The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Peptidomic analysis of hippocampal tissue for explore leptin neuroprotective effect on the preterm ischemia-hypoxia brain damage model rats.

The most common injury of preterm infants is periventricular leukomalacia (PVL) and to date there is still no safe and effective treatment. In our previous studies, leptin has been found to have neuroprotective effects on the preterm ischemia-hypoxia brain damage model rats in animal behavior. To gain insight into the neuroprotective mechanisms of leptin on preterm brain damage model rats, we constructed a comparative peptidomic profiling of hippocampal tissue between leptin-treated after model and preterm ischemia-hypoxia brain damage model rats using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by nano liquid chromatography tandem mass spectrometry. We identified and quantified 4164 peptides, 238 of which were differential expressed in hippocampal tissue in the two groups. A total of 150 peptides were up regulated and 88 peptides were down regulated. These peptides were imported into the Ingenuity Pathway Analysis (IPA) and identified putative roles in nervous system development, function and diseases. We concluded that the preterm ischemia-hypoxia brain damage model with leptin treatment induced peptides changes in hippocampus, and these peptides, especially for the peptides associated "microtubule-associated protein 1b (MAP1b), Elastin (Eln), Piccolo presynaptic cytomatrix protein (Pclo), Zinc finger homeobox 3(Zfhx3), Alpha-kinase 3(Alpk3) and Myosin XVA(Myo15a) ", could be candidate bio-active peptides and participate in neuroprotection of leptin. These may advance our current understanding of the mechanism of leptin's neuroprotective effect on preterm brain damage and may be involved in the etiology of preterm brain damage. Meanwhile, we found that repression of ILK signaling pathway plays a significant role in neuroprotection of leptin. A better understanding of the role of ILK signaling pathway in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious drug for therapy against human nervous system disorders.

Regulation by walnut protein hydrolysate on the components and structural degradation of photoaged skin in SD rats.

Skin photoaging induced by consecutive exposure of skin to ultraviolet radiation is primarily responsible for skin aging and preparation of food-derived ingredients with anti-aging functions has been the hot topic worldwide. Dietary consumption of food supplements has been found to modulate skin functions and can be useful in the prevention of skin aging. To evaluate the effect of walnut protein hydrolysate (WPH) on photoaged skin, Sprague-Dawley rats (SD rats) were orally administered with WPH and then were regularly exposed to ultraviolet radiation (UV-R). After a consecutive UV-R for 18 weeks, the delaying efficiency of WPH against elasticity degradation was examined and the mechanical mechanism was explored subsequently. The contents of hydroxyproline (Hyp) and hyaluronic acid (HA) in the extracellular matrix (ECM) were measured by biochemical reactions and color rendering procedures; the levels of types I and III collagen (Col I and III) and the activity of matrix metalloproteinase-1 (MMP-1) were detected by enzyme-linked immunosorbent assay (ELISA); the protein levels of elastin and fibrillin-1 were examined by western blotting. Moreover, the histological change in the skin structure was illustrated by hematoxylin & eosin (HE) and Masson staining. The results revealed that WPH evidently enhanced the elasticity of photoaged skin and stimulated the biosynthesis of ECM components Col I, Hyp and HA in the dermal layer; meanwhile WPH inhibited the MMP-1 activity, alleviated epidermal hyperplasia, and repaired the damaged skin mechanical structure in a dose-dependent manner. In particular, in comparison with the UV-R group, the WPH group in which WPH was administered at a high-dose level showed significantly improved skin appearance, ECM components and structure (P < 0.05). Taken together, the elasticity improvement caused by WPH against the skin photoaging process can be attributed to the regulation of the metabolism of the components and repair of the damaged mechanical structure of the ECM. This research proved the potential of WPH as a functional ingredient for the development of anti-photoaging foods.

Rise and fall of elastic fibers from development to aging. Consequences on arterial structure-function and therapeutical perspectives.

