Three-dimensional synthetic niche components to control germ cell proliferation

Spermatogonial stem cells (SSCs) are increasingly studied for potential use in tissue regeneration due to their ability to dedifferentiate into embryonic stem cell-like cells. For their successful therapeutic use, these cells must first be expanded in vitro using an appropriate culture system. We hypothesized that a hydrogel with proper biochemical and biomechanical properties may mimic the composition and structure of the native basement membrane onto which SSCs reside, thus allowing us to control SSC proliferation. This hypothesis was examined in two-dimensional (2D) and three-dimensional (3D) cultures using hydrogels formed from calcium cross-linked alginate molecules conjugated with synthetic oligopeptides containing the Arg-Gly-Asp sequence (RGD peptides). The RGD peptide density (N(RGD)) in gel matrices was controlled by mixing alginate molecules modified with RGD peptides and unmodified alginate molecules at varied ratios. The mechanical stiffness was controlled with the cross-linking density of gel matrices. Interestingly, the RGD peptide density modulated cell proliferation in both 2D and 3D cultures as well as the number and size of SSC colonies formed in 3D cultures. In contrast, cell proliferation was minimally influenced by mechanical stiffness in 2D cultures. Overall, the results of this study elucidate an important factor regulating SSC proliferation and also present a bioactive hydrogel that can be used as a 3D synthetic basement membrane. In addition, the results of this study will be broadly useful in controlling the proliferation of various stem cells.     

Expression of the vascular endothelial growth factor receptor-2/Flk-1 in breast carcinomas: correlation with proliferation

Vascular endothelial growth factor (VEGF) and its receptor Flk-1/KDR play an important role in vascular permeability and tumor angiogenesis. Prompted by the hypothesis that VEGF/Flk-1 system may have regulatory roles in breast carcinogenesis, we investigated the expression of Flk-1 in 141 invasive breast carcinomas in correlation with clinical and immunohistochemical prognostic parameters, including proliferation indices like Ki-67 and Topoisomerase IIalpha (Topo-IIalpha). The immunohistochemical avidin-biotin-peroxidase method was performed on paraffin sections for the detection of Flk-1, p53, Bcl-2, c-erbB-2, Ki-67, Topo-IIalpha, ER, and PR. Flk-1 was detected in 91 of 141 (64.5%) of invasive breast carcinomas showing a widespread cytoplasmic expression in most of the neoplastic cells. Flk-1 expression was correlated with the menopausal status (P = 0.051) of the patient and the nuclear grade of the invasive breast carcinoma (P = 0.003), but demonstrated no correlation with histologic grade, stage, and patient survival. It is interesting that Flk-1 expression demonstrated a significant correlation with 2 well-established proliferation indices, Ki-67 (P = 0.037) and topo-IIalpha (P = 0.009), whereas there was no correlation with the expression of ER, PR, p53, Bcl-2, and c-erbB-2. Moreover, Flk-1 expression showed an inverse correlation with TIMP-1 mRNA localization in intratumoral stromal cells (P = 0.013). In conclusion, the significant correlation of Flk-1 expression in invasive breast carcinomas with proliferation indices like Ki-67 and topo-IIalpha suggests that VEGF may exert a growth factor activity on mammary cancer cells through its receptor Flk-1. On the other hand, the inverse correlation of Flk-1 with TIMP-1 mRNA in intratumoral stromal cells supports the notion that TIMP-1 may have an inhibitory role on angiogenesis.     

T-kininogen induces endothelial cell proliferation

Basal proliferation of endothelial cells increases with age, and this might play a role in the etiology of age-related vascular diseases, as well as angiogenesis. Serum kininogen levels increase during aging in rats and humans, and T-kininogen (T-KG) can affect proliferative homeostasis in several cell models. Both kinins and kininogens have been shown previously to be angiogenic through activation of endothelial cell proliferation, and here we show that exposure of endothelial cells to T-KG results in vigorous cell proliferation, accompanied by ERK/AKT activation. In our experiments, the proliferative response requires B1 and B2 kinin receptors, even though kinins are not released from the precursor. We hypothesize that the age-related increase in T-KG could play a significant role in the age-related dysregulation of vascular physiology and function.     

