Selenium Deficiency Promotes Dilatation of the Aorta by Increasing Expression and Activity of Vascular Smooth Muscle Cell Derived Matrix Metalloproteinase-2.

OBJECTIVE: Selenium (Se) is a key part of the body's oxidation defence system. However, it is unclear whether Se affects the development of aortic aneurysm (AA). An animal experiment was conducted to clarify the role of Se in AA development. METHODS: C57BL/6N male mice were fed with a Se deficient (Se-D, < 0.05 mg/kg), Se adequate (Se-A, 0.2 mg/kg), or Se supplemented (Se-S, 1 mg/kg) diet for 8 weeks. Subsequently, an AA murine model (Se-D, n = 11; Se-A, n = 12; Se-S, n = 15) was established using angiotensin II (Ang II, 1 mg/kg/min) for four weeks plus ß-aminopropionitrile (BAPN, 1 mg/mL) for the first two weeks. Saline replaced Ang II, and BAPN was removed during the modelling process for sham mice (Se-A, n = 9). To determine whether Se deficiency promoted aortic dilation via matrix metalloproteinase-2 (MMP-2), the non-specific MMP inhibitor doxycycline (Dox, 100 mg/kg/day) was given to Se-D AA mice (n = 7) for two weeks. RESULTS: The maximum aortic diameter in Se-D AA model mice was significantly increased compared with Se-A AA model mice. MMP-2 expression and activity in the aortic media of Se-D AA model mice was significantly increased compared with Se-A AA model mice. A large number of vascular smooth muscle cells (VSMCs) were found aggregating in the media of the non-dilated aorta of Se-D AA model mice, which was completely inhibited by Dox. The percentage of VSMCs in aortic media of Se-D AA model mice was significantly higher than in Se-A AA model mice. The maximum aortic diameter and occurrence rate of AA in Se-D AA model mice with Dox were significantly reduced compared with Se-D AA model mice. CONCLUSION: Se deficiency promoted dilatation of the aorta in AA model mice by increasing expression and activity of VSMC derived MMP-2, causing abnormal aggregation and proliferation of VSMCs in aortic media.

A Polymeric Extracellular Matrix Nanoremodeler for Activatable Cancer Photo-Immunotherapy.

Cancer immunotherapy has shown tremendous potential to train the intrinsic immune system against malignancy in the clinic. However, the extracellular matrix (ECM) in tumor microenvironment is a formidable barrier that not only restricts the penetration of therapeutic drugs but also prevents the infiltration of antitumor immune cells. We herein report a semiconducting polymer-based ECM nanoremodeler (SPNcb) to combine photodynamic antitumor activity with cancer-specific inhibition of collagen-crosslinking enzymes (lysyl oxidase (LOX) family) for activatable cancer photo-immunotherapy. SPNcb is self-assembled from an amphiphilic semiconducting polymer conjugated with a LOX inhibitor (ß-aminopropionitrile, BAPN) via a cancer biomarker (cathepsin B, CatB)-cleavable segment. BAPN can be exclusively activated to inhibit LOX activity in the presence of the tumor-overexpressed CatB, thus blocking collagen crosslinking and decreasing ECM stiffness. Such an ECM nanoremodeler synergizes immunogenic phototherapy and checkpoint blockade immunotherapy to improve the tumor infiltration of cytotoxic T cells, inhibiting tumor growth and metastasis.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

The Effect of Polydeoxyribonucleotide Extracted from Salmon Sperm on the Restoration of Bisphosphonate-Related Osteonecrosis of the Jaw.

Bisphosphonates (BPs) used for treating skeletal diseases can induce bisphosphonate-related osteonecrosis of the jaw (BRONJ). Despite much effort, effective remedies are yet to be established. In the present study, we investigated the feasibility of polydeoxyribonucleotide (PDRN) extracted from salmon sperm for the treatment of BRONJ, in a BRONJ-induced rat model. Compared with BRONJ-induced samples, PDRN-treated samples exhibited lower necrotic bone percentages and increased numbers of blood vessels and attached osteoclast production. Moreover, local administration of PDRN at a high concentration (8 mg/kg) remarkably resolved the osteonecrosis. Findings from this study suggest that local administration of PDRN at a specific concentration may be considered clinically for the management of BRONJ.

Pre-clinical evaluation of small molecule LOXL2 inhibitors in breast cancer.

Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.

Wound-healing modulation in upper airway stenosis-Myths and facts.

