Inhibition Of Lysyl Oxidase Activity Research Articles

Extracellular matrix remodeling is associated with the survival of cardiomyocytes in the subendocardial region of the ischemic myocardium.

A significant amount of cardiomyocytes in subendocardial region survive from ischemic insults. In order to understand the mechanism by which these cardiomyocytes survive, the present study was undertaken to examine changes in these surviving cardiomyocytes and their extracellular matrix. Male C57BL/6 mice aged 8-12 weeks old were subjected to a permanent left anterior descending coronary artery ligation to induce ischemic injury. The hearts were collected at 1, 4, 7, or 28 days after the surgery and examined by histology. At day 1 after left anterior descending ligation, there was a significant loss of cardiomyocytes through apoptosis, but a proportion of cardiomyocytes were surviving in the subendocardial region. The surviving cardiomyocytes were gradually changed from rod-shaped to round-shaped, and appeared disconnected. Connexin 43, an important gap junction protein, was significantly decreased, and collagen I and III deposition was significantly increased in the extracellular matrix. Furthermore, lysyl oxidase, a copper-dependent amine oxidase catalyzing the cross-linking of collagens, was significantly increased in the extracellular matrix, paralleled with the surviving cardiomyocytes. Inhibition of lysyl oxidase activity reduced the number of surviving cardiomyocytes. Thus, the extracellular matrix remodeling is correlated with the deformation of cardiomyocytes, and the electrical disconnection between the surviving cardiomyocytes due to connexin 43 depletion and the increase in lysyl oxidase would help these deformed cardiomyocytes survive under ischemic conditions.

Forkhead box C1 promotes metastasis and invasion of non-small cell lung cancer by binding directly to the lysyl oxidase promoter.

Increasing evidence indicates that human forkhead box C1 (FOXC1) plays important roles in tumor development and metastasis. However, the underlying molecular mechanism of FOXC1 in non–small cell lung cancer (NSCLC) metastasis remains unclear. Here, we identified FOXC1 as an independent prognostic factor in NSCLC and showed clear biological implications in invasion and metastasis. FOXC1 overexpression enhanced the proliferation, migration and invasion of NSCLC cells, whereas FOXC1 silencing impaired the effects both in vitro and in vivo. Importantly, we found a positive correlation between FOXC1 expression and lysyl oxidase (LOX) expression in NSCLC cells and patient samples. Downregulation of LOX or LOX activity inhibition in NSCLC cells inhibited the FOXC1‐driven effects on cellular migration and invasion. Xenograft models showed that inhibition of LOX activity by β‐aminopropionitrile monofumarate decreased the number of lung metastases. Mechanistically, we demonstrated a novel FOXC1‐LOX mechanism that was involved in the invasion and metastasis of NSCLC. Dual‐luciferase assay and ChIP identified that FOXC1 bound directly in the LOX promoter region and activated its transcription. Collectively, the present study offered new insight into FOXC1 in the mediation of NSCLC metastasis through interaction with the LOX promoter and further revealed that targeted inhibition of LOX protein activity could prevent lung metastasis in murine xenograft models. These data implicated FOXC1 as a potential therapeutic strategy for the treatment of NSCLC metastasis.

Lysyl oxidase blockade ameliorates anovulation in polycystic ovary syndrome.

Is overexpression of lysyl oxidase (LOX), an enzyme responsible for the cross-linking of collagens, a cause of anovulation in polycystic ovary syndrome (PCOS)? LOX overexpression was present in PCOS ovaries, due at least in part to interleukin-1β (IL-1β), and inhibition of LOX activity with β-aminopropionitrile (BAPN) ameliorated polycystic ovary morphology and anovulation. Aberrant ovarian extracellular matrix (ECM) remodeling and inflammation may contribute to the development of PCOS. It remains unknown whether proinflammatory IL-1β is a contributing factor for LOX overexpression in PCOS ovaries and whether inhibition of LOX can improve PCOS conditions. LOX and IL-1β abundance in the granulosa cells and follicular fluid was compared between non-PCOS (n = 30) and PCOS (n = 39) patients. The effect and mechanism of IL-1β on LOX expression was examined in cultured primary human granulosa cells. The improvements in PCOS conditions by LOX inhibition with BAPN was investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model. The abundance of LOX and IL-1β was measured with quantitative real-time polymerase chain reaction (qRT-PCR), LOX activity assays and enzyme-linked immunosorbent assays (ELISA), respectively. The effect of IL-1β on LOX expression was examined in the presence or absence of inhibitors for signaling molecules and small interfering RNA-mediated knockdown of the putative transcription factor. Chromatin immunoprecipitation assays were conducted to further identify the responsible transcription factor. The role of LOX in ovulation was investigated in a DHEA-induced PCOS rat model with administration of the LOX inhibitor BAPN. The numbers of retrieved total oocytes and MII oocytes were recorded upon ovarian stimulation. Increased abundance of LOX (P < 0.05) and IL-1β (P < 0.05) was observed in the granulosa cells and follicular fluid in PCOS patients. IL-1β increased LOX expression via activation of ERK1/2 and JNK and subsequent activation of the transcription factor c-Jun. Inhibition of LOX with BAPN ameliorated irregular estrous cyclicity (P < 0.05), polycystic ovary morphology and anovulation (P < 0.05) in PCOS rats, but appeared to be ineffective in the improvement of oocyte quality. N/A. Ovarian tissue-directed specific inhibition of LOX in combination with oocyte quality-improving drugs may be more effective in the treatment of PCOS. Inflammation of the ovary is a contributing factor for the aberrant expression of LOX in the PCOS ovary, and inhibition of LOX together with anti-inflammatory therapy may improve the core features of PCOS. This work was supported by National Key R & D Program of China (2017YFC1001403) and Doctorial Innovation Fund of Shanghai Jiao Tong University School of Medicine (BXJ201718). The authors declare no competing financial interests.

Inhibition of Lysyl Oxidase Activity Prevents Volume Overload Diastolic and Systolic Dysfunction

The cardiac extracellular matrix (ECM) is a structural framework which bridges cardiomyocytes to the cystoskeletal myofibril, preserving alignment, while providing tensile strength to the myocardium. Abnormalities in the architectural scaffolding of the ECM have been noted during various cardiac pathologies. The major structural components of the cardiac ECM are collagens, which are produced by fibroblasts. The relative expression of collagens and the degree of cross‐linking dictate the passive mechanical properties of the heart. Lysyl oxidase (LOX) is a copper‐dependent amine oxidase that plays a critical role in covalently cross‐linking fibrillar collagens, which determines the insolubility, stiffness and resistance to degradation of the resulting collagen fibrils. In our previous studies we have shown marked increases in LOX expression and activity in failing hearts relative to compensated and unstressed hearts. Our goal of this study was to determine whether LOX inhibition, initiated during the early stages of cardiac remodeling, could attenuate fibrosis and cardiac dysfunction.We used the aortocaval fistula (ACF) surgical model on Sprague‐Dawley rats to induce biventricular volume overload (VO). Two weeks after surgery, both sham‐operated and VO rats were treated with the LOX inhibitor, beta‐aminoproprionitrile (BAPN; 100 mg/kg/d), for 12 weeks. BAPN was administered via an osmotic pump surgically inserted into the abdominal cavity. After a total of 14 weeks of VO, left ventricular (LV) pressure‐volume catheterization was used to assess alterations in cardiac function. Western blot protein analyses were performed to assess alterations in LOX, collagens I and III, matrix metalloproteinases (MMPs), and their inhibitors (TIMPs). Further, LV strain and stiffness were calculated to determine alterations in diastolic function.Our data demonstrate that chronic VO caused a significant increase in LV wall stress at both diastole and systole, and produced significant LV hypertrophy (183%, 60%, and 92% increase, respectively vs. control; p&lt;0.05). Further, VO induced significant increases in LV strain and stiffness (126% and 77% increase, respectively vs. control), as well as significant increases in the expression of fibrotic proteins including LOX, collagen I, collagen III, MMP2, and TIMP1 (135%, 58.4%, 26.7%, 39%, and 63% increase, respectively vs. control; p&lt;0.05). Finally, VO also caused systolic dysfunction, as evident by decreases in cardiac contractility and ejection fraction (51% and 26% decrease, respectively vs. control; p&lt;0.05). Further, our data highlight the cardioprotective role of LOX inhibition against adverse cardiac remodeling due to VO. LOX inhibition completely prevented VO‐induced increases in LV wall stress and ventricular hypertrophy. Further, LOX inhibition prevented VO‐induced fibrosis and diastolic dysfunction, as evident by normalized levels of collagens, LOX, MMPs, TIMPs, and LV stiffness. Finally, LOX inhibition prevented the VO‐induced decline in cardiac contractility. Overall, our data indicate that LOX inhibition prevented adverse ECM remodeling and cardiac dysfunction in the volume overloaded heart.Support or Funding InformationSupport for this project provided by the American Heart Association Southeast Affiliate and the NIH Heart, Lung and Blood Institute: Grant‐in‐Aid (#16GRNT30440008‐JDG), AHA Predoctoral Fellowship (# 16PRE29150010‐EEH), and F31 (#1F31HL134263‐ 01A1‐EEH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Screening of lysyl oxidase (LOX) and lysyl oxidase like (LOXL) enzyme expression and activity in preterm prelabor rupture of fetal membranes.