In the arteries of vertebrates, evolution has given rise to resilient macromolecular structures, elastin and elastic fibers, capable of sustaining an elevated blood pressure and smoothening the discontinuous blood flow and pressure generated by the heart. Elastic fibers are produced only during development and childhood, before being progressively degraded by mechanical stress and enzymatic activities during adulthood and aging. During this period, arterial elastic fiber calcification and loading of lipids also occur, all of these events conducting to arteriosclerosis. This leads to a progressive dysfunction of the large elastic arteries inducing elevated blood pressure as well as altered hemodynamics and organ perfusion, which induce more global malfunctions of the body during normal aging. Additionally, some arterial conditions occur more frequently with advancing age, such as atherosclerosis or aneurysms, which are called age-related diseases or pathological aging. The physiological or pathological degradation of elastic fibers and function of elastic arteries seemed to be rather inevitable over time. However, during the recent years, different molecules - including several ATP-dependent potassium channel openers, such as minoxidil - have been shown to re-induce elastin production and elastic fiber assembly, leading to improvements in the arterial structure and function or in organ perfusion. This review summarizes the changes in the arterial elastic fibers and structure from development until aging, and presents some of the potential pharmacotherapies leading to elastic fiber neosynthesis and arterial function improvement.

The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Heparan sulfates facilitate harmless amyloidogenic fibril formation interacting with elastin-like peptides.

Heparan sulfates (HSs) modulate tissue elasticity in physiopathological conditions by interacting with various matrix constituents as tropoelastin and elastin-derived peptides. HSs bind also to protein moieties accelerating amyloid formation and influencing cytotoxic properties of insoluble fibrils. Interestingly, amyloidogenic polypeptides, despite their supposed pathogenic role, have been recently explored as promising bio-nanomaterials due to their unique and interesting properties. Therefore, we investigated the interactions of HSs, obtained from different sources and exhibiting various degree of sulfation, with synthetic amyloidogenic elastin-like peptides (ELPs), also looking at the effects of these interactions on cell viability and cell behavior using in vitro cultured fibroblasts, as a prototype of mesenchymal cells known to modulate the soft connective tissue environment. Results demonstrate, for the first time, that HSs, with differences depending on their sulfation pattern and chain length, interact with ELPs accelerating aggregation kinetics and amyloid-like fibril formation as well as self-association. Furthermore, these fibrils do not negatively affect fibroblasts' cell growth and parameters of redox balance, and influence cellular adhesion properties. Data provide information for a better understanding of the interactions altering the elastic component in aging and in pathologic conditions and may pave the way for the development of composite matrix-based biomaterials.

neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the Expression of Extracellular Matrix Components in Human Dermal Fibroblasts.

Eleven neo-clerodane diterpenoids (1-11) including the new analogues 1, 2, and 10, and 3',5,6,7-tetrahydroxy-4'-methoxyflavone (12) were isolated from the aerial parts of Salvia polystachya. Polystachyne G (1) and 15-epi-polystachyne G (2) were isolated as an epimeric mixture, containing a 5-hydroxyfuran-2(5H)-one unit in the side chain at C-12 of the neo-clerodane framework. Polystachyne H (10) contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy group. The structures of these compounds were established by analysis of their NMR spectroscopic and MS spectrometric data. The absolute configurations of compounds 3, 4, and 10 were determined through single-crystal X-ray diffraction analysis. The antibacterial, antifungal, and phytotoxic activities of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of nine of the isolates (1-8 and 11) was assayed. Compounds 1-4, 8, and 11 increased the expression of the genes codifying for type I, type III, and type V collagens and for elastin.

Mechanical, structural, and dynamical modifications of cholesterol exposed porcine aortic elastin.

Elastin is a protein of the extracellular matrix that contributes significantly to the elasticity of connective tissues. In this study, we examine dynamical and structural modifications of aortic elastin exposed to cholesterol by NMR spectroscopic and relaxation methodologies. Macroscopic measurements are also presented and reveal that cholesterol treatment may cause a decrease in the stiffness of tissue. 2H NMR relaxation techniques revealed differences between the relative populations of water that correlate with the swelling of the tissue following cholesterol exposure. 13C magic-angle-spinning NMR spectroscopy and relaxation methods indicate that cholesterol treated aortic elastin is more mobile than control samples. Molecular dynamics simulations on a short elastin repeat VPGVG in the presence of cholesterol are used to investigate the energetic and entropic contributions to the retractive force, in comparison to the same peptide in water. Peptide stiffness is observed to reduce following cholesterol exposure due to a decrease in the entropic force.

Adipose-derived Stem Cells Counteract Urethral Stricture Formation in Rats.

