Lactate Promotes Synthetic Phenotype in Vascular Smooth Muscle Cells.

RATIONALE: The phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. OBJECTIVE: To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs. METHODS AND RESULTS: Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L-) medium. Compared with the L- medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues. CONCLUSIONS: These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Marathon performance but not BMI affects post-marathon pro-inflammatory and cartilage biomarkers.

We tested the hypothesis that changes in serum cartilage oligomeric matrix protein (COMP), tumour necrosis factor α (TNF-α), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) concentration after regular endurance training and running a marathon race depend on body mass index (BMI) and/or on marathon performance. Blood samples were collected from 45 runners of varying BMI and running experience before and after a 10-week marathon training programme and before, immediately and 24 h after a marathon race. Serum biomarker concentrations, BMI and marathon finishing time were measured. The mean (95% confidence interval (CI)) changes from before to immediately after the marathon were COMP: 4.09 U/L (3.39-4.79 U/L); TNF-α: -1.17 mg/L (-2.58 to 0.25 mg/L); IL-6: 12.0 pg/mL (11.4-12.5 pg/mL); and hsCRP: -0.08 pg/mL (-0.14 to -0.3 pg/mL). The mean (95% CI) changes from immediately after to 24 h after the marathon were COMP: 0.35 U/L (-0.88 to 1.57 U/L); TNF-α: -0.43 mg/L (-0.99 to 0.13 mg/L); IL-6: -9.9 pg/mL (-10.5 to -9.4 pg/mL); and hsCRP: 1.52 pg/mL (1.25-1.79 pg/mL). BMI did not affect changes in biomarker concentrations. Differences in marathon finishing time explained 32% of variability in changes in serum hsCRP and 28% of variability in changes in serum COMP during the 24 h recovery after the marathon race (P < 0.001). Slower marathon finishing time but not a higher BMI modulates increases in pro-inflammatory markers or cartilage markers following a marathon race.

Cell matrix contact modifies endothelial major histocompatibility complex class II expression in high-glucose environment.

Atherosclerosis is a chronic inflammatory disease. Cardiovascular risk factors such as hyperglycemia, hyperlipidemia, and arterial hypertension induce endothelial dysfunction with alterations in endothelial biosecretion and immune behavior. The aim of this study is to elucidate whether glucose-induced modifications of endothelial biosecretory and immune functions are regulated by interactions of endothelial cells (ECs) with their extracellular matrix [ECs plated on polystyrene-coated tissue culture plates (TC-EC) vs. ECs embedded within three-dimensional (3-D) collagen-based matrixes (3D-EC)]. In the absence of glucose, IFN-γ-induced phosphorylation of JAK and STAT proteins and human leukocyte antigen (HLA)-DR expression were lower in 3D-EC compared with TC-EC. Inversely, the expression of suppressor of cytokine signaling proteins (SOCS)-1 and -3 were significantly higher in naïve 3D-EC compared with naïve TC-EC. IFN-γ-induced upregulation of SOCS proteins was further amplified by the 3-D environment. Glucose significantly augmented IFN-γ-dependent signaling pathways in TC-EC. IFN-γ-induced phosphorylation of JAK and STAT proteins as well as HLA-DR expression by ECs in low- and high-glucose medium was significantly lower in 3-D than in two-dimensional environment. Glucose increased SOCS expression in TC-EC and 3D-EC to the same extent, such that expression levels in 3D-EC exceeded SOCS-1 and -3 expression in TC-EC by 1.6-2.5-fold. In conclusion, low- and high-glucose concentrations amplify IFN-γ-induced signaling pathways in TC-EC. Increased SOCS expression raises the threshold for IFN-γ to induce HLA-DR expression in a 3-D environment. This immunoprotective effect is maintained even in states of experimental hyperglycemia.

Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment.

