Serum Lysyl Oxidase Is a Potential Diagnostic Biomarker for Kidney Fibrosis.

BACKGROUND: Kidney fibrosis is the ultimate consequence of advanced stages of chronic kidney disease (CKD); however, there are currently no reliable biomarkers or noninvasive diagnostic tests available for the detection of kidney fibrosis. Lysyl oxidase (LOX) promotes collagen cross-linking, and serum LOX levels have been shown to be elevated in patients with fibrosis of the heart, lungs, and liver. However, serum LOX levels have not been reported in patients with kidney fibrosis. We explored whether serum LOX levels are associated with kidney fibrosis. METHOD: Overall, 202 patients with kidney disease underwent renal biopsy, scoring of kidney fibrosis, and determination of the area of kidney fibrosis. LOX levels were measured in serum and in kidney tissues. We analyzed the association of circulating LOX and tissue LOX levels with the scores and areas of kidney fibrosis. LOX expression was also investigated with in vitro and in vivo kidney fibrosis models. RESULTS: Serum LOX levels were higher in patients with kidney fibrosis than in those without kidney fibrosis (p < 0.001) and higher in patients with moderate-severe kidney fibrosis than in patients with mild kidney fibrosis (p < 0.001). Both serum LOX and renal tissue LOX levels correlated with the area of kidney fibrosis (r = 0.748, p < 0.001; r = 0.899, p < 0.001, respectively). Receiver operating characteristic curve analysis of serum LOX levels showed an area under the curve of 0.80 (95% CI: 0.74-0.86). The optimal serum LOX level cutoff point was 253.34 pg/mL for the prediction of kidney fibrosis and 306.56 pg/mL for the prediction of moderate-severe kidney fibrosis. LOX expression levels were significantly upregulated (2.3-2.6 and 6-fold, respectively) in in vitro and in vivo interstitial fibrosis models. CONCLUSIONS: Both serum LOX and tissue LOX levels correlated with the presence and degree of kidney fibrosis in patients with CKD. These results suggest that serum LOX levels could potentially serve as a noninvasive diagnostic biomarker for kidney fibrosis and may further potentially serve as a stratified biomarker for the identification of mild and moderate-severe kidney fibrosis.

Is cardiovascular disease in patients with diabetes associated with serum levels of MMP-2, LOX, and the elastin degradation products ELM and ELM-2?

BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression in diabetic patients, namely matrix metalloproteinase 2 (MMP-2), lysyl oxidase (LOX) and elastin itself. In this study we investigate whether the serum concentrations of elastin-related proteins correlate to signs of CVD in patients with T2DM. METHODS: Blood samples from 302 type 2 diabetic patients were analysed for MMP-2, LOX, and the elastin degradation products ELM and ELM2. The results were investigated for correlations to signs of CVD in different vascular territories, as determined by myocardial perfusion scintigraphy, carotid artery thickness and ankle-brachial blood pressure index. RESULTS: T2DM patients with peripheral arterial disease (low ankle-brachial index) (PAD) display higher levels of MMP-2 and ELM compared to patients without PAD. However, none of the proteins or degradation products correlated with myocardial ischemia or a combined measure of CVD-signs, including myocardial ischemia, increased carotid thickness and decreased ankle-brachial blood pressure. CONCLUSIONS: Our results suggest that the diabetic environment affects the circulating amounts of MMP-2 and ELM in patients with PAD. However, the same connection could not be seen in diabetic patients with CVD broadly identified in three vascular territories. LOX and ELM-2 did not correlate to any type of CVD. Overall, serum levels of elastin-related molecules are only remotely related to CVD in type 2 diabetes.

Blocking Surgically Induced Lysyl Oxidase Activity Reduces the Risk of Lung Metastases.

Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distant metastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX) activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery.

