Milena Armacki - Academia.edu (original) (raw)

Papers by Milena Armacki

Science Advances

Implanted electronic sensors, compared with conventional medical imaging, allow monitoring of adv... more Implanted electronic sensors, compared with conventional medical imaging, allow monitoring of advanced physiological properties of soft biological tissues continuously, such as adhesion, pH, viscoelasticity, and biomarkers for disease diagnosis. However, they are typically invasive, requiring being deployed by surgery, and frequently cause inflammation. Here we propose a minimally invasive method of using wireless miniature soft robots to in situ sense the physiological properties of tissues. By controlling robot-tissue interaction using external magnetic fields, visualized by medical imaging, we can recover tissue properties precisely from the robot shape and magnetic fields. We demonstrate that the robot can traverse tissues with multimodal locomotion and sense the adhesion, pH, and viscoelasticity on porcine and mice gastrointestinal tissues ex vivo, tracked by x-ray or ultrasound imaging. With the unprecedented capability of sensing tissue physiological properties with minimal i...

Frontiers in Immunology, Mar 2, 2023

Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the ... more Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B 4 (LTB 4). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and Eselectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB 4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB 4 biosynthesis enzyme LTA 4 hydrolase (LTA 4 H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA 4 H-positive sEVs in the serum of polytrauma patients, peaking 12 h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during posttraumatic inflammation.

Viszeralmedizin 2021 Gemeinsame Jahrestagung Deutsche Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten (DGVS), Sektion Endoskopie der DGVS, Deutsche Gesellschaft für Allgemein und Viszeralchirurgie (DGAV), 2021

74. Jahrestagung der Deutschen Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten mit Sektion Endoskopie – 13. Herbsttagung der Deutschen Gesellschaft für Allgemein- und Viszeralchirurgie gemeinsam mit den Arbeitsgemeinschaften der DGAV, 2019

Zeitschrift für Gastroenterologie, 2011

Zeitschrift für Gastroenterologie, 2007

Advanced Science, 2021

Abstract Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma... more Abstract Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma‐related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro‐inflammatory cargo. These sEVs transfer transcripts for ICAM‐1, VCAM‐1, E‐selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil‐endothelium interactions, and destabilize barrier integrity. Inhibition of sEV‐release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT‐plasma‐sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism.

American Journal of Physiology-Gastrointestinal and Liver Physiology, 2021

All-time preservation of an intact mucosal barrier is crucial to ensuring intestinal homeostasis ... more All-time preservation of an intact mucosal barrier is crucial to ensuring intestinal homeostasis and, hence, the organism's overall health maintenance. This complex process relies on an equilibrated signaling system between the intestinal epithelium and numerous cell populations inhabiting the gut mucosa. Any perturbations of this delicate crosstalk, particularly regarding the immune cell compartment and microbiota, may sustainably debilitate the intestinal barrier function. As a final joint event, a critical rise in epithelial permeability facilitates the exposure of submucosal immunity to microbial antigens, resulting in uncontrolled inflammation, collateral tissue destruction and dysbiosis. Organoid-derived intestinal co-culture models have established themselves as convenient tools to re-enact such pathophysiological events, explore interactions between selected cell populations and assess their roles with a central focus on intestinal barrier recovery and stabilization.

Gastroenterology, 2020

BACKGROUND & AIMS Pancreatic tumor cells release extracellular vesicles (sEVs, exosomes) that... more BACKGROUND & AIMS Pancreatic tumor cells release extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas (PDACs). We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form pre-metastatic niches for pancreatic cancer cells in mice. METHODS We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and non-tumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative PCR; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative PCR, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. RESULTS Levels of PRKD1 were reduced in human PDAC tissues compared with non-tumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs, compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs-a process that required CD82. CONCLUSIONS Human PDAC have reduced levels of PRKD1 compared with non-tumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Journal of Clinical Investigation, 2018

Zeitschrift für Gastroenterologie, 2015

Zeitschrift für Gastroenterologie, 2013

PLoS ONE, 2011

Muscle differentiation is a highly conserved process that occurs through the activation of quiesc... more Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1,-2, and-3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

