Expression Of Fibulin-5 Research Articles

Regulation of Fibulin-2 Gene Expression by Integrin α3β1 Contributes to the Invasive Phenotype of Transformed Keratinocytes

The laminin-binding integrin α3β1 is highly expressed in epidermal keratinocytes where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, roles for α3β1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by α3β1 in immortalized keratinocytes. α3β1-responsive genes included several that are involved in extracellular matrix proteolysis or remodeling, including fibulin-2 and SPARC. However, α3β1-dependent induction of specific target genes was influenced by the genetic lesion that triggered immortalization, as α3β1-dependent fibulin-2 expression occurred in cells immortalized by either SV40 large T antigen or p53-null mutation, while α3β1-dependent SPARC expression occurred only in the former cells. Interestingly, qPCR arrays did not reveal strong patterns of α3β1-dependent gene expression in freshly isolated primary keratinocytes, suggesting that this regulation is acquired during immortalization. p53-null keratinocytes transformed with oncogenic RasV12 retained α3β1-dependent fibulin-2 expression, and RNAi-mediated knockdown of fibulin-2 in these cells reduced invasion, although not their tumorigenic potential. These findings demonstrate a prominent role for α3β1 in immortalized/transformed keratinocytes in regulating fibulin-2 and other genes that promote matrix remodeling and invasion.

Effect of Fibulin-5 on cell proliferation and invasion in human gastric cancer patients

ObjectivesTo explore the effect of Fibulin-5 expression on cell proliferation and invasion in human gastric cancer patients. MethodsFibulin-5 expression was detected in 56 samples of surgically resected gastric cancer and paired noncancerous tissues using qRT-PCR and immunoblotting. Fibulin-5 was knocked down by Fibulin-5 shRNA in MGC-803 cells, then BrdU cell proliferation and transwell invasion assays were used to determine cell proliferation and invasion. ResultsThe level of Fibulin-5 mRNA in gastric cancer tissues was significantly higher as compared with that in normal tumor-adjacent tissues (P<0.05). Otherwise, the level of Fibulin-5 protein in cancer and noncancerous tissues was consistent with mRNA expression (P<0.05). Fibulin-5 protein expression in tumor tissues with poorly differentiated, lymph node metastasis and advanced TNM tumor stage was significantly higher (P<0.05, respectively). Fibulin-5 was obviously knocked down by Fibulin-5 shRNA (P<0.05), and Fibulin-5 knockdown significantly inhibited cell proliferation and invasion in MGC-803 cells (P<0.05, respectively). ConclusionsThe high-expression of Fibulin-5 is associated with the malignant clinicopathologic parameters in gastric cancer and Fibulin-5 knockdown inhibits cell proliferation and invasion in MGC-803 cells, suggesting Fibulin-5 may act as a key factor in the progression of gastric cancer.

Temporospatial expression of fibulin-1 after acute spinal cord injury in rats

ObjectiveFibulin-1 is a matricellular protein that plays important roles in motility inhibition in a variety of cells and blocks the proliferation of cultured neural stem cells. The biological function of fibulin-1 in the spinal cord has not been fully elucidated.MethodsTo clarify the expressions and possible functions of fibulin-1 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats. Our work studied the temporospatial expression patterns of fibulin-1.ResultsWestern blot analysis revealed that fibulin-1 levels significantly increased 5 days after spinal cord contusion. Immunohistochemistry confirmed an increased number of fibulin-1 immunopositive cells about 2 mm from the lesion site. Moreover, double immunofluorescence labeling suggested that these changes were especially prominent in neurons and microglia.ConclusionThese findings suggest that fibulin-1 may be involved in neuronal apoptosis and microglial activation after SCI.

Reduced Fibulin-2 Contributes to Loss of Basement Membrane Integrity and Skin Blistering in Mice Lacking Integrin α3β1 in the Epidermis

Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3β1, a major receptor in epidermis for adhesion to laminin-332, plays critical roles in basement membrane organization during skin development. In the current study, we identify a role for α3β1 in promoting stability of nascent epidermal basement membranes through induction of fibulin-2, a matrix-associated protein that binds laminin-332. We demonstrate that mice lacking α3β1 in epidermis display ruptured basement membrane beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3-null mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3β1 in basement membrane organization is recapitulated during wound healing. Mice lacking epidermal α3β1 also have reduced fibulin-2 expression, and fibulin-2-null mice display perinatal skin blisters similar to those in α3β1-deficient mice. Interestingly, α3-null wound epidermis or keratinocytes also show impaired processing of the laminin-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3β1 in basement membrane stability through fibulin-2 induction, both in neonatal skin and adult wounds.