The stress hormone-regulating hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the causality as well as the treatment of depression. To investigate a possible association between genes regulating the HPA axis and response to antidepressants and susceptibility for depression, we genotyped single-nucleotide polymorphisms in eight of these genes in depressed individuals and matched controls. We found significant associations of response to antidepressants and the recurrence of depressive episodes with single-nucleotide polymorphisms in FKBP5, a glucocorticoid receptor-regulating cochaperone of hsp-90, in two independent samples. These single-nucleotide polymorphisms were also associated with increased intracellular FKBP5 protein expression, which triggers adaptive changes in glucocorticoid receptor and, thereby, HPA-axis regulation. Individuals carrying the associated genotypes had less HPA-axis hyperactivity during the depressive episode. We propose that the FKBP5 variant-dependent alterations in HPA-axis regulation could be related to the faster response to antidepressant drug treatment and the increased recurrence of depressive episodes observed in this subgroup of depressed individuals. These findings support a central role of genes regulating the HPA axis in the causality of depression and the mechanism of action of antidepressant drugs.

Modulation of dendritic cells and toll-like receptors by marathon running.

The focus of this study was to assess exercise-induced alterations of circulating dendritic cell (DC) subpopulations and toll-like receptor (TLR) expression after marathon running. Blood sampling was performed in 15 obese non-elite (ONE), 16 lean non-elite (LNE) and 16 lean elite (LE) marathon runners pre- and post-marathon as well as 24 h after the race. Circulating DC-fractions were measured by flow-cytometry analyzing myeloid DCs (BDCA-1+) and plasmacytoid DCs (BDCA-2+). We further analyzed the (TLR) -2/-4/-7 in peripheral blood mononuclear cells (rt-PCR/Western Blot) and the cytokines CRP, IL-6, IL-10, TNF-α and oxLDL by ELISA. After the marathon, BDCA-1 increased significantly in all groups [LE (pre/post): 0.35/0.47%; LNE: 0.26/0.50% and ONE: 0.30/0.49%; all p < 0.05]. In contrast, we found a significant decrease for BDCA-2 directly after the marathon (LE: 0.09/0.01%; LNE: 0.12/0.03% and ONE: 0.10/0.02%; all p < 0.05). Levels of TLR-7 mRNA decreased in all groups post-marathon (LE 44%, LNE 67% and ONE 52%; all p < 0.01), with a consecutive protein reduction (LE 31%, LNE 52%, ONE 42%; all p < 0.05) 24 h later. IL-6 and IL-10 levels increased immediately after the run, whereas increases of TNF-α and CRP-levels were seen after 24 h. oxLDL levels remained unchanged post-marathon. In our study population, we did not find any relevant differences regarding training level or body weight. Prolonged endurance exercise induces both pro- and anti-inflammatory cytokines. Anti-inflammatory cytokines, such as IL-10, may help to prevent excessive oxidative stress. Marathon running is associated with alterations of DC subsets and TLR-expression independent of training level or body weight. Myeloid and plasmacytoid DCs are differently affected by the excessive physical stress. Immunomodulatory mechanisms seem to play a key role in the response and adaptation to acute excessive exercise.

oxLDL downregulates the dendritic cell homing factors CCR7 and CCL21.

INTRODUCTION: Dendritic cells (DCs) and oxLDL play an important role in the atherosclerotic process with DCs accumulating in the plaques during plaque progression. Our aim was to investigate the role of oxLDL in the modulation of the DC homing-receptor CCR7 and endothelial-ligand CCL21. METHODS AND RESULTS: The expression of the DC homing-receptor CCR7 and its endothelial-ligand CCL21 was examined on atherosclerotic carotic plaques of 47 patients via qRT-PCR and immunofluorescence. In vitro, we studied the expression of CCR7 on DCs and CCL21 on human microvascular endothelial cells (HMECs) in response to oxLDL. CCL21- and CCR7-mRNA levels were significantly downregulated in atherosclerotic plaques versus non-atherosclerotic controls [90% for CCL21 and 81% for CCR7 (P < 0.01)]. In vitro, oxLDL reduced CCR7 mRNA levels on DCs by 30% and protein levels by 46%. Furthermore, mRNA expression of CCL21 was significantly reduced by 50% (P < 0.05) and protein expression by 24% in HMECs by oxLDL (P < 0.05). CONCLUSIONS: The accumulation of DCs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands, which are known to regulate DC migration. oxLDL induces an in vitro downregulation of CCR7 and CCL21, which may play a role in the reduction of DC migration from the plaques.

Magnetic resonance imaging of myocardial injury and ventricular torsion after marathon running.

