17ß-Estradiol Regulates mTORC2 Sensitivity to Rapamycin in Adaptive Cardiac Remodeling

Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17β-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERβ-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal “female advantage”

Molekulare Mechanismen adaptiver und maladaptiver kardiovaskulärer Remodellingprozesse

Kenntnisse über die molekularen pathogenetischen Mechanismen bilden die Grundlage neuer zielspezifischer Therapien und können wichtige Hinweise auf Wirkungen und unerwünschte Wirkungen liefern. Dabei sind die Vorgänge bei kardiovaskulären Remodellingvorgängen sehr komplex und umfassen nicht nur eine Vielfalt von Verknüpfungen intrazellulärer Signalkaskaden, sondern auch ein Wechselspiel zwischen unterschiedlichen Zelltypen, einer breiten Dynamik von Zelltransformationen, Zytokinen, Wachstumsfaktoren und anderen Zellaktivatoren, sowie systemischen Regulationen durch Vesikel, Mikro- und Nichtkodierende RNA, genetischen Prädispositionen und epigenetischen Veränderungen. Experimentelle Arbeiten in diesem Bereich erfordern ein Fokussieren auf einzelne Aspekte, um grundlegende Interaktionen tiefgehender verstehen zu können. Die vorliegenden Arbeiten haben wesentliche Aspekte zum Verständnis der molekularen Mechanismen des Urokinase-/Urokinaserezeptorsystems im Kontext der Atherogenese beigetragen. Erstmalig konnte die Arbeitsgruppe zusätzlich zur bekannten Rolle von STAT-Proteinen bei der transkriptionellen Regulation VSMC die Beteiligung von Januskinasen und der PI3-K in der intrazellulären Signalvermittlung des uPAR nachweisen und deren Bedeutung für die Migration VSMC. Weitere Arbeiten deckten die Rolle monozytär exprimierter Urokinase an der uPA/uPAR-induzierten Wachstumshemmung von VSMC vermittelt durch STAT1 auf und beschrieben Auswirkungen von direktem und indirektem Kontakt von VSMC und Monozyten auf die Proliferationsrate von VSMC. Als zusätzlicher essentieller Faktor für die Regulation von STAT1, der in VSMC einen Proliferationsstop vermittelte, wurde das Tight Junction Protein ZO-2 identifiziert, das in atherosklerotischen Läsionen hochreguliert ist. Die Bedeutung dieses Proteins an transkriptionellen Regulationen, Signaltransduktion, Zelldifferenzierung und Morphogenese waren zwar bereits in anderen Zellen zusätzlich zum klassischen Einfluss auf die parazelluläre Permeabilität beschrieben worden, nicht jedoch in VSMC. Eine Studie mit direkterer pharmakologisch translationaler Bedeutung waren die Untersuchungen des Statins Rosuvastatin auf die Regulation des uPA/uPAR-Systems und die funktionellen Konsequenzen bezüglich Neointimabildung. Wir konnten einen neuen pleiotropen Effekt dieses Statins in VSMC aufzeigen, der durch eine Herunterregulation des uPAR und damit verbundenen Erhalt des kontraktilen Phänotyps mit Hemmung von Migration und Proliferation gekennzeichnet war. Des Weiteren erfolgten Studien zur Rolle des mTOR-Signalweges bei der phänotypischen Modifikation VSMC und adaptiver und maladaptiver Remodellingprozesse am Herzen, die erstmalig auf die Bedeutung geschlechtsspezifischer Aspekte des mTOR-Signalweges hinwiesen und mechanistisch weiter aufklärten. Die Erkenntnisse dieser Studien haben grundlegend zum Verständnis der nicht-fibrinolytischen Funktionen des uPA/uPAR-Systems, sowie der mTOR-Signaltransduktion bei kardio-vaskulärem Remodelling mit der Bedeutung des Einflusses von Geschlecht und spezifischer Hormonrezeptoren beigetragen. Diese Arbeiten bekräftigen die Notwendigkeit, im heutigen Zeitalter der Präzisionsmedizin, Studien mit neuen molekularen Therapieansätzen immer auch unter Berücksichtigung von Geschlecht und Alter durchzuführen

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

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

Measurement of functional L-type Cav1.2 Ca2+ channels in response to growth factors.

<p>Functional L-type Cav1.2 Ca²⁺ channels measured as nimodipine sensitive Ca²⁺-influx upon depolarisation with 60 mmol/L KCl in response to treatment with systemic sclerosis microenvironment defining growth factors for 6 days. <b>A,</b> Bars represent the mean+SEM of nimodipine sensitive Ca²⁺-influx from 3 independent experiments. Control was set to 1. *P<0.05, **P<0.01, ***P<0.001. <b>B-F,</b> Representative recordings of Ca²⁺ dependent intracellular fluorescence intensities in response to depolarisation with KCl. Mean±SEM F/F0 of 30–40 cells per growth factor are plotted against time. Black lines represent tracings without the specific L-type calcium channel blocker nimodipine, grey lines those obtained after preincubation with 1 mmol/L nimodipine.</p

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