Lysyl Oxidase Deficiency Research Articles

Lysyl oxidase-mediated elastin upregulation promotes the proliferation and migration of human retinal endothelial cells.

Proliferative diabetic retinopathy (PDR) is a major cause of irreversible blindness in the working age population. The dysfunction of retinal vascular endothelial cells (RVECs) is the primary cause of PDR. Extracellular matrix (ECM) accumulation promotes intracellular signaling required for RVEC proliferation, migration, survival, and tube morphogenesis. This study aimed to investigate the role of lysyl oxidase (LOX) in the cellular function of RVECs and PDR pathogenesis and to identify the underlying mechanisms. Protein expression was determined with western blot. The interaction between LOX and elastin (ELN) was detected using a co-immunoprecipitation (Co-IP) assay, and the Cell Counting Kit-8 (CCK-8) assay evaluated cell viability. A colony formation assay was employed to assess the proliferation of human RVECs (hRVECs), and a transwell assay to determine their migration ability. Streptozotocin was used to establish PDR in mice in vivo. A histological analysis was conducted using hematoxylin and eosin (H&E) staining. The results showed that LOX was overexpressed in PDR patients. The LOX knockdown suppressed ECM formation and hRVEC proliferation and migration. Additionally, LOX upregulated ELN expression. However, overexpressed ELN promoted hRVEC proliferation and migration. In vivo experiments showed that curcumin-mediated LOX deficiency restored retinal tissue structure. The LOX-knockdown suppressed ECM formation and hRVEC proliferation and migration by inactivating ELN. Therefore, LOX/ELN signaling may be a potential PDR biomarker.

Inhibition of lysyl oxidase stimulates TGF-β signaling and metalloproteinases-2 and -9 expression and contributes to the disruption of ascending aorta in rats: protection by propylthiouracil.

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of β-aminoproprionitrile (BAPN). We investigated the mechanisms underlying the damage to the ascending thoracic aorta induced by LOX deficiency and evaluated whether 6-propylthiouracil (PTU) can afford protection in rats. BAPN administration caused disruption of the ascending aortic wall, increased the number of apoptotic cells, stimulated TGF-β signaling (increase of nuclear p-SMAD2 staining), and up-regulated the expression of metalloproteinases-2 and -9. In BAPN-treated animals, PTU reduced apoptosis, p-SMAD2 staining, MMP-2, and -9 expression, and markedly decreased the damage to the aortic wall. Our results suggest that, as in some heritable vascular diseases, enhanced TGF-β signaling and upregulation of MMP-2 and -9 can contribute to the pathogenesis of ascending aorta damage caused by LOX deficiency. We have also shown that PTU, a drug already in clinical use, protects against the effects of LOX inhibition. MMP-2 and -9 might be potential targets of new therapeutic strategies for the treatment of vascular diseases caused by LOX deficiency.

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.

PP.04.09

Objective: Lysyl oxidase (LOX) is a key enzyme in extracellular matrix (ECM) remodelling. Because LOX deficiency is lethal, we have developed a transgenic mouse model to study the impact of LOX overexpression on cardiovascular remodelling. Design and method: A new mouse model that over-expresses human LOX was generated by conventional methods. Transgene expression was determined by real time PCR in 8 different tissues including aorta, heart, kidney, white adipose tissue (WAT), brown adipose tissue, lung, liver and skeletal muscle. We tested the impact of LOX over-expression on cardiovascular remodelling in TgLOX mice and their wild-type (WT) littermates mice after chronic infusion with Ang II (1.4 μg/kg/min) or saline (n = 10 per group). Aortic diameter and cardiac function were evaluated by ultrasonography. Results: The maximum expression of human LOX was found in aorta followed by heart and WAT. Neither the expression of endogenous LOX nor that of LOX-Like (LOXL) isoenzymes was modified by transgene expression in these tissues. Ang II-induced aortic diameter dilation was similar in TgLOX and WT mice after Ang II infusion. However, the mortality rate due to aortic rupture was higher in WT mice (20 %) compared to TgLOX mice (0 %). The quantification of heart function showed that Ang II infusion decreased ejection fraction (EF) and fractional shortening (FS) in TgLOX mice, while they were augmented in WT mice. A stronger hypertrophic response induced by Ang II occurred in TgLOX mice as evidenced by an increased LV mass and left ventricle posterior wall (LVPW) thickness. Accordingly, the left ventricular inner diameter (LVID) was significantly lowered in TgLOX mice. After Ang II infusion, cardiac output, stroke volume and mRNA levels of hypertrophic markers such as ANP and B-HMC remained similar in both, transgenic and WT mice. Finally, LOX overexpression increased the Ang II-induced expression of pro-inflammatory (Emr-1, IL6, Mmp-9) and fibrosis-related (Serpin-1, Col-1a1) markers in cardiac tissue compared with WT mice Conclusions: We have developed a valuable model to improve our knowledge about LOX biology in the cardiovascular system. Our data evidence that LOX over-expression impairs cardiac function under hypertensive conditions.

