Expanding multimodal microscopy by high spectral resolution coherent anti-Stokes Raman scattering imaging for clinical disease diagnostics.

Over the past years fast label-free nonlinear imaging modalities providing molecular contrast of endogenous disease markers with subcellular spatial resolution have been emerged. However, applications of these imaging modalities in clinical settings are still at the very beginning. This is because single nonlinear imaging modalities such as second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) have only limited value for diagnosing diseases due to the small number of endogenous markers. Coherent anti-Stokes Raman scattering (CARS) microscopy on the other hand can potentially be added to SHG and TPEF to visualize a much broader range of marker molecules. However, CARS requires a second synchronized laser source and the detection of a certain wavenumber range of the vibrational spectrum to differentiate multiple molecules, which results in increased experimental complexity and often inefficient excitation of SHG and TPEF signals. Here we report the application of a novel near-infrared (NIR) fiber laser of 1 MHz repetition rate, 65 ps pulse duration, and 1 cm(-1) spectral resolution to realize an efficient but experimentally simple SGH/TPEF/multiplex CARS multimodal imaging approach for a label-free characterization of composition of complex tissue samples. This is demonstrated for arterial tissue specimens demonstrating differentiation of elastic fibers, triglycerides, collagen, myelin, cellular cytoplasm, and lipid droplets by analyzing the CARS spectra within the C-H stretching region only. A novel image analysis approach for multispectral CARS data based on colocalization allows correlating spectrally distinct pixels to morphologic structures. Transfer of this highly precise but compact and simple to use imaging approach into clinical settings is expected in the near future.

A compact microscope setup for multimodal nonlinear imaging in clinics and its application to disease diagnostics.

The past years have seen increasing interest in nonlinear optical microscopic imaging approaches for the investigation of diseases due to the method's unique capabilities of deep tissue penetration, 3D sectioning and molecular contrast. Its application in clinical routine diagnostics, however, is hampered by large and costly equipment requiring trained staff and regular maintenance, hence it has not yet matured to a reliable tool for application in clinics. In this contribution implementing a novel compact fiber laser system into a tailored designed laser scanning microscope results in a small footprint easy to use multimodal imaging platform enabling simultaneously highly efficient generation and acquisition of second harmonic generation (SHG), two-photon excited fluorescence (TPEF) as well as coherent anti-Stokes Raman scattering (CARS) signals with optimized CARS contrast for lipid imaging for label-free investigation of tissue samples. The instrument combining a laser source and a microscope features a unique combination of the highest NIR transmission and a fourfold enlarged field of view suited for investigating large tissue specimens. Despite its small size and turnkey operation rendering daily alignment dispensable the system provides the highest flexibility, an imaging speed of 1 megapixel per second and diffraction limited spatial resolution. This is illustrated by imaging samples of squamous cell carcinoma of the head and neck (HNSCC) and an animal model of atherosclerosis allowing for a complete characterization of the tissue composition and morphology, i.e. the tissue's morphochemistry. Highly valuable information for clinical diagnostics, e.g. monitoring the disease progression at the cellular level with molecular specificity, can be retrieved. Future combination with microscopic probes for in vivo imaging or even implementation in endoscopes will allow for in vivo grading of HNSCC and characterization of plaque deposits towards the detection of high risk plaques.

Mortality prediction after cardiac surgery: blood lactate is indispensible.

