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Objectives: In the current study, we aimed to investigate the effect of administration of resveratrol (RES) and beta-aminopropionitrile (BAPN) separately and together on the liver fibrosis progression via regulation of the gene expression and protein level of lysyl oxidase (LOX).Materials and methods: The six-week old Wistar rats received carbon tetrachloride (CCl4) intraperitoneally and RES and BAPN were administrated orally for eight weeks. The hepatoprotective effects of RES, BAPN, and combination treatment were evaluated. Then the hepatic protein and gene expression levels of LOX were measured.Results: Both RES and BAPN showed the antifibrotic effect through the reduction of collagen fiber bundles, hepatic hydroxyproline content, and protein level of LOX. The antifibrotic effect increased when RES and BAPN up-taken together.Conclusion: The co-administration of RES and BAPN can be considered as a promising therapeutic approach via targeting LOX.

Introduction: Thoracic Aortic Dissection (TAD) are associated with matrix changes and biochemical changes in the aortic wall. Several studies proved a relationship between inflammatory markers, in example IL-1s, and these matrix changes. However, the exact mechanism is yet still unknown. Therefore, we investigated the role of IL-1s, MMP-2, MMP-9, smooth muscle cell (SMC) apoptosis and elastic fiber fracture in the development of TAD in rat. Methods: TAD was investigated in 96 male Sprague-Dawley rats which were divided in 4 groups equally (Control, BAPN, BAPN IL-1s and BAPN IL-1s antibody). Rats were monitored for 6 weeks in which systolic blood pressure, survival and the development of TAD were measured. After termination aortic tissue was harvested. Expression of IL-1s, MMP-2 and MMP-9 was measured by western blot. Apoptosis was measured by TUNEL assay and aorta elastin concentration by Victoria blue staining. Additionally, biomechanical characteristics were measured in-vitro. Results: Blood pressures were equal between groups. Mortality differed significantly between groups (control 0%, BAPN; 54.2%, BAPN IL-1s 75% and BAPN anti-IL-1s 37.5%), as was the occurrence of TAD (control 0%, BAPN 45.8, BAPN IL- 75%, anti-IL-1s 37.5%). Aneurysm diameters varied between groups (control 1.13±0.05mm, BAPN 3.48±0.42mm, BAPN IL-1s 5.33±0.42mm, BAPN anti-IL-1s 2.4±0.35mm). Relative elastin area to the total vascular wall significantly differed between groups (control 6.53±0.58%, BAPN 3.47±0.50%, BAPN IL1s 2.98±0.57%, BAPN anti-IL-1s 3.87±.058%) Conclusion: In this study IL-1s may have led to increased formation of TAD. On the contrary anti-IL-1s therapy significantly reduced death in rats, which may be explained by a reduction in the expression of MMP-2, MMP-9 and degradation the aortic wall matrix. These results can be seen as the basis for selective drug treatment of TAD. Funding Statement: This work was supported by National Natural Science Foundation of 15 China (81200225) and Talent Development Fund of Shanghai(2017038) Declaration of Interests: "None declared." Ethics Approval Statement: All the procedures on the rats were approved by the Ethics Committee for Animal Experimentation and conducted according to the Guidelines for Animal Experimentation of our institutes.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p, Highlights ► Alterations in collagen cross-links have previously been reported in humans sustaining fracture. ► An animal model of lathyrism was employed. ► An array of analytical techniques was used to determine structural and material properties of bone. ► Even anatomically localized changes in collagen cross-links affect bone strength, independent of mineral characteristics.

Lysyl oxidase (Lox) is a copper-dependent amine oxidase that catalyzes the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM). In mammals, four closely related Lox-like enzymes have been described that share a highly conserved catalytic domain with Lox. We have characterized Xenopus laevis cDNAs for Lox, Loxl-1, and Loxl-3, and show that they are expressed during early embryonic development. Using RT-PCR we detected maternal transcripts for Xloxl-1, but levels remained low until tailbud stages. Transcripts for Xlox and Xloxl-3 were not detected until early neurulae, although transcripts for Xlox remained at low levels until tailbud stages. Whole mount in situ hybridization showed that transcripts for Xloxl-1 and Xloxl-3 are localized in the notochord, while transcripts for Xlox are found in the notochord, somites, and head. X. laevis Lox-like enzymes were inhibited by incubating embryos, from cleavage stages to tadpole stages, in beta-aminopropionitrile, a specific inhibitor of the catalytic domain. The resulting embryos appeared to differentiate normally but suffered from poor collagen fiber formation. Defects included kinks in the notochord, a posterior shift of the somites, abnormal gut coiling, and the formation of edemas. Our data suggest that Lox-related enzymes are required for the proper formation of the ECM during X. laevis development.

