IsoChichibabin desmosine-13C3,15N1 synthesis and quantitative LC-MS/MS analysis of desmosine and isodesmosine in human skin.

Desmosine and isodesmosine are crosslinking amino acids of elastin, which is an essential component of the dermal extracellular matrix protein. Quantitative analysis of crosslinker desmosines in human skin dermis has not been fully achieved due to the insoluble nature of elastin protein. In the present study, chemical synthesis of isotopically labeled desmosine, desmosine-13C3,15N1, was carried out via isoChichibabin pyridinium synthesis starting from corresponding isotopically labeled amino acids. Isotope-dilution LC-MS/MS analysis of desmosine and isodesmosine utilizing synthetic desmosine-13C3,15N1 enabled the quantitative analysis of desmosines in human skin for the first time. Thus, ca. 1.43 µg of desmosines was detected from analysis of 1 mg of dry human skin.

Lysyl Oxidase Enhances the Deposition of Tropoelastin through the Catalysis of Tropoelastin Molecules on the Cell Surface.

The cross-linking of elastin by lysyl oxidase (LOX) family members is essential for the integrity and elasticity of elastic fibers, which play an important role in the characteristic resilience of various tissues. However, the temporal sequence of oxidation by LOX during elastic fiber formation is still incompletely understood. Here, we demonstrate that the cross-linking of tropoelastin molecules by LOX occurs concurrent with elastin deposition. Our data show that LOX deficiency or the inhibition of LOX enzyme activity leads to the loss of elastin deposition in skin fibroblast. Moreover, overexpression of LOX promotes the deposition and alignment of tropoelastin, whereas the addition of recombinant active-form of LOX in culture medium caused abnormal elastic fiber assembly. Immunoblotting and immunofluorescence show that LOX and tropoelastin are present together with fibronectin on the cell surface of preconfluent cultures. Further, fluorescence activated cell sorting (FACS) analysis for the localization of LOX on the cell surface reveals that the transfer of LOX to the extracellular space occurs in association with elastic fiber formation. In conclusion, our results support the view that LOX and tropoelastin are present on the cell surface and suggests the possibility that lysine oxidation by LOX precedes tropoelastin deposition onto microfibrils.

Effect of ß-agonist on the dexamethasone-induced expression of aromatase by the human monocyte cells.

Emerging evidence suggests that sex steroids are important for human skin health. In particular, estrogen improves skin thickness, elasticity and moisture of older women. The major source of circulating estrogen is the ovary; however, local estrogen synthesis and secretion have important roles in, for example, bone metabolism and breast cancer development. We hypothesized that infiltrated peripheral monocytes are one of the sources of estrogen in skin tissues. We also hypothesized that, during atopic dermatitis under stress, a decline in the hypothalamus-pituitary-adrenal axis (HPA) and facilitation of the (hypothalamus)-sympathetic-adrenomedullary system (SAM) attenuates estrogen secretion from monocytes. Based on this hypothesis, we tested aromatase expression in the human peripheral monocyte-derived cell line THP-1 in response to the synthetic glucocorticoid dexamethasone (Dex), the synthetic ß-agonist isoproterenol (Iso) and the ß-antagonist propranolol (Pro). Dex mimics glucocorticoid secreted during excitation of the HPA, and Iso mimics catecholamine secreted during excitation of the SAM. We found that aromatase activity and the CYP19A1 gene transcript were both upregulated in THP-1 cells in the presence of Dex. Addition of Iso induced their downregulation and further addition of Pro rescued aromatase expression. These results may suggest that attenuation of estrogen secretion from peripheral monocytes could be a part of the pathology of stress-caused deterioration of atopic dermatitis. Further examination using an in vitro human skin model including THP-1 cells might be a valuable tool for investigating the therapeutic efficacy and mechanism of estrogen treatment for skin health.

Asnovolins A-G, Spiromeroterpenoids Isolated from the Fungus Aspergillus novofumigatus, and Suppression of Fibronectin Expression by Asnovolin E.

Seven novel spiromeroterpenoids, asnovolins A-G (1-7), one of which was shown to suppress fibronectin expression, were isolated from Aspergillus novofumigatus CBS117520 along with a known compound, novofumigatonin (8). The structures of asnovolins A-G were elucidated using MS and 2D-NMR data. Asnovolin E (5) suppressed fibronectin expression by normal human neonatal dermal fibroblast cells.

MiR-29-mediated elastin down-regulation contributes to inorganic phosphorus-induced osteoblastic differentiation in vascular smooth muscle cells.

Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification.