BACKGROUND: Blood lactate is accepted as a mortality risk marker in intensive care units (ICUs), especially after cardiac surgery. Unfortunately, most of the commonly used ICU risk stratification scoring systems did not include blood lactate as a variable. We hypothesized that blood lactate alone can predict the risk of mortality after cardiac surgery with an accuracy that is comparable to those of other complex models. We therefore evaluated its accuracy at mortality prediction and compared it with that of other widely used complex scoring models statistically. METHODS: We prospectively collected data of all consecutive adult patients who underwent cardiac surgery between January 1, 2007, and December 31, 2009. By using χ2 statistics, a blood lactate-based scale (LacScale) with only four cutoff points was constructed in a developmental set of patients (January 1, 2007, and May 31, 2008). LacScale included five categories: 0 (≤ 1.7 mmol/L); 1 (1.8-5.9 mmol/L), 2 (6.0-9.3 mmol/L), 3 (9.4-13.3 mmol/L), and 4 (≥ 13.4 mmol/L). Its accuracy at predicting ICU mortality was evaluated in another independent subset of patients (validation set, June 1, 2008, and December 31, 2009) on both study-population level (calibration analysis, overall correct classification) and individual-patient-risk level (discrimination analysis, ROC statistics). The results were then compared with those obtained from other widely used postoperative models in cardiac surgical ICUs (Sequential Organ Failure Assessment [SOFA] score, Simplified Acute Physiology Score II [SAPS II], and Acute Physiology and Chronic Health Evaluation II [APACHE II] score). RESULTS: ICU mortality was 5.8% in 4,054 patients. LacScale had a reliable calibration in the validation set (2,087 patients). It was highly accurate in predicting ICU mortality with an area under the ROC curve (area under curve [AUC]; discrimination) of 0.88. This AUC was significantly larger than that of all the other models (SOFA 0.83, SAPS II: 0.79 and APACHE II: 0.76) according to DeLong's comparison. Integrating the LacScale in those scores further improved their accuracy by increasing their AUCs (0.88, 0.81, and 0.80, respectively). This improvement was also highly significant. CONCLUSION: Blood lactate accurately predicts mortality at both individual patient risk and patient cohort levels. Its precision is higher than that of other commonly used "complex" scoring models. The proposed LacScale is a simple and highly reliable model. It can be used (at bedside without electronic calculation) as such or integrated in other models to increase their accuracy.

Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.

Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs.

In vivo characterization of atherosclerotic plaque depositions by Raman-probe spectroscopy and in vitro coherent anti-stokes Raman scattering microscopic imaging on a rabbit model.

Visualization as well as characterization of inner arterial plaque depositions is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable visual information but cannot deliver information about the chemical composition of individual plaques. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions of arterial walls on a rabbit model in vivo, using a miniaturized filtered probe with one excitation and 12 collection fibers integrated in a 1 mm sleeve. Rabbits were treated with a cholesterol-enriched diet. The methodology can improve the efficiency of animal experiments and shows great potential for applications in cardiovascular research. In order to further characterize the plaque depositions visually, coherent anti-Stokes Raman scattering (CARS) microscopy images have been acquired and are compared with the Raman-probe results.

Left atrial endocarditis as a rare complication of mitral valve endocarditis: a clinical case.

BACKGROUND: Infective Endocarditis (IE) is considered as a multifaceted problem in every aspect from etiology and presentation to diagnosis and management. Early recognition of this disease and especially its complications, remain a critical task for the cardiologist. Atrial endocarditis is a rare and sometimes unrecognized complication of mitral valve endocarditis. CASE PRESENTATION: We present a 48 year-old male patient who was admitted to our clinic because of recent onset of malaise, fever, jaundice and peripheral edema. Important physical findings were peripheral stigmata of IE in addition to holosystolic murmur over the left sternal border. Transthoracic and transesophageal echocardiophy revealed a severe eccentric MR due to a flailed posterior mitral valve caused by IE. The presence of atrial septal endocarditis caused by jet streaming was also observed. Blood culture was positive for streptococcus oralis and antibiotic therapy was immediately initiated. Considering the large burden of infective tissue, the patient was planned for an early surgical intervention. A minimally invasive resection of the atrial mass, direct closure of the defect, resection of the diseased portions of mitral leaflets and implantation of a biological mitral valve prosthesis was performed. Intra-operative and histological findings confirmed provisional diagnosis by echocardiography. CONCLUSIONS: Together with comprehensive echocardiographic evaluation, attention should be placed on mural vegetations and excluded among all cases of mitral valve endocarditis, particularly those with severe eccentric regurgitant jets.

In patients with aortic stenosis increased flow-mediated dilation is independently associated with higher peak jet velocity and lower asymmetric dimethylarginine levels.