Lysyl Oxidase as a Serum Biomarker of Liver Fibrosis in Patients with Severe Obesity and Obstructive Sleep Apnea.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is associated with the progression of nonalcoholic fatty liver disease (NAFLD). We hypothesized that the hypoxia of OSA increases hepatic production of lysyl oxidase (LOX), an enzyme that cross-links collagen, and that LOX may serve as a biomarker of hepatic fibrosis. DESIGN: Thirty-five patients with severe obesity underwent liver biopsy, polysomnography, and serum LOX testing. A separate group with severe OSA had serum LOX measured before and after 3 mo of CPAP or no therapy, as did age-matched controls. LOX expression and secretion were measured in mouse hepatocytes following exposure to hypoxia. SETTING: The Johns Hopkins Bayview Sleep Disorders Center, and the Hypertension Unit of the Heart Institute at the University of São Paulo Medical School. MEASUREMENTS AND RESULTS: In the bariatric cohort, the apnea-hypopnea index was higher in patients with hepatic fibrosis than in those without fibrosis (42.7 ± 30.2 events/h, versus 16.2 ± 15.5 events/h; P = 0.002), as was serum LOX (84.64 ± 29.71 ng/mL, versus 45.46 ± 17.16 ng/mL; P < 0.001). In the sleep clinic sample, patients with severe OSA had higher baseline LOX than healthy controls (70.75 ng/mL versus 52.36 ng/mL, P = 0.046), and serum LOX decreased in patients with OSA on CPAP (mean decrease 20.49 ng/mL) but not in untreated patients (mean decrease 0.19 ng/mL). Hypoxic mouse hepatocytes demonstrated 5.9-fold increased LOX transcription (P = 0.046), and enhanced LOX protein secretion. CONCLUSIONS: The hypoxic stress of obstructive sleep apnea may increase circulating lysyl oxidase (LOX) levels. LOX may serve as a biomarker of liver fibrosis in patients with severe obesity and nonalcoholic fatty liver disease.

Lysyl oxidase genetic variants affect gene expression in cervical cancer.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the homeostasis of tumors. The aim of this study was to investigate the association between LOX polymorphisms and cervical cancer, and the effect of these polymorphisms on gene expression. We evaluated two polymorphisms of LOX, rs1800449G/A (G473A) and rs2278226C/G, in 262 cervical cancer cases and 298 healthy controls in the Chinese population. Results showed that the prevalence of rs1800449AA genotype was significantly increased in cases than in controls (p=0.004). Individuals who carried the rs1800449A allele had a 1.56-fold increased risk for cervical cancer than those with the rs1800449G allele (p=0.003). The rs2278226CG genotype also revealed a significantly higher proportion in cases (20.6%) than in controls (7.7%, p<0.001). Interestingly, when analyzing these two polymorphisms with the serum level of LOX, we identified that cervical cancer patients carrying the rs2278226CG genotype had a significantly elevated level of LOX than those with rs2278226CC wild type, whereas the same phenomenon was not observed in controls. The rs1800449 polymorphism did not affect the LOX serum level in either controls or patients. These results suggest that the polymorphisms in the LOX gene may be involved in the development of cervical cancer through various mechanisms.

Brief report: lysyl oxidase is a potential biomarker of fibrosis in systemic sclerosis.

OBJECTIVE: Fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc). Levels of lysyl oxidase (LOX), an extracellular enzyme that stabilizes collagen fibrils, have been found to be elevated in the skin of SSc patients, but have not been evaluated in the serum or correlated with the clinical parameters. We undertook this study to evaluate serum LOX levels in SSc patients and to correlate these levels with clinical parameters of SSc. METHODS: SSc patients were evaluated for demographic features, clinical manifestations, routine laboratory tests, serum autoantibodies, serum LOX concentrations, and nailfold capillaroscopy patterns. They underwent pulmonary function testing, echocardiography, and high-resolution computed tomography scans of the lung, assessment of skin fibrosis by the modified Rodnan skin thickness score (MRSS), and assessment of disease severity and activity by the Medsger severity scale and the Valentini activity index. RESULTS: Twenty-six SSc patients were evaluated and compared with 25 healthy controls and with 9 disease control patients with primary myelofibrosis. Almost 62% of the SSc patients (16 of 26) had limited cutaneous SSc (lcSSc), while 38% had diffuse cutaneous SSc (dcSSc) (10 of 26); 31% of the patients (8 of 26) had lung involvement. The LOX concentration in SSc patients was higher than that in healthy controls and similar to that in disease controls (P < 0.0001), and it was significantly higher in patients with dcSSc than in those with lcSSc (P = 0.006). The LOX concentration correlated with the MRSS in patients without lung fibrosis. CONCLUSION: This study is the first to demonstrate high serum LOX levels in SSc patients that correlate specifically with skin fibrosis. These correlations suggest that LOX levels may serve as a novel biomarker of fibrosis. Future studies are warranted to determine whether LOX is a potential therapeutic target in SSc.

Evaluation of serum lysyl oxidase as a blood test for colorectal cancer.

AIMS: Lysyl oxidase (LOX) expression is elevated in colorectal cancer (CRC) tissue and associated with disease progression. A blood test may form a more acceptable diagnostic test for CRC although LOX has not previously been measured in the serum. We therefore sought to determine the clinical usefulness of a serum LOX test for CRC in a symptomatic population. METHODS: Adult patients referred to a hospital colorectal clinic with bowel symptoms completed a questionnaire and provided a blood sample for serum LOX measurement. Associations between presenting symptoms, serum LOX concentrations and outcomes of investigations were tested by univariate and multivariate analyses to determine if serum LOX was clinically useful in the prediction of CRC. LOX expression in CRC and adjacent colon biopsies was evaluated by ELISA and immunohistochemistry. RESULTS: Thirty-one cases of colorectal cancer and 16 high-risk polyps were identified from a total of 962 participants. There was no association between serum LOX concentration and the presence of CRC, high-risk polyps or cancers at any site. LOX expression was significantly increased in CRC tissue compared to adjacent colon. CONCLUSION: Despite overexpression of LOX in CRC tissue, elevated serum levels could not be demonstrated. Serum LOX measurement is therefore not a clinically useful test for CRC.

The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Increased aortic stiffness and attenuated lysyl oxidase activity in obesity.

OBJECTIVE: One potential mechanism through which obesity exerts adverse effects on the vascular system is by increasing aortic stiffness, a change known to be predictive of increased cardiovascular mortality. The aim of this study was to investigate the pathophysiology that links obesity to aortic stiffening. APPROACH AND RESULTS: Obese (ob/ob) mice were used to examine physical, morphological, and molecular changes in the aorta in response to obesity. ob/ob mice had increased aortic pulse wave velocity and tissue rigidity. ob/ob aorta exhibited decreases of lysyl oxidase (LOX) activity and cross-linked elastin, and increases of elastin fragmentation and elastolytic activity. The aortas of ob/ob mice were surrounded by a significant amount of proinflammatory and pro-oxidative perivascular adipose tissue. In vitro studies revealed that the conditioned medium from differentiated adipocytes or the perivascular adipose tissue of ob/ob mice attenuated LOX activity. Furthermore, inhibition of LOX in wild-type lean mice caused elastin fragmentation and induced a significant increase in pulse wave velocity. Finally, we found that obese humans had stiffer arteries and lower serum LOX levels than do normal-weight humans. CONCLUSIONS: Our results demonstrated that obesity resulted in aortic stiffening in both humans and mice, and established a causal relationship between LOX downregulation and aortic stiffening in obesity.

Zinc and copper status and blood pressure.

In order to elucidate the relationships between Zn and Cu and blood pressure, the present case-control study was carried out. Zn and Cu status was evaluated in 60 subjects, pharmacologically untreated, affected by mild stable hypertension and in 60 normotensives matched for sex, age and smoking habits. Different markers of Zn and Cu status, including serum, erythrocyte and urine levels of the two trace elements and activities of some Zn- or Cu-dependent enzymes (alkaline phosphatase, lactic dehydrogenase, superoxide dismutase and lysyl oxidase) were evaluated. No significant difference between hypertensives and normotensives was observed in the mean levels of Zn and Cu as well as in Zn- or Cu-dependent enzymes, though higher levels of serum copper were associated with increased risk of hypertension. Interesting relationships between the biological parameters investigated were observed in the hypertensive subjects. Inverse correlations between blood pressures and serum Zn were observed. Furthermore, blood pressure was inversely related to lysyl oxidase activity. These findings give further support to the hypothesis that an imbalance of Zn and Cu bioavailability may be associated to hypertensive condition.

Zinc, copper, and zinc- or copper-dependent enzymes in human hypertension.

Imbalance of zinc and copper status has been hypothesized in human hypertension. A case-control study was carried out to elucidate the possible relationship between zinc and copper status and essential hypertension. Thirty-one subjects affected by mild stable hypertension, pharmacologically untreated, were investigated together with 31 normotensive controls individually matched for sex, age, and smoking habits. Zinc and copper in serum and urine wee measured, and serum activities of alkaline phosphatase (AP), lactic dehydrogenase (LDH), copper-zinc superoxide dismutase (Cu-Zn SOD), lysyl oxidase (LOX), and monoamine oxidase (MAO) were evaluated. No significant difference in serum and urine zinc and copper content as far as in serum activity of zinc (AP and LDH) or copper (Cu-Zn SOD, LOX, and MAO)-dependent enzymes was found between hypertensives and normotensives. Positive relationships were found in normotensives between serum and urine levels of zinc (r = 0.577; p = 0.001) and copper (r = 0.394; p = 0.028), and between serum copper and Cu-Zn SOD (r = 0.534; p = 0.002). In normotensives, diastolic blood pressure and serum zinc were positively related (r = 0.370; p = 0.041). In hypertensives, inverse correlations were observed between diastolic blood pressure and AP (r = -0.498; p = 0.004) and Cu-Zn SOD (r = 0.452; p = 0.011), and between systolic blood pressure and LOX (r = -0.385; p = 0.033). Diastolic blood pressure was related to LDH inversely in hypertensives (r = -0.357; p = 0.049) and positively in normotensives (r = 0.457; p = 0.010). In normotensives, diastolic blood pressure was inversely related with MAO (r = -0.360; p = 0.046). These findings support the hypothesis that an imbalance of zinc and copper status might be involved in human hypertension.