Increased frequency of congenital heart defects in Menkes disease

ATP7A is a copper-transporting ATPase critical for central and peripheral nervous system function. Mutations in ATP7A cause Menkes disease and occipital horn syndrome (OHS), allelic X-linked recessive conditions that feature vascular abnormalities ascribed to low activity of lysyl oxidase, a copper-dependent enzyme. From a recently created Menkes disease/OHS patient registry, we identified four of 95 patients with major congenital heart defects (4.2%), a proportion exceeding the general population prevalence (≈1%). In conjunction with mouse models of Menkes disease, OHS, and lysyl oxidase deficiency (which feature aortic aneurysms, irregular attachment between vascular endothelium and mesoderm, and other defects of embryological development) our observation suggests an important role of copper metabolism in cardiac development. Congenital heart disease may be an under-appreciated abnormality in Menkes disease, and should be considered in a broad differential diagnosis of cardiac defects found prenatally in male fetuses. Conversely, newborn infants with suspected or confirmed Menkes disease should be evaluated for heart disease by careful clinical examination and echocardiography, if indicated.

Kinky Hair, Kinky Vessels, and Bladder Diverticula in Menkes Disease

Menkes disease (MD) is an infantile-onset X-linked recessive neurodegenerative disorder caused by deficiency or dysfunction of a copper-transporting ATPase, ATP7A. The effect of altered transportation of copper may affect various enzymatic functions differently. Among all enzymatic functions, lysyl-oxidase enzymatic activity, which is crucial in the formation of the lysine-derived cross-links in collagen and elastin, is the most sensitive to the copper transport alterations. Pili torti, tortuous intracranial vessels and bladder diverticula are clinical aspects strictly related to the connective tissue alterations dependent on the lysyl-oxidase deficiency. Despite a pleiotropic clinical appearance of MD patients, we observed tortuous intracranial vessels and bladder diverticula in 4 consecutive Menkes patients at different stages of the disease. We speculate that these findings are present at early stages and could be considered suggestive findings in MD.

Lysyl oxidase enhances elastin synthesis and matrix formation by vascular smooth muscle cells

Lysyl oxidase (LOX) is a copper-dependent enzyme that initiates covalent crosslinking of elastin precursors by oxidizing peptidyl lysine to aminoadipic semi-aldehydes. Previous studies have shown LOX deficiency to affect crosslinking of elastin and collagen in vivo, resulting in disorganized connective tissue formation. In this study, we investigated the utility of exogenously supplemented LOX peptides (50-100 microl/well) to elastin synthesis, crosslinking efficiency and matrix deposition in adult rat aortic smooth muscle cell (RASMC) cultures. Additionally, we also examined the role of LOX peptides on SMC proliferation and matrix metalloproteinase (MMP) synthesis in these cultures. Highly purified bovine aorta LOX peptide was found to increase matrix elastin synthesis by 40-80% to that in control cultures in a dose-dependent manner, while the crosslinking efficiency significantly (as measured by the ratio of matrix elastin protein to the total elastin protein synthesized) improved to 45-55% of total elastin synthesized under these conditions. However, LOX peptides affected neither SMC proliferation relative to controls, nor elastin precursor (tropoelastin) synthesis, nor the total elastin synthesis on a per-cell basis. In general, LOX peptides also did not affect MMP-2 and MMP-9 activities relative to control cultures, except for MMP-9 activity suppression at a higher LOX dose, suggesting that these LOX peptide cues could be safely used to enhance tropoelastin crosslinking into matrix structures and elastin matrix yield, within tissue-engineered constructs, a major challenge in the field.

Downregulation of lysyl oxidase in cadmium-resistant fibroblasts.