BACKGROUND: Recently, it has been shown that endothelial dysfunction and aortic stenosis (AS) share several risk factors. Endothelial function represents a crucial factor for the regulation of vascular tonus and its malfunction influences the formation of thrombosis and inflammation. However, the role of endothelial dysfunction in AS remains unclear. METHODS: Echocardiographic, clinical, and laboratory data of 34 patients (age 74.5 ± 7.9 years, 20 men) with at least moderate AS (peak jet velocity 3.8 ± 0.8 m/s) were collected. In all patients, endothelial function was determined by brachial artery flow-mediated dilation (FMD). Patients with rheumatic or endocarditic valve disease, bicuspid valves, a left ventricular ejection fraction of ≤40%, and coronary artery disease were excluded. Sixteen volunteers (age 69.3 ± 9.4 years, 10 men) without valve disease served as controls. RESULTS: Patients with AS had a trend toward a lower FMD than controls with a comparable risk profile (5.4% ± 3.6% vs 7.4% ± 4.1%, P = .1). Univariate correlates of FMD in patients with AS were peak jet velocity, medication with angiotensin-converting enzyme inhibitor, diabetes, diastolic blood pressure, and asymmetric dimethylarginine. Backward elimination identified peak jet velocity (ß = 0.51, P = .001), and asymmetric dimethylarginine (ß = -0.45, P = .003) as independent predictors of FMD in multivariate analysis. CONCLUSIONS: In patients with AS, we found a strong positive relation between the peak jet velocity and a higher FMD. This effect might be mediated by nitric oxide release due to turbulent poststenotic blood flow or the rising transvalvular gradient, and the increasing pulse pressure may be counteracted by a parallel increase in FMD.

Comparative analysis of oncofetal fibronectin and tenascin-C expression in right atrial auricular and left ventricular human cardiac tissue from patients with coronary artery disease and aortic valve stenosis.

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17 × AVS; 13 × AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r = 0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r = 0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.

Association of waist circumference, traditional cardiovascular risk factors, and stromal-derived factor-1 in adolescents.

BACKGROUND: Overweight and the metabolic syndrome (MS) represent dramatically increasing problems in children and adolescents. Waist circumference (WC) is an important factor to determine MS. So far, WC is a predictor of blood pressure, high-density lipoprotein (HDL), insulin concentration, and visceral fat in adolescents. We investigated whether WC and body mass index standard deviation score (BMI-SDS) are predictors of adiponectin, stromal-derived factor (SDF-1), and soluble E-selectin (sE-selectin) as parameters for beginning insulin resistance and endothelial damage. METHODS: Seventy-nine male Caucasian adolescents were studied, aged 13-17 yr. Thirty-eight (48%) of them had a WC above 90th age percentile. All participants were enrolled in one consultation, recording various parameters and collecting one blood sample. RESULTS: Differences in systolic blood pressure, HDL, high sensitive C-reactive protein, and hemoglobin A1c could be found between groups above or below the 90th WC percentile. Linear regression analysis revealed that WC and BMI-SDS predict traditional risk factors, as well as reduced adiponectin, lower SDF-1, and higher sE-selectin levels. Multiple linear regression analyses show that SDF-1 is in closest correlation to WC and BMI-SDS. CONCLUSIONS: WC and BMI-SDS predict various alterations of traditional and new cardiovascular risk factors. SDF-1 might be a new marker for diagnosis of obesity-related diseases and help understand pathophysiologic mechanisms.

Evaluation of the microcirculation during extracorporeal membrane-oxygenation.

Extracorporeal membrane oxygenation (ECMO) provides pulmonary and circulatory support in critically ill patients (Cardiopulmonary Resuscitation, Acute Respiratory Distress Syndrome, or low output syndrome). Hemodynamic parameters are used for evaluation of the macrocirculation, while the microcirculation is monitored by blood-lactate as a surrogate parameter. We evaluated the microcirculation by orthogonal polarization spectral imaging in a patient during ECMO support. This method was initially proposed to quantify changes of microcirculation in patients with septic shock. However, we were able to non-invasively monitor microcirculatory changes at the bedside during temporary intentional arrest of ECMO due to an exchange of the oxygenator. Using a computerized analyzation model, the flow after ECMO stop in vessels (10-100 microm) in the sublingual mucosa was acutely absent or intermittent, respectively. 120 s after restart, microflow was improved with new ECMO settings compared to baseline, while macrocirculation with a mean arterial pressure of 75 mmHg was present after 60 s. The application of orthogonal polarization spectral imaging might be a valuable technique for evaluation of the microcirculation during extracorporeal circulation. It is rapidly implementable, can be used in vivo, and no invasive probes are required.

