Basement Membrane Preparation Research Articles

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

Prognostic impact of kallikrein-related peptidase transcript levels in prostate cancer.

We aimed to study mRNA levels and prognostic impact of all 15 human kallikrein-related peptidases (KLKs) and their targets, proteinase-activated receptors (PARs), in surgically treated prostate cancer (PCa). Seventy-nine patients with localized grade group 2-4 PCas represented aggressive cases, based on metastatic progression during median follow-up of 11 years. Eighty-six patients with similar baseline characteristics, but no metastasis during follow-up, were assigned as controls. Transcript counts were detected with nCounter technology. KLK12 protein expression was investigated with immunohistochemistry. The effects of KLK12 and KLK15 were studied in LNCaP cells using RNA interference. KLK3, -2, -4, -11, -15, -10 and -12 mRNA, in decreasing order, were expressed over limit of detection (LOD). The expression of KLK2, -3, -4 and -15 was decreased and KLK12 increased in aggressive cancers, compared to controls (P < .05). Low KLK2, -3 and -15 expression was associated with short metastasis-free survival (P < .05) in Kaplan-Meier analysis. PAR1 and -2 were expressed over LOD, and PAR1 expression was higher, and PAR2 lower, in aggressive cases than controls. Together, KLKs and PARs improved classification of metastatic and lethal disease over grade, pathological stage and prostate-specific antigen combined, in random forest analyses. Strong KLK12 immunohistochemical staining was associated with short metastasis-free and PCa-specific survival in Kaplan-Meier analysis (P < .05). Knock-down of KLK15 reduced colony formation of LNCaP cells grown on Matrigel basement membrane preparation. These results support the involvement of several KLKs in PCa progression, highlighting, that they may serve as prognostic PCa biomarkers.

Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation.

BackgroundVascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr‐1 (early growth response‐1). Egr‐1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type.Methods and ResultsA CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr‐1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr ‐1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr‐1, or Egr‐1 deletion, perturbed endothelial cell proliferation in models of cell counting or real‐time growth using the xCELLigence System. We found that Ser26 mutation or Egr‐1 deletion ameliorated endothelial cell migration toward VEGF‐A165 (vascular endothelial growth factor‐A) in a dual‐chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr‐1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr‐1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr‐1 deletion increased VE‐cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis.ConclusionsThese findings not only indicate that Egr‐1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr‐1. This highlights the importance of Ser26 in Egr‐1 for a range of proangiogenic processes.

Collagen type-IV Langmuir and Langmuir–Schäfer layers as model biointerfaces to direct stem cell adhesion

In biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement Membranes (BM), a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network-like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air–water (A–W) interface. Transferring these layers onto cell culture substrates using the Langmuir–Schäfer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A–W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mN m−1. Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mN m−1 onto poly(ethylene terephthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the areal Col-IV density by LS transfer at 10, 15 and 20 mN m−1 on PET is shown. The LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.

Fracture Healing Effects of Locally-Administered Adipose Tissue-Derived Cells.

PurposeAlthough the applications of adipose tissue-derived cells (ADCs) in regenerative medicine have been investigated, the role of ADCs in fracture healing remains unclear. In this study, we examined the fracture-healing effects and survival of transplanted ADCs using micro-computed tomography (CT) and bioluminescence imaging (BLI).Materials and MethodsLuciferase-expressing ADCs were suspended in solubilized basement membrane preparation (SBMP) and xenografted on defects in the right femur of nude mice (n=5). SBMP alone was grafted on a defect in the contralateral femur. Serial in vivo micro-CT and BLI were performed for 20 days. Ex vivo BLI images of both femurs were obtained. Differences in the Hounsfield unit (HU), HUratio, and luciferase activities were compared using Wilcoxon signed-rank tests and non-parametric longitudinal analyses (p<0.05).ResultsIn vivo BLI revealed a signal drop on day 2, reconstitution on day 5, and continuous decrement thereafter. Ex vivo BLI revealed residual activity in the ADC-implanted and adjacent areas. No activity was detected in the contralateral femur. The overall increment rate of normalized HUs was higher for ADC-treated femurs than for SBMP-treated femurs. Cell migration to distant injury sites was not detected.ConclusionEnhanced bone density in the implant area suggests that ADCs have fracture-healing effects.

