Targeting proinflammatory cytokines ameliorates calcifying phenotype conversion of vascular progenitors under uremic conditions in vitro

Severe vascular calcification develops almost invariably in chronic kidney patients posing a substantial risk to quality of life and survival. This unmet medical need demands identification of novel therapeutic modalities. We aimed to pinpoint components of the uremic microenvironment triggering differentiation of vascular progenitors to calcifying osteoblast-like cells. In an unbiased approach, assessing the individual potency of 63 uremic retention solutes to enhance calcific phenotype conversion of vascular progenitor cells, the pro-inflammatory cytokines IL-1 beta and TNF-alpha were identified as the strongest inducers followed by FGF-2, and PTH. Pharmacologic targeting of these molecules alone or in combination additively antagonized pro-calcifying properties of sera from uremic patients. Our findings stress the importance of pro-inflammatory cytokines above other characteristic components of the uremic microenvironment as key mediators of calcifying osteoblastic differentiation in vascular progenitors. Belonging to the group of "middle-sized molecules", they are neither effectively removed by conventional dialysis nor influenced by established supportive therapies. Specific pharmacologic interventions or novel extracorporeal approaches may help preserve regenerative capacity and control vascular calcification due to uremic environment

Adhesion dynamics in the neocortex determine the start of migration and the post-migratory orientation of neurons.

peer reviewedThe neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development

mTOR regulates adaptive cell fate programs to balance regeneration versus calcification in mesenchymal progenitors