[Surgical therapy of infective endocarditis. General aspects and case report]. / Indikationen zur herzchirurgischen Therapie bei Endokarditis. Fallbeschreibung und allgemeine Aspekte.

BACKGROUND: Infective endocarditis due to viridans streptococci is associated with a mortality of 5-10%. Even today, it remains difficult to diagnose it at an early stage, to select a sufficient antibiotic therapy and to choose the right time for surgical intervention. CASE REPORT: A 37-year-old male patient presented with anemia, fever, adynamia and a loud systolic murmur over the base of the heart. Blood culture data were positive for Streptococcus mitis. Transthoracic echocardiography revealed an endocarditis of the aortic and mitral valve with regurgitations as well as a hypertrophic obstructive cardiomyopathy. The hemodynamically stable patient was treated with penicillin G, gentamicin and verapamil. Because of an extension of valve vegetations and a decline in the hemodynamic situation with an incipient sepsis, the patient was surgically treated urgently by replacement of the aortic and mitral valve as well as a Morrow septal myectomy. A postoperative sepsis required the application of high catecholamine doses. Because of a respiratory insufficiency, a prolonged mechanical ventilation was required. Finally, the patient could be discharged for in-hospital rehabilitation. CONCLUSION: The indication for surgical therapy in patients with endocarditis of the aortic and mitral valve as well as hypertrophic obstructive cardiomyopathy should be critically discussed with regard to the patient's age, the aims of conservative therapy, and the consequences of a surgical intervention. If there are any indices of a disease progress in spite of antibiotic therapy, patients should be subjected to cardiac surgery immediately.

Detection and characterization of early plaque formations by Raman probe spectroscopy and optical coherence tomography: an in vivo study on a rabbit model.

Intravascular imaging techniques provide detailed specification about plaque appearance and morphology, but cannot deliver information about the biochemical composition of atherosclerotic plaques. As the biochemical composition is related to the plaque type, important aspects such as the risk of a plaque rupture and treatment are still difficult to assess. Currently, various spectroscopic techniques are tested for potential applications for the chemical analysis of plaque depositions. Here, we employ Raman spectroscopy in combination with optical coherence tomography (OCT) for the characterization of plaques on rabbits in vivo. Experiments were carried out on New Zealand white rabbits treated with a fat- and cholesterol-enriched diet, using a Raman probe setup with a 785-nm multimode laser as an excitation source. Subsequently, OCT images were acquired with a swept source at 1305±55 nm at 22.6 mW. Raman spectra were recorded from normal regions and regions with early plaque formations. The probe positioning was monitored by x-ray angiography. The spectral information identified plaque depositions consisting of lipids, with triglycerides as the major component. Afterward, OCT images of the spectroscopically investigated areas were obtained. The spectral information correlates well with the observed intravascular morphology and is in good agreement with histology. Raman spectroscopy can provide detailed biochemical specification of atherosclerotic plaques.

Stimulation of serum- and glucocorticoid-regulated kinase-1 gene expression by endothelin-1.

The serum- and glucocorticoid-regulated kinase-1 (SGK1) participates in the regulation of sodium homeostasis and blood pressure by mineralocorticoids. Aldosterone rapidly induces SGK1 transcription, which contributes to the activation of renal epithelial sodium channels. Another important regulator of blood pressure is the vasoactive hormone endothelin-1 (ET-1) that is systemically upregulated in chronic renal failure. In the present study, we investigated whether ET-1 modulates SGK1 expression, and thereby might explain some of its hypertensive effects. As assessed by real-time PCR analysis, ET-1 triggered the rapid increase of SGK1 mRNA levels in A-10 smooth muscle cells and also in intact aortas of adult rats. In A-10 cells transcriptional activation was associated with a more than 6-fold upregulation of SGK1 protein expression and in similar range as found after treatment with aldosterone. A stimulatory effect of ET-1 was not only observed in isolated cells, but also in an animal model. Upon subtotal nephrectomy (SNX) of rats, myocardial ET-1 levels strongly increased, which was followed by a more than 2-fold induction of SGK1 expression in the left ventricle. The myocardial upregulation of SGK1 was completely abrogated by a specific ET(A) receptor antagonist, thereby substantiating the in vivo role of ET-1 in SGK1 expression. Thus, these data demonstrate that ET-1 increases expression of SGK1 in vivo and in vitro, and therefore indicate that SGK1 upregulation might be involved in ET-1-dependent regulation of blood pressure and cardiac modelling during mild renal failure.

