospital. In one patient, an acute exacerbation of the disease led to preterm delivery in the 31st week of pregnancy. The affected patient died postpartum due to generalised disease and septic complications. In general, perinatal mortality was found to be 25% (excluding early abortion). The number of spontaneous abortions, premature deliveries and small for date babies was elevated in our group of patients, in comparison to the normal group. As a result of our own observations in serological controlled pregnancies and of an extensive review of the literature, we came to the following conclusions: Uncomplicated SLE is no contraindication for pregnancy. However, an SLE nephritis represents a relative or even absolute contraindication, depending on the clinical course. Recent prospective studies permit us to conclude, that a pregnancy will not lead to an aggravation of SLE. On the other hand, SLE can cause complications in pregnancy with a subsequent rise in maternal and foetal morbidity and mortality. Most frequent are preeclampsia, premature labour, foetal maldevelopment and flare-ups of the underlying disease. For monitoring the disease, frequent determinations of complement proteins C3/C4 are helpful. The measurement of the C3 turnover can be used to distinguish between the development of preeclampsia and exacerbation of the disorder.(ABSTRACT TRUNCATED AT 250 WORDS)

[The difference between the mineral content and the width of the radius on each side and its significance for handedness determination of skeletal material]. / Die Seitendifferenz des Mineralgehaltes und der Breite am Radius und ihre Bedeutung für die Händigkeitsbestimmung an Skelettmaterial.

The objective of our work was to investigate whether the side-different use of the upper extremities due to handedness produces detectable differences in bone-mineral content (BMC) and bone width (BW). For this purpose 251 recent individuals whose handedness was established, were examined by means of the 125I-photon-absorption technique. Highly significant right-left differences in BMC and BW were found on the midshaft and distal radius. Discrimination functions based on BMC and BW were carried out, allowing for the classification into the appropriate handedness categories. Applying the same method we tried to diagnose the handedness of the skeletal material of 40 medieval and 27 neolithic individuals.

Correlations between endogenous prostaglandin E formation in the bone and spongiosa density.

The formation of prostaglandin (PG) E in the femoral head and the lumbar vertebral body of rats under ex vivo conditions after a linoleic acid (LA) rich diet (13.3 J%) has been compared to that of rats after an LA poor diet (0.5 J%). LA rich diet increased the formation of PGE in the lumbar vertebral body. In the femoral head were a similar, but statistically insignificant effect. The spongiosa density in the femoral head and the lumbar vertebral body increased after LA rich diet in contrast to LA poor fed rats. We conclude that the spongiosa density could be modulated by the LA content of the diet possible via alterations of endogenous PG biosynthesis.

Regional lactate production in early canine endotoxin shock.

High serum lactate may not reflect the severity of endotoxin shock: the lactate load could even be formed immediately after the endotoxin challenge. During the first 30 min after endotoxin injection (Escherichia coli; 1.5 mg/kg iv) into anesthetized dogs (4 mg.kg-1.h-1 etomidate, n = 19) we studied arterial lactate concentration; contributions of portal and splanchnic (n = 6), renal and pulmonary (n = 7), and femoral (n = 6) vascular beds to the early lactate rise; and regional O2 extraction and blood flow (microspheres). In control dogs (n = 5, no endotoxin), we found no significant hemodynamic and biochemical changes. Endotoxin caused an immediate decrease in blood pressure, cardiac output, and organ perfusion, followed by recovery after approximately 5 min to approximately 75% of preshock values at t = 30 min (except for renal blood flow, which remained low). Arterial lactate concentration started to increase almost immediately after endotoxin and increased rapidly until t = 15 min (to 300%) and then leveled off, but in spite of the hemodynamic recovery it remained elevated. A major part of the early increase in lactate concentration can be explained by splanchnic lactate production. The total splanchnic bed released more lactate than the portal bed, indicating that the liver produces lactate. We conclude that the lactate concentration later in canine endotoxin shock depends on events that occur during early shock in which the liver may play a crucial role.

Growth patterns and hormonal sensitivity of primary tumor, abdominal metastasis and ascitic fluid from human epithelial ovarian carcinomas in the tumor colony-forming assay.

The human tumor colony forming assay was used to evaluate the response of ovarian carcinoma cells from primary tumors, ascitic fluids and metastasis to hormonal treatment. In 12/35 patients a sufficient colony formation (greater than 30 colonies/dish) was obtained in order to perform a simultaneous drug testing. The plating efficiency of the metastatic samples (0.12%) was significantly higher (P less than 0.053) than those from the primary tumor (0.076%) or those that were derived from the ascitic fluid (0.082%). Colonies from the metastatic tissues could be evaluated 2-4 days earlier than those from primary tumors. These discrepancies may be due to a heterogeneity in the clonable tumor cell compartment of primary tumor and metastasis. The antiproliferative properties of the antiestrogen tamoxifen and the progestin gestoneron were studied. In 9/12 cases a significant, dose-dependent reduction of colony formation (greater than 70-90% of the controls) was observed after continuous exposure to 1 mumole tamoxifen. No correlation between the dose response and the content of steroid receptors was found. Even estrogen receptor negative tumor samples showed a maximal antiproliferative effect of tamoxifen.

In vitro responsiveness of ovarian epithelial carcinomas to endocrine therapy.

As previously reported, ovarian epithelial carcinomas may respond to endocrine therapy. We examined the direct effect of progesterone, medroxyprogesteroneacetate, gestoneron, 17-beta-estradiol, tamoxifen, 4-OH-tamoxifen, or N-desmethyltamoxifen on the proliferative capacity of ovarian carcinoma cells by means of the colony assay described by Hamburger and Salmon. The growth rate of 25 tested tumors (ascitic fluid, primary tumor, metastases) was 68%. The plating efficiency was 0.078%. Beside the drug testing estrogen and progesterone receptor levels were determined. The inhibition of colony survival was slightest with 17-beta-estradiol, more pronounced with medroxyprogesteroneacetate, gestoneron, N-desmethyltamoxifen, and progesterone, and greatest with 4-OH-tamoxifen and tamoxifen. Significant and dose-dependent inhibition of greater than 70% was observed with tamoxifen and 4-OH-tamoxifen in 80% of the tested tumors. There was no significant correlation between the in vitro responsiveness and the level of hormonal act not only via an estrogen receptor but also via an antiestrogen-binding site.