Obwohl Herz-Kreislauf-Erkrankungen (CVD) die häufigste Todesursache weltweit darstellen, fehlen trotz intensiven Bemühungen durchgreifende Behandlungskonzepte und überzeugende Präventionsstrategien. Ziel dieser Arbeit war es, die zugrunde liegenden Mechanismen der vaskulären Kalzifizierung, eine der wichtigsten Ursachen für CVD, zu analysieren und eine therapeutische Option auszuarbeiten. Um den fehlgeleiteten Prozess der extraossären Kalzifizierung in der Gefäßwand zu ergründen, wurde in einem in vitro Modell untersucht, wie Programme kontrolliert werden, welche die Differenzierung und Entwicklung von humanen Vorläuferzellen des vaskulären Systems steuern. Als potente und zentrale Regulationseinheit der Zelle um auf Stress,Wachstumsfaktoren und Nährstoffe zu reagieren wurde das mTOR-Netzwerk mit seinen beiden strukturell und funktionell verschiedenen Multiproteinkomplexen für die steuernden Befehle in Betracht gezogen und zur Beeinflussung der Prozesse anvisiert. Rapamycin diente der gezielten pharmakologischen Modulation des Netzwerks während der osteoblastären Differenzierung, die ebenso wie die Menge der Kalziumablagerung, die Aktivität der mTOR-Komplexe und die der Zellschicksalsprogramme während des 21-tägigen Prozesses der Kalzifizierung kontinuierlich analysiert wurden. Die Blockade von mTORC1 offenbarte eine zentrale Rolle von mTORC2 für die Induktion protektiver Zellprogramme, die durch den Einsatz eines pharmakologischen Inhibitors und durch genetischen Knockout bestätigt wurde. Der benefizielle Einfluss der mTORC2-Aktivität auf die zellulären Prozesse der Gefäßwand konnte in-vivo durch die Behandlung von Mäusen mit Rapamycin über 37 Tage mit anschließender immunohistologischer Untersuchung der Aorten bestätigt werden. In einem Medium-Transfer-Modell wurde außerdem der durch Rapamycin ausgelöste günstige parakrine Effekt von MSCs auf glatte Muskelzellen bei osteoblastärer Differenzierung untersucht. Die Modulation des mTOR-Netzwerks führte über die Induktion von Autophagie und Verminderung von Seneszenz und Apoptoseprozessen zu einer Reduktion der Kalzifizierung, die durch gezielte Inaktivierung des mTOR-Komplexes 2 verhindert werden konnte. Die Kontrolle der mTORC1-Aktivität zusammen mit einer Verstärkung der mTORC2-Funktionen bietet die Möglichkeit, schützende Zellprogramme und endogene parakrine Effekte für die Behandlung fortschreitender Arteriosklerose einzusetzen. Außerdem konnte festgestellt werden, dass die Modulation des mTOR-Netzwerks das zelleigene regenerative Potential schützt, was, ebenso wie die gezielte Aktivierung von mTORC1, bedeutsam für die spezifische Induktion von Knochenbildung im Rahmen einer lokalen Therapie zur Knochenregeneration bei Osteoporosefrakturen sein könnte.Cardiovascular disease (CVD) is the most prominent contributor to global mortality and concepts for treatment as well as compelling prevention strategies are missing. In our study, we provide new insights in the underlying processes for vascular calcification, one basic pillar of CVD, by analysing cell fate programs and their regulation in human cells involved in vascular biology. Accelerated calcifying arteriosclerosis features osteoblastic transformation of vascular smooth muscle cells (VSMCs) and their progenitors, mesenchymal stromal cells (MSCs). Targeting signaling pathways controlling cell differentiation could shift maladaptive cell fate programs towards protective and prevent from vascular calcification. mTOR kinase contained in two functionally and structurally distinct multiprotein complexes mTORC1 and mTORC2 integrates extracellular stimuli into cell differentiation and growth responses. We hypothesize distinct roles for mTORC1 and mTORC2 in regulation of cell fate programs in a temporally controlled sequence. Rapamycin served for pharmacologic mTOR targeting during osteoblastic differentiation of MSCs. Matrix calcium deposition, mTORC1 and mTORC2 targets, and cell fate programs were followed for 21 days. Central role of mTORC2 in induction of protective cell fate programs was determined by AKT blockade, genetic disruption of mTORC2 function and autophagy inhibition. Beneficial Effects of mTORC2 acitivation on vascular cells induced by mTORC1 blockade were confirmed in vivo by low dose-rapamycin injection into mice and immunohistochemical analysis of aortas after 37 days treatment. Paracrine effects of rapamycin conditioned MSCs on VSMCs undergoing osteoblastic transformation were assessed by medium transfer. Attenuation of mTORC1 and activation of mTORC2 downstream signaling effectors inhibited calcification via induction of autophagy and down-regulation of proteins mediating apoptosis and cell senescence. Negative interference with mTORC2 function or autophagy disrupted protective programs via induction of apoptosis and cell senescence. Secretome of rapamycin conditioned MSCs mitigated osteoblastic transformation of VSMCs. Control of mTORC1 activation together with enhancement of mTORC2 function leads to induction of autophagy and maintenance of protective cell fate programs during osteoblastic transformation. Regenerative approaches aiming to translate our findings hold promise for treatment of accelerated arteriosclerosis. Furthermore, modulation of the mTOR signalling protects the endogenous regeneration capacity of osteoblast precursor cells and provides new options for specific induction of bone formation in osteoporosis

mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells

Vascular regeneration depends on intact function of progenitors of vascular smooth muscle cells such as pericytes and their circulating counterparts, mesenchymal stromal cells (MSC). Deregulated MSC differentiation and maladaptive cell fate programs associated with age and metabolic diseases may exacerbate arteriosclerosis due to excessive transformation to osteoblast-like calcifying cells. Targeting mTOR, a central controller of differentiation and cell fates, could offer novel therapeutic perspectives. In a cell culture model for osteoblastic differentiation of pluripotent human MSC we found distinct roles for mTORC1 and mTORC2 in the regulation of differentiation towards calcifying osteoblasts via cell fate programs in a temporally-controlled sequence. Activation of mTORC1 with induction of cellular senescence and apoptosis were hallmarks of transition to a calcifying phenotype. Inhibition of mTORC1 with Rapamycin elicited reciprocal activation of mTORC2, enhanced autophagy and recruited anti-apoptotic signals, conferring protection from calcification. Pharmacologic and genetic negative interference with mTORC2 function or autophagy both abolished regenerative programs but induced cellular senescence, apoptosis, and calcification. Overexpression of the mTORC2 constituent rictor revealed that enhanced mTORC2 signaling without altered mTORC1 function was sufficient to inhibit calcification. Studies in mice reproduced the in vitro effects of mTOR modulation with Rapamycin on cell fates in vascular cells in vivo. Amplification of mTORC2 signaling promotes protective cell fates including autophagy to counteract osteoblast differentiation and calcification of MSC, representing a novel mTORC2 function. Regenerative approaches aimed at modulating mTOR network activation patterns hold promise for delaying age-related vascular diseases and treatment of accelerated arteriosclerosis in chronic metabolic conditions.Peer Reviewe

