Markus Essler

Thrombin Inactivates Myosin Light Chain Phosphatase via Rho and Its Target Rho Kinase in Human Endothelial Cells

The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Cyclic AMP Blocks Bacterial Lipopolysaccharide-Induced Myosin Light Chain Phosphorylation in Endothelial Cells Through Inhibition of Rho/Rho Kinase Signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.

Increased 18F-fluorodeoxyglucose uptake in abdominal aortic aneurysms in positron emission/computed tomography is associated with inflammation, aortic wall instability, and acute symptoms

OBJECTIVE With the established computed tomographic (CT)- morphologic parameters, only the relative, but not the individual rupture risk of abdominal aortic aneurysm (AAA), can be determined. So far, increased aortic 18F-fluorodeoxyglucose (FDG) metabolism measured by positron emission tomography (PET) has been reported in AAA with increased rupture risk. The aim of the study was to analyze the histopathologic changes in AAA wall correlated with increased FDG uptake for further implications on aortic wall stability and AAA rupture risk. METHODS Fifteen patients with asymptomatic (n = 12) and symptomatic (n = 3) AAA underwent FDG-PET/CT, followed by open AAA repair. FDG-PET/CT was used for precise localization of maximum FDG uptake, and the maximum standard uptake values (SUV(max)) were calculated. Biopsies of the AAA wall were operatively collected from areas with maximum FDG uptake, immunohistologically stained, and semiquantitatively analyzed for inflammatory infiltrates, vascular smooth muscle cells (VSMC), matrix metalloproteinase (MMP)-2 and -9 expression, as well as for elastin and collagenous fibers. RESULTS Symptomatic AAA showed significantly increased FDG uptake compared with asymptomatic AAA (SUV(max), 3.5 +/- 0.6 vs 7.5 +/- 3; P < .001). Thus, increased FDG uptake was correlated with higher densities of inflammatory infiltrates (r = +0.87, P < .01) and macrophage and T-cell infiltrations (r = +0.95, P < .01 and r = +0.66, P < .05), with higher MMP-9 expressions (r = +0.86; P < .01), and with reduction of collagen fiber (r = -0.76; P < .01) and VSMCs (r = -0.71; P < .01). Consecutive correlations were found for total inflammatory infiltrates, T lymphocytes, and macrophages with MMP-9 expression (r = +0.79, +0.79 and +0.74; P < .01). Moreover, MMP-9 expression was correlated with decreasing collagen fiber content (r = -0.53, P < .05) and VSMC density (r = -0.57, P < .05). CONCLUSIONS Maximum aortic FDG uptake correlated significantly with inflammation, followed by increased MMP expression and histopathologic characteristics of aneurysm wall instability and clinical symptoms. Therefore, FDG-PET/CT might be a new diagnostic technique to study AAA disease in vivo and may contribute to improve prediction of individual AAA rupture risk.

YopE of Yersinia, a GAP for Rho GTPases, selectively modulates Rac‐dependent actin structures in endothelial cells

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, ‘in vivo’ targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects ‘life‐like’ amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine‐1‐phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)‐infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine‐1‐phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac‐ but not Rho‐ or CDC42‐regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac‐mediated signalling pathways in primary target cells of Yersinia.

Bacterial toxins block endothelial wound repair. Evidence that Rho GTPases control cytoskeletal rearrangements in migrating endothelial cells.

We investigated the effect of bacterial toxins that modify and inactivate Rho GTP-binding proteins on the migratory response of endothelial cells to wounding. C3-transferase from Clostridium botulinum, EDIN from Staphylococcus aureus, and toxin A from Clostridium difficile blocked migration of human umbilical vein endothelial cells (HUVECs) in an in vitro wound repair assay. Migrating HUVECs expressed actin microspikes (maximum at 10 minutes after wounding), ruffles (maximum at 12 hours), and fibers (maximum at 24 hours), and within these actin structures, vinculin-containing focal complexes/adhesions were formed. C3-Transferase ADP ribosylated RhoA, RhoB, and RhoC in HUVECs and abolished the formation of actin stress fibers/focal adhesions but had no effect on expression of microspikes, ruffles, or the associated vinculin-containing focal complexes. Similar results were obtained with EDIN and toxin A. These results indicate that endothelial cells migrating into a wounded area express distinct combinations of actin/vinculin structures in a spatially and temporally coordinated manner. The GTPase Rho selectively controls the formation of actin fibers/focal adhesions that occurs 2 to 24 hours after wounding. A mechanism is proposed by which Rho-specific bacterial toxins could influence vascular repair, angiogenesis, or atherosclerosis.

German Multicenter Study Investigating 177Lu-PSMA-617 Radioligand Therapy in Advanced Prostate Cancer Patients