Summary Objective Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies. Methods Explant cultures of adult bovine articular cartilage were radiolabeled with [ 14 C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, β-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking. Tissue was hydrolyzed and analyzed for [ 14 C]metabolites in the forms of lysine, hydroxylysine, dehydrodihydroxylysinonorleucine (ΔDHLNL), and hydroxylysyl pyridinoline (HP). Results Explant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, ΔDHLNL and HP. During chase, [ 14 C]hydroxylysine maintained steady-state levels, [ 14 C]DHLNL rose to a plateau, and [ 14 C]HP increased gradually. Addition of BAPN inhibited formation of [ 14 C]DHLNL. Analysis of raw data and that normalized to [ 14 C]hydroxylysine gave characteristic time constants for formation of ΔDHLNL and HP crosslinks of 1–2 and 7–30 days, respectively. The distribution of [ 14 C]lysine metabolites in collagen crosslinks was described by peak values in [ 14 C]DHLNL/[ 14 C]hydroxylysine of 0.047–0.064 and in [ 14 C]HP/[ 14 C]hydroxylysine of 0.03. Conclusion Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.

In vitro reactivity for each of four osteolathyrogens with a model compound for the lysyl oxidase (LO) cofactor was evaluated and coupled with mixture toxicity testing to evaluate agent-cofactor reactivity as a potential mechanism of action for osteolathyrism. Reactivity of the model cofactor (mLTQ: 4-butylamino-5-methyl-o-quinone), with each of two ureides, semicarbazide (SC) and thiosemicarbazide (TSC), and each of two aminonitriles, aminoacetonitrile (AAN) and beta-aminopropionitrile (betaAPN), was assessed using UV-vis spectrophotometry; both in the absence and presence of Cu(II)-bipyridine (bipy) complex. Two sets of mixture toxicity experiments were conducted using a frog embryo assay that assessed the incidence of osteolathyrism in the notochord of tadpoles after 96-h exposure. The resulting concentration-response curves for each set were evaluated (chi(2) goodness-of-fit test) against theoretical curves for two combined effects models: dose-addition and independence, to determine the combined effect of each osteolathyrogen combination. The agents SC, TSC and AAN each showed rapid, irreversible reactivity with mLTQ, both in the absence and presence of Cu(II)-bipy complex, as indicated by bleaching of the mLTQ peak (504 nm) and formation of an adduct at 350 nm. betaAPN showed no apparent reactivity in the absence of prolonged incubation with mLTQ, whether Cu(II)-bipy complex was present or not. After prolonged incubation (24-144 h) a new peak formed at 350 nm, suggesting that betaAPN reacts weakly with the cofactor, but in a manner different from the other agents examined. The toxicity tests indicated a dose-additive combined effect for the SC:TSC, AAN:SC and AAN:SC:TSC mixtures (0.1P0.9; with slope and EC(50) additivity quotient values between 0.93-1.14), suggesting these agents induce osteolathyrism via a common mechanism. The betaAPN:SC and betaAPN: SC:TSC mixtures showed a greater-than dose-additive combined effect (P0.001; slope and EC(50) additivity quotient values from 0.74 to 0.89), suggesting a different or additional mechanism of osteolathyrism for betaAPN. Taken together the results indicate that SC, TSC and AAN induce osteolathyrism primarily, if not completely, by binding to the LO cofactor, thereby inhibiting proper connective tissue fiber cross-linking. While betaAPN also has the potential to react with the cofactor, the nature of the reactivity observed suggests that betaAPN-cofactor binding is, at best, a secondary mechanism for induction of osteolathyrism in vivo.

Collagenase was injected into the Achilles tendon of both hind legs of 10 clinically normal adult male New Zealand white rabbits. One month after induction of the injury, beta-aminoproprionitrile (BAPN) or hyaluronic acid (HA) was injected into the tendon core of the right hind leg of each rabbit, the left hind leg being left untreated. The treatment effects were evaluated by electron microscopy and analysis of the glycosaminoglycan (GAG) content of samples at 2 and 6 months post-treatment. At 2 months, collagen fibrils in tendons from both hind legs were relatively small in diameter, irregularly arranged, and interspersed with abundant active tenocytes as compared with those in normal tendon uninjured by collagenase. In the matrix, the amount of HA increased, but chondroitin-6-sulphate was eliminated. At 6 months, BAPN-treated tendons had small-diameter, regularly arranged collagen fibrils. HA-treated tendons, on the other hand, had large diameters, as well as regularly arranged collagen fibrils by comparison with non-treated tendon. The results suggest that HA, unlike BAPN, promoted healing.

Aortic dissection, as a life-threatening cardiovascular disease, is characterized by remodeling of extracellular matrix (ECM) and inflammatory response. ADAMTS1 (A Disintegrin and Metalloproteinase with Thrombospondin Motifs 1) is able to degrade ECM proteins versican and aggrecan in blood vessels

