Lysyl Oxidase Protein Expression Research Articles

Attenuation of palmitic acid-induced lysyl oxidase overexpression in the ovary contributes to the improvement of ovulation in obesity by metformin

Abstract STUDY QUESTION Does palmitic acid (PA), the most common saturated free fatty acid (FFA) in individuals with obesity, contribute to anovulation through upregulation of the collagen-crosslinking enzyme lysyl oxidase (LOX) in the ovary? SUMMARY ANSWER Increased PA in individuals with obesity can cause LOX upregulation via the activation of hypoxia-inducible factor-1α (HIF-1α), resulting in abnormal collagen deposition in the ovary and anovulation, which can be ameliorated by metformin therapy. WHAT IS KNOWN ALREADY The underlying cause of anovulation in individuals with obesity is poorly defined, and accumulating evidence indicates that hormonal disturbance, insulin resistance, and inflammation may all play a role in the development of ovulation disorders in individuals with obesity. However, it remains to be determined whether PA plays a role in the regulation of LOX expression, thus disrupting ovarian extracellular matrix (ECM) remodelling in the ovary and resulting in impaired ovulation in individuals with obesity. STUDY DESIGN, SIZE, DURATION PA concentration and LOX protein abundance and activity in follicular fluid and ovarian tissue were compared between control (n = 21) subjects, patients with obesity with ovulation (n = 22), and patients with obesity with anovulation (n = 16). The effect of PA on LOX protein expression, and the underlying mechanism, was examined in primary human granulosa cells in vitro. The improvements in obesity conditions induced by LOX inhibition combined with metformin were investigated in a high-fat diet-induced obese rat model. PARTICIPANTS/MATERIALS, SETTING, METHODS The abundance of PA concentration and LOX activity was measured via a LOX activity assay and ELISA, respectively. The effect of PA on LOX protein expression was examined in the presence or absence of inhibitors of signalling molecules and siRNA-mediated knockdown of the putative transcription factor. Chromatin immunoprecipitation assays were subsequently conducted to further identify the responsible transcription factor. The role of metformin in the treatment of anovulation by LOX inhibition was investigated in a high-fat diet (HFD)-induced obese rat model. The numbers of retrieved total oocytes and metaphase II oocytes were recorded upon ovarian stimulation. Masson’s trichrome staining was used to measure the total collagen content, and immunohistochemical staining and western blotting were used to measure LOX, HIF-1α, and collagen I and IV in the ovary. MAIN RESULTS AND THE ROLE OF CHANCE Significantly increased FFA, LOX, and collagen abundance were observed in the ovaries of obese women with anovulation, compared to healthy controls or obese women with ovulation. In a HFD-induced obese rat model, metformin corrected the distortion of ovarian morphology by decreasing LOX and collagen protein abundance in the ovary and improving oestrous cyclicity and ovulation. PA increased LOX expression via the activation of HIF-1α in human granulosa cells, which was attenuated by metformin. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Several other saturated and polyunsaturated FFAs, such as stearic acid and arachidonic acid, are also increased in the blood of individuals with obesity, and increased levels of other FFAs may also contribute to the development of anovulation in individuals with obesity, which needs to be further verified in the future. WIDER IMPLICATIONS OF THE FINDINGS Elevated PA in individuals with obesity can cause LOX dysregulation via activation of HIF-1α, resulting in abnormal collagen deposition in the ovary and anovulation. This dysregulation can be ameliorated by metformin therapy through its local effect on ECM remodelling in the ovary, which is independent of its systemic effect on insulin sensitivity and chronic inflammation. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (grant numbers 82101730, 82130046, and 31900598) and Innovative Research Team of High-level local Universities in Shanghai (SHSMU-ZLCX20210201). All the authors declare no conflicts of interest in relation to this work.

Short‐term effect of rose bengal photodynamic therapy on collagen I, collagen V, nuclear factor kappa B, lysyl oxydase, transforming growth factor beta and interleukin 6 expression of human corneal fibroblasts, in vitro

Aims/Purpose: To investigate collagen I, collagen V, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), lysyl oxidase (LOX), transforming growth factor β (TGF‐β) and interleukin‐6 (IL‐6) expression in healthy human corneal fibroblasts (HCFs) and keratoconus fibroblasts (KC‐HCFs), 24 h after rose bengal photodynamic therapy (RB‐PDT).Methods: HCFs were isolated from healthy human corneal donors (n = 5) and KC‐HCFs from elective penetrating keratoplasties (n = 5). Both cell cultures underwent RB‐PDT (0.001% rose bengal concentration, 0.17 J/cm2 fluence) and 24 h later collagen I, collagen V, NF‐κB, LOX, TGF‐β and IL‐6 mRNA and protein expression has been determined using qPCR and Western blot, compared to untreated cultures. IL‐6 concentration in the cell culture supernatant has been determined by ELISA.Results: TGF‐β mRNA expression was significantly lower (p = 0.02) and IL‐6 mRNA expression was significantly higher in HCFs 24 h after treatment (p = 0.01), than in controls. COL1A1, COL5A1 and TGF‐β mRNA expression was significantly lower (p = 0.048; p = 0.02 and p = 0.02) and IL‐6 mRNA expression was significantly higher (p = 0.02) in KC‐HCFs 24 h after RB‐PDT, than in untreated KC‐HCFs. TGF‐β protein expression in HCFs was significantly higher 24 h after treatment, than in controls (p = 0.03). IL‐6 protein concentration in the HCF and KC‐HCF culture supernatant was significantly higher 24 h after treatment, than in controls (p = 0.02; p = 0.03). Nevertheless, collagen I, collagen V protein, NF‐κB and LOX mRNA and protein expression did not differ significantly between any of the RB‐PDT treated and untreated groups.Conclusions: In conclusion, our study demonstrates that RB‐PDT reduces collagen I, collagen V and TGF‐β mRNA expression, while increasing IL‐6 mRNA and protein expression in KC‐HCFs. In HCFs, RB‐PDT increases TGF‐β and IL‐6 protein level after 24 h. These observations may provide a basis for future research in this field.

The chaperone protein GRP78 released from MPN cells increases the expression of lysyl oxidase in a human stromal cell line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found increased expression of the chaperone protein glucose-regulated protein (GRP) 78, a central regulator of ER stress, in megakaryocytes from primary myelofibrosis or postessential thrombocythemia myelofibrosis patients. GRP78 was overexpressed in JAK2V617F-harboring cell lines; however, inhibitors of ER stress did not affect the expression levels of GRP78. In contrast, ruxolitinib, a well-known inhibitor of JAK2V617F, clearly blocked GRP78 expression in these cells through downregulation of transcription factor 4 (ATF4). Interestingly, GRP78 was secreted from HEL and SET-2 cells into culture media. Coculture of these cells with HS-5 cells, a human bone marrow stroma-derived cell line, induced enhanced expression of lysyl oxidase (LOX), which mediates cross-linking of collagen fibers and induces tissue fibrosis, in HS-5 cells. An anti-GRP78 neutralizing antibody abrogated LOX elevation; in contrast, recombinant GRP78 protein induced LOX protein expression in HS-5 cells. Our observations suggest that the oncogenic protein JAK2V617F induces overexpression and release of GRP78, which may induce a fibrotic phenotype in surrounding bone marrow stromal cells.

TLR4 antagonism ameliorates persistent post-operative cognitive decline

Collagen cross-linking is mediated by lysyl oxidase (LOX) enzyme in the extracellular matrix (ECM) of mitral valve leaflets. Alterations in collagen content and LOX protein expression in the ECM of degenerative mitral valve may enhance leaflet expansion and disease severity.Twenty posterior degenerative mitral valve leaflets from patients with severe mitral regurgitation were obtained at surgery. Five normal posterior mitral valve leaflets procured during autopsy served as controls. Valvular interstitial cells (VICs) density was quantified by immunohistochemistry, collagen Types I and III by picro-sirius red staining and immunohistochemistry, and proteoglycans by alcian blue staining. Protein expression of LOX and its mediator TGFβ1 were quantified by immunofluorescence and gene expression by PCR.VIC density was increased, structural Type I collagen density was reduced, while reparative Type III collagen and proteoglycan densities were increased (P<.0001) with an increase in spongiosa layer thickness in myxomatous valves. These changes were associated with a reduction in LOX (P<.0001) and increase in TGFβ1 protein expression (P<.0001). However, no significant change was seen in gene expression. Linear regression analysis identified a correlation between Type I collagen density and LOX grade (R2=0.855; P<.0001).Reduced Type I collagen density with a simultaneous increase in Type III collagen and proteoglycan densities possibly contributes to spongiosa layer expansion resulting in incompetent mitral valve leaflets. Observed changes in Type I and III collagen densities in Degenerative Mitral Valve Disease may be secondary to alterations in LOX protein expression, contributing to disorganization of ECM and disease severity.

Role of the lysyl oxidase enzyme family in cardiac function and disease

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation.

Abstract 156: Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells

Abstract Chondrosarcoma, a common primary bone malignancy that affects the joints and bones, is associated with metastases in the lung, especially in cases of high-grade chondrosarcoma. The protein lysyl oxidase (LOX) can contribute to tumor growth and metastasis, while the adipokine visfatin is pro-angiogenic and facilitates tumorigenesis as well as metastasis. However, it is unclear as to whether any association exists between visfatin and LOX expression in human chondrosarcoma metastasis. We have discovered that visfatin induces the migration, invasion and increased expression of LOX mRNA and protein in human chondrosarcoma cell lines (JJ012 and SW1353). Pretreatment of these cell lines with Src and Akt inhibitors, or transfecting them with small interfering RNAs (siRNAs), inhibited visfatin-induced chondrosarcoma metastasis and LOX expression. Thus, visfatin promotes chondrosarcoma metastasis by inducing LOX expression via the Src/Akt signaling pathway in the JJ012 cell line. microRNA analysis revealed that visfatin-induced Src/Akt signaling is responsible for downregulating mature miR-26b-5p, which targets the 3' untranslated region (3`UTR) of LOX but does not affect primary and precursor miR-26b-5p expression. This downregulation of miR-26b-5p expression by visfatin was dose-dependent. Pretreatment with Src and Akt inhibitors or transfection with siRNA reversed miR-26b-5p expression. The migration and invasion ability of JJ012 cells was reduced when they were transfected with the miR-26b-5p mimic. In summary, our data demonstrate that visfatin induces LOX expression and promotes chondrosarcoma cell migration and invasion through the Src/Akt signaling pathway, by inhibiting miR-26b-5p. Citation Format: Cheng-Chieh Yu, Chih-Yang Lin, Chih-Hsin Tang. Visfatin induces LOX expression and promotes tumor metastasis through the Src/Akt signaling pathway in human chondrosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 156.

Upregulated LOX and increased collagen content associated with aggressive clinicopathological features and unfavorable outcome in oral squamous cell carcinoma.

Collagen is a core protein that maintains the homeostasis of the extracellular matrix (ECM), and its dysregulation in human cancers has attracted increasing attention. In tumors, increased lysyl oxidase (LOX)-catalyzed collagen cross-linking plays a critical role in collagen dysregulation. However, the expression patterns of LOX and collagen and their clinicopathological significance in oral squamous cell carcinoma (OSCC) have not been well established. The LOX mRNA expression in OSCC was measured by RT-PCR and bioinformatics analysis. LOX protein expression and total collagen content were identified by immunohistochemistry or Masson's trichrome staining in a retrospective cohort of primary OSCC samples, respectively. Moreover, the associations between LOX and collagen expression and various clinicopathological parameters or patient survival were assessed. LOX mRNA was overexpressed in OSCC samples. Higher expression of LOX, collagen content or co-overexpression of LOX and collagen was significantly associated with aggressive clinicopathological features. Importantly, aberrant expression of LOX, collagen content, or both were markedly correlated with decreased overall and disease-free survival (P < 0.05). Moreover, univariate and multivariate Cox models analyses indicated that LOX, collagen content or their combination could serve as an independent prognostic predictor for OSCC patients. ROC analysis further revealed that the combination of LOX and collagen was superior to parameter alone as a prognostic predictor. Our findings reveal that elevated LOX and collagen content significantly corelate with aggressiveness and worse prognosis in OSCC.

Abstract 316: Chronic Intermittent Hypoxia Attenuates BAPN Induced Thoracic Aortic Aneurysms in Mice

Objective: Aortic aneurysm (AA) is a life-threatening cardiovascular disorder due to the predisposition for dissection and rupture. Chronic intermittent hypoxia (CIH) has protective effects on heart and brain against ischemia injury. However, whether CIH prevents against AA formation was not elucidated. The present study aimed to investigate the effect of CIH treatment on thoracic aortic aneurysms (TAA) in mice. Approach and Results: Seventy-five C57BL/6 mice 3 weeks of age were divided into three groups, i.e. Control group ( 21% O 2 , 24 h per day, 4 weeks, n = 15); β-aminopropionitrile (BAPN) group (oral administration BAPN 1g / kg / day, 21% O 2 , 24 h per day, 4 weeks, n = 30); CIH+BAPN group ( 5% -21% O 2 90s / cycle, 8h per day, 4 weeks; BAPN 1g / kg / day, n = 30). We found that CIH significantly reduced the incidence of TAA (18 of 30 versus 9 of 30; p&lt;0.05), and decreased maximal aortic diameter in BAPN group by hematoxylin and eosin staining (1.71 ± 0.35 versus 1.23± 0.15 mm; p &lt;0.05). CIH significantly reduced elastin breaks in BAPN group by Verhoeff-van Gieson staining. The apoptosis of vascular smooth muscle cells was detected by TUNEL staining increased in BAPN group and markedly reversed by CIH. Moreover, RNA-Seq analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway. CIH&amp;nbsp;significantly down-regulated the expression of inflammation signaling pathways, up-regulated the expression of PI3K/AKT survival pathway and extracellular matrix homeostasis related pathway in BAPN induced TAA mice. Confirmed by real-time quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) assays, CIH&amp;nbsp;significantly inhibited the expression of IL-1β, TNF-α, MMP-9 and Cathepsin S, and increased the expression of hypoxia inducible factor-1α (HIF-1α), Lysyl oxidase (LOX), ITGB1 and COL1A2. Our findings also showed that the expression of MMP-9, MMP-2, cathepsin S, IL-1β, MCP-1 and Mac-2 increased in BAPN group and markedly decreased in CIH+BAPN group by immunohistochemistry staining. Besides, CIH also significantly increased the protein expression of HIF-1α, LOX and p-AKT by western blot analysis. Conclusions: The findings from our study suggest that CIH prevented the TAA development of BAPN-induced mice.

Hypoxia Inducible Factors Modify Collagen I Fibers in MDA-MB-231 Triple Negative Breast Cancer Xenografts

Hypoxia inducible factors (HIFs) are transcription factors that mediate the response of cells to hypoxia. HIFs have wide-ranging effects on metabolism, the tumor microenvironment (TME) and the extracellular matrix (ECM). Here we investigated the silencing effects of two of the three known isoforms, HIF-1α and HIF-2α, on collagen 1 (Col1) fibers, which form a major component of the ECM of tumors. Using a loss-of-function approach for HIF-1α or 2α or both HIF-1α and 2α, we identified a relationship between HIFs and Col1 fibers in MDA-MB-231 tumors. Tumors derived from MDA-MB-231 cells with HIF-1α or 2α or both HIF-1α and 2α silenced contained higher percent fiber volume and lower inter-fiber distance compared to tumors derived from empty vector MDA-MB-231 cells. Depending upon the type of silencing, we observed changes in Col1 degrading enzymes, and enzymes involved in Col1 synthesis and deposition. Additionally, a reduction in lysyl oxidase protein expression in HIF-down-regulated tumors suggests that more non-cross-linked fibers were present. Collectively these results identify the role of HIFs in modifying the ECM and the TME and provide new insights into the effects of hypoxia on the tumor ECM.

Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia

ABSTRACTPurpose: Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM).Methods: Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus β-aminopropionitrile (BAPN) group, an FDM plus TGF-β1 (TGF-β1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-β1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured.Results: In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-β1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-β1-treated eyes compared with the FDM eyes. The ultimate stress and Young’s modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-β1 group. The ultimate strain (%) of the sclera was lowest in the TGF-β1 group, followed by the FDM group and the BAPN group.Conclusion: LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

Association of altered collagen content and lysyl oxidase expression in degenerative mitral valve disease.

Collagen cross-linking is mediated by lysyl oxidase (LOX) enzyme in the extracellular matrix (ECM) of mitral valve leaflets. Alterations in collagen content and LOX protein expression in the ECM of degenerative mitral valve may enhance leaflet expansion and disease severity. Twenty posterior degenerative mitral valve leaflets from patients with severe mitral regurgitation were obtained at surgery. Five normal posterior mitral valve leaflets procured during autopsy served as controls. Valvular interstitial cells (VICs) density was quantified by immunohistochemistry, collagen Types I and III by picro-sirius red staining and immunohistochemistry, and proteoglycans by alcian blue staining. Protein expression of LOX and its mediator TGFβ1 were quantified by immunofluorescence and gene expression by PCR. VIC density was increased, structural Type I collagen density was reduced, while reparative Type III collagen and proteoglycan densities were increased (P<.0001) with an increase in spongiosa layer thickness in myxomatous valves. These changes were associated with a reduction in LOX (P<.0001) and increase in TGFβ1 protein expression (P<.0001). However, no significant change was seen in gene expression. Linear regression analysis identified a correlation between Type I collagen density and LOX grade (R2=0.855; P<.0001). Reduced Type I collagen density with a simultaneous increase in Type III collagen and proteoglycan densities possibly contributes to spongiosa layer expansion resulting in incompetent mitral valve leaflets. Observed changes in Type I and III collagen densities in Degenerative Mitral Valve Disease may be secondary to alterations in LOX protein expression, contributing to disorganization of ECM and disease severity.

MicroRNA-19b is a potential biomarker of increased myocardial collagen cross-linking in patients with aortic stenosis and heart failure

This study analyzed the potential associations of 7 myocardial fibrosis-related microRNAs with the quality of the collagen network (e.g., the degree of collagen fibril cross-linking or CCL) and the enzyme lysyl oxidase (LOX) responsible for CCL in 28 patients with severe aortic stenosis (AS) of whom 46% had a diagnosis of chronic heart failure (HF). MicroRNA expression was analyzed in myocardial and blood samples. From the studied microRNAs only miR-19b presented a direct correlation (p < 0.05) between serum and myocardium. Compared to controls both myocardial and serum miR-19b were reduced (p < 0.01) in AS patients. In addition, miR-19b was reduced in the myocardium (p < 0.01) and serum (p < 0.05) of patients with HF compared to patients without HF. Myocardial and serum miR-19b were inversely correlated (p < 0.05) with LOX, CCL and LV stiffness in AS patients. In in vitro studies miR-19b inhibition increased (p < 0.05) connective tissue growth factor protein and LOX protein expression in human fibroblasts. In conclusion, decreased miR-19b may be involved in myocardial LOX up-regulation and excessive CCL, and consequently increased LV stiffness in AS patients, namely in those with HF. Serum miR-19b can be a biomarker of these alterations of the myocardial collagen network in AS patients, particularly in patients with HF.

Abstract 5012: Nuclear expression of lysyl oxidase enzyme is an independent prognostic factor in rectal cancer patients

Abstract Emerging evidence implies that the complex and seemingly paradoxical role of lysyl oxidase (LOX) in cancer may depend on its subcellular localization. LOX expression has been reported extracellularly, as well as intracellularly in both the cytoplasmic and nuclear compartments. In this study we analyze previously unreported LOX expression patterns in the nucleus of rectal cancer patient samples and determine the clinical significance of this expression. LOX expression was examined by immunohistochemistry in 137 primary rectal cancer patients who participated in the randomized Swedish rectal cancer trial of preoperative radiotherapy between 1987 and 1990. Location and expression of LOX protein were detected by confocal microscopy and Western blot in three colon cancer cell lines. LOX nuclear expression was high in normal mucosal and showed a significant decrease in primary rectal cancer (P&amp;lt;0.001). Furthermore, a significant increase was observed from primary cancer to lymph node metastasis (P&amp;lt;0.01). High nuclear LOX expression was correlated with a high rate of distant metastasis and total recurrence (P = 0.046, P = 0.048, respectively). It was also confirmed that LOX (50-kDa and 32-kDa isoforms) are located in the nucleus of in vitro colon cancer cells. High nuclear LOX expression was found in the metastatic cell line SW620 compared to the parental primary cancer cell line SW480. Radiotherapy had no effect on LOX expression and localization in cancer tissues as well as in in vitro culture. Moreover, multivariable analysis showed that high nuclear LOX expression correlated with poor overall survival (P = 0.003) and disease free survival (P = 0.006), independent of age, gender, TNM stage, differentiation and radiotherapy treatment. Nuclear LOX also correlated with other biological factors (such as NF-κB) known to be associated with patient survival suggesting a possible mechanism. Cytoplasmic LOX is high in primary cancer and metastasis compared with normal tissue, but not prognostic. So here we firstly proved the nuclear expression of LOX enzyme both in patient rectal cancer tissues and in in vitro colon cancer cells. The strong correlation of nuclear LOX expression to survival offers a promising prognostic biomarker in rectal cancer. Citation Format: Na Liu, Thomas R. Cox, Weiyingqi Cui, Gunnar Adell, Birgitta Holmlund, Janine T. Erler, Xiaofeng Sun. Nuclear expression of lysyl oxidase enzyme is an independent prognostic factor in rectal cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5012.

Abstract 764: LOX and LOXL2 inhibition as a treatment for ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women. Despite advances in our understanding of the molecular biology of EOC, patient survival has not significantly improved in decades and new treatments are needed. There is increased interest in therapeutic targeting of the tumor microenvironment (TME). The lysyl oxidase (LOX) family of enzymes are involved in extracellular matrix remodelling through crosslinking of collagen and elastin. They have been shown to play an important role in the TME of other cancers and are regulated by hypoxia, however little is known about their role in EOC. Therefore we aimed to investigate the role of LOX and LOXL2 in EOC using in vitro models. Tumor epithelial (n = 2) and matched cancer-associated fibroblast cell lines (n = 2) derived from an SV40-induced mouse model of EOC were generated. MTT assays were performed on all cell lines to assess the effect of LOX and LOXL2 inhibition on cell viability. The effect of LOX and LOXL2 inhibition on cancer cell migration and invasion towards cancer-associated fibroblast conditioned media was also investigated using transwell assays. RNA and protein were isolated and analysed from cells cultured in normoxic and hypoxic conditions to assess whether LOX and LOXL2 gene and protein expression is regulated by hypoxia. Inhibition of LOX and LOXL2 did not affect cell viability. LOX and LOXL2 inhibition significantly reduced the migration and invasion of EOC cells towards cancer-associated fibroblast conditioned media in vitro (p&amp;lt;0.05). Gene expression analysis comparing Lox and Loxl2 expression in all cell lines demonstrated a significant increase in Lox and Loxl2 expression in cells cultured in hypoxia (p&amp;lt;0.05). Similarly, cellular and secreted LOX and LOXL2 protein expression was increased in cells cultured in hypoxic conditions, supporting hypoxia as a regulator of LOX and LOXL2 expression. This study demonstrates a role for LOX and LOXL2 in promoting migration and invasion of EOC cells in vitro. The upregulation of LOX and LOXL2 in hypoxia suggests a role in facilitating tumor growth and progression within hypoxic tumors. These results provide support for the utility of LOX and LOXL2 inhibition as promising and novel therapeutic strategies in EOC. Citation Format: Angela Cho, Amanda L. Hudson, Samuel Yuen, Nham Tran, Viive M. Howell, Emily K. Colvin. LOX and LOXL2 inhibition as a treatment for ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 764.

Suppression of Phosphatidylinositol 3-Kinase/Akt Signaling Attenuates Hypoxia-Induced Pulmonary Hypertension Through the Downregulation of Lysyl Oxidase.

Lysyl oxidase (LOX) is a copper-dependent enzyme that catalyzes covalent cross-linking of collagen. In response to hypoxia, phosphatidylinositol 3-kinase (PI3K) pathway is activated and contributes to pulmonary arterial hypertension (PAH). However, potential role of LOX in hypoxia-induced PAH is poorly understood. In this study, we explored the mechanism responsible for the development of hypoxia-induced PAH. Potent inhibitors of PI3K/Akt and LOX, wortmannin and β-aminopropionitrile (β-APN), were administrated in rat model of hypoxia-induced PAH. The cross-linking of collagen was assessed by the determination of hydroxyproline. LOX, LOXL-1, LOXL-2, LOXL-3, LOXL-4, Akt, and phospho-Akt expression was detected by real-time polymerase chain reaction and western blot analysis. We observed that collagen cross-linking and LOX activity were elevated in hypoxia-exposed rat lung tissue, but these effects were reversed by β-APN and wortmannin. In addition, exposure to hypoxia enhanced mRNA and protein expression and activity of LOX and LOXL-1 in a PI3K/Akt-dependent manner and induced the development of PAH. After the administration of wortmannin, the upregulation of LOX and cross-linking of collagen were significantly reversed in hypoxia-exposed rat pulmonary artery tissue. Taken together, the present study demonstrated that the upregulation of LOX expression and collagen cross-linking is PI3K/Akt dependent in rat with hypoxia-induced PAH. Suppression of PI3K/Akt pathway may alleviate hypoxia-induced PAH through the downregulation of LOX.

Lysyl oxidase expression in cardiac fibroblasts is regulated by α2β1 integrin interactions with the cellular microenvironment

Lysyl oxidase (LOX) catalyzes crosslink formation between fibrillar collagens and elastins and an increase in LOX activity has been associated with cardiac fibrosis following myocardial infarction (MI). It has been previously reported that LOX expression is regulated by growth factors and cytokines including transforming growth factor (TGF-β1); however, it is unclear how the biophysical and biochemical properties of the cellular microenvironment affect LOX expression. In this study, we isolated rat cardiac fibroblasts (CF) and infarct cardiac fibroblasts (ICF), from healthy and 1-week post-MI left ventricular tissue respectively, and cultured them under varied substrate conditions in vitro to assess their influence on LOX expression. Culture of ICF on collagen I-coated plates increased LOX expression versus uncoated plates with an additional increase observed with the presence of TGF-β1. To further investigate the effect of integrin interactions with collagen I on LOX expression, we inhibited the α2β1 integrin from binding to collagen I and found gene and protein expression of LOX to be downregulated. Together, this demonstrates that the interaction of α2β1 integrin to collagen I in the cellular microenvironment can regulate expression of LOX. Further studies investigating additional integrin interactions may identify therapeutic targets for treating cardiac fibrosis.

LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

Abstract 19428: TRAF3IP2 Mediates Aldosterone-induced Cardiac Hypertrophy and Fibrosis

Background: Aldosterone (Aldo) is a key regulator of blood pressure and electrolyte balance. Aldo/salt has been shown to induce oxidative stress, inflammation, cardiac hypertrophy and fibrosis. TRAF3 interacting protein 2 (TRAF3IP2) is a cytoplasmic redox-sensitive adaptor molecule and an upstream regulator of NF-κB and AP-1. It plays a critical role in the regulation of various proinflammatory cytokines that exert prohypertrophic and profibrotic effects. Therefore, we hypothesized that TRAF3IP2 is a critical mediator of Aldo/salt-induced cardiac hypertrophy and fibrosis. Methods: TRAF3IP2-null and wild type (WT) mice (male, 12-13 weeks) were infused continuously with Aldo for 4 weeks via mini-osmotic pumps (0.2 mg/Kg/day; n = 6-8). Mice were fed 1% NaCl in drinking water. Salt alone served as control. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography, myocardial function by echocardiography, cardiac hypertrophy by heart weight to body weight ratios, cardiac fibrosis by trichrome Gomori, and gene and protein expression by RT-qPCR, immunoblotting and ELISA. Results: Aldo/salt elevated SBP to a similar extent in both TRAF3IP2-null and WT mice. Aldo/salt upregulated TRAF3IP2 expression in left ventricular homogenates from WT mice, and TRAF3IP2 gene deletion (vs. WT) blunted cardiac hypertrophy by 54% (p &lt; 0.05), suppressed ANP expression by 3.5 fold (p &lt; 0.05), prevented concentric LV remodeling (decreased LVPWs; 93%; p &lt; 0.01, LVPWd; 95%; p &lt; 0.05, LVAWs; 29.8%; p &lt; 0.05 and LVAWd; 26.2%, and increased LVIDs; 19.2%; p &lt; 0.01 and LVIDd; 8.53%) and markedly reduced both interstitial and perivascular fibrosis. Further, TRAF3IP2 gene deletion inhibited Aldo/salt-induced p65 and c-Jun activation, and expression of inflammatory cytokine (IL-18 and IL-6), matrix metalloproteinase (MMP2) and extracellular matrix proteins (collagen Iα1 and IIIα1). TRAF3IP2 gene deletion also blunted mRNA and protein expression of lysyl oxidase, an enzyme involved in collagen crosslinking. Conclusions: TRAF3IP2 gene deletion blunts Aldo/salt-induced cardiac hypertrophy and fibrosis by inhibiting proinflammatory, prohypertrophic, and profibrotic effects, signifying its potential as a therapeutic target in hypertrophic heart diseases.

Exercise training decreases myocardial collagen III and heart failure signs rate, and improves physical performance in spontaneously hypertensive rats (LB522)

The aim of this study was to evaluate the influence of physical exercise on physical performance, heart failure (HF) signs rate, and myocardial collagen I and III and lysyl oxidase protein expression in spontaneously hypertensive rats (SHR). Methods: Sixteen‐month‐old SHR were divided into two groups: sedentary (SED, n=8) and exercise (EXE, n=8). Physical training was performed 5 times per week for 16 weeks in a treadmill. Physical performance was assessed by exercise test on a treadmill with a gradual speed increase. At the moment of euthanasia, HF signs were evaluated: tachypnea in vivo, and ascyte, pleural effusion, atrial thrombi, right ventricular hypertrophy, and liver and lung congestion post mortem. Collagen I and III and lysyl oxidase protein expression were analyzed by Western blot. Statistics: The Student's t test and the Goodman test were used to compare the groups (p&lt;0.05). Results: The EXE group showed greater physical capacity and lower HF signs rate than SED group. Collagen III protein expression was lower in EXE group (1.00±0.58 vs. 0.73±0.43, p&lt;0.05). Collagen I and lysyl oxidase protein expression were not significantly different between groups. Conclusion: Physical exercise improves physical performance and reduces heart failure signs rate and myocardial collagen III expression in spontaneously hypertensive rats.Grant Funding Source: Supported by CAPES and CNPq

Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.