Lysyl oxidase (LOX) and LOX like enzymes (LOXL1-4) physiologically remodel extracellular matrix and pathologically contribute to cellular senescence under oxidative stress (OS). We characterized LOX and LOXL expressions and activity in human fetal membranes. Human fetal membranes from women with uncomplicated pregnancies at term, preterm birth with intact membranes (PTB) or preterm prelabor rupture of membranes (pPROM), and in vitro fetal membranes stimulated with water-soluble cigarette smoke extract (CSE), an OS inducer, were analyzed by real-time PCR and immunohistochemistry for LOX and LOXL (1-4) expression and localization. LOX activity was measured by fluorometric assay. LOX gene expression was ∼2.5-fold higher in fetal membranes from pPROM compared to PTB and term (P=0.02). LOX and LOXL1, 2 and 4 were localized to both amniotic and chorionic cells, whereas LOXL3 was limited to chorion. LOX and LOXL isoform expressions were not different between CSE treated and untreated groups, while LOX activity was increased in the presence of an antioxidant (P=0.02). Increase of LOX expression in pPROM, an OS-related disease, and the apparent inhibition of LOX activity by CSE restored by antioxidant treatment suggest that reactive oxygen species might influence LOX-mediated tissue remodeling in fetal membranes. Balanced antioxidant supplementation during pregnancy may reduce the risk of pPROM by increasing LOX activity.

Inhibition of lysyl oxidase activity reverses fibrosis and improves cardiac function (1152.8)

The collagen crosslinking enzyme, lysyl oxidase (LOX), is elevated in human dilated cardiomyopathy and heart failure, but it is not known if LOX plays a causative role in disease. However, clinical evidence shows that diuretics with LOX inhibitory properties are more effective at slowing the progression of heart failure. Using the aortocaval fistula (ACF) rat model of volume overload, we assessed the efficacy of LOX inhibition to reverse cardiac fibrosis and dysfunction in rodents with established cardiac disease. Serial echocardiography was used to evaluate cardiac structural and functional changes. LOX inhibition (100 mg/kg/d BAPN; osmotic minipumps) was initiated 8 wks post‐surgery in ACF and SHAM‐operated rats. Treatment continued for 6 wks and hearts were collected for analysis. Left ventricular (LV) sections were stained with PSR for collagen volume fraction (CVF). Protein expression was assessed in LV homogenates by WB. Untreated ACF had significantly increased LV LOX expression (65% increase), concomitant with LV dilatation (43% increase), depressed fractional shortening (26% decrease), increased collagen cross‐linking (52% increase), and interstitial fibrosis (CVF; 135% increase) relative to SHAM (p&lt;0.05). LOX inhibition reversed fibrosis (CVF; Treated ACF vs SHAM; p=NS) and partially restored cardiac function (20% increase vs Untreated ACF). However, there was no significant reversal of LV hypertrophy or dilatation. LV NADPH oxidase‐4 was increased in untreated ACF (52% increase vs SHAM), but was not different in treated ACF, indicating a possible role for oxidative stress in the observed beneficial effects of LOX inhibition. These data identify inhibition of LOX activity as a potential therapeutic approach for the regression of cardiac fibrosis and improvement of function in heart failure patients.Grant Funding Source: Supported by LSUSOM REF grant (jdg) and the American Heart Association GSA 11GRNT7700002 (jdg)

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.

N-Acetylcysteine Downregulation of Lysyl Oxidase Activity Alleviating Bleomycin-Induced Pulmonary Fibrosis in Rats

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease without beneficial therapy, except for lung transplantation. A high oral dose of N-acetylcysteine (NAC) added to prednisone and azathioprine has been found to improve lung function in IPF patients, though the mechanism of action remains poorly understood. Objective: Based on our previous findings showing elevation of glutathione (GSH) content associated with downregulation of lysyl oxidase (LOX) activity, which is essential for collagen deposition, the aim of the present study was to test the hypothesis that NAC alleviates IPF by regulating LOX function. Methods: We firstly analyzed the time course of collagen deposition in lung tissue, hydroxyproline content, LOX activity, GSH levels, and transforming growth factor-β₁ (TGF-β₁) and α-smooth muscle actin (α-SMA) expression in bleomycin (BLM)-induced pulmonary fibrosis in a rat model. Then, we focused our studies on NAC modulation of LOX activity. Results: LOX activity was increased on day 9 and peaked 14 days after BLM administration, while TGF-β₁ protein peaked on day 9. Interestingly, NAC treatment for 14 days from day 0 reversed LOX activity to normal levels and increased GSH levels in the lung of BLM-dosed rats. Consistently, NAC partially attenuated pulmonary fibrosis and inhibited TGF-β₁ and α-SMA expression in this model. Conclusions: Our study supports a novel mechanism of NAC alleviating IPF by inhibition of LOX activity via elevation of lung GSH in BLM-induced pulmonary fibrosis. The TGF-β₁/α-SMA pathway may also play an important role in modulation of LOX activity.

Abstract 2456: TGF-Beta regulates tumor ECM remodeling and progression to metastasis

Abstract To understand how loss of TGF-β signaling in cancer cells promotes tumor progression, we are evaluating modifications to the extracellular matrix in TGF-β type II receptor KO mammary tumors. Previous work from our lab supports a stromal expansion and increased TGF-β availability in PyMT tumors lacking TGF-β signaling in the epithelium. This expansion is characterized by increased activated fibroblasts, collagen deposition, and MDSC infiltration. We hypothesize that the increased TGF-β availability and fibroblast activation in TGF-β receptor II knock out tumors will also lead to increased ECM remodeling. Crosslinking of collagen fibers increases the overall stiffness of affected tissue providing the mechanical force necessary for tumor cell migration and invasion. Lysyl oxidase is a potent extracellular enzyme that crosslinks collagen and elastin fibers. The study of human breast cancer cell lines shows that HIF-1a upregulates lysyl oxidase in highly invasive cells and treatment with a lysyl oxidase inhibitor significantly decreases cellular invasion. Past work also supports TGF-B driving lysyl oxidase expression in the stroma of many tissue types. The interaction between the TGF-β secreted by infiltrating myeloid cells and stromal fibroblasts has yet to be elucidated and the combination of these effects on ECM remodeling and tumor metastasis will be the focus of this study. In this study we utilize transgenic mice which express PyMT under a mammary epithelial specific promoter, MMTV, and have the type II TGF-β receptor conditionally deleted from mammary epithelial cells. Prior to tumor palpation, we see an increase in collagen deposition as well as tissue tensile strength indicating a higher degree of collagen crosslinking. In PyMT tumors originating from the RII KO mammary glands, we see an increased deposition of collagen as well as an increased expression of lysyl oxidase. In our model, both in vitro and in vivo evidence supports stromal expression of lysyl oxidase by fibroblasts. Increased levels of stromal lysyl oxidase are correlated with increased levels of available TGF-β in the RII KO tumors which is primarily derived from infiltrating myeloid cells. Upon inhibition of lysyl oxidase activity during development of PyMT tumors in vivo, we observe a dramatic decrease in the ability of the cells to metastasize primarily through a loss of the ability to intravasate into the circulation. These results suggest that loss of the TGF-β type II receptor in mammary carcinoma cells promotes invasion through increases in lysyl oxidase activity and tissue stiffness. Future studies will focus on cellular response to variability in tissue stiffness and how the changes relate to in vivo tumors and their ability to enter the blood stream. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2456. doi:1538-7445.AM2012-2456

Control of Megakaryocyte Expansion and Bone Marrow Fibrosis by Lysyl Oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.

Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

Developmental toxicity of cartap on zebrafish embryos

Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144 h post-fertilization, to a range of concentrations of 25–1000 μg/l. Results of the experiment indicated that cartap concentrations of 100 μg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25 μg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis.

Hypoxia-Inducible Factor 1α Induces Fibrosis and Insulin Resistance in White Adipose Tissue

Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1alpha (HIF1alpha) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1alpha, we determined that HIF1alpha fails to induce the expected proangiogenic response. In contrast, we observed that HIF1alpha initiates adipose tissue fibrosis, with an associated increase in local inflammation. "Trichrome- and picrosirius red-positive streaks," enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1alpha and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by beta-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Inhibition of lysyl oxidase activity can delay phenotypic modulation of chondrocytes in two-dimensional culture

Chondrocytes frequently de-differentiate in two-dimensional (2D) culture, especially in the presence of serum. To examine the role of lysyl oxidase (LOX) induced cross-linking in this phenomenon, the effect of the specific LOX inhibitor beta-aminopropionitrile (BAPN) was studied in 2D chondrocyte culture. Chick embryo sternal chondrocytes (both proliferative and hypertrophic, from caudal and cranial zones, respectively) were cultured in the presence and absence of BAPN. The production and activities of LOX and LOX-like (LOXL) were assessed by enzyme assay and the use of specific antibodies. Seventeen batches of serum of different origin were compared. Chondrocyte phenotype was assessed both morphologically and biochemically, the latter by quantitative analysis of production of radiolabeled cartilage collagens II, IX, X and XI, and the de-differentiation marker collagen I, for up to 4 weeks in culture. LOX and LOXL were identified, by Western blotting and immunofluorescence, and LO activity was measured in the medium, with both proliferative and hypertrophic chondrocytes. Inhibition of LO activity prevented or delayed chondrocyte de-differentiation, as characterized by changes in cell shape and synthesis of the five different collagen types, from the first days of culture for up to 4 weeks, depending on the origin of the serum added to the culture medium. LO activity may be involved in the control of chondrocyte phenotype, in addition to serum factors. Inhibition of LO activity by BAPN may be useful for the maintenance of the chondrocyte phenotype in 2D culture. Specific variations in the relative proportions of collagens II, IX and XI could be involved in the mechanism underlying these observations.

High levels of homocysteine inhibit lysyl oxidase (LOX) and downregulate LOX expression in vascular endothelial cells

Background: Hyperhomocysteinemia, an independent risk factor for cardiovascular disease and atherothrombosis, alters endothelial function through a mechanism not fully understood. Downregulation of lysyl oxidase (LOX), an enzyme involved in extracellular matrix maturation, impairs the endothelial barrier function and could be involved in homocysteine (HC)-induced endothelial dysfunction. Objective: The aim of this study was to analyze the effect of HC on LOX regulation in vascular endothelial cells. Results: HC at pathophysiological concentrations (35 μM) inhibited LOX activity in porcine aortic endothelial cells. Homocysteine thiolactone and related molecules containing sulfhydryl groups (cysteine), but not methionine or homocystine (non-containing thiol-group) inhibited LOX. In addition, the blockade of HC-sulfhydryl group by N-ethylmaleimide abrogated HC-induced LOX downregulation. This process was triggered by oxidative stress since superoxide dismutase and vitamin C reverted LOX inhibition caused by HC. On the contrary, the effect was not mediated through the induction of endoplasmic reticulum stress. Finally, higher doses of HC (200 μM), common in severe hyperhomocysteinemia, decreased LOX mRNA levels (≈2-fold) and LOX promoter activity in transient transfection experiments. Conclusions: These findings suggest that LOX inhibition contributes to the endothelial dysfunction associated with hyperhomocysteinemia. This effect was dependent on a mechanism involving both an inhibition of LOX activity and a reduction of LOX expression.

