Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells.

1. In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock. 2. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques. 3. In vascular SMC exposed to 1 micrograms ml-1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]-angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]-vasopressin binding was observed. 4. The pharmacological characterization of angiotensin II binding sites in control and LPS-exposed cells demonstrated that LPS induced an increase in the AT1 subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS. 5. The effect of LPS on the angiotensin II receptor was dose-, time- and protein-synthesis dependent and was associated with an increased expression of the receptor gene. 6. The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO-synthase. 7. These results suggest that, besides inducing factors such as cytokines and NO-synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non-specific LPS-related induction of genes.

Expression of smoothelin and smooth muscle actin in the skin.

INTRODUCTION: Smoothelin is a cytoskeletal protein of differentiated smooth muscle cells with contractile capacity, distinguishing it from other smooth muscle proteins, such as smooth muscle actin (SMA). OBJECTIVE: To evaluate the expression of smoothelin and SMA in the skin in order to establish specific localizations of smoothelin in smooth muscle cells with high contractile capacity and in the epithelial component of cutaneous adnexal structures. Methods: Immunohistochemical analysis (smoothelin and SMA) was performed in 18 patients with normal skin. RESULTS: SMA was expressed by the vascular structures of superficial, deep, intermediate and adventitial plexuses, whereas smoothelin was specifically expressed in the cytoplasm of smooth muscle cells of the deepest vascular plexus and in no other plexus of the dermis. The hair erector muscle showed intense expression of smoothelin and SMA. Cells with nuclear expression of smoothelin and cytoplasmic expression of SMA were observed in the outer root sheath of the inferior portion of the hair follicles and intense cytoplasmic expression in cells of the dermal sheath to SMA. CONCLUSIONS: We report the first study of smoothelin expression in normal skin, which differentiates the superficial vascular plexus from the deep. The deep plexus comprises vessels with high contractile capacity, which is important for understanding dermal hemodynamics in normal skin and pathological processes. We suggest that the function of smoothelin in the outer root sheath may be to enhance the function of SMA, which has been related to mechanical stress. Smoothelin has not been studied in cutaneous pathology; however we believe it may be a marker specific for the diagnosis of leiomyomas and leiomyosarcomas of the skin. Also, smoothelin could differentiate arteriovenous malformations of cavernous hemangioma of the skin

KAM theory for conformally symplectic systems

We present a KAM theory for some dissipative systems (geometrically, these are conformally symplectic systems, i.e. systems that transform a symplectic form into a multiple of itself). For systems with n degrees of freedom depending on n parameters we show that it is possible to find solutions with n-dimensional (Diophantine) frequencies by adjusting the parameters. We do not assume that the system is close to integrable, but we use an a-posteriori format. Our unknowns are a parameterization of the solution and a parameter. We show that if there is a sufficiently approximate solution of the invariance equation, which also satisfies some explicit non–degeneracy conditions, then there is a true solution nearby. We present results both in Sobolev norms and in analytic norms. The a–posteriori format has several consequences: A) smooth dependence on the parameters, including the singular limit of zero dissipation; B) estimates on the measure of parameters covered by quasi–periodic solutions; C) convergence of perturbative expansions in analytic systems; D) bootstrap of regularity (i.e., that all tori which are smooth enough are analytic if the map is analytic); E) a numerically efficient criterion for the break–down of the quasi–periodic solutions. The proof is based on an iterative quadratically convergent method and on suitable estimates on the (analytical and Sobolev) norms of the approximate solution. The iterative step takes advantage of some geometric identities, which give a very useful coordinate system in the neighborhood of invariant (or approximately invariant) tori. This system of coordinates has several other uses: A) it shows that for dissipative conformally symplectic systems the quasi–periodic solutions are attractors, B) it leads to efficient algorithms, which have been implemented elsewhere. Details of the proof are given mainly for maps, but we also explain the slight modifications needed for flows and we devote the appendix to present explicit algorithms for flows.

Is ENP good news for the transfer of environmental norms? Incentives and socialization for Eastern neighbours

The launching of the European Neighbourhood Policy has created some expectations. Cooperation between the EU and its partners is expected to get deeper, to the point that neighbouring countries have been promised to share “everything but institutions” with the EU. Moreover, cooperation is also expected to be broader, as it has been presented as including more and more issue areas. In other words, the ENP has the vocation of being a universal instrument to promote the transfer of EU norms. This paper focuses on one single issue area, the environment, and one group of ENP partners, the Western Newly Independent States and the South Caucasus, to revise to what extent neighbourhood policy can provide the mechanisms to encourage rule transfer. Are incentives and disincentives powerful enough? Can the ENP promote the socialization of neighbours into EU environmental norms?