Abstract 4066: Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells

Abstract Glycodelin is a lipocalin protein mainly expressed in well-differentiated epithelial cells in reproductive tissues. Previously, glycodelin has been shown to induce cell differentiation in endometrial and breast cancer cells. We have used glycodelin-transfected MCF-7 breast cancer cells to study the glycodelin-induced differentiation. Glycodelin expression changed the cellular morphology towards normal, less malignant direction. In addition, glycodelin-producing cells formed significantly smaller tumors in xenograft mice. We have found that the effects of glycodelin are at least partly mediated by repressed PKCδ activity, rendering the cells resistant to activation of phorbol 12-myristate 13-acetate (PMA), a known activator of PKCδ. Here we continued these previous studies by addressing the effect of glycodelin on the invasion and migration of MCF-7 breast cancer cells, and studying the affected signaling route in more detail. Activation of signaling proteins was detected with an antibody array of 43 phospho kinases. Cell migration was studied using wound healing test. The cells were grown on chamber slides until they formed a monolayer and then a scratch was made using a pipet tip. After addition of PMA or DMSO control, the migration of the cells was monitored under microscope. In some of the control cells, PKCδ was down-regulated with RNAi prior the migration assay. For invasion through Matrigel basement membrane preparation, a Boyden chamber assay was used. The cells were grown with and without PMA and the amount of invasive cells was quantified. Phospho-kinase array did not reveal any widespread changes in the amount of phosphorylated signaling molecules between the glycodelin-producing and control cells irrespective of the PMA treatment. However, in addition to PKCδ some changes were found in the levels of phosphoproteins such as p38α and p27. In wound healing test PMA caused a two-fold increase in the migration of the control cells, but had no effect on the glycodelin-producing cells. When the expression of PKCδ was blocked in the control cells using siRNA, the addition of PMA did not increase the migration of the cells. PMA also increased the invasiveness of the cells, but that was detected in both cells clones regardless of glycodelin expression. Without PMA treatment both glycodelin-expressing and control cells behaved similarly in wound healing and invasion tests. In conclusion, glycodelin abolished PMA-induced migration in MCF-7 breast cancer cells, while it did not have any effect on the invasion of the cells. Our results suggest that the effect of glycodelin in migration is mediated by reduced activation of PKCδ. Citation Format: Laura C. Hautala, Riitta Koistinen, Hannu Koistinen. Glycodelin abolishes PMA-induced migration of MCF-7 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2014-4066

Matrigel induces L-plastin expression and promotes L-plastin-dependent invasion in human cholangiocarcinoma cells.

The function of the extracellular matrix (ECM) in the tumor microenvironment is not limited to forming a barrier against tumor invasion. As demonstrated in pathological specimens, cholangiocarcinoma samples exhibit an enrichment of the ECM surrounding the tumor cells. In this study, we examined involvement of the ECM in the regulation of the invasiveness of cholangiocarcinoma cells. The RMCCA1 cholangiocarcinoma cell line was cultured in culture plates either with or without a coating of reconstituted ECM basement membrane preparation (BD Matrigel matrix). In vitro invasion assays were then performed. In addition, the protein expression profile of the cell line was examined using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. The proteins expressed and their functional associations with cancer progression were determined. Culturing the RMCCA1 cell line in the BD Matrigel matrix induced cell invasion. Numerous proteins were induced by culturing the RMCCA1 cells in the matrix gel. The expression of L-plastin, an actin-binding protein, was significantly upregulated. The knockdown of L-plastin expression by siRNA silencing significantly suppressed the cellular response to matrix gel-stimulated cancer cell invasion. The ECM promotes the invasiveness of cholangiocarcinoma cells by upregulating L-plastin. These findings suggest the potential exploitation of this mechanism as a means of inhibiting the invasiveness of cholangiocarcinoma cells.

Abstract 3265: PMA induces malignant characteristics in MCF-7 breast cancer cells via MAPK-pathway

Abstract Phorbol 12-myristate 13-acetate (PMA) is a well known tumor promoter, the effect of which is mainly mediated by protein kinase C (PKC). The effect of PMA varies depending on the cell line and growth conditions. In MCF-7 breast cancer cells PMA has been found to induce growth arrest and resistance to apoptosis. Here, we studied the effects of PMA on differentiation and malignant characteristics of MCF-7 cells. The cells were grown on Matrigel besement membrane preparation or on plastic/glass surface with and without PMA, rottlerin or bryostatin. The cell proliferation assay was carried out using the cells grown on 96-well plates. Wound healing test, on glass surface, was used to detect the differences in cell migration. Activation of MAPK-pathway proteins was studied with Phospho-kinase array. When grown on Matrigel, the addition of PMA to cell culture medium increased the motility of the cells and induced dramatic morphological changes, leading to the formation of web-like multicellular structures. These changes were not observed when the cells were grown on plastic or glass surface. However, PMA caused a two-fold increase in the migration of the cells on glass surface. Furthermore, the PMA treated cells had lower proliferation rate than the untreated cells. Preincubation of the cells with PKC-inhibitors rottlerin or bryostatin abolished the effects of PMA, suggesting that these effects are mediated by PKC. PMA also induced several changes in the phosphorylation of different proteins involved in MAPK-pathway. Our results confirm the previous findings showing that PMA induces malignant characteristics in MCF-7 breast cancer cells. Morphological differentiation was only seen when the cells were grown on basement membrane preparation, recapitulating the physiological growth environment of the cells better than the artificial plastic surface. Furthermore, our results indicate that the effects of PMA on MCF-7 cells are mediated by MAPK-pathway. 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 3265. doi:1538-7445.AM2012-3265

Abstract 3537: Manipulation of local and systemic immune suppression by GPI-anchored immune stimulatory proteins

Abstract Each day in the United States alone, approximately 3400 people are diagnosed while another 1500 people die from cancer. Breast cancer is the most common form of cancer among females and accounts for the second leading cause of death among female cancer patients. In order to be effective in a therapeutic setting, current immunotherapies must be able to overcome the multiple layers of tumor-induced immune suppression such as modulation of MHC expression and costimulatory molecules (CD80/CD86), expression of inhibitory molecules PDL-1 and GITRL and the secretion of immunosuppressive factors such as VEGF and TGF-β into the tumor microenvironment. These immunosuppressive factors promote the recruitment, differentiation and expansion of regulatory immune cells such as myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the tumor microenvironment. Using the 4T07 murine breast cancer model we investigated the effects of expressing GPI-anchored immune stimulatory molecules (GPI-ISMs), namely cytokines (IL-2, IL-12) and the costimulatory protein B7-1, on the surface of the tumor cells. BALB/c mice were challenged subcutaneously (s.c.) with either wild-type 4T07 cells (4T07-WT) or 4T07 cells expressing GPI-ISMs. We observed significant splenomegaly in the mice challenged with 4T07-WT cells relative to the mice challenged with 4T07 cells expressing GPI-ISMs. This observed splenomegaly correlated with tumor size and a 4-5 fold increase in the percentage of splenic CD11b+Gr1+ MDSCs indicating the role of active immune suppression in the tumorigenicity of 4T07 breast cancer cells. We then conducted studies to analyze the effect of GPI-ISMs on infiltrating cells into the tumor microenvironment as well as in the spleen and draining lymph nodes (dLNs). Three groups of mice were challenged (s.c.) with the following cells mixed in a 1:1 ratio with BD Matrigel™ (a solubilized basement membrane preparation derived from a mouse sarcoma): 4T07-WT, 4T07-B7/IL-12 or PBS (control). Seven days post challenge, the Matrigel/tumor, spleen and dLNs were harvested from the mice, digested and analyzed for cellular infiltrates by flow cytometry. We observed that the expression of GPI-ISMs on the surface of tumor cells led to reduced angiogenesis as evidenced by a reduced level of blood vessels and decreased presence of CD4+CD25+FOXP3+ regulatory T cells and CD11b+Gr1+ MDSCs locally at the tumor site and dLNs as well as systemically in the spleen. Additionally, there was a decrease in CD8+PD1+ exhausted T cells at the tumor site. Along with the inhibition of immune suppressive cell populations, the GPI-ISMs increased the presence of CD4+ and CD8+ T cells as well as dendritic cells and B cells. These observations suggest that components of the active immune suppression evident in this model can be inhibited by expressing GPI-ISMs on the surface of the 4T07 tumor cells and could be effective in a therapeutic setting. 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 3537. doi:1538-7445.AM2012-3537

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice

Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

Abstract 1501: Prostate-specific antigen cleaves nidogen-1

Abstract Prostate-specific antigen (PSA or kallikrein-related peptidase 3, KLK3) is a chymotrypsin-like serine protease expressed in the prostate and secreted into seminal fluid. Several studies have demonstrated that PSA exerts anti-angiogenic activity in vitro and in vivo. Using a human umbilical vein endothelial cell (HUVEC) tube formation model, where the cells were grown on Matrigel™ basement membrane preparation, we recently showed that the enzymatic activity of PSA is essential for its anti-angiogenic function. Previously, PSA has been shown to cleave components of extracellular matrix (ECM), e.g., laminin and fibronectin, as well as unidentified proteins in Matrigel™. Proteolytic products of some ECM proteins, such as collagen XVIII-fragment endostatin, are known to be anti-angiogenic. Cleavage of ECM is also crucial for cancer cell invasion. We studied the PSA-generated protein fragments derived from Matrigel™ and aimed to characterize their anti-angiogenic activity. PSA was purified from seminal fluid and incubated with Matrigel™ for 24 h at 37°C. The resulting proteolytic fragments were analyzed by SDS-PAGE and mass spectrometry. Recombinant human nidogen-1 (also known as entactin) was incubated with PSA for 18 h at 37°C and the fragments were analyzed by SDS-PAGE followed by silver staining. The effect of nidogen-1 fragments was studied in the HUVEC tube formation model. We found that PSA cleaves nidogen-1 in Matrigel™. This was confirmed by using recombinant nidogen-1. The nidogen-1 fragments, however, did not have any anti-angiogenic effect on HUVECs in the tube formation model. We conclude that the cleavage of nidogen-1 by PSA may lead to disruption of basement membrane structure and, thus, may inhibit HUVEC tube formation as well as facilitate cancer cell invasion. The nidogen-1 fragments per se do not mediate the anti-angiogenic function of PSA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1501. doi:10.1158/1538-7445.AM2011-1501

Extracellular Trypanosoma cruzi calreticulin in the host–parasite interplay

Calreticulin (CRT) from vertebrates is a calcium-binding protein present mainly in the endoplasmic reticulum (ER). There, it directs the conformation of proteins and controls calcium levels. This review will focus on several extracellular roles of Trypanosoma cruzi CRT (TcCRT) in relation to its capacity to inhibit the complement system, mediate parasite infectivity, interfere with angiogenesis and, as a possible consequence, with tumor growth. The TcCRT antiangiogenic effect parallels with the capacity of T. cruzi infection to inhibit tumor development in vivo. Thus, the TcCRT, complement, and endothelial cell interactions seem to be an evolutionary adaptation to promote prolonged parasite-host relationships.

The soluble form of urokinase receptor promotes angiogenesis through its Ser⁸⁸-Arg-Ser-Arg-Tyr⁹² chemotactic sequence.

The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² internal sequence. To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² sequence. In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr⁹² chemotactic sequence, the maximal effect being reached at 10 nmol L⁻¹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) β₃ vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² chemotactic sequence.

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Advantages and difficulties in culturing human pluripotent stem cells in growth factor-defined serum-free medium

Axon Regeneration through Scaffold into Distal Spinal Cord after Transection

We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0–15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 μm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30–50 μm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1–10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.

Axon Regeneration Through Scaffold Into Distal Spinal Cord After Transection

We employed Fast Blue (FB) axonal tracing to determine the origin of regenerating axons after thoracic spinal cord transection injury in rats. Schwann cell (SC)-loaded, biodegradable, poly(lactic-co-glycolic acid) (PLGA) scaffolds were implanted after transection. Scaffolds loaded with solubilized basement membrane preparation (without SCs) were used for negative controls, and nontransected cords were positive controls. One or 2 months after injury and scaffold implantation, FB was injected 0-15 mm caudal or about 5 mm rostral to the scaffold. One week later, tissue was harvested and the scaffold and cord sectioned longitudinally (30 microm) on a cryostat. Trans-scaffold labeling of neuron cell bodies was identified with confocal microscopy in all cell-transplanted groups. Large (30-50 microm diameter) neuron cell bodies were predominantly labeled in the ventral horn region. Most labeled neurons were seen 1-10 mm rostral to the scaffold, although some neurons were also labeled in the cervical cord. Axonal growth occurred bidirectionally after cord transection, and axons regenerated up to 14 mm beyond the PLGA scaffolds and into distal cord. The extent of FB labeling was negatively correlated with distance from the injection site to the scaffold. Electron microscopy showed myelinated axons in the transverse sections of the implanted scaffold 2 months after implantation. The pattern of myelination, with extracellular collagen and basal lamina, was characteristic of SC myelination. Our results show that FB labeling is an effective way to measure the origin of regenerating axons.

Junctional Adhesion Molecule: An Expression in Human Endometrial Carcinoma

Junctional adhesion molecule A (JAM-A) is involved in cell-cell contact and tight junction formation. Loss of cell adhesion molecules may be associated with high histologic grade and invasiveness of endometrial carcinoma. We attempted to determine JAM-A expression in human endometrial carcinoma and its correlations with pathologic features, stage, and survival. Junctional adhesion molecule A expression in human endometrial carcinoma was evaluated by immunohistochemistry. In addition, we cultured human well and poorly differentiated endometrial adenocarcinoma cell lines, Ishikawa cells, and KLE in 3-dimensional basement membrane preparation, and JAM-A expression in these cells was assessed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Junctional adhesion molecule A immunostaining intensity was negatively correlated with histologic grade (tau = -0.420, P < 0.0001), myometrial invasion (tau = -0.306, P < 0.01), and stage (tau = -0.383, P < 0.0001). Low JAM-A immunostaining intensity was associated with positive vascular space involvement (P < 0.01). Moreover, low immunostain intensity was significantly (P < 0.0001) related to low overall survival rate and progression-free survival rate. Additionally, in our 3-dimensional epithelial cell culture, JAM-A expression in poorly differentiated adenocarcinoma was significantly lower than that in well-differentiated adenocarcinoma (P < 0.001). Junctional adhesion molecule A expression seems to be reduced in high-grade or advanced endometrial carcinoma and may be a prognostic factor.

Novel Egg White–based 3-D Cell Culture System

Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.

Formation of Cardiac Fibers in Matrigel Matrix

We report a simple in vitro model of cardiac tissue that mimics three-dimensional (3-D) environment and mechanical load conditions and, as such, may serve as a convenient method to study stem cell engraftment or address developmental questions such as cytoskeleton or intercalated disk maturation. To create in vitro cardiac fibers we used Matrigel, a commercially available basement membrane preparation. A semisolid pillow from concentrated Matrigel was overlaid with a suspension of rat neonatal cardiomyocytes in a diluted Matrigel solution. This created an environment in which the multicellular fibers continuously contracted against a mechanical load. The described approach allows continuous structural and functional monitoring of 20-300-micron-thick cardiac fibers and provides easy access to epitopes for immunostaining purposes.