Non-linear imaging and characterization of atherosclerotic arterial tissue using combined SHG and FLIM microscopy.

Atherosclerosis is one of the leading causes of death in the Western World and its characterization is extremely interesting from the diagnostic point of view. Here, we employed combined SHG-FLIM microscopy to characterize arterial tissue with atherosclerosis. The shorter mean fluorescence lifetime measured within plaque depositions (1260 ± 80 ps) with respect to normal arterial wall (1480 ± 100 ps) allowed discriminating collagen from lipids. SHG measurements and image analysis demonstrated that the normal arterial wall has a more anisotropic Aspect Ratio (0.37 ± 0.02) with respect to plaque depositions (0.61 ± 0.02) and that the correlation length can be used for discriminating collagen fibre bundles (2.0 ± 0.6 µm) from cholesterol depositions (4.1 ± 0.6 µm). The presented method has the potential to find place in a clinical setting as well as to be applied in vivo in the near future. Graphic composition of SHG and FLIM images representing normal arterial wall and plaque depositions.

Early outcome of carotid angioplasty and stenting with and without cerebral protection devices: a systematic review of the literature.

BACKGROUND: Carotid angioplasty and stenting (CAS) is increasingly being used for treatment of symptomatic and asymptomatic carotid artery disease (CAD). To evaluate the efficacy of cerebral protection devices in preventing thromboembolic complications during CAS, we conducted a systematic review of studies reporting on the incidence of minor stroke, major stroke, or death within 30 days after CAS. SUMMARY OF REVIEW: We searched for studies published between January 1990 and June 2002 by means of a PubMed search and a cumulative review of reference lists of all relevant publications. In 2357 patients a total of 2537 CAS procedures had been performed without protection devices, and in 839 patients 896 CAS procedures had been performed with protection devices. Both groups were similar with respect to age, sex distribution, cerebrovascular risk factors, and indications for CAS. In many studies the periprocedural complication rates had not been presented separately for patients with symptomatic and asymptomatic CAD. The combined stroke and death rate within 30 days in both symptomatic and asymptomatic patients was 1.8% in patients treated with cerebral protection devices compared with 5.5% in patients treated without cerebral protection devices (chi2=19.7, P<0.001). This effect was mainly due to a decrease in the occurrence of minor strokes (3.7% without cerebral protection versus 0.5% with cerebral protection; chi2=22.4, P<0.001) and major strokes (1.1% without cerebral protection versus 0.3% with cerebral protection; chi2=4.3, P<0.05), whereas death rates were almost identical (approximately 0.8%; chi2=0.3, P=0.6). CONCLUSIONS: On the basis of this early analysis of single-center studies, the use of cerebral protection devices appears to reduce thromboembolic complications during CAS. These technical aspects should be taken into account before the initiation of further randomized trials comparing CAS with carotid endarterectomy.

Modulation of the beta-adrenergic receptor system of vascular smooth muscle cells in vitro and in vivo by chronically elevated endothelin-1 levels.

Endothelin-1 (ET-1) levels are chronically elevated in several cardiovascular diseases and correlate with an increased mortality. However, in contrast to acute biological activities such as vasoconstriction, little is known about long-term effects of ET-1. In this study we determined the effects of ET-1 on the beta(2)-adrenergic receptor (AR) system. Incubation of smooth muscle cells with ET-1 for 72 hr led to increased beta(2)AR density as determined by radioligand binding. Experiments with inhibitors of protein and RNA synthesis as well as RT-PCR revealed that beta(2)AR upregulation required de novo synthesis. In addition, protein kinase C but neither NO nor prostaglandin metabolism were involved in this effect. The enhanced expression of beta(2)AR was associated with an increased expression of its stimulatory G-protein and the receptor's ability to stimulate adenylyl cyclase. To study chronic effects of ET-1 in vivo, rats were infused with ET-1 for 3 weeks. Similarly as in cultured cells, prolonged ET-1 exposure led to increased betaAR expression in vivo. As a consequence, beta(2)AR-induced vasodilatation was increased in aortic rings from ET-1-treated animals. Our results therefore suggest that chronically elevated ET-1 levels in vitro and in vivo induce counterregulatory mechanisms by increasing betaARs that attenuate the vasoconstrictive effects of ET-1.