Serum lysyl oxidase activity in chronic liver disease in comparison with serum levels of prolyl hydroxylase and laminin.

Lysyl oxidase was partially purified from serum by a diethylaminoethyl batch procedure in the presence of 6 mol/L urea and dialyzed against 3 mol/L KSCN. Using this method, we determined serum lysyl oxidase activity in 52 patients with liver disease and in 14 healthy controls, and we examined usefulness of serum lysyl oxidase in assessing liver fibrogenesis. For this purpose, serum lysyl oxidase activity in chronic liver disease was compared with serum levels of prolyl hydroxylase and laminin P1. As compared with controls, serum lysyl oxidase activity increased 1.6-fold in chronic persistent hepatitis, 4.4-fold in chronic active hepatitis and 11.8-fold in cirrhosis, indicating an increase in concert with the development of liver fibrosis. In hepatocellular carcinoma, the serum activity, although significantly increased, was lower than that in cirrhosis. Serum prolyl hydroxylase was significantly increased in chronic active hepatitis, in liver cirrhosis and in hepatocellular carcinoma. Serum laminin P1 was significantly increased in chronic active hepatitis, in cirrhosis and in hepatocellular carcinoma. Serum lysyl oxidase activity did not correlate significantly with serum levels of prolyl hydroxylase and laminin P1 in any subject or in any subgroup. The magnitude of the increase and the abnormal percentage of serum lysyl oxidase activity were larger than those for serum prolyl hydroxylase and laminin P1. These results suggest that serum lysyl oxidase activity is a more sensitive indicator of liver fibrosis than serum prolyl hydroxylase and laminin P1.

[The diagnostic importance of research on the lysyl oxidase activity in children with circumscribed scleroderma]. / Diagnosticheskoe znachenie issledovanii aktivnosti liziloksidazy u detei, bol'nykh ogranichennoi sklerodermiei.

Blood serum lysyl oxidase activity considerably varies, these variations correlating with the skin process dissemination, in children suffering from focal scleroderma. This enzyme was undetectable in normal subjects and patients with dermatoses whose clinical symptoms are similar to those of focal scleroderma. Blood serum lysyl oxidase activity elevation is a specific sign, characteristic of patients with focal scleroderma; it therefore may be used for the laboratory diagnosis of this dermatosis, assessment of its dissemination and severity.

Increased activity of serum amine oxidases in granuloma annulare, necrobiosis lipoidica and diabetes.

Serum monoamine oxidase, diamine oxidase and lysyl oxidase-like activity were measured in patients with granuloma annulare (GA), necrobiosis lipoidica (NL) and diabetes mellitus. In diabetes, all enzyme measurements were raised by a factor of about 2 X 2, and in NL by a factor of about 1 X 5. The rise in patients with GA was small and only significant in the case of benzylamine monoamine oxidase. "Stiff' collagen would seem to link these three disorders and the present results suggest that these amine oxidases could be useful in monitoring collagen abnormality in diabetes and diabetes-associated disorders, particularly in the absence of chronic liver disease. A negative correlation was found between enzyme activity and blood glucose levels, thus collagen changes in these conditions may occur independently of elevated blood glucose levels. Possible involvement of these enzymes in angiopathy remains to be elucidated.

[Copper-containing amine oxidases of blood vessels (review of the literature)]. / Med'soderzhashchie aminoksidazy krovenosnykh sosudov (obzor).

Modern data on types, physico-chemical properties, physiological role of copper-containing amine oxidase of blood vessels are reviewed. Properties of lysyl oxidase--specific copper-containing amine oxidase--are described. Importance of the enzyme for normal functions of connective tissue as well as alterations in the enzymatic activity under various pathological conditions are discussed. At the same time, properties and physiological functions of another copper-containing amine oxidase of blood vessels, which uses polyamines as substrate, are considered. Effect of the copper levels on content of polyamines in body is discussed.

The existence of inhibited lysyl oxidase and the presence of multiple forms.

Considerable difficulty in puriying tissue lysyl oxidase has been previously encountered and the cause of this difficulty has now been ascertained. For these studies, we have used blood plasma lysyl oxidase as a model system. Blood plasma has been shown to contain considerable lysyl oxidase activity. The enzyme exists in a partially or completely inhibited state. Homogeneous preparations of the enzyme from the blood have been obtained by two different methods. Method A yields one form of lysyl oxidase while Method B yields three forms of the enzyme. The methods for the purification of the enzyme and some properties of the enzyme and preliminary data on the lysyl oxidase inhibitor will be discussed.