Christian Maasch

The Jak/Stat pathway and urokinase receptor signaling in human aortic vascular smooth muscle cells.

The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53–60, 85–90, and 130–140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59 fyn , p53/56 lyn , p53/59 hck , and p55 fgr . Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-γ activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.

High Glucose Concentrations Increase Endothelial Cell Permeability via Activation of Protein Kinase Cα

Endothelial cell permeability is impaired in diabetes mellitus and may be increased by high extracellular glucose concentrations. High glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. We tested the hypothesis that high glucose concentration activates PKC in endothelial cells and leads to an increase in endothelial cell permeability via distinct PKC isoforms. Porcine aortic endothelial cells were used, and the PKC isoforms alpha, delta, epsilon, zeta, and theta were identified in these cells. Glucose caused a rapid dose-dependent increase in endothelial cell permeability, with an EC50 of 17.5 mmol/L. Phorbol 12-myristate 13-acetate (TPA) induced an increase in permeability very similar to that elicited by glucose. The effect of glucose and TPA was totally reversed by preincubating the cells with the PKC inhibitors staurosporine (10(-8) mol/L) and Goe 6976 (10(-8) mol/L). Downregulation of PKC by preincubation with TPA for 24 hours also abolished the effect of glucose and TPA on endothelial cell permeability. High glucose (20 mmol/L) caused an increase in PKC activity at 2, 10, and 30 minutes. Cell fractionation and Western blot analysis showed a glucose-induced translocation of PKC alpha and PKC epsilon. Confocal microscopy confirmed the translocation and showed an association of PKC alpha and PKC epsilon with nuclear structures and the cell membrane. Specific antisense oligodesoxynucleotides (ODNs) against PKC alpha reduced the expression of the isoform, abolished the effects of glucose on endothelial cell permeability completely, and reduced the TPA effect significantly. In contrast, specific antisense ODNs against PKC epsilon had no effect on glucose-induced permeability and only a minor effect on the TPA-induced increase in permeability. We conclude that an increase in extracellular glucose leads to a rapid dose-dependent increase in endothelial cell permeability via the activiation of PKC and that this effect is mediated by the PKC isoform alpha.

Integrin-Induced Protein Kinase Cα and Cε Translocation to Focal Adhesions Mediates Vascular Smooth Muscle Cell Spreading

The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-L-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms alpha, delta, epsilon, and zeta were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKCalpha immunoreactivity was transiently increased. PKC isoforms a and epsilon were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKCalpha and PKCepsilon, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms alpha and epsilon with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform a is translocated to the nucleus, whereas PKC isoforms alpha and epsilon are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.

Requirement for Protein Kinase C in Reactive Oxygen Species–Induced Apoptosis of Vascular Smooth Muscle Cells

BACKGROUND Vascular smooth muscle cell (VSMC) apoptosis is a component of a variety of cardiovascular diseases and may be related to reactive oxygen species (ROS). This study was designed to determine the role of protein kinase C (PKC) in ROS-induced VSMC apoptosis. METHODS AND RESULTS Rat aortic VSMCs were exposed to H(2)O(2), and the nature of cell death was characterized in the absence or presence of different PKC inhibitors. The results demonstrate that exposure of VSMCs to H(2)O(2) led to a dose-dependent (25 to 100 micromol/L) and time-dependent (peak at 2 minutes) activation of PKC. Among the PKC isoforms alpha, beta, delta, epsilon, and zeta, only PKC-alpha and PKC-epsilon were found to change their intracellular distribution on H(2)O(2) treatment. Apoptosis was the predominant form of cell death when PKC had been activated by H(2)O(2) alone or by H(2)O(2) in the presence of 50 nmol/L phorbol 12-myristate 13-acetate. In contrast, necrosis became the predominant form of cell death when PKC had been downregulated by prolonged exposure to 200 nmol/L phorbol 12,13-dibutyrate or inhibited by 50 nmol/L staurosporine, 100 nmol/L calphostin C, or 30 micromol/L H-7. In addition, caspase-3 was activated in H(2)O(2)-induced VSMC apoptosis but not when PKC was downregulated or inhibited. Inhibition of caspase-3 by 50 micromol/L Ac-DEVD-CHO could significantly attenuate H(2)O(2)-induced apoptosis and was not associated with the induction of necrosis. CONCLUSIONS We conclude that in VSMCs, PKC converts the ROS-induced signals from necrotic cell death to the activation of an apoptotic cell death program. These data imply a novel and important role of PKC for the pathogenesis of such vascular diseases as atherosclerosis, restenosis, and hypertension.

The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGFs proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGFs angiogenic effects.

