Emerging Roles of Lysyl Oxidases in the Cardiovascular System: New Concepts and Therapeutic Challenges.

Lysyl oxidases (LOX and LOX-likes (LOXLs) isoenzymes) belong to a family of copper-dependent enzymes classically involved in the covalent cross-linking of collagen and elastin, a pivotal process that ensures extracellular matrix (ECM) stability and provides the tensile and elastic characteristics of connective tissues. Besides this structural role, in the last years, novel biological properties have been attributed to these enzymes, which can critically influence cardiovascular function. LOX and LOXLs control cell proliferation, migration, adhesion, differentiation, oxidative stress, and transcriptional regulation and, thereby, their dysregulation has been linked to a myriad of cardiovascular pathologies. Lysyl oxidase could modulate virtually all stages of the atherosclerotic process, from endothelial dysfunction and plaque progression to calcification and rupture of advanced and complicated plaques, and contributes to vascular stiffness in hypertension. The alteration of LOX/LOXLs expression underlies the development of other vascular pathologies characterized by a destructive remodeling of the ECM, such as aneurysm and artery dissections, and contributes to the adverse myocardial remodeling and dysfunction in hypertension, myocardial infarction, and obesity. This review examines the most recent advances in the study of LOX and LOXLs biology and their pathophysiological role in cardiovascular diseases with special emphasis on their potential as therapeutic targets.

Lysyl oxidase may play a critical role in hypoxia-induced NSCLC cells invasion and migration.

Lysyl oxidase (LOX), a copper-dependent amine oxidase known to function both intracellularly and extracellularly, is implicated in promoting tumor progression and hypoxic metastasis in certain malignancies. Nonsmall cell lung cancer (NSCLC) is a highly aggressive cancer with poor prognosis worldwide. However, the role and molecular mechanism by which LOX involving in hypoxic NSCLC invasion and migration are poorly understood. This study explores the effect of LOX on invasion and migration of NSCLC cells under hypoxic conditions. Small interfering RNA (siRNA) targeting LOX was used to silence LOX expression of hypoxic NSCLC cells, SPCA1 and A549. Cellular invasive and migratory potentials were determined by matrigel invasion and migration assays. Expression of LOX, Src, Src activation (Tyr418 phosphorylation of Src), and Snail were evaluated by real-time PCR and western blot, respectively. The results showed that LOX mRNA and protein expression were upregulated under hypoxic conditions in NSCLC cells. Knockdown of LOX led to inhibition of hypoxia-induced invasion and migration. Phosphorylated Src (Tyr418) and Snail proteins were decreased along with LOX downregulation. Our data provide molecular evidences that LOX is mechanistically linked to increased invasion and migration of hypoxic NSCLC cells, and may serve as an antimetastasis target of human NSCLC.

Silencing of lysyl oxidase gene expression by RNA interference suppresses metastasis of breast cancer.

OBJECTIVE: The aim of this study was to investigate possible mechanisms of LOX gene effects on invasion and metastasis of breast cancer cells by RNA interference. METHODS: LOX-RNAi-LV was designed, synthesized, and then transfected into a breast cancer cell line (MDA-MB-231). Expression of LOX, MMP-2 and MMP-9 was determined by real-time PCR, and protein expression of LOX by Western blotting. Cell migration and invasiveness were assessed with Transwell chambers. A total of 111 cases of breast cancer tissues, cancer-adjacent normal breast tissues, and 20 cases of benign lesion tissues were assessed by immunohistochemistry. RESULTS: Expression of LOX mRNA and protein was suppressed, and the expression of MMP-2 and MMP-9 was significantly lower in the RNAi group than the control group (P<0.05), after LOX-RNAi-LV was transfection into MDA-MB-231 cells. Migration and invasion abilities were obviously inhibited. The expression of LOX protein in breast cancer, cancer-adjacent normal breast tissues and benign breast tumor were 48.6% (54/111), 26.1% (29/111), 20.0% (4/20), respectively, associations being noted with clinical stage, lymph node metastasis, tumor size and ER, PR, HER2, but not age. LOX protein was positively correlated with MMP-2 and MMP-9. CONCLUSION: LOX displayed an important role in invasion and metastasis of breast cancer by regulating MMP-2 and MMP-9 expression which probably exerted synergistic effects on the extracellular matrix (ECM).

Lysyl oxidase (lox) gene deficiency affects osteoblastic phenotype.

