Angelika Kusch

Non-HLA-antibodies targeting Angiotensin type 1 receptor and antibody mediated rejection.

Antibody-mediated mechanisms directed against non-HLA related targets may exert negative impact on allograft function and survival. Angiotensin type 1 receptor (AT(1)R) emerges as a functional target for non-HLA allo- and autoantibodies (AT(1)R-Abs) comprising of IgG1 and IgG3 subclasses. Proof of concept for pathophysiologic relevance of AT(1)R-Abs in antibody mediated rejection (AMR) in renal transplants was provided by passive transfer studies in animal model and therapeutic rescue of patients. Although AT(1)R-Abs may belong to complement fixing IgG subclasses, C4d positivity in renal transplant biopsies was not frequently detected implicating complement independent mechanisms of injury. AT(1)R-Abs exert direct effects on endothelial and vascular smooth muscle cells by induction of Erk1/2 signaling and increased DNA binding of transcription factors associated with pro-inflammatory and pro-coagulatory responses. Establishment of enzyme-linked immunosorbent assay employing extracts of cells overexpressing AT(1)R in its native conformation was instrumental for recent studies in independent cohorts. Assessing the AT(1)R-Ab-status along with the HLA-antibodies may help to identify patients at particular risk for irreversible acute or chronic allograft injuries and improve overall outcomes. This review summarizes the current state of research in AT(1)R biology, development in diagnostic strategies, discusses recent clinical studies, and provides perspectives on further refinements in understanding AT(1)R-Ab-actions.

Estrogen Receptor-β Signals Left Ventricular Hypertrophy Sex Differences in Normotensive Deoxycorticosterone Acetate-Salt Mice

We found earlier that deoxycorticosterone acetate-salt treatment causes blood pressure–independent left ventricular hypertrophy, but only in male mice. To test the hypothesis that the estrogen receptor-&bgr; (ER&bgr;) protects the females from left ventricular hypertrophy, we treated male and female ER&bgr;-deficient (ER&bgr;−/−) mice and their male and female littermates (wild-type [WT]) with deoxycorticosterone acetate-salt and made them telemetrically normotensive with hydralazine. WT males had increased (+16%) heart weight/tibia length ratios compared with WT females (+7%) at 6 weeks. In ER&bgr;−/− mice, this situation was reversed. Female WT mice had the greatest heart weight/tibia length ratio increases of all of the groups (+23%), even greater than ER&bgr;−/− males (+10%). Echocardiography revealed concentric left ventricular hypertrophy in male WT mice, whereas ER&bgr;−/− females developed dilative left ventricular hypertrophy. The hypertrophic response in female ER&bgr;−/− mice was accompanied by the highest degree of collagen deposition, indicating maladaptive remodeling. ER&bgr;+/+ females showed robust protective p38 and extracellular signal–regulated kinase 1/2 signaling relationships compared with other groups. Calcineurin A&bgr; expression and its positive regulator myocyte-enriched calcineurin-interacting protein 1 were increased in deoxycorticosterone acetate-salt female ER&bgr;−/− mice, yet lower than in WT males. Endothelin increased murine cardiomyocyte hypertrophy in vitro, which could be blocked by estradiol and an ER&bgr; agonist. We conclude that a functional ER&bgr; is essential for inducing adaptive p38 and extracellular signal–regulated kinase signaling, while reducing maladaptive calcineurin signaling in normotensive deoxycorticosterone acetate female mice. Our findings address the possibility of sex-specific cardiovascular therapies.

Sex differences in exercise-induced physiological myocardial hypertrophy are modulated by oestrogen receptor beta

AIMS Oestrogen receptor alpha (ERα) and beta (ERβ) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action. METHODS AND RESULTS We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERβ-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERβ-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERβ deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERβ-deleted mice. CONCLUSION The sex-specific response of the heart to exercise is modulated by ERβ. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERβ.

mTOR Regulates Vascular Smooth Muscle Cell Differentiation From Human Bone Marrow–Derived Mesenchymal Progenitors

Objective—Vascular smooth muscle cells (VSMCs) and circulating mesenchymal progenitor cells (MSCs) with a VSMC phenotype contribute to neointima formation and lumen loss after angioplasty and during allograft arteriosclerosis. We hypothesized that phosphoinositol-Akt-mammalian target of rapamycin-p70S6 kinase (PI3K/Akt/mTOR/p70S6K) pathway activation regulates VSMC differentiation from MSCs. Methods and Results—We studied effects of PI3K/Akt/mTOR signaling on phenotypic modulation of MSC and VSMC marker expression, including L-type Ca(2+) channels. Phosphorylation of Akt and p70S6K featured downregulation of VSMC markers in dedifferentiated MSCs. mTOR inhibition with rapamycin at below pharmacological concentrations blocked p70S6K phosphorylation and induced a differentiated contractile phenotype with smooth muscle (sm)-calponin, sm-&agr;-actin, and SM protein 22-alpha (SM22&agr;) expression. The PI3K inhibitor Ly294002 abolished Akt and p70S6K phosphorylation and reversed the dedifferentiated phenotype via induction of sm-calponin, sm-&agr;-actin, SM22&agr;, and myosin light chain kinase. Rapamycin acted antiproliferative without impairing MSC viability. In VSMCs, rapamycin increased a homing chemokine for MSCs, stromal cell–derived factor-1–alpha, at mRNA and protein levels. The CXCR4-mediated MSC migration toward conditioned medium of rapamycin-treated VSMCs was enhanced. Conclusions—We describe novel pleiotropic effects of rapamycin at very low concentrations that stabilized differentiated contractile VSMCs from MSCs in addition to exerting antiproliferative and enhanced homing effects.

Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model.

Abstract Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinasetype plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral bloodderived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPAinduced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture fivefold, whereas VSMC display an increased expression of cell surfaceassociated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

Stat1 Nuclear Translocation by Nucleolin upon Monocyte Differentiation

Background Members of the signal transducer and activator of transcription (Stat) family of transcription factors traverse the nuclear membrane through a specialized structure, called the nuclear pore complex (NPC), which represents a selective filter for the import of proteins. Karyophilic molecules can bind directly to a subset of proteins of the NPC, collectively called nucleoporins. Alternatively, the transport is mediated via a carrier molecule belonging to the importin/karyopherin superfamily, which transmits the import into the nucleus through the NPC. Methodology/Principal Findings In this study, we provide evidence for an alternative Stat1 nuclear import mechanism, which is mediated by the shuttle protein nucleolin. We observed Stat1-nucleolin association, nuclear translocation and specific binding to the regulatory DNA element GAS. Using expression of nucleolin transgenes, we found that the nuclear localization signal (NLS) of nucleolin is responsible for Stat1 nuclear translocation. We show that this mechanism is utilized upon differentiation of myeloid cells and is specific for the differentiation step from monocytes to macrophages. Conclusions/Significance Our data add the nucleolin-Stat1 complex as a novel functional partner for the cell differentiation program, which is uniquely poised to regulate the transcription machinery via Stat1 and nuclear metabolism via nucleolin.

Recovery of Senescent Endothelial Cells From Injury

Percutaneous coronary intervention is increasingly performed in elderly patients. Because the procedure is associated with endothelial cell (EC) denudation, we compared recovery of young and old ECs from scratch injuries inflicted in culture. Although senescent ECs displayed markedly reduced potential to proliferate and migrate, they repopulated the wounds as fast as young cells. Morphometric analysis revealed that senescent cells were significantly larger and as a result far fewer senescent cells managed to cover the lesion. Compared with young EC, senescent cells displayed increased expression of senescence-associated β-galactosidase, nitric oxide synthase (eNOS), and AKT kinase, and secreted increased amounts of growth factors (VEGF, TGF-β), cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1), and matrix proteins (fibronectin). This secretory phenotype rather than the rate of wound closure per se may contribute to unfavorable vascular remodeling in the elderly undergoing coronary catheterization.

Sex-Specific mTOR Signaling Determines Sexual Dimorphism in Myocardial Adaptation in Normotensive DOCA-Salt Model

The deoxycorticosterone acetate (DOCA)-salt mouse model exhibits adverse cardiac remodeling in male mice and cardiac protection in female mice, even when blood pressure is normalized. We hypothesized that intact mammalian target of rapamycin (mTOR) signaling is necessary for cardiac protection in females. We first tested sex differences and intracellular signaling after mTOR targeting with rapamycin in wild-type mice. Radio-telemetric blood pressure was maintained at normal for 6 weeks. Rapamycin significantly reduced left ventricular hypertrophy, preserved ejection fraction, inhibited fibrosis, and maintained capillary structure in male mice. Decreased mTORC1 and increased mTORC2 activity were detected in rapamycin-treated male mice compared with vehicle controls. In contrast, female mice developed dilative left ventricular hypertrophy, cardiac fibrosis, and capillary loss similar to DOCA-salt females lacking the estrogen receptor &bgr; (ER&bgr;−/−) that we described earlier. Because rapamycin downregulated ER&bgr; in female mice, we next studied ER&bgr;−/− normotensive DOCA-salt females. Vehicle-treated wild-type females maintained their high constitutive mTORC1 and mTORC2 in response to DOCA-salt. In contrast to males, both mTORCs were decreased by rapamycin, in particular mTORC2 by 60%. ER&bgr;−/− DOCA-salt females showed similar mTORC1 and mTORC2 response patterns. We suggest that ER&bgr;-dependent regulation involves sex-specific use of mTOR signaling branches. Maintenance of both mTORC1 and mTORC2 signaling seems to be essential for adaptive cardiac remodeling in females and supports a rationale for sex-specific therapeutic strategies in left ventricular hypertrophy.

Protein kinase C inhibition ameliorates posttransplantation preservation injury in rat renal transplants

Background Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca2+-dependent and Ca2+-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation. Methods Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry. Results Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal–regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action. Conclusions The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

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”.