177Lu-labeled PSMA-617 is a promising new therapeutic agent for radioligand therapy (RLT) of patients with metastatic castration-resistant prostate cancer (mCRPC). Initiated by the German Society of Nuclear Medicine, a retrospective multicenter data analysis was started in 2015 to evaluate efficacy and safety of 177Lu-PSMA-617 in a large cohort of patients. Methods: One hundred forty-five patients (median age, 73 y; range, 43–88 y) with mCRPC were treated with 177Lu-PSMA-617 in 12 therapy centers between February 2014 and July 2015 with 1–4 therapy cycles and an activity range of 2–8 GBq per cycle. Toxicity was categorized by the common toxicity criteria for adverse events (version 4.0) on the basis of serial blood tests and the attending physician’s report. The primary endpoint for efficacy was biochemical response as defined by a prostate-specific antigen decline ≥ 50% from baseline to at least 2 wk after the start of RLT. Results: A total of 248 therapy cycles were performed in 145 patients. Data for biochemical response in 99 patients as well as data for physician-reported and laboratory-based toxicity in 145 and 121 patients, respectively, were available. The median follow-up was 16 wk (range, 2–30 wk). Nineteen patients died during the observation period. Grade 3–4 hematotoxicity occurred in 18 patients: 10%, 4%, and 3% of the patients experienced anemia, thrombocytopenia, and leukopenia, respectively. Xerostomia occurred in 8%. The overall biochemical response rate was 45% after all therapy cycles, whereas 40% of patients already responded after a single cycle. Elevated alkaline phosphatase and the presence of visceral metastases were negative predictors and the total number of therapy cycles positive predictors of biochemical response. Conclusion: The present retrospective multicenter study of 177Lu-PSMA-617 RLT demonstrates favorable safety and high efficacy exceeding those of other third-line systemic therapies in mCRPC patients. Future phase II/III studies are warranted to elucidate the survival benefit of this new therapy in patients with mCRPC.

Rapid stiffening of integrin receptor-actin linkages in endothelial cells stimulated with thrombin: a magnetic bead microrheology study.

By using magnetic bead microrheology we study the effect of inflammatory agents and toxins on the viscoelastic moduli of endothelial cell plasma membranes in real time. Viscoelastic response curves were acquired by applying short force pulses of ~500 pN to fibronectin-coated magnetic beads attached to the surface membrane of endothelial cells. Upon addition of thrombin, a rapid stiffening of the membrane was observed within 5 s, followed by recovery of the initial deformability within 2 min. By using specific inhibitors, two known pathways by which thrombin induces actin reorganization in endothelial cells, namely activation of Ca2+-calmodulin-dependent myosin light chain kinase and stimulation of Rho/Rho-kinase, were excluded as possible causes of the stiffening effect. Interestingly, the cytotoxic necrotizing factor of Escherichia coli, a toxin which, in addition to Rho, activates the GTPases Rac and CDC42Hs, also induced a dramatic stiffening effect, suggesting that the stiffening may be mediated through a Rac- or Cdc42Hs-dependent pathway. This work demonstrates that magnetic bead microrheometry is not only a powerful tool to determine the absolute viscoelastic moduli of the composite cell plasma membrane, but also a valuable tool to study in real time the effect of drugs or toxins on the viscoelastic parameters of the plasma membrane.

Postacquisition Detection of Tumor Motion in the Lung and Upper Abdomen Using List-Mode PET Data: A Feasibility Study

One of the main degrading factors in the quality of oncologic PET images of the thorax and upper abdomen is respiratory motion of tumors. One method to reduce this effect is the acquisition of PET data in gated mode. A second method is the correction of studies for motion. Motion registration is essential in both cases. We report a method using list-mode data to detect the craniocaudal (z) movement of thoracic and abdominal lesions without using any external gating device. The aim of this study was to show the feasibility of applying this method to patient data. Methods: For 10 patients with lesions in the lung or upper abdominal organs, images for short time bins of 250, 500, and 750 ms were reconstructed. A volume of interest, which was manually defined in a summed image around the structure of interest, was projected to each time bin. The center of mass of the activity distribution in this volume of interest was determined for each case. The curves of the z-coordinate of the center of mass (zCOM) over the time were analyzed and compared with respiration curves obtained by a pressure-sensitive belt. Results: In 7 of the 10 patients, movement of the lesion was registered in good accordance with the pressure belt. In the 3 remaining patients, no changes in the center of mass due to respiration could be detected, most likely because of minimal respiratory motion. The maximal difference in zCOM for a lesion that was detected within the 10-min acquisition was 18.5 mm. For 7 of the 10 patients, the mean value for each respiration amplitude was between 11.0 and 2.0 mm. Conclusion: We have shown the feasibility of registering movement of high-uptake lesions without the use of any external device that may restrain the patient. Furthermore, unlike external sensors, this method quantifies internal motion and, thus, is a promising base for correction methods.

Therapeutic response and side effects of repeated radioligand therapy with 177 Lu-PSMA-DKFZ-617 of castrate-resistant metastatic prostate cancer

Prostate-specific membrane antigen (PSMA) is highly expressed on prostate epithelial cells and strongly up-regulated in prostate cancer (PC), making it an optimal target for the treatment of metastasized PC. Radioligand therapy (RLT) with 177Lu-PSMA-DKFZ-617 (Lu-PSMA) is a targeted therapy for metastatic PC. In this study, we retrospectively analyzed the side effects and the response rate of 24 hormone and/or chemorefractory PC patients with a mean age of 75.2 years (range: 64–82) with distant metastases and progressive disease according to the PSA level, who were treated with Lu-PSMA. Median PSA was 522 ng/ml (range: 17–2360). Forty-six cycles of Lu-PSMA were performed. Of the 24 patients, 22 received two cycles. Eight weeks after the first cycle of Lu-PSMA therapy 79.1% experienced a decline in PSA level. Eight weeks after the second cycle of Lu-PSMA therapy 68.2% experienced a decline in PSA relative to the baseline value. Apart from two cases of grade 3 anemia, there was no relevant hemato- or nephrotoxicity (grade 3 or 4). These results confirmed that Lu-PSMA is a safe treatment option for metastatic PC patients and has a low toxicity profile. A positive response to therapy in terms of decline in PSA occurs in about 70% of patients.