7-Ketocholesterol-induced lysosomal dysfunction exacerbates vascular smooth muscle cell calcification via oxidative stress.

Vascular calcification is known to reduce the elasticity of aorta. Several studies have suggested that autophagy-lysosomal pathway (ALP) in vascular smooth muscle cells (VSMCs) is associated with vascular calcification. A major component of oxidized low-density lipoproteins, 7-ketocholesterol (7-KC), has been reported to promote inorganic phosphorus (Pi)-induced vascular calcification and induce ALP. The aim of this study was to unravel the relationship between ALP and the progression of calcification by 7-KC. Calcification of human VSMCs was induced by Pi stimulation in the presence or absence of 7-KC. FACS analysis showed that 7-KC-induced apoptosis at a high concentration (30 µM), but not at a low concentration (15 µM). Interestingly, 7-KC promoted calcification in VSMCs regardless of apoptosis. Immunoblotting and immunostaining showed that 7-KC inhibits not only the fusion of autophagosomes and lysosomes but also causes a swell of lysosomes with the reduction of cathepsin B and D. Moreover, lysosomal protease inhibitors exacerbated the apoptosis-independent calcification by 7-KC although inhibition of autophagosome formation by Atg5 siRNA did not. Finally, the 7-KC-induced progression of calcification was alleviated by the treatment with antioxidant. Taken together, our data showed that 7-KC promotes VSMC calcification through lysosomal-dysfunction-dependent oxidative stress.

Domain 36 of tropoelastin in elastic fiber formation.

Elastic fiber assembly is a complex stepwise process involving multiple different proteins and enzymes. Domain 36, encoded by the last exon of the elastin gene, is recognized to be an important domain for deposition onto microfibrils, an essential step in elastic fiber assembly. However, the role of domain 36 in elastic fiber assembly has not been clarified. Here, we utilized our established in vitro assembly model to identify the importance of domain 36 for the assembly process. Our results showed that the lack of domain 36 in bovine tropoelastin results in deficient elastic fiber assembly. A similar result was obtained with the point mutation of two cysteine residues and the deletion of the Lysine-Arginine-Lysine-Arginine (RKRK) sequence in domain 36. Double immunofluorescence of tropoelastin and fibrillin-1, a main component of microfibrils, demonstrated reduced localization of these mutant tropoelastin molecules on fibrillin-1 fibers. Moreover, the binding affinity of these mutants to fibrillin-1 and microfibril-associated glycoprotein (MAGP) was significantly decreased. These data indicate that domain 36 of tropoelastin facilitates elastic fiber assembly by interacting with microfibrils via two cysteine residues and the RKRK sequence.

5-aza-2'-Deoxycytidine, a DNA methyltransferase inhibitor, facilitates the inorganic phosphorus-induced mineralization of vascular smooth muscle cells.

AIM: Vascular calcification, an independent risk factor for cardiovascular disease in patients with chronic kidney disease(CKD), refers to the mineralization of vascular smooth muscle cells(VSMCs) caused by phenotypic changes toward osteoblast-like cells. DNA methylation, mediated by DNA methyltransferases(DNMTs), plays an important role in the differentiation of osteoblasts. We herein assessed the effects of a DNMT inhibitor on phenotypic changes in VSMCs and the development of vascular calcification. METHODS: The effects of 5-aza-2'-deoxycytidine(5-aza-dC), a DNMT inhibitor, on human aortic smooth muscle cells(HASMCs) were evaluated. The expression and DNA methylation status of osteogenic genes were determined using RT-qPCR and bisulfite sequencing, respectively. Mineralization of HASMCs was induced by high concentrations of inorganic phosphate(Pi), as confirmed by quantitation of the calcium levels and von Kossa staining. Moreover, we examined the effects of the suppression of DNMT1 and/or alkaline phosphatase(ALP) on the mineralization of HASMCs. RESULTS: 5-aza-dC increased the expression and activity of ALP and reduced the DNA methylation levels of the ALP promoter region in the HASMCs. In addition, both treatment with 5-aza-dC and downregulation of the DNMT1 expression promoted the Pi-induced mineralization of HASMCs. Moreover, both treatment with phosphonoformic acid(PFA), a sodium-dependent phosphate transporter inhibitor, and suppression of the ALP expression inhibited the 5-aza-dC-promoted mineralization of HASMCs. CONCLUSIONS: The present study showed that DNMT inhibitors facilitate the Pi-induced development of vascular calcification via the upregulation of the ALP expression along with a reduction in the DNA methylation level of the ALP promoter region.

Trichostatin A, an HDAC class I/II inhibitor, promotes Pi-induced vascular calcification via up-regulation of the expression of alkaline phosphatase.

AIM: Vascular calcification, a major complication of chronic kidney disease (CKD), refers to the mineralization of vascular smooth muscle cells (VSMCs), resulting from a phenotypic change towards osteoblast-like cells. Histone deacetylase inhibitors (HDIs), potential therapeutic agents for CKD, are known to promote the differentiation and mineralization of osteoblasts. In this study, we aimed to determine the effects of an HDI on the phenotypic change of VSMCs and the development of vascular calcification. METHODS: The effect of trichostatin A (TSA), an HDI, on human aortic smooth muscle cells (HASMCs) was determined. The mineralization of HASMCs was induced by inorganic phosphorus (Pi), and was confirmed by quantitation of Ca levels and by von Kossa staining. Furthermore, we examined the effect of alkaline phosphatase (ALP) suppression using siRNA on Pi-induced vascular calcification in the presence or absence of TSA. RESULTS: TSA increased the expression and activity of ALP in HASMCs at a concentration which showed an inhibitory effect of histone deacetylase (HDAC) activity but not on cell viability. Moreover, TSA promoted the Pi-induced mineralization of HASMCs. In addition, both phosphonoformic acid (PFA), which is a sodium-dependent phosphate transporter inhibitor, and suppression of ALP expression by siRNA markedly inhibited the TSA-promoted mineralization of HASMCs. CONCLUSION: These data show that inhibition of HDAC activity promotes Pi-induced vascular calcification via the up-regulation of ALP expression. Taken together, HDIs may increase the risk of vascular calcification in CKD patients.

Galectin-7 and actin are components of amyloid deposit of localized cutaneous amyloidosis.

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowen's disease. The proteins were resolved on one- or two-dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50-67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14-kDa (pI = 7.0) and 42-kDa (pI = 5.4) proteins reacted with the antibody against galectin-7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin-7 by MALDI-TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin-7 and actin were detected by immunoblot assay in the water-soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor-associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin-7 and F-actin. Galectin-7 and actin, which contain considerable amount of ß-sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.

N(ɛ)-(carboxymethyl)lysine modification of elastin alters its biological properties: implications for the accumulation of abnormal elastic fibers in actinic elastosis.

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(ɛ)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.

Quinazolinobenzodiazepine Derivatives, Novobenzomalvins A-C: Fibronectin Expression Regulators from Aspergillus novofumigatus.

Three new quinazolinobenzodiazepine derivatives, novobenzomalvins A (1), B (2), and C (3), have been isolated as fibronectin expression regulators from Aspergillus novofumigatus CBS117520. The structures of 1 to 3 were established by spectroscopic and physicochemical analysis, and chemical investigation including the total synthesis of 1. Treatment with novo-benzomalvins A (1), B (2), C (3), and N-methylnovobenzomalvin A (5) increased the expression of fibronectin in normal human neonatal dermal fibroblast cells.

New insights into the pathogenesis of autosomal-dominant cutis laxa with report of five ELN mutations.

Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28-34 of the ELN gene in five probands with ADCL features and found five de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5), and c.2124del25 (CL-6). Four probands (CL-1,-2,-3,-6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGF-ß) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications, associated with increased TGF-ß signaling and mutation-specific differences in endoplasmic reticulum stress and apoptosis.

Effects of Bidens pilosa L. var. radiata Scherff on experimental gastric lesion.

Bidens pilosa L. var. radiata Scherff: (BP) is a plant used as a traditional folk medicine. BP, cultivated with only green manure on Miyako Island, Okinawa prefecture, was processed to powder and is referred to as MMBP. We have reported that MMBP has antioxidant, anti-inflammatory, and anti-allergy properties. In this study, we investigated the effects of MMBP on several experimental gastric lesions induced by HCl/EtOH, a non-steroidal anti-inflammatory drug, or cold-restraint stress, comparing these results with those of rutin or anti-ulcerogenic drugs (cimetidine or sucralfate) based on the lesion index and hemorrhage from the gastric lesions. Orally administered MMBP prevented the progression of the gastric lesions. Moreover, treatment with MMBP, rutin, or sucralfate, which had potent antioxidative activity, inhibited increases in the levels of thiobarbituric acid reactive substances (TBARS) in the gastric mucosal lesions. The inhibition of the gastric mucosal TBARS content by MMBP may have been due to the antioxidant effects of MMBP. These results indicate that MMBP prevents the progression of acute gastric mucosal lesions, possibly by suppressing oxidative stress in the gastric mucosa.

DANCE/fibulin-5 promotes elastic fiber formation in a tropoelastin isoform-dependent manner.