Calcium Antagonists Ameliorate Ischemia-Induced Endothelial Cell Permeability by Inhibiting Protein Kinase C

BACKGROUND Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells. METHODS AND RESULTS Ischemia was induced by potassium cyanide/deoxyglucose, and permeability was measured by albumin flux. Ion channels were characterized by patch clamp. [Ca2+]i was measured by fura 2. PKC activity was measured by substrate phosphorylation after cell fractionation. PKC isoforms were assessed by Western blot and confocal microscopy. Nifedipine prevented the ischemia-induced increase in permeability in a dose-dependent manner. Ischemia increased [Ca2+]i, which was not affected by nifedipine. Instead, ischemia-induced PKC translocation was prevented by nifedipine. Phorbol ester also increased endothelial cell permeability, which was dose dependently inhibited by nifedipine. The effects of non-calcium-channel-binding dihydropyridine derivatives were similar. Analysis of the PKC isoforms showed that nifedipine prevented ischemia-induced translocation of PKC-alpha and PKC-zeta. Specific inhibition of PKC isoforms with antisense oligodeoxynucleotides demonstrated a major role for PKC-alpha. CONCLUSIONS Nifedipine exerts a direct effect on endothelial cell permeability that is independent of calcium channels. The inhibition of ischemia-induced permeability by nifedipine seems to be mediated primarily by PKC-alpha inhibition. Anti-ischemic effects of dihydropyridine calcium antagonists could be due in part to their effects on endothelial cell permeability.

Endothelial-cell permeability and protein kinase C in pre-eclampsia

BACKGROUND Oedema and vascular leakage play a part in the pathogenesis of pre-eclampsia. We tested the hypothesis that serum from pre-eclamptic patients increases endothelial-cell permeability and examined possible signal-transduction pathways. METHODS We studied eight patients with pre-eclampsia, eight normotensive pregnant women, eight non-pregnant women, five pregnant patients with pre-existing hypertension, and four hypertensive non-pregnant women. Cultured human umbilical-vein endothelial-cell monolayers were used and permeability was measured by albumin flux. The part played by protein kinase C (PKC) signalling was examined by down-regulation with phorbol ester and with the inhibitors Goe 6976 and staurosporine. PKC isoforms were assessed by western blot and confocal microscopy. Antisense oligodesoxynucleotides (ODN) were used to test for specific PKC isoforms. FINDINGS Serum from pre-eclamptic women increased endothelial permeability significantly (by 100%, p<0.01). The change in permeability decreased rapidly after delivery. Serum from normotensive pregnant women and non-pregnant women had no effect. Permeability was not influenced by serum from patients with essential hypertension or pregnant patients with pre-existing hypertension. Serum from pre-eclamptic patients induced a translocation of PKC isoforms alpha and epsilon within the cells. Goe 6976 and staurosporine (10(-8) mol/L) inhibited the increase in permeability induced by serum from pre-eclamptic patients. Down-regulation of PKC alpha and, to a lesser extent, PKC epsilon by antisense ODN also inhibited the pre-eclampsia-induced permeability increase. INTERPRETATION Serum from pre-eclamptic patients contains a factor or factors that increase endothelial-cell permeability. The effect of pre-eclamptic serum may be mediated by PKC alpha and epsilon.

Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca2+]i regulate translocation of PKCα and that different modes of Ca2+ and Ca2+ release play a role in PKCα targeting. We constructed deletion mutants of PKCα to analyze the Ca2+‐sensitive domains and their role in targeting. Confocal microscopy was used and [Ca2+]i was measured by fluo‐3. The fusion protein PKCα‐GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wildtype PKCα protein. The Ca2+ ionophore ionomycin induced a speckled cytosolic PKCα‐GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage‐operated Ca2+ channel opening led to a localized accumulation of PKCα‐GFP near the plasma membrane. Opening Ca2+ stores with InsP3, thapsigargin, or ryanodine induced a specific PKCα‐GFP targeting to distinct intracellular areas. The G‐protein‐coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca2+ influx and led to a linear PKCα‐GFP membrane association. PKCα‐GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca2+‐induced PKCα targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCα can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca2+]i. The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca2+]i changes determine the spatial and temporal targeting of PKCα.–Maasch, C., Wagner, S., Lindschau, C., Alexander, G., Buchner, K., Gollasch, M., Luft, F. C., Haller, H. Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i. FASEB J. 14, 1653–1663 (2000)

Crystal structure of a mirror-image L -RNA aptamer (Spiegelmer) in complex with the natural L- protein target CCL2

We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokines activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide·L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamers intramolecular contacts and permits a detailed analysis of its structure–function relationship. Furthermore, the analysis of the intermolecular drug–target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

Targeting complement component 5a promotes vascular integrity and limits airway remodeling

Increased microvascular dilatation and permeability is observed during allograft rejection. Because vascular integrity is an important indicator of transplant health, we have sought to limit injury to blood vessels by blocking complement activation. Although complement component 3 (C3) inhibition is known to be vasculoprotective in transplantation studies, we recently demonstrated the paradoxical finding that, early in rejection, C3−/− transplant recipients actually exhibit worse microvascular injury than controls. In the genetic absence of C3, thrombin-mediated complement component 5 (C5) convertase activity leads to the generation of C5a (anaphylatoxin), a promoter of vasodilatation and permeability. In the current study, we demonstrated that microvessel thrombin deposition is significantly increased in C3−/− recipients during acute rejection. Thrombin colocalization with microvessels is closely associated with remarkably elevated plasma levels of C5a, vasodilatation, and increased vascular permeability. Administration of NOX-D19, a specific C5a inhibitor, to C3−/− recipients of airway transplants significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia, even in the absence of conventional T-cell–directed immunosuppression. As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit.