Lysyl Oxidase-like 1 Expression Research Articles

LOXL1 promotes tumor cell malignancy and restricts CD8 + T cell infiltration in colorectal cancer

BackgroundColorectal cancer (CRC) is a leading cause of cancer mortality globally. Lymph node metastasis and immunosuppression are main factors of poor prognosis in CRC patients. Lysyl oxidase like 1 (LOXL1), part of the lysyl oxidase (LOX) family, plays a yet unclear role in CRC. This study aimed to identify effective biomarkers predictive of prognosis and efficacy of immunotherapy in CRC patients, and to elucidate the prognostic value, clinical relevance, functional and molecular features, and immunotherapy predictive role of LOXL1 in CRC and pan-cancer.MethodsWeighted gene co-expression network analysis (WGCNA) was employed to explore gene modules related to tumor metastasis and CD8 + T cell infiltration. LOXL1 emerged as a hub gene through differential gene expression and survival analysis. The molecular signatures, functional roles, and immunological characteristics affected by LOXL1 were analyzed in multiple CRC cohorts, cell lines and clinical specimens. Additionally, LOXL1's potential as an immunotherapy response indicator was assessed, along with its role in pan-cancer.ResultsTurquoise module in WGCNA analysis was identified as the hub module associated with lymph node metastasis and CD8 + T cell infiltration. Aberrant elevated LOXL1 expression was observed in CRC and correlated with poorer differentiation status and prognosis. Molecular and immunological characterization found that LOXL1 might mediate epithelial-mesenchymal transition (EMT) process and immunosuppressive phenotypes of CRC. Functional study found that LOXL1 enhanced tumor cell proliferation, migration and invasion. Moreover, high LOXL1 levels corresponded to reduced CD8 + T cell infiltration and predicted poor clinical outcomes of immunotherapy. Similar trends were also observed at the pan-cancer level.ConclusionsOur findings underscore the critical role of LOXL1 in modulating both malignancy and immunosuppression in CRC. This positions LOXL1 as a promising biomarker for predicting prognosis and the response to immunotherapy in CRC patients.Graphical

Selective Inhibition of Hepatic Stellate Cell and Fibroblast-derived LOXL1 Attenuates BDL and Mdr2-/- Induced Cholestatic Liver Fibrosis.

Lysyl oxidase-like 1 (LOXL1) proteins are amine oxidases that play a crucial role in extracellular matrix remodeling due to their collagen cross-linking and intracellular functions. The role of LOXL1 in cholestatic liver fibrosis remains unexplored. We measured LOXL1 expression in two murine models of cholestasis (Mdr2 knockout [Mdr2-/-] and bile duct ligation [BDL]). We used adeno-associated virus (AAV) serotype 6-mediated hepatic delivery against LOXL1 (AAV2/6-shLoxl1) to investigate the therapeutic efficacy of targeting LOXL1 in cholestatic liver fibrosis. NIH-3T3 murine fibroblasts were used to investigate the function and regulatory mechanisms of LOXL1 in vitro. LOXL1 expression was significantly upregulated in Mdr2 -/- and BDL mice compared to their corresponding controls, predominantly in collagen-rich fibrous septa and portal areas. AAV2/6-shLoxl1 significantly reduced LOXL1 levels in Mdr2-/- and BDL mice, mainly located in desmin-positive hepatic stellate cells (HSCs) and fibroblasts. Concomitant with reduced LOXL1 expression, there was reduced ductular reaction, inflammation, and fibrosis in both Mdr2 -/- and BLD mouse models. Additionally, Loxl1 intervention decreased Ki-67 positive cells in the desmin-positive areas in both Mdr2 -/- and BDL mice. Overexpression of LOXL1 significantly promoted fibroblast proliferation by activating the platelet-derived growth factor receptor and extracellular signal-regulated kinase signaling pathways in vitro. Our findings demonstrated that selective inhibition of LOXL1 derived from HSCs/fibroblasts attenuated cholestatic liver/biliary fibrosis, inflammation, ductal reaction, and HSC/fibroblast proliferation. Based on our findings LOXL1 could be a potential therapeutic target for cholestatic fibrosis.

Commentary: The genetics of pseudoexfoliation syndrome/glaucoma.

Genetics forms the basis of all life and evolution. The study of genetics primarily deals with trait inheritance and gene expression. Medical genetics has revolutionized the understanding of health and disease and opened several new prospects in prevention, diagnostics, and therapy. Genetic determinants of several ocular diseases are now well elucidated, with work in progress in many others. Ocular pseudoexfoliation (PXF) results from the accumulation of abnormal fibrillar aggregates in the anterior segment of the eye, blocking the trabecular meshwork and leading to glaucoma. These fibrils consist of elastic fiber components of elastin, fibrillin-1, fibulins, latent TGF-ß binding proteins, and the crosslinking enzyme lysyl oxidase-like 1 (LOXL1), produced by the epithelial cells in the anterior segment (lens epithelial cells, ciliary epithelial cells, and trabecular meshwork cells).[1] The LOXL1 locus on chromosome 15q24.1 has shown the most significant genetic association in PXF glaucoma, similar to the current study’s findings.[23] LOXL1 encodes a member of the lysyl oxidase (LOX) family of enzymes (LOX and LOXL1-4) required for lysine-derived covalent crosslinking in collagen and elastin, contributing to the tensile strength and elasticity of connective tissues. The LOXL1 isoform catalyzes the crosslinking of soluble tropoelastin into insoluble elastin in elastic fibers. Two common nonsynonymous protein-coding variants in exon 1 of the LOXL1 gene, rs1048661 (p.R141L) and rs3825942 (p.G153D), have consistently been associated with PXF in numerous population-based and well-powered genome-wide association studies (GWASs).[3] However, the phenomenon of allele reversal of these two variants in different ethnic populations reflects our ignorance of the exact biological mechanism underlying PXF and the need for further work in elucidating it.[4] Studies in transgenic mice models showed an inverse relationship between LOXL1 expression and intraocular pressure (IOP), with increased IOP in Loxl1-/- mice from elevated episcleral venous pressure.[5] LOXL1 animal models have been unsuccessful in duplicating the PXF phenomenon seen in humans, indicating additional unknown mechanisms. A deep resequencing approach has identified a single common variant (minor allele G of the intergenic sequence variant rs7173049: A>G) downstream of LOXL1 that downregulates two genes affecting vitamin A metabolism in ocular tissues in PXF patients. This allele did not show any reversal in different populations and suggested potential therapeutic targets.[6] The GWAS method identified another polymorphism outside of LOXL1, at 19p13, rs4926244, in the intronic region of CACNA1A, affecting calcium metabolism.[7] Another locus at 13q12 (proteasome maturation protein, POMP) showed increased strength of genetic association with increasing distance from the equator, consistent with the observed prevalence of ocular PXF, indicative of gene-environment interaction.[4] It is interesting to note that several other single nucleotide polymorphisms (SNPs) associated with PXF glaucoma have been identified in different ethnic groups. Multiple genetic loci identified in PXF open the prospect of developing polygenic risk scores (PRS) for PXF in the future. The prospect of gene–environment interactions has spawned several epigenetic investigations with exciting results. These include DNA hypermethylation in the promoter of the LOXL1 gene, downregulation of HSP70 (heat shock protein 70), long non-coding RNAs such as LOXL1 antisense RNA 1 (evidence for regulatory function in immortalized lens epithelial cells), and microRNAs, particularly miR-122-5p (shown to regulate the TGF-b/Smad signaling pathway and lipoprotein metabolism).[3] MicroRNAs are involved in the epigenetic regulation of gene expression and are potential biomarkers of disease identifiable in blood and aqueous humor. Mass spectrometric proteomic analyses of various biological samples have also revealed cellular stress/inflammation, lipid metabolism, coagulation, and dietary factors (altered vitamin D and retinol-binding proteins) contributing to PEX development. Specific metabolomic profiles in plasma and aqueous humor have been identified and validated using machine-learning prediction models.[3] These analyses implicate oxidative stress in the pathophysiology of PXF. However, there is a significant knowledge gap in the exact pathomechanism of the PXF syndrome that requires well-designed and extensive functional investigations. The vast proportion of work done in genetics in PXF comes from the developed world. Studies from India are extremely few, underlining the need to cover a lot of ground, particularly as genetics and epigenetics vary with ethnicity and region. Studies similar to this one and more extensive/multicentric investigations from different parts of India will go a long way in unraveling the specifics of PXF pathobiology among South Asians.[2] The primary challenge in conducting genetic research is the relative scarcity of geneticists/trained researchers in the developing world and funding requirements. Stalwarts in the field have called for equity in genomics.[8] The onus of responsibility lies on clinician-scientists to convince policy-makers and funding agencies for more significant funding in core research in this field.

MiR-15b and miR-16 suppress TGF-β1-induced proliferation and fibrogenesis by regulating LOXL1 in hepatic stellate cells

Activation of hepatic stellate cells (HSCs) is an important event during the progress of liver fibrosis. MicroRNA (miR)-15b and miR-16 have been found to be involved in activation of HSCs. However, the roles of miR-15b/16 in liver fibrosis remain unclear. The expression of miR-15b/16 was decreased in TGF-β1-stimulated LX-2 cells. Overexpression of miR-15b/16 in LX-2 cells suppressed TGF-β1-induced cell proliferation and the expression levels of tissue inhibitor of metalloproteinase type 1, collagen type I, and α-smooth muscle actin. The activation of Smad2/3 caused by TGF-β1 was repressed by miR-15b/16 overexpression. The two miRNAs directly bound to the 3′-UTR of lysyl oxidase-like 1 (LOXL1) and suppressed the LOXL1 expression. Furthermore, knockdown of LOXL1 attenuated miR-15b/16 downregulation-induced cell proliferation, fibrogenic response and phosphorylation of Smad2/3. Collectively, miR-15b/16 exhibited anti-fibrotic activity through regulation of Smad2/3 pathway.

Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50–80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 μM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

Abstract 1547: LOXL1 is a novel responsible gene for peritoneal dissemination potentially via promoting EMT and motility in gastric cancer

Abstract Background. In gastric cancer (GC), peritoneal dissemination (PD) occurs frequently and is incurable. We have identified 25 PD-associated genes in GC by in silico analysis in previous research. In this study, we focused on Lysyl oxidase-like 1 (LOXL1), one of the 25 PD-associated genes, because LOXL1 is a member of the lysyl oxidase (LOX) gene family and may have the ability to promote EMT in GC. Materials and Methods. We assessed LOXL1 expression using a public GC dataset (GSE15459) and a highly-disseminated peritoneal GC cell line (RT-qPCR). Next, we performed gene expression analysis, including gene set enrichment analysis (GSEA) with two public GC datasets (GSE15459 and TCGA). Moreover, we performed a series of in vitro experiments using GC cells. Finally, we evaluated the clinicopathological significance of LOXL1 expression using RT-qPCR and immunohistochemistry staining in the Kyushu cohort. We also performed survival analyses of LOXL1 expression in the Kyushu cohort, GSE15459, and TCGA datasets. Results. The expression of LOXL1 was higher in GC patients with PD compared with GC patients without PD in the GSE15459 dataset. LOXL1 was also overexpressed in highly-disseminated peritoneal GC cells relative to its control cells. Moreover, LOXL1 expression positively correlated with the EMT gene set in GSEA. Additionally, LOXL1 overexpression reduced CDH1 expression, increased VIM expression, CDH2 expression, SNAI2 expression, and PLS3 expression, and promoted migration capacity in GC cells. Finally, high LOXL1 expression was associated with poorly-differentiated, lymph node metastasis, and PD in the Kyushu cohort, and was a poor prognostic factor in three GC datasets. Conclusions. LOXL1 was a novel responsible gene for PD in GC, possibly through the activation of EMT and cell motility. Citation Format: Qingjiang Hu, Takaaki Masuda, Yoko Zaitsu, Yasuo Tsuda, Yuichi Hisamatsu, Yuichiro Nakashima, Koji Ando, Yasue Kimura, Eiji Oki, Masaki Mori, Koshi Mimori. LOXL1 is a novel responsible gene for peritoneal dissemination potentially via promoting EMT and motility in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1547.

Integral role for lysyl oxidase-like-1 in conventional outflow tissue function and behavior.

Lysyl oxidase-like-1 (LOXL1), a vital crosslinking enzyme in elastin fiber maintenance, is essential for the stability and strength of elastic vessels and tissues. Variants in the LOXL1 locus associate with a dramatic increase in risk of exfoliation syndrome (XFS), a systemic fibrillopathy, which often presents with ocular hypertension and exfoliation glaucoma (XFG). We examined the role of LOXL1 in conventional outflow function, the prime regulator of intraocular pressure (IOP). Using Loxl1-/- , Loxl1+/- , and Loxl1+/+ mice, we observed an inverse relationship between LOXL1 expression and IOP, which worsened with age. Elevated IOP in Loxl1-/- mice was associated with a larger globe, decreased ocular compliance, increased outflow facility, extracellular matrix (ECM) abnormalities, and dilated intrascleral veins, yet, no dilation of arteries or capillaries. Interestingly, in living Loxl1-/- mouse eyes, Schlemm's canal (SC) was less susceptible to collapse when challenged with acute elevations in IOP, suggesting elevated episcleral venous pressure (EVP). Thus, LOXL1 expression is required for normal IOP control, while ablation results in altered ECM repair/homeostasis and conventional outflow physiology. Dilation of SC and distal veins, but not arteries, is consistent with key structural and functional roles for elastin in low-pressure vessels subjected to cyclical mechanical stress.

LOXL1 Is Regulated by Integrin α11 and Promotes Non-Small Cell Lung Cancer Tumorigenicity.

Integrin α11, a stromal collagen receptor, promotes tumor growth and metastasis of non-small cell lung cancer (NSCLC) and is associated with the regulation of collagen stiffness in the tumor stroma. We have previously reported that lysyl oxidase like-1 (LOXL1), a matrix cross-linking enzyme, is down-regulated in integrin α11-deficient mice. In the present study, we investigated the relationship between LOXL1 and integrin α11, and the role of LOXL1 in NSCLC tumorigenicity. Our results show that the expression of LOXL1 and integrin α11 was correlated in three lung adenocarcinoma patient datasets and that integrin α11 indeed regulated LOXL1 expression in stromal cells. Using cancer-associated fibroblast (CAF) with either a knockdown or overexpression of LOXL1, we demonstrated a role for LOXL1 in collagen matrix remodeling and collagen fiber alignment in vitro and in vivo in a NSCLC xenograft model. As a consequence of collagen reorganization in NSCLC tumor stroma, we showed that LOXL1 supported tumor growth and progression. Our findings demonstrate that stromal LOXL1, under regulation of integrin α11, is a determinant factor of NSCLC tumorigenesis and may be an interesting target in this disease.

P3.03-19 The Lysyl Oxidase like-1 Promotes NSCLC Tumorigenecity Through Increased Matrix Reorganization and Stiffness

The importance of the stroma in tumorigenicity has been increasingly recognized in the past few years and targeting tumor stroma has become a new strategy for the development of new therapies. The lysyl oxidase like-1 (LOXL1) is a matrix cross-linking enzyme, essentially secreted by cancer-associated fibroblasts (CAFs) in the stroma. We previously showed that LOXL1 expression strongly correlated with integrin α11 expression, a collagen receptor that promotes NSCLC tumorigenecity, suggesting that LOXL1 may also contribute to lung tumor growth and metastasis by modifying the stromal collagen matrix. Primary CAFs (CAF094) isolated from a NSCLC resection specimen and immortalized with hTERT were infected with full length LOXL1 (LOXL1 overexpression), LOXL1 shRNA (LOXL1 knockdown) or with their respective controls, using a lentiviral system. To investigate the impact of LOXL1 on collagen matrix reorganization, we allowed CAFs expressing different levels of LOXL1 to contract collagen matrices. Contracted collagen matrices have then been submitted to second harmonic generation to analyze collagen fibers. We established the role of LOXL1 in lung carcinoma invasion and growth using an in vitro organotypic model and an in vivo xenograft model, respectively. LOXL1-overexpressing CAFs embedded in collagen lattices displayed greater ability to reorganize the collagen matrix than those with lower LOXL1 expression. Furthermore, analysis of the collagen matrix demonstrated that LOXL1 contributed to more linear and dense collagen fibers organization. As a consequence of collagen matrix reorganization, invasion of the lung carcinoma H661 cell line significantly increased in an organotypic model in presence of LOXL1-overexpressing CAFs. Moreover, we showed that loss of Loxl1 in mice inhibited subcutaneous growth of the lung adenocarcinoma H4006 cell line compared to the mouse wild-type counterparts. We demonstrated that overexpression of LOXL1 in NSCLC tumor stroma results in an increased of tumor growth and invasion, due to a change in the matrix reorganization. Thus, LOXL1 appears as an interesting target to improve the outcome of lung cancer.

Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking

Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1) levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing arrested expression of α-SMA, elastin and collagen Ι.Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51% decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response.

Alternative mRNA splicing coupled to nonsense-mediated decay (NMD) is a common mRNA surveillance pathway also known to dynamically modulate gene expression in response to cellular stress. Here, we investigated the involvement of this pathway in the regulation of lysyl oxidase-like 1 (LOXL1) expression in response to pseudoexfoliation (PEX)-associated pathophysiologic factors. Transcript levels of LOXL1 isoforms were determined in ocular tissues obtained from donor eyes without and with PEX syndrome. Pseudoexfoliation-relevant cell types, including human Tenon's capsule fibroblasts (hTCF) and trabecular meshwork cells (hTMC), were exposed to puromycin, caffeine, TGF-β1, homocysteine, IL-6, retinoic acid, UV-B radiation, oxidative stress, and mechanical stress for up to 48 hours. Western blot analysis was carried out using antibodies against LOXL1, (phosphorylated-) eukaryotic initiation factor 2-α (eIF2-α), and regulator of nonsense transcripts 2 (UPF2). RNA interference was used to knockdown UPF1-3 and Serine/threonine-protein kinase (SMG1). Constitutive expression of wild-type LOXL1 and alternatively spliced LOXL1-a transcripts was detected in all ocular tissues showing highest levels in trabecular meshwork and differential expression between PEX and control specimens. LOXL1-a transcripts were upregulated in hTCF and hTMC by NMD inhibitors puromycin and caffeine (≥6-fold; P < 0.01) or after knockdown of NMD core factors (≥2-fold; P < 0.05), whereas mRNA and protein levels of LOXL1 were reduced (≤0.8 fold; P < 0.05). Exposure of cells to various PEX-associated (stress) factors, including TGF-β1, UV-B light, oxidative stress, mechanical stress, and retinoic acid enhanced LOXL1-a transcript levels (≥1.5-fold; P < 0.05), while partially downregulating LOXL1 levels (≤0.7-fold; P < 0.05). Stress-induced inhibition of NMD was dependent on phosphorylation of eIF2α. These findings provide evidence for a functional role of alternative splicing coupled to NMD in the posttranscriptional regulation of LOXL1 gene expression and suggest this mechanism to represent a dynamic mode of adapting LOXL1 expression to PEX-associated environmental and nutritional cues.

Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

Although lysyl oxidase-like 1 (LOXL1) is known as the principal genetic risk factor for pseudoexfoliation (PEX) syndrome, a major cause of glaucoma and cardiovascular complications, no functional variants have been identified to date. Here, we conduct a genome-wide association scan on 771 German PEX patients and 1,350 controls, followed by independent testing of associated variants in Italian and Japanese data sets. We focus on a 3.5-kb four-component polymorphic locus positioned spanning introns 1 and 2 of LOXL1 with enhancer-like chromatin features. We find that the rs11638944:C>G transversion exerts a cis-acting effect on the expression levels of LOXL1, mediated by differential binding of the transcription factor RXRα (retinoid X receptor alpha) and by modulating alternative splicing of LOXL1, eventually leading to reduced levels of LOXL1 mRNA in cells and tissues of risk allele carriers. These findings uncover a functional mechanism by which common noncoding variants influence LOXL1 expression.

Vaginal Expression of LOXL1 in Premenopausal and Postmenopausal Women With Pelvic Organ Prolapse.

This study aimed to compare cellular expression of lysyl oxidase-like 1 (LOXL1), a key enzyme in elastin metabolism, of premenopausal women with pelvic organ prolapse (POP) compared with premenopausal controls without POP and postmenopausal women with POP. In addition, we examined whether variation of LOXL1 expression was dependent on biopsy site. A standardized protocol was utilized to obtain vaginal biopsies from 30 women (10 premenopausal POP, 10 postmenopausal POP, and 10 premenopausal non-POP). Expression levels of messenger RNA (mRNA) and protein of LOXL1 were determined using real-time quantitative polymerase chain reactions and enzyme-linked immunosorbant assays. Analysis was performed to determine if there were differences between group or biopsy site. Significant differences in LOXL1 mRNA expression were found between patient groups (P = 0.0033). LOXL1 mRNA expression (relative to 18S) was upregulated in the postmenopausal POP group (54.5 ± 14.7) compared with the premenopausal POP group (5.2 ± 14.7, P = 0.0034) and the premenopausal non-POP group (23 ± 18, P = 0.0359). No significant differences in LOXL1 protein expression (nanogram/milliliter per microgram total protein) were seen between groups (premenopausal POP, 3.2 × 10 ± 6.3 × 10; postmenopausal POP, 4.3 × 10 ± 6.3 × 10; premenopausal non-POP, 5.0 × 10 ± 7.7 × 10; P = 0.15). No differences in mRNA expression were seen between sites (P = 0.74), but significant variation was noted in protein expression (P = 0.001). Premenopausal and postmenopausal women with POP exhibit differential expression of LOXL1 suggesting different pathways in the pathogenesis of POP. The role of biopsy location on LOXL1 expression requires further investigation.

The pathophysiology of pseudoexfoliation syndrome is affected by interaction of TGF‐β1 and LOXL1

PurposeThe cross‐linking enzyme lysyl oxidase‐like 1 (LOXL1) and profibrotic transforming growth factor (TGF)‐ß1 play key roles in pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma. The purpose of this study was to investigate the interaction between LOXL1 and TGF‐ß1 with respect to the PEX‐specific disordered matrix metabolism.MethodsPrimary human Tenon's capsule fibroblasts (hTCF) obtained from patients were treated with TGF‐ß1 (0‐10 ng/ml) for 12‐72 hours without or with preincubation with inhibitors of TGF‐β signalling pathways. Expression of LOXL1 and PEX‐specific extracellular matrix components was examined by using quantitative RT‐PCR and Western immunoblot analysis. Direct binding of LOXL1 to TGF‐ß1 was analyzed by blot overlay assay and solid phase ELISA using purified LOXL1, recombinant human TGF‐ß1, TGFß1‐LAP. The effect of LOXL1 on TGF‐ß1 signaling was analyzed using TGF‐ß receptor signaling real time PCR assays (BioRad) after transient transfection of hTCF with a full‐length pCMV6‐LOXL1 vector construct/with empty vector .ResultsTGFß1 significantly increased LOXL1 expression, secretion and enzymatic activity and correlated with enhanced expression of BMP‐1, elastin, fibrillin‐1, fibulin‐4 and fibulin‐5 with peak effects at 10 ng/ml for 48 hours. This induction was blocked by TGF‐β receptor inhibitors and inhibitors of the canonical Smad and non‐canonical signaling pathways. Direct binding between LOXL1 and TGFß1‐LAP was demonstrated by Blot overlay assays and ELISA. LOXL1 overexpression temporarily upregulates different transcriptional regulators and some protein kinases of p38‐MAPK signalling pathway after 12 to 24 hours post‐transfection.ConclusionsThe results of this study indicate that the interaction of LOXL1 and TGF‐ß1 plays an important role in the PEX‐associated abnormal matrix metabolism and fibrosis.

Expression and Regulation of LOXL1 and Elastin-related Genes in Eyes With Exfoliation Syndrome

Exfoliation syndrome (XFS) is a complex, late-onset disorder of the elastic fiber system and the most common identifiable cause of open-angle glaucoma. Strong genetic risk is conferred by the lysyl oxidase-like 1 (LOXL1) gene, but additional comodulating factors are necessary for the manifestation of the disease. The aim of this study was to establish a comprehensive expression profile of LOXL1 and elastic proteins in XFS eyes and to analyze their regulation in an in vitro cell culture system. Eyes with XFS with and without glaucoma, and normal control eyes were analyzed for major elastic fiber components (elastin, fibrillin-1, fibulin-4), and lysyl oxidase (LOX) enzymes by real-time PCR, immunohistochemistry, and electron microscopy. Cultured human Tenon's capsule fibroblasts were exposed to transforming growth factor-β1, IL-6, homocysteine, oxidative stress, hypoxia, or ultraviolet radiation, and changes in the expression of LOXL1 and elastic components of XFS material were assessed by real-time PCR, immunohistochemistry, and Western blotting. LOXL1 expression in anterior eye tissues was significantly increased in early XFS stages but was decreased in advanced stages as compared with controls. LOXL1 was also found to be a major component of XFS material and to colocalize with elastin, fibrillin-1, and fibulin-4, which were upregulated in parallel to LOXL1. In contrast, in most posterior segment tissues, LOXL1 and elastic fiber proteins displayed no differential expression. Interestingly, lamina cribrosa specimens of early and late XFS stages without and with glaucoma revealed a selective downregulation of LOXL1 and elastic fiber components on the mRNA and protein level, which was associated with pronounced ultrastructural alterations of the laminar elastic fiber network in XFS eyes. Treatment of cultured cells with XFS-associated pathogenetic stimuli induced a significant increase in the expression of LOXL1 and elastic proteins and resulted in their assembly into XFS-like fibrils in vitro. The findings support the notion that both genetic and nongenetic factors may cooperate in the stable accumulation of XFS aggregates and provide evidence for a XFS-specific elastinopathy of the lamina cribrosa, possibly rendering XFS eyes more vulnerable to pressure-induced optic nerve damage and glaucoma development.

