Molecular mechanisms of ovarian hyperstimulation syndrome: paracrine reduction of endothelial claudin 5 by hCG in vitro is associated with increased endothelial permeability.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation associated with severe vascular hyperpermeability. Primary co-cultures of human luteinized granulosa cells (LGCs) and human umbilical vein endothelial cells (HUVECs) were used as a model of steroidgenic/endothelial cell interaction in OHSS. METHODS: hCG and the vascular endothelial growth factor (VEGF) inhibitor, Flt-1Fc, were added to co-cultures of LGCs and HUVECs separated by a micropore membrane. Endothelial permeability to labeled bovine serum albumin was measured and the expression of the endothelial cell-specific adhesion protein claudin 5 was investigated using immunocytochemistry and western blotting. RESULTS: The addition of hCG increased HUVEC permeability in the presence of LGCs (P < 0.05). hCG increased VEGF concentrations in both chambers of the co-culture system (P < 0.05). The increased permeability in the presence of LGCs and hCG was inhibited when VEGF was blocked by Flt-1Fc (P < 0.05). Endothelial membrane claudin 5 protein was reduced in the presence of hCG and LGCs, as measured by immunocytochemistry (P < 0.05) and western blotting (P < 0.05) and this reduction was inhibited by Flt-1Fc. hCG had no direct effects on endothelial cell claudin 5. CONCLUSIONS: For OHSS, this novel paradigm suggests that hCG can increase endothelial permeability by up-regulating VEGF in LGCs which causes reduction in endothelial claudin 5 expression.

Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells.

This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin-angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.

Potential role of Renin-Angiotensin-system for tumor angiogenesis in receptor negative breast cancer.

OBJECTIVE: This study examined the potential role of Angiotensin II for the regulation of angiogenesis associated genes in receptor positive and negative human breast cancer. METHODS: Expression of different Renin-Angiotensin system (RAS) components in human breast cancer tissue was investigated using immunofluorescence, and in a receptor positive (MCF-7) and receptor negative (MDA-MB 468) breast cancer cell line by performing immunocytochemistry and RT-PCR. Both cell lines were stimulated with Angiotensin II and Angiotensin II receptor type 1 (At(1)R) blocker Candesartan, and gene expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 and 2 (Ang-1 and Ang-2), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2alpha (HIF-2alpha) were quantified by TaqMan-Real-Time PCR analysis. RESULTS: RAS components, Angiotensinogen, Renin, Angiotensin I-converting enzyme (ACE), and At(1)R and At(2)R were expressed in hormone-receptor negative and positive human breast cancer tissue as well as in MDA-MB 468 and in MCF-7 human breast cancer cells. In addition, we found expression of VEGF, Ang-1, TIMP-1, and HIF-2alpha in both cell lines. However, only in receptor negative MDA-MB 468 cells, did Angiotensin II significantly increase gene expression of VEGF, HIF-2alpha, and TIMP-1. This effect was completely inhibited by Candesartan. CONCLUSION: In conclusion, it is hypothesized that Angiotensin II may be involved in regulation of tumor angiogenesis especially in receptor negative breast cancer by regulation of angiogenesis associated genes via At(1)R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At(1)R in receptor negative human breast cancer cells and may lead to new therapeutical anticancer strategies based upon inhibition of At(1)R.

Regulation of tight junction proteins occludin and claudin 5 in the primate ovary during the ovulatory cycle and after inhibition of vascular endothelial growth factor.

Ovarian follicular and corpus luteum development, including angiogenesis, are characterized by cell-cell rearrangements that may require dynamic changes in cell-cell adhesion. The present study investigates the expression of tight junction proteins occludin and claudin 5 during follicular and luteal development in the primate ovary and after inhibition of vascular endothelial growth factor (VEGF) by VEGF trap treatment. Occludin was localized to the plasma membrane of granulosa cells. During follicular development occludin staining decreased significantly (P < 0.05) and disappeared completely by the ovulatory stage. After inhibition of VEGF, occludin staining was significantly (P < 0.05) higher in the granulosa of secondary and tertiary follicles compared with controls. Claudin 5 was exclusively localized to the theca vasculature. A significant (P < 0.05) increase in staining was detected from the pre-antral to the antral and ovulatory stage. However, dual staining with CD31 revealed that within the theca endothelium the amount of claudin 5 remained constant during follicular development. Treatment with VEGF trap throughout the follicular phase revealed a lack of claudin 5 staining in the theca interna but no difference was observed in the remaining theca externa vasculature. In the corpus luteum, claudin 5 was also localized in the vasculature. Treatment with VEGF trap in the mid-luteal phase resulted in a significant increase in staining (P < 0.05). These results led us to hypothesize that tight junctions are involved in regulation of follicular growth, antrum transition and follicular angiogenesis which is compromised by VEGF inhibition. VEGF may influence luteal vascular permeability by regulation of the endothelial specific tight junction protein claudin 5.

Anatomic relation between the rectus capitis posterior minor muscle and the dura mater.

STUDY DESIGN: Anatomic study of the suboccipital region, specifically the deep muscles of the suboccipital triangle, was performed in cadaveric specimens. OBJECTIVE: To observe and describe the relationship between the deep suboccipital musculature and the spinal dura. SUMMARY OF BACKGROUND DATA: A review of the literature revealed no reports describing a physical connection between suboccipital musculature and the spinal dura. METHODS: Dissections of the suboccipital region were performed in 10 embalmed and one fresh sagittally hemisected head and neck specimens. RESULTS: A connective tissue bridge between the rectus capitis posterior minor muscle and the dorsal spinal dura at the atlanto-occipital junction was observed in every specimen. The fibers of the connective tissue bridge were oriented primarily perpendicular to the dura. This arrangement of fibers appears to resist movement of the dura toward the spinal cord. CONCLUSIONS: Awareness of the physical relation between the rectus capitis posterior minor muscle and spinal dura via this connective tissue bridge should lessen the potential risk of dural damage during surgery. This connective tissue bridge may help resist dural infolding during head and neck extension.

Anatomical observation of a craniomandibular muscle originating from the skull base: the sphenomandibularis.

This study describes a hitherto unreported, functionally distinct craniomandibular muscle as observed in 25 cadaveric specimens and MRI scans of clinical patients. The muscle was consistently observed as originating from the maxillary surface of the sphenoid bone and inserting on the temporal crest (internal oblique line) of the mandible. This anatomic observation should broaden our understanding of craniomandibular function and may have clinical implications regarding retro-orbital pain.

[What does the anesthetist expect from the obstetrician?]. / Was erwartet der Anästhesist vom Geburtshelfer?

The anaesthetist would ideally like the following to be observed in a timely order in the antenatal phase: 1. early provision of information to the patient regarding epidural catheter for pain relief in labour, 2. informing the anaesthetist about relevant points in the patient history and examination findings, 3. knowledge of the contraindications to lumbar regional anaesthesia, 4. clear information as to the urgency of a caesarian section, 5. provision of aspiration prophylaxis. In the intrapartum phase it is necessary through clear communication to agree early on: 1. monitoring instructions for epidurals in the delivery room, 2. instructions for epidural top ups, 3. whether the presence of relatives in theatre in the case of a caesarian section is appropriate. In the postnatal phase anaesthetists expect to be actively involved in the resuscitation of newborns to fulfill educational requirements, to have clear decisions about responsibilities for monitoring of patients post caesarian sections and detailed information about early symptoms of possible complications of lumbar regional anaesthesia.

[Suspension cultures of Ehrlich ascites tumor cells as a test system for the study of cytotoxic effects of anaesthetics (author's transl)]. / Suspensionskulturen von Ehrlich-Ascitestumorzellen als Arbeits-Modell zum Nachweis zelltoxischer Anaesthetikawirkungen.

Cultures of a permanently in suspension growing line of Ehrlich ascites tumor cells (EATC) were studied regarding their use as a test system for the determination of cytotoxic effects of anaesthetics. These cells, having a comparatively high reduplication rate, are cultured at 37 degrees C in vitro in a chemically defined liquid medium supplemented by 15% horse serum without agitation in a closed system. In order to detect drug effects on the cultures various cellular parameters can be determined due to the suspension character of the cultures without complicated preparatory measures, e.g. cell number or multiplication rate, cell volume, cell volume distribution. Moreover biochemical parameters, such as protein or nucleic acid content, may be estimated after centrifugation of the cultures separately in the cell sediment and the medium supernatant. Applying drugs of various pharmacological groups (cytostatic and anti-inflammatory drugs, local and general anaesthetics) the usefulness of some of these parameters for the detection of cytotoxic effects was studied.

[Artificial respiration for the postoperative care after major surgery (author's transl)]. / Respiratorbehandlung als Beginn der postoperativen Rehabilitation nach grossen chirurgischen Eingriffen

An analysis was made of the postoperative course of 158 cases of major surgery performed during 1971-1973. The operations comprised thoraco-abdominal resection of the oesophageal and cardia, abdominal duodenopancreatectomy, partial gastrectomy and enterectomy and partial hepatectomy. 63 per cent of the patients were given artificial respiration either prophylactically immediately after the operation (47 per cent) or at a later stage (53 per cent) on account of respiratory complications (arterial hypoxia below 55 mm Hg pO2). Postoperative mortality for the group of patients who did not have artificial respiration was 46 per cent, as compared with 32 per cent for the artifically ventilated patients - a difference of 14 per cent. The causes for this discrepancy, and especially the role of non-respiratory complications are discussed and the indications for prophylactic postoperative artificial respiration are reviewed.

[Anesthesiologic peculiarities in bilateral pheochromocytoma and suspected multiple endocrine neoplasia]. / Anästhesiologische Besonderheiten bei beidseitigem Phäochromozytom und Verdacht auf multiple endokrine Neoplasie.

Based on a case report with surgical removal of bilateral giant phaeochromocytomas with heredofamilial affliction and suspicion of multiple endocrine neoplasia type II (MEN-II syndrome) and of malignancy, the anaesthetic implications are discussed in a patient inadvertently not prepared by antiadrenergic treatment. It is pointed out that in MEN II syndrome, a combination of phaeochromocytoma and medullary carcinoma of the thyroid, surgical removal of the phaeochromocytoma merits utmost priority before thyreoidectomy. The most important laboratory parameters in the diagnosis of MEN II syndrome are pre- and postoperative determinations of calcitonin and carcinoembryonal antigen (CEA).

[Renin-angiotensin system under extracorporeal circulation during heart valve surgery]. / Das Renin-Angiotensin-System unter extrakorporaler Zirkulation während Herzklappenoperationen.

Angiotensin I (A I), angiotensin II (A II) and the activity of angiotensin-converting enzyme (ACE) were measured in 15 patients undergoing cardiopulmonary bypass for mitral or aortic valve replacement. During cardiopulmonary bypass A I, A II, A I/II ratio and arteriovenous A II--difference decreased markedly, whereas the activity of ACE fell only during a small 15 min period after start of extracorporeal circulation. Possible reasons for these effects are discussed.

[Diagnostic and intensive care problems in hemolytic uremic syndrome]. / Diagnostische und intensivmedizinische Probleme beim Hämolytisch-Urämischen Syndrom (HUS).

We observed a young man with apparently septic appendicitis who finally could be diagnosed as suffering from haemolytic uraemic syndrome (HUS) when spuriously normalising platelet counts were identified as a cell counter artifact. This case stresses the fact that automatically counted abnormal thrombocyte numbers should be examined by microscopy. Therapy is empirically based on corticosteroids, plasmapheresis and fresh frozen plasma. The application of thrombocyte concentrates should be avoided because of the risk of aggravating the disease process.

