Evaluation of smooth muscle and collagen subtypes in normal newborns and those with bladder exstrophy

PURPOSE: Many patients who undergo bladder exstrophy closure as newborns, subsequent epispadias repair and later bladder neck reconstruction become completely continent yet complications can occur. After successful initial exstrophy closure and later epispadias repair some patients may fail to gain sufficient capacity for bladder neck reconstruction or satisfactory capacity and continence after bladder neck reconstruction. In an attempt to understand the pathogenesis of these failures we compared bladder biopsies from normal neonates and those with exstrophy. MATERIALS AND METHODS: Bladder biopsies obtained from the midline of the bladder wall just above the base of the trigone from 12 newborns with exstrophy were compared to bladder sections from 9 neonatal cadavers. All bladder specimens were stained with monoclonal antibodies against type I, III or IV collagen and a subset was further stained with Masson's trichrome to define the extracellular matrix. All specimens were then analyzed using a color digital image analysis system. RESULTS: At initial examination of the extracellular matrix there was an increase in the collagen-to-smooth muscle ratio from 0.38 in controls to 1.2 in newborns with exstrophy, comprising an increase in collagen and decrease in smooth muscle. The collagen component of the extracellular matrix was then further defined to quantitate the amount of each collagen type (I, III and IV) deposited. We then evaluated the ratio of collagen type-to-total collagen sampled. Compared to control bladders there was no statistical difference in the amount of type I or IV in the bladders of newborns with exstrophy at initial closure. However, there was a 3-fold increase in type III collagen (0.14 +/- 0.05 to 0.46 +/- 0.2%, p < 0.001) in the bladders of neonatal controls versus newborns with exstrophy. CONCLUSIONS: This alteration in collagen makeup may represent an earlier developmental stage of the exstrophy bladder at birth, which then remodels and changes after successful initial closure. Further studies are underway to examine the collagen composition of bladders at bladder neck reconstruction, failed closures and augmentation.     

Purinergic modulation of rat urinary bladder detrusor smooth muscle

This paper reviews the use of economic evaluations in the Swedish health care system. The most important actors are defined and examples are given how economic evaluations have played a role in the decision making process. The introduction of extracorporal shock-wave lithotripsy (ESWL) is used as an example on how economic evaluation was used for recommendations to the county councils to adopt this technology. Mammography is used as an example of how the evaluation is used in the political process following an initiative in the parliament. A study of the cost-effectiveness of hypertension treatment illustrates how economic evaluations are included as part of a medical technology assessment by SBU, the Swedish Council on Technology Assessment in Health Care. The role of economic evaluations for drug reimbursement and pricing is also reviewed. The main conclusions are that economic evaluations are one of several factors influencing a decision making process that have a strong strive for consensus. It is thus difficult to make a definitive statement of the contribution of such study to the outcome of the decision making process, and there is no evidence that the evaluation was the decisive factor. However, a number of changes in the way resources are allocated in the Swedish health care system speaks for an increasing role for such studies in the future. The county councils are identified as the main target for economic evaluations, and SBU has a key role in supplying the county councils with high quality assessment of new and old technologies.     

Identification of myoglobin in human smooth muscle

Myoglobin (Mb) has been believed to be absent generally from mammalian smooth muscle tissue. Examination of human rectal, uterine, bladder, colon, small intestine, arterial, and venous smooth muscle by immunohistochemical techniques shows that each of these tissues is immunopositive for both smooth muscle myosin and human Mb. Mb-specific primers were used for the polymerase chain reaction to generate cDNA from smooth muscle tissues. Southern hybridization with a Mb-specific probe gave a very strong signal with the cDNA from rectum, weaker signals from small intestine and uterus, a faint signal from colon, and no signal from bladder tissue. High performance liquid chromatography analysis coupled with sequence determination has shown that contaminating heme-binding serum albumin as well as hemoglobin in extracts of smooth muscle seriously compromise any heme-based or spectrophotometric assay of Mb. Combined affinity and size exclusion chromatography, however, provide the necessary resolution. The cDNA-derived amino acid sequence of human smooth muscle Mb was found to be identical to that of Mb from striated muscle.     

Temporal and spatial expression of distinct troponin T genes in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only < 60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.     