Lysyl oxidase (LOX) catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. The present study examined the role of Lox gene deficiency for the osteoblast phenotype in primary calvarial osteoblasts from E18.5 Lox knockout (Lox ( -/- )) and wild type (wt) (C57BL/6) mice. Next to Lox gene depletion, mRNA expression of Lox isoforms, LOXL1-4, was significantly downregulated in Lox ( -/- ) bone tissue. A significant decrease of DNA synthesis of Lox ( -/- ) osteoblasts compared to wt was found. Early stages of osteoblastic apoptosis studied by annexin-V binding as well as later stages of DNA fragmentation were not affected. However, mineral nodule formation and osteoblastic differentiation were markedly decreased, as revealed by significant downregulation of osteoblastic markers, type I collagen, bone sialoprotein, and Runx2/Cbfa1.

Lysyl oxidase deficiency: a new cause of human arterial dissection.

Spontaneous coronary artery dissection is a rare cause of myocardial ischaemia. The underlying mechanism is unknown but some dissections are associated with extracellular matrix disorganisation of genetic origin. A deficiency in extracellular matrix protein cross links has rarely been studied. A single clinical case of spontaneous coronary artery dissection is reported. Lysyl oxidase (LOX) and extracellular matrix organisation were investigated by skin immunohistology and polymerase chain reaction of LOX expression. Both approaches found a dramatic LOX decrease. LOX deficiency has a major role in human arterial wall organisation during development. The suspected mechanism is an elastin or collagen polymer cross linking deficiency.

Reduction of LOX- and LOXL2-mRNA expression in head and neck squamous cell carcinomas.

BACKGROUND: Besides other molecular functions, the lysyl oxidase gene (LOX) has been assumed and shown to have a tumor suppressive function in vitro and in vivo. The cancer-related functions of both LOX and LOXL2 have not yet been investigated in squamous cell carcinoma of the head and neck (HNSCC). MATERIALS AND METHODS: We examined the mRNA levels of LOX and LOXL2 in ten malignantly transformed cell lines and sixteen malignant tissue samples of different graded and staged HNSCC by RT-PCR. RESULTS: The LOX-mRNA level in both cell lines and tissues of HNSCC was markedly reduced as opposed to benign keratinocyte cell lines and mucosal tissue samples of the upper aerodigestive tract. Similar results were shown for LOXL2-mRNA levels in cell lines, whereas no reduction of LOXL2-mRNA levels was found in the malignantly transformed tissues. CONCLUSION: These findings support the presumption that LOX is involved in tumor suppressive processes and also of LOXL2 playing a role in malignant transformation.

Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice.

BACKGROUND: The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. METHODS AND RESULTS: Murine Lox gene was disrupted by routine methods. Lox(-/-) mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox(-/-) aortas were observed, and electron microscopy of the aortic walls of the Lox(-/-) fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox(-/-) fetuses. The wall of the aorta in the Lox(-/-) fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox(+/+) aortas. In Lox(-/-) fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. CONCLUSIONS: Lox has an essential role in the development and function of the cardiovascular system. Inactivation of the Lox gene causes structural alterations in the arterial walls, leading to abnormalities in the cardiovascular functions. Alterations in LOX activity may also play a critical role in certain human cardiovascular diseases.

Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors.

Lysyl oxidase (LOX), a copper-dependent amine oxidase, has been implicated in tumor suppression and cell growth regulation. The chromosomal locus of LOX, 5q23, is affected by loss of heterozygosity (LOH) in colon cancer, suggesting that the LOX gene could be affected by LOH and consequently, loss or reduction of LOX function contribute to the tumorigenic process. Identification of microsatellite markers within the LOX locus has allowed us to map the LOX gene within the 5q23.1 region. Analysis of this locus and flanking loci in matched tumor and blood DNA samples from a panel of colorectal cancer patients, demonstrated that 38% (16/42) of informative samples were affected by LOH or allelic imbalance. Furthermore, 75% (6/8) of these tumor samples were shown to have significantly reduced LOX mRNA levels. Similar reduction in LOX levels were detected in a panel of matched normal colon and colon tumor samples. Tumor samples demonstrating LOH by RFLP, were subject to mutational analysis, including RT-PCR, exonic deletion detection by PCR, cDNA and genomic DNA sequencing, and were found to have a spectrum of alterations and mutations affecting the LOX gene. These results confirm that loss or reduction of LOX function during tumor development is a direct consequence of somatic mutations and is associated with colon tumor pathogenesis.

Metabolic and molecular bases of Menkes disease and occipital horn syndrome.