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Expectativas, actitudes y normas percibidas respecto al uso del preservativo por los jóvenes de Mozambique = Expectations, attitudes and perceived norms regarding condom use in Mozambique young people

Objetivos: Conocer la prevalencia del uso auto informado del preservativo en la última relación sexual, así como algunas actitudes, creencias y percepciones sobre su uso para la prevención de la transmisión del VIH por vía heterosexual, en jóvenes de las ciudades de Nampula, Bemba y Lichinga, en Mozambique. Diseño: Estudio transversal de encuesta. Participantes: Seiscientos treinta y dos estudiantes de secundaria (56,8% varones), con edades comprendidas entre 15 y 24 años, seleccionados mediante un muestreo aleatorio estratificado proporcional. El porcentaje de participación es del 79%. Mediciones principales: Mediante un cuestionario, se evalúan en una escala de 0 a 10 las actitudes hacia el uso del preservativo, expectativas de resultados y de autoeficacia y aceptación percibida sobre su utilización. Asimismo se evalúa el uso auto informado del preservativo en la última relación sexual. Resultados: Sólo un 47,4% (IC 0,95 = 42,0:52,8) de los jóvenes sexualmente activos utilizó el preservativo en la última relación sexual. En general las mujeres tienen una actitud más favorable respecto a su uso y creen más en su eficacia que los hombres, pero lo utilizan menos y se sienten poco capaces de pedir su uso al varón, principalmente si se trata de su actual pareja. La autoeficacia percibida para utilizar o pedir el uso del preservativo aumenta en el caso de una pareja ocasional tanto en hombres como en mujeres. Conclusiones: Se requieren acciones institucionales y sociales que promocionen el uso del preservativo entre los jóvenes de Mozambique. Los programas preventivos deberían insistir en el cambio de actitudes y creencias, y enfatizar los beneficios que se derivan de su uso adecuado y sistemático desde la primera relación coital

Developing Smooth, Quiet, Safe Portland Cement Concrete Pavements, March 2004

The concrete paving industry has spent large amounts of time working to provide safe, quiet, and smooth pavements for the traveling public as their needs and driving habits have changed since the advent of the automobile. During that time, the efforts of research, design, and construction were directed at one of the problems at a time. Current public surveys indicate that the traveling public wishes to have safe, quiet, and smooth pavements. This report identifies the problems remaining in the areas of developing smooth, quiet, and safe portland cement concrete pavement in each pavement we build. It develops the research framework that can be used to bring the existing information together with additional research in each area. The resulting answers can be used in each pavement design for a quiet, safe, and smooth pavement that is also long lasting.

Upregulation of vasopressin V1A receptor mRNA and protein in vascular smooth muscle cells following cyclosporin A treatment.

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

Focused time-lapse inversion of radio and audio magnetotelluric data

Geoelectrical techniques are widely used to monitor groundwater processes, while surprisingly few studies have considered audio (AMT) and radio (RMT) magnetotellurics for such purposes. In this numerical investigation, we analyze to what extent inversion results based on AMT and RMT monitoring data can be improved by (1) time-lapse difference inversion; (2) incorporation of statistical information about the expected model update (i.e., the model regularization is based on a geostatistical model); (3) using alternative model norms to quantify temporal changes (i.e., approximations of l(1) and Cauchy norms using iteratively reweighted least-squares), (4) constraining model updates to predefined ranges (i.e., using Lagrange Multipliers to only allow either increases or decreases of electrical resistivity with respect to background conditions). To do so, we consider a simple illustrative model and a more realistic test case related to seawater intrusion. The results are encouraging and show significant improvements when using time-lapse difference inversion with non l(2) model norms. Artifacts that may arise when imposing compactness of regions with temporal changes can be suppressed through inequality constraints to yield models without oscillations outside the true region of temporal changes. Based on these results, we recommend approximate l(1)-norm solutions as they can resolve both sharp and smooth interfaces within the same model. (C) 2012 Elsevier B.V. All rights reserved.

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being ~0.35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Strong smooth muscle differentiation is dependent on myocardin gene amplification in most human retroperitoneal leiomyosarcomas.

Myocardin (MYOCD), a serum response factor (SRF) transcriptional cofactor, is essential for cardiac and smooth muscle development and differentiation. We show here by array-based comparative genomic hybridization, fluorescence in situ hybridization, and expression analysis approaches that MYOCD gene is highly amplified and overexpressed in human retroperitoneal leiomyosarcomas (LMS), a very aggressive well-differentiated tumor. MYOCD inactivation by shRNA in a human LMS cell line with MYOCD locus amplification leads to a dramatic decrease of smooth muscle differentiation and strongly reduces cell migration. Moreover, forced MYOCD expression in three undifferentiated sarcoma cell lines and in one liposarcoma cell line confers a strong smooth muscle differentiation phenotype and increased migration abilities. Collectively, these results show that human retroperitoneal LMS differentiation is dependent on MYOCD amplification/overexpression, suggesting that in these well-differentiated LMS, differentiation could be a consequence of an acquired genomic alteration. In this hypothesis, these tumors would not necessarily derive from cells initially committed to smooth muscle differentiation. These data also provide new insights on the cellular origin of these sarcomas and on the complex connections between oncogenesis and differentiation in mesenchymal tumors.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.