Mirjam Schuchardt

The Sphingosine-1-Phosphate Analogue FTY720 Reduces Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—The sphingosine-1-phosphate (S1P) analogue FTY720 is a potent immunosuppressive agent currently in Phase III clinical trials for kidney transplantation. FTY720 traps lymphocytes in secondary lymphoid organs thereby preventing their migration to inflammatory sites. Previously, we have identified FTY720 as a potent activator of eNOS. As both inhibition of immune responses and stimulation of eNOS may attenuate atherosclerosis, we administered FTY720 to apolipoprotein E−/− mice fed a high-cholesterol diet. Methods and Results—FTY720 dramatically reduced atherosclerotic lesion volume (62.5%), macrophage (41.8%), and collagen content (63.5%) after 20 weeks of high-cholesterol diet. In isolated aortic segments and cultured vascular smooth muscle cell, FTY720 potently inhibited thrombin-induced release of monocyte chemoattractant protein-1. This effect was mediated by the S1P3 sphingolipid receptor as FTY720 had no effect on thrombin-induced monocyte chemoattractant protein-1 release in S1P3−/− mice. In contrast to S1P receptors on lymphocytes, FTY720 did not desensitize vascular S1P receptors as arteries from FTY720-treated mice retained their vasodilator response to FTY720-phosphate. Conclusions—We suggest that FTY720 inhibits atherosclerosis by suppressing the machinery involved in monocyte/macrophage emigration to atherosclerotic lesions. As vascular S1P receptors remained functional under FTY720 treatment, S1P agonists that selectively target the vasculature and not the immune system may be promising new drugs against atherosclerosis.

Mass-Spectrometric Identification of a Novel Angiotensin Peptide in Human Plasma

Objective—Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. Methods and Results—Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A / Ang II was higher in end-stage renal failure patients. Conclusion—Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.

High-density lipoprotein loses its anti-inflammatory capacity by accumulation of pro-inflammatory-serum amyloid A

AIMS High-density lipoprotein (HDL) is known to have potent anti-inflammatory properties. Monocyte chemoattractant protein-1 is an important pro-inflammatory cytokine in early atherogenesis. There is evidence that HDL can lose its protective function during inflammatory disease. In patients with end-stage renal disease (ESRD), epidemiological studies have documented that the inverse correlation between HDL-cholesterol and cardiovascular risk is lost. Many structural modifications leading to reduced HDL function have been characterized, but the functional consequences are not fully understood. METHODS AND RESULTS We showed that HDL from patients with ESRD has a lower anti-inflammatory potential by reduced inhibition of monocyte chemoattractant protein-1 formation in vascular smooth muscle cells. Via a proteomic approach, we identified proteins in HDL from ESRD patients exerting pro-inflammatory actions. By chromatographic separation of proteins and mass-spectrometric analysis, we found serum amyloid A (SAA) to be one molecule acting as a potent pro-inflammatory protein. SAA is enriched in HDL from ESRD patients, correlating with reduced anti-inflammatory capacity. In SAA signal transduction, activation of formyl-peptide receptor 2 is involved. SAA enrichment in HDL of healthy subjects reduced the anti-inflammatory capacity of HDL and correlated with its decreased function. CONCLUSION These results suggest that SAA enrichment of HDL during disease conditions contributes to the decreased protective function. It is a novel finding that SAA acts as a pro-inflammatory molecule to reduce the anti-inflammatory properties of HDL.

Enhancement of the endothelial NO synthase attenuates experimental diastolic heart failure

BackgroundDiastolic heart failure is a rising problem with a high incidence and similar mortality and morbidity compared to patients with systolic heart failure. Nevertheless, the underlying pathophysiology is still debated.AimWe investigated the effect of pharmacological enhancement of endothelial nitric oxide synthase (eNOS) on experimental diastolic heart failure (DHF).MethodsDHF was induced in 60 DAHL salt-sensitive rats by salt diet in 8-week-old animals. 30 were treated with the eNOS enhancer AVE3085 (DHFeNOS) and 30 with placebo (DHF). Rats with normal salt intake served as controls.Results and conclusionDiastolic dysfunction with increased diastolic stiffness constant and increased left ventricular (LV) pressure was analyzed by invasive pressure–volume loop measurements in the DHF group compared to controls. Cardiac hypertrophy as indicated by LV mass measurements by echocardiography, and increased cardiac collagen content as measured by immunohistochemistry were associated with an increased activation state of calcineurin, AKT, ERK½, but not JNK and p38 kinases. Titin isoforms were not altered in this model of DHF. Treatment with AVE3085 significantly increased eNOS mRNA and protein levels in the cardiac tissue and decreases NAD(P)H oxidase subunits p22phox and gp91phox. Diastolic dysfunction was attenuated and cardiac hypertrophy and fibrosis were improved in comparison with untreated DHF animals. This was associated with a normalized activation state of calcineurin, AKT and ERK½. Therefore, we suggest that targeting the NO system might yield a future therapeutic aim for the treatment of DHF.

Differential effects of uridine adenosine tetraphosphateon purinoceptors in the rat isolated perfused kidney

BACKGROUND AND PURPOSE Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up4A) as a novel and highly potent endothelial‐derived contracting factor. Up4A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X1 receptor activation. However, other purinoceptors are also involved and were analysed here.

Arteriosclerosis and vascular calcification: causes, clinical assessment and therapy.

Arteriosclerosis is a pathological, structural (media vascular calcification) and physiological (modified vascular smooth vessel cells; increased arterial stiffness) alteration of the vessel wall. Through improved assessment methods (functional and imaging), it has become a well‐known phenomenon in recent decades. However, its clinical importance was underestimated until recently.

