Prevention of vascular calcification by the endogenous chromogranin A-derived mediator that inhibits osteogenic transdifferentiation.

The adrenal glands participate in cardiovascular (CV) physiology and the pathophysiology of CV diseases through their effects on sodium and water metabolism, vascular tone and cardiac function. In the present study, we identified a new adrenal compound controlling mesenchymal cell differentiation that regulates osteoblastic differentiation in the context of vascular calcification. This peptide was named the "calcification blocking factor" (CBF) due to its protective effect against vascular calcification and is released from chromogranin A via enzymatic cleavage by calpain 1 and kallikrein. CBF reduced the calcium content of cells and thoracic aortic rings under calcifying culture conditions, as well as in aortas from animals treated with vitamin D and nicotine (VDN animals). Furthermore, CBF prevented vascular smooth muscle cell (VSMC) transdifferentiation into osteoblast-like cells within the vascular wall via the sodium-dependent phosphate transporter PIT-1 and by inhibition of NF-κB activation and the subsequent BMP2/p-SMAD pathway. Pulse pressure, a marker of arterial stiffness, was significantly decreased in VDN animals treated with CBF. In line with our preclinical data, CBF concentration is significantly reduced in diseases characterized by increased calcification, as shown in patients with chronic kidney disease. In preparation for clinical translation, the active site of the native 19-AS long native CBF was identified as EGQEEEED. In conclusion, we have identified the new peptide CBF, which is secreted from the adrenal glands and might prevent vascular calcification by inhibition of osteogenic transdifferentiation. The anti-calcific effects of CBF and short active site may therefore promote the development of new tools for the prevention and/or treatment of vascular calcification.

A Novel Long-Term ex vivo Model for Studying Vascular Calcification Pathogenesis: The Rat Isolated-Perfused Aorta.

The investigation of vascular calcification and its underlying cellular and molecular pathways is of great interest in current research efforts. Therefore, suitable assays are needed to allow examination of the complex calcification process under controlled conditions. The current study describes a new ex vivo model of isolated-perfused rat aortic tissue with subsequent quantification and vessel staining to analyze the calcium content of the aortic wall. A rat aorta was perfused ex vivo with control and calcification media for 14 days, respectively. The calcification medium was luminally perfused and induced a significant increase in calcium deposition within the media of the vessel wall detected alongside the elastic laminae. Perfusion with control medium induced no calcification. In addition, the mRNA expression of the osteogenic marker bone morphogenetic protein 2 (BMP-2) increased in aortic tissue after perfusion, while SM22α as smooth muscle marker decreased. This newly developed ex vivo model of isolated-perfused rat aorta is suitable for vascular calcification studies testing inducers and inhibitors of vessel calcification and studying signaling pathways within calcification progression.

The impact of blood pressure variability and pulse pressure on graft survival and mortality after kidney transplantation.

BACKGROUND: Blood pressure variability and pulse pressure are strong and independent predictors of cardiovascular morbidity and mortality in the general population. So far, there are no data on the impact of blood pressure variability on mortality and graft survival after renal transplantation. METHODS: We performed a retrospective analysis of 877 patients who underwent kidney transplantation between 1997 and 2011 in two transplant centers in Germany (Berlin and Bochum) with a follow-up of 12-266 months. Visit-to-visit blood pressure variability over the first 12 months after transplantation (3 visits) and during the first 120 months after transplantation (7 visits) was calculated as the coefficient of variation (CV = standard deviation (SD)/mean blood pressure). Patient and graft survival was defined as composite endpoint. RESULTS: Cumulative survival was significantly higher for those patients with lower systolic blood pressure and pulse pressure within both the first 12 months and the 120 months posttransplant. After adjustment of data for gender, age, body mass index, and coronary artery disease, the cumulative incidence of the combined endpoint did not significantly differ between patients with lower vs higher CV (12 months CV hazard ratio (HR) (95% CI) = 0.90 (0.66-1.23), P = 0.51; 120 months CV HR (95% CI) = 0.92 (0.67-1.26), P = 0.60). A lower systolic blood pressure remained highly predictive for better survival in adjusted analyses. CONCLUSION: Visit-to-visit blood pressure variability is not associated with mortality or graft loss after kidney transplantation in this retrospective analysis. In analogy to the general population, however, there is an inverse relationship of survival and pulse pressure as a marker of arterial stiffness.

