Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI.

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.

Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles.

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.

Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells.

The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-ß, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)-derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.

Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT-1 expression.

Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. 'Uraemic' and control EV (EVUR ; EVCTRL ) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL , EC- and plasma-derived EVUR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EVUR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EVUR . Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EVUR . In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD.

Non-contact monitoring of agitation and use of a sheltering device in patients with dementia in emergency departments: a feasibility study.

BACKGROUND: Agitation is common in geriatric patients with cognitive impairment, e.g. in persons with dementia (PWD), who are admitted to an emergency department (ED). It might be a first sign of upcoming delirium and is associated with a higher risk of an unfavorable clinical course. Hence, monitoring of vital signs and enhanced movement as indicators of upcoming agitation is essential in these patients during their stay in the ED. Since PWD rarely tolerate fixed monitoring devices, a novel developed non-contact monitoring system (NCMSys) might represent an appropriate alternative. Aim of this feasibility study was to test the validity of a NCMSys and of the tent-like "Charité Dome" (ChD), aimed to shelter PWD from the busy ED environment. Furthermore, effects of the ChD on wellbeing and agitation of PWD were investigated. METHODS: Both devices were attached to patient's bed. Tests on technical validity and safety issues of NCMSys and ChD were performed at the iDoc institute with six healthy volunteers. A feasibility study evaluating the reliability of the NCMSys with and without the ChD was performed in the real-life setting of an ED and on a geriatric-gerontopsychiatric ward. 19 patients were included, ten males and nine females; mean age: 77.4 (55-93) years of which 14 were PWD. PWD inclusion criteria were age ≥ 55 years, a dementia diagnosis and a written consent (by patients or by a custodian). Exclusion criteria were acute life-threatening situations and a missing consent. RESULTS: Measurements of heart rate, changes in movement and sound emissions by the NCMSys were valid, whereas patient movements affected respiratory rate measurements. The ChD did not impact patients' vital signs or movements in our study setting. However, 53% of the PWD (7/13) and most of the patients without dementia (4/5) benefited from its use regarding their agitation and overall wellbeing. CONCLUSIONS: The results of this feasibility study encourage a future controlled clinical trial in geriatric ED patients, including PWD, to further evaluate if our concept of non-contact measurement of vital signs and movement combined with the "Charité Dome" helps to prevent upcoming agitation in this vulnerable patient group in the ED. TRIAL REGISTRATION: ICTRP: "Charité-Dome-Study - DRKS00014737" (retrospectively registered).

Arterial tissue transcriptional profiles associate with tissue remodeling and cardiovascular phenotype in children with end-stage kidney disease.

Chronic kidney disease (CKD) greatly increases the risk for cardiovascular disease (CVD). However, molecular mechanisms underlying CKD-induced arterial remodeling are largely unknown. We performed a systematic analysis of arterial biopsies from children with stage 5 predialysis CKD participating in the Cardiovascular Comorbidity in Children with Chronic Kidney Disease (4 C) study. For comparison, we studied biopsies from children without CKD, coronary bypass vessels from adults with atherosclerotic coronary heart disease without CKD and aortic sections of subtotally nephrectomized rats. In pediatric CKD patients, gene expression was correlated to the cardiovascular phenotype assessed by surrogate end-points. The arterial calcium content correlated with the intima-media thickness (IMT) of biopsied vessels from pediatric CKD patients, was markedly increased compared to biopsies from children without CKD and comparable to adult coronary bypass patients. Significant transcriptional changes included ECM components, pro-calcifying factors, and physiological calcification inhibitors; most were highly accordant with changes observed in adults with atherosclerosis and in uremic rats. Individual gene expression levels were significantly associated with the left ventricular mass index and carotid intima media thickness. Thus, inflammatory processes (TNF, IL-10), calcification inhibitors (CA2), the Wnt-pathway (FGF-2) and foremost, ECM components (HMGA1, VNN1, VCAN), impact pathobiological responses in arteries from children with CKD.