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 its 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-β we found a strongly reduced migration signal after Up(4)A stimulation in the PDGFR-β 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.

Adenosine 5′-Tetraphosphate Is a Highly Potent Purinergic Endothelium-Derived Vasoconstrictor

Besides 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 still remains uncertain. The supernatant from mechanically stimulated human microvascular endothelial cells elicited a potent vasoconstrictive response in the isolated perfused rat kidney. Whereas a nonselective purinoceptor blocker blocked this vasoactivity most potently, the inhibition of the endothelin receptor by BQ123 weakly affected that vasoconstrictive response. As a compound responsible for that vasoconstrictive effect, we have isolated from HMECs and identified the mononucleotide adenosine 5′-tetraphosphate (AP4). This nucleotide proved to be the most potent vasoactive purinergic mediator identified to date, exerting the vasoconstriction predominantly through activation of the P2X1 receptor. The intraarterial application of AP4 in a Wistar–Kyoto rat induced a strong increase of the mean arterial pressure. The plasma concentration of AP4 is in the nanomolar range, which, in vivo, induces a significant change in the mean arterial pressure. To our knowledge, AP4, which exerts vasoactive effects, is the most potent endogenous mononucleotide identified to date in mammals. The effects of AP4, the plasma concentration of AP4, and its release suggest that this compound functions as an important vasoregulator.

Iohexol plasma clearance measurement in older adults with chronic kidney disease—sampling time matters

BACKGROUND Accurate and precise measurement of GFR is important for patients with chronic kidney disease (CKD). Sampling time of exogenous filtration markers may have great impact on measured GFR (mGFR) results, but there is still uncertainty about optimal timing of plasma clearance measurement in patients with advanced CKD, for whom 24-h measurement is recommended. This satellite project of the Berlin Initiative Study evaluates whether 24-h iohexol plasma clearance reveals a clinically relevant difference compared with 5-h measurement in older adults. METHODS In 104 participants with a mean age of 79 years and diagnosed CKD, we performed standard GFR measurement over 5 h (mGFR300) using iohexol plasma concentrations at 120, 180, 240 and 300 min after injection. With an additional sample at 1440 min, we assessed 24-h GFR measurement (mGFR1440). Study design was cross-sectional. Calculation of mGFR was conducted with a one compartment model using the Brochner-Mortensen equation to calculate the fast component. mGFR values were compared with estimated GFR values (MDRD, CKD-EPI, BIS1, Revised Lund-Malmö and Cockcroft-Gault). RESULTS In all 104 subjects, mGFR1440 was lower than mGFR300 (23 ± 8 versus 29 ± 9 mL/min/1.73 m(2), mean ± SD; P < 0.001). mGFR1440 was highly correlated with mGFR300 (r = 0.9). The mean absolute difference mGFR300 - mGFR1440 was 5.9 mL/min/1.73 m(2) corresponding to a mean percentage difference of 29%. In individuals with eGFRCKD-EPI ≤ 30 mL/min/1.73 m(2), percentage difference of mGFR300 and mGFR1440 was even higher (35%). To predict mGFR1440 from mGFR300, we developed the correction formula: mGFR1440 = -2.175 + 0.871 × mGFR300 (1-fold standard error of estimate: ±2.3 mL/min/1.73 m(2)). The GFR estimating equation with the best accuracy and precision compared with mGFR300 and mGFR1440 was the Revised Lund Malmö. CONCLUSIONS In elderly CKD patients, measurement of iohexol clearance up to 5 h leads to a clinically relevant overestimation of GFR compared with 24-h measurement. In clinical care, this effect should be bore in mind especially for patients with considerably reduced GFR levels. A new correction formula has been developed to predict mGFR1440 from mGFR300. For accurate GFR estimates in elderly CKD patients, we recommend the Revised Lund Malmö equation.

Cystatin C standardization decreases assay variation and improves assessment of glomerular filtration rate.

BACKGROUND Cystatin C is increasingly used in glomerular filtration rate (GFR) estimation equations. The dependence of cystatin C results upon the analytical method has been a major source of controversy. METHODS Cystatin C was measured with non-standardized turbidimetric Roche Generation 1 and standardized nephelometric Siemens assays in 3666 and additionally with standardized Roche Generation 2 and Siemens in 567 blood samples of the Berlin Initiative Study. Cystatin C-based GFR was assessed with CKD-EPIcys (Chronic Kidney Disease Epidemiology) and CAPA (Caucasian, Asian, Pediatric, Adult) equations and the impact of the assays on GFR estimation was determined. Equation performance compared to measured GFR was evaluated. RESULTS Concordance of Roche Gen2 and Siemens was high with median difference of 0.003 ± 0.13 mg/L (limits of agreement: -0.12 to 0.12) and Passing Bablok correlation was essentially perfect. Roche Gen1 assay showed worse concordance with Siemens: median difference was 0.08 ± 0.13 mg/L (limits of agreement: -0.18 to 0.34) and correlation was inferior. Mean difference (± SD) of estimated GFRCKD-EPIcys was 0 ± 4 mL/min/1.73 m(2) for Gen2 and Siemens compared to -5 ± 8 with Gen1. Performance of GFR estimating equations was not influenced by the choice of Siemens or Gen2 assays. CONCLUSIONS Standardization of Roche Gen2 assay improved accuracy of cystatin C measurement compared to Siemens. It suggests only negligible method bias and results in equal performance of both assays when estimating GFR indicating that successful calibration has led to major progress in cystatin C analysis.