OBJECTIVE: To investigate a function of fibulin-5 in the elastic fiber formation, we studied the molecular interactions among elastin, fibrillin-1, and fibulin-5 in the extracellular space and the maturation of tropoelastin using retinal pigment epithelial cells (ARPE-19). DESIGN AND METHODS: Bacterial recombinant tropoelastin (rTE) was added to ARPE-19 cells overexpressing V5-tagged fibulin-5 (ARPE-Fibulin-5). These elastic fibers were evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: Immunoprecipitation assays revealed that fibulin-5 is able to separately interact with tropoelastin or fibrillin-1 in the culture medium. Moreover, immunofluorescent staining showed that elastin, fibrillin-1, and fibulin-5 co-localize in the extracellular matrix. Desmosine levels were significantly increased in ARPE-Fibulin-5 relative to untransfected cells in spite of equal deposition of tropoelastin by enzyme-linked immunosorbent assay. The addition of a tropoelastin isoform, which lacked the peptide encoded by exon 26A (Delta26A) and could bind to fibulin-5 strongly, led to a larger increase in cross-linking amino acids compared to tropoelastin containing the exon 26A peptide sequence. CONCLUSION: These data provide new insights into the initial steps of elastic fiber assembly and identify fibulin-5 and tropoelastin isoforms as potential targets for the regeneration of elastic fibers in vivo.

7-ketocholesterol, a major oxysterol, promotes pi-induced vascular calcification in cultured smooth muscle cells.

AIM: Oxysterols are found in high concentrations in advanced atherosclerotic plaques and are considered as an important factor in the development of vascular calcification. The purpose of this study was to investigate the effect of 7-ketocholesterol (7kc), a major oxysterol in plaques, on in vitro arterial calcification. METHODS: Bovine vascular smooth muscle cells (VSMCs) were cultured with inorganic phosphate (Pi) in the presence or absence of 7kc. Calcium deposition was determined by Calcium C-test Wako and von Kossa staining. Phenotypic change was evaluated by mRNA expression using semi-quantitative reverse transcription-polymerase chain reaction. Cell apoptosis was determined by in situ DNA fragmentation assay. RESULTS: 7kc significantly enhanced the calcium deposition, phenotypic change of VSMCs, and apoptosis in the presence of Pi. Treatment with risedronate, a bisphosphonate, or Y-27632, an Rho kinase inhibitor, completely or partially prevented the effects induced by 7kc in the presence of Pi, respectively. CONCLUSION: These results suggest that 7kc, a major oxysterol, significantly accelerates vascular calcification in the presence of Pi via the mevalonate pathway and Rho-ROCK signaling pathway. Our present data provide beneficial information on the development of a therapeutic approach for arterial calcification, especially in patients with a mineral imbalance, including hypocalcaemia, hyperphosphatemia, and hypercholesterolemia.

[Variance of expressions of extracellular matrix components and effects of anti-osteoporotic drugs on Mönckeberg's arteriosclerosis].

Mönckeberg-type arteriosclerosis occurs as a complication in diabetic, uremic patients and in postmenoposal women. It has been shown that arterial calcification generates loss of elasticity in tunica media. We have already reported that the expression of tropoelastin (TE), the precursor protein of elastin, is suppressed by arterial calcification, although no changes of mRNA expression of the other elastic fiber components, such as fibrillins, was observed. We examined the effects of bisphosphonates, known as anti-osteoporotic drugs, in inorganic phosphate (Pi)-induced calcified bovine aortic smooth muscle cells (BASMCs) (in vitro arterial calcification model). Treatment with the bisphosphonate risedronate, significantly inhibited calcium deposition in the arterial calcification model. Risedronate also inhibited suppression of TE mRNA expression and the progression of osteopontin (OPN) and core binding factor-alpha1 (Cbfa1), an osteogenic transcription factor, by BASMCs calcification. Basically, bisphosphonates could inhibit phenotypic transition such as SMC to osteoblast-like cell. Inhibitory effects of bisphosphonates were also shown in female Sprague-Dawley rats with calcinosis induced by administration of an over-dose of vitamin D2 (in vivo arterial calcification model). It is known that arterial calcification is accelerated by oxidative low-density lipoprotein (oxLDL). Therefore we examined the effects of 7-ketocholesterol (7kc), a component of oxLDL, on in vitro arterial calcification. Thereupon, it was revealed that 7kc drastically accelerated Pi-induced calcification, and risedronate completely restored the calcification and mRNA expression accelerated by 7kc.

Characterization of the molecular interaction between tropoelastin and DANCE/fibulin-5.

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation.

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.