Detection of Acute Tubular Necrosis Using Blood Oxygenation Level-Dependent (BOLD) MRI.

BACKGROUND/AIMS: To date, there is no imaging technique to assess tubular function in vivo. Blood oxygen level-dependent magnetic resonance imaging (BOLD MRI) measures tissue oxygenation based on the transverse relaxation rate (R2*). The present study investigates whether BOLD MRI can assess tubular function using a tubule-specific pharmacological maneuver. METHODS: Cross sectional study with 28 participants including 9 subjects with ATN-induced acute kidney injury (AKI), 9 healthy controls, and 10 subjects with nephron sparing tumor resection (NSS) with clamping of the renal artery serving as a model of ischemia/reperfusion (I/R)-induced subclinical ATN (median clamping time 15 min, no significant decrease of eGFR, p=0.14). BOLD MRI was performed before and 5, 7, and 10 min after intravenous administration of 40 mg furosemide. RESULTS: Urinary neutrophil gelatinase-associated lipocalin was significantly higher in ATN-induced AKI and NSS subjects than in healthy controls (p=0.03 and p=0.01, respectively). Before administration of furosemide, absolute medullary R2*, cortical R2*, and medullary/cortical R2* ratio did not significantly differ between ATN-induced AKI vs. healthy controls and between NSS-I/R vs. contralateral healthy kidneys (p>0.05 each). Furosemide led to a significant decrease in the medullary and cortical R2* of healthy subjects and NSS contralateral kidneys (p<0.05 each), whereas there was no significant change of R2* in ATN-induced AKI and the NSS-I/R kidneys (p>0.05 each). CONCLUSION: BOLD-MRI is able to detect even mild tubular injury but necessitates a tubule-specific pharmacological maneuver, e.g. blocking the Na+-K+-2Cl- transporter by furosemide.

Identification of the Vasoconstriction-Inhibiting Factor (VIF), a Potent Endogenous Cofactor of Angiotensin II Acting on the Angiotensin II Type 2 Receptor.

BACKGROUND: The renin-angiotensin system and especially the angiotensin peptides play a central role in blood pressure regulation. Here, we hypothesize that an as-yet unknown peptide is involved in the action of angiotensin II modulating the vasoregulatory effects as a cofactor. METHODS AND RESULTS: The peptide with vasodilatory properties was isolated from adrenal glands chromatographically. The effects of this peptide were evaluated in vitro and in vivo, and the receptor affinity was analyzed. The plasma concentration in humans was quantified in patients with chronic kidney disease, patients with heart failure, and healthy control subjects. The amino acid sequence of the peptide from bovine adrenal glands was HSSYEDELSEVL EKPNDQAE PKEVTEEVSSKDAAE, which is a degradation product of chromogranin A. The sequence of the peptide isolated from human plasma was HSGFEDELSEVLENQSSQAELKEAVEEPSSKDVME. Both peptides diminished significantly the vasoconstrictive effect of angiotensin II in vitro. Therefore, we named the peptide vasoconstriction-inhibiting factor (VIF). The vasoregulatory effects of VIF are mediated by the angiotensin II type 2 receptor. VIF impairs angiotensin II-induced phosphorylation of the p38 mitogen-activated protein kinase pathway but not of extracellular-regulated kinase 1/2. The vasodilatory effects were confirmed in vivo. The plasma concentration was significantly increased in renal patients and patients with heart failure. CONCLUSIONS: VIF is a vasoregulatory peptide that modulates the vasoconstrictive effects of angiotensin II by acting on the angiotensin II type 2 receptor. It is likely that the increase in VIF may serve as a counterregulatory effect to defend against hypertension. The identification of this target may help us to understand the pathophysiology of renal and heart failure and may form a basis for the development of new strategies for the prevention and treatment of cardiovascular disease.

Analysis of Transpulmonary Thermodilution Data Confirms the Influence of Renal Replacement Therapy on Thermodilution Hemodynamic Measurements.

