Detlev Drenckhahn

Pericyte involvement in capillary sprouting during angiogenesis in situ

SummaryTo investigate the participation of microvascular pericytes in the process of capillary sprouting, we examined whole-mount preparations of the rat mesentery by use of a double immunofluorescence approach. Angiogenesis was induced by intraperitoneal injections of either the mast cell-degranulating substance compound 48/80 or tumor cell-conditioned medium. Capillary sprouts were visualized by staining with rhodaminconjugated phalloidin and pericytes were simultaneosly stained by an antibody to the intermediate filament protein desmin. Developing pericytes were negative for the smooth-muscle isoform of α-actin, bbut were clearly reactive for desmin. Pericytes appear to be involved in the carliest stages of capillary sprouting. Pericytes were regularly found lying at and in front of the advancing tips of endothelial sprouts. At many sites pericytes were seen to bridge the gap between the leading edges of opposing endothelial sprouts, which were apparently preparing to merge, suggesting that pericytic processes may serve as guiding structures aiding outgrowth of endothelial cells.

Differential effects of hydrocortisone and TNFα on tight junction proteins in an in vitro model of the human blood-brain barrier

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood–brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE‐cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration of glucocorticoids induced expression of the targets occludin 2.75 ± 0.04‐fold and claudin‐5 up to 2.32 ± 0.11‐fold, which is likely to contribute to the more than threefold enhancement of transendothelial electrical resistance reflecting barrier tightness. Our analyses further provide direct evidence that the GC hydrocortisone prevents endothelial barrier breakdown in response to pro‐inflammatory stimuli (TNFα administration), which could be demonstrated to be partly based on maintenance of occludin levels. Our studies strongly suggest stabilization of BBB function as a mode of GC action on a molecular level in the human brain vasculature.

Occludin as direct target for glucocorticoid-induced improvement of blood–brain barrier properties in a murine in vitro system

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood–brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm2. Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand‐dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

Inhibition of Rho A activity causes pemphigus skin blistering

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV–immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)–mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG–triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice

Previous experiments using cultured endothelial monolayers indicate that Rho‐family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin‐based contractility and junctional distribution of the Ca2+‐dependent endothelial cell adhesion molecule, VE‐cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng ml−1) inactivated 50–90 % of all endothelial Rho proteins within 60–90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F‐actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE‐cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y‐27632 (30 μm) for 90–120 min induced F‐actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE‐cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (Lp) but did not attenuate the transient increase in Lp of microvessels exposed to bradykinin. Perfusion of microvessels with Y‐27632 (30 μm) for up to 100 min reduced basal Lp but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B‐mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA‐mediated contractile mechanisms are not involved in bradykinin‐ or PAF‐induced hyperpermeability of intact microvessels.

Pemphigus foliaceus IgG causes dissociation of desmoglein 1–containing junctions without blocking desmoglein 1 transinteraction

Autoantibodies against the epidermal desmosomal cadherins desmoglein 1 (Dsg1) and Dsg3 have been shown to cause severe to lethal skin blistering clinically defined as pemphigus foliaceus (PF) and pemphigus vulgaris (PV). It is unknown whether antibody-induced dissociation of keratinocytes is caused by direct inhibition of Dsg1 transinteraction or by secondary cellular responses. Here we show in an in vitro system that IgGs purified from PF patient sera caused cellular dissociation of cultured human keratinocytes as well as significant release of Dsg1-coated microbeads attached to Dsg-containing sites on the keratinocyte cellular surface. However, cell dissociation and bead release induced by PF-IgGs was not caused by direct steric hindrance of Dsg1 transinteraction, as demonstrated by single molecule atomic force measurements and by laser trapping of surface-bound Dsg1-coated microbeads. Rather, our experiments strongly indicate that PF-IgG-mediated dissociation events must involve autoantibody-triggered cellular signaling pathways, resulting in destabilization of Dsg1-based adhesive sites and desmosomes.

Ischemia-induced phosphorylation and translocation of stress protein αB-crystallin to Z lines of myocardium

It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein αB-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of αB-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of αB-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemiareperfusion injury was accompanied by a rapid shift of a fraction of αB-crystallin to a more acidic isoelectric point. This shift is caused by αB-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic αB-crystallin form with the phosphorylated B1 form of lenticular αB-crystallin. In view of the chaperone-like function of αB-crystallin in conjunction with its high level of constitutive expression in the myocardium (1-2% of soluble protein content), we consider αB-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.

Identification of the taste cell G-protein, α-gustducin, in brush cells of the rat pancreatic duct system

Abstract The major pancreatic excretory ducts have been shown to contain a large number of specialized epithelial cells, named brush cells, that are characterized by an apical tuft of stiff microvilli. The function of pancreatic brush cells is unknown. Because of some structural similarities to taste receptor cells of the tongue, we addressed the question whether pancreatic brush cells contain the taste cell-specific GTP-binding protein, α-gustducin, and hence might be considered to be involved in intraductal chemoreception. By immunostaining, we show that ductal brush cells of the rat pancreatic duct system contain α-gustducin, which is concentrated in the apical tuft of microvilli and is also found along the basolateral cell surface. A further outcome of this study is that brush cells are concentrated in the terminal portions of extralobular ducts and in the major pancreatic duct where brush cells comprise up to 22% of the ductal epithelium. Immunoblotting of the major pancreatic duct revealed a 42-kDa band that comigrated with α-gustducin of the rat tongue. In view of our previous observation that the ductal brush cells are particularly rich in nitric oxide synthase-I, there is reason to assume that these cells might play a role in certain aspects of chemoreceptive signalling. Thus, chemosensory control of pancreatic secretion might occur at two independent sites, the intestine and the terminal portions of the excretory duct system.

Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific

Endothelial cells (ECs) from different vascular beds display certain common qualities, but each subtype is uniquely adapted to meet the demands of the underlying tissues. The structural peculiarities of intercellular junctions are, for instance, considered to account for the differences in permeability displayed by various vascular beds: strong occludin expression is unique to cerebral ECs and considered to account for the high electrical resistance and low paracellular permeability of brain microvessels which constitute the blood–brain barrier (BBB). The integrity of the BBB is compromised in many disorders of the human CNS; therapeutic strategies include treatment with glucocorticoids (GCs), which improve barrier properties of the BBB. In contrast, positive effects of GCs on peripheral vascular permeability could not be demonstrated clearly, while side‐effects of prolonged GC treatment are considerable. In an effort to elucidate this difference, we analysed the expression of occludin and the glucocorticoid receptor (GR) in BBB and non‐BBB (myocardium) endothelial cells. Our results demonstrate complete GR downregulation by GCs in murine non‐BBB endothelial cells in vivo, whereas GC administration led to nuclear concentration of GRs in BBB endothelium. In correlation with these in vivo data, the use of cerebral and myocardial endothelial cell lines proved GR downregulation in non‐BBB cells in vitro in response to GC treatment. Divergent transactivating activity of GRs in the BBB and non‐BBB endothelial cellular context could be demonstrated after transfection of endothelial cells with a model GC‐responsive test promoter plasmid in the presence and absence of dexamethasone. Our results thus suggest differential signalling mechanisms involved in endothelial barrier regulation, arguing for the development of tissue‐specific drugs for therapeutic applications.

A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.