Cytoskeletal and cytocontractile protein composition of smooth muscle cells in developing and obstructed rabbit bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, alpha-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM alpha-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

A comparison of the beta-blocking activities of practolol, sotalol, and propranolol in human and guinea pig tracheal smooth muscle

Beta adrenergic blockade was studied in vitro with human tracheal muscle strips and guinea pig tracheal chains. It was shown in isolated smooth muscle from both man and guinea pig that the order of potency for the three beta-blocking agents studied was: propranolol greater than sotalol greater than practolol. Under the conditions of this study, propranolol was about 30,000 times and sotalol about 30 times as potent as practolol. The order of potency suggests that the nature of adrenergic blockade induced by practolol on tracheal smooth muscle is only weakly beta2-relative to the blocking effects of propranolol and sotalol. Beta adrenergic blockade by propranolol, sotalol, and practolol produced different degrees of increased histamine lethality in mice. Whereas both propranolol at 0.01 mg/kg and sotalol at 1.0 mg/kg resulted in 100% histamine-induced lethality, practolol at 50 mg/kg resulted in only 50% histamine-induced lethality. These data, when added to those from our previous studies, suggest that the mechanisms responsible for resistance to the effects of histamine in untreated mice are at least partially mediated by the beta2-adrenergic system. Thus, in three different tissues, the blocking activity of practolol was shown to be less than that of sotalol or propranolol.     

Phenotypic changes in the regenerating rabbit bladder muscle. Role of interstitial cells and innervation on smooth muscle cell differentiation

It was not Julius Caesar who was born by Caesarean section, as generally assumed, but Scipio Cornelius Africanus, who subdued Spain 100 years before Caesar's time. In chambers with walls of porphyrite, the Byzantine empresses used to give birth to the heirs to the throne. In England, the infertility of Queen Anne, who suffered from porphyria, led to the succession of the Protestant House of Hannover following the Catholic Stuarts. Christina of Sweden, called 'queen of baroque, rebel and scholar', was born in the 'caul'. At the age of 39 years, Johanna of Pfirt, married to Albrecht the Lame, secured the continuation of the Habsburg dynasty by giving birth to Rudolf the Founder. Maria Theresia, who had 16 children, was called 'mother-in-law of Europe'. She was delivered of her first child at the age of 19. The death of her sister Maria-Anna in childbed was one of the reasons why Gerard van Swieten was called to Vienna. Elisabeth of Württemberg, first wife of Franz I of Austria, died, not as a consequence of. but after a forceps operation carried out by Johann Lukas Bo?r. In England, Princess Charlotte, daughter of George IV, and her baby son died at the delivery; Sir Richard Croft, who had not used the forceps, committed suicide after this tragic incident. Being the next in succession, Victoria ascended the throne. The term 'narcose au chloroforme' (first used by James Young Simpson) was changed to 'narcose à la reine' after this method had been used at the birth of Victoria's eighth child by John Snow. It was Queen Victoria, who passed on haemophilia in European dynasties. When Marie Louise of Habsburg had her first child, Napoleon's son, the later Duke of Reichstadt, Antoine Dubois had to perform a turning of the transverse presentation and use the forceps on the head following after. The birth of Napoleon himself was a case of precipitate labour. Johann Klein, the successor of Bo?r, applied the forceps when Archduchess Sophie was delivered of her first child, the later Emperor Franz Joseph I of Austria, the first of the four 'salt princes'. The later Emperor Wilhelm II of Prussia was delivered by Eduard Arnold Martin the Elder, the obstetrician of Princess Victoria, the eldest daughter of Queen Victoria; the breech presentation became even more complicated by the raised arms of the child. Both latter monarchs had been 'asphyctic' after their birth. Johann Wolfgang von Goethe was also among those who were apparently dead after their birth.     

Arachidonic acid-induced calcium signalling in human airway smooth muscle cells

Until now computer-assisted parasite identification was based on database applications requiring data specification on an individual basis, thus limiting the ability of the system to handle rule-based knowledge as humans are used to do. A new Expert PArasite IdentificatiON (EPAION: Greek term for expert) system was developed to serve as an interface between the database and the user, where the database is a repository for bionomic and morphological facts about the parasites for the expert system. The system was developed by using a logic-based computer language which allows the definition of rules and facts to assist the creation of queries to the database. The components of the system are the knowledge base, the multimedia data base, the inference mechanism, and the graphical user interface. The operational modules of the system are the Parasite Identifier and the system Utilities. This expert system facilitates knowledge incorporation in a manner simulating the natural mental process, thus allowing the checking of the accuracy of the information that the user feeds to the computer and the creation of intelligent queries to the database. These characteristics accelerate focusing and optimize the parasite identification scheme regardless of the user's profile of competency.     

Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells

OBJECTIVE: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. METHODS: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. RESULTS: Thrombin (5 nmol/l), thrombin receptor activating protein (3 mumol/l) and low density lipoprotein (33 nmol/l) induce a 652 +/- 80%, 593 +/- 80% and a 316 +/- 60% increase in [3H]thymidine incorporation into DNA (mean +/- SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245 +/- 160% or 1200 +/- 40% increase of DNA synthesis (mean +/- SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22 +/- 8.4% and a 29% +/- 6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63 +/- 14% increase in cell number (mean +/- SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538 +/- 54 to 923 +/- 75 nmol/l (mean +/- SD, n = 4). CONCLUSION: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.     