[Gas chromatographic determination of the solubility coefficient for volatile anaesthetics (author's transl)]. / Gaschromatographische Bestimmung des Verteilungskoeffizienten für volatile Anaesthetika.

For the determination of the solubility coefficient of volatile anaesthetics in Schindler's liquid culture, modified by Karzel, a gas-chromatographic analysis with direct-injection-method was used. A well defined volume with a known concentration of the anaesthetic was added to a vessel with known volume. After equilibration the concentration of the inhalational anaesthetics was determined in the liquid at various temperatures. The solubility coefficient and the temperature coefficient were then calculated from these data. We obtained the following solubility coefficients at -4 degrees C (37 degrees): for halothane 1,07 (0,77), for enflurane 1,14 (0,70), for methoxyflurane 4,92 (3,23) and for isoflurane 1,13 (0,82). In Schindler's liquid culture modified by Karzel the values at 24 degrees (37 degrees) were: for halothane 1,33 (0,92), for enflurane 1,32 (0,87), for methoxyflurane 6,61 (4,48) and for isoflurane 1,19 (0,98). The temperature coefficient for this temperature range were in water (in Schindler's liquid culture) for halothane: -2,31 . 10(-2) K(-1) (-3,15 . 10(-2) K(-1)), for methoxyflurane: -13,0 . 10(-2) K(-1) (-16,48 . 10(-2) K(-1)), for enflurane: -3,38 . 10(-2) K(-1) (-3,46 . 10(-2) K(-1)) and for isoflurane: -2,38 . 10(-2) K(-1) (-1,62 . 10(-2) K(-1)).

[Anesthesia in aortocoronary bypass surgery: comparison of effects on hemodynamics of recent technics of total intravenous anesthesia]. / Zur Anästhesie bei aortokoronaren Bypassoperationen: Vergleich der hämodynamischen Einflüsse neuerer Techniken der totalen intravenösen Anästhesie.

20 patients electively scheduled for operation were enrolled in the study and anaesthetized randomly with one of the two techniques etomidate + fentanyl (I) or flunitrazepam + fentanyl (II). Haemodynamics were assessed by continuously measuring blood pressure in the systemic and pulmonary circulations, as well as the heart rate, and intermittently determining the cardiac output. It could be shown that systemic arterial blood pressure, in spite of an initial drop after induction of anaesthesia, remained within an acceptable safety margin throughout the investigation period. Whereas heart rate remained almost constant in both groups, the rate-pressure product which may serve as a measure of myocardial oxygen consumption in the clinical setting, decreased considerably in both groups, although more appreciably under the influence of the anaesthetic technique flunitrazepam + fentanyl. Although the diastolic blood pressure as the most significant individual factor determining coronary blood flow was not changed appreciably in both groups, it remained at a higher level under the influence of the anaesthetic technique etomidate + fentanyl (I). We conclude that both anaesthetic regimes described here may be safely applied for the anaesthesia of patients with reduced coronary perfusion and cardio-haemodynamic reserve potential.

[Hemodynamic effects of an etomidate-flunitrazepam or midazolam- fentanyl combination for induction of anesthesia in patients with heart valve diseases]. / Hämodynamische Effekte einer Kombination von Etomidat, Flunitrazepam oder Midazolam mit Fentanyl zur Anästhesie-Einleitung bei Patienten mit Herzklappenvitien.

