Collagen Gel Culture Research Articles

Identification of ETFB as a candidate protein that participates in the mechanoregulation of fibroblast cell number in collagen gel culture

Fibroblast activation is strongly influenced by mechanical environment in the wound-healing process, especially in fibrosis. Mechanically stressed three-dimensional collagen embedded culture is a useful model representing fibroblasts in morphological as well as biochemical situations encountered during fibrosis. To find key proteins involved in reducing the number of fibroblasts during mechanical stress, we performed two-dimensional gel electrophoresis (2DE)-based differential display and siRNA-based functional screening with collagen gel culture focusing on the differences between attached and detached culture environments. Membrane extracts of fibroblasts from 1 day of attached or detached cultures were subjected to 2DE. We compared protein expression levels and identified the attached-culture-dominant proteins by MALDI-TOF-MS. Next, fibroblasts were transfected with siRNA and embedded in collagen gel. Cell number was counted after 3 days in culture. Eight attached culture dominant proteins were identified with MALDI-TOF-MS. Transfection of siRNA against these proteins demonstrated that electron transfer flavoprotein β subunit (ETFB)-specific siRNA reduced the cell number in the attached culture without a decrease in the detached culture. ETFB participates in the mechanoregulation of fibroblast cell number in collagen gel culture.

Hepatocytic differentiation of rhesus monkey embryonic stem cells promoted by collagen gels and growth factors

rES (rhesus monkey embryonic stem) cells have similar characteristics to human ES (embryonic stem) cells, and might be useful as a substitute model for preclinical research. Before their clinical application, it is critical to understand the roles of factors that control the differentiation of ES cells into hepatocytes. Here, we analysed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of DE (definitive endoderm) cells were generated from rES cells after being treated with activin A. The DE cells were then plated on to collagen gels or type I collagen-coated wells with growth factors to induce hepatocyte differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed, including the expression of immature hepatic genes and the generation of 15±3% AFP (alpha fetoprotein)/CK (cytokeratin)18 double-positive cells. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver-specific genes. The mRNA expression of AFP (immature hepatic gene) was detected at day 11 but decreased at day 18. In contrast, mRNA expression of albumin (mature hepatic gene) was detected at day 11 and increased at day 18. Compared with type I collagen systems, significantly higher AFP/CK18 double-positive cells (68±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES.

Culture Models for Studying Thyroid Biology and Disorders

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

Slit‐Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5-E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Abstract 3360: Epithelial-mesenchymal transition (EMT) stimulates tumor cells to produce invadopodia-like protrusions in collagen matrix, suppressing cell growth

Abstract Epithelial tumor cells often change their epithelial phenotype to mesenchymal one when they invade into surrounding stromal tissue. This change, called epithelial-mesenchymal transition (EMT), is thought to be a crucial event in tumor progression. EMT is known to be regulated by environmental factors. However, it is unclear how EMT contributes to invasive growth of tumor cells. To address this question, we characterized EMT-induced tumor cells under various conditions in vitro. EMT was induced in three human carcinoma cell lines of the lung, pancreas and stomach by treating with TGF-beta and/or TNF-alpha. The EMT treatment of these cell lines induced expression of the invasion marker laminin gamma2 chain and MT1-MMP, in addition to other well-known phenotypes such as the morphological change and vimentin expression. EMT induction enhanced cell motility in monolayer culture. Although EMT cells showed comparable cell growth with the control cells in monolayer culture, their growth rates were extremely suppressed in collagen gel and soft agar culture conditions. Most characteristically, EMT-induced tumor cells commonly and markedly extended invadopodia-like protrusions in collagen gel. Such protrusion formation in collagen gel was effectively blocked by the TGF-beta/Smad pathway inhibitor SB431542 but scarcely suppressed by the synthetic and natural MMP inhibitors, TAPI-1 and TIMP-2, respectively. These data suggest that EMT may enhance interstitial tumor infiltration without cell growth by promoting prominent invadopodia formation and cell motility. The TGF-beta-induced cytoskeletal change seems to play a more critical role than MMP expression in the tumor invasion. 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 3360. doi:10.1158/1538-7445.AM2011-3360