Ion channels in freshly isolated and cultured human bronchial smooth muscle cells

Voltage-gated ion currents were studied in human bronchial airway smooth muscle (ASM) cells. Proliferating or growth-arrested cells in culture were compared with freshly isolated cells. Three types of charybdotoxin (ChTX)-sensitive K+ channel were observed in all cell types, with conductances in symmetrical 140 mM KCl solutions ([Ca2+]i < 0.1 nM) of 206 +/- 14 pS (n = 32), 144 +/- 11 pS (n = 27) and 109 +/- 5 pS (n = 25). The relative proportion of each channel type differed substantially between the three groups of cells. In freshly isolated ASM cells large conductance K+ channels were represented almost entirely by a conductance of 206 pS, which was found in all twenty-three patches studied. In contrast, in most patches from proliferating cells the majority of channels had conductances of either 144 pS (14 of 21 patches) or 109 pS (8 of 21 patches). Cultured cells that had been growth arrested by serum depletion revealed the same set of channels as the proliferating cells, but the occurrence of the 109 pS channel was much more frequent (16 of 19 patches). As has been shown previously, 206 pS channels were active at a physiological membrane potential (-60 to -20 mV) even at a very low free [Ca2+]. The 144 pS channels could be recorded only at depolarized potentials (+80 to +100 mV), whereas 109 pS channels were active over a wide range of potentials (-60 to +100 mV), but only in the presence of GTP. In a proportion of cultured cells a tetrodotoxin-sensitive Na+ current and a hyperpolarization-induced inwardly rectifying K+ current were also observed (15 and 21%, respectively, of all cells examined). Neither of these currents were observed in freshly isolated cells. Whole-cell outward current in all groups was sensitive to tetraethylammonium, ChTX, and iberiotoxin, but not to 4-aminopyridine. In summary, it is clear that during proliferation there are considerable changes in the expression of ionic channels in ASM that have profound functional significance. In particular, these changes would tend to make the tissue more excitable, and may be of relevance to the proliferative process itself.     

Quantitative analysis of smooth muscle fibers in corpus cavernosum of human fetuses

PURPOSE: Quantify objectively the normative distribution and the percentage of smooth muscle fibers in the corpus cavernosum of human fetuses with 24 weeks post-conception (WPC) of gestational age. MATERIAL AND METHODS: We studied 7 penises taken from 7 fresh human fetuses. We analyzed 5 randomized sections from each penis and in every section we analyzed 3 fields, totaling 15 fields per penis and 105 fields for the final results. Immunohistological staining for the smooth muscle fibers was used to accentuate the differences between the intracavernous structures (smooth muscle fibers and collagen fibers). The fields studied were digitized with a final magnification of 450X and a computerized analysis of the smooth muscle fibers was performed with image analyzer software. The percentage of smooth muscle fibers per standard square area was estimated and the mean value was used for each penis. RESULTS: The distribution of smooth muscle fibers in the corpus cavernosum of human fetuses with 24 WPC of gestational age ranged from 17.52% to 27.76% of the total area. The mean value was 22.72% and the standard deviation was 3.56. CONCLUSIONS: Our results show that the percentage of smooth muscle cells in corpus cavernosum of human fetuses with 24 WPC of gestational age is significantly smaller when compared with the data available for adult cadavers.     

Oxytocin analogues with combined high smooth muscle and negligible antidiuretic activities. Investigation of position 7 in neurohypophyseal hormones

The proline residue in position 7 of oxytocin occupies one of the four corner positions in the two beta turns proposed for the preferred conformation of the pituitary hormone. It has been suggested that synthetic modifications of the residues in these corner positions will yield analogues in which one or more of the biological activities of the parent hormone is highly accentuated in terms of potency relative to other activities. In a continued effort to test this hypothesis the following analogues of oxytocin were prepared: [7-glycine]oxytocin, [1-beta-mercaptopropionic acid,7-glycine]oxytocin, [7-alanine]oxytocin, and [1-beta-mercaptopropionic acid,7-alanine]oxytocin. These peptides were found to possess the following specific activities, respectively: rat uterotonic, 65 +/- 2, 355 +/- 3, 22 +/- 1, 123 +/- 4; avian vasodepressor, 5.3 +/- 0.8, 17 +/- 0.4, 4.8 +/- 0.1, 9.8 +/- 0.5; rat antidiuretic, less than0.01, 0.062, 0.081 +/- 0.01, 0.17 +/- 0.01; rat pressor, 0.3, 0.5, 0.4, 0.5 unit/mg. Thus the analogues retain high uterotonic activity but exhibit strongly diminished renal and vascular activities relative to oxytocin. Especially noteworthy is [1-beta-mercaptopropionic acid,7-glycine]oxytocin with its high uterotonic activity but very low antidiuretic and pressor activities. The activity profile of this analogue combined with the fact that it is only slowly enzymatically degraded warrants further investigations of this peptide for clinical applications.     

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.     

Degeneration and regeneration of striated skeletal muscle fibers in Duchenne muscular dystrophy

Like all other muscular dystrophies, Duchenne muscular dystrophy is characterized by the coexistence of degenerative lesions of the muscle fibers and of regenerative changes. The present study has been carried out in order to precise the degree of regeneration at different stages of the disease, by analyzing the expression of several markers of cell proliferation and of muscular differentiation. In the two affected foetuses of our series, the m. quadriceps is histologically normal, except for the absent expression of immunoreactive dystrophin. The quadriceps from the eight children of our series (20 months-16 years) all present clear dystrophic changes. Muscle regeneration is characterized by activation of the satellite cells, by their multiplication followed by their fusion giving birth to regenerative fibers. By studying the expression of muscular markers (vimentin, desmin, isoforms of the myosin heavy chains), it has been possible to define more precisely the degree of maturation and of differentiation of these regenerative fibers. Our results suggest that an abortive regeneration of the muscle fibers in Duchenne muscular dystrophy can explain, at least partly, the progressive evolution of this disease.     

Expression of cyclo-oxygenase-2 in human airway smooth muscle is associated with profound reductions in cell growth

1. It is now accepted that uncontrolled proliferation of human airway smooth muscle (HASM) cells contributes, in many cases, to the chronic stages of asthma. However, the physiological and pathophysiological processes regulating cell growth and division in the airway are not clear. We have recently shown that the immediate early gene, cyclo-oxygenase-2, is induced by cytokines in HASM cells. Since cyclo-oxygenase metabolites, such as prostaglandin (PG) E2 have been shown to modulate HASM cell growth, we have investigated any autocrine action of endogenously released cyclo-oxygenase-1/2 products on the proliferative responses in these cells. 2. HASM cells were cultured from healthy tissue obtained at lung or heart/lung transplantation. HASM cell proliferation was measured by [3H]-methyl thymidine uptake by cells and by cell counts. Cyclo-oxygenase-2 expression was measured by Western blot analysis and activity measured by the release of PGE2, by radioimmunoasay. 3. HASM cells proliferated in response to foetal calf serum, a response that was greatly inhibited when cyclo-oxygenase-2 was induced with either interleukin-1beta plus tumour necrosis factor-alpha or interleukin-1beta, tumour necrosis factor alpha plus interferon gamma (each at 10 ng ml(-1)). The inhibitory effect of cytokines on HASM cell proliferation was reversed in a concentration dependent manner by either the mixed cyclo-oxygenase-1/-2 inhibitor, indomethacin or the selective cyclo-oxygenase-2 inhibitor, L-745,337 (each at 10 microM). 4. PGE2 or the stable analogue of prostacyclin, cicaprost concentration-dependently (0.1 pmol to 1 microM) inhibited serum induced proliferation of HASM cells. By contrast, the TP receptor agonist, U46619 stimulated proliferation of HASM cells when cells were cultured without but not with serum. Other cyclo-oxygenase products, PGD2, PGF2alpha had no effect on cellular proliferation at concentrations up to 1 microM. 5. These observations illustrate a profound inhibitory effect of cyclo-oxygenase-2 induction on HASM cell proliferation, possibly via IP or EP receptor activation. Cyclo-oxygenase-2 induction has, thus far, been associated with the pro-inflammatory responses of plasma exudation and oedema formation and is assumed to be an enzyme worthy of selective inhibition in many disease states. However, our observations suggest that cyclo-oxygenase-2 can have an anti-inflammatory, anti-proliferative function in the airways. These observations may have importance in the use and development of therapies for airway disease such as asthma.     

Ultrastructural observations of the human uterine smooth muscle cells during gestation

At term pregnancy, the myometrium consists of bundles of smooth muscle cells bound together by varying amounts of connective tissue. Each bundle contains both dark and light muscle cells. During uterine contractions it is believed that the smooth muscle cells become darker, decrease in volume, and exhibit changes in diameter. This is accompanied by widening of the interspaces and by a decrease in the areas of cellular contact. Between contractions, there are more light cells which become arranged closer to each other and exhibit large areas of interdigitation. The significance of these observations in the mechanism of uterine contraction and retraction is discussed. Cell believed to be modified smooth muscle cells occupy the myoendometrial junction and the decidua basalis. They are irregular in shape, poor in myofilament content, and rich in other cytoplasmic organelles and form a loosely arranged layer of cells between the myometrium and the trophoblast. The possible functional significance of these cells is also discussed.