Recent reports provide indirect evidence of myocardial injury and ventricular dysfunction after prolonged exercise. However, existing data is conflicting and lacks direct verification of functional myocardial alterations by CMR [cardiac MR (magnetic resonance)]. The present study sought to examine structural myocardial damage and modification of LV (left ventricular) wall motion by CMR imaging directly after a marathon. Analysis of cTnT (cardiac troponin T) and NT-proBNP (N-terminal pro-brain natriuretic peptide) serum levels, echocardiography [pulsed-wave and TD (tissue Doppler)] and CMR were performed before and after amateur marathon races in 28 healthy males aged 41 ± 5 years. CMR included LGE (late gadolinium enhancement) and myocardial tagging to assess myocardial injury and ventricular motion patterns. Echocardiography indicated alterations of diastolic filling [decrease in E/A (early transmitral diastolic filling velocity/late transmitral diastolic filling velocity) ratio and E' (tissue Doppler early transmitral diastolic filling velocity)] postmarathon. All participants had a significant increase in NT-proBNP and/or cTnT levels. However, we found no evidence of LV LGE. MR tagging demonstrated unaltered radial shortening, circumferential and longitudinal strain. Myocardial rotation analysis, however, revealed an increase of maximal torsion by 18.3% (13.1 ± 3.8 to 15.5 ± 3.6 °; P=0.002) and maximal torsion velocity by 35% (6.8 ± 1.6 to 9.2 ± 2.5 °·s-1; P<0.001). Apical rotation velocity during diastolic filling was increased by 1.23 ± 0.33 °·s-1 after marathon (P<0.001) in a multivariate analysis adjusted for heart rate, whereas peak untwist rate showed no relevant changes. Although marathon running leads to a transient increase of cardiac biomarkers, no detectable myocardial necrosis was observed as evidenced by LGE MRI (MR imaging). Endurance exercise induces an augmented systolic wringing motion of the myocardium and increased diastolic filling velocities. The stress of marathon running seems to be better described as a burden of myocardial overstimulation rather than cardiac injury.

Immunoregulatory effects of the flavonol quercetin in vitro and in vivo.

PURPOSE: Atherosclerosis is known to be an inflammatory disease. Dendritic cells (DCs) are essential for the regulation of the immune system. Up to 10% of the cells in atherosclerotic plaques are DCs. The cardiovascular protective effects of flavonoids (tea, wine) may be mediated by anti-inflammatory mechanisms that affect DC regulation. We aimed to characterize the impact of the flavonol quercetin on DC activity and differentiation in vitro and in vivo. METHODS: For the in vitro experiments, we used murine DCs and endothelial cells to study adhesion properties. For all other experiments (DC phagocytosis capacity, DC maturation, DC differentiation (BDCA-1/-2) and NF-kB-activation), human monocyte-derived DCs were used. The cells were incubated with quercetin (10 µmol/L) ± oxLDL (10 µg/mL) between 24 and 48 h. For in vivo experiments, eight healthy male volunteers took 500 mg of quercetin twice daily over 4 weeks, five healthy male volunteers served as control. Before and after intake, blood samples were collected. Peripheral blood leukocytes were isolated (analyses of DC differentiation), and plasma was immediately frozen. RESULTS: Quercetin reduced DC adhesion (-42%; p < 0.05) and expression of CD11a (-21%; p < 0.05). OxLDL-induced DC differentiation was partially inhibited by quercetin (BDCA-1-29%; BDCA-2-33%; p < 0.05). These effects were achieved by compensation of oxLDL-induced up-regulation of NF-kB by quercetin. The 4-week treatment with quercetin resulted in relevant plasma levels (2.47 µmol/L) and reduced BDCA-2 + DCs in the peripheral blood by 42% (p < 0.05) as well as systemic levels of the NO-synthase inhibitor asymmetric dimethylarginine (-31%, p < 0.05). CONCLUSION: In vitro, quercetin reduced DC adhesion and oxLDL-induced DC differentiation. In vivo, quercetin reduced circulating plasmacytoid DCs and systemic ADMA-levels. The immunoregulatory effects of quercetin may contribute to the anti-atherosclerotic potential of flavonols.

Immunomodulatory effects of aerobic training in obesity.

INTRODUCTION: Physical inactivity and obesity are independent risk factors for atherosclerosis. We analyzed the immunomodulatory capacity of 10-week intensified exercise training (ET) in obese and lean athletes. Markers of the innate immune response were investigated in obese (ONE: ET≤40 km/week) and lean athletes (LNE: ET≤40 km/week and LE: ET≥55 km/week). METHODS: Circulating dendritic cells (DC) were analyzed by flow-cytometry for BDCA-1/-2-expression. TLR-2/-4/-7 and MyD88 were analyzed by RT-PCR and Western blot. Circulating oxLDL levels were analyzed by ELISA. RESULTS: BDCA-1 expression at baseline was lower in ONE compared to both other groups (ONE 0.15%; LNE 0.27%; LE 0.33%; P < .05), but significantly increased in ONE after training (+50%; P < .05). In contrast, BDCA-2 expression at baseline was higher in ONE (ONE 0.25%; LNE 0.11%; LE 0.09%; P < .05) and decreased in ONE after the 10-week training period (-27%; P < .05). Gene activations of TLR-4 and TLR-7 with corresponding protein increase were found for all three groups (P < .01/P < .05) compared to pre training. A reduction of oxLDL levels was seen in ONE (-61%; P < .05). CONCLUSIONS: Intensified exercise induces an increase of BDCA-1+ DCs and TLR-4/-7 in obese athletes. We hereby describe new immune modulatory effects, which-through regular aerobic exercise-modulate innate immunity and pro-inflammatory cytokines in obesity.

Cardiac structure and function in response to a multi-stage marathon over 4486 km.

AIMS: To investigate whether participation in the Trans Europe Foot Race 2009 (TEFR), an ultramarathon race held over 64 consecutive days and 4486 km, led to changes in cardiac structure and function. METHODS: Cardiac magnetic resonance imaging was performed in 20 of 67 participating runners (two women; mean ± SD age 47.8 ± 10.4 years) at three time points (baseline scan at 294 ± 135 km (B), scan two at 1735 ± 86 km (T1) and scan three at 3370 ± 90 km (T2)) during the TEFR. Imaging included an assessment of left ventricular structure (mass) and function (strain). In parallel, cardiac troponin I, NT-pro-BNP, myostatin and GDF11 were determined in venous blood samples. A subsample of ten runners returned for a follow-up scan eight months after the race. RESULTS: Left ventricular mass increased significantly (B, 158.5 ± 23.8 g; T1, 165.1 ± 23.2 g; T2, 167 ± 24.6 g; p < 0.001) over the course of the race, although no significant change was seen in the remaining structural and functional parameters. Serum concentrations of cardiac troponin I and NT-proBNP significantly increased 1.5 - and 3.5-fold, respectively, during the first measurement interval, with no further increase thereafter (cardiac troponin I, 6.8 ± 3.1 (B), 16.9 ± 10.4 (T1) and 17.1 ± 9.7 (T2); NT-proBNP, 30.3 ± 22.8 (B), 135.9 ± 177.5 (T1) and 111.2 ± 87.3 (T2)), whereas the growth markers myostatin and GDF11 did not change. No association was observed with functional parameters, including the ejection fraction and the volume of both ventricles. The follow-up scans showed a reduction to baseline values (left ventricular mass 157 ± 19.3 g). CONCLUSIONS: High exercise-induced cardiac volume load for >2 months in ultra-endurance runners results in a physiological structural adaptation with no sign of adverse cardiovascular remodelling.

Clinical characteristics and treatment outcome in a representative sample of depressed inpatients - findings from the Munich Antidepressant Response Signature (MARS) project.

Depression is a common and often difficult-to-treat clinical condition with a high rate of patients showing insufficient treatment response and persistence of symptoms. We report the characteristics of a representative sample of depressed inpatients participating in the Munich Antidepressant Response Signature (MARS) project. Eight hundred and forty-two inpatients admitted to a psychiatric hospital for treatment of a major depressive episode, recurrent or bipolar depression were thoroughly characterized with respect to demographic factors, clinical history, and the degree of HPA-axis dysregulation evaluated by means of combined dex/CRH tests, and the predictive value of these factors for treatment outcome is investigated. 80.8% of patients responded to treatment (i.e., improvement in symptom severity of at least 50%) and 57.9% reached remission (i.e., near absence of residual depressive symptoms) at discharge after a mean treatment period of 11.8 weeks. Regression analysis identified early partial response (within 2 weeks) as the most important positive predictor for achieving remission. Previous ineffective treatment trials in the current episode and presence of a migration background are potent negative predictors for treatment outcome. In addition, remitters were characterized by a more pronounced normalization of an initially dysregulated HPA-axis. We could show that a large majority of inpatients suffering from depression benefits from antidepressant treatment during hospitalization. However, a considerable number of patients failed to achieve remission. We demonstrated that this subgroup can be characterized by a set of demographic, clinical and neuroendocrine variables allowing to predict unfavorable outcome at an early stage of treatment.