Adult human CD133/1 + kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin + and CD133/1 + cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1 + cells. Isolated CD133/1 + papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1 + progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Simvastatin as a Modulator of Tissue Remodeling through Inhibition of Matrix Metalloproteinase (MMP) Release from Human Lung Fibroblasts

Methods: This study, using an in-vitro model (three-dimensional collagen gel contraction system), evaluated the effect of cytokines (tumor necrosis factor-α, TNF-a and interleukin-1β, IL-1b) on the MMP release and MMP activation from human lung fibroblasts. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Results: In three-dimensional collagen gel cultures (3D cultures) where cytokines (TNF-a and IL-1b) can induce the production of MMPs by fibroblasts, it was found that simvastatin inhibited MMP release. In 3D cultures, cytokines together with NE induced collagen degradation and can lead to activation of the MMP, which was inhibited by simvastatin. Conclusion: Simvastatin may play a role in regulating human lung fibroblast functions in repair and remodeling processes by inhibiting MMP release and the conversion from the latent to the active form of MMP.

Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.

Alignment of Astrocytes Increases Neuronal Growth in Three-Dimensional Collagen Gels and Is Maintained Following Plastic Compression to Form a Spinal Cord Repair Conduit

After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transglutaminase

We aimed to develop cell-embedded gel-filled macroporous scaffold (CGS) culture technology as a fundamental technology for the construction of large-scale tissue-like structures, which will be indispensable for practical liver tissue engineering. Tissue transglutaminase (tTGase) from guinea pig liver showed no cytotoxicity toward primary hepatocytes, and hepatocyte-embedded gelatin gel cultures could be realized. The albumin production activities in tTGase-mediated hepatocyte-embedded gelatin gel cultures were similar to those in collagen gel cultures, representing an established hepatocyte culture method for the expression of liver-specific functions. Therefore, it was expected that a hepatocyte-embedded gelatin gel culture system enabling high liver-specific function expression could be created. The CGS culture system was created by incubating a suspension of hepatocytes and tTGase-containing gelatin-filled hydrophilic-treated scaffolds at 37 °C. The albumin production activities in CGS with poly( l-lactic acid) macroporous scaffold (porosity = 68.4%) were inferior to those in tTGase–gelatin gel cultures because of a diffusion problem. However, the activities were similar between CGS with macroporous polyurethane foam (porosity = 98.8%) and tTGase–gelatin gel cultures, even under stationary conditions. On the other hand, further functional improvements of hepatocytes were achieved in HGF- or HGF/heparin-containing gel cultures. Based on these results, tTGase-mediated CGS cultures are expected to become a fundamental technology for the creation of engineered liver tissues.

Mevalonate-Ras Homology (Rho)/Rho-Associated Coiled-Coil-Forming Protein Kinase (ROCK)-Mediated Signaling Pathway as a Therapeutic Target for the Treatment of Endometriosis-Associated Fibrosis

a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Histologically, this disease is characterized by endometrial gland and stroma with surrounding dense fibrous tissue. During the development of endometriotic lesions, excess fibrosis may lead to scarring and to altera- tion of the tissue function. It has been suggested that type I collagen is a major contributor to endometriosis-associated fibrosis. One approach to understanding the pathogenesis of endometriosis is to investigate the mechanisms underlying fibrogenesis associated with this disease. Using three-dimensional collagen gel culture model, we have evaluated the extracellular matrix contractility and myofibroblastic differentiation of endometriotic stromal cells. Endometriotic stromal cells showed enhanced extracellular matrix contractility in comparison with normal endometrial stromal cells. Activation of the Ras homology (Rho)/Rho- associated coiled-coil-forming protein kinase (ROCK)-mediated signaling pathway with simultaneously enhanced myofi- broblastic differentiation is involved in this mechanism. We have proposed the mevalonate-Rho/ROCK-mediated signal- ing pathways as possible therapeutic targets for the treatment of endometriosis-associated fibrosis. This paper is a review of the recent information concerning the mechanism of endometriosis-associated fibrosis, focusing on the contractility of endometriotic stromal cells. The modulators of mevalonate-Rho/ROCK signaling pathways were also evaluated as promising agents for the treatment and prevention of endometriosis-associated fibrosis.

Abstract 1375: Amlexanox inhibits FGF-mediated paracrine and autocrine activity of tumor-derived melanoma cell lines and cell strains

Abstract Melanoma is a highly invasive disease characterized by widespread lymphatic and systemic metastases. Although angiogenesis is critical for this to occur, the responsible mechanisms within melanoma cells remain unclear. The purpose of this study is to identify factors secreted by tumor-derived melanoma cells which can mediate these processes. Serum-free conditioned medium (CM) isolated from two melanoma cell lines and four newly derived melanoma cell strains promote both the growth in culture and angiogenic outgrowth activity of HUVEC and HMEC1 in 3-dimensional (3-D) collagen gel culture. Substantial amounts of both FGF1 (acidic) and FGF2 (basic) are found in the conditioned medium (CM). The addition of neutralizing antibodies to FGF1 (5 μg/ml) and FGF2 (3 μg/ml) to the melanoma CM significantly decreased the proliferation of HUVEC and HMEC1 by (20%) and (40%), respectively, and the angiogenic activity of HMEC1 by (20%). Amlexanox (AMX) is an anti-inflammatory agent that binds intracellular S100A13, one component of the putative FGF1-secretion complexes. AMX suppresses constitutive release of FGF1, but not FGF2, from these melanoma cells. AMX-treated melanoma-CM results in significant reduction of: HUVEC mitogenic activity, endothelial migration into 3-D collagen gel as well as melanoma cell proliferation. These AMX-mediated effects correlate with a concomitant decrease of FGFR1 phosphorylation in melanoma cells. The sum of these results point to the importance of the FGF-1 pathway and nominate AMX as a significant anti-melanoma agent by virtue of its impact on both paracrine and autocrine activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1375.

Organotypic culture of human bone marrow adipose tissue

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was <0.8 ng/mL under all culture conditions. Dexamethasone promoted adiponectin gene expression, while insulin inhibited it. This finding suggests that dexamethasone, but not insulin, may serve as a powerful adipogenic factor for BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 microg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 microg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 microg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.

Creation of a long-lifespan ciliated epithelial tissue structure using a 3D collagen scaffold

We describe a method of using a 3D collagen gel scaffold applied at the air–liquid interface to culture dissociated primary tracheal-bronchial ciliated cells into a ciliated epithelial tissue structure (CETS). This 3D collagen gel culture system enables the induction of ciliogenesis and continuously provides support, maintenance, development, differentiation and propagation for the growth of cilia into the CETS. The CETS developed by this system resembles the ciliary metachronal motility and morphological, histological and physiopharmacological characteristics of cells found in native and in vivo ciliated epithelia. The CETS can be sustained for months with a straightforward and simple maintenance protocol. The integrity of the functional ciliary activity of this CETS enables the evaluation of long-term effects of many pulmonary drug candidates without using animals.

Comparison of two different methods for rat adrenocortical cells culture

Objective To investigate a novel 3-dimensional cell culture model in collagen gel, and its influence on morphology and function of adrenocortical cells. Methods Adrenocortical cells of ne-onate rats were isolated,and then cultured in self-made collagen type Ⅰ gel for 14 days. This 3-dimension-al cell culture model was compared with the classic plane culture model on the morphology, cell growth rate,protein expression and secretory functions. Results Adrenocortical cells expressed specific cyto-chrome P450 :CYP11 B1 and CYP11 B2 under both culture models. At the 8th day after primary culture,the growth rate of adrenocortical cells in 3-dimensional cell culture model was higher than the classic plane culture model significantly. At the 14th day, the growth rate in 3-dimensional cell culture model and the classic plane culture model was 16.6% and 2.9% respectively. The secretory functions of adrenocortical cells were more sustainable in 3-dimensional collagen gel. At the 14th day, corticosterone and aldosterone levels in the medium of 3-dimensional cell culture model were (278.33 ± 38.20) μg/L and (133.85 ±38.20) ng/L,while those were (160.35±28.12) μg/L and (74.03 ± 12.12) ng/L in the medium of plane euhure model, with the differences being statistically significant (P ＜ 0.05). Conclusion Three-dimensional culture in collagen gel is favorable for the maintenance of adrenocortical ceils functions. Key words: Collagen Type Ⅰ ; Adrenocortical cells; Culture

Statins inhibit matrix metalloproteinase release from human lung fibroblasts

Pleiotropic effects of statins have been reported to include inhibition of matrix metalloproteinase (MMP) release from macrophages and endothelial cells. We evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodelling processes. Monolayer and three-dimensional (3D) collagen gel cultures of fibroblasts were used. Cytokines (tumour necrosis factor-alpha and interleukin-1alpha) were used to induce MMP release and mRNA expression. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Atorvastatin inhibited MMP-1 and -3 release and mRNA expression in both culture systems. Similar results were obtained with simvastatin and fluvastatin. In 3D cultures where cytokines also stimulated MMP-9 release, atorvastatin also inhibited MMP-9 release. In 3D cultures, cytokines together with NE induced collagen degradation, which was also inhibited by atorvastatin. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate. The current data suggest that statins may modulate remodelling processes mediated by fibroblasts by inhibiting MMP release.

Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms

The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that gamma-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-kappaB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model

Background:The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7).Methods:Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10 μg ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture.Results:MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 μg ml–1 mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3 μg ml–1), which caused neuron death equivalent to other cell types.Conclusion:Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Vascular regression and survival are differentially regulated by MT1-MMP and TIMPs in the aortic ring model of angiogenesis

This study was designed to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in the reabsorption of neovessels in collagen gel cultures of rat and mouse aortic rings. Aortic angiogenesis was associated with collagen lysis and production of the matrix-degrading enzymes MMP-2, MMP-9, and membrane-type MMP (MT1-MMP, or MMP-14). Vascular growth and regression were not affected by disruption of MMP-2 or MMP-9. In addition, no effect on vascular regression was observed by blocking plasmin, a protease implicated in the activation of MMPs, with epsilon-aminocaproic acid or by adding plasminogen, which caused a modest increase in vascular proliferation. Conversely, angiogenesis was blocked and vessels stabilized by inhibiting MT1-MMP with neutralizing antibodies, TIMP-2, TIMP-3, or TIMP-4. TIMP-1, which blocks MMP-2 and MMP-9 but is a poor inhibitor of MT1-MMP, had no antiangiogenic effect. However, TIMP-1 prolonged the survival of neovessels following angiogenesis. Vascular regression was accelerated in aortic cultures from TIMP-1- and TIMP-2-deficient mice. The vascular survival effect of anti-MT1-MMP antibodies and TIMPs with MT1-MMP inhibitory activity was associated with complete inhibition of collagen lysis. In contrast, TIMP-1 had no anticollagenolytic effect. These results indicate that MT1-MMP plays a critical role not only in angiogenesis but also in vascular regression and demonstrate that TIMPs with anti-MT1-MMP activity have opposite effects on angiogenic outcomes depending on the stage of the angiogenic process. This study also suggests the existence of a TIMP-1-mediated alternate pathway of vascular survival that is unrelated to MT1-MMP inhibitory activity.