Ulrich F. Mondorf

Oxidized LDL Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration by an Inhibitory Effect on the Akt/Endothelial Nitric Oxide Synthase Pathway

BackgroundOxidized LDL (oxLDL) inhibits endothelial cell (EC) migration. Stimulating ECs with vascular endothelial growth factor (VEGF) leads to the activation of Akt/protein kinase B, which in turn activates endothelial nitric oxide synthase (eNOS) by phosphorylation on serine 1177. VEGF-induced cell migration is dependent on the generation of nitric oxide (NO). Therefore, we investigated whether oxLDL affects EC migration by an inhibitory effect on the Akt/eNOS pathway. Methods and ResultsDuring an in vitro “scratched wound assay,” oxLDL dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells. Western blot analysis revealed that oxLDL dose- and time-dependently led to dephosphorylation and thus deactivation of Akt. Moreover, oxLDL inhibited the VEGF-induced generation of NO, as detected and quantified using a fluorescent NO indicator, 4,5-diaminofluorescein diacetate. Overexpression of a constitutively active Akt construct (Akt T308D/S473D) or a phosphomimetic eNOS construct (eNOS S1177D) almost completely reversed the inhibitory effect of oxLDL on VEGF-induced EC migration and NO generation. ConclusionsOur data indicate that oxLDL-induced dephosphorylation of Akt, followed by impaired eNOS activation, reduces the intracellular level of NO and thereby inhibits VEGF-induced EC migration.

Contribution of angiotensin i converting enzyme gene polymorphism and angiotensinogen gene polymorphism to blood pressure regulation in essential hypertension

The renin-angiotensin system (RAS) is involved in the pathogenesis of essential hypertension. In the present study we examined the genotype frequencies of the insertion/deletion polymorphisms of the ACE gene and the M235T polymorphism of the Angiotensinogen (Agt) gene in patients with essential hypertension in comparison with normotensive subjects. In hypertensive patients functional effects of blood pressure response to ACE inhibition were investigated. A total of 121 patients with essential hypertension (group 1) and 125 normotensive control subjects (group 2) were included in this study. All patients were genotyped by polymerase chain reactions (PCR) for the insertion/deletion (I/D) polymorphism of the ACE gene and the M235T polymorphism of the Agt gene. To analyze possible functional impacts on blood pressure regulation 50 mg of captopril was administered to hypertensive patients. No significant association of essential hypertension with polymorphisms of the Agt and ACE gene was found. The ACE serum levels in patients with the DD-genotype of the ACE I/D polymorphism were higher than in patients with the II-genotype (P < .01). In patients with the ID-genotype the ACE serum levels were in-between. A captopril test was performed in hypertensive patients. The patients were further divided into subgroups according to the diastolic and systolic blood pressure response. Group 1a consisted of patients with a diastolic blood pressure drop of > 5 mm Hg and group 1b with < or =5 mm Hg. Group 1c consisted of patients with a systolic blood pressure drop of > 10 mm Hg and group 1d with < or =10 mm Hg. Twice as many patients with the DD genotype of the ACE gene were found in group 1a compared to group 1b (chi(2) = 5.673; P = .017). No association of systolic blood pressure change to the investigated polymorphisms was found. Our results do not support the hypothesis that the investigated polymorphisms contribute to essential hypertension. Furthermore, no major impact of these polymorphisms on blood pressure response to captopril were detected. We conclude that the investigated genotypes have no influence on blood pressure level and homeostasis.

Involvement of the platelet-derived growth factor receptor in angiotensin II-induced activation of extracellular regulated kinases 1 and 2 in human mesangial cells

In mesangial cells angiotensin II (Ang II) has been shown to activate extracellular regulated kinases 1 and 2 (ERK1/2). Here, we studied the role of the epidermal growth factor receptor (EGFR) and platelet‐derived growth factor receptor (PDGFR) in Ang II‐induced ERK1/2 activation in human mesangial cells. Ang II induced activation of ERK1/2 via the AT1 receptor, and this response was blocked by the PDGFR‐selective tyrosine kinase inhibitor AG1295, but not by AG1478, an EGFR‐selective tyrosine kinase inhibitor, indicating participation of the PDGFR, but not of the EGFR in Ang II‐induced ERK1/2 activation. In agreement with this assumption, Ang II caused tyrosine phosphorylation of the PDGFR and the adapter protein Shc in an AG1295‐sensitive fashion. In conclusion, our data show that Ang II‐induced activation of mitogenic signalling cascade in human mesangial cells involves ligand‐independent activation of the PDGFR, but not of the coexpressed EGFR.