Menkes disease and occipital horn syndrome (OHS) are related disorders of copper transport that involve abnormal neurodevelopment, connective tissue problems, and often premature death. Location of the gene responsible for these conditions on the X chromosome was indicated by pedigree analysis from the time of these syndromes' earliest descriptions. Characterization of an affected female with an X-autosomal translocation was used to identify the Menkes/OHS gene, which encodes a highly evolutionarily conserved, copper-transporting P-type ATPase. The gene normally is expressed in nearly all human tissues, and it localizes to the trans-Golgi network of cells. However, in over 70% of Menkes and OHS patients studied, expression of this gene has been demonstrated to be abnormal. Major gene deletions detectable by Southern blotting account for 15-20% of patients, and an interesting spectrum of other mutations is evident among 58 families whose precise molecular defects have been reported as of this writing. The center region of the gene seems particularly prone to mutation, and those that influence mRNA processing and splicing appear to be relatively common. Further advances in understanding the molecular and cell biological mechanisms involved in normal copper transport may ultimately yield new and better approaches to the management of these disorders.

Congenital cutis laxa and lysyl oxidase deficiency.

We report two phenotypically similar patients with primary cutis laxa associated with deficiency of lysyl oxidase, an extracellular copper enzyme the gene for which is located on chromosome 5. Previous reports of this condition have had characteristic occipital projections, abnormality of copper metabolism and X-linked inheritance. The two reported patients have no occipital projections, normal copper metabolism, Wormian bones, and a pattern of inheritance consistent with the autosomal recessive inheritance of the lysyl oxidase gene.

Expression of mRNAs for lysyl oxidase and type III procollagen in cultured fibroblasts from patients with the Menkes and occipital horn syndromes as determined by quantitative polymerase chain reaction.

The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a reduction in the activity of lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme that initiates the crosslinking of collagens and elastin. We report here that the amount of lysyl oxidase mRNA, as studied by Northern blotting, and the number of lysyl oxidase mRNA molecules per picogram of RNA, as determined by a quantitative PCR method, were decreased in three cultured skin fibroblast lines from patients with the Menkes syndrome and two from patients with the occipital horn syndrome compared with four control cell lines. The decreased lysyl oxidase activity found in these disorders thus appears to be a least in part due to a pretranslational mechanism. No decrease was found in the number of the beta-actin mRNA molecules in the Menkes cell lines, but rather a slight increase, whereas a decrease was found in these molecules in the occipital horn cell lines. An additional abnormality found in the Menkes cell lines was a significant increase in the number of mRNA molecules for type III procollagen in two of the three cell lines investigated. The present and previous data indicate that the Menkes syndrome may involve several abnormalities in the expression of genes for connective tissue proteins.

Markedly reduced activity of lysyl oxidase in skin and aorta from a patient with Menkes' disease showing unusually severe connective tissue manifestations.

In Menkes' disease, a severe disturbance of copper handling appears to render copper unavailable for copper-requiring processes. We have measured the activity of lysyl oxidase, the copper-dependent enzyme that initiates the cross-linking of collagen and elastin, in extracts of skin and aorta obtained at autopsy from a patient with unusually marked connective tissue manifestations, and found it to be only 6-12% of normal, thus suggesting a basis for these alterations.

Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome.

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.

Abnormal copper metabolism and deficient lysyl oxidase activity in a heritable connective tissue disorder.

Biochemical abnormalities were studied in two brothers with bladder divericulas, inguinal hernias, slight skin laxity, and hyperelasticity and skeletal abnormalities including occipital exostoses. Lysyl oxidase activity was low in the medium of cultured skin fibroblasts, this abnormality being accompanied by reduced conversion of the newly synthesized collagen into the soluble form. Copper concentrations were markedly elevated in the cultured skin fibroblasts, but decreased in the serum and hair. Serum cerulophasmin levels were also low. The reduced lysyl oxidase activity is suggested to be responsible for ther clinical manifestations, but the deficiency in this copper-dependent enzyme may be secondary to the abnormalities in the metabolism of the cation. Nevertheless, a mutation directly affecting both lysyl oxidase and an intracellular copper transport protein cannot be excluded. The disease is tentatively classified as one subtype of the Ehlers-Danlos syndrome.

[Osteogenesis imperfecta. Up-to-date review (author's transl)]. / Osteogénesis imperfecta. I. Estado actual.

Based on review of recent literature and in their own experience, authors point out basic concepts on alterations of collagen, emphasizing clinical and radiological data which differ types II and III in neonates.