Objective—To investigate effects of β-aminopropionitrile and a combination of insulin-like growth factor (IGF)-I and β-aminopropionitrile on metabolism of equine tendon fibroblasts. Sample Population—Flexor tendon explants from 3 horses. Procedure—Explants received 1 of 4 treatments (control, IGF-I, β-aminopropionitrile, and IGF-I/β-aminopropionitrile) for 10 days, and message expression for collagen types I and III was assessed by use of in situ hybridization. Histologic findings, new protein production, and quantitative determinations of glycosaminoglycan, DNA, and de novo collagen synthesis were made. Results—Insulin-like growth factor-I stimulated an anabolic response in tendon. Collagen synthesis and glycosaminoglycan and DNA content of explants were all increased. β-Aminopropionitrile significantly suppressed collagen synthesis, which was not ameliorated by concurrent IGF-I treatment. β-Aminopropionitrile caused alterations in cell morphology characterized by large round cells with eccentric nuclei and decreased density of collagen fibers. Protein production and collagen type-III mRNA expression were reduced in these cells. Conclusion and Clinical Relevance—Treatment with β-aminopropionitrile resulted in decreased production of protein and collagen synthesis, which could be expected to suppress tendon healing. The negative effects of β-aminopropionitrile could not be abrogated by addition of IGF-I to the medium. Treatment resulted in alterations in cell morphology and matrix consistency, which could further delay tendon healing. β-Aminopropionitrile may impair tendon healing at a cellular level by decreasing collagen production or increasing rate of degradation of existing matrix. Because of reduced crosslinking during β- aminopropionitrile treatment, in combination with transiently decreased tensile strength, alterations in collagen content and structure may weaken the healing tendon. (Am J Vet Res 2001;62:1557–1562)

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Collagen, a naturally birefringent biopolymer and key structural component of the periodontal ligament (PDL), is altered substantially with regard to its molecular cross-linked structure by the dietary lathyrogen, beta-amino-proprionitrile (beta-APN). Our purpose in studying beta-APN-fed animals was to learn if the strength of birefringence of periodontal collagen, measured microscopically by the Senarmont compensator method, correlates with the molecular changes known to occur in these lathyritic animals, and to explore this technique for the periodontal ligament. Five experimental animals were fed a diet containing 0.25% beta-APN for two weeks, and 3 control animals were fed normal rat chow. Tissues were decalcified and the mandibular incisors were cut in cross section at 4 microns and stained with eosin Y. The Senarmont compensator, an attachment for the polarizing microscope, was used to measure phase retardation. Ten measurements were taken from each of the three sides (mesial, lateral and lingual) of the triangularly shaped incisor sections. The collagen fibers of the beta-APN-fed rats had lower values of phase retardation than the controls (p0.001), indicating reduced molecular organization. In addition, minor but significant regional differences were revealed, supporting the method for structural studies on the periodontal collagen.

Objective. The aim of this study was to investigate the efficacy of beta-aminopropionitrile (BAPN) and prednisolone on the prevention of esophageal damage and stricture formation after caustic esophageal burn. Method. Twenty-eight rats were divided into four equal groups. In groups 1, 2, and 3, caustic esophageal burns were generated by applying NaOH to the 1.5 cm segment of the abdominal esophagus. Group 4 was for the sham. Normal saline to group 1, BAPN to group 2, and prednisolone to group 3 were administered intraperitoneally as a single daily dose. Results. Treatment with BAPN decreased the stenosis index (SI) and histopathologic damage score (HDS) seen in caustic esophageal burn rats. The SI in group 4 was significantly lower compared with groups 1, 2, and 3. Group 2 had the minimum SI value in corrosive burn groups. The differences related to SI between groups 1, 2, and 3 were not statistically significant. The HDS was significantly lower in group 4 compared with groups 1, 2, and 3. The HDS in group 2 was significantly lower compared with groups 1 and 3. Conclusion. This study demonstrated that BAPN was able to decrease the development of stenosis and tissue damage better than prednisolone.

This study was designed to assess the potential relationship between the late loss of angiographic luminal diameter and biochemical abnormalities of arterial wall collagen in rabbits subjected to angioplasty, and to test the hypothesis that beta-aminopropionitrile (beta APN), an inhibitor of lysyl oxidase, would inhibit such changes when administered orally for 1 mo after angioplasty. Endovascular injury was induced in rabbit iliac arteries by ipsilateral balloon angioplasty (BA) and by contralateral balloon angioplasty accompanied by exposure to continuous wave neodymium: yttrium aluminum garnet laser radiation (LBA). Computer measurement of angiographic luminal diameter demonstrated significant vessel narrowing at 1 and 6 mo after both procedures. By quantitative histology, the majority of the 1-mo loss in angiographic diameter could not be attributed to neointimal thickening. Analysis of collagen cross-linking by HPLC in collagen obtained from the LBA-injured segments of the arteries 1 mo after angioplasty revealed a significant increase, relative to values from uninjured arteries (P < 0.05), in the difunctional cross-link dihydroxylysinonorleucine (DHLNL). 6 mo after angioplasty, the content of hydroxypyridinium, the trifunctional maturational product of DHLNL, was significantly elevated in both BA- and LBA-treated arteries compared with values from uninjured arteries (P < 0.05). In animals administered beta APN, luminal narrowing at 1 mo, compared with controls, was attenuated (P < 0.01) and DHLNL content was decreased (P < 0.05) in arteries subjected to LBA, but not in arteries subjected to BA. The results suggest that lathyrogenic agents may be efficacious in favorably modulating LBA-induced alterations in vessel diameter and mural connective tissue.

β-aminopropionitrile (BAPN), a compound that inhibits crosslinking of collagen, has been widely used to examine the function of collagen in developing tissues. We used BAPN to examine the function of collagen during the formation and patterning of feather rudiments in embryonic chicken skin. Organ cultures of skin were treated with different concentrations of BAPN and examined for changes in morphology, histology, and extent of collagen crosslinkage. Increasing concentrations of BAPN led to striped patterns of feather rudiments, increases in feather rudiment diameter, and increases in skin thickness in cultured skin. These changes in morphology were paralleled by a progressive decrease in collagen crosslinkage as the concentration of BAPN was increased. However, these changes were also paralleled by an increase in cell death in the epidermis. Culture of monolayers of separated epidermal and dermal cells in the same concentration range of BAPN that altered feather development in the organ cultures showed that BAPN killed epidermal cells but not dermal cells. Our reults suggest that developmental alterations caused by BAPN may, in some cases, be due to acute toxicity to specific cell types, and not simply to inhibition of collagen crosslinkage. © 1994 Wiley-Liss, Inc.

Background We previously described the production and clinical outcomes of tissue-engineered buccal mucosa (TEBM) used to treat recurrent urethral strictures. In this study, two patients developed a recurrent stricture and there was also evidence of graft contraction. Objective Assess possible preclinical methods to reduce contraction of TEBM. Design, setting and participants Using the model of TEBM in use clinically (ie, oral keratinocytes and fibroblasts cultured on de-epidermised acellular dermal scaffold), three methods of reducing TEBM contraction were investigated in vitro. Interventions The techniques assessed were pretreatment of de-epidermised dermis (DED) with glutaraldehyde, culture with β-aminopropionitrile (β-APN; a lysyl oxidase inhibitor), and physical restraint of TEBM grafts during culture. Measurements Contraction was assessed using serial digital image analysis. The cytotoxicity of the pharmacologic manipulations was assessed using monolayer cultures of oral mucosa cells. Results and limitations Control TEBM lost a mean of 45.4% of its original surface area over 28 d of culture. Treating TEBM with glutaraldehyde, β-APN, or mechanical restraint during culture all significantly inhibited graft contraction. Glutaraldehyde treatment was most effective (only 5.5% loss of area with 0.1% glutaraldehyde), followed by mechanical restraint for at least 7 d (21.4% loss of area), and then β-APN (28.7% loss of area). None of the treatments had any significant effect on cell viability. This in vitro study identifies solutions for graft contracture to explore in the clinic. Conclusions Glutaraldehyde pretreatment and restraint of TEBM grafts during culture both reduce graft contraction.

To study the relationship between the mechanical properties of collagen and the bone turnover, 2-week-old Balb/C mice were fed on a diet containing 0.25% Beta aminopropionitrile (B-APN), a potent inhibitor of collagen crosslink formation, for 3 weeks. Mandibular incisor socket was selected for the analysis of bone formation and resorption parameters. Plastic embedded sections stained with toluidine blue and cut at 4 microns were used to analyze the average area of bone lamellae, bone-forming surface, and the number of osteoblasts/mm of forming surface. Similar sections were used to localize acid phosphatase on resorbing surfaces and within the osteoclasts, while bone alkaline phosphatase was determined by a colorimetric method. Morphometric analyses showed that the area of newly formed bone lamellae, total forming surface, number of osteoblasts and the Alk. Pase activity were significantly lower in B-APN-fed mice as compared to the controls. There was a concomitant smaller, but significant, reduction in total resorption surface, active resorption surface and the number of osteoclasts. These results suggest that the regulation of bone formation and resorption at this site, which is independent of systemic regulation, is influenced by the mechanical properties of the collagenous matrix, which in turn may have a significant effect on the existing pool of bone-forming cells, but may not influence the recruitment of new cells.

A chronological account of observations on the nature of the shell membrane protein system is presented. The review indicates that although the major protein in the shell membrane is not elastin, i...

In two experiments, commercial laying pullets and hens were fed diets containing either low or high concentrations of beta-aminopropionitrile (BAPN), the toxin present in some legume seeds in the genus Lathyrus. BA PN was fed as mono-beta-aminopropionitrile fumarate. The main effects of feeding BAPN to laying birds appear to be on shell formation, egg weight and egg production. Heavier eggs, abnormal in shape and shell texture, were frequently obtained when BAPN was included in the diet. Effects on both egg weight and egg malformation were observed with or without a reduction in egg production. The toxic level of BA PN fumarate in the diet of laying birds appears to lie between 0.1 and 0.2 g kg−1. Considering BAPN content in seeds of different Lathyrus species and the level of active constituent of BAPN fumarate which had adverse effects, it was found that Lathyrus seeds which contain BAPN as a toxic constituent have margins of safety and could be utilised to constitute a proportion of the layer diets without any deleterious effects.

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.