Chronic kidney disease induces a systemic microangiopathy, tissue hypoxia and dysfunctional angiogenesis.

Chronic kidney disease (CKD) is associated with excessive mortality from cardiovascular disease (CVD). Endothelial dysfunction, an early manifestation of CVD, is consistently observed in CKD patients and might be linked to structural defects of the microcirculation including microvascular rarefaction. However, patterns of microvascular rarefaction in CKD and their relation to functional deficits in perfusion and oxygen delivery are currently unknown. In this in-vivo microscopy study of the cremaster muscle microcirculation in BALB/c mice with moderate to severe uremia, we show in two experimental models (adenine feeding or subtotal nephrectomy), that serum urea levels associate incrementally with a distinct microangiopathy. Structural changes were characterized by a heterogeneous pattern of focal microvascular rarefaction with loss of coherent microvascular networks resulting in large avascular areas. Corresponding microvascular dysfunction was evident by significantly diminished blood flow velocity, vascular tone, and oxygen uptake. Microvascular rarefaction in the cremaster muscle paralleled rarefaction in the myocardium, which was accompanied by a decrease in transcription levels not only of the transcriptional regulator HIF-1α, but also of its target genes Angpt-2, TIE-1 and TIE-2, Flkt-1 and MMP-9, indicating an impaired hypoxia-driven angiogenesis. Thus, experimental uremia in mice associates with systemic microvascular disease with rarefaction, tissue hypoxia and dysfunctional angiogenesis.

Collagen XIV and a related recombinant fragment protect human vascular smooth muscle cells from calcium-/phosphate-induced osteochondrocytic transdifferentiation.

Transdifferentiation of vascular smooth muscle cells (VSMC) promotes the development of vascular calcifications such as arteriosclerosis. The aim was to investigate effects of specific extracellular matrix (ECM) components on transdifferentiation of VSMC to identify novel ECM-based therapeutic tools. Human collagens I & IV (CI, CIV) along with collagen XIV (CXIV) and a CXIV-derived fragment (CXIV-F), both of which induce differentiation, were applied in an in-vitro model of calcium-/phosphate (Ca/P)-induced osteochondrocytic transdifferentiation of human and murine VSMC. Transdifferentiation was determined by RT-PCR and calcium contents of VSMC cultures. Signaling pathways involved were determined by western-blot and luciferase reporter plasmid assays. Under normal culture conditions, CI induced VSMC proliferation and a more epithelioid/synthetic phenotype while CIV and predominantly CXIV provoked opposite effects. CIV and CXIV further blocked Ca/P-induced osteochondrocytic transdifferentiation of VSMC displayed e.g. by reduced gene expressions of Runx2, Sox9, osterix and increased expressions of αSMA and SM22α. This involved impaired activation of ERK1/2, NF-ĸB and Wnt-signaling. Similar preventive effects were achieved by applying CXIV-F. Impaired preventive effects of CXIV by co-treatment with a cluster of differentiation (CD)44 agonist propose CD44 as a CXIV-target structure on VSMC. In conclusion, CXIV and CXIV-F interfere with osteochondrocytic transdifferentiation of VSMC and should be further explored as potential therapeutic tools in vascular calcification.

Reduced Microvascular Density in Omental Biopsies of Children with Chronic Kidney Disease.