Impact of coronary endothelial dysfunction on adverse long-term outcome after heart transplantation.

BACKGROUND: Coronary vasomotor dysfunction is a common finding in cardiac transplant recipients and is an early marker for the development of graft atherosclerosis. The present prospective study tested whether endothelial dysfunction independently predicts cardiovascular-related events and death after heart transplantation (HTx). METHODS: Functional and structural coronary changes were evaluated in 185 consecutive patients 25+/-33 months after HTx. The following potential risk factors for graft survival were assessed at baseline: hypertension, diabetes, dyslipidemia, donor and recipient characteristics (age, gender, cytomegalovirus-infection, human leukocyte antigen-mismatch), pretransplantation diagnosis, ischemic time, treated rejection episodes, immunosuppressive regimens, and medication.The prespecified prospectively defined endpoints were cardiovascular-related events with progressive heart failure, acute myocardial infarction, coronary revascularization, retransplantation, and death. Patients were followed-up for 60+/-17 months. RESULTS: Event-free survival for the entire group was 73% (25 cardiovascular-related events, 25 deaths). Using multivariate analysis, epicardial endothelial dysfunction (relative risk [RR] 1.97; P=0.028), angiographic cardiac allograft vasculopathy (RR 2.11; P=0.023), diabetes (RR 2.32; P=0.022), high serum levels of CyA (RR 3.54; P=0.006) and Tac (RR 6.82; P=0.002), uncommon reasons for transplantation (RR 4.69; P=0.002), and the absence of statin therapy (RR 0.33; P=0.025) were detected as independent predictors of cardiovascular-related events and death. CONCLUSION: This is the first study showing that epicardial endothelial dysfunction independently predicts outcome in HTx patients providing functional and prognostic information that complete angiographic risk factor assessment.

Impact of polyphenols on physiological stress and cardiac burden in marathon runners - results from a substudy of the BeMaGIC study.

Both physiologic stress and chronic heart disease are associated with increased systemic levels of chromogranin A (CGA) and NT-proBNP. Marathon running causes physiological stress and imposes a significant cardiac burden. Polyphenol-rich Mediterranean and Asian diets have been demonstrated to exert beneficial effects on the cardiovascular system. In this study we investigated whether pretreatment with a polyphenol beverage could attenuate the physiological and cardiac stress associated with a marathon. In the BeMaGIC trial, 277 athletes were randomized into 2 groups in a double-blinded fashion, receiving 1-1.5 L/day of the same beverages either with (study beverage) or without (placebo) polyphenol enrichment (approximately 400 mg of gallic acid equivalents per day of a complex mixture of polyphenols). Blood samples were taken 3 weeks and 1 day before, and immediately, 24 h, and 72 h after running a marathon. In our current substudy, CGA and NT-proBNP levels were analyzed by ELISA in the fastest 18 and the slowest 22 runners. CGA and NT-proBNP levels increased significantly immediately after the marathon and returned to baseline at 72 h after the marathon. Neither CGA nor NT-proBNP differed significantly between athletes receiving study beverage versus placebo. Separating our cohort into fast and slow runners did not reveal any significant difference regarding CGA or NT-proBNP levels between groups. Our study provides no evidence that polyphenol supplementation attenuates marathon running-induced physiological stress and cardiac burden in fast or slow runners.

Influence of polyphenol-rich diet on exercise-induced immunomodulation in male endurance athletes.