Wound contraction is a clinically important biological process because it frequently results in contractures, strictures, and stenosis. If collagen synthesis could be altered to minimize the contracture, then the outcome could be improved. Lathyrism produces poorly cross-linked collagen in healing anastomosis, keeping a larger portion of the synthesized collagen soluble. Ultimately, the amount of contracting collagen is reduced, lowering the bulk and lessening the contracture. The aim of this study was to observe the effects of a lathyrogen, beta-aminopropionitrile (BAPN), on the healing of colonic anastomosis. Thirty rats were divided into three groups. Colostomy and anastomosis were performed on all rats. Intraperitoneal saline solution (control) and either intraperitoneal (ip) or oral (po) BAPN were administered. The rats were killed 1 week later. Anastomotic healing was assessed by bursting pressure and the hydroxyproline content of the anastomotic tissues. Granulation tissue thickness, number of fibroblasts, inflammatory cells, and growing capillaries in granulation tissue per unit area were determined. Collagen fibril diameters were estimated, and spatial arrangements of fibrils were examined by an electron microscope. All results were evaluated by Mann-Whitney U-test. The analyses of anastomotic tissues from BAPN-treated rats showed a significantly reduced mean bursting pressure (158.9 +/- 12.3, 171.3 +/- 13.9, ip and po, respectively), hydroxyproline content (8.9 +/- 2.6, 10.1 +/- 2.7), granulation tissue thickness (24.3 +/- 2.6, 16.1 +/- 5.2), number of inflammatory cells (37.8 +/- 4.3, 25 +/- 4.3), fibroblasts (3.2 +/- 1.1, 2.8 +/- 0.7), and a significantly reduced collagen fiber diameter (15 +/- 2, 20 +/- 3) compared with those of control group (236.9 +/- 9, 14 +/- 4.4, 26.8 +/- 4.8, 39 +/- 2.6, 6.9 +/- 1.1, and 35 +/- 5, respectively). As a result, collagen fibers were flimsy, and lost their regular parallel alignment in the BAPN groups. On the other hand, a number of growing capillaries were found to be significantly increased in these groups (16.5 +/- 1.1, 18.2 +/- 0.7) compared to the control (6.7 +/- 1.3). Thus, it is suggested that BAPN may be useful in the prevention of gastrointestinal stricture formation.

Introduction Autologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells’ immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues. Methods Mitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA). Results Co-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α. Conclusions Equine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.

Fibroblasts derived from the involved skin of scleroderma patients frequently display a phenotype of supernormal collagen expression when cultured. Fibroblasts displaying this phenotype derived from seven patients were treated with relaxin (1-100 ng/ml) and interferon-gamma (1-100 U/ml), individually and in combination, to assess the relative abilities of these cytokines to down-modulate collagen synthesis and secretion. Scleroderma fibroblasts displayed varying sensitivities to both relaxin and interferon-gamma. Relaxin (100 ng/ml) decreased expression of collagen by six of seven lines tested from 8 to 59% compared to untreated cultures. Interferon-gamma (100 U/ml) depressed collagen secretion by all seven lines in a range from 7 to 89%. When relaxin and interferon-gamma were used in combination, relaxin augmented IFN-gamma-induced decreases in collagen secretion in four of seven lines. In three of these lines, the use of relaxin in conjunction with suboptimal doses of interferon-gamma resulted in decreases equivalent to or greater than that seen with a tenfold higher concentration of interferon-gamma. This study demonstrates the ability of relaxin to directly alter the excessive collagen-producing phenotype of scleroderma fibroblasts. In addition, in some cases, combining relaxin and interferon-gamma resulted in a cooperative effect in decreasing collagen expression by scleroderma cells in vitro.

A hypothetical basis for control of surface scar in human beings is: lathyrism produces poorly cross-linked collagen in healing wounds; poorly corss-linked collagen is more susceptible to digestion by tissue collagenase than is normally cross-linked collagen; and colchicine stimulates tissue collagenase activity. Therefore, treatment of patients with abnormal deposits of surface scar by excising the scar, inducing lathyrism, and administering colchicine should tend to correct abnormal balance between collagen synthesis and collagenolysis and result in a small scar with improved physical properties. Ten patients with massive keloids, resistant to conventional therapy by excision, grafting, and/or intralesional injection of steroids, have been treated by excising the keloid, grafting the defect, inducing lathyrism with Beta aminopropionitrile fumurate or penicillamine and administering colchicine. Patients were followed for 18 months to five years. No toxicity or untoward side effects from therapy were observed. No patients developed recurrent keloids while undergoing treatment. All patients showed some change in the amount of scar which persisted during the period of study. This data supports the hypothesis that lathyrism and colchicine therapy exert a measurable beneficial effect on surface scar in human beings.

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.