JunB contributes to the epithelial‐mesenchymal transition in response to TGF‐β (LB277)

The process of epithelial‐mesenchymal transition (EMT) is important in tissue fibrosis, wound healing and cancer. Transforming growth factor beta (TGF‐β) induces EMT and fibrotic proteins in a variety of responsive cells via a mechanism that is not fully understood. This study identifies a critical role of JunB in the EMT and fibrotic process in response to TGF‐β. Knock‐down of the immediate‐early response gene JunB by siRNA abrogates TGF‐β‐induced disruption of epithelial cell junctions, formation of actin fibers, focal adhesions and expression of fibrotic proteins. JunB contributes to Smad‐mediated repression of Id2 helix‐loop‐helix protein but does not affect the induction of Hmga2, Snail and Slug. Knock‐down of JunB abolishes up‐regulation of ATF3, which is required for Id2 repression. Importantly, JunB contributes to the TGF‐β‐induced fibrotic response by regulating expression of fibronectin, fibulin‐2, tropomyosin (Tpm1) and β3‐integrin, which play critical roles in matrix deposition, cell‐matrix adhesion and actin stress‐fibers. In summary, JunB provides important input in setting the transcriptional program of the EMT and fibrotic responses to TGF‐β. Thus, JunB represents an important target in diseases associated with EMT, including cancer and fibrosis.Grant Funding Source: Supported by R01 CA95263

Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium.

Cell-matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2-3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell-matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.

Enhanced bioactive myocardial transforming growth factor-β in advanced human heart failure.

Transforming growth factor (TGF)-β activation is known to play a central role in progressive ventricular remodeling in advanced heart failure in animal models, but there has been no direct evidence of increased TGF-β activity in the myocardium of patients with advanced human heart failure. METHODS AND RESULTS: Using a recently developed bioassay that measures TGF-β bioactivity rather than TGF-β abundance, we measured bioactive TGF-β in human myocardium from control non-failing donors (NF), and patients with ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM). Both free and total soluble TGF-β were significantly increased in ICM and DCM compared with NF. Free TGF-β had an excellent correlation with phosphorylated Smad2 (R(2)=0.55, P<0.0001), a downstream marker of TGF-β signaling. Collagen type I and type III were significantly upregulated in DCM compared with NF, consistent with histological evidence of myocardial fibrosis. Expression of fibulin-2, a positive modulator of TGF-β, was significantly increased in DCM compared with NF, and the free TGF-β level was correlated with fibulin-2 mRNA (R(2)=0.24, P<0.006). Although both free and total soluble TGF-β are significantly increased in ICM and DCM compared with NF, the superior correlation of free TGF-β with downstream signaling suggests that this is the most functionally relevant form. The present findings suggest that sustained TGF-β activation in both ICM and DCM contributes to excess myocardial fibrosis.

Diverse functions of fibulin-5 in tumors

Altered interactions between the extracellular matrix and cells play an important part in tumorigenesis and metastasis. As a member of matricellular glycoprotein, fibulin-5 is expressed in elastin-rich tissues and organizes the matrix structures by interacting with many extracellular proteins. Fibulin-5 expression is closely associated with normal embryonic development and organogenesis. Mice deficient for the fibulin-5 gene exhibit systemic elastic fiber defects with manifestation of loose skin, emphysematous lung and tortuous vessels. Additionally, fibulin-5 null mice exhibited increased angiogenesis after wound healing or PVA sponge implantation and matrigel implantation experiments show fibulin-5 inhibited vessel formation, suggesting fibulin-5 functions as an angiogenesis inhibitor. Fibulin-5 also plays critical roles in proliferation, migration and invasion of certain tumors, and the effect of fibulin-5 on tumorigenesis appears to be largely context-dependent. This effect might involve the inhibiting action of fibulin-5 on angiogenesis. This review focuses on recent advances in our understanding of the roles of fibulin-5 in tumorigenesis: both tumor promoting and suppressing activity of fibulin-5 are reviewed, and the emerging evidences of its promising potential as therapeutic options and/or targets in the treatment of cancer also highlighted.

