Administration Of Beta-aminopropionitrile Research Articles

Collagen Content and Cross‐links Scale with Passive Stiffness in Dystrophic Mouse Diaphragm, but are not Altered with Administration of Collagen Cross‐linking Inhibitor Beta‐aminopropionitrile

In fibrotic skeletal muscle, such as that seen in Duchenne Muscular Dystrophy (DMD), there is upregulation of extracellular matrix (ECM) material, primarily collagen, and a severe change in muscle mechanics that often leads to contracture. Collagen cross-links, which can make the ECM stiffer, are increased in fibrotic models, such as the mdx mouse model of DMD. The objective of this study was to test the effects of cross-link inhibitor beta-aminopropionitrile (BAPN) on skeletal muscle ECM of wildtype (wt) and mdx mice. We hypothesized that BAPN treatment would reduce the amount of collagen cross-links and passive stiffness in skeletal muscle. Wt (N=9) and mdx (N=13) mice were given daily BAPN or phosphate-buffered saline (PBS) injections starting at 16-19 weeks of age for 4 weeks up to sacrifice. Soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DP) muscles were collected for muscle active and passive mechanical measurement and imaged for collagen alignment data. Muscle collagen content and cross-linking were analyzed via hydroxyproline and collagen solubility assay. Across genotypes and muscles, BAPN treatment had no significant effects on collagen content or cross-links. Mdx mice showed reduced specific tension across muscles as expected (Fig 1A). The mdx DP had markedly higher dynamic stiffness than wt (+149%), without genotype differences in EDL and SOL, but BAPN increased stiffness in the mdx SOL (+100%) (Fig 1B). Total collagen content was increased across muscles in mdx mice, with the most dramatic effect again in the DP (+210%) compared to EDL (+62%) and SOL (+30%) (Fig 1C). Mdx mice also had significantly greater collagen cross-linking in DP (+9.3%) and EDL (+38%) muscles as measured by collagen solubility (Fig 2A). The dynamic stiffness of DP muscles was significantly related to total collagen content (R2 = 0.25) (Fig 1D), and cross-linking index of solubility also significantly scaled with dynamic stiffness across the DP (R2=0.19) (Fig 2B). BAPN treatment increased orientation index, a measure for collagen alignment, in wt SOL (+162%), but did not have any other effects on alignment. Orientation index was significantly increased in mdx EDL and SOL compared to wt (EDL: +76%; SOL: +45%), but not in the DP (Fig 2C). Dynamic stiffness across all muscles was significantly related to orientation index (R2 = 0.06), but this relationship was not significant in the DP (Fig 2D). These data show that the relationship between passive muscle stiffness and collagen is muscle-dependent. The DP had the strongest relationships with more collagen and cross-linking related to increased stiffness across conditions while also being the most fibrotic muscle. However, collagen alignment in the DP was not related to stiffness, implicating other factors contributing to stiffness in fibrosis. Overall, administration of BAPN had little or no impact on muscle stiffness, collagen content, cross-linking, and collagen alignment. Thus, the delivery or action of BAPN was not effective in reducing collagen cross-links within muscle.

Cardiomyocyte-specific transgenic expression of lysyl oxidase-like protein-1 induces cardiac hypertrophy in mice

