Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.

Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Brucine suppresses breast cancer metastasis via inhibiting epithelial mesenchymal transition and matrix metalloproteinases expressions.

OBJECTIVE: To examine the effect of brucine on the migration, invasion, adhesion and expressions of epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) in the highly metastatic breast cancer cell lines MDA-MB-231 and Hs578-T. METHODS: MDA-MB-231 and Hs578-T cells were divided to 4 groups: the control group (0.1% DMSO), and 25, 50 and 100 µmol/L brucine groups. The cell viability was determined using a CellTiter-Glo® luminescent cell viability. The scratch wound healing assay and tanswell migration assay were used to determine the migration ability of these cells treated by different concentrations of brucine. The proliferation rate, invasive potential and adhesive ability were respectively performed by colony formation assay, transwell invasion assay and adhension assay. The protein and mRNA expressions of EMT biomarkers, MMP-2 and MMP-9 were investigated by real-time reverse transcription polymerase chain reaction and Western blot. RESULTS: Compared with the control group, brucine had little effect on cell viability or proliferation (P>0.05), but led to a dose-dependent decrease on migration, invasion, adhension of MDA-MB-231 and Hs578-T cells (P<0.01). Furthermore, brucine increased the protein and mRNA levels of EMT markers such as E-cadherin and ß-catenin in MDA-MB-231 and Hs578-T cells, and decreased the protein and mRNA levels of mesenychmal markers such as vimentin and fibronectin, as well as the expressions of MMP-2 and MMP-9 (all P<0.01). CONCLUSION: Brucine inhibited triple negative breast cancer cells metastasis potentially through EMT reversion and MMP-2 and MMP-9 inhibition.

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function.

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1ß and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

Pro-angiogenic activity of notoginsenoside R1 in human umbilical vein endothelial cells in vitro and in a chemical-induced blood vessel loss model of zebrafish in vivo.

OBJECTIVE: This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. METHODS: The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. RESULTS: R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. CONCLUSION: R1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

Effects of elevated magnesium and substrate on neuronal numbers and neurite outgrowth of neural stem/progenitor cells in vitro.

Because a potential treatment for brain injuries could be elevating magnesium ions (Mg(2+)) intracerebrally, we characterized the effects of elevating external Mg(2+) in cultures of neonatal murine brain-derived neural stem/progenitor cells (NSCs). Using a crystal violet assay, which avoids interference of Mg(2+) in the assay, it was determined that substrate influenced Mg(2+) effects on cell numbers. On uncoated plastic, elevating Mg(2+) levels to between 2.5 and 10mM above basal increased NSC numbers, and at higher concentrations numbers decreased to control or lower levels. Similar biphasic curves were observed with different plating densities, treatment durations and length of time in culture. When cells were plated on laminin-coated plastic, NSC numbers were higher even in basal medium and no further effects were observed with Mg(2+). NSC differentiation into neurons was not altered by either substrate or Mg(2+) supplementation. Some parameters of neurite outgrowth were increased by elevated Mg(2+) when NSCs differentiated into neurons on uncoated plastic. Differentiation on laminin resulted in increased neurites even in basal medium and no further effects were seen when Mg(2+) was elevated. This system can now be used to study the multiple mechanisms by which Mg(2+) influences neuronal biology.

Alumina-zirconia composites functionalized with laminin-1 and laminin-5 for dentistry: effect of protein adsorption on cellular response.

The present paper describes a study on laminin interaction with the surface of two alumina-zirconia composites with different percentages of ZrO2, both with submicrometric grain size. As major molecules within the basement membrane (BM), laminins are important protein fragments for epithelial cell adhesion and migration. On the other hand, alumina-zirconia composites are very attractive materials for dental applications due to their esthetic and mechanical properties. X-Ray photoelectron spectroscopy and atomic force microscopy were used to study the adsorption of two types of laminin, laminin-1 (Ln-1) and laminin-5 (Ln-5), onto the ceramics surfaces. The in vitro cell response was determined by intracellular phosphorylation of major kinases. Ceramics samples functionalized with laminins showed better cellular activation than untreated specimens; furthermore, cellular activation was found to be greater for the composite with higher percentage in zirconia when functionalized with Ln-5, whereas the adsorption of Ln-1 resulted in a greater activation for the alumina-rich oxide.

Xiongshao capsule promotes angiogenesis of HUVEC via enhancing cell proliferation and up-regulating the expression of bFGF and VEGF.

OBJECTIVE: To evaluate the angiogenic effect of the Xiongshao capsule (XSC) in human umbilical vein endothelial cells (HUVEC), and to investigate the possible molecular mechanisms mediating its biological effect. METHODS: Serum pharmacology was applied in this study, in which different doses of XSC were administrated to rats orally and then XSC-containing serum (XSC-S) was collected for the following in vitro experiments. The viability of HUVEC was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell density was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with propidium iodide staining was performed to determine cell cycle phase. Cell migration was determined by wound-healing method. Capillary tube formation by HUVEC was examined using ECMatrix gel-based assay. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression levels were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) analyses. RESULTS: XSC-S dose-dependently stimulated proliferation of HUVEC by promoting the cell cycle G1 to S progression. In addition, XSC-S treatment dramatically increased the migration and capillary tube formation of HUVEC in a dose-dependent manner. Moreover, XSC-S enhanced the expression of VEGF and bFGF at both mRNA and protein levels. CONCLUSION: XSC can promote several features of angiogenesis in endothelial cells through up-regulating the expression of bFGF and VEGF, suggesting that XSC may be a potential novel therapeutic agent for the treatment of ischemic heart diseases.

A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line.

Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.

Effects of extracellular matrix molecules on the growth properties of oligodendrocyte progenitor cells in vitro.

The extracellular matrix (ECM) is a component of neural cell niches and regulates multiple functions of diverse cell types. To date, limited information is available concerning its biological effects on the growth properties of oligodendrocyte progenitor cells (OPCs). In the present study, we examined effects of several ECM components, i.e., fibronectin, laminin, and Matrigel, on the survival, proliferation, migration, process extension, and purity of OPCs isolated from embryonic day 15 rat spinal cords. All three ECM components enhanced these biological properties of the OPCs compared with a non-ECM substrate, poly-D-lysine. However, the extents of their effects were somewhat different. Among these ECMs, fibronectin showed the strongest effect on almost all aspects of the growth properties of OPCs, implying that this molecule is a better substrate for the growth of OPCs in vitro. Because of its survival- and growth-promoting effects on OPCs, fibronectin may be considered as a candidate substrate for enhancing OPC-mediated repair under conditions when exogenous delivery or endogenous stimulation of OPCs is applied.

BDNF and extracellular matrix regulate differentiation of mice neurosphere-derived cells into a GABAergic neuronal phenotype.

Differentiation of neurosphere-derived cells is regulated by extracellular cues, namely, growth factors and proteins of the extracellular matrix (ECM). In this study we analyzed the influence of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), retinoic acid plus potassium chloride (RA-KCl), and the nonsynthetic ECMs laminin (LN) and fibronectin (FN) versus the synthetic adhesion substrate poly-L-lysine (PLL) in the in vitro differentiation of postnatal neurosphere cells. BDNF increased the number of differentiated neurons and decreased the number of neuronal precursors (nestin-positive cells) compared with NGF or RA-KCl. Moreover, cells treated with BDNF plus B27 supplement acquired a gamma-aminobutyric acid (GABA)-ergic phenotype and showed increased survival. No significant differences were found in the number of differentiated neurons in the presence of the ECMs alone. Nevertheless, FN or PLL in combination with BDNF promoted the acquisition of a GABAergic phenotype. The results obtained in this study highlight the importance of growth factors and ECM proteins for the potential of neurosphere cells to differentiate into neurons.

A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA. hy926 endothelial cell line

Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.

[Programmed application of extracellular matrix promotes neural differentiation of mouse embryonic stem cells].

OBJECTIVE: To study the role of extracellular matrix (ECM) in neural differentiation of mouse embryonic stem cells (ESCs). METHODS: Mouse ESCs were incubated in the ESC conditioned medium, and the formation of embryonic bodies (EBs) were induced in bacteriological dishes using high-concentration all-trans retinoic acid (RA). The EBs were seeded on different matrixes (gelatin, fibronectin, and laminin/poly-L-ornithine) to test their impact on neural differentiation of the ESCs using immunofluorescence assay. The effect of laminin/poly-L-ornithine on the growth of neurites was evaluated with fluorescence microscopy. RESULTS: High-concentration RA activated and accelerated the differentiation of ESCs toward nestin-positive neural progenitor cells. Fibronectin supplement in the matrix dose-dependently promoted ESC differentiation into neural progenitor cells, while laminin/poly-L-ornithine increased the growth of the neurites and induced the maturation of the differentiated neural cells. CONCLUSION: ECM plays an important role in neural differentiation of mouse ESCs, and application of FN produces the most conspicuous effect during the differentiation of the ESCs into the neural progenitor cells;laminin/poly-L-ornithine is the most effective during their differentiation into neural cells.

[Effect of huangqi and dangshen extraction on angiogenesis in Matrigel implant of mice].

AIM: To study the inhibitory effect of huangqi and dangshen extraction (SQ) on angiogenesis induced by b-FGF. METHODS: Matrigel implant assay was used. Matrigel(500 microL) containing b-FGF and heparin was injected subcutaneously into the abdomens of mice and harvested 5 d later. The amount of hemoglobin and micro-vascular area present in the implant were measured and compared. The mice were given different dosage of SQ (experimental group) or the same volume of glucose (vehicle group) once a day by intraperitoneal injection. Inhibitory experiment started 3 d before Matrigel implant and continued until the end of study. RESULTS: SQ in lower dosage (< or = 50% V/V) increased hemoglobin content and micro-vascular area in Matrigel implant while SQ in higher dosage (> or = 60%, V/V) reduced hemoglobin content and micro-vascular area in Matrigel implant. The effect of enhance ment and inhibition was in a limited concentration-effect manner. CONCLUSION: SQ in different dosage has different effects on angiogenesis. We should use different dosage in different purpose.

Laminin-1-derived scrambled peptide AG73T disaggregates laminin-1-induced ovarian cancer cell spheroids and improves the efficacy of cisplatin.

We found previously that the laminin-1-derived synthetic peptide AG73 (LQVQLSIR) promoted ovarian cancer cell metastasis in vivo. We have now studied the role of this metastasis-promoting peptide in vitro using TAC3 ovarian cancer cells, which display anchorage-independent growth and form multicellular spheroids. Our goal is to better understand how this peptide can regulate metastasis in vivo. We found that the exogenous addition of either laminin-1 or peptide AG73 stimulated the formation and growth of the spheroids. Western blot analysis indicated that laminin-1 enhanced the expression of integrin beta1, and that AG73 peptide enhanced expression of syndecan-1 and downstream effectors, including mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase (ERK), and also phosphatidylinositol (PI)-3 kinase/AKT activity signaling. The soluble peptide AG73T, which is a scramble peptide of AG73, was able to disaggregate the laminin-1-induced spheroids. Furthermore, the disaggregated cells were twice as sensitive to cisplatin as the intact spheroids. The AG73T peptide in the presence of laminin-1 suppressed expression of integrin beta1 and its downstream effectors, including MAPK/ERK and PI3/AKT activity signaling. The MEK inhibitor U0126 reduced TAC3 cell growth more effectively in the presence of both laminin-1 and AG73T than in the presence of laminin-1 alone. Inhibition of the PI3-K cascade with LY294002 was also more effective in the presence of laminin-1 and AG73T. The increased sensitivity to cisplatin in the presence of AG73T may be due to the greater bioavailability of the drug to the free-floating cells over the spheroids. These findings suggest a novel function of AG73T in ovarian cancer and help to define mechanisms important in ovarian cancer spheroid formation and spread.