Effect of nebivolol on left ventricular function in patients with chronic heart failure: a pilot study.

BACKGROUND: Sympathetic activity is a significant predictor of a poor prognosis in heart failure. Beta-blockers have been shown to improve the prognosis of patients with heart failure. AIM: This pilot study examined the tolerability and efficacy of the new beta-blocker nebivolol on left ventricular function in patients with chronic heart failure. METHODS AND RESULTS: Twelve patients with an ejection fraction of 13-39% were included in this double blind, placebo-controlled randomized trial of nebivolol administered in addition to standard therapy. Exercise time, heart rate, left ventricular function and tolerability were examined at baseline and after 3 months of orally administered nebivolol (2.5 and 5 mg, n = 6) or placebo (n = 6). Nebivolol was well tolerated and the NYHA class improved in four patients. Heart rate decreased while the maximal exercise duration and performance remained stable. Left ventricular function increased (ejection fraction 31.5 +/- 10.11 to 42.0 +/- 10.99%, P < or = 0.01) after 12 weeks of nebivolol. The left ventricular end-systolic diameter decreased in the nebivolol-group from 56.5 +/- 9.40 to 50.2 +/- 9.43 mm (P < or = 0.02). CONCLUSION: These data indicate that nebivolol might improve cardiac function in patients with reduced left ventricular function.

Endothelin-1 regulates protein kinase C isoforms differently in smooth muscle cells and in cardiomyocytes.

Endothelin-1 (ET-1) is known to increase the mitotic response of different growth factors but also to different vasoactive hormones already at threshold concentrations. The aim of this study was to investigate the influence of chronic elevated ET-1 concentrations on protein kinase C (PKC) isoforms in vitro and in vivo. Smooth muscle cells were incubated with ET-1 at a concentration of 10 mol/L. The incubation lasted for 1-96 hours. For in vivo studies, rats were chronically infused with ET-1 for 4 weeks using minipumps. Specific antibodies were used to determine the amount of PKC isoform in western blots. Incubation of smooth muscle cells with ET-1 revealed an increase in PKC-alpha (48 hours, 314 +/- 31.7%). In vivo PKC-alpha was augmented to 166 +/- 17.5%. In vitro PKC-epsilon showed an elevated concentration after 18 hours of 213 +/- 15.9% (maximum, 339 +/- 4.5% at 72 hours). In vivo PKC-epsilon was elevated to 162 +/- 4.2%. In the immunofluorescence microscopy after 48 hours of ET-1 incubation PKC-alpha was localized in the cytoplasm. Addition of angiotensin II resulted in a translocation of it into the nucleus. These data show that chronic elevated ET-1 concentrations modulate PKC isoforms. This might explain the increased response to different vasoactive hormones after ET-1 incubation.

Endothelin-1 increases serum-glucocorticoid-regulated kinase-1 expression in smooth muscle cells.

The mechanism of salt-sensitive hypertension is not fully understood. Several studies point to a possible role of endothelin (ET)-1 in this form of hypertension. Serum-regulated and glucocorticoid-regulated kinase-1 (SGK1) mediates trafficking of the renal epithelial sodium channel. The aim of the study was to find out whether ET-1 regulates SGK1. Rat smooth muscle cells were incubated with ET-1 (10(-7) M, 0-120 minutes). After 30 minutes a significant increase in SGK1 mRNA was found (122 +/- 4.2%), and a maximum was reached after 120 minutes (217 +/- 7.6%). Incubation of smooth muscle cells with ET-1 (10(-7) mol/L) in the presence of an ETA receptor antagonist inhibited SGK1 gene transcription (93 +/- 3.7%). Western blot analysis showed a time-dependent increase in SGK1 protein in smooth muscle cells. These data indicate that ET-1 increases SKG1 mRNA and protein concentration. Inhibition of ET-1 by ET antagonism prevented a SGK1 increase. Therefore, ET antagonism might influence blood pressure by regulating the sodium balance through reducing SGK1 gene expression.