Inhibition of the equilibrative nucleoside transporter 1 and activation of A2A adenosine receptors by 8-(4-chlorophenylthio)-modified cAMP analogs and their hydrolytic products

Cyclic AMP analogs containing hydrophobic modification of C8 at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2′-O-methyl-adenosine (8-pCPT-2′-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2′-O-methyl-ado, and, to a smaller extent, 8-pCPT-2′-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A2A adenosine receptor (A2AR)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A2AR-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A2AR-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.

Lipoprotein (a) stimulates mitogen activated protein kinase in human mesangial cells.

Evidence suggests an important role of elevated serum lipoproteins in the progression of renal glomerulosclerosis. We report here that lipoprotein (a) (Lp(a)) increased phosphorylation and activity of mitogen activated protein kinase (MAPK) in human mesangial cells. When protein kinase C (PKC) was depleted by long‐term incubation with the phorbol 12‐O‐myristate 13‐acetate the effect of Lp(a) on MAPK activation was completely inhibited. Forskolin, a stimulator of the adenylyl cyclase, and dibutyryl‐cAMP reduced the effect of Lp(a) on MAPK phosphorylation and activation. We conclude that Lp(a) stimulates the MAPK cascade via activation of PKC and that activation of protein kinase A counteracts Lp(a) induced MAPK activation in human mesangial cells.

Propeptide big-endothelin, N-terminal-pro brain natriuretic peptide and mortality. The Ludwigshafen risk and cardiovascular health (LURIC) study

Abstract Context: The endothelin system (Big-ET-1) is a key regulator in cardiovascular (CV) disease and congestive heart failure (CHF). Objectives: We have examined the incremental value of Big-ET-1 in predicting total and CV mortality next to the well-established CV risk marker N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP). Methods: Big-ET-1 and NT-proBNP were determined in 2829 participants referred for coronary angiography (follow-up 9.9 years). Results: Big-ET-1 is an independent predictor of total, CV mortality and death due to CHF. Discussion: The conjunct use of Big-ET-1 and NT-proBNP improves the risk stratification of patients with intermediate to high risk of CV death and CHF. Conclusions: Big-ET-1improves risk stratification in patients referred for coronary angiography.

Influence of diuresis on enzymuria.

Urinary excretion of renal brush border enzymes may serve as an early marker of renal injury. However, the distinction between physiological and pathological levels remains controversial, since enzymuria is affected by physiological parameters. To clarify the influence of diuresis, we investigated the urinary excretion of alanine-aminopeptidase (AAP; EC 3.4.11.2) as function of diuretic state. 17 healthy volunteers of both sexes were subjected to protocols with sudden or prolonged water load preceded and followed by a thirst period. Urinary excretion of AAP was measured using an enzyme kinetic assay. As expected AAP excretion increased with urine flow, the increments diminished yielding an overall excretion pattern that resembled saturation kinetics. This function is described by a mathematical model. This model assumes, that AAP is released in proximal tubules at a constant rate and reabsorbed or inactivated in the distal tubule and collecting duct. Non-linear fits of the model equation to our data allowed two parameters, χ and μ, to be defined. χ describes the rate of AAP release independent of urinary flow, and μ the ratio of distal tubular reabsorption or inactivation. If a substrate is not reabsorbed at all, μ approximates zero. Since μ fitted for AAP differed significantly from zero, this indicates reabsorption or inactivation of AAP in the distal nephron. Therefore, our study supports the theory of flow-dependent reabsorption or inactivation of AAP in the distal nephron.

Blood Pressure Control in Essential Hypertension and Impairment of Renal Function with Age

Hypertension has been recognized to be an important cause for the development of end-stage renal disease (ESRD). We assessed the quality of blood pressure control in 103 patients with essential hypertension and correlated renal function and age. Patients were stratified into three subgroups by their blood pressure level under current medication. Group 1 were hypertensive patients with normalized blood pressure (<140/90 mmHg, n = 25), group 2 patients with mild hypertension (140-159/90-99 mmHg, n = 43) and group 3 patients with moderate to severe hypertension (>160/100 mmHg, n = 35). A negative correlation between age and creatinine clearance (CCr) could be confirmed for patients of group 1 (correlation coefficient r1 = -0.56; p1 < 0.01) and group 2 (r2 = -0.55; p2 < 0.001). Furthermore the regression coefficient (m) of decline in CCr versus age was higher in group 2 patients (m2 = -1.83) than in group 1 (m1 = -1.30). In group 3 we found no correlation of renal function with age, indicating that age may not be the leading variable. Patients in group 1 were all within normal limits of age adjusted CCr, but 12% in group 2 and 23% in group 3 had impaired CCr. Furthermore proteinuria was found to be 20% (group 1), 26% (group 2) and 31% (group 3). This analysis provides further evidence of the importance of blood pressure control in essential hypertension to preserve renal function.