A comparison of the haemodynamic effects of 3 anaesthetic techniques, the combinations etomidate + fentanyl (I), flunitrazepam + fentanyl (II) or midazolam + fentanyl (III), respectively was carried out in 45 patients undergoing various types of cardiac valve replacement surgery. Haemodynamics were assessed by continuously measuring the heart rate as well as the blood pressure in the systemic and pulmonary circulations whereas the cardiac output was measured intermittently. In the first 30 minutes after induction of anaesthesia, a mean arterial blood pressure drop of 10% (I), 20% (II) or 15% (III) respectively, was observed; at the same time, the rate did not change significantly. Cardiac index, however, fell significantly in all 3 groups by 33% (I) 30% (II) or 28% (III), respectively. Pulmonary pressure, wedge pressure and systemic vascular resistance rose only in groups I and III and decreased in group II (flunitrazepam + fentanyl). On the other hand, pulmonary vascular resistance as well as left ventricular work index were significantly decreased in all 3 groups. We conclude that all 3 anaesthetic techniques investigated here may be effectively applied for safe induction of anaesthesia in patients with valvular lesions of the heart. On account of the effect of the combination flunitrazepam + fentanyl on decreasing pulmonary artery pressure and wedge pressure, this technique seems to be preferable in patients with pulmonary hypertension.

[In vitro studies on the influence of volatile anaesthetics on cell number, cell volume, and cellular protein, DNA, and RNA content (author's transl)]. / In Vitro-Untersuchungen zum Einfluss von volatilen Anaesthetika auf Zellvermehrung, Zellvolumen sowie zellulären Protein- und Nucleinsäurestoffwechsel.

The aim of the present investigations was to see, if halothane (h), enflurane (e), isoflurane (i), and methoxyflurane (m) exert cytostatic or cytotoxic effects. The experiments were performed on suspension cultures of an established line of Ehrlich ascites tumor cells, which were gased by a mixture of N2 (78%), O2 (20%), and CO2 (2%) to which the volatile anaesthetics in 5 different concentrations were added by vaporizers. Under standardized conditions (incubation time: 24 or 48 hrs; initial cell density: 2 X 10(5) cells/ml culture medium) the following results were obtained: 1. All anaesthetics exerted dose dependent cytostatic effects, which were reversible up to concentrations of 2.0 vol% (m), 3.0 vol% (h), and 5.0 vol% (e, i). The cytostatic ID50 values (mmol/l) were found to be 0.50 (h), 1.00 (e), 0.72 (i), 1.25 (m). 2. In cell cultures, which were exposed to anaesthetics for 48 hrs cytotoxic effects were observed after doses of 2.0 vol% (m), 3.0 and 4.0 vol% (h), 4.0 and 5.0 vol% (e and i). 3. In contrast to colchicine, which was studied in comparison to the volatile anaesthetics, the mean cell volume of anaesthetic treated cultures showed only minimal deviations from control values. 4. With exception of m, which produced a significant decrease of the cellular protein content in the dose range 1.5-2.0 vol% and of the cellular DNA content in all concentrations applied the 3 other anaesthetics caused an increase of the cellular protein content and a somewhat smaller increase of the cellular nucleic acids content. The obtained results indicate that the effect of anaesthetic agents on dividing cells is not due to the same mode or site of action, but to an influence on different stage of the cell cycle, particularly the interphase. Colchicine-like c-mitosis were not obtained.

[The course of the catecholamine excretion during combination anaesthesia with halothane and thalamonal (author's transl)]. / Verhalten der Harnkatecholamine bei der Kombinationsnarkose mit Halothan-Thalamonal

On 10 patients who had to undergo a ca. 4 hour operation of the lower abdominal region, the pattern of catecholamine excretion before, during and after operation was traced. 1. A decrease of systolic blood pressure on average of 80 mm Hg, in correlation to the concentration of Halothane and Thalamonal, was recorded. 2. The excretion of adrenaline and noradrenalin was significantly lower during anaesthesia as compared with the initial value, suggesting a depression of sympathoadrenal system. 3. The postoperative amount of adrenaline and especially noradrenaline increased markedly, when anaesthesia worn off, postoperative shivering started, and surgical wounds caused pain. 4. The excretion of urine during operation was slightly reduced, the renal output showed normal amounts, when calculated up to 24 hours. The results show, that the combined use of halothane an thalamonal because of its depressant effects on the sympathoadrenal system is capable of reducing the liberation of catecholamines during anaesthesia.