Vera Krump-Konvalinkova

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 after several months of proliferation. Only those cells that were transfected with both plasmids acquired the capacity to grow in vitro without demonstrating major crisis, and these cells have been in culture for 24 months. HPMEC isolated from two different donors were used, generating two populations of immortalized cells, HPMEC-ST1 and HPMEC-ST2. Single cell–derived clones of the immortalized cells HPMEC-ST1 exhibited growth characteristics that were similar to those of the parental HPMEC. One selected clone, HPMEC-ST1.6R, displayed all major constitutively expressed and inducible endothelial phenotypic markers, including platelet endothelial cell adhesion molecule (PECAM-1, CD31), von Willebrand factor (vWF), and the adhesion molecules, intercellular adhesion molecule (ICAM-1), vascular adhesion molecule (VCAM-1), and E-selectin. In addition, an angiogenic response was demonstrated by sprout formation on a biological extracellular matrix (Matrigel). The HPMEC-ST1.6R cells did not form tumors in nude mice. The microvascular endothelial cell line, HPMEC-ST1.6R, will be a valuable tool for the study of microvascular endothelial physiology and pathology including gene expression, angiogenesis, and tumorigenesis.

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 (e.g. metal ions, particulate debris) factors on the regulation of the inflammatory and angiogenic response. A central feature is the use of microvascular endothelial cells (MEC), which can be used in both 2-and 3-dimensional (3-D) assays. We have also used genetic manipulation to develop a permanent MEC line from the human lung (HPMEC-ST1), which is being tested for its suitability to study cell-biomaterial interactions. In addition, suitable in vitro techniques are being developed in order to investigate drug delivery systems (DDS). Of particular interest is the targeting of the central nervous system, our approach being to establish a human model of the blood-brain barrier (BBB). A mainstay of our scientific philosophy is that such in vitro methods can make an important contribution to understanding biological reactions at the tissue-biomaterial interface and thus further a causal approach to tissue engineering (TE) and drug delivery applications.

Stable Knock-Down of the Sphingosine 1-Phosphate Receptor S1P1 Influences Multiple Functions of Human Endothelial Cells

Objectives—Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein–coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. Methods and Results—All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress–mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet–endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-α. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. Conclusions—Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

Experimental approaches to study vascularization in tissue engineering and biomaterial applications

The success of tissue engineering and biomaterial applications is not only dependent on the growth and functioning of the organ- or tissue-specific cells on the biomaterial but is entirely dependent in most cases on a successful vascularization after implantation. The process of vascularization involves angiogenesis; the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. We have established in vitro methods using human microvascular endothelial cells to evaluate novel biomaterials for endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. These in vitro studies can be used to rapidly evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response.

Endothelial Accumulation of Hydroxyethyl Starch and Functional Consequences on Leukocyte-Endothelial Interactions

To date, accumulation of hydroxyethyl starch (HES) has been studied mainly in skin specimens, but there are no detailed reports available regarding starch accumulation in the endothelium. Because endothelial cells play an essential role during shock, we studied the accumulation of HES in human umbilical venous endothelial cells (HUVEC). HUVEC (n = 9) were incubated with a fluorescein-conjugated HES 200/0.5 (FITC-HES) at 0.5–20 mg/ml for 1–72 h. FITC-HES was internalized dose- and time-dependently by pinocytosis into secondary lysosomes. Asymptotic elimination curves showed that 50% of the formerly ingested molecules could not be eliminated. Despite accumulation, starch molecules did not attenuate the expression of E-selectin, ICAM-1 or VCAM-1 on TNF-α-activated HUVEC. However, apart from adhesion molecule expression, perfusion studies showed that HES reduced neutrophil adhesion by direct inhibition of integrin-mediated interactions.

Tumorigenic conversion of endothelial cells

Tumors of endothelial origin develop rarely. Until now, only two angiosarcoma (AS)-derived endothelial cell lines have been be isolated, ISO-HAS and AS-M. Both AS-derived endothelial cell lines presented the typical endothelial characteristics, such as the expression of CD31 and von Willebrand factor, but differed from normal endothelial cells in a nuclear expression of p53, in a delayed angiogenic reaction, and a reduced expression of caveolin. In addition, differences in the expression of cytokines and cell adhesion molecules responsive to proinflammatory stimuli were observed. While AS-M showed an expression pattern similar to that of human umbilical vein endothelial cells (HUVEC), ISO-HAS clearly differed from primary endothelial cells by a constitutive expression of E-selection, granulocyte macrophage colony-stimulating factor, and vascular endothelial cell adhesion molecule-1. Even though the telomeres of both AS-derived established cell lines were longer than telomeres of freshly isolated HUVEC, neither transcripts encoding telomerase nor telomerase activity were detected, suggesting that the tumor cells of endothelial origin may use a telomerase-independent mechanism for maintaining the telomere length. This observation has implications for development of therapeutic approaches for angiosarcoma.

Pathomechanismen der gestörten Wundheilung durch metallische Korrosionsprodukte

Hintergrund. Im Dentalbereich werden seit langer Zeit metallische Werkstoffe unterschiedlicher chemischer Zusammensetzung verwendet. Bei einigen Patienten kommt es nach der Implantatinsertion zu Wundheilungsstörungen. In diesem Artikel werden neben Grundlagen der eng verwandten Entzündungs- und Reparationsvorgänge die Pathomechanismen einer gestörten Wundheilung erörtert. Wundheilungsstörungen. Die Modulation der Wundheilung kann über die Effekte der durch Korrosion freigesetzten Metallionen, aber auch durch die durch Abrieb entstandenen Mikropartikel auf die an der Heilung beteiligten Zelltypen (z. B. Endothelzellen) ausgeübt werden. Modelle. In diesem Zusammenhang werden In-vitro-Modelle vorgestellt, mit deren Hilfe die komplexen Geschehnisse der Werkstoff-Gewebe-Grenzfläche in isolierte Aspekte zerlegt werden können. Darüber hinaus werden neu entwickelte, computergestützte Methoden angesprochen, welche die objektive Quantifizierung von biomaterial- bzw. korrosionsprodukt-induzierten Effekten auf komplexe Vorgänge, z. B. die Angiogenese in vitro, erlauben. Wegen der zentralen Bedeutung der Titanimplantate für Anwendungen in der Mund-, Kiefer- und Gesichtschirurgie werden erste experimentelle Ansätze zur Untersuchung möglicher negativer Auswirkungen vorgestellt. Der Beitrag schließt mit einer Diskussion über die Relevanz solcher Studien für die klinische Implantologie. Background. Metallic materials of variable chemical composition have been used in dental practice for a long time. Complications with respect to tissue healing after insertion of implants are well documented. In this paper we present relevant aspects of the related fields of inflammation and repair processes and focus on the pathomechanisms of this impaired healing response. Modulation of wound healing. This latter process is modulated by specific metal ions released by corrosion activity as well as by wear particles, which influence the function of the participating cell types (e.g. endothelial cells). In vitro models. In this context, in vitro models are presented that permit study of isolated aspects of the complex sequence of events at the biomaterial-tissue interface. Furthermore, newly developed, computer-assisted methods allowing an objective quantification of biomaterial/corrosion product-induced effects on complex processes, such as angiogenesis in vitro, are demonstrated. Because of the central importance of titanium implants in maxillofacial surgery, new experimental approaches to study possible negative effects are presented. Finally, the relevance of such studies for clinical implantology is evaluated.