Lysyl oxidase (LOX) and LOX-like protein-1 (LOXL-1) are extracellular matrix-embedded amine oxidases that have critical roles in the cross-linking of collagen and elastin. LOX family proteins are abundantly expressed in the remodeled heart of animals and humans and are implicated in cardiac fibrosis; however, their role in cardiac hypertrophy is unknown. In this study, in vitro stimulation with hypertrophic agonists significantly increased LOXL-1 expression, LOX enzyme activity and [(3)H] leucine incorporation in neonatal rat cardiomyocytes. A LOX inhibitor, beta-aminopropionitrile (BAPN), inhibited agonist-induced leucine incorporation in cardiomyocytes in vitro, suggesting the involvement of LOXL-1 in cardiomyocyte hypertrophy. Abdominal aortic constriction in rats produced left ventricular hypertrophy in parallel with LOXL-1 mRNA upregulation. And BAPN administration significantly inhibited angiotensin II-induced cardiac hypertrophy in vivo. These results suggest a role of LOXL-1 in cardiac hypertrophy in vivo. We generated transgenic mice with cardiomyocyte-specific expression of LOXL-1. LOXL-1 transgenic mice pups were born normally and grew to adulthood without increased mortality; these mice exhibited a greater left ventricle to body weight ratio, larger myocyte diameter, and more brain natriuretic peptide expression than their wild-type littermates. Echocardiography revealed that the LOXL-1 transgenic mice also had greater wall thickness with preserved cardiac contraction. Our results indicate a possible fundamental role of LOXL-1 in cardiac hypertrophy.

The Lysyl Oxidase Inhibitor, β-Aminopropionitrile, Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.

Inhibitors of collagen synthesis and hypophysectomy: effects on androgen induced growth of ventral prostate in rats.

To elucidate the effect of decreased amounts of collagen on prostatic growth, beta-aminopropionitrile, one of the agents that selectively inhibit collagen formation, was administered, with or without exogenous androgen and/or hypophysectomy, to rats of varying ages. The weight of the ventral prostate and the collagen content and the ratio of type III to type I collagen in the ventral prostate were examined. Administration of beta-aminopropionitrile, with or without exogenous androgen, reduced the collagen content in the ventral prostate in any age groups, but increased the weight of the ventral prostate only in young rats. Administration of beta-aminopropionitrile to hypophysectomized rats did not change the weight and collagen content. Administration of beta-aminopropionitrile prevented androgen-induced growth of prostatic collagen, resulting in increased prostatic weight in young rat. The ratio of type III to type I collagen in the rat ventral prostate remained unchanged by administration of beta-aminopropionitrile, exogenous androgen, hypophysectomy, or advancing age. It would appear that in young rats prostatic collagen plays an important role in suppressing prostatic growth. The ratio of type III to type I collagen is stable under various experimental conditions in the ventral prostate.

Type II collagen during cartilage and corneal development: immunohistochemical analysis with an anti-telopeptide antibody.

We have examined the pattern of immunoreactivity of a monoclonal antibody, II-5B2, with specificity for an epitope which resides within the NH2-terminal extension peptide (telopeptide) of the avian type II collagen molecule. This epitope is available in regions of matrix where de novo synthesis of the molecule is ongoing, but not where synthesis has ceased and maturation and crosslink formation have occurred. Within the cartilaginous growth plate, the epitope disappears from the matrix soon after the chondrocytes become hypertrophic; within the cornea, the epitope disappears subjacent to the epithelium. The II-5B2 epitope is not made available by a variety of procedures shown to remove potentially masking substances and to disrupt fibrillar organization. It is rendered available, however, when covalent crosslink formation between collagen molecules is blocked through administration of beta-aminopropionitrile or penicillamine. In contrast, the epitope of another monoclonal antibody against type II collagen, II-II6B3, which resides in the triple-helical domain of the molecule, in cartilage is present throughout the growth plate including the hypertrophic zone, and in cornea extends for a considerable distance into the stroma. Thus, it is available for antibody binding regardless of fibril maturation and crosslinking. These data suggest that the II-5B2 epitope becomes unavailable when the telopeptide becomes crosslinked. By using these two monoclonal antibodies in serial sections, one can establish the crosslinking pattern of type II collagen in the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung lysyl oxidase and prolyl hydroxylase: increases induced by cadmium chloride inhalation and the effect of beta-aminopropionitrile in rats.

Industrial workers who are accidentally exposed to cadmium fumes often develop severe lung damage leading to widespread peribronchiolar scarring. This study examined the effect of a single exposure of cadmium chloride aerosol on rat lung lysyl oxidase and prolyl hydroxylase, both markers of connective tissue biosynthesis. Rats were killed at 2, 4, 7, 10, and 21 days after a 2-h exposure to 0.1% Cdcl2 aerosol. Total lung lysyl oxidase was increased 14.8 times that a saline control animals by 4 days and returned to near normal values by 10 days. Interestingly, a small amount of lysyl oxidase activity was also detectable in the lung lavage of unexposed animals and was markedly elevated at the earlier times after cadmium exposure. Total lung prolyl hydroxylase activity paralleled that of lung lysyl oxidase and increased 7.4-fold by the fourth day. A significant increase in total lung hydroxyproline could be demonstrated. Administration of beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, prevented much of the increase in lysyl oxidase activity and the accumulation of collagen. The altered tissue amounts of lysyl oxidase and prolyl hydroxylase after cadmium inhalation correlated well with the marked interstitial cell hyperplasia 4 to 5 days after exposure and suggested that connective tissue protein synthesis is activated in the interstitial cell fibroblasts soon after cadmium exposure.

Effect of metabolic alteration of periodontal fibers on orthodontic tooth movement: An experimental study

In the present study orthodontic force was applied to the molars of rats treated with the lathyrogen beta-aminopropionitrile (BAPN). New bone formation was measured at two alveolar locations after 9 days of force application. Observation resulted in the following conclusions: 1. New alveolar bone formation in response to orthodontic force in BAPN-treated rats statistically exceeded corresponding bone formation in control animals when measured at two tension sites in the periodontal ligament. 2. BAPN administration produced disorganization of the collagenous fibers of the periodontium of experimental animals. Multiple eosinophilic cell-free areas were found distributed throughout the radicular portions of affected periodontal ligaments. Normal ligament function architecture was disrupted in treated animals. The areas of periodontium surrounding orthodontically treated teeth exhibited relatively normal organization under these conditions, while the periodontium of adjacent nonorthodontically treated teeth was markedly disorganized. Orthodontic stimulation of the periodontium of BAPN-treated rats may have disrupted the formation of eosinophilic cell-free areas characteristically seen in the periodontium of the experimental group. 3. The present results suggest that the typical histologic response to orthodontic force application can occur in the presence of a chemically and physically altered periodontium. The quantitative data collected infer that fiber tension on the alveolus may not be absolutely necessary to stimulate bone formation. Distortion of the alveolus related to force application may be a more important factor initiating bone response. However, the fibers of the periodontium may play a passive role in transferring orthodontic force to the alveolus.

Beta-aminopropionitrile as a radiation reaction preventive agent.

Beta-aminopropionitrile (BAPN), an inhibitor of collagen maturation, was tested as a radiation reaction preventive agent (RRPA) using radiation-induced lung fibrosis in rats and mice as a model. Following pulmonary irradiation, treatment with BAPN significantly prevented an increase in collagen content as measured by hydroxyproline analysis. This effect persisted during BAPN maintenance for periods of up to 10 weeks but was lost when the drug was discontinued. BAPN administration did not increase the mouse lung LD50/160 in the drug doses employed in this study. Other possible RRPAs deserving further study include D-penicillamine and beclomethasone dipropionate.

Remodelling of periosteal lesions induced by surgical stimulation or beta-aminopropionitrile administration.

Remodelling of periosteal lesions induced by surgical stimulation or beta-aminopropionitrile administration.

Influence of beta-aminopropionitrile on dissecting aneurysm and on plasma amino acids in the Turkey

Increasing levels of beta-aminopropionitrile (BAPN) from 0 to 1.17 per cent in the diet of turkeys 6 weeks of age decreased blood plasma free proline and alanine and increased valine, isoleucine and leucine, suggesting that altered circulating amino acid levels should be considered as a possible causative factor in the weakening of connective tissue due to BAPN administration. Aortic rupture occurred within 2 days after BAPN feeding was begun, indicating a rapid weakening of aortal connective tissue. Determination of BAPN in plasma using an amino acid chromatography-ninhydrin reaction procedure suggests that dietary fish meal results in higher circulating BAPN levels.