Short- and long-term cultivation of embryonic and neonatal murine keratinocytes

Studies using cultured cells allow one to dissect complex cellular mechanisms in greater detail than when studying living organisms alone. However, before cultured cells can deliver meaningful results they must accurately represent the in vivo situation. Over the last three to four decades considerable effort has been devoted to the development of culture media which improve in vitro growth and modeling accuracy. In contrast to earlier large-scale, non-specific screening of factors, in recent years the development of such media has relied increasingly on a deeper understanding of the cell's biology and the selection of growth factors to specifically activate known biological processes. These new media now enable equal or better cell isolation and growth, using significantly simpler and less labor-intensive methodologies. Here we describe a simple method to isolate and cultivate epidermal keratinocytes from embryonic or neonatal skin on uncoated plastic using a medium specifically designed to retain epidermal keratinocyte progenitors in an undifferentiated state for improved isolation and proliferation and an alternative medium to support terminal differentiation.

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions

Vascular endothelial growth factor (VEGF) has potent angiogenic and neuroprotective effects in the ischemic brain. Its effect on axonal plasticity and neurological recovery in the post-acute stroke phase was unknown. Using behavioral tests combined with anterograde tract tracing studies and with immunohistochemical and molecular biological experiments, we examined effects of a delayed i.c.v. delivery of recombinant human VEGF(165), starting 3 days after stroke, on functional neurological recovery, corticorubral plasticity and inflammatory brain responses in mice submitted to 30 min of middle cerebral artery occlusion. We herein show that the slowly progressive functional improvements of motor grip strength and coordination, which are induced by VEGF, are accompanied by enhanced sprouting of contralesional corticorubral fibres that branched off the pyramidal tract in order to cross the midline and innervate the ipsilesional parvocellular red nucleus. Infiltrates of CD45+ leukocytes were noticed in the ischemic striatum of vehicle-treated mice that closely corresponded to areas exhibiting Iba-1+ activated microglia. VEGF attenuated the CD45+ leukocyte infiltrates at 14 but not 30 days post ischemia and diminished the microglial activation. Notably, the VEGF-induced anti-inflammatory effect of VEGF was associated with a downregulation of a broad set of inflammatory cytokines and chemokines in both brain hemispheres. These data suggest a link between VEGF's immunosuppressive and plasticity-promoting actions that may be important for successful brain remodeling. Accordingly, growth factors with anti-inflammatory action may be promising therapeutics in the post-acute stroke phase.

Exogenous gonadotropins have little impact on follicular but considerable effect on serum cytokine concentrations – a comparison between Natural Cycle and stimulated IVF using a multiplexed assay platform

Introduction: Throughout follicular growth and subsequent corpus luteum formation the leukocyte number increases and follicular vascularisation changes. These processes are enhanced under exogenous stimulation with gonadotropins. Cytokines released by leukocytes contribute to further recruitment and vascularisation of the follicle, and they play an important role in regulating ovarian steroidogenesis by influencing theca and granulosa–lutein cell function. Changes in cytokine and vascular endothelial growth factor (VEGF) concentrations in the ovary as a consequence of gonadotropin stimulation may negatively influence oocyte quality. In this project we have compared the intrafollicular production of inflammatory cytokines and growth factors between natural IVF cycles (NC) and classical, gonadotropin-stimulated IVF cycles (gsIVF). Material and Methods: Serum on the day of oocyte retrieval and follicular fluid (FF) were collected in 37 NC and 39 gsIVF cycles. Thirteen women within this population underwent one NC and one gsIVF cycle each. A total of 14 cytokines from Bio-Plex panels I and II were determined in matched serum and FF samples using Luminex xMAP technology on the Bio-Plex(R) platform, using the serum protocol. Results: Tumour necrosis factor-alpha, RANTES, eotaxin and interferon-gamma-induced protein-10 levels were lower in FF than in serum, and thus not further investigated. Interleukin (IL)-6, -8, -10, -15, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median concentrations in FF than in serum, indicating possible ovarian production. Moreover, most of these showed higher evels in the gsIVF than in the NC groups in the serum, but not in the follicular fluid. IL-8 was reduced in gsIVF cycles. Conclusion: The fact that serum but not FF levels of the studied cytokines were higher in the stimulated than in the natural cycles can be attributed to the increased number of active follicles present after controlled ovarian stimulation.

Translation from research to applications

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering-the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

Mechanisms of airway obliteration after lung transplantation

Post-transplant bronchiolitis obliterans, also called bronchiolitis obliterans syndrome, affects up to 50-60% of patients who survive 5 yr after surgery according to its clinical definition, which is based on the degree of obstructive airway disease. Alloimmune-independent and -dependent mechanisms produce injuries and inflammation of epithelial cells and subepithelial structures, leading to aberrant tissue repair. The triggering of innate immunity by various infections or chemical injuries after, for example, gastroesophageal reflux, may lead to the release of danger signals that are able to activate dendritic cells, a crucial link with adaptive immunity. Inflammation can also increase the expression and display of major histocompatibility alloantigens and thus favor the initiation of rejection episodes. These phenomena may be limited in time and location or may be protracted. Reducing the risk of alloimmune-independent factors may be as important as treating acute episodes of lung rejection. Excessive immunosuppression may be deleterious by increasing the risk of infection, thereby triggering innate and adaptive immunity. New potential therapeutic targets are emerging from the research performed on leukotriene receptors, chemokine receptors, and growth factors. Neutralizing these molecules reduces the initial mononuclear and polynuclear infiltrates or the subsequent fibroproliferative process and the neovascular changes, feeding this process.

Coronary collateral function long after drug-eluting stent implantation

OBJECTIVES: This study was designed to compare coronary collateral function in patients after bare-metal stent (BMS) or drug-eluting stent (DES) implantation. BACKGROUND: Drug-eluting stents have an inhibitory effect on the production of cytokines, chemotactic proteins, and growth factors, and may therefore negatively affect coronary collateral growth. METHODS: A total of 120 patients with long-term stable coronary artery disease (CAD) after stent implantation were included. Both the BMS group and the DES group comprised 60 patients matched for in-stent stenosis severity of the vessel undergoing collateral flow index (CFI) measurement at follow-up and for the duration of follow-up. The primary end point of the investigation was invasively determined coronary collateral function 6 months after stent implantation. Collateral function was assessed by simultaneous aortic, coronary wedge, and central venous pressure measurements (yielding CFI) and by intracoronary electrocardiogram during balloon occlusion. RESULTS: There were no differences between the groups regarding age, gender, body mass index, frequency of cardiovascular risk factors, use of cardiovascular drugs, severity of CAD, or site of coronary artery stenoses. Despite equal in-stent stenosis severity (46 +/- 34% and 45 +/- 36%) and equal follow-up duration (6.2 +/- 10 months and 6.5 +/- 5.4 months), CFI was diminished in the DES versus BMS group (0.154 +/- 0.097 vs. 0.224 +/- 0.142; p = 0.0049), and the rate of collaterals insufficient to prevent ischemia during occlusion (intracoronary electrocardiographic ST-segment elevation > or =0.1 mV) was higher with 50 of 60 patients in the DES group and 33 of 60 patients in the BMS group (p = 0.001). CONCLUSIONS: Collateral function long after coronary stenting is impaired with DES (sirolimus and paclitaxel) when compared with BMS. Considering the protective nature of collateral vessels, this could lead to more serious cardiac events in the presence of an abrupt coronary occlusion.

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Modulation of signal transduction by vitamin E

The ability of vitamin E to modulate signal transduction and gene expression has been observed in numerous studies; however, the detailed molecular mechanisms involved are often not clear. The eight natural vitamin E analogues and synthetic derivatives affect signal transduction with different potency, possibly reflecting their different ability to interact with specific proteins. Vitamin E modulates the activity of several enzymes involved in signal transduction, such as protein kinase C, protein kinase B, protein tyrosine kinases, 5-, 12-, and 15-lipoxygenases, cyclooxygenase-2, phospholipase A2, protein phosphatase 2A, protein tyrosine phosphatase, and diacylglycerol kinase. Activation of some these enzymes after stimulation of cell surface receptors with growth factors or cytokines can be normalized by vitamin E. At the molecular level, the translocation of several of these enzymes to the plasma membrane is affected by vitamin E, suggesting that the modulation of protein-membrane interactions may be a common theme for vitamin E action. In this review the main effects of vitamin E on enzymes involved in signal transduction are summarized and the possible mechanisms leading to enzyme modulation evaluated. The elucidation of the molecular and cellular events affected by vitamin E could reveal novel strategies and molecular targets for developing similarly acting compounds.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice?

The role of platelets as inflammatory cells is demonstrated by the fact that they can release many growth factors and inflammatory mediators, including chemokines, when they are activated. The best known platelet chemokine family members are platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), which are synthesized in megakaryocytes, stored as preformed proteins in alpha-granules and released from activated platelets. However, platelets also contain many other chemokines such as interleukin-8 (IL-8), growth-regulating oncogene-alpha(GRO-alpha), epithelial neutrophil-activating protein 78 (ENA-78), regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and monocyte chemotactic protein-3 (MCP-3). They also express chemokine receptors such as CCR4, CXCR4, CCR1 and CCR3. Platelet activation is a feature of many inflammatory diseases such as heparin-induced thrombocytopenia, acquired immunodeficiency syndrome, and congestive heart failure. Substantial amounts of PF4, beta-TG and RANTES are released from platelets on activation, which may occur during storage. Although very few data are available on the in vivo effects of transfused chemokines, it has been suggested that the high incidence of adverse reactions often observed after platelet transfusions may be attributed to the chemokines present in the plasma of stored platelet concentrates.