Bones from 2-week-old chicks, fed a diet containing 0.05% β-aminopro-pionitrile (β-APN), were studied for 3H-tetracycline and β-proline incorporation at 7, 14 and 21 days of the experiment. Liquid scintillation counts showed a comparable amount of 3H-proline incorporation in the controls and experimentals. However, the incorporation of 3H-tetracycline was significantly lower in experimentals at all intervals. Fluorescent microscopy after cold tetracycline injections at similar intervals showed a comparable amount of linear bone apposition in controls and β-APN-fed chicks. The bone ash content in β-APN-fed chicks was lower at all intervals whereas the serum calcium level was similar to the controls. Microradiography showed that cortical and marrow areas, in cross sections, were not significantly different in the controls and β-APN-fed chicks. On the basis of these data it is suggested that β-APN at the dose used does not inhibit the bone matrix synthesis but inhibits its mineralization.

Authors examined the effects of B -aminopropionitrile (BAPN) and prednisolone, added to the drinking water at 1 mg/ml and 2μg/ml, respectively, on survival of 2-month old male LAF/J mice. In one experiment BAPN, prednisolone, or both were administered throughout the entire lifespan, with no significant differences from control in either mean lifespan, 28–29 months, or maximum lifespan, 33–35 months. In a second experiment, BAPN was administered for 0, 3, 6, 9 or 12 months only. Median lifespan of 26 months for control animals was significantly increased for 3 of the 4 test groups, the animals treated with BAPN for 12 months showing the greatest increase, i.e. median 31 months. Maximum lifespan of control animals was 33 months, and was 1–3 months longer for BAPN treated groups.

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.

Antibodies raised in rabbits to tropoelastin isolated from lathyritic chick aorta were conjugated with ferritin and used to identify and locate elastin-containing elements in sections of aortic tissue from developing chicks. The ferritin-antibody conjugate was associated with a network of fine filaments with a diameter of 3-5 nm which, in the 8-10 day old chick embryo aorta, was distributed between and around small but recognizable elastic fibers. In older aortic tissue this ferritin-labelled material was seen principally in loose association with the periphery of developing elastic fibers. The microfibrils did not interact with the ferritin-antibody conjugate. Treatment of chicks with beta aminopropionitrile did not interfere with the accumulation of ferritin-labelled filaments on these small elastic fibers, suggesting that the mechanism of addition of new material onto growing fibers may be independent of cross-linking.

Lysyl oxidase activity was measured in aortas of spontaneously hypertensive (SHR) and deoxycorticosterone acetate-salt (DOCA-salt) induced hypertensive rats. Lysyl oxidase, the enzyme which catalyzes the initial step in cross-linking of collagen and elastin, was significantly elevated in aortas of both models of hypertension. Aortic collagen synthesis was increased in both models and collagen was significantly elevated in aortas of DOCA-salt hypertensive rats. Administration of β-aminopropionitrile (BAPN), an inhibitor of lysyl oxidase, before the onset of DOCA-hypertension prevented the development of hypertension and reduced lysyl oxidase and collagen content to control values. These results suggest that lysyl oxidase is instrumental in determining levels of aortic collagen content and that inhibitors of collagen and elastin crosslinking may be effective antihypertensive agents.

Um den Einflus vonD-Penicillamin (DPA) undβ-Aminopropionitril (BAPN), sowie einer Kombination beider Substanzen (Gewichtsverhaltnis 1:1) auf das Kollagen der Haut, der Aorta und der distalen Femur-Epiphysenfuge zu untersuchen, wurden die mechanischen Eigenschaften dieser bindegewebigen Organe uberpruft, nachdem Untersuchungen zum Wirkungseintritt und Abklingen der Wirkung, gemessen an der Abnahme der Reiskraft exzidierter Hautstreifen, mit den beiden Einzelverbindungen durchgefuhrt waren. Die Wirkung von 320 mg/kg DPA tritt innerhalb von 7 Tagen ein und hat nach 14 Tagen ihr Maximum erreicht. Nach Absetzen der Substanz werden innerhalb von 15 Tagen die Kontrollwerte erreicht. BAPN erreicht am 18. Versuchstag seine maximale Wirkung. Nach Absetzen der Behandlung wird innerhalb von 35 Tagen der Kontrollwert nicht erreicht. DPA and BAPN wirken organspezifisch: DPA beeinflust das Kollagen der Haut wesentlich starker als das Kollagen der Aorta und hat unter den vorliegenden Versuchsbedingungen keinen Einflus auf das Kollagen der distalen Femur-Epiphysenfuge. BAPN beeinflust verglichen mit DPA in geringerem Mase das Kollagen der Haut, etwa gleich stark das Kollagen der Aorta und stark das Kollagen der distalen Femur-Epiphysenfuge. Werden beide Substanzen gleichzeitig verabreicht, bleiben die organspezifischen Wirkungen von DPA einerseits und BAPN andererseits erhalten. Es wird erortert, ob die unterschiedliche chemische Zusammensetzung des Kollagens (Typ I–IV) mit der organspezifischen Wirkung der Prufsubstanzen in Zusammenhang gebracht werden kann.

The biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size.

Beta-aminopropionitrile (BAPN) is an inhibitor of pig plasma benzylamine oxidase. BAPN is oxidized by benzylamine oxidase. Inhibition develops in a time-dependent fashion upon incubation of BAPN with the enzyme in the absence of substrate. The product of oxidation of BAPN by benzylamine oxidase, cyanacetaldehyde, was identified and prepared by synthesis. It is an irreversible inhibitor of the enzyme.