Placental expression of vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 correlates with severity of clinical preeclampsia and villous hypermaturity

Overexpression of soluble vascular endothelial growth factor receptor-1 has been linked to preeclampsia and is thought to be secondary to placental insufficiency caused by hypoxia. Villous hypermaturity, characterized by presence of increased syncytial knots, has been associated with syndromes of placental insufficiency, particularly when severe. This study was undertaken to determine whether there is a link between soluble vascular endothelial growth factor receptor-1 expression, villous hypermaturity, and clinical severity of preeclampsia. We conducted a retrospective cohort study in which 48 placentas were selected from pathology archives (hypertensive group). Of these, 6 had chronic hypertension, 15 had mild preeclampsia, 14 had severe preeclampsia, and 13 had hemolysis, elevated liver enzymes, and low platelets syndrome. These were compared with 55 placentas from normotensive patients (control group). One representative section of placental parenchyma from each case was stained with an antibody to vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 and given a score based on extent and intensity of staining, representing expression level. Assignment of staining score was done, blinded to clinical history and pathologic diagnosis. Vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 staining was seen in placental syncytiotrophoblasts and was particularly strong in syncytial knots. There was a positive association between vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 staining score and severity of clinical hypertensive state, small placental size, and villous hypermaturity. The association between vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 score and small placentas did not persist after controlling for hypermaturity. Vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 overexpression in the placenta strongly correlates with both severity of hypertensive disease and villous hypermaturity. The correlation with villous hypermaturity further links hypoxia to vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 production in the placenta.     

血管内皮生长因子受体-1促进肝癌细胞侵袭和转移的初步研究

目的 检测不同转移潜能的肝癌细胞中有无血管内皮生长因子受体-1(VEGFR-1)及其配体(VEGF)的表达以探讨VEGFR-1在肝癌侵袭转移中的作用.方法 分别用RT-PCR、ELISA和/或Western blot法检测四种肝癌细胞株MHCC97-H、MHCC97-L、SMMC 7721和HepG 2有无VEGFR-1 mRNA和蛋白表达,用VEGFR-1的特异性配体VEGF-B处理MHCC97-H细胞以研究VEGFR-1活化对肝癌细胞迁移、侵袭和增殖等的影响.结果 MHCC97-H、MHCC97-L和SMMC 7721有VEGFR-1 mRNA和蛋白表达,但4种肝癌细胞都有VEGFR-1的配体VEGF-A和VEGF-B的表达;VEGFR-1活化能促进肝癌细胞MHCC97-H侵袭和迁移;VEGFR-1依赖的侵袭和迁移能被VEGFR-1的中和抗体18F1阻断,VEGFR-1活化不能促进细胞增殖和存活.结论 VEGFR-1及其配体的表达与肝癌细胞的侵袭转移有关,肝癌中的VEGF过表达可以自分泌的方式激活表达VEGFR-1的肝癌细胞,VEGFR-1及其配体可能是防治原发性肝癌侵袭转移的新靶点.     

Endocrinology: The effects of natural and synthetic sex steroids on human decidual endothelial cell proliferation

The effects of natural and synthetic sex steroids on the proliferationof human decidual capillary endothelial cells in culture wereinvestigated. Oestradiol at 5.0 ng/ml stimulated culture growth.Lower concentrations of oestradiol inhibited growth, while higherconcentrations had no effect. Low concentrations of progesterone(5.0 and 10.0 ng/ml) had no effect on growth, while higher concentrations(20.0 and 40.0 ng/ml) inhibited growth. Combinations of oestradioland progesterone usually reproduced the effects of progesteronealone. 3-Ketodesogestrel and levonorgestrel both inhibited endothelialcell growth, while medroxyprogesterone acetate had no effect.These findings suggest that (i) oestradiol and progesteronehave direct effects on endothelial cells and are probably involvedin the cyclical growth and regression of endometrial blood vesselsin vivo, and (ii) the actions of certain progestogen-only contraceptivesmay have direct effects on endometrial blood vessels.     

Selective cell proliferation can be controlled with CPC particle coatings

To develop implantable, engineered, cartilage constructs supported by a scaffold, techniques to encourage rapid tissue growth into, and on the scaffold are essential. Preliminary studies indicated that human endothelial cells proliferated at different rates on different calcium phosphate ceramic (CPC) particles. Judicious selection of particles may encourage specific cell proliferation, leading to an ordered growth of tissues for angiogenesis, osteogenesis, and chondrogenesis. The goal of this study was to identify CPC surfaces that encourage bone and vascular cell growth, and other surfaces that support chondrocyte growth while inhibiting proliferation of vascular cells. Differences in bone and vascular cell proliferation were observed when using epoxy without embedded CPCs to encourage bone cells, and when three CPCs were tested, which encouraged vascular cell proliferation. One of these (CPC 7) also substantially depressed cartilage cell proliferation. Only one small-diameter crystalline CPC (CPC 2) supported rapid chondrocyte proliferation, and maintained the cartilage cell phenotype.     

Selective endothelial cell attachment to peptide-modified terpolymers

In a previous report we screened a combinatorial peptide library to identify novel ligands that bind with high affinity and specificity to human blood outgrowth endothelial cells (HBOEC). In this study we demonstrate the use of the phage display-selected-HBOEC-specific peptides as a tool to direct and modulate endothelial cell (EC) behavior with a focus on designing functional biomaterials intended for use in cardiovascular applications. First, we ensured that our peptide ligands did not interfere with EC function as tested by proliferation, migration, tube formation, and response to vascular endothelial growth factor. Second, peptides that supported EC function were incorporated into methacrylic terpolymers via chain transfer free radical polymerization. The HBOEC-specific peptide, TPSLEQRTVYAK, when covalently coupled to a terpolymer matrix, retained binding affinity towards HBOEC in a serum-free medium. Under the same binding conditions, the attachment of human umbilical vein endothelial cells (HUVEC) was limited, thus establishing HBOEC specificity. To our knowledge, this is the first report demonstrating specificity in binding to peptide-modified biomaterials of mature EC, i.e., HUVEC, and EC of progenitor origin such as HBOEC. The findings from this work could facilitate the development of autologous cell therapies with which to treat cardiovascular disease.     

Selective human endothelial cell activation by chemokines as a guide to cell homing

An original model of organo-specific, immortalized and stabilized endothelial cell lines was used to delineate the part played by some chemokines (CCL21, CX3CL1, CCL5 and CXCL12) and their receptors in endothelium organo-specificity. Chemokine receptor expression and chemokine presentation were investigated on organo-specific human endothelial cell lines. Although the chemokines showed distinct binding patterns for the various endothelial cell lines, these were not correlated with the expression of the corresponding receptors (CX3CR1, CXCR4, CCR5 and CCR7). Experiments with CCL21 on peripheral lymph node endothelial cells demonstrated that the chemokine did not co-localize with its receptor but was associated with extracellular matrix components. The specific activity of chemokines was clearly shown to be related to the endothelial cell origin. Indeed, CX3CL1 and CCL21 promoted lymphocyte recruitment by endothelial cells from the appendix and peripheral lymph nodes, respectively, while CX3CL1 pro-angiogenic activity was restricted to endothelial cells from the appendix and skin. The high specificity of the chemokine/endothelium interaction allowed the design of a direct in vitro endothelial cell targeting assay. This unique cellular model demonstrated a fundamental role for chemokines in conferring on the endothelium its organo-specificity and its potential for tissue targeting through the selective binding, presentation and activation properties of chemokines.     

fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C. elegans as revealed by a nonbiased synthetic screen

We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans. Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23. We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels.     

Vascular endothelial growth factor receptor-2-mediated mitogenesis is negatively regulated by vascular endothelial growth factor receptor-1 in tumor epithelial cells

Vascular endothelial growth factor (VEGF) receptors are present on nonendothelial cells suggesting that VEGF may mediate nonendothelial effects during organogenesis and tumorigenesis. Here we show that VEGF receptor-1 (VEGFR-1) negatively regulates VEGFR-2-mediated proliferation via nitric oxide (NO) in an epithelial cancer cell line ECV304. Cell proliferation was assessed by [(3)H]thymidine incorporation, fluorescent-activated cell-sorting analysis, and cell number using a Coulter Counter. Total NO generated by the action of nitric oxide synthase was measured by Seivers NOA 280 Nitric Oxide Chemiluminescence Analyser. VEGF (1 ng/ml) stimulated DNA synthesis and increased ECV304 cell number in a manner that was inhibited by a neutralizing anti-VEGFR-2 antibody. In contrast, VEGF (50 ng/ml) stimulated NO release in a manner that was inhibited by functionally neutralizing anti-VEGFR-1 antibody. Blockage of the VEGFR-1 receptor signal with anti-VEGFR-1 stimulated DNA synthesis and increased cell number. Cell-cycle analysis showed that inhibition of VEGFR-1 increased the transition from G(1) to S phase whereas inhibition of VEGFR-2 blocked the VEGF-mediated transition from G(1) to S phase. Finally, the addition of NO donors suppressed both VEGF-mediated proliferation and the increase in growth after blockade of VEGFR-1. Conversely, inhibition of VEGF mediated NO release by nitric oxide synthase inhibitor, L-monomethyl-L-arginine, restored the mitogenic effect of VEGF. These findings identify a dose-dependent reciprocal regulatory mechanism for VEGF via its two receptors. It shows that VEGFR-1 induces cell cytostasis via NO and as such is a suitable target for molecular strategies suppressing tumorigenesis.     

Cinematographic analysis of endothelial cell proliferation in culture

Summary A method is described for the application of time-lapse cinematographic techniques (TLC) to analysis of the behavior of individual cells and clones in endothelial cultures. Using TLC, detailed and precise information on cell cycle time, proliferative state, and migration activity can be obtained on several hundred individual cells in a given filmed sequence of proliferating endothelial cultures.     

不同剪切力对内皮细胞表面凝集素样氧化低密度脂蛋白受体1表达的影响

目的 研究不同剪切力对内皮细胞表面凝集素样氧化低密度脂蛋白受体1(LOX-1)表达的影响.方法 体外培养人脐静脉内皮细胞,将其种植于载玻片上,放入流室系统分别施加以不同的剪切力.按施加剪切力的不同分为实验组和对照组:实验组分为低剪切力组(4.2 dyne/cm2,n=5)、中等剪切力组(8.4 dyne/cm2,n=5)和高剪切力组(15.0 dyne/cm2,n=5);对照组(n=5)静态条件下培养,不施加剪切力.剪切力加载1、2、4、8 h后RT-PCR测定各组内皮细胞LOX-1 mRNA的表达.结果 低剪切力(4.2 dyne/cm2)作用下,内皮细胞LOX-1表达随时间延长而增加.与对照组比较,中等剪切力(8.4dyne/cm2)作用2、4、8 h内皮细胞LOX-1的表达水平均降低[2 h:0.2346±0.0397比0.2948±0.0128,4 h:0.1747±0.0585比0.2985±0.0204,8 h:0.0256±0.0067比0.2968±0.0175,均P＜0.05].高剪切力(15.0dyne/cm2)作用下,血管内皮细胞LOX-1表达一直维持在极低水平.结论 不同剪切力可以影响内皮细胞的LOX-1表达水平.     

Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells

Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.     

Extracellular matrix tenascin-X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation

BACKGROUND: An extracellular matrix tenascin-X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels. The precise in vivo role of TNX remains to be elucidated. To identify proteins that interact with TNX in the extracellular environment, we searched for TNX-binding proteins using a yeast two-hybrid system. RESULTS: We used mouse TNX-specific fibronectin type III repeats (mTNX/FNIII13-25) as a bait for the screening. We found that vascular endothelial growth factor B (VEGF-B) binds to mTNX/FNIII13-25. This interaction was confirmed by pull-down assays and co-immunoprecipitation assays. The full-length mTNX, as well as mTNX/FNIII13-25, interacted with both alternative splice isoforms VEGF-B186 and VEGF-B167. Furthermore, the full-length mTNX also bound to VEGF-A. The minimal region of TNX that interacts with VEGF-B was mapped to the FNIII repeats (FNIII13-25) but not to the other characteristic domains of TNX. The TNX-binding site of VEGF-B was located in the N-terminal 115-amino acid region. mTNX/FNIII13-25 did not prevent the interaction of VEGF-B with VEGFR-1 (VEGF receptor 1), and VEGF-B could simultaneously bind to both mTNX/FNIII13-25 and VEGFR-1. A conditioned medium from transfected 293T cells coexpressing full-length TNX and VEGF-B could promote DNA synthesis in bovine endothelial cells in which VEGFR-1 were expressed. VEGFR-1 phosphorylation triggered by VEGF-B186 were increased in cells plated with mTNX/FNIII13-25 or full-length mTNX, compared with cells plated with VEGF-B186 alone. CONCLUSION: TNX interacts with VEGF-B and enhances the ability of VEGF-B to stimulate cell proliferation. This enhanced mitogenecity is caused by increased signals mediated by the VEGFR-1 receptor. This finding suggests a role for TNX in the regulation of the development of blood vessels such as vasculogenesis and angiogenesis.     

可溶性血管内皮生长因子受体1对动脉粥样硬化斑块的作用

BACKGROUND: Angiogenesis in atherosclerotic plaque occurred on the basis of atherosclerotic lesions, and the new formed blood vessels promoted the development of angiogenesis. Soluble vascular endothelial growth factor receptor 1 (sFlt-1) gene transfection reduces neointimal formation after vascular injury in rabbits, also reduces early vascular inflammation and proliferation, and the formation of neointima lately.     

Endometrial endothelial cell proliferation during the menstrual cycle

Adult endometrium is a tissue in which physiological angiogenesisoccurs regularly and provides an accessible source of materialfor study. Endometrial biopsies and immunohistochemistry wereused to test two hypotheses: firstly, that there are peaks ofendothelial cell proliferation in human endometrium during themenstrual cycle, and secondly that in-vitro endothelial cellmigratory signal production by human endometrium (measured ina previous study) accompanies endothelial cell proliferativeactivity in vivo. Proliferating cells were identified usinganti-proliferating cell nuclear antigen, and endothelial cellswere identified using anti-CD34. The method was validated bya smaller, separate study using bromodeoxyuridine incorporationand detection with formalin-fixed biopsies, and comparison withproliferating cell nuclear antigen staining. The main study(n=50) showed that significant peaks of endometrial endothelialcell proliferation could not be identified, due to the largevariability in endothelial cell proliferative activity betweenindividuals at the same stage of the menstrual cycle. Endometrialendothelial cell migratory signal production did not correlatewith endothelial cell proliferation (n = 27). Results suggestthat blood vessel growth in the endometrium may occur by a processthat differs from the traditional concept of angiogenesis, whichhas been derived mainly from in-vitro or in-vivo experimentalstudies     

Inhibition of T cell proliferation by antibodies to synthetic peptides

While T cell proliferation to antigen in the presence of antigen-presenting cells is well known to be readily inhibited by antibodies directed against Class II major histocompatibility complex (MHC) (Ia/HLA-DR) products, it has not been possible to inhibit proliferation by antibodies directed against the antigen. Because of the implications of these observations for targets of T cell recognition, this phenomenon was reinvestigated using human T cell clones, recognizing a small (24 amino acid) synthetic peptide (termed p20) derived from the influenza hemagglutinin-1 molecule. It was found that proliferation of clones to p20 was inhibited efficiently (less than 90%), using p20 as antigen, and rabbit anti-p20. Inhibition was possible either by coculturing p20 antigen and antibody to p20 with cloned T cells and antigen-presenting (E-) cells, or by pulsing antigen-presenting cells with antigen prior to a brief incubation with antibody before washing the E- cells and using them to stimulate cloned T cells. These results do not indicate why previous attempts had failed, but in view of the different techniques available now (cloned T cells, small synthetic polypeptides, and antibody raised against polypeptide) we investigated the influence of these parameters. It was found that, using cloned T cells, the form of the antigen was of importance, as antibody inhibition of the response to hemagglutinin or whole influenza A was much less apparent. These differences were interpreted as being due to greater access of anti-p20 to p20 than to hemagglutinin or influenza. If uncloned T cell lines were used, inhibition was also much harder to detect. This was interpreted as masking of inhibition of the response of some clones in the line by interleukin 2-induced recruitment.     

An assay for macrophage-mediated regulation of endothelial cell proliferation

We have developed an assay that quantifies the potential of macrophages to regulate proliferation of endothelial cells. We show that young mice macrophages can be distinguished from old mice macrophages by their ability to inhibit vascular endothelial cell proliferation. While young mice macrophages robustly inhibit proliferation, old mice macrophages fail to do so and actually promote the proliferation of endothelial cells. In this report, we outline a technique that directly assesses the effect of macrophages on modulation of endothelial cell proliferation. This assay will help us in understanding the mechanisms of macrophage function in several disease states characterized by abnormal angiogenesis including cancers, angiogenic eye disease and atherosclerotic heart disease.