Expression and significance of lysyl oxidase-like 1 and fibulin-5 in the cardinal ligament tissue of patients with pelvic floor dysfunction

Pelvic organ prolapse (POP) is a disabling disorder in women characterized by a loss of pelvic floor support, leading to the herniation of the uterus into or through the vagina. POP is a complex problem that likely involves multiple mechanisms with limited therapies available, and is associated with defects in connective tissue including elastic fibers. This study was designed to investigate the expression of fibulin-5 and lysyl oxidase-like 1 (LOXL1) in the cardinal ligament in samples taken from the POP group compared to the non-POP group. Specimens were obtained during abdominal hysterectomy from the cardinal ligament of 53 women with POP and 25 age- and parity- matched women with non-POP among post-menopausal women with benign gynecologic pathology. Protein expression was evaluated using the immunohistochemical staining method. For statistical analyses, chi-square test and Spearman's correlation were used with the statistical package SPSS13.0 system. Our results showed that both fibulin-5 and LOXL1 expressions were decreased in the cardinal ligament in the POP group compared to the non-POP group (P < 0.05). The expression of fibulin-5 and LOXL1 were correlated closely with the stage of POP, accompanied by stress urinary incontinence and frequency of vaginal delivery (P < 0.05), but had no relationship with post-menopausal state (P > 0.05). The expression of fibulin-5 was positively associated with LOXL1 in POP (P < 0.05). We conclude that changes in fibulin-5 and LOXL1 expression may play a role in the development of POP.

Epigenetic Silencing of Lysyl Oxidase-Like-1 through DNA Hypermethylation in an Autosomal Recessive Cutis Laxa Case

We have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation.

Genotype-Correlated Expression of Lysyl Oxidase-Like 1 in Ocular Tissues of Patients with Pseudoexfoliation Syndrome/Glaucoma and Normal Patients

Pseudoexfoliation (PEX) syndrome is a generalized disease of the extracellular matrix and the most common identifiable cause of open-angle glaucoma. Two single nucleotide polymorphisms in the lysyl oxidase-like 1 (LOXL1) gene (rs1048661 and rs3825942) have been recently identified as strong genetic risk factors for both PEX syndrome and PEX glaucoma. Here we investigated the expression and localization of LOXL1, LOXL2, and lysyl oxidase (LOX) in tissues of PEX syndrome/glaucoma patients and controls in correlation with their individual single nucleotide polymorphism genotypes and stages of disease. LOXL1 ocular expression was reduced by approximately 20% per risk allele of rs1048661, whereas risk alleles of rs3825942, which were highly overrepresented in PEX cases, did not affect LOXL1 expression levels. Irrespective of the individual genotype, LOXL1 expression was significantly increased in early PEX stages but was decreased in advanced stages both with and without glaucoma compared with controls, whereas LOX and LOXL2 showed no differences between groups. LOXL1 was also found to be a major component of fibrillar PEX aggregates in both intra- and extraocular locations and to co-localize with various elastic fiber components. These findings provide evidence for LOXL1 involvement in the initial stages of abnormal fibrogenesis in PEX tissues. Alterations of LOXL1 activation, processing, and/or substrate specificity may contribute to the abnormal aggregation of elastic fiber components into characteristic PEX fibrils.