Wound healing plays a major role in the development of acquired subglottic stenosis. Pharmacologic treatment of subglottic stenosis must address both physiologic and pathologic healing processes. The relevant Pubmed and Ovid databases from 1960 to 2007 were systematically searched. Several modulating agents have been tested. Most of them were poorly investigated. Three modalities were thoroughly studied-steroids and antibiotics, mitomycin, and antireflux medications. However, there are conflicting data regarding their role in preventing and treating subglottic stenosis. Current data support to some extent the textbook suggestions of antibiotics, steroids, and antireflux treatment. As no other treatment options exist, we recommend using these modalities for pharmacologic modulation of subglottic stenosis. Mitomycin should still be considered as an unproven treatment; its use may be considered as an adjunct.

Effects of hydrocortisone and beta-aminopropionitrile on stress-strain and stress-relaxation behaviors, and birefringent retardation of collagen fibers in the rat incisor periodontal ligament.

Three groups of male Wistar rats received daily subcutaneous injections of 10 mg/kg of hydrocortisone (HC group), 300 mg/kg of beta-aminopropionitrile (BAPN group), or saline (control group), for 10 days. The shear stress-strain and stress-relaxation properties of the incisor periodontal ligament were examined in transverse sections from dissected mandibles. Both the maximum shear stress and failure strain energy density increased significantly following the administration of hydrocortisone. The maximum shear stress decreased following the administration of BAPN. However, the stress-relaxations in the initial 10 min did not show significant differences among the three groups. Polarized light microscopic analysis revealed that the retardation value of the collagen fibers was highest in the HC group and lowest in the BAPN group for the bone-related area, but not for the tooth-related and middle areas of the ligament. It is suggested that the changes induced by hydrocortisone or BAPN occurred mainly in the elastic components and to a minor extent in the viscous components although the physical and biomechanical properties are determined by the interaction of all the various components. We also suggest that the main response to the drugs occurred in the collagen fibers in the bone-related area of the ligament.

Collagen fibrillogenesis by chondrocytes in alginate.

Collagen is the primary structural component in connective tissue. The poor mechanical properties of most cell-seeded cartilage grafts used for cartilage repair can be attributed to the low level of collagen synthesized compared with native cartilage. In this study, the synthesis and assembly of collagen by chondrocytes in hydrogels were investigated, with particular attention paid to the role of cross-link formation in this process. Primary bovine chondrocytes were seeded in alginate and collagen synthesis was assessed in the presence and absence of beta-aminopropronitrile (BAPN), a potent inhibitor of the enzyme lysyl oxidase and collagen cross-link formation. Cultures on days 21, 35, and 49 were evaluated by stereology, biochemistry, and real-time reverse transcriptase-polymerase chain reaction. All measures of collagen synthesis (except hydroxyproline) significantly increased in the presence of 0.25 mM BAPN. By 35 days of culture, the average collagen fibril diameter was 62 +/- 10 nm in control cultures and 109 +/- 20 nm with BAPN supplementation. The collagen volume density increased from 5 +/- 3% in control cultures to 17 +/- 1% in the presence of BAPN. Likewise, the expression of cartilage-specific collagens (type II and XI) and aggrecan increased significantly as a result of BAPN culture. These findings demonstrate the prominent role of collagen cross-linking in collagen fibrillogenesis and suggest approaches by which collagen synthesis and assembly could be controlled in tissue-engineered constructs.

[Effects of 764-3 on collagen deposition in hypertensive pulmonary arteries in hypoxic rats].

OBJECTIVE: To investigate the changes of collagen deposition in pulmonary arteries in chronic hypobaric hypoxic rats and the roles of 764-3. METHODS: Wistar rats were divided into 10 groups. The hypoxic (50.7 kPa) rats were treated with 764-3 (20 mg/kg, once daily) and beta-aminopropionitrile (BAPN, 150 mg/kg, twice daily), respectively. At the end of treatment, the hemodynamic morphological and chemical parameters were measured. RESULTS: Comparing with the normals, hydroxyproline (HYP) content of extrapulmonary artery (EPA) increased obviously in hypoxic rats, the smooth muscle cells (SMC) in the media of muscular intra-acinar pulmonary artery showed hypertrophy with change in phenotype, and a striking increment of collagen accumulation (P < 0.001); 764-3 could reduce HYP content of EPA (P < 0.05-0.001), and most of SMC in the media became thin and elongated with reversion of change of SMC phenotype, and collagen accumulation was reduced (P < 0.001). In addition, BAPN was more effective than 764-3 (P < 0.05). CONCLUSIONS: Excessive collagen accumulation in pulmonary arteries plays an important role in the maintenance of hypoxic pulmonary hypertension. 764-3 can partially inhibit collagen deposition in pulmonary arteries, attenuate hypoxic pulmonary hypertension, and has a moderate influence on collagen deposition in compared with BAPN.

Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with inherited connective tissue disorders.

Lysyl oxidase (LO) is an extracellular copper-dependent enzyme that catalyzes the initial reaction in the formation of lysine or hydroxylysine-derived crosslinks during collagen biosynthesis. We have isolated a cDNA for human LO from skin fibroblast poly(A+)RNA by PCR using primers based on the recently published sequence of human LO. This cDNA probe detects a major mRNA of 4.2 kb on Northern blots of RNA from normal fibroblasts. The level of LO mRNA was not significantly affected by cell density or by ascorbate treatment. Treatment of skin fibroblasts with hydralazine (50 microM), which increases the mRNAs for both the alpha and the beta subunits of prolyl hydroxylase (PH) and the mRNAs for lysyl hydroxylase, also increased LO mRNA by fourfold over a 72-h time course. In contrast, hydralazine dramatically decreased the mRNAs for alpha 1(I) collagen. Administration of minoxidil (500 microM), which specifically decreases LH activity without affecting PH activity or collagen biosynthesis in skin fibroblasts, stimulated the level of LO mRNA. Neither the administration of penicillamine (100 microM), which interferes with collagen cross-linking, nor the administration of beta-aminopropionitrile, which is a strong irreversible inhibitor of LO, to fibroblasts significantly changed the levels of LO mRNA over a 72-h time course. However, bleomycin (0.6 microgram/ml) significantly decreased the 4.2-kb LO mRNA in contrast to the levels of the alpha 1(I) collagen mRNAs, which were unchanged. No significant change was observed in the steady-state levels of LO mRNAs in fibroblasts isolated from patients with certain connective tissue disorders, including Marfan syndrome, Menkes disease, cutis laxa, and pseudoxanthoma elasticum.

Unusual occupational toxins.

Occupational and environmental medicine affords encounters with many unusual toxins, ranging from exotic metals to rocket fuels. Twelve of the most unusual industrial toxins are reviewed here and their clinical manifestations and treatments explored: acetonitrile, acrylonitrile, boron hydrides, dimethylaminopropionitrile, dimethylformamide, hydrazines, methyl isocyanate, 2-nitropropane, phosphine, Stalinon, tellurium, and vanadium.

Synergistic vascular toxicity and fatty acid anilides in the toxic oil syndrome.

The underlying etiology of the toxic oil syndrome may be related to any of several toxic contaminants. The hypothesis is made that two or more toxic compounds may act synergistically to cause vascular damage in the toxic oil syndrome. To support this hypothesis, previous studies are reviewed concerning the remarkable synergistic toxic action of allylamine and beta-aminopropionitrile on the media of blood vessels. Although these toxins are not directly related to the toxic oil syndrome, this previous experimental work emphasizes the possibility that unexplored synergistic actions may be important. Furthermore, the hypothesis that contaminating fatty acid anilides in toxic oil undergo alterations during cooking is supported by high pressure liquid chromatographic analysis. The theoretic metabolism of fatty acid anilides is discussed. Recent data concerning the toxic actions of the anilides of oleic and linoleic acid are given. These data suggest that these anilides induce immunologic alterations that may be similar to those seen in the toxic oil syndrome. In addition, the heated anilides appear to have increased toxicity, supporting the concept that the use of toxic oil in cooking may increase its toxicity.

Atherosclerosis mouse model induced by a high-cholesterol diet supplemented with beta-aminopropionitrile: effects of various anti-atherosclerotic agents on the biochemical parameters.

A mouse model of atherosclerosis was produced by feeding a 1.5% cholesterol diet with 0.4% beta-aminopropionitrile (BAPN) fumarate, a chemical lathyrogen, for 10 weeks, and the pharmacological sensitivity and specificity of this model were evaluated biochemically with various hypolipidemic drugs and calcium antagonists. Histological findings on this model showed typical angiolathyrism with foam cells in the media of the thoracic aorta. Uniform and marked accumulation of cholesterol, notably esterified cholesterol, in the aorta was observed, although it was much less in mice receiving a high-cholesterol diet or BAPN alone. The reduction in elastin contents in the aorta was a characteristic feature of this model. Clofibrate, cetaben and elastase tended to prevent the increase of cholesterol contents in the aorta, together with their significant hypocholesterolemic effects. Nifedipine, diltiazem and verapamil showed a slight preventive effect on the cholesterol accumulation and on the reduction of elastin content in the aorta without a cholesterol lowering effect in the serum. MgCl2 was more effective than other calcium antagonists and even had a hypocholesterolemic effect. The results indicate that this mouse atherosclerosis model may be usable for primary drug evaluation.

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid.

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

Spontaneous arterial lesions involving breaks in the internal elastic lamina in the rat: effects of beta-aminopropionitrile and familial distribution.

We have studied the effects of beta-aminopropionitrile (BAPN) administration on the formation of spontaneous arterial lesions, characterized principally by a rupture in the internal elastic lamina (IEL) in the caudal and renal arteries of the Wistar rat. Treatment with BAPN (an inhibitor of lysyl oxidase) increased the formation of these lesions in rats up to 12 weeks of age but had differential effects on the caudal and renal artery in older rats. Administration of the nitrile to weanling rats led to the premature formation of lesions in caudal arteries of both male and female rats which morphologically resemble lesions which form spontaneously later in life. Dietary supplements of copper or pyridoxine were without effect on the formation of spontaneous caudal artery lesions when given from 5 wks of age but a copper supplement from midgestation slightly inhibited lesion formation only in male rats. This suggests that if copper deficiency is involved in spontaneous lesion formation, it is only a contributory factor. Quantification of either caudal or renal artery lesions within different litters of Wistar rats showed that there exists a familial aggregation in the frequency of spontaneous lesion formation, certain litters showing significantly higher levels of lesions than others. Adult Sprague-Dawley rats also appear to be more susceptible to the development of renal artery IEL defects than Wistar rats. The possibility of a hereditary disorder leading to a minor defect in elastic fibre structure which could be responsible for the spontaneous lesions is discussed.

Effects of topical treatment with beta-aminopropionitrile after radial keratotomy in the rabbit.

This study assessed the effect of beta-aminopropionitrile treatment on the long-term curvature and compliance of corneal tissue subjected to radial keratotomy (RK). beta-Aminopropionitrile is known to inhibit cross-linking of collagen and is expected to enhance the flexibility of scar tissue, thereby reducing wound contracture and regression after RK. Seventeen adult New Zealand rabbits, weighing about 4.5 kg each, underwent RK in both eyes (eight incisions, 90% deep, 3-mm pupillary zone). Their mean preoperative corneal curvature was 44.25 diopters (+/- 0.32D at 95% confidence level). Nine of the rabbits were treated topically with beta-aminopropionitrile ointment (33 weight % in petroleum three times daily), while the control group received the petrolatum base only. The remainder of the animals received bland petrolatum gel as a control. The animals were given periodic keratometric examinations and were killed after six to eight weeks. At that time, the beta-aminopropionitrile group showed a mean reduction of 1.85 +/- 0.13 D in corneal curvature, compared with 1.18 +/- 0.08 D in the control group. The compliance and strength of the corneas were measured in vitro immediately after death. In the pressure range from 10 to 40 mm Hg, the beta-aminopropionitrile-treated corneas changed in curvature by an average of 1.4 D as compared with 0.5-D flattening for the controls. These results indicate the effectiveness of beta-aminopropionitrile treatment in enhancing longterm compliance and reducing refractive regression after RK.

Enhancement of radial keratotomy by chemical inhibition of collagen cross-linkages: a preliminary report.

beta-Aminopropionitrile, a lysyl oxidase and collagen cross-linkage inhibitor, resulted in a 0.90 diopter or 20% enhancement of radial keratotomy in rabbits.

An ultrastructural study of fibroblasts derived from bovine ligamentum nuchae and their capacity for elastogenesis in culture.

Fibroblast cultures were readily propagated from fetal bovine ligamentum nuchae. The ligament cells were easily cultured by standard techniques and were maintained in culture flasks for up to 57 days. During this time they accumulated an extensive extracellular matrix which contained the main structural elements of the parent tissue, namely collagen and elastic fibres. Elastogenesis was seen to proceed in two phases: the formation of parallel bundles of 10--12 nm wide microfibrils followed by the deposition within these bundles of amorphous elastin-like material. Elastic fibres were not produced in cultures that were supplemented with ascorbic acid either in the absence or presence of the lathyrogen BAPN.

Detection of teratogenic compounds using differentiating embryonic cells in culture.

Methods are described for screening for teratogenic compounds using differentiating neural crest and prechondrogenic limb bud mesenchyme cells in culture. Substances to be tested are either added directly to the culture medium or are combined in a dialysis bag with the postmitochondrial fraction from rat liver and certain cofactors. In the latter case, the compound and its metabolites are gradually released into the medium from the dialysis bag. The results obtained with 14 compounds demonstrate a positive relationship between teratogenicity in vivo and alterations in the growth or the differentiation of the cultured cells.