Stress is associated with increased susceptibility to infection. We investigated if the mechanism involves immunomodulation of dendritic cells and whether this can be inhibited by a polyphenol-rich diet. Blood samples were taken from a total of 100 male endurance athletes at 5 time points around a marathon run: 4 weeks before; 1 week before; and immediately, 24 h, and 72 h after. Participants were randomized into 2 double-blinded groups. One group received a polyphenol-rich beverage during a 3-week training phase before marathon while the other group received a placebo beverage. Flow cytometric analysis of dendritic cell (DC) counts and subpopulation counts (myeloid, plasmocytoid DCs) was performed. Levels of viral antigen presenting toll-like receptor (TLR) 7 messenger RNA was measured by real-time polymerase chain reaction. Marathon running induced a significant increase of circulating myeloid DCs (0.2% vs. 0.33% of whole-blood leukocytes (wbl); p < 0.01) and a significant decrease of plasmozytoid DCs (0.12% vs. 0.03% of wbl; p < 0.01) and TLR7 expression (decline of 60%; p < 0.01). Polyphenol supplementation did not significantly affect mobilization of dendritic cells but showed beneficial effects on regeneration of TLR7 expression in wbl at 3 days postmarathon (decline of 40% vs. increase of 1000%; p < 0.05). In conclusion, physical stress affects circulating DCs, with an increase of myeloid and a decrease of plasmozytoid DCs. This may partially explain the susceptibility to viral infections after strenuous exercise. These detrimental effects are not attenuated by polyphenol supplementation. However, polyphenols support regeneration of viral antigen presenting TLR7 after strenuous exercise.

Drugs modulating endothelial function after transplantation.

Endothelial cells play a vital role in the success or failure of a transplant procedure. The procedure itself can be viewed as a series of insults that damages the endothelium thereby triggering an inflammatory cascade that may, if uncontrolled, drive the proliferative and fibrotic processes characteristic of chronic graft vasculopathy. Unfortunately, many immunosuppressant agents contribute to this process. Glucocorticoids and the calcineurin inhibitor cyclosporine induce endothelial dysfunction, and although tacrolimus may not have the same disruptive effects on endothelial function as cyclosporine, its endothelial activity is still being established. In contrast, antiproliferative agents slow the proliferation and migration of endothelial cells and so help protect against graft vasculopathy. Researchers agree that endothelial cell dysfunction is a potentially treatable stage in the multifactorial process of graft vasculopathy and rejection. A number of cardiovascular agents (statins, angiotensin converting enzyme inhibitors, calcium channel blockers), immunoregulatory drugs, and dietary compounds have been shown to have beneficial effects on endothelial function. We briefly review the evidence supporting their use as protection for endothelial cells in transplant recipients.

Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols.

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.

MicroRNAs as Biomarkers for Acute Atrial Remodeling in Marathon Runners (The miRathon Study--A Sub-Study of the Munich Marathon Study).

INTRODUCTION: Physical activity is beneficial for individual health, but endurance sport is associated with the development of arrhythmias like atrial fibrillation. The underlying mechanisms leading to this increased risk are still not fully understood. MicroRNAs are important mediators of proarrhythmogenic remodeling and have potential value as biomarkers in cardiovascular diseases. Therefore, the objective of our study was to determine the value of circulating microRNAs as potential biomarkers for atrial remodeling in marathon runners (miRathon study). METHODS: 30 marathon runners were recruited into our study and were divided into two age-matched groups depending on the training status: elite (ER, ≥55 km/week, n = 15) and non-elite runners (NER, ≤40 km/week, n = 15). All runners participated in a 10 week training program before the marathon. MiRNA plasma levels were measured at 4 time points: at baseline (V1), after a 10 week training period (V2), immediately after the marathon (V3) and 24h later (V4). Additionally, we obtained clinical data including serum chemistry and echocardiography at each time point. RESULTS: MiRNA plasma levels were similar in both groups over time with more pronounced changes in ER. After the marathon miR-30a plasma levels increased significantly in both groups. MiR-1 and miR-133a plasma levels also increased but showed significant changes in ER only. 24h after the marathon plasma levels returned to baseline. MiR-26a decreased significantly after the marathon in elite runners only and miR-29b showed a non-significant decrease over time in both groups. In ER miRNA plasma levels showed a significant correlation with LA diameter, in NER miRNA plasma levels did not correlate with echocardiographic parameters. CONCLUSION: MiRNAs were differentially expressed in the plasma of marathon runners with more pronounced changes in ER. Plasma levels in ER correlate with left atrial diameter suggesting that circulating miRNAs could potentially serve as biomarkers of atrial remodeling in athletes.

Treatment with a CRH-1-receptor antagonist (R121919) does not affect weight or plasma leptin concentration in patients with major depression.

Weight gain during treatment with psychotropic drugs is frequently observed and is assumed to be responsible for non-compliance and for an elevated risk to develop a number of somatic co-morbidities including cardiovascular disorders and type 2 diabetes. Absence of weight inducing effects is therefore a major objective for the development of new compounds. Recently, R121919, the first corticotropin releasing hormone receptor 1 (CRH1R) antagonist, was tested in major depression. Clinical efficacy, safety, and tolerability of this compound could be demonstrated. Since CRH is discussed to be involved in the regulation of appetite and weight, directly and via interaction with leptin, CRH1R antagonists are suspected to influence body weight. Effects of 30 days of treatment with the CRH1R antagonist R121919 on weight and leptin levels in 20 patients suffering from major depression were investigated. No significant weight changes during treatment with R121919 were observed. Furthermore, noeffects on plasma leptin concentrations were found. We conclude that treatment with the CRH1R antagonist R121919 does not affect weight or plasma leptin concentrations in patients with major depression. Together with previous findings indicating safety, tolerability, and clinical efficacy CRH1R antagonists are highly promising as a new treatment option in depression.

The combined dexamethasone/CRH test as a potential surrogate marker in depression.

There is compelling evidence that impaired corticosteroid receptor function is the key mechanism in the pathogenesis of depression resulting in a dysfunctional stress hormone regulation, which can be most sensitively detected with the combined dexamethasone (dex)/corticotropin releasing hormone (CRH) test. Treatment with different kinds of antidepressants is associated with a reduction of the hormonal responses to the combined dex/CRH test suggesting normalization of impaired corticosteroid receptor signaling as the final common pathway of these drugs. Consequently, the combined dex/CRH test is suggested as a screening tool to decide whether new compounds designed as antidepressants provide sufficient efficacy to normalize corticoid receptor signaling in depressed patients. We summarize own data and findings from the literature suggesting that (1) the neuroendocrine response to the combined dex/CRH test is elevated during a major depressive episode, but (2) tends to normalize after successful treatment. (3) Favorable response to antidepressant treatment can be predicted by determining the dex suppresser status on admission. For optimal prediction of non-response to antidepressant treatment, however, the results of a second dex/CRH test are necessary. These findings, together with the fact that impaired corticosteroid receptor signaling is considered as key mechanism of the pathogenesis in depression, support the suitability of the combined dex/CHR test as a surrogate marker for treatment response in depression. In conclusion, the combined dex/CRH test is a promising candidate to serve as a screening tool for the antidepressive effects of new compounds in clinical drug trials. Furthermore, the test appears to be capable of predicting the individual likelihood to respond to a current antidepressant treatment. If a drug treatment fails to normalize the outcome of the combined dex/CRH test, a change of the treatment strategy is recommended. Further systematic research is required and already ongoing to confirm the suitability of the combined dex/CRH test as a surrogate marker in depression.

Treatment of depression with the CRH-1-receptor antagonist R121919: endocrine changes and side effects.

A dysregulation of the hypothalamus-pituitary-adrenocortical (HPA) system has been hypothesized to account for a myriad of cardinal symptoms of affective disorders. Specifically, increased CRH signalling via CRH type 1 receptors is thought to be an important factor in the pathogenesis of major depression and anxiety disorders. Consequently, a number of drugs have been developed in order to target the postulated increase in CRH/CRH 1 receptor signalling. One of these compounds, R121919, binds with high affinity to CRH1 receptors antagonising the action of CRH. R121919 was recently tested in an open-label study conceptualized as a safety and tolerability study. As part of this study, a thorough endocrine evaluation and detailed clinical laboratory analysis were assessed several times during 30 days of treatment with two different dose regimens of R121919 (5-40 mg vs. 40-80 mg) in 24 patients with a major depressive episode. During treatment with the experimental drug no serious side effects were noted. In particular, there were no adverse effects or impairment of the hypothalamic-pituitary-gonadal system, the hypothalamic-pituitary-thyroid axis, the renin-angiotensin system, prolactin or vasopressin secretion. Furthermore, no changes in the serum corticotropin and cortisol concentrations and in the responsivity of corticotropin and cortisol following a CRH stimulation test were noted. No effects of R121919 on clinical laboratory parameters including liver enzymes, EEG and ECG were observed. These results encourage the development of other CRH-1-R antagonists as a novel class of antidepressive drugs.