Lysyl oxidase, a copper-dependent metalloenzyme, plays a central role in crosslinking of collagen and elastin in the extracellular matrix. Notably, lung lysyl oxidase activity is markedly stimulated in rats exposed to cadmium vapors. To further understand the mechanism of cadmium toxicity, the mRNA expression, synthesis, post-translational processing, and catalytic activity of lysyl oxidase were examined in cadmium-resistant (CdR) cells and the cadmium-sensitive Swiss mouse 3T3 cells from which they were derived. These CdR cells synthesized and accumulated markedly elevated levels of metallothionein, a known marker for cadmium resistance, whereas the expression of lysyl oxidase was reduced considerably. In comparison to the parental, cadmium-sensitive cells, the suppression of enzyme production in the CdR cells was seen at the mRNA level, at the levels of intracellular proprotein production and mature enzyme secreted into the medium, and in terms of total enzyme activity in the culture. The presence of cupric chloride in the culture medium during the incubation of the CdR cells for 16 h significantly enhanced lysyl oxidase activity accumulating in the medium, suggesting that lysyl oxidase deficiency in CdR cells may be related to abnormal copper metabolism.

Aortic Elastin Abnormalities in Osteogenesis Imperfecta Type II

Skin and aortic samples from two patients who died by lethal perinatal Osteogenesis Imperfecta (O. I.) were studied by optical and electron microscopy and compared with similar samples from two normal human fetuses and one newborn child. No significant abnormalities were observed in the dermis of O. I. patients apart from small differences in the diameter of reticular collagen fibrils. On the contrary, in the aortas of both patients collagen fibrils were significantly smaller than in the controls; moreover, elastin lamellae were deeply altered and consisted of roundish aggregates of elastin, massively permeated by cytochemically recognizable glycosaminoglycans. As identical features were described in experimental lathyrism by using inhibitors of the enzyme lysyl oxidase (Pasquali Ronchetti et al., 1984), the conclusion is reached that in the two cases of lethal perinatal O. I. examined, a severe lysyl oxidase deficiency could account for the observed ultrastructural abnormalities of elastin and that, besides defects of collagen type I, additional alterations of cellular metabolism might be responsible for the clinical heterogeneity of the diesease.

X-linked cutis laxa: defective cross-link formation in collagen due to decreased lysyl oxidase activity.

We studied several members of a family with an X-linked form of cutis laxa; the affected males have mild skin laxity, a characteristic facies, skeletal abnormalities, structural abnormalities of the genitourinary tract, and low serum copper levels. The activity of lysyl oxidase, a copper-dependent enzyme involved in cross-link formation in collagen, was decreased in skin-biopsy specimens (13 to 26 per cent of normal) and in culture medium from cells to two affected males (15 to 20 per cent of normal). Immunoreactive lysyl oxidase from skin of both patients was virtually undetectable by immunodiffusion assay. The amounts of lysyl-derived aldehydes (the product formed in collagen and elastin by lysyl oxidase) and of cross-links formed from these products were decreased in dermal fibroblasts in culture. Collagen extractability from these cells was increased in culture. These findings suggest that lysyl oxidase deficiency provides the biochemical basis of the X-linked form of cutis laxa.

Lysyl oxidase deficiency in lung and fibroblasts from mice with hereditary emphysema

Lysyl oxidase activity was measured in the lungs and from cultured fibroblasts of Blotchy mice. A marked decrease in lysyl oxidase activity was observed in lungs of affected mice as compared to normal litter mates. Fibroblasts cultured from Blotchy mice were also deficient in lysyl oxidase, producing less than half of normal enzyme levels. Normal and Blotchy fibroblasts which had been maintained in culture for several months and had undergone spontaneous transformation, continued to show the same magnitude of difference in lysyl oxidase levels. The data suggest that the deficiency of lysyl oxidase is inherent in Blotchy fibroblasts and support the idea that the deficiency of this enzyme is the metabolic lesion that leads to the connective tissue defects observed in these animals.

Collagen cross-linking: the substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that contains copper and is irreversibly inhibited by compounds called lathyrogens (1,2,3). 3-Aminopropionitrile (BAPN) is the most widely studied and best known of these compounds. When growing animals are either fed lathyrogens or made copper deficient they develop a wide variety of connective tissue and skeletal abnormalities such as dissecting aneurysms of the thoracic aorta, kyphoscoliosis and abdominal hernias. The pathogenesis of these abnormalities is related to decreased collagen cross-linking secondary to decreased lysyl oxidase activity which results from either lack of the copper cofactor(4) or irreversible inhibition by BAPN. Since clinical abnormalities in these disorders have been noted as a result of deranged biosynthesis in both elastin and collagen with lysyl oxidase deficiency, it seemed appropriate to define the substrate specificity of highly purified lysyl oxidase in more detail and to determine whether the same enzyme is active on collagen and elastin substrates(5,6,7,8).