Low density lipoproteins downregulate lysyl oxidase in vascular endothelial cells and the arterial wall

Objective— Hypercholesterolemia induces endothelial dysfunction, a hallmark of the atherosclerotic process, modulating the expression of key genes in vascular endothelial cells. Methods and Results— By differential display analysis, we have studied the effect of high concentrations of native low density lipoprotein (LDL) on endothelial gene expression. mRNA levels of lysyl oxidase (LOX), an enzyme involved in collagen and elastin cross-linking, were downregulated by LDL treatment in endothelial cells in a dose- and time-dependent manner (80% of inhibition by 180 mg/dL LDL for 24 hours). This reduction of LOX expression was associated with a decrease in LOX activity (40% and 54% of inhibition after 24 and 48 hours of LDL treatment, respectively). LOX mRNA half-life was not modified by LDL, but transcriptional inhibition blocked the effect of LDL. Inhibition of LOX activity by either LDL or β-aminopropionitrile, an inhibitor of LOX, increased endothelial permeability (192±0.19- and 3.37±0.74-fold, respectively). Interestingly, a reduction in LOX expression (3.5-fold) was observed in vivo in the vascular wall of hypercholesterolemic pigs. Conclusions— These findings suggest that LDL downregulation of LOX could contribute to the endothelial dysfunction caused by hypercholesterolemia, thus contributing to atherosclerotic plaque formation.

Low density lipoproteins downregulate lysyl oxidase in vascular endothelial cells and the arterial wall.

Hypercholesterolemia induces endothelial dysfunction, a hallmark of the atherosclerotic process, modulating the expression of key genes in vascular endothelial cells. By differential display analysis, we have studied the effect of high concentrations of native low density lipoprotein (LDL) on endothelial gene expression. mRNA levels of lysyl oxidase (LOX), an enzyme involved in collagen and elastin cross-linking, were downregulated by LDL treatment in endothelial cells in a dose- and time-dependent manner (80% of inhibition by 180 mg/dL LDL for 24 hours). This reduction of LOX expression was associated with a decrease in LOX activity (40% and 54% of inhibition after 24 and 48 hours of LDL treatment, respectively). LOX mRNA half-life was not modified by LDL, but transcriptional inhibition blocked the effect of LDL. Inhibition of LOX activity by either LDL or beta-aminopropionitrile, an inhibitor of LOX, increased endothelial permeability (192+/-0.19- and 3.37+/-0.74-fold, respectively). Interestingly, a reduction in LOX expression (3.5-fold) was observed in vivo in the vascular wall of hypercholesterolemic pigs. These findings suggest that LDL downregulation of LOX could contribute to the endothelial dysfunction caused by hypercholesterolemia, thus contributing to atherosclerotic plaque formation.

Abnormal Collagen Assembly, though Normal Phenotype, in Alginate Bead Cultures of Chick Embryo Chondrocytes

The collagens produced by chick embryo chondrocytes cultured in alginate beads were investigated both biochemically and ultrastructurally. The cartilage phenotype is maintained for at least 14 days, as indicated by the production of the cartilage-specific collagens II, IX, and XI and the absence of collagen I. There were differences in the distributions of collagens among the three different compartments analyzed (cells and their associated matrix, further-removed matrix (released by alginate solubilization), and culture medium), with large amounts of collagen IX (mainly in proteoglycan form) in the culture medium. Inhibition of lysyl oxidase activity by β-aminopropionitrile led to an overall decrease in collagen production. In contrast to the biochemical observations, collagen ultrastructure in the extracellular matrix of alginate cultures was not in the form of the expected 64-nm banded fibrils, but rather in the form of segment-long-spacing-like crystallites. This abnormal structure is likely to be a result of alginate disrupting normal assembly. We conclude that, in this system, the native fibrillar structure of the collagenous matrix is not essential for the maintenance of the differentiated phenotype of chondrocytes.