Apoptosis causes lumen formation during angiogenesis in vitro.

Endoplasmic reticulum‐resident chaperones modulate the inflammatory and angiogenic responses of endothelial cells

SummaryBackground Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. Objectives To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. Methods Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on thei…

High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells

Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shea…

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on postimplantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structura…

Effects of nano-scaled particles on endothelial cell function in vitro: studies on viability, proliferation and inflammation.

Recent studies give support for a connection between the presence of inorganic particles (of microm and nm size) in different organs and tissues and the development of inflammatory foci, called granulomas. As the potential source of particles (e.g. porcelain dental bridges) and the location of particle detection were topographically far apart, a distribution via the blood stream appears highly probable. Thus, endothelial cells, which line the inner surface of blood vessels, would come into direct contact with these particles, making particle-endothelial interactions potentially pathogenically relevant. The objective of this study was to evaluate the effects that five different nano-scaled p…

The role of oxidative stress in pro-inflammatory activation of human endothelial cells on Ti6Al4V alloy

Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjec…

Growth of human cells on polyethersulfone (PES) hollow fiber membranes.

A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 microm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoa…

Molecular basis of endothelial dysfunction in sepsis.

Sepsis is one of the major causes of mortality in critically ill patients and develops as a result of the host response to infection. A complex network of events is set into motion in the body by the infection and results in the pathogenesis of sepsis. This review article focuses on the molecular mechanisms and components involved in the pathogenesis of sepsis with a major emphasis on the endothelium. This includes sepsis-inducing bacterial components (e.g. endotoxins), cellular targets of these molecules and their responses, host reactions, intracellular and cytokine networks, individual susceptibility and new therapeutic targets in sepsis treatment.

Generation of human pulmonary microvascular endothelial cell lines.

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis aft…

Improving cytocompatibility of Co28Cr6Mo by TiO 2 coating: gene expression study in human endothelial cells

Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO 2 ) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol–gel TiO 2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showe…

Vascularization and gene regulation of human endothelial cells growing on porous polyethersulfone (PES) hollow fiber membranes.

Abstract Open-cell hollow fibers made of polyethersulfone (PES) manufactured in the absence of solvents with pore diameters smaller than 100 μm were examined for vascularization by human endothelial cells. The goal of this study was to determine whether the 3-D porous character of the PES surface affected human endothelial cell morphology and functions. Freshly isolated human endothelial cells from the skin (HDMEC), from the lung (HPMEC) and from umbilical cords (HUVEC) and two human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS.c1 were added to PES fibers and cell adherence and growth was followed by confocal laser scanning microscopy. Prior coating of PES with gelatin or fibronectin wa…

Impact of Ceramic and Metallic Nano-scaled Particles on Endothelial Cell Functionsin vitro

The sections in this article are Introduction Origin of Particles in the Human Environment Evidence for Size-dependent Toxicity of Particles Dissemination and Interferences of Nanoparticles within the Body Endothelial Cells and Nanoparticle Exposure Testing of Nanoparticle-induced Effects on Human Endothelial Cells In Vitro Materials and Methods Cell Culture Particles Transmission Electron Microscopy (TEM) Cytotoxicity Assay Detection of Ki67 Expression Quantification of IL-8 Release in Cell Culture Supernatant Quantification of E-selectin Cell Surface Protein Expression Fluorescence Staining Statistical Analysis Results Discussion Particle Internalization Particle Cytotoxicity Pro-inflamma…

The Role of Oxidative Stress in the Response of Endothelial Cells to Metals

The involvement of endothelial cells in inflammation and blood vessel formation (angiogenesis) makes them important for the integration of metal implants. Metal degradation products can, however, influence these processes, possibly leading to ineffective wound healing, prolonged inflammation and eventually aseptic loosening of the implant. Different metal degradation processes have been shown to lead to ROS formation. Oxidative stress, therefore, can mediate the reactions of the human body to the implant. While the response of endothelial cells to oxidative stress has been well studied, the effects of ROS produced as the result of metal degradation have not been addressed as yet. Therefore,…

The effects of metal implants on inflammatory and healing processes

Abstract Metal implants are known for their superior mechanical properties. However, cases of implant failure mainly due to aseptic loosening do occur. The formation of particulate wear debris and corrosion products, such as metal ions and reactive oxygen species, are considered to be crucial factors leading to the failure of metal implants. These metal degradation and corrosion products can induce inflammatory responses, mediated among others by neutrophils, macrophages and endothelial cells. Furthermore, these degradation products may affect blood vessel formation, one of the central processes in wound healing after implantation. Such events can lead to the aseptic loosening of implants c…

Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials

The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. However, the mechanisms involved in the context of the complex healing microenvironment are poorly understood. To evaluate the vascularization potential of bone biomaterials, angiogenic stimuli were added to human dermal microvascular endothelial cells (HDMEC) growing on three-dimensional (3-D) bone biomaterials consisting of porous hydroxyapatite, porous calcium phosphate, porous nickel-titanium, successfully being used in humans, and also silk fibroin nets. HDMEC did not migrate to form microcapillary-like structures as they did on cell culture plastic. In cocultures of HDMEC…

Tissue response and biomaterial integration: the efficacy of in vitro methods

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material…

Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay

AbstractEndotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC)…

Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility

Healing of soft tissue trauma and bone discontinuities following implantation involves acute inflammatory reactions and the formation of blood vessels (angiogenesis). During angiogenesis new capillary vessels arise from the existing vasculature. Endothelial cells (EC) are the major cell type involved in angiogenesis. Corrosion of orthopaedic metallic implant materials (e.g. CoCr alloys) can cause locally high concentrations of heavy metal ions in the peri-implant tissues. Some divalent metal ions (Co2+, Ni2+, Zn2+) lead to the activation of EC in vitro. Upon exposure to these ions. EC release cytokines and chemokines and increase the expression of cell surface adhesion molecules, which repr…

Material‐Induced Cellular Interactions

In vitro methodologies to evaluate biocompatibility: status quo and perspective

Abstract The increasing use of biomaterials in clinical medicine to augment or replace failing organ function has heightened the need to apply relevant test systems to study the safety and efficacy of new medical devices. This becomes all the more important as the field of "tissue engineering" develops, in which the aim is to reconstruct tissue and organ function, for example, by using the patient's own cells seeded on to a three-dimensional (3-D) scaffold structure. In the biomaterial research field, there has been a necessary expansion of the concept of biocompatibility to address not only the biosafety issue, that is, the exclusion of cytotoxic and other deleterious effects of biomateria…

Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells

Abstract We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps bet…

Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering.

We have examined a novel biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons for its ability to support the growth of human cells. Various human cells of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were examined for adherence and growth on the nets by confocal laser microscopy after staining of the cells with calcein-AM and by electron microscopy. All the cells readily adhered and spread over the individual fibers of the nets. Most of the cells were able to grow and survive on the nets for at least 7 weeks and growth not only covered the individual fibers of the net but generally bridged the ga…

In Vitro Expression of the Endothelial Phenotype: Comparative Study of Primary Isolated Cells and Cell Lines, Including the Novel Cell Line HPMEC-ST1.6R

Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed the characteristics defining distinctive endothelial phenotypes in the cell lines EA.hy926, ECV304, EVLC2, HAEND, HMEC-1, ISO-HAS-1 and a cell line recently generated in our laboratory, HPMEC-ST1.6R, and have compared these phenotypes with those found in primary human endothelial cells isolated from umbilical vein (HUVEC), …

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro.

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to sho…

Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro

Particulate matter is associated with different human diseases affecting organs such as the respiratory and cardiovascular systems. Very small particles (nanoparticles) have been shown to be rapidly internalized into the body. Since the sites of internalization and the location of the detected particles are often far apart, a distribution via the blood stream must have occurred. Thus, endothelial cells, which line the inner surface of blood vessels, must have had direct contact with the particles. In this study we tested the effects of metallic nanoparticles (Co and Ni) on oxidative stress and proinflammatory response in human endothelial cells in vitro. Exposure to both nanoparticle types…

Cell culture models of higher complexity in tissue engineering and regenerative medicine.

Cell culture techniques have tended to be used in biomaterial research as a screening method prior to embarking on specific in vivo experimentation. This presentation aims at showing that it is possible to develop more sophisticated in vitro systems using primary human cells in co-culture with other cell types and biomaterials in a three-dimensional setting. While the predictive value of such systems is still not proven these models can be employed to unravel the complexity of biological systems in order to understand molecular mechanisms of cell-cell and cell-material interactions. The brief overview is under the headings of basic principles of relevant culture systems, the study of inflam…

Migration of renal carcinoma cells is dependent on protein kinase Cδ via β1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesi…

In vitro methodologies to evaluate biocompatibility: Status quo and perspectives

Visions for regenerative medicine: interface between scientific fact and science fiction.

This article gives a brief overview of the authors' views on the future development of tissue engineering with respect to the challenges both to the materials and life sciences. Emphasis will be placed on the advantages of three-dimensional bioresorbable polymers in combination with relevant molecular cues and the application of autologous stem or progenitor cells. There is a requirement for much more diversity in the synthesis of so-called "intelligent" materials, which respond to external stimuli, as well as the development of novel drug and gene delivery systems. In addition, much more basic research is necessary in developmental biology and the application of modern cell and molecular b…

Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal…

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tis…

The effect of electrochemically simulated titanium cathodic corrosion products on ROS production and metabolic activity of osteoblasts and monocytes/macrophages.

Nowadays aseptic loosening is the most common cause of orthopaedic implant failure. Some of its reasons have already been described up to now; however, others remain still hypothetical. Besides the inflammatory response to wear particles originating at different sources, the role of reactive oxygen species as products of cellular reactions and/or as a result of the process of corrosion of an implant leading to implant failure has recently been discussed too. In the present study, we used a galvanostatic polarization to simulate the cathodic partial reaction of the corrosion process at a titanium alloy surface. With respect to cells occurring at the interface of a metal implant, the behaviou…

Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines

Adaptation to hypoxia, a universal hallmark of carcinomas, is a critical step for tumor cell survival and growth. One of the principal regulators of hypoxia-responsive pathways is the transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha). Currently, it is known that tumoral production of members of the vascular endothelial growth factor (VEGF)-family (VEGFs) may promote tumor growth and progression by acting on carcinoma cells that express the cognate receptors (VEGFRs). However, the influence of hypoxia in the formation of such a tumoral VEGF/VEGFR loop is not completely understood. In the present study we examined the potential existence of a HIF-1 alpha/VEGF/VEGFR autocrine…

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy.

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H(2)O(2) and compared this with the response of endothelial cells gr…