BACKGROUND: Endothelial dysfunction is an early manifestation of cardiovascular disease (CVD) and consistently observed in patients with chronic kidney disease (CKD). We hypothesized that CKD is associated with systemic damage to the microcirculation, preceding macrovascular pathology. To assess the degree of "uremic microangiopathy", we have measured microvascular density in biopsies of the omentum of children with CKD. PATIENTS AND METHODS: Omental tissue was collected from 32 healthy children (0-18 years) undergoing elective abdominal surgery and from 23 age-matched cases with stage 5 CKD at the time of catheter insertion for initiation of peritoneal dialysis. Biopsies were analyzed by independent observers using either a manual or an automated imaging system for the assessment of microvascular density. Quantitative immunohistochemistry was performed for markers of autophagy and apoptosis, and for the abundance of the angiogenesis-regulating proteins VEGF-A, VEGF-R2, Angpt1 and Angpt2. RESULTS: Microvascular density was significantly reduced in uremic children compared to healthy controls, both by manual imaging with a digital microscope (median surface area 0.61% vs. 0.95%, p<0.0021 and by automated quantification (total microvascular surface area 0.89% vs. 1.17% p = 0.01). Density measured by manual imaging was significantly associated with age, height, weight and body surface area in CKD patients and healthy controls. In multivariate analysis, age and serum creatinine level were the only independent, significant predictors of microvascular density (r2 = 0.73). There was no immunohistochemical evidence for apoptosis or autophagy. Quantitative staining showed similar expression levels of the angiogenesis regulators VEGF-A, VEGF-receptor 2 and Angpt1 (p = 0.11), but Angpt2 was significantly lower in CKD children (p = 0.01). CONCLUSIONS: Microvascular density is profoundly reduced in omental biopsies of children with stage 5 CKD and associated with diminished Angpt2 signaling. Microvascular rarefaction could be an early systemic manifestation of CKD-induced cardiovascular disease.

Wnt signaling contributes to vascular calcification by induction of matrix metalloproteinases.

BACKGROUND: Vascular calcifications such as arteriosclerosis, which is characterized by a calcificiation of the tunica media, represent major comorbidities e.g. in patients with chronic kidney disease (CKD). An essential step during the development of arteriosclerosis is the transdifferentiation/calcification of vascular smooth muscle cells (VSMC) resembling osteogenesis. The matrix metalloproteinases (MMP)-2 and -9 were shown to promote these VSMC calcifications and their inhibition is of therapeutic value to prevent arteriosclerosis in preclinical studies. Aiming for an understanding of the underlying regulatory mechanisms of MMPs we here investigated, if the MMP-mediated VSMC calcification involves altered signaling of the Wnt pathway, which is known to impact osteogenesis. METHODS: We used an experimental in vitro model of vascular calcification. Transdifferentiation/calcification of murine VSMC was induced by elevated calcium and phosphorus levels. Calcification was assessed by calcium and alkaline phosphatase measurements. Activation/activity of the gelatinases MMP-2 and MMP-9 was assessed by conversion of fluorescence-labelled substrates. Activation of the Wnt pathway was analysed by a reporter gene assay. RESULTS: Besides pro-calcifying culture conditions, also activation of Wnt signaling by a specific agonist (under normal culture conditions) stimulated VSMC-calcification accompanied by enhanced expression and secretion of the gelatinases MMP-2 and -9. Vice versa, recombinant MMP-2 and -9 induced a time-delayed activation of Wnt signaling after 72 h in VSMC but showed no direct effects after 24-48 h. These effects were blocked by pharmacological inhibition of MMPs or of Wnt signaling. CONCLUSIONS: Our study suggests that the pro-calcifying environment in CKD induces Wnt signaling in VSMC which in turn contributes to the induction of MMPs which then foster the development of arteriosclerosis. Thus, besides MMP inhibition, the inhibition of Wnt signaling in VSMC might represent a therapeutic target for the prevention of vascular calcifications.

The matrix metalloproteinases 2 and 9 initiate uraemic vascular calcifications.

BACKGROUND: The matrix metalloproteinases (MMP) MMP-2 and MMP-9 are physiological regulators of vascular remodelling. Their dysregulation could contribute to vascular calcification. We examined the role of the MMP-2 and MMP-9 in uraemic vascular calcification in vivo and in vitro. METHODS: The impact of pharmacological MMP inhibition on the development of media calcifications was explored in an aggressive animal model of uraemic calcification. In addition, the selective effects of addition and inhibition, respectively, of MMP-2 and MMP-9 on calcium-/phosphate-induced calcifications were studied in a murine cell line of vascular smooth muscle cells (VSMCs). RESULTS: High-dose calcitriol treatment of uraemic rats given a high phosphate diet induced massive calcifications, apoptosis and increased gene expressions of MMP-2, MMP-9 and of osteogenic transcription factors and proteins in aortic VSMC. The MMP inhibitor doxycycline prevented the VSMC transdifferentiation to osteoblastic cells, suppressed transcription of mediators of matrix remodelling and almost completely blocked aortic calcifications while further increasing apoptosis. Similarly, specific inhibitors of either MMP-2 or -9, or of both gelatinases (Ro28-2653) and a selective knockdown of MMP-2/-9 mRNA expression blocked calcification of murine VSMC induced by calcification medium (CM). In contrast to MMP inhibition, recombinant MMP-2 or MMP-9 enhanced CM-induced calcifications and the secretion of gelatinases. CONCLUSIONS: These data indicate that both gelatinases provide essential signals for phenotypic VSMC conversion, matrix remodelling and the initiation of vascular calcification. Their inhibition seems a promising strategy in the prevention of vascular calcifications.

A Lindera obtusiloba Extract Blocks Calcium-/Phosphate-Induced Transdifferentiation and Calcification of Vascular Smooth Muscle Cells and Interferes with Matrix Metalloproteinase-2 and Metalloproteinase-9 and NF-κB.

Vascular calcifications bear the risk for cardiovascular complications and have a high prevalence among patients with chronic kidney disease. Central mediators of vascular calcifications are vascular smooth muscle cells (VSMC). They transdifferentiate into a synthetic/osteoblast-like phenotype, which is induced, for example, by elevated levels of calcium and phosphate (Ca/P) due to a disturbed mineral balance. An aqueous extract from Lindera obtusiloba (LOE) is known to exert antifibrotic and antitumor effects or to interfere with the differentiation of preadipocytes. Using murine and rat VSMC cell lines, we here investigated whether LOE also protects VSMC from Ca/P-induced calcification. Indeed, LOE effectively blocked Ca/P-induced calcification of VSMC as shown by decreased VSMC mineralization and secretion of alkaline phosphatase. In parallel, mRNA expression of the calcification markers osterix and osteocalcin was reduced. Vice versa, the Ca/P-induced loss of the VSMC differentiation markers alpha smooth muscle actin and smooth muscle protein 22-alpha was rescued by LOE. Further, LOE blocked Ca/P-induced mRNA expressions and secretions of matrix metalloproteinases-2/-9 and activation of NF-κB, which are known contributors to vascular calcification. In conclusion, LOE interferes with the Ca/P-induced transdifferentiation/calcification of VSMC. Thus, LOE should be further analysed regarding a potential complementary treatment option for cardiovascular diseases including vascular calcifications.

K⁺-channel inhibition reduces portal perfusion pressure in fibrotic rats and fibrosis associated characteristics of hepatic stellate cells.

BACKGROUND & AIMS: In liver fibrosis, activated hepatic stellate cells (HSC) secrete excess extracellular matrix, thus, represent key targets for antifibrotic treatment strategies. Intermediate-conductance Ca(2) (+) -activated K(+) -channels (KCa3.1) are expressed in non-excitable tissues affecting proliferation, migration and vascular resistance rendering KCa3.1 potential targets in liver fibrosis. So far, no information about KCa3.1 expression and their role in HSC exists. Aim was to quantify the KCa3.1 expression in HSC depending on HSC activation and investigation of antifibrotic properties of the specific KCa3.1 inhibitor TRAM-34 in vitro and in vivo. METHODS: KCa3.1 expression and functionality were studied in TGF-ß1-activated HSC by quantitative real time PCR, western-blot and patch-clamp analysis respectively. Effects of TRAM-34 on HSC proliferation, cell cycle and fibrosis-related gene expression were assessed by [(3) H]-thymidine incorporation, FACS-analysis and RT-PCR respectively. In vivo, vascular resistance and KCa3.1 gene and protein expression were determined in bile duct ligated rats by in situ liver perfusion, Taqman PCR and immunohistochemistry respectively. RESULTS: Fibrotic tissues and TGF-ß1-activated HSC exhibited higher KCa3.1-expressions than normal tissue and untreated cells. KCa3.1 inhibition with TRAM-34 reduced HSC proliferation by induction of cell cycle arrest and reduced TGF-ß1-induced gene expression of collagen I, alpha-smooth muscle actin and TGF-ß1 itself. Furthermore, TRAM-34 blocked TGF-ß1-induced activation of TGF-ß signalling in HSC. In vivo, TRAM-34 reduced the thromboxane agonist-induced portal perfusion pressure. CONCLUSION: Inhibition of KCa3.1 with TRAM-34 downregulates fibrosis-associated gene expression in vitro, and reduces portal perfusion pressure in vivo. Thus, KCa3.1 may represent novel targets for the treatment of liver fibrosis.

Inhibition of vascular calcification by block of intermediate conductance calcium-activated potassium channels with TRAM-34.

Vascular calcifications are a hallmark of advanced cardiovascular disease in patients with chronic kidney disease. A key event is the transition of contractile vascular smooth muscle cells (VSMC) into an osteoblast-like phenotype, promoting a coordinated process of vascular remodeling resembling bone mineralization. Intermediate-conductance calcium-activated potassium channels (KCa3.1) are expressed in various tissues including VSMC. Aiming for novel therapeutic targets in vascular calcification, we here studied effects of KCa3.1-inhibition on VSMC calcification by the specific KCa3.1 inhibitor TRAM-34. Calcification in the murine VSMC cell line MOVAS-1 and primary rat VSMC was induced by calcification medium (CM) containing elevated levels of PO4(3-) and Ca(2+). Cell signaling, calcification markers, and release of nitric oxide and alkaline phosphatase were assessed by luciferase reporter plasmids, RT-PCR and specific enzymatic assays, respectively. KCa3.1 gene silencing was achieved by siRNA experiments. TRAM-34 at 10nmol/l, decreased CM-induced calcification and induced NO release of VSMC accompanied by decreased TGF-ß signaling. The CM-induced mRNA expressions of osterix, osteocalcin, matrix-metalloproteinases (MMP)-2/-9 were reduced by TRAM-34 while osteopontin expression was increased. Further, TRAM-34 attenuated the CM- and TNF-α-induced activation of NF-κB and reduced the release of MMP-2/-9 by VSMC. Finally, TRAM-34 abrogated CM-induced apoptosis and KCa3.1 gene silencing protected VSMC from CM-induced onset of calcification. In summary, TRAM-34 interferes with calcification relevant signaling of NF-κB and TGF-ß thereby blocking the phenotypic transition/calcification of VSMC. We conclude that the results provide a rationale for further studies regarding a possible therapeutic role of KCa3.1 inhibition by TRAM-34 or other inhibitors in vascular calcification.

Wideband MRE and static mechanical indentation of human liver specimen: sensitivity of viscoelastic constants to the alteration of tissue structure in hepatic fibrosis.

Despite the success of elastography in grading hepatic fibrosis by stiffness related noninvasive markers the relationship between viscoelastic constants in the liver and tissue structure remains unclear. We therefore studied the mechanical properties of 16 human liver specimens with different degrees of fibrosis, inflammation and steatosis by wideband magnetic resonance elastography (MRE) and static indentation experiments providing the specimens׳ static Young׳s modulus (E), dynamic storage modulus (G') and dynamic loss modulus (G″). A frequency-independent shear modulus µ and a powerlaw exponent α were obtained by fitting G' and G″ using the two-parameter sprinpot model. The mechanical parameters were compared to the specimens׳ histology derived parameters such as degree of Fibrosis (F), inflammation score and fat score, amount of hydroxyproline (HYP) used for quantification of collagen, blood markers and presurgery in vivo function tests. The frequency averaged parameters G', G″ and µ were significantly correlated with F (G': R=0.762, G″: R=0.830; µ: R=0.744; all P<0.01) and HYP (G': R=0.712; G″: R=0.720; µ: R=0.731; all P<0.01). The powerlaw exponent α displayed an inverse correlation with F (R=-0.590, P=0.034) and a trend of inverse correlation with HYP (R=-0.470, P=0.089). The static Young׳s modulus E was less correlated with F (R=0.587, P=0.022) and not sensitive to HYP. Although inflammation was highly correlated with F (R=0.773, P<0.001), no interaction was discernable between inflammation and mechanical parameters measured in this study. Other histological and blood markers as well as liver function test were correlated with neither F nor the measured mechanical parameters. In conclusion, viscoelastic constants measured by wideband MRE are highly sensitive to histologically proven fibrosis. Our results suggest that, in addition to the amount of connective tissue, subtle structural changes of the viscoelastic matrix determine the sensitivity of mechanical tissue properties to hepatic fibrosis.

The inhibitor of Ca(2+)-dependent K+ channels TRAM-34 blocks growth of hepatocellular carcinoma cells via downregulation of estrogen receptor alpha mRNA and nuclear factor-kappaB.

Hepatocellular carcinoma (HCC) is the most common liver malignancy still demanding for novel therapeutic options. Since the ion channel inhibitor TRAM-34 (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole) was shown to block growth in various cancer cells, we investigated anti-tumor effects of TRAM-34 in human HCC cell lines. We found that TRAM-34 reduced HCC cell proliferation without induction of apoptosis. This was due to a decreased mRNA expression of estrogen receptor alpha (ESR1) and a reduced activation of NF-kappaB, which both are implicated in the development of HCC. Therefore, TRAM-34 might represent a novel therapeutic target for the treatment of HCC.

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPARγ and induction of iNOS.

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC50 of 10µM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and ß-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.

(+)-Episesamin exerts anti-neoplastic effects in human hepatocellular carcinoma cell lines via suppression of nuclear factor-kappa B and inhibition of MMP-9.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Treatment options, especially in advanced tumor stages, are still limited. Inhibition of signaling cascades involved in the pathogenesis of HCC - such as NF-ĸB - offer a promising therapeutic approach. Aim of this study was to examine anti-neoplastic effects of (+)-episesamin which has been isolated from an anti-fibrotic extract of Lindera obtusiloba on human HCC cells with particular interest in activation of NF-κB. The human HCC cell lines HepG2, Huh-7 and SK-Hep1 were treated with (+)-episesamin. Beside measurement of proliferation, invasion and apoptosis, effects of (+)-episesamin on NF-κB-activity, VEGF secretion and enzymatic MMP-9 activity were determined. Anti-inflammatory effects were assessed by IL-6 ELISA using HCC cells and RAW264.7 macrophages. 10 µM (+)-episesamin reduced the proliferation of HCC cells by ~50%, suppressed invasion and induced apoptosis. DNA-binding ELISA experiments revealed that (+)-episesamin treated HCC cells showed a suppressed basal and TNFα-induced activation of NF-κB and a subsequent suppression of TNFα- and LPS-induced IL-6 production. Further, (+)-episesamin exhibited inhibitory effects on the enzymatic activity of recombinant MMP-9 and the secretion of MMP-9 and VEGF by HCC cells into their supernatants. Our findings show that anti-neoplastic effects of (+)-episesamin are mediated via suppressed activation of NF-κB which entails a decreased release of pro-inflammatory IL-6. In addition, (+)-episesamin inhibits MMP-9, which is strongly expressed in invasive HCC, and the production of proangiogenic VEGF. We conclude that (+)-episesamin has the potential to be further explored as a complementary treatment for HCC.