The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40 ng/ml in the culture medium, but decreased at 80 ng/ml. Under CoCl2- induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or antiangiogenic activities of BMSCs.

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。

非洲爪蟾早期胚胎发育中PEDF和PEDF-R的功能以及vestigial-like家族表达图式的研究

PEDF 蛋白（Pigment epithelium-derived factor）又名“色素上皮源因子”或 “色素上皮衍生因子”，为一个多功能性分泌糖蛋白，前人研究表明PEDF 蛋白 具有神经保护性、免疫调节、抑制新生血管生成以及抑制肿瘤恶化等多种功能。 PEDF-R 是PEDF 的受体， 属于PNPLA2 （ Patatin-like phopholipase domain-containing 2 family）蛋白家族的一个新成员，PEDF 蛋白与其结合后会激 活PEDF-R 的磷脂酶A2 活性。本研究中，我们描述了非洲爪蟾PEDF 和PEDF-R 基因的表达图式及其在胚胎发育中的可能功能。RT-PCR 结果显示PEDF 是非母 源性表达，而PEDF-R 则是母源性表达的。原位杂交实验表明它们均在神经系统 中特异表达，但PEDF-R 的表达区域更加广泛，在鳃弓、眼泡和耳泡中也有表达。 通过mRNA 过表达和Morpholino(MO)阻断蛋白合成等手段发现，PEDF 功能获 得和功能缺失后胚胎几乎不受影响。然而PEDF-R 过表达后胚胎向注射一侧弯 曲，TUNEL 凋亡检测实验发现这些胚胎在注射一侧发生了凋亡。这两个基因神 经表达的特异性表明它们可能在早期神经发育中有重要功能。TUNEL 结果暗示 着PEDF-R 可能是一个与凋亡信号通路相关的受体。PEDF 功能获得和缺失并未 导致胚胎明显的表型，这表明PEDF 在非洲爪蟾中可能还存在其他的受体来行使 与PEDF-R 不同功能的途径。 果蝇的vestigial 基因编码一个转录辅助因子，在果蝇中只有一个成员，即 vestigial(vg)基因。在脊椎动物中有四个vestigial 同源基因，即vestigial-like 非洲爪蟾早期胚胎发育中PEDF 和PEDF-R 的功能以及vestigial-like 家族表达图式的研究 2 1,2,3,4_(vgl-1,2,3,4)。Vestigial 蛋白能作为辅助因子与果蝇中的Scalloped(Sd)蛋白 或者哺乳动物中的TEF 蛋白结合成复合体，通过Sd/TEF 蛋白的TEA/ATTS 结构 域与DNA 结合，从而调节下游基因的转录。本研究中，我们克隆了非洲爪蟾 vestigial-like 家族的四个成员，并对其在爪蟾胚胎发育过程中的表达进行研究。 RT-PCR 显示vgl-2 和vgl-3 是合子型表达的，vgl-1、vgl-4 则是母源性表达。原位 杂交显示：vgl-1 主要在神经管背部、耳泡和眼泡中表达；vgl-2 则是在肌肉、第 一二鳃弓、脊索中特异表达；vgl-3 神经胚时期在后脑有强的表达信号，从神经 胚后期到尾芽期后脑部位的表达几乎消失了，而在胚胎的头部以及神经管中开始 有微弱的表达；vgl-4 的表达较广泛，在神经管、眼泡、耳泡、肌肉以及脊索中 均有表达。在爪蟾中这四个成员的表达图式各不相同，提示它们有可能与其行使 组织特异性基因调控的功能相关，上述结果将有助于对vestigial-like 家族基因在 胚胎发育中的功能研究。

Physiological investigation of periosteum structure and its application in periosteum tissue engineering

Although many different materials, techniques and methods, including artificial or engineered bone substitutes, have been used to repair various bone defects, the restoration of critical-sized bone defects caused by trauma, surgery or congenital malformation is still a great challenge to orthopedic surgeons. One important fact that has been neglected in the pursuit of resolutions for large bone defect healing is that most physiological bone defect healing needs the periosteum and stripping off the periosteum may result in non-union or non-healed bone defects. Periosteum plays very important roles not only in bone development but also in bone defect healing. The purpose of this project was to construct a functional periosteum in vitro using a single stem cell source and then test its ability to aid the repair of critical-sized bone defect in animal models. This project was designed with three separate but closely-linked parts which in the end led to four independent papers. The first part of this study investigated the structural and cellular features in periostea from diaphyseal and metaphyseal bone surfaces in rats of different ages or with osteoporosis. Histological and immunohistological methods were used in this part of the study. Results revealed that the structure and cell populations in periosteum are both age-related and site-specific. The diaphyseal periosteum showed age-related degeneration, whereas the metaphyseal periosteum is more destructive in older aged rats. The periosteum from osteoporotic bones differs from normal bones both in terms of structure and cell populations. This is especially evident in the cambial layer of the metaphyseal area. Bone resorption appears to be more active in the periosteum from osteoporotic bones, whereas bone formation activity is comparable between the osteoporotic and normal bone. The dysregulation of bone resorption and formation in the periosteum may also be the effect of the interaction between various neural pathways and the cell populations residing within it. One of the most important aspects in periosteum engineering is how to introduce new blood vessels into the engineered periosteum to help form vascularized bone tissues in bone defect areas. The second part of this study was designed to investigate the possibility of differentiating bone marrow stromal cells (BMSCs) into the endothelial cells and using them to construct vascularized periosteum. The endothelial cell differentiation of BMSCs was induced in pro-angiogenic media under both normoxia and CoCl2 (hypoxia-mimicking agent)-induced hypoxia conditions. The VEGF/PEDF expression pattern, endothelial cell specific marker expression, in vitro and in vivo vascularization ability of BMSCs cultured in different situations were assessed. Results revealed that BMSCs most likely cannot be differentiated into endothelial cells through the application of pro-angiogenic growth factors or by culturing under CoCl2-induced hypoxic conditions. However, they may be involved in angiogenesis as regulators under both normoxia and hypoxia conditions. Two major angiogenesis-related growth factors, VEGF (pro-angiogenic) and PEDF (anti-angiogenic) were found to have altered their expressions in accordance with the extracellular environment. BMSCs treated with the hypoxia-mimicking agent CoCl2 expressed more VEGF and less PEDF and enhanced the vascularization of subcutaneous implants in vivo. Based on the findings of the second part, the CoCl2 pre-treated BMSCs were used to construct periosteum, and the in vivo vascularization and osteogenesis of the constructed periosteum were assessed in the third part of this project. The findings of the third part revealed that BMSCs pre-treated with CoCl2 could enhance both ectopic and orthotopic osteogenesis of BMSCs-derived osteoblasts and vascularization at the early osteogenic stage, and the endothelial cells (HUVECs), which were used as positive control, were only capable of promoting osteogenesis after four-weeks. The subcutaneous area of the mouse is most likely inappropriate for assessing new bone formation on collagen scaffolds. This study demonstrated the potential application of CoCl2 pre-treated BMSCs in the tissue engineering not only for periosteum but also bone or other vascularized tissues. In summary, the structure and cell populations in periosteum are age-related, site-specific and closely linked with bone health status. BMSCs as a stem cell source for periosteum engineering are not endothelial cell progenitors but regulators, and CoCl2-treated BMSCs expressed more VEGF and less PEDF. These CoCl2-treated BMSCs enhanced both vascularization and osteogenesis in constructed periosteum transplanted in vivo.

A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

胎牛血清促进色素上皮衍生因子在皮肤角质形成细胞和成纤维细胞中的表达

下载PDF阅读器目的:探讨表皮角质形成细胞和真皮成纤维细胞中色素上皮衍生因子(PEDF) 表达的影响因素.方法:体外培养角质形成细胞和成纤维细胞,并以10%FBS(胎牛血清)刺激,通过免疫荧光和Western blot检测PEDF的表达.结果:PEDF大多位于细胞浆中,但在细胞核中也有少量表达.细胞浆中PEDF并非均质型分布,而是呈细颗粒状集聚.10%FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达,而且这一分布形式不受组胺和佛波肉豆蔻醋酸(PMA) 的影响.结论:10% FBS促进角质形成细胞和成纤维细胞中PEDF的集聚和表达.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 microg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kappaB (NF-kappaB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO(-)) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kappaB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kappaB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO(-) formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.

Increased serum pigment epithelium-derived factor is associated with microvascular complications, vascular stiffness and inflammation in Type 1 diabetes

To determine in Type 1 diabetes patients if levels of pigment epithelium-derived factor (PEDF), an anti-angiogenic, anti-inflammatory and antioxidant factor, are increased in individuals with complications and positively related to vascular and renal dysfunction, body mass index, glycated haemoglobin, lipids, inflammation and oxidative stress.

Increased serum pigment epithelium derived factor levels in Type 2 diabetes patients

Serum PEDF levels (mean (S.D.)) were increased in 96 Type 2 diabetic vs. 54 non-diabetic subjects; 5.3 (2.8) vs. 3.2 (2.0)mug/ml, p

Clinical correlates of serum pigment epithelium-derived factor in type 2 diabetes patients

Aim: To determine if serum pigment epithelium-derived factor (PEDF) levels in Type 2 diabetes are related to vascular risk factors and renal function. Methods: PEDF was quantified by ELISA in a cross-sectional study of 857 male Veterans Affairs Diabetes Trial (VADT) subjects, and associations with cardiovascular risk factors and renal function were determined. In a subset (n = 246) in whom serum was obtained early in the VADT (2.0 ± 0.3 years post-randomization), PEDF was related to longitudinal changes in renal function over 3.1 years. Results: Cross-sectional study: In multivariate regression models, PEDF was positively associated with serum triglycerides, waist-to-hip ratio, serum creatinine, use of ACE inhibitors or angiotensin receptor blockers, and use of lipid-lowering agents; it was negatively associated with HDL-C (all p < 0.05). Longitudinal study: PEDF was not associated with changes in renal function over 3.1 years (p > 0.09). Conclusions: Serum PEDF in Type 2 diabetic men was cross-sectionally associated with dyslipidemia, body habitus, use of common drugs for blood pressure and dyslipidemia, and indices of renal function; however, PEDF was not associated with renal decline over 3.1 years.

Cord blood adipokines, neonatal anthropometrics and postnatal growth in offspring of Hispanic and Native American women with diabetes mellitus

BACKGROUND: Offspring of women with diabetes mellitus (DM) during pregnancy have a risk of developing metabolic disease in adulthood greater than that conferred by genetics alone. The mechanisms responsible are unknown, but likely involve fetal exposure to the in utero milieu, including glucose and circulating adipokines. The purpose of this study was to assess the impact of maternal DM on fetal adipokines and anthropometry in infants of Hispanic and Native American women.

METHODS: We conducted a prospective study of offspring of mothers with normoglycemia (Con-O; n = 79) or type 2 or gestational DM (DM-O; n = 45) pregnancies. Infant anthropometrics were measured at birth and 1-month of age. Cord leptin, high-molecular-weight adiponectin (HMWA), pigment epithelium-derived factor (PEDF) and C-peptide were measured by ELISA. Differences between groups were assessed using the Generalized Linear Model framework. Correlations were calculated as standardized regression coefficients and adjusted for significant covariates.

RESULTS: DM-O were heavier at birth than Con-O (3.7 ± 0.6 vs. 3.4 ± 0.4 kg, p = 0.024), but sum of skinfolds (SSF) were not different. At 1-month, there was no difference in weight, SSF or % body fat or postnatal growth between groups. Leptin was higher in DM-O (20.1 ± 14.9 vs. 9.5 ± 9.9 ng/ml in Con-O, p < 0.0001). Leptin was positively associated with birth weight (p = 0.0007) and SSF (p = 0.002) in Con-O and with maternal hemoglobin A1c in both groups (Con-O, p = 0.023; DM-O, p = 0.006). PEDF was positively associated with birth weight in all infants (p = 0.004). Leptin was positively associated with PEDF in both groups, with a stronger correlation in DM-O (p = 0.009). At 1-month, HMWA was positively associated with body weight (p = 0.004), SSF (p = 0.025) and % body fat (p = 0.004) across the cohort.

CONCLUSIONS: Maternal DM results in fetal hyperleptinemia independent of adiposity. HMWA appears to influence postnatal growth. Thus, in utero exposure to DM imparts hormonal differences on infants even without aberrant growth.

Characterization of pro- and anti-angiogenic factors in models of diabetic retinopathy

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015