BACKGROUND: Transpulmonary thermodilution (TPTD) is used frequently in the intensive care unit to determine cardiac index (CI), intrathoracic blood volume index (ITBVI), and extravascular lung volume index (EVLWI). Renal replacement therapy (RRT) influences TPTD results, but the underlying mechanisms are not completely understood. We hypothesized that RRT blood flow induces errors in TPTD measurements. METHODS: We analyzed TPTD data available from the PiCCO® plus hemodynamic measurement device on a personal computer using a proprietary Pulsion Medical Systems software. By using the dialysis catheter to inject the thermal indicator, 20 measurement series were performed in 12 intensive care unit patients determining CI, ITBVI, and EVLWI during RRT with the blood pump stopped, and at flows of 100 and 200 mL/min, respectively. RESULTS: Data export was successful in 17 measurement series and showed a significant decrease in measured CI (6.5 ± 2.5 vs 5.4 ± 1.9 L/min/m, P < 0.001) and ITBVI (1358.8 ± 274.5 vs 1132.8 ± 218.3 mL/m, P < 0.001) with RRT and a significant increase in EVLWI (8.6 ± 4.4, 10.2 ± 4.5 mL/kg, P < 0.001). Blood temperature before and the temperature decrease after injection of the thermal indicator were unchanged by RRT. Mean transit time and downslope time of the thermodilution curve, however, were both increased with the RRT blood pump running (P ≤ 0.001). CONCLUSIONS: Analysis of TPTD data shows that thermodilution curve forms are modified with RRT, resulting in an erroneous calculation of thermodilution-derived hemodynamic parameters.

Interaction of human serum albumin with short polyelectrolytes: a study by calorimetry and computer simulations.

We present a comprehensive study of the interaction of human serum albumin (HSA) with poly(acrylic acid) (PAA; number average degree of polymerization: 25) in aqueous solution. The interaction of HSA with PAA is studied in dilute solution as a function of the concentration of added salt (20-100 mM) and temperature (25-37 °C). Isothermal titration calorimetry (ITC) is used to analyze the interaction and to determine the binding constant and related thermodynamic data. It is found that only one PAA chain is bound per HSA molecule. The free energy of binding ΔGb increases with temperature significantly. ΔGb decreases with increasing salt concentration and is dominated by entropic contributions due to the release of bound counterions. Coarse-grained Langevin computer simulations treating the counterions in an explicit manner are used to study the process of binding in detail. These simulations demonstrate that the PAA chains are bound in the Sudlow II site of HSA. Moreover, ΔGb is calculated from the simulations and found to be in very good agreement with the measured data. The simulations demonstrate clearly that the driving force of binding is the release of counterions in full agreement with the ITC-data.

Absolute quantification of endogenous angiotensin II levels in human plasma using ESI-LC-MS/MS.

BACKGROUND: Angiotensin II acts as a peptide hormone and component of renin-angiotensin- system (RAS) regulating the blood pressure, and seems to be involved in renal and vascular disorders. There is no reliable quantification method for angiotensin II available until now and the angiotensin II plasma levels described in the literature are correspondingly strongly divergent. Therefore, we developed and validated a sensitive, selective and reliable LC-ESI-MS/MS method for absolute quantification of angiotensin II concentration in human plasma based on the AQUA strategy. METHODS: Plasma samples were extracted using MAX Oasis cartridges and were subjected to a further immunoaffinity-purification using immobilized anti-angiotensin II antibodies in order to isolate endogenous angiotensin II. Stable isotope ((13)C- and (15) N-) labeled angiotensin II was used as an internal standard. The fractionated samples were analysed using LC-ESI-MS/MS. RESULTS: The calibration curve was established in plasma in the concentration range 6-240 pM (r(2) > 0.999). The developed and validated method was successfully applied for quantification of endogenous angiotensin II in human plasma of healthy volunteers and chronic kidney disease (CKD-5D) patients. The mean plasma angiotensin II levels were found to be 18.4 ± 3.3 pM in healthy subjects and 64.5 ± 32.4 pM in CKD-5D patients (each n =9). CONCLUSION: The LC-ESI-MS/MS-based method for quantification of angiotensin II levels in human plasma was successfully evaluated within the study. This method is applicable for clinical applications aiming at the validation of the impact of highly physiologically and pathophysiologically active angiotensin II.

Modulation of NADPH oxidase activity by known uraemic retention solutes.

BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. CONCLUSIONS: The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress.

Urinary calprotectin: a new diagnostic marker in urothelial carcinoma of the bladder.