BACKGROUND: A medical treatment for urethral stricture (US) is not yet available. OBJECTIVE: To evaluate if local injection of human adipose tissue-derived stem cells (hADSC) prevents urethral fibrosis in a rat model of US. DESIGN, SETTING, AND PARTICIPANTS: Male rats were divided into three groups: sham, US, and hADSC (n=12 each). Sham rats received a vehicle injection in the urethral wall. US and hADSCs were incised and injected with the fibrosis-inducer transforming growth factor-ß1 in the urethral wall. INTERVENTION: One day later, hADSCs were injected in the urethral wall of hADSC rats whereas sham and US rats were injected with the vehicle. After 4 wk, the rats underwent cystometries and tissues were then harvested for functional and molecular analyses. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cystometry, microultrasound, histochemistry, organ bath studies, reverse transcription polymerase chain reaction, and western blot. RESULTS AND LIMITATIONS: US rats exhibited 49-51% shorter micturition intervals, 35-51% smaller micturition volumes and bladder capacity, 33-62% higher threshold pressures and flow pressures, and 35-37% lower bladder filling compliance compared with hADSC-treated rats and sham rats (p<0.05). By ultrasound, US rats had hyperechogenic and thick urethral walls with narrowed lumen compared with sham rats, whereas hADSC rats displayed less extensive urethral changes. Isolated detrusor from US rats exhibited 34-55% smaller contractions than detrusor from sham rats (p<0.05). Corresponding values were 11-35% for isolated detrusors from hADSC rats. Collagen and elastin protein expression were increased in the penile urethras of US rats compared with sham and hADSC groups (p<0.05). Endothelial and inducible nitric oxide synthase expressions were higher (p<0.05) in the hADSC group. Compared with US rats, hADSC rats demonstrated decreased expression of several fibrosis-related genes. Administration of hADSCs was performed at an early stage of US development, which we consider a limitation of the study. CONCLUSIONS: Local injection of hADSCs prevents stricture formation and urodynamic complications in a new rat model for US. PATIENT SUMMARY: Stem cell therapy is effective for preventing urethral stricture in an experimental setting.

Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

BACKGROUND: We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. OBJECTIVE: Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). RESULTS: We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. CONCLUSION: We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin.

Elastin Modification by 4-Hydroxynonenal in Hairless Mice Exposed to UV-A. Role in Photoaging and Actinic Elastosis.

Chronic exposure to ultraviolet (UV) radiation causes oxidative stress, which is involved in photoaging and actinic elastosis. UV and reactive oxygen species generate lipid peroxidation products, including the α, ß-unsaturated carbonyl compounds such as acrolein or 4-hydroxynonenal (4-HNE). These aldehydes can modify proteins of the extracellular matrix, but their role in the pathogenesis of photoaging is not clarified. The aim of this study was to investigate whether these aldehydes contribute to alter elastin metabolism and whether topical carbonyl scavengers delay UV-induced skin photoaging. Hairless mice (4-6-week old) daily exposed to UV-A (20 J cm(-2) per day, up to 600 J cm(-2)) exhibited the typical features of photoaging, associated with a significant increase in 4-HNE- and acrolein-adduct content, and elastotic material deposition. Immunofluorescence studies showed the accumulation of 4-HNE adducts on elastin in the dermis of UV-A-exposed mice. This was mimicked in vitro by incubating orcein-elastin with 4-HNE or acrolein, which altered its digestion by leukocyte-elastase, a feature possibly involved in the accumulation of elastotic material. A daily topical application of carnosine completely reversed the development of photoaging alterations and 4-HNE-adduct formation on elastin. These data emphasize the role of 4-HNE and acrolein in the mechanism of photoaging, and the preventive effect of carbonyl scavengers.

Vigna angularis water extracts protect against ultraviolet b-exposed skin aging in vitro and in vivo.

Exposure to ultraviolet (UV) radiation induces various pathological changes, such as thickened skin and wrinkle formation. In particular, UVB irradiation increases matrix metalloproteinase (MMP)-1 production and collagen degradation, leading to premature aging, termed photoaging. The azuki bean (Vigna angularis; VA) has been widely used as a food product as well as a traditional medicine. However, its activity needs additional study to confirm its functional application in foods and cosmetics for protecting skin. In this study, hot-water extract from VA (VAE) and its active component, rutin, were investigated to determine their antiphotoaging effects. VAE was found to have antioxidant activity. In UVB-exposed normal human dermal fibroblasts cells with VAE and rutin treatments, MMP-1 production was significantly suppressed (90% and 47%, respectively). The effects of both topical and oral administration of VAE were tested in UVB-irradiated hairless mice. VAE suppressed wrinkle formation and skin thickness by promoting elastin, procollagen type I, and TGF-ß1 expression (118%, 156%, and 136%, respectively) and by diminishing MMP-1 production. These results suggest that VAE may be effective for preventing skin photoaging accelerated by UVB radiation.

