Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be regulated by basic fibroblast growth factor and interleukin-4 tissue expression, respectively.

Mechanical Alterations in airway smooth muscle after interaction with indoor pollutant – A mathematical model.

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully understood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholine (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle ¯bers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, incorporates the phenomenology of both MCh and FA and reproduces experimental results observed with in vitro exposure of smooth muscle to FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells in a tissue level model. The model can also be used in different biological scales.

Mechanical alterations in airway smooth muscle after interaction with indoor pollutant - A mathematical model

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully w1derstood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholinc (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle fibers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, ir1corporates the phenomenology of both MCh and FA and reproduces experirnental results observed with ir1 vitro exposure of smooth muscle to .FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells ir1 a tissue level model. The model can also be used in different biological scales.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Expression and function of 5-hydroxytryptamine 4 receptors in smooth muscle preparations from the duodenum, ileum, and pelvic flexure of horses without gastrointestinal tract disease

OBJECTIVE: To evaluate the expression of the 5-hydroxytryptamine 4 (5-HT4) receptor subtype and investigate the modulating function of those receptors on contractility in intestinal tissues obtained from horses without gastrointestinal tract disease. SAMPLE POPULATION: Smooth muscle preparations from the duodenum, ileum, and pelvic flexure collected immediately after slaughter of 24 horses with no history or signs of gastrointestinal tract disease. PROCEDURES: In isometric organ baths, the contractile activities of smooth muscle preparations in response to 5-hydroxytryptamine and electric field stimulation were assessed; the effect of tegaserod alone or in combination with 5-hydroxytryptamine on contractility of intestinal specimens was also investigated. Presence and distribution of 5-HT4 receptors in intestinal tissues and localization on interstitial cells of Cajal were examined by use of an immunofluorescence technique. RESULTS: Widespread 5-HT4 receptor immunoreactivity was observed in all intestinal smooth muscle layers; 5-HT4 receptors were absent from the myenteric plexus and interstitial cells of Cajal. In electrical field-stimulated tissue preparations of duodenum and pelvic flexure, tegaserod increased the amplitude of smooth muscle contractions in a concentration-dependent manner. Preincubation with tegaserod significantly decreased the basal tone of the 5-HT-evoked contractility in small intestine specimens, compared with the effect of 5-HT alone, thereby confirming that tegaserod was acting as a partial agonist. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, 5-HT4 receptors on smooth muscle cells appear to be involved in the contractile response of the intestinal tract to 5-hydroxytryptamine. Results suggest that tegaserod may be useful for treatment of reduced gastrointestinal tract motility in horses.

Abolishing myofibroblast arrhythmogeneicity by pharmacological ablation of α-smooth muscle actin containing stress fibers

Rationale: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes. Objective: We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast–cardiomyocyte cross-talk. Methods and Results: Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. Conclusions: The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling.

Leiomyomatoid angiomatous neuroendocrine tumor (LANT) of the pituitary: a distinctive biphasic neoplasm with primitive secretory phenotype and smooth muscle-rich stroma

We describe a hitherto undocumented variant of dimorphic pituitary neoplasm composed of an admixture of neurosecretory cells and profuse leiomyomatous stroma around intratumoral vessels. Radiologically perceived as a macroadenoma of 3.8 cm in diameter, this pituitary mass developed in an otherwise healthy 43-year-old female. At the term of a yearlong history of amenorrhea and progressive bitemporal visual loss, subtotal resection was performed via transsphenoidal microsurgery. Discounting mild hyperprolactinemia, there was no evidence of excess hormone production. Histologically, solid sheets, nests and cords of epithelial-looking, yet cytokeratin-negative cells were seen growing in a richly vascularized stroma of spindle cells. While strong immunoreactivity for NCAM, Synaptophysin and Chromogranin-A was detected in the former, the latter showed both morphological and immunophenotypic hallmarks of smooth muscle, being positive for vimentin, muscle actin and smooth muscle actin. Architectural patterns varied from monomorphous stroma-dominant zones through biphasic neuroendocrine-leiomyomatous areas, to pseudopapillary fronds along vascular cores. Only endothelia were labeled with CD34. Staining for S100 protein and GFAP, characteristics of sustentacular cells, as well as bcl-2 and c-kit was absent. Except for alpha-subunit, anterior pituitary hormones tested negative in tumor cells, as did a panel of peripheral endocrine markers, including serotonin, somatostatin, calcitonin, parathormone and vasoactive intestinal polypeptide. Mitotic activity was absent and the MIB-1 labeling index low (1-2%). While assignment of this lesion to any established neoplastic entity is not forthcoming, we propose it is being considered as a low-grade neuroendocrine tumor possibly related to null cell adenoma.

Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma

Continuous changes in the length of smooth muscles require a highly organized sarcolemmal structure. Yet, smooth muscle cells also adapt rapidly to altered environmental cues. Their sarcolemmal plasticity must lead to profound changes which affect transmembrane signal transduction as well as contractility. We have established porcine vascular and human visceral smooth muscle cultures of epithelioid and spindle-shaped morphology and determined their plasma membrane properties. Epithelioid cells from both sources contain a higher ratio of cholesterol to glycerophospholipids, and express a less diverse range of lipid-associated annexins. These findings point to a reduction in efficiency of membrane segregation in epithelioid cells. Moreover, compared to spindle-shaped cells, cholesterol is more readily extracted from epithelioid cells with methyl-beta-cyclodextrin and its synthesis is more susceptible to inhibition with lovastatin. The inability of epithelioid cells to process vasoactive metabolites, such as angiotensin or nucleotides further indicates that contractile properties are impaired. Phenotypic plasticity extends beyond the loss of smooth muscle cell marker genes. The plasma membrane has undergone profound functional changes which are incompatible with cyclic foreshortening, but might be important in the development of vascular disease.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

OBJECTIVE: To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. SAMPLE POPULATION: Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. PROCEDURES: Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A(max)), and area under the curve (AUC) were evaluated. RESULTS: Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M(3) antagonists (more commonly for basal tone than for A(max) and AUC). The M(2) receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M(3) receptor antagonists were generally more potent than the M(2) receptor antagonists. In a protection experiment, an M(3) receptor antagonist was less potent than when used in combination with an M(2) receptor antagonist. Receptor antagonists for M(1) and M(4) did not affect contractility variables. CONCLUSIONS AND CLINICAL RELEVANCE: Bethanechol acting on muscarinic receptor sub-types M(2) and M(3) may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.

Early loss of arteriolar smooth muscle cells: more than just a pericyte loss in diabetic retinopathy

Incipient diabetic retinopathy is characterized by increased capillary permeability and progressive capillary occlusion. The earliest structural change is the loss of pericytes (PC) from the retinal capillaries. With the availability of the XLacZ mouse, which expresses the LacZ reporter in a PC/vascular smooth muscle cell (vSMC) specific fashion, we quantitatively assessed the temporal dynamics of smooth muscle cells in arterioles under hyperglycemic conditions. We induced stable hyperglycemia in XLacZ mice. After 4, 8, and 12 weeks of diabetes retinae were isolated and beta-galactosidase/lectin stained. The numbers of smooth muscle cells were counted in retinal whole mounts, and diameters of retinal radial and branching arterioles and venules were analyzed at different distances apart from the center of the retina. After eight weeks of diabetes, the numbers of vSMCs were significantly reduced in radial arterioles 1000 microm distant from the optic disc. At proximal sites of branching arterioles (400 microm distant from the center), and at distal sites (1000 microm), vSMC were significantly reduced already after 4 weeks (to a maximum of 31 %). These changes were not associated with any measurable variation in vessel diameters. These data indicate quantitatively that hyperglycemia not only causes pericyte loss, but also loss of vSMCs in the retinal vasculature. Our data suggest that arteriolar vSMC in the eye underlie similar regulations which induce early pericyte loss in the diabetic retina.

In vitro effects of bethanechol on smooth muscle preparations from abomasal fundus, corpus, and antrum of dairy cows

Abomasal displacement has been associated with gastric hypomotility. The supply of prokinetic drugs available to address this problem is insufficient. The goal of the study was to investigate the effect of the muscarinic agonist bethanechol (BeCh) on contractility parameters of smooth muscle preparations from several regions of the bovine abomasum (fundus, corpus, and antrum). Cumulative concentration-response curves were constructed using BeCh in vitro with and without pre-incubation with antagonists targeted at M(2) and M(3) muscarinic acetylcholine receptor (mAChR) subtypes. In all preparations investigated, BeCh induced a significant and concentration-dependent increase in all contractility parameters investigated. The maximal attainable effect (V(max)) was more pronounced in circular specimens, and V(max) of antral specimens in circular orientation were significantly lower when compared to the other preparations. Both antagonists caused a rightward shift of the concentration-response curve, suggesting that the effect of BeCh is mediated at least partly by M(2) and M(3) AChRs.