Oncogene, 2013

Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily o... more Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily of the CAMK super-family. PKDs have a critical role in cell motility, migration and invasion of cancer cells. Expression of PKD isoforms is deregulated in various tumours and PKDs, in particular PKD2, have been implicated in the regulation of tumour angiogenesis. In order to further elucidate the role of PKD2 in tumours, we investigated the signalling context of this kinase by performing an extensive substrate screen by in vitro expression cloning (IVEC). We identified a novel splice variant of calcium and integrin-binding protein 1, termed CIB1a, as a potential substrate of PKD2. CIB1 is a widely expressed protein that has been implicated in angiogenesis, cell migration and proliferation, all important hallmarks of cancer, and CIB1a was found to be highly expressed in various cancer cell lines. We identify Ser 118 as the major PKD2 phosphorylation site in CIB1a and show that PKD2 interacts with CIB1a via its alanine and proline-rich domain. Furthermore, we confirm that CIB1a is indeed a substrate of PKD2 also in intact cells using a phosphorylation-specific antibody against CIB1a-Ser 118. Functional analysis of PKD2-mediated CIB1a phosphorylation revealed that on phosphorylation, CIB1a mediates tumour cell invasion, tumour growth and angiogenesis by mediating PKD-induced vascular endothelial growth factor secretion by the tumour cells. Thus, CIB1a is a novel mediator of PKD2-driven carcinogenesis and a potentially interesting therapeutic target.

Molecular Biology of the Cell, 2010

Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Gol... more Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacyl...

Molecular Biology of the Cell, 2013

Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the develo... more Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix–bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. ...

Journal of Cell Science, 2012

Cell migration and invasion are largely dependent on the complex organization of the various cyto... more Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this ...

Cellular Signalling, 2009

Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edg... more Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase. Using a mass spectrometric approach, we found that PKD phosphorylates cortactin on Ser 298 in the 6th cortactin repeat region and on Ser 348, right before the helical-proline rich domain of cortactin. We developed phosphospecific antibodies against these phosphorylated sequences, and used them as tools to follow the in vivo phosphorylation of cortactin by PKD. Examination of cortactin phosphorylation kinetics revealed that Ser 298 serves as a priming site for subsequent phosphorylation of Ser 348. Src, a well-known cortactin kinase, strongly potentiated the in vivo PKD mediated cortactin phosphorylation. This Src effect is neither mediated by prephosphorylation of cortactin nor by activation of PKD by Src. Phosphorylation of cortactin by PKD does not affect its subcellular localization, nor does it affect its translocation to podosomes or membrane ruffles. Moreover, there was no effect of PKD mediated cortactin phosphorylation on EGF receptor degradation and LPA induced migration. Taken together, these data establish cortactin as a novel PKD substrate and reveal a novel connection between Src and PKD.

Frontiers in Immunology, 2020

Trauma represents a major socioeconomic burden worldwide. After a severe injury, hemorrhagic shoc... more Trauma represents a major socioeconomic burden worldwide. After a severe injury, hemorrhagic shock (HS) as a frequent concomitant aspect is a central driver of systemic inflammation and organ damage. The kidney is often strongly affected by traumatic-HS, and acute kidney injury (AKI) poses the patient at great risk for adverse outcome. Recently, thirty-eight-negative kinase 1 (TNK1) was proposed to play a detrimental role in organ damage after trauma/HS. Therefore, we aimed to assess the role of TNK1 in HS-induced kidney injury in a murine and a post hoc analysis of a non-human primate model of HS comparable to the clinical situation. Mice and non-human primates underwent resuscitated HS at 30 mmHg for 60 min. 5 h after the induction of shock, animals were assessed for systemic inflammation and TNK1 expression in the kidney. In vitro, murine distal convoluted tubule cells were stimulated with inflammatory mediators to gain mechanistic insights into the role of TNK1 in kidney dysfunction. In a translational approach, we investigated blood drawn from either healthy volunteers or severely injured patients at different time points after trauma (from arrival at the emergency room and at fixed time intervals until 10 days post injury; identifier: NCT02682550, https://clinicaltrials.gov/ct2/show/NCT02682550). A pronounced inflammatory response, as seen by increased IL-6 plasma levels as well as early signs of AKI, were observed in mice, non-human primates, and humans after trauma/HS. TNK1 was found in the plasma early after trauma-HS in trauma patients. Renal TNK1 expression was significantly increased in mice and non-human primates after HS, and these effects with concomitant induction of apoptosis were blocked by therapeutic inhibition of complement C3 activation in non-human primates. Mechanistically, in vitro data suggested that IL-6 rather than C3 cleavage products