High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells

Abstract Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

INTRODUCTION:Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. METHODS:We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-β1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-β receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. RESULTS:VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-β1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-β1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-β1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-β receptor 1 and enhanced canonical and noncanonical TGF-β signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. CONCLUSIONS:Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-β1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients

Cell functions of mesenchymal stromal cells upon treatment with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Chemotactic response towards a 5 ng/ml gradient of each growth factor. Bars represent the mean+SEM of 3 independent experiments, 3 replicates each. Control was set to 1. <b>B,</b> Proliferation measured by BrdU incorporation after stimulation with 10 ng/ml of each growth factor for 24 h. Bars represent the mean+SEM of 3 independent experiments, 5 replicates each. Control was set to 1. <b>C,</b> Total collagen content in cell culture supernatants after 6 days of treatment with 10 ng/ml of each growth factor. Bars represent the mean+SEM of 6 independent experiments, 2 replicates each. *P<0.05, **P<0.01, ***P<0.001. <b>D,</b> Illustration of the induction of specific cell types of vascular smooth muscle cell (VSMC) and fibroblast lineages by systemic sclerosis microenvironment defining growth factors.</p

Analysis of the Transforming growth factor-β1 signaling network.

<p><b>A,</b> Expression of TGF-β1 mRNA and <b>B,</b> secreted TGF-β1 protein for assessment of the autocrine TGF-β feedback loop. Bars represent the mean+SEM of 6 independent experiments, 3 replicates each. <b>C,</b> Expression of CTGF mRNA. Quantitative real-time PCR analysis of mRNA transcripts. Bars represent the mean+SEM of 6 independent experiments quantified with the ΔΔCt method in duplicates. <b>D,</b> Expression of TGF-β receptor 1 protein. <b>E-G,</b> Induction of canonic and non-canonic TGF-β signaling. <b>E,</b> Phosphorylation of SMAD3 at Ser423/425. <b>F,</b> Phosphorylation of AKT at Ser473. <b>G,</b> Phosphorylation of ERK1/2 at Thr202/Tyr204. Analysis of all experiments was performed after 6 days treatment with 10 ng/ml transformin growth factor -β1. Representative western blots of 6 independent experiments are shown. Bars represent the mean+SEM of densitometrically determined band intensities after normalization to GAPDH (TGFBR1, pSMAD3) or total kinase (pAKT, pERK). Control was set to 1. *P<0.05, **P<0.01, ***P<0.001.</p

Phenotypic modulation upon long-term stimulation (6 days) with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Representative phase contrast photomicrographs from a total of 6 independent experiments are shown. (original magnification x200, scale bar = 50 μm) <b>B,</b> Representative immunofluorescence images from a total of 6 independent experiments are shown. (green = smooth muscle-α-actin, blue = nuclear DNA stained with 4',6-diamidino-2-phenylindole (DAPI), original magnification x400, scale bar = 50 μm).</p

Characterization of mesenchymal stromal cells from healthy controls and patients with systemic sclerosis.

<p><b>A,</b> Representative FACS analysis of an MSC defining surface marker panel confirming homogeneity of isolated cells. <b>B,</b> Osteoblastic differentiation of MSCs after incubation for 21 days with osteoblast-induction medium. (Alizarin red S staining, original magnification x100, scale bars = 50 μm) <b>C,</b> Adipocytic differentiation of MSCs after incubation for 21 days with adipocyte-induction medium. (Oil-Red-O staining, original magnification x200, scale bars = 50 μm) <b>D,</b> Chondroblastic differentiation of MSCs after incubation for 32 days with chondroblast induction medium. Western blot analysis for chondrocyte specific type II collagen.</p