Beta-aminopropionitrile, an inhibitor of collagen cross-linking, effectively limited the degree of posttraumatic vitreous proliferation in rabbits three weeks after double perforating injury. Light microscopic examination of the sites of perforating injury after five weeks showed minimal intravitreal fibrous proliferation in treated animals. Electron micrographs of the cicatricial vitreous collagen showed fragmentation of collagen into smaller fibrils and disintegration into amorphous areas. Inhibition of collagen cross-linking may undermine the strength of the vitreous scaffold by making vitreous and cicatricial collagen more sensitive to tissue collagenases produced during inflammation. The failure of fibrovascular membranes to grow into the vitreous may be a secondary effect of weakened vitreous support.

SummaryEnzymatic conversion of the lathyrogen, β-aminopropionitrile (BAPN) to the deaminated, nonlathyrogenic metabolite, cyanoacetic acid (CAA) by rat liver homogenates was found to be less than 1%. Urine of rats injected with 400 mg/kg of BAPN ip contained 32% unchanged BAPN in 24 hr, with negligible recovery thereafter. BAPN-derived CAA showed 12 and 22% recovery in the urine in 24 and 48 hr, respectively. These data suggest that significant amounts of unchanged BAPN may not exist in rat tissues after 24 hr, and that BAPN-derived CAA in such tissues is gradually excreted. The observation that radioactivity in rat brain and liver 24 hr after [14C]BAPN injection is present exclusively as [14C]CAA supports this interpretation.

Changes in the ultimate loads required to extract the mandibular and maxillary 1st, 2nd and 3rd molars from the dissected jaws were examined 1, 2, 4, 8, 16 and 24 days following oral administration of a lathyrogen, beta;-aminopropionitrile (BAPN). The average amount of water taken by the rats was fairly constant but because their body weight increased during the experimental period, the average dose of BAPN decreased gradually from 331 to 192mg/Kg b. w. In the control groups, the ultimate loads increased gradually from the beginning to the end of the experimental period. They were 34.4, 26.9 and 13.6N (newtons) in the mandibular 1st, 2nd and 3rd molars and were 34.2, 30.6 and 18.0N in the maxillary 1st, 2nd and 3rd molars respectively at the end of the experimental pe-riod. In the experimental groups, they were 9.9, 10.2 and 8.4 N in the mandibular 1st, 2nd and 3rd molars and were 13.5, 14.6 and 10.9N in the maxillary 1st, 2nd and 3rd molars respectively at the end of the experimental period. The biggest values of the percentage reductions of the ultimate loads were 73, 71 and 51 in the mandibular 1st, 2nd and 3rd molars and were 64, 58 and 64 in the maxillary 1st, 2nd and 3rd molars respectively during the experimental period. From an analysis of the results, it is suggested that the effects of BAPN appeared rather rapidly in the teeth with imm ature periodontal ligaments and that the functioning teeth tended to be affected more severely by the drug

Increased resorption rates and decreased fetal weight were produced by intubating pregnant rats with various single (35001–4000 mg/kg) or multiple (2500–3000 mg/kg) doses of β-aminopropionitrile (BAPN) during days 8–17 of gestation. The most embryotoxic gestational intervals were those that included day 15. Resorption (55% ) and ectocardia and/or gastroschisis (16%) resulted when 2500 mg/kg BAPN was administered on days 14–15, whereas no gross congenital malformations occurred when BAPN was administered before day 14. Higher doses of BAPN, such as 5000 m/kg, resulted mainly in fetal resorptions; 2500 mg/kg or greater on day 16 caused only cleft palate in most fetuses. Severe degrees of kyphoscoliosis, bony curvatures, and exostoses at muscleinsertion sites were observed when BAPN was given after day 16. The pathogenesis of these malformations is compatible with the known biochemical effect of BAPN — inhibition of crosslinking of both collagen and elastin — and in doing so: (a) could decrease the tensile strength of the anterior chest wall or abdominal wall to produce ectocardia and/or gastroschisis; (b) interfere with palate closure; and (c) produce osteolathyritic effects late in gestation which resemble the skeletal effects caused postnatally in young weanling animals given this agent.

The static elastic properties of the aorta ad systolic blood pressure in the caudal artery have been measured in 55 spontaneously hypertensive rats of the AS strain. Animals were divided into five groups and treated from the age of 4 to 12 weeks as follows: (1) 0.25 mm silver clip positioned around the right renal artery; (2) renal artery clipped as group (1) followed by daily injection of beta-aminopropionitrile fumarate (beta-APN); (3) daly injection of beta-APN without clipping; (4) daily injection of 0.09% saline; (5) untreated. Mean systolic blood pressures at 12 weeks of age were Group (1) 21.8; Group (2) 16.2; Group (3) 17.0; Group (4) 18.0; Group (5) 17.5 (units kPa). Thus, administration of beta-APN prevents the development of renal hypertension in clipped animals while producing no significant alteration in animals with intact renal arteries. At pressures within the physiological range the functional distensibility of all the treated animals was greater than that of the controls (pressure strain elastic modulus significantly lower). Similar changes were observed in the variation of structural distensibility (incremental elastic modulus) with pressure. At any level of total strain greater than 0.7 the incremental elastic modulus of both groups of animals treated with beta-APN was significantly less than control values. These observations are consistent with reports in the literature that administration of beta-APN causes a reduction in either the amount of cross-linked or normal scleroprotein in the aorta.

The effects of the teratogenic drug betaaminopropionitrile (BAPN) on the induction of sister chromatid exchanges (SCEs) in utero was evaluated. The frequency of sister chromatid exchanges increased with increasing doses of BAPN in the day 10 mouse embryo. The present study warns of the possible clastogenic activity of BAPN.

The effects of diabetes on collagen cross-link formation and solubility were investigated in granulation tissue collagen induced by polyester fabric implanted subcutaneously in rats at the same time diabetes was produced by injection of streptozotocin. Thus, all the collagen analyzed was formed in a diabetic milieu. Ten days later the implants were removed and the total collagen content as well as the fraction soluble in 0.5 M acetic acid was determined. Predominantly type I collagen accumulated in the implants. Total collagen content was the same in diabetics and controls; however, the acid-soluble fraction in diabetic animals was only half that of controls (8.5% and 17.7%, respectively), and the ratio of β chains to a chains in the acid-soluble fraction was higher in diabetics (0.89) than in controls (0.69). In animals treated with β-aminopropionitrile or D-penicillamine the acid-soluble fraction of collagen from diabetics equaled that from controls. These observations indicate that both intramolecular and intermolecular cross-links are increased in type I collagen from diabetic animals. Since these cross-links interfere with degradation of collagen by collagenase, they may contribute to accelerated intimal sclerosis of arteries and to capillary basement membrane thickening in diabetes.

Synopsis In two experiments, 4‐ and 9‐week‐old broiler chicks were fed on diets containing either 0.025% or 0.1% β‐aminopropionitrile for 1 week. In both experiments the higher level caused some retardation of growth. There was, however, no differential effect on the size of the pectoralis muscle or on its collagen content. Enzyme tests showed little evidence of gross liver or bone damage but intramuscular collagen became more susceptible to solubilisation. It is probable that feeding the lathyrogen in low concentration will make intramuscular collagen more amenable to study.

SummaryThe process or bone mineralization in the tibial diaphysis, in response to treatment with a lathyrogen, βAminopropionitrile (BAPN), at a dose known to inhibit collagen crosslinking, was studied in growing rats. BAPN treatment impaired the initial rate of mineralization but not its onset. In addition, in the BAPN treated group, there was a 5% decrease in diaphyseal ash weight, a decrement estimated to be greater than expected from the decreased rate of mineralization. This suggests that the maximal level of mineral deposition was also decreased as a result of BAPN treatment.

beta-Aminopropionitrile (BAPN) is a potent irreversible inhibitor of lysyl oxidase, the enzyme which initiates cross-linkage formation in elastin and collagen. The initial interaction of BAPN with aortic lysyl oxidase is competitive with elastin or alkyl amine substrates. Irreversible inhibition develops in a time- and temperature-dependent fashion upon incubation of enzyme with BAPN in the absence of substrate with a limiting inactivation rate constant of 0.16 min-1 and a KI of 6 microM at 37 degrees C. The labeled carbons of [1,2-14C]BAPN and [3-14C]BAPN covalently bind to the enzyme to equivalent extents and in parallel with the development of inactivation, negating the possibility that the nitrile moiety is eliminated from BAPN by enzymatic action. The copper content of the enzyme is not significantly altered upon interaction with BAPN. The extent of labeling by [14C]BAPN is reduced by prior treatment of the enzyme with carbonyl-modifying reagents, suggesting the possibility of enzyme-inhibitor Schiff base formation. However, BAPN is not processed to a free aldehyde product upon incubation with lysyl oxidase. A mechanism of inhibition is postulated which involves the formation of a covalent bond between an enzyme nucleophile and a ketenimine formed from BAPN by enzyme-assisted beta-proton abstraction.

We evaluated the effect of beta-aminopropionitrile (beta APN) on the nitrogen dioxide (NO2) animal model of emphysema. Rats maintained on a beta APN-supplemented or a regular diet were exposed to 30 ppm NO2 for intervals ranging from 1 to 8 wk. Emphysema development was assessed by histologic evaluation and by changes in lung volume and mean linear intercept values. Evidence of pathologic changes were also documented by clinical and radiographic findings of osteolathyrism. The induction of centriacinar emphysema was attributed specifically to NO2 exposure. Neither the severity of the emphysema nor the time course of its development was altered by the beta APN-supplemented diet. These findings are in marked contrast to those observed with the exogenous elastase model of the disease, and they suggest that elastin synthesis and repair may not modulate elastin destruction in the NO2 model of emphysema.