PURPOSE: Recently, a proteomic study of sera from patients with bladder cancer identified S100A8 and S100A9 as tumor-associated proteins. The present cross-sectional study investigates whether calprotectin, the heterodimer of S100A8/S100A9 may serve as a urinary biomarker for the detection of urothelial bladder cancer. METHODS: Urinary calprotectin concentrations were assessed in a population of 181 subjects including 46 cases of bladder cancer. 41 cases of renal cell cancer, 54 cases of prostate cancer, and 40 healthy subjects served as control. Acute kidney injury, urinary tract infection, previous BCG-treatment and secondary transurethral resection of the bladder tumor were defined as exclusion criteria. Assessment was performed by enzyme-linked immunosorbent assay and immunohistochemistry detecting calprotectin. RESULTS: Median calprotectin concentrations (ng/ml) were significantly higher in patients with bladder cancer than in healthy controls (522.3 vs. 51.0, p < 0.001), renal cell cancer (90.4, p < 0.001), and prostate cancer (71.8, p < 0.001). In urothelial carcinoma prominent immunostaining occurred in a subset of tumor cells and in infiltrating myeloid cells. Receiver operating characteristic analysis provided an area under the curve of 0.88 for the differentiation of bladder cancer and healthy control. A cut-off value of 140 ng/ml (determined by Youden's index) resulted in sensitivity and specificity values of 80.4 and 92.5 %. Low grade tumors were associated with significantly lower calprotectin concentrations than high grade tumors (351.9 vs. 1635.2 ng/ml, p = 0.004). CONCLUSIONS: Urothelial malignancies are associated with highly increased concentrations of calprotecin in the urine. In absence of renal failure and pyuria, calprotectin constitutes a promising biomarker for the detection of bladder cancer.

Differential impact of belatacept and cyclosporine A on central aortic blood pressure and arterial stiffness after renal transplantation.

Calcineurin inhibitors (CNI) are potent vasoconstrictors and induce an acceleration of arteriosclerosis, thus contributing to the cardiovascular risk after renal transplantation. The study compares the impact of belatacept and cyclosporine A (CsA) on arterial stiffness and central aortic blood pressure. We performed a case-control study in 46 patients (23 on belatacept and 23 on CsA) matched for age, body mass index, time after transplantation, and time on dialysis prior to transplantation. Pulse wave analysis (SphygmoCor, AtCor(®) ) was used to assess central aortic blood pressure, aortic augmentation pressure, and pulse wave velocity (PWV) as a marker of arterial stiffness. Assessment of vascular function was performed after a minimum of 20 months and a median follow-up of 81 months post-transplant. Peripheral systolic and diastolic blood pressure did not significantly differ in the two groups (p > 0.05 each). The central aortic augmentation pressure was higher in the CsA group (12.7 mmHg vs. 7.3 mmHg, p = 0.048). PWV as a measure of arterial stiffness did not differ in the two groups. Thus, belatacept is not associated with a significant difference in arterial stiffness compared to CsA after a median of 81 months post-transplant. It is associated, however, with a lower aortic augmentation pressure, a strong independent cardiovascular risk factor.

Characterization of polymer membranes by MALDI mass-spectrometric imaging techniques.

For physical and chemical characterization of polymers, a wide range of analytical methods is available. Techniques like NMR and X-ray are often combined for a detailed characterization of polymers used in medical applications. Over the past few years, MALDI mass-spectrometry has been developed as a powerful tool for space-resolved analysis, not least because of its mass accuracy and high sensitivity. MALDI imaging techniques combine the potential of mass-spectrometric analysis with imaging as additional spatial information. MALDI imaging enables the visualization of localization and distribution of biomolecules, chemical compounds, and other molecules on different surfaces. In this study, surfaces of polymeric dialyzer membranes, consisting of polysulfone (PS) and polyvinylpyrrolidone (PVP), were investigated, regarding chemical structure and the compound's distribution. Flat membranes as well as hollow fiber membranes were analyzed by MALDI imaging. First, analysis parameters like laser intensity and laser raster step size (spatial resolution in resulting image) were established in accordance with polymer's characteristics. According to the manufacturing process, luminal and abluminal membrane surfaces are characterized by differences in chemical composition and physical characteristics. The MALDI imaging demonstrated that the abluminal membrane surface consists more of polysulfone than polyvinylpyrrolidone, and the luminal membrane surface displayed more PVP than PS. The addition of PVP as hydrophilic modifier to polysulfone-based membranes increases the biocompatibility of the dialysis membranes. The analysis of polymer distribution is a relevant feature for characterization of dialysis membranes. In conclusion, MALDI imaging is a powerful technique for polymer membrane analysis, regarding not only detection and identification of polymers but also localization and distribution in membrane surfaces.

Removal of protein-bound, hydrophobic uremic toxins by a combined fractionated plasma separation and adsorption technique.

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.

Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification.

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up(4)A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up(4)A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up(4)A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up(4)A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up(4)A-mediated process indicated that P2Y(2/6) receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up(4)A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.

Uridine adenosine tetraphosphate (Up4A) is a strong inductor of smooth muscle cell migration via activation of the P2Y2 receptor and cross-communication to the PDGF receptor.

The recently discovered dinucleotide uridine adenosine tetraphosphate (Up(4)A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up(4)A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up(4)A. Indeed, we found a strong chemoattractant effect of Up(4)A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up(4)A mediates it's migratory signal mainly via the P2Y(2). The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-ß we found a strongly reduced migration signal after Up(4)A stimulation in the PDGFR-ß knockdown cells compared to control cells. In this study, we present substantiate data that Up(4)A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein OPN. We further suggest that the initiation of the migration process occurs predominant through direct activation of the P2Y(2) by Up(4)A and via transactivation of the PDGFR.

Ramipril-based versus diuretic-based antihypertensive primary treatment in patients with pre-diabetes (ADaPT) study.

BACKGROUND: Previous randomized controlled trials demonstrated a protective effect of renin angiotensin system blocking agents for the development of type-2 diabetes in patients with pre-diabetes. However, there are no real-world data available to illustrate the relevance for clinical practice. METHODS: Open, prospective, parallel group study comparing patients with an ACE inhibitor versus a diuretic based treatment. The principal aim was to document the first manifestation of type-2 diabetes in either group. RESULTS: A total of 2,011 patients were enrolled (mean age 69.1±10.3 years; 51.6% female). 1,507 patients were available for the per-protocol analysis (1,029 ramipril, 478 diuretic group). New-onset diabetes was less frequent in the ramipril than in the diuretic group over 4 years. Differences were statistically different at a median duration of 3 years (24.4% vs 29.5%; p<0.05). Both treatments were equally effective in reducing BP (14.7±18.0/8.5±8.2 mmHg and 12.7±18.1/7.0±8.3 mmHg) at the 4 year follow-up (p<0.001 vs. baseline; p=n.s. between groups). In 38.6% and 39.7% of patients BP was below 130/80 mmHg (median time-to-target 3 months). There was a significant reduction of cardiovascular morbidity and mortality in favour of ramipril (p=0.033). No significant differences were found for a change in HbA1c as well as for fasting blood glucose levels during follow-up. The rate of adverse events was higher in diuretic treated patients (SAE 15.4 vs. 12.4%; p<0.05; AE 26.6 vs. 25.6%; p=n.s). CONCLUSIONS: Ramipril treatment is preferable over diuretic based treatment regimens for the treatment of hypertension in pre-diabetic patients, because new-onset diabetes is delayed.

Relationship between magnesium and clinical biomarkers on inhibition of vascular calcification.

BACKGROUND: Arteriosclerosis and cardiovascular disease are strongly associated with vascular calcification. Hyperphosphatemia is an essential risk factor for increased vascular calcification. End-stage renal disease (ESRD) patients could serve as an in vivo model for accelerated calcification. This study focuses on the most likely protective effects of magnesium ion (Mg(2+)) on phosphate-induced vascular calcification ex vivo/in vitro. Furthermore, plasma Mg(2+) concentrations of ESRD and healthy controls were investigated for association with surrogate parameters of vascular calcification in vivo. METHODS: Aortic segments of male Wistar-Kyoto rats were incubated and the phosphate concentration of the medium was elevated. The aortic segments were incubated in the absence and presence of MgCl(2); tissue calcification was quantified by different methods. Serum Mg(2+) concentrations of patients with chronic kidney disease (CKD stage 5; ESRD) and patients without CKD (controls) were associated with carotid intima media thickness (IMT) and aortic pulse wave velocity (PWV) as surrogate parameter for arteriosclerosis and arterial stiffening. RESULTS: Incubation of aortic segments in the presence of ß-glycerophosphate and NaH(2)PO(4) caused an increased tissue Ca(2+) deposition compared to control conditions. This increased amount of Ca(2+) in the aortic rings was significantly decreased in the presence of Mg(2+). In CKD patients, but not in controls, magnesium serum concentration was associated with the IMT of the carotid arteries. In addition, CKD patients with higher magnesium serum concentration had a significantly lower PWV. DISCUSSION AND CONCLUSION: Elevated phosphate concentrations in the culture media induce ex vivo/in vitro medial calcification in intact rat aortic rings in the presence of alkaline phosphatase. Mg(2+) ions reduced ex vivo/in vitro vascular calcification despite increased phosphate concentration. This hypothesis is additionally based on the fact that CKD patients with high Mg(2) serum levels had significantly lower IMT and PWV values, which may result in a lower risk for cardiovascular events and mortality in these patients. Therefore, Mg(2+) supplementation may be an option for treatment and prevention of vascular calcification resulting in a reduction of cardiovascular events in CKD patients.

