Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Immunohistochemical evidence for myofibroblastlike cells associated with liver injury induced by Aflatoxin B1 in rainbow trout (Oncorhynchus mykiss)

In mammalian species, profibrogenic cells are activated to become myofibroblasts in response to liver damage. Few studies have examined hepatic myofibroblasts and their role in liver damage in teleosts. The aim of the present study was to investigate the involvement of myofibroblast-like cells in rainbow trout (Oncorhynchus mykiss) with hepatic damage induced by aflatoxin B1 (AFB1). Histopathological and immunohistochemical analyses characterized alterations in the liver stroma during the carcinogenic process. Anti-human a-smoothmuscle actin (SMA) and anti-human desmin primary antibodies were used in immunohistochemistry. Only the anti-SMA reagent labelled cells in trout liver. In the livers of control fish, only smooth muscle in blood vessels and around bile ducts was labelled. In the livers from AFB1-treated fish, SMA-positive cells were present in the stroma surrounding neoplastic lesions and in areas of desmoplastic reaction. These observations indicate that in teleosts, as in mammals, the myofibroblast-like cell is involved in fibrosis associated with liver injury. Chronic liver injury induced in trout by aflatoxin may provide a useful model system for study of the evolution of such mechanisms.

Quantification of cellular action fibers alignment from confocal microscopic images.

The cellular rheology has recently undergone a rapid development with particular attention to the cytoskeleton mechanical properties and its main components - actin filaments, intermediate filaments, microtubules and crosslinked proteins. However it is not clear what are the cellular structural changes that directly affect the cell mechanical properties. Thus, in this work, we aimed to quantify the structural rearrangement of these fibers that may emerge in changes in the cell mechanics. We created an image analysis platform to study smooth muscle cells from different arteries: aorta, mammary, renal, carotid and coronary and processed respectively 31, 29, 31, 30 and 35 cell image obtained by confocal microscopy. The platform was developed in Matlab (MathWorks) and it uses the Sobel operator to determine the actin fiber image orientation of the cell, labeled with phalloidin. The Sobel operator is used as a filter capable of calculating the pixel brightness gradient, point to point, in the image. The operator uses vertical and horizontal convolution kernels to calculate the magnitude and the angle of the pixel intensity gradient. The image analysis followed the sequence: (1) opens a given cells image set to be processed; (2) sets a fix threshold to eliminate noise, based on Otsu's method; (3) detect the fiber edges in the image using the Sobel operator; and (4) quantify the actin fiber orientation. Our first result is the probability distribution II(Δθ) to find a given fiber angle deviation (Δθ) from the main cell fiber orientation θ0. The II(Δθ) follows an exponential decay II(Δθ) = Aexp(-αΔθ) regarding to its θ0. We defined and determined a misalignment index α of the fibers of each artery kind: coronary αCo = (1.72 ‘+ or =’ 0.36)rad POT -1; renal αRe = (1.43 + or - 0.64)rad POT -1; aorta αAo = (1.42 + or - 0.43)rad POT -1; mammary αMa = (1.12 + or - 0.50)rad POT -1; and carotid αCa = (1.01 + or - 0.39)rad POT -1. The α of coronary and carotid are statistically different (p < 0.05) among all analyzed cells. We discussed our results correlating the misalignment index data with the experimental cell mechanical properties obtained by using Optical Magnetic Twisting Cytometry with the same group of cells.

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

Mutations in myosin light chain kinase cause familial aortic dissections.

Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.

Thoracic aortic disease in tuberous sclerosis complex: molecular pathogenesis and potential therapies in Tsc2+/- mice.

Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes.

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-Swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4 M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.

Mechanotransduction pathway activation in familial thoracic aortic aneurysms and dissections

Thoracic aortic aneurysms and dissections (TAAD) are autosomal dominantly inherited in 19% of patients. Mapping studies determined that the disease is genetically heterogeneous with multiple loci and genetic mutations accounting for familial TAAD. However, regardless of the specific mutation, resulting pathology is consistently medial degeneration, characterized by increased proteoglycans and loss of elastic fibers. We tested the hypothesis that genetic mutations leading to familial TAAD alter common pathways in aortic smooth muscle cells (SMCs). Identification of mutations at R460 in TGFBR2 reveals a 5% contribution to TAAD, however downstream analysis of Smad2 phosphorylation in the TGF-β pathway is not commonly altered in familial or sporadic disease when compared to controls. Expression profiling using Illumina's Sentrix HumanRef 8 Expression Beadchip array was done on RNA isolated from SMCs explanted from 6 patients with inherited TAAD with no identified mutation and 3 healthy controls obtained from the International Institute for the Advancement of Medicine. Significant increases in expression of proteoglycan genes in patients' SMCs, specifically lumican, podocan, and decorin were confirmed using Q-PCR and tissue immunofluorescence. NCI's Ingenuity Pathway Analysis predicted alterations in the ERK, insulin receptor and SAPK/JNK pathways (p<0.001), which SMCs activate in response to cyclic stretch. Immunoblotting indicated increased phosphorylation of ERK and GSK-3β, a protein from the insulin receptor pathway, in explanted patient SMCs, also confirmed by increased immunoreactivity against phosphorylated ERK and GSK-3β in the sub-intimal SMCs from patient tissue compared to controls. To determine if mechanotransduction pathway activation was responsible for the medial degeneration a specific inhibitor of GSK-3β, SB216763 was incubated with control cells and significantly increased the expression levels of proteoglycans. Mechanical strain was also applied to control SMCs confirming pathways stimulation with stretch. Incubation with pathway inhibitors against insulin receptor and ERK pathways identify, for the first time that stretch induced GSK-3β phosphorylation may increase proteoglycan expression, and ERK phosphorylation may regulate the expression of MMP2, a protein known to degrade elastic fibers. Furthermore, specific mutations in SMC-specific β-myosin heavy chain and α-actin, in addition to upregulation of pathways activated by cyclic stretch suggest that SMC response to hemodynamic factors, play a role in this disease. ^

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Primary Mesenchymal Cells Isolated from SPARC-null Mice Exhibit Altered Morphology and Rates of Proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo.

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation.