Localization of AU-rich element-containing mRNA in cytoplasmic granules containing exosome subunits

Eukaryotic mRNAs can be degraded in either decapping/5'-to-3' or 3'-to-5' direction after deadenylation. In yeast and mammalian cells, decay factors involved in the 5'-to-3' decay pathway are concentrated in cytoplasmic processing bodies (P bodies). The mechanistic steps and localization of mammalian mRNA decay are still not completely understood. Here, we investigate functions of human mRNA decay enzymes in AU-rich element (ARE)-mediated mRNA decay (AMD) and find that the deadenylase, poly(A) ribonuclease PARN, and enzymes involved in the 5'-to-3' and 3'-to-5' decay pathways are required for AMD. The ARE-containing reporter mRNA accumulates in discrete cytoplasmic granular structures, which are distinct from P bodies and stress granules. These granules consist of poly(A)-specific ribonuclease, exosome subunits, and decay-promoting ARE-binding proteins. Inhibition of AMD increases accumulation of ARE-mRNA in these granules. We refer to these structures as cytoplasmic exosome granules and suggest that some AMD may occur in these granules.     

The relationship between wall shear stress distributions and intimal thickening in the human abdominal aorta

Purpose

The goal of this work was to determine wall shear stress (WSS) patterns in the human abdominal aorta and to compare these patterns to measurements of intimal thickness (IT) from autopsy samples.

Methods

The WSS was experimentally measured using the laser photochromic dye tracer technique in an anatomically faithful in vitro model based on CT scans of the abdominal aorta in a healthy 35-year-old subject. IT was quantified as a function of circumferential and axial position using light microscopy in ten human autopsy specimens.

Results

The histomorphometric analysis suggests that IT increases with age and that the distribution of intimal thickening changes with age. The lowest WSS in the flow model was found on the posterior wall inferior to the inferior mesenteric artery, and coincided with the region of most prominent IT in the autopsy samples. Local geometrical features in the flow model, such as the expansion at the inferior mesenteric artery (common in younger individuals), strongly influenced WSS patterns. The WSS was found to correlate negatively with IT (r² = 0.3099; P = 0.0047).

Conclusion

Low WSS in the abdominal aorta is co-localized with IT and may be related to atherogenesis. Also, rates of IT in the abdominal aorta are possibly influenced by age-related geometrical changes.

     

Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture

The expression of actin-isoform mRNAs in the smooth muscle cells (SMC) of the aortic media in rats has been studied by Northern-blot hybridization, using a general actin-cRNA probe, and two cRNA probes specific for beta- and gamma-cytoplasmic actins, during: (1) development, (2) intimal thickening after endothelial injury induced by balloon catheterization, and (3) growth in culture. In 5-day-old rats, the ratio between alpha-smooth-muscle-actin mRNA and beta- and gamma-cytoplasmic-actin mRNAs was close to 1. It increased to about 4 in 6-week-old rats. Replicating SMC from regions of intimal thickening 15 days after endothelial injury, and SMC growing in culture contained a predominance of cytoplasmic actin mRNAs. Intimal SMC 60 days after endothelial injury (at which time the endothelium had fully regenerated) demonstrated a pattern of actin mRNAs similar to that of normal media. Functional mRNA measured by translation in a reticulocyte lysate showed increases in the level of alpha-actin and decreases in beta-actin in rats from 5 days to 6 weeks of age. These results suggest that during development, under pathological conditions, and in cell culture, the expression of actin isoforms in arterial SMC depends on many factors, including the amount and translation efficiency of mRNAs, and the relative stabilities of the proteins involved.     

Direct correlation between calcium content and blood pressure: a study of aortic coarctation in rats

The calcium content of aorta was measured by atomic absorption after coarctation in the rat. At 7 and 14 days, the calcium content was elevated on the proximal side of the coarctation, where pressure was increased significantly. On the distal, low pressure side of the aortic coarctation, calcium was reduced significantly. There is a direct correlation between the blood pressure and the content of calcium (r = 0.69, P less than 0.001). The width of the aortic media on the high pressure side was increased significantly at 7 and 14 days after coarctation, whereas no significant changes in width were present on the low pressure side of the constriction. We conclude that pressure regulates the aortic calcium content, likely acting through a local effect.     

Determination of layer-specific mechanical properties of human coronary arteries with nonatherosclerotic intimal thickening and related constitutive modeling

At autopsy, 13 nonstenotic human left anterior descending coronary arteries [71.5 +/- 7.3 (mean +/- SD) yr old] were harvested, and related anamnesis was documented. Preconditioned prepared strips (n = 78) of segments from the midregion of the left anterior descending coronary artery from the individual layers in axial and circumferential directions were subjected to cyclic quasi-static uniaxial tension tests, and ultimate tensile stresses and stretches were documented. The ratio of outer diameter to total wall thickness was 0.189 +/- 0.014; ratios of adventitia, media, and intima thickness to total wall thickness were 0.4 +/- 0.03, 0.36 +/- 0.03, and 0.27 +/- 0.02, respectively; axial in situ stretch of 1.044 +/- 0.06 decreased with age. Stress-stretch responses for the individual tissues showed pronounced mechanical heterogeneity. The intima is the stiffest layer over the whole deformation domain, whereas the media in the longitudinal direction is the softest. All specimens exhibited small hysteresis and anisotropic and strong nonlinear behavior in both loading directions. The media and intima showed similar ultimate tensile stresses, which are on average three times smaller than ultimate tensile stresses in the adventitia (1,430 +/- 604 kPa circumferential and 1,300 +/- 692 kPa longitudinal). The ultimate tensile stretches are similar for all tissue layers. A recently proposed constitutive model was extended and used to represent the deformation behavior for each tissue type over the entire loading range. The study showed the need to model nonstenotic human coronary arteries with nonatherosclerotic intimal thickening as a composite structure composed of three solid mechanically relevant layers with different mechanical properties. The intima showed significant thickness, load-bearing capacity, and mechanical strength compared with the media and adventitia.     

Simultaneous staining of cytoplasmic iron and collagen matrix in human liver biopsy specimens

One of the major liver fibrogenic activators is the cellular iron overload that can severely damage parenchymal and non-parenchymal cells. The aim of this study was to investigate a histochemical staining technique that allows the simultaneous detection of the irregular deposition of matrix collagen and both the amount and distribution of iron in liver cells on the same histological section. The method was evaluated using 3-microm histological sections obtained from ten standard liver biopsy specimens taken from patients with hepatitis C virus-related diseases and simultaneous liver cell iron overload. The results indicate that the double-staining technique is simple, sensitive and rapid, and can be routinely applied to liver biopsy specimens for diagnostic purposes. Furthermore, it may also facilitate the study of the complex relationship between hepatic fibrosis and iron overload during common genetic or secondary liver metabolic disorders.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often spotty distribution of collagen X with increasing cartilage degeneration associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and spotty collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

Summary The injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone.

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes.

Sendai virus induces human peripheral blood leukocytes to produce high levels of tumor necrosis factor (TNF) mRNA. TNF mRNA can represent as much as 0.6% of the total mRNA. Kinetic studies indicate that the level of TNF mRNA peaks about 2 hours before that of IFN-alpha mRNA produced in the same system. Although the peak levels of TNF and IFN-alpha mRNA were similar, TNF in the culture supernatants was at a 200 fold lower level than IFN-alpha. Cloning and sequence analysis of TNF cDNA isolated from peripheral blood leukocytes RNA showed that normal human cells in response to Sendai virus produce TNF identical to that previously isolated and cloned from tumor-derived cell lines. A bacterial expression system was used to produce the cloned TNF at a maximum level of 2 X 10(6) units per ml of culture.     

Permeability of human medial collateral ligament in compression transverse to the collagen fiber direction

This study quantified the apparent and intrinsic hydraulic permeability of human medial collateral ligament (MCL) under direct permeation transverse to the collagen fiber direction. A custom permeation device was built to apply flow across cylindrical samples of ligament while monitoring the resulting pressure gradient. MCLs from 5 unpaired human knees were used (donor age 55 +/- 16 yr, 4 males, 1 female). Permeability measurements were performed at 3 levels of compressive pre-strain (10%, 20% and 30%) and 5 pressures (0.17, 0.34, 1.03, 1.72 and 2.76 MPa). Apparent permeability was determined from Darcy's law, while intrinsic permeability was determined from the zero-pressure crossing of the pressure-permeability curves at each compressive pre-strain. Resulting data were fit to a finite deformation constitutive law [Journal of Biomechanics 23 (1990) 1145-1156]. The apparent permeability of human MCL ranged from 0.40 +/- 0.05 to 8.60 +/- 0.77 x 10(-16) m(4)/Ns depending on pre-strain and pressure gradient. There was a significant decrease in apparent permeability with increasing compressive pre-strain (p=0.024) and pressure gradient (p<0.001), and there was a significant interaction between the effects of compressive pre-strain and pressure (p<0.001). Intrinsic permeability was 14.14 +/- 0.74, 6.30 +/- 2.13 and 4.29 +/- 1.71 x 10(-16) m(4)/Ns for compressive pre-strains of 10%, 20% and 30%, respectively. The intrinsic permeability showed a faster decrease with increasing compressive pre-strain than that of bovine articular cartilage. These data provide a baseline for investigating the effects of disease and chemical modification on the permeability of ligament and the data should also be useful for modeling the poroelastic material behavior of ligaments.     

Functional medial thickening and folding of the internal elastic lamina in coronary spasm

Although there are a number of studies on vasospastic angina, the structural changes at the cellular level that occur in the coronary arterial wall during spasm are not well known. Coronary spasm was induced by brushing the coronary adventitia in nine anesthetized beagles, and structural changes in the spastic coronary segments were examined by light and electron microscopy, making comparisons with the adjacent nonspastic segments. The % diameter stenosis of the spastic segments as measured angiographically was 79.4±12% (mean±SD). Light microscopic changes in the spastic and nonspastic segments were as follows: medial thickness 1,512 vs. 392 μm (P<0.0001) and % diameter and % area stenoses of spastic segment 81.0% and 96.5%, respectively, indicating that spasm was induced by medial thickening. Circular smooth muscle cells (SMCs) in the media were arranged in parallel with the internal (IEL) and external (EEL) elastic lamina in nonspastic segments but radially rearranged in spastic segments. SMCs were classified by their patterns of connection to IEL into six types by electron microscopy. Of these, three contracted and pulled the IEL toward the EEL, causing folding of the IEL and waving of EEL resulting in thickening of the media and narrowing of the lumen. We conclude that coronary spasm was elicited by radial rearrangement of the medial SMCs due to their own contraction and resultant medial thickening and folding of IEL, creating a piston effect to narrow the lumen, i.e., spasm.     

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections

 Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of α1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro α1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for α1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN. Accepted: 21 July 1998     

RNA processing: splicing and the cytoplasmic localisation of mRNA

An unexpected link has been discovered between pre-mRNA splicing in the nucleus and mRNA localisation in the cytoplasm. The new findings suggest that recruitment of the Mago Nashi and Y14 proteins upon splicing of oskar mRNA is an essential step in the localisation of the RNA to the posterior pole of the Drosophila oocyte.     

Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.     

Relative contribution of wall shear stress and injury in experimental intimal thickening at PTFE end-to-side arterial anastomoses

BACKGROUND: Intimal hyperplastic thickening (IHT) is a frequent cause of prosthetic bypass graft failure. Induction and progression of IHT is thought to involve a number of mechanisms related to variation in the flow field, injury and the prosthetic nature of the conduit. This study was designed to examine the relative contribution of wall shear stress and injury to the induction of IHT at defined regions of experimental end-to-side prosthetic anastomoses. METHODS AND RESULTS: The distribution of IHT was determined at the distal end-to-side anastomosis of seven canine Iliofemoral PTFE grafts after 12 weeks of implantation. An upscaled transparent model was constructed using the in vivo anastomotic geometry, and wall shear stress was determined at 24 axial locations from laser Doppler anemometry measurements of the near wall velocity under conditions of pulsatile flow similar to that present in vivo. The distribution of IHT at the end-to-side PTFE graft was determined using computer assisted morphometry. IHT involving the native artery ranged from 0.0+/-0.1 mm to 0.05+/-0.03 mm. A greater amount of IHT was found on the graft hood (PTFE) and ranged from 0.09+/-0.06 to 0.24+/-0.06 mm. Nonlinear multivariable logistic analysis was used to model IHT as a function of the reciprocal of wall shear stress, distance from the suture line, and vascular conduit type (i.e. PTFE versus host artery). Vascular conduit type and distance from the suture line independently contributed to IHT. An inverse correlation between wall shear stress and IHT was found only for those regions located on the juxta-anastomotic PTFE graft. CONCLUSIONS: The data are consistent with a model of intimal thickening in which the intimal hyperplastic pannus migrating from the suture line was enhanced by reduced levels of wall shear stress at the PTFE graft/host artery interface. Such hemodynamic modulation of injury induced IHT was absent at the neighboring artery wall.