Angioprotectin: an angiotensin II-like peptide causing vasodilatory effects.

The family of angiotensin peptides has been steadily growing in recent years. Most are fragments of angiotensin II (Ang II) with different affinities to the known angiotensin receptors. Here, we describe a novel endogenous Ang II-like octapeptide in plasma from healthy humans and patients with end-stage renal failure, which acts as a stronger agonist at Mas receptors than Ang 1-7. Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an Ang II-like octapeptide, angioprotectin, with the sequence Pro-Glu-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Pro¹ and Glu² instead of Asp¹ and Arg². Pro-Glu-Val-Tyr-Ile-His-Pro-Phe in angioprotectin is most likely generated enzymatically from Ang II. Angioprotectin antagonized the contractile actions of Ang II on rat aortic rings. The physiological antagonism of vasoconstrictor actions of Ang II by angioprotectin is mediated by the Mas receptor. Angioprotectin has a stronger affinity to the Mas receptor than Ang-1-7. Plasma concentrations were ~15% of plasma Ang II concentrations in healthy volunteers and up to 50% in patients with renal failure. A commercially available Ang II antibody did not discriminate between angioprotectin and Ang II; thus, angioprotectin can contribute to Ang II concentrations measured by antibody-based assays. This novel peptide is likely to be a relevant component of the human renin-angiotensin-system.

A biomarker for severity of Alzheimer's disease: 1H-NMR resonances in cerebrospinal fluid correlate with performance in mini-mental-state-exam.

CONTEXT: There is no valid biomarker for severity of Alzheimer's disease (AD) available until now. OBJECTIVE: Therefore, we investigated (1)H-NMR spectroscopy for specific resonances as biomarkers for severity of AD. MATERIALS AND METHODS: Cerebrospinal fluid (CSF) of patients with diagnosed AD and healthy control subjects was analysed by one-dimensional water-suppressed (1)H-NMR spectroscopy. The resonances were correlated with the cognitive performance of the patients and controls. RESULTS: Specific (1)H-NMR resonances showed higher intensities in AD patients than in control subjects. Mini-mental-state-exam scores correlated with (1)H-NMR resonances in AD patients. DISCUSSION AND CONCLUSION: (1)H-NMR resonances of CSF are obviously valid biomarker for severity of AD, despite the lack of knowledge of the underlying molecular structure. Successful isolation and identification of these substances will most likely provide details to the pathophysiology of AD.

Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor.

Beyond serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors (EDCFs) remains unclear. The supernatant obtained from mechanically stimulated human endothelial cells obtained from dermal vessels elicited a vasoconstrictive response in an isolated perfused rat kidney. A purinoceptor blocker had a greater effect than an endothelin receptor blocker in decreasing endothelially derived vasoconstriction in the isolated perfused rat kidney. The nucleotide uridine adenosine tetraphosphate (Up(4)A) was isolated from the supernatant of stimulated human endothelium and identified by mass spectrometry. Up(4)A is likely to exert vasoconstriction predominantly through P2X1 receptors, and probably also through P2Y2 and P2Y4 receptors. Plasma concentrations of Up(4)A that cause vasoconstriction are found in healthy subjects. Stimulation with adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), acetylcholine, endothelin, A23187 and mechanical stress releases Up(4)A from endothelium, suggesting that Up(4)A contributes to vascular autoregulation. To our knowledge, Up(4)A is the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. We conclude that Up(4)A is a novel potent nonpeptidic EDCF. Its vasoactive effects, plasma concentrations and its release upon endothelial stimulation strongly suggest that Up(4)A has a functional vasoregulatory role.