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

BackgroundNasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.Material and MethodsStandard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.ResultsOur results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.ConclusionHigher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.

Twist2 functions as a tumor suppressor in murine osteosarcoma cells

The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.

Fibulin-1 is Down-Regulated Through Promoter Hypermethylation and Suppresses Renal Cell Carcinoma Progression

Renal cell carcinoma is one of the most common cancers worldwide but the molecular mechanisms that underlie it are not fully understood. Fibulin-1, a multi-functional extracellular matrix protein, is involved in many types of cancer but its function in renal cell carcinoma is unclear. We investigated fibulin-1 expression and function in renal cell carcinoma. We performed real-time polymerase chain reaction, Western blot analysis and immunohistochemistry to determine fibulin-1 expression in renal cell carcinoma cells and patient tissues. Methylation specific polymerase chain reaction and quantitative sequencing were done to examine the methylation status of the FBLN1 gene promoter. Eukaryotic expression plasmid and a lentiviral vector were used to over express fibulin-1 in ACHN and 786-O renal cell carcinoma cells to investigate its function in vitro and in vivo. Fibulin-1 was significantly down-regulated in renal cell carcinoma cell lines and patient tissues. Dysregulation was associated with renal cell carcinoma progression. The FBLN1 gene promoter region was hypermethylated and its methylation status correlated with fibulin-1 expression. Fibulin-1 over expression led dramatically to decreased cell growth, enhanced tumor cell apoptosis, decreased cell motility and angiogenesis in cultured renal cell carcinoma cells and in xenograft tumors in nude mice. Fibulin-1 is down-regulated in renal cell carcinoma through promoter hypermethylation. It functions as a tumor suppressor and angiogenesis inhibitor in renal cell carcinoma.

CXCL1/GROα increases cell migration and invasion of prostate cancer by decreasing fibulin-1 expression through NF-κB/HDAC1 epigenetic regulation

Inflammatory tumor microenvironments play pivotal roles in the development of cancer. Inflammatory cytokines such as CXCL1/GROα exert cancer-promoting activities by increasing tumor angiogenesis. However, whether CXCL1/GROα also plays a role in the progression of prostate cancer, particularly in highly invasive castration-resistant prostate cancer (CRPC), has not been investigated. We explored whether CXCL1/GROα enhances cell migration and invasion in PC-3 and DU145 CRPC. Induction of PC-3 and DU145 cancer progression by CXCL1/GROα is associated with increased AKT activation and IκB kinase α (IKKα) phosphorylation, resulting in nuclear factor-kappaB (NF-κB) activation. Activated NF-κB interacts with histone deacetylase 1 (HDAC1) to form a gene-silencing complex, which represses the expression of fibulin-1D by decreasing the acetylation of histone H3 and H4 on the NF-κB-binding site of the fibulin-1D promoter. Blockade of AKT2 by small hairpin RNA (shRNA) decreases IKKα phosphorylation, NF-κB nuclear translocation and cell migration, indicating that AKT is required in CXCL1/GROα-mediated NF-κB activation and cell migration. In addition, NF-κB and HDAC1 shRNA decrease the effect of CXCL1/GROα on fibulin-1D downregulation, migration and invasion, suggesting that the NF-κB/HDAC1 complex is also involved in CXCL1/GROα-mediated cancer progression. Our findings provide the first evidence that CXCL1/GROα decreases fibulin-1D expression in prostate cancer cells and also reveals novel insights into the mechanism by which CXCL1/GROα regulates NF-κB activation through the AKT pathway. Our results also clearly establish that co-operation of NF-κB and HDAC1 regulates fibulin-1D expression by epigenetic modification. Our study suggests that inhibition of CXCL1/GROα-mediated AKT/NF-κB signaling may be an attractive therapeutic target for CRPC.

Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells

To investigate whether heart and neural crest derivatives expressed transcript 2 (HAND2) regulates fibulin-1 (FBLN1) expression during decidualization of human endometrial stromal cells (ESCs). Invitro experiment. Research laboratory. Twenty-four patients undergoing hysterectomy for benign reasons. ESCs were cultured with various progestins (medroxyprogesterone acetate [MPA], norethisterone, levonorgestrel, dienogest, and P), E(2), dexamethasone, and/or 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP). HAND2 and FBLN1 were silenced by small interfering RNA technology. HAND2 and FBLN1 expression levels were assessed by real-time polymerase chain reaction and Western blot analysis. MPA or E(2) + MPA increased HAND2 mRNA levels in ESCs in a time- and dose-dependent manner, and this stimulatory effect was blocked by RU-486 (P receptor antagonist). HAND2 was increased by E(2) + MPA earlier than FBLN1. Simultaneous MPA and 8-Br-cAMP treatment synergistically enhanced HAND2 mRNA levels. P and all the progestins significantly increased HAND2 mRNA levels, whereas E(2), 8-Br-cAMP, or dexamethasone alone had no effect. Silencing of HAND2 expression significantly reduced FBLN1 expression, whereas FBLN1 silencing had no effect on HAND2 expression. These results suggest that progestin-induced HAND2 contributes to FBLN1 expression in human ESCs.

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.

삼백초(Saururus chinensis) 추출물의 항산화능 및 생리활성 연구

약용소재로 사용되고 있는 삼백초의 잎과 줄기로부터 유기 용매의 극성에 따른 순차적 분획물을 얻어 항산화 효과와 연관하여 피부의 탄력 및 혈관형성에 미치는 영향을 조사하였다. 삼백초 추출 분획물에 대한 항산화 활성은 시료의 농도가 증가할수록 DPPH에 대한 전자공여능도 증가하였으며, <TEX>$ED_{50}$</TEX>은 EtOAc 분획에서 합성 항산화제인 BHT의 27.22 <TEX>${\mu}g/ml$</TEX>보다 12.84 <TEX>${\mu}g/ml$</TEX>로 높게 나타났다. 또한 <TEX>$H_2O_2$</TEX>에 의해 유도된 HDF 세포사멸(<TEX>$IC_{50}$</TEX>)에 대하여 삼백초 추출 50 <TEX>${\mu}/ml$</TEX>의 EtOAc 분획에서 89.39%의 가장 높은 세포 생존율을 나타내었으며, 동량의 n-BuOH 분획에서도 67.98%의 유의적인 세포 생존율을 보였다. 한편 삼백초 추출 분획물은 피부 탄력과 관련있는 fibulin-5 mRNA의 발현율을 감소시켰으며, 항산화 활성이 높은 삼백초 추출 EtOAc 분획에 대한 CAM assay 결과, 혈관형성을 억제하는 것으로 나타났다. 이러한 삼백초 추출 EtOAc 분획은 향후 항산화 활성 및 해독과 관련된 혈관형성 억제의 약재개발 가능성이 있음을 암시하는 것으로 이의 작용기작에 대한 더욱 자세한 연구가 필요하다고 생각한다. Saururus chinensis has long been widely used in oriental folk medicines to treat diseases. In the current study, organic solvent fractions obtained from the main methanolic extract of S chinensis were evaluated for their antioxidative and related physiological activities. The antioxidant activity of the fractions was measured using DPPH free radical scavenging activity, increased in a dose-dependent manner, and the <TEX>$ED_{50}$</TEX> of the ethyl acetate fractions exhibited a value of 12.84 <TEX>${\mu}g/ml$</TEX> higher than 27.22 <TEX>${\mu}g/ml$</TEX> compared to the BHT. Also, the cell viability of S. chinensis on <TEX>$H_2O_2$</TEX>-induced HDF cell death (<TEX>$IC_{50}$</TEX>) showed the highest cell viability of 89.39% in 50 <TEX>${\mu}g/ml$</TEX> of ethyl acetate fraction and 67.98% of visible cell survival rate in n-butanolic fraction. Meanwhile, all fractions of the S. chinensis extract led to a slight down regulation of the mRNA expression of fibulin-5, which is related to skin elasticity, and the ethyl acetate fraction having high antioxidant activity showed a markedly inhibitory effect on chick embryonic angiogenesis using the CAM assay. These results suggest that the ethyl acetate fraction of S. chinensis extract could be a good material in therapeutic application for antioxidant and related anti-angiogenesis activities.

Abstract 2330: Murine interferon-α inhibits aortic endothelial outgrowth

Abstract Although Interferon-α exerts anti-angiogenic effects, it has proven clinically useful in only a handful of tumor types. IFNα has a high degree of species specificity, and this can complicate mechanistic studies in animal models. We reported that human IFNα causes premature senescence of endothelial cells through effects on interferon regulatory factor 1 (IRF-1). We hypothesize that IFNα may also lead to increased expression of anti-angiogenic factors. Treatment of human endothelial cells with IFNα at 0.5µg/ml or 1.0µg/ml led to cell death with increased expression of ANG-2 and fibulin-5 in surviving cells. To establish the activity of a murine IFNα synthesized by our group for expanded study with mouse models, 2H11 murine endothelial cells were treated with murine IFNα at 0.05µg/ml, 0.5µg/ml, or 1.0µg/ml. Growth was inhibited at 0.05µg/ml and not at the higher doses. The expression of IRF-1, a downstream target of IFNα, was also upregulated in these cells upon treatment with 0.05µg/ml IFNα. The purpose of this study was to characterize the angiogenic response to murine IFNα in the more physiologically relevant mouse aortic outgrowth assay. Aortas were removed from 4 week old C57B/6 mice immediately following euthanasia and sectioned in 1mm increments. Sections from a single mouse were used for each replicate, and treatments were replicated 5 times. Sections were washed in basal medium, plated on a thin layer of Matrigel (50µl) in a 96-well plate, covered with 50µl Matrigel and incubated at 37°C. 24 hours after plating, sections were treated with IFNα at 0.5µg/ml, 1.0µg/ml, and 10µg/ml, or EGM-2 only. Sections were imaged at days 5 and 7. Photographs were digitally analyzed) to determine area and density of outgrowth and maximum vessel length. H&amp;E stained paraffin-embedded sections were examined to evaluate vessel morphology. All IFNα treatments significantly decreased the area and density of aortic outgrowth. The effect was most pronounced at lower doses. Successful outgrowth was achieved in a similar number of treated and untreated sections. Maximum vessel length was decreased at both time points Outgrowths were characterized by linear growth perpendicular to the surface of the aorta, and robust branching. Capillary-type structure with lumen was evident in H&amp;E sections. Vessel morphology did not differ with treatment. These data suggest that our newly synthesized murine IFNα has an antiangiogenic effect on endothelial cells. These results agree with our preliminary data showing murine endothelial cells undergo growth inhibition upon treatment with IFNα. Interestingly the response is only to the lower doses of murine IFNα. Higher doses have no effect in 2H11 murine endothelial cells and reduced effect on aortic outgrowth. Further studies are underway in our laboratory to demonstrate the factors responsible for this effect. 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 2330. doi:1538-7445.AM2012-2330

Decreased expression of elastin, fibulin‐5 and lysyl oxidase‐like 1 in the uterosacral ligaments of postmenopausal women with pelvic organ prolapse

Pelvic organ prolapse is associated with defects in connective tissues, including elastic fibers. The purpose of this study was to investigate the expression of fibulin-5 and lysyl oxidase-like 1 (LOXL1), which play an essential role in the synthesis and assembly of elastic fibers, in the uterosacral ligaments of women with advanced pelvic organ prolapse (POP) compared with controls. Specimens were obtained prospectively during transvaginal or abdominal hysterectomy from 30 women with advanced pelvic organ prolapse and 30 controls matched to the POP group for age and parity among postmenopausal women with benign gynecologic diseases. The expressions of elastin, fibulin-5 and LOXL1 in uterosacral ligaments were measured by immunohistochemistry. We detected a decreased, sometimes absent, expression of fibulin-5 and LOXL1 in the uterosacral ligaments of women with POP, despite a positive expression of elastin. There was a decrease in positive percentage of LOXL1 in the POP group (23.3%) compared with the controls (60%) (P = 0.004). With immunolabeling intensity classified as negative, weak, moderate or strong, there was a decrease in the expression of fibulin-5 in the POP group (P = 0.049). We also detected a significantly decreased expression of LOXL1 in the POP group (P = 0.001). There was decreased expression of fibulin-5 and LOXL1 in the uterosacral ligaments of patients with pelvic organ prolapse, while the elastin expression was equivalent, which may suggest the possibility of defects in elastic fiber remodeling in the postpartum period and contribute to POP.

Overexpression of Fibulin-5 in Retinal Pigment Epithelial Cells Inhibits Cell Proliferation and Migration and Downregulates VEGF, CXCR4, and TGFB1 Expression in Cocultured Choroidal Endothelial Cells

Purpose of the study: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss. Fibulin-5 (FBLN5) plays a pleiotropic role in the pathogenesis of AMD. We examined whether the in vitro overexpression of FBLN5 in retinal pigment epithelial (RPE) cells alters the proliferation and migration of cocultured choroidal endothelial cells (CECs) and explored the possible mechanisms involved.Materials and methods: A recombinant lentiviral vector carrying the Fbln5 gene was generated to transduce rat RPE cells. The expression of FBLN5 in transduced RPE cells was detected by quantitative real-time PCR and Western blot. The transduced RPE cells were then cocultured with rhesus macaque CECs in a Transwell coculture system. The impact of overexpression of FBLN5 in RPE cells on CEC proliferation and migration was assessed, as well as the impact on the mRNA expressions of vascular endothelial growth factor (VEGF), C-X-C chemokine receptor type 4 (CXCR4), and transforming growth factor β1 (TGFB1).Results: Our results showed that a recombinant lentivirus carrying the Fbln5 gene, which could induce overexpression of FBLN5 in RPE cells, was successfully generated. Overexpression of FBLN5 in RPE cells inhibited cell proliferation and migration and downregulated the mRNA expressions of VEGF, CXCR4, and TGFB1 in cocultured CECs.Conclusions: These findings suggest that FBLN5 may interfere with choroidal neovascularization by downregulating VEGF, CXCR4, and TGFB1 expression and inhibiting CEC proliferation and invasion, intensifying interest in FBLN5 as a target for therapeutic intervention in neovascular AMD.

Effect of mechanical stretch on the expressions of elastin, LOX and Fibulin-5 in rat BMSCs with ligament fibroblasts co-culture

The occurrence of PFD is closely related with elasticity, toughness, and functional changes of the connective tissue of the pelvic support tissue. This study aims to evaluate the effect of mechanical stretch on the differentiation of BMSCs with a co-culture with pelvic ligament fibroblasts. BMSCs was isolated and identified from 7day SPF SD rats. Rat pelvic ligament fibroblasts were obtained from rat pelvic ligament. The fourth passage of fibroblasts was subjected to 10% deformation with 1Hz, 12h periodic one-way mechanical stretch stimulation, and the cells were then co-cultured with BMSCs. The longer co-culture and co-culture with mechanical stretch (i.e. 6 and 12days) induced more expression of elastin, LOX, and Fibulin-5, compared to the groups without stimulation. Compared to co-culture group each, Co-culture with mechanical stretch stimulation group induced significant expression of elastin, LOX, and Fibulin-5, both in 3, 6 and 12days co-culture groups (P<0.05). However, there were no significant differences among 3, 6, and 12days control groups. These results suggest that in an indirect co-culture system, pelvic ligament fibroblasts with mechanical stretch stimulation can promote BMSCs differentiation, reflecting in the increased expression of elastin, LOX, and Fibulin-5.

Loss of fibulin-2 protects against progressive ventricular dysfunction after myocardial infarction

Remodeling of the cardiac extracellular matrix (ECM) is an integral part of wound healing and ventricular adaptation after myocardial infarction (MI), but the underlying mechanisms remain incompletely understood. Fibulin-2 is an ECM protein upregulated during cardiac development and skin wound healing, yet mice lacking fibulin-2 do not display any identifiable phenotypic abnormalities. To investigate the effects of fibulin-2 deficiency on ECM remodeling after MI, we induced experimental MI by permanent coronary artery ligation in both fibulin-2 null and wild-type mice. Fibulin-2 expression was up-regulated at the infarct border zone of the wild-type mice. Acute myocardial tissue responses after MI, including inflammatory cell infiltration and ECM protein synthesis and deposition in the infarct border zone, were markedly attenuated in the fibulin-2 null mice. However, the fibulin-2 null mice had significantly better survival rate after MI compared to the wild-type mice as a result of less frequent cardiac rupture and preserved left ventricular function. Up-regulation of TGF-β signaling and ECM remodeling after MI were attenuated in both ischemic and non-ischemic myocardium of the fibulin-2 null mice compared to the wild type counterparts. Increase in TGF-β signaling in response to angiotensin II was also lessened in cardiac fibroblasts isolated from the fibulin-2 null mice. The studies provide the first evidence that absence of fibulin-2 results in decreased up-regulation of TGF-β signaling after MI and protects against ventricular dysfunction, suggesting that fibulin-2 may be a potential therapeutic target for attenuating the progression of ventricular remodeling.