Nikola Golenhofen

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.

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.

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.

Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells

In endothelial monolayers agonist-induced influx of Ca2+ and activities of the actin cytoskeleton have been shown to be crucially involved in regulation of barrier properties. By laser tweezer application we demonstrated that the strength of adhesion of VE-cadherin-coated microspheres to the surface of cultured endothelial monolayers is significantly reduced by treatment with two well-established permeability-increasing compounds, cytochalasin D and the Ca2+-ionophore A23187, which shows that both compounds directly affect cadherin-mediated adhesion. Cytochalasin D and A23187 caused considerable decay of F-actin (30-60%). Stabilisation of F-actin by jasplakinolide completely blocked drug-induced weakening of bead adhesion showing that attenuation of cadherin-cadherin trans-interaction induced by cytochalasin D and A23187 depends largely on downregulation of F-actin. Single molecule fluorescence microscopy demonstrated that drug-induced weakening of adhesion is accompanied by an increase in lateral mobility of cadherins as well as by dispersal of cadherin-enriched plasmalemmal microdomains. However, the lifetime (≈700 milliseconds, koff≈1.4 second–1) and apparent on-rate of cadherin trans-interaction (relative frequency of binding) remained unchanged in response to cytochalasin D and A23187 indicating that cadherin-mediated adhesion is not modulated by inside-out changes of the affinity but, rather, appears to be controlled by actin-dependent tethering and compartmentalization of cadherins.

Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons.

The so‐called stress response involving up‐regulation of heat shock proteins (Hsps) is a powerful mechanism of cells to deal with harmful conditions to which they are exposed throughout life, such as hyperthermia, hypoxia, or oxidative stress. Some members of the group of small Hsps (sHsps) seem to play a neuroprotective role in the brain. Here we analyzed the expression of all 11 sHsps in the rat brain by using RNA in situ hybridization and quantitative real‐time RT‐PCR. Additionally, we investigated sHsps in cultured neurons exposed to heat shock. We found seven sHsps to be expressed in the rat brain, with HspB5 (αB‐crystallin), HspB6 (Hsp20), and HspB11 (Hsp16.2) showing the highest expression levels (4–24% of reference genes) followed by HspB1 (Hsp25) and HspB8 (Hsp22; 0.1–2% of reference genes), all being widely expressed in the brain areas investigated. HspB2 (MKBP) and HspB3, however, showed selective expression in only some regions (B2: cortex and hippocampus, B3: cortex and cerebellum). Whereas HspB5 was expressed mainly in the white matter, HspB6 showed the greatest expression in the cerebellar cortex, and HspB11 was widely distributed over the whole brain. In cultured hippocampal neurons, heat shock led to an increase of HspB1 and HspB8 mRNA and additionally HspB5 protein. Our data indicate that the sHsps induced by heat shock, HspB1, B5, and B8, might be especially involved in neuroprotection under stress conditions. The other sHsps showing constant neuronal expression may play a constitutive role or may be up‐regulated and important in types of stresses other than heat shock.

Ischemia-induced increase of stiffness of αB-crystallin/HSPB2-deficient myocardium

The two small heat shock proteins (sHSPs), αB-crystallin and HSPB2, have been shown to translocate within a few minutes of cardiac ischemia from the cytosol to myofibrils; and it has been suggested that their chaperone-like properties might protect myofibrillar proteins from unfolding or aggregation during stress conditions. Further evidence of an important role for HSPs in muscle function is provided by the fact that mutations of the αB-crystallin gene cause myopathy and cardiomyopathy. In the present study, we subjected isolated papillary muscles of αB-crystallin/HSPB2-deficient mice to simulated ischemia and reperfusion. During ischemia in αB-crystallin/HSPB2-deficient muscles, the development of contracture started earlier and reached a higher value compared to the wildtype mice. The recovery of contracture of αB-crystallin/HSPB2-deficient muscles was also attenuated during the simulated reperfusion period. However, twitch force was not significantly altered at any time of the experiment. This suggests that during ischemic insults, αB-crystallin/HSPB2 may not be important for the contraction process itself, but rather serve to maintain muscular elasticity.

Differential role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous.

The growing world of small heat shock proteins : From structure to functions

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).

Erratum to: Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.

αB-crystallin/HspB5 regulates endothelial–leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin

AbstractαB-crystallin is a small heat shock protein, which has pro-angiogenic properties by increasing survival of endothelial cells and secretion of vascular endothelial growth factor A. Here we demonstrate an additional role of αB-crystallin in regulating vascular function, through enhancing tumor necrosis factor α (TNF-α) induced expression of endothelial adhesion molecules involved in leukocyte recruitment. Ectopic expression of αB-crystallin in endothelial cells increases the level of E-selectin expression in response to TNF-α, and enhances leukocyte–endothelial interaction in vitro. Conversely, TNF-α-induced expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin is markedly inhibited in endothelial cells isolated from αB-crystallin-deficient mice. This is associated with elevated levels of IκB in αB-crystallin deficient cells and incomplete degradation upon TNF-α stimulation. Consistent with this, endothelial adhesion molecule expression is reduced in inflamed vessels of αB-crystallin deficient mice, and leukocyte rolling velocity is increased. Our data identify αB-crystallin as a new regulator of leukocyte recruitment, by enhancing pro-inflammatory nuclear factor κ B-signaling and endothelial adhesion molecule expression during endothelial activation.