Role of interleukin-18 in destabilization of the atherosclerotic plaque in humans.

Precise location of IL-18 in cell and tissue elements of the atherosclerotic lesions in humans and its role in destabilization of the atherosclerotic plaque were detected. The data suggested a hypothesis on indirect involvement of IL-18 in destruction of the elastic and collagen fibers in an unstable plaque due to this cytokine capacity to induce the production of IFN-γ in T cells and macrophages, this eventually leading to inhibition of collagen and elastin synthesis in smooth muscle cells of the vascular wall and to loosening of the plaque cap.

Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. MATERIAL AND METHODS: Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. RESULTS: Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB. CONCLUSION: These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.

Effects of elastin degradation and surrounding matrix support on artery stability.

Tortuous arteries are often associated with aging, hypertension, atherosclerosis, and degenerative vascular diseases, but the mechanisms are poorly understood. Our recent theoretical analysis suggested that mechanical instability (buckling) may lead to tortuous blood vessels. The objectives of this study were to determine the critical pressure of artery buckling and the effects of elastin degradation and surrounding matrix support on the mechanical stability of arteries. The mechanical properties and critical buckling pressures, at which arteries become unstable and deform into tortuous shapes, were determined for a group of five normal arteries using pressurized inflation and buckling tests. Another group of nine porcine arteries were treated with elastase (8 U/ml), and the mechanical stiffness and critical pressure were obtained before and after treatment. The effect of surrounding tissue support was simulated using a gelatin gel. The critical pressures of the five normal arteries were 9.52 kPa (SD 1.53) and 17.10 kPa (SD 5.11) at axial stretch ratios of 1.3 and 1.5, respectively, while model predicted critical pressures were 10.11 kPa (SD 3.12) and 17.86 kPa (SD 5.21), respectively. Elastase treatment significantly reduced the critical buckling pressure (P < 0.01). Arteries with surrounding matrix support buckled into multiple waves at a higher critical pressure. We concluded that artery buckling under luminal pressure can be predicted by a buckling equation. Elastin degradation weakens the arterial wall and reduces the critical pressure, which thus leads to tortuous vessels. These results shed light on the mechanisms of the development of tortuous vessels due to elastin deficiency.

Optimal proportions of gelatin-resorcin-formalin components in aortic surgery.

BACKGROUND: Gelatin-resorcin-formalin (GRF) glue, a haemostatic agent often employed for aortic surgery, has beneficial effects on early results in surgery for acute aortic dissection but may have late adverse effects, probably due to excess use of the activators such as formaldehyde and glutaraldehyde. The purpose of this study was to determine the optimal proportions of GRF components that minimise toxicity to human aortic smooth muscle cells and elastin with acceptable adhesive strength. METHODS: (1) The degree of polymerisation was examined at various proportions (activator/gelatin+resorcinol=0%, 2%, 4%, 6%, 8% and 10%) to estimate adhesive strength. (2) (i) The toxicity of the activator was confirmed 24h after its supplementation to human aortic smooth muscle cells in various proportions (activator/human aortic smooth muscle cell=0%, 0.5%, 1%, 1.5%, 2% and 2.5%). (ii) The toxicity of GRF glue to human aortic smooth muscle cells was evaluated 1h and 60 h after its supplementation (activator/gelatin+resorcinol=0%, 2%, 4%, 6%, 8% and 10%). Another set of experiments in the same study was also performed. The only difference was that GRF glues were washed after polymerisation to exclude non-polymerised constituents. (3) Effects of 8%-GRF glue on toxicity to elastin derived from human aortic wall cells were investigated using an immunoblotting method. RESULTS: (1) The polymerisation area increased dose dependently and that of the 10% activator/gelatin+resorcinol mixture was significantly wider than those of 6%, 4%, 2% and 0%, but had no significant difference from that of 8%. (2) (i) Human aortic smooth muscle cell death occurred in all dishes except activator-free dishes. (ii) Sixty hours after exposure to GRF glue, human aortic smooth muscle cell death occurred only in the 10% dish. In a washed GRF glue study, no human aortic smooth muscle cell death occurred in any dishes. (3) Toxicity to elastin was not significantly different between 8%-GRF glue and the control, whereas toxicity of elastase to elastin was significantly higher than for both the glue and the control. CONCLUSIONS: An 8%-GRF glue provides lower toxicity to human aortic smooth muscle cells and elastin with an acceptable degree of polymerisation, and thus seems to be an optimal proportion for GRF glue.

Effects of angiotensin on the expression of fibrosis-associated cytokines, growth factors, and matrix proteins in human lung fibroblasts.

BACKGROUND AND OBJECTIVES: Angiotensin (Ang) II plays an important role in fibrogenesis in various organs, including the lung. The aim of this study is to elucidate (i) the effects of Ang II on the expression of cytokines, growth factors or matrix proteins in normal human lung fibroblasts, and (ii) the inhibitory effects of an Ang II type 1 (AT1) receptor blocker, candesartan. METHODS: Normal human adult lung fibroblasts were cultured. Candesartan was added and the cells were incubated. All the cells in culture dishes were collected at day 0 and 2, and the cell numbers were counted using a Neubauer haemocytometer (Clay-Adams, Parsippany, NJ, USA). The cell proliferation rates at day 2 were calculated in comparison to those at day 0. Total cellular RNA was extracted for real-time quantitative PCR, or the culture supernatant was collected for either a Sircol assay or enzyme-linked immunosorbent assay (ELISA). Laser scanning confocal microscopy was used for analyzing the cells with and without prior exposure to candesartan. Comparisons between the means of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test or Games-Howell's test. Values of P < 0*05 were considered to be statistically significant. RESULTS: Among the 12 fibrosis-associated cytokines and growth factors, mRNA expressions of interleukin (IL)-4, IL-7, and platelet-derived growth factor-D were significantly modulated by Ang II, and suppressed by candesartan. Soluble collagen and elastin levels were significantly elevated by Ang II, and suppressed by candesartan. Under confocal microscopy, the intracellular distribution of elastin was significantly increased by Ang II, and suppressed by candesartan. CONCLUSION: These data indicate that Ang II promotes lung fibrosis by increasing the matrix formation, which was suppressed by AT1 receptor blocker.

Biomechanical characterization of decellularized and cross-linked bovine pericardium.

BACKGROUND AND AIM OF THE STUDY: Although bovine pericardium has been used extensively in cardiothoracic surgery, its degeneration and calcification are important limiting factors in the continued use of this material. The study aims were to decellularize bovine pericardium and to compare the biomechanical properties of fresh and decellularized bovine pericardia to those treated with different concentrations of glutaraldehyde (GA). METHODS: An established protocol for decellularization using sodium dodecyl sulfate was used, and histological analysis performed to validate the adequacy of decellularization. Contact cytotoxicity was used to study the in-vitro biocompatibility of variously treated pericardia. Mechanical testing involved uniaxial testing to failure. Mechanical properties of the fresh and decellularized pericardia (untreated and treated with 0.5% and 0.05% GA) were compared. RESULTS: Histological analysis of decellularized bovine pericardium did not show any remaining cells or cell fragments. The histoarchitecture of the collagen-elastin matrix appeared well preserved. Untreated decellularized pericardium was biocompatible in contact cytotoxicity tests with smooth muscle and fibroblast cells. The GA-treated tissue was cytotoxic. There were no significant differences in the mechanical properties of fresh and decellularized pericardia, but there was an overall tendency for GA-treated pericardia to be stiffer than their untreated counterparts. CONCLUSION: An acellular matrix, cross-linked with a reduced concentration of GA, can be produced using bovine pericardium. This biomaterial has excellent biomechanical properties and, potentially, may be used in the manufacture of heart valves and pericardial patches for clinical application.

Elastin stabilization for treatment of abdominal aortic aneurysms.

BACKGROUND: Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development. METHODS AND RESULTS: Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl2-mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells. CONCLUSIONS: Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs.

Vitamin A and emphysema.

Within the last several years, research scientists and clinicians have been intrigued with the potential use of an active form of vitamin A, retinoic acid (RA), for the treatment and prevention of emphysema. The interest in this area can be largely attributed to the work of Massaro and Massaro (1996, 1997, 2000) in which they presented evidence that RA partially protects against and to some degree restores elastase-induced emphysema in rats. The mechanism for this protective effect of RA is in part related to elastin metabolism. RA also inhibits inflammation, an upstream event that may lead to the development of emphysema. Although there is evidence of this protective effect in young rats and a mechanistic explanation, more studies are needed in humans in order to establish a role for vitamin A in protecting against emphysema. Too many unanswered questions remain to definitively state that vitamin A protects against this disease in humans. Nevertheless, the potential for this novel approach in prevention and treatment of emphysema is an exciting area of research.