Susann Patschan

Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells

Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100-F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4-5 days, it was nearly undetectable. Early induction of autophagosomes was associated with their fusion with lysosomes, a phenomenon that later became subverted. Autophagic cell death can be triggered by the products of damaged plasma membrane, sphingolipids, and ceramide. We observed a clustering of membrane rafts shortly after exposure to GC; later, after 24 h, we observed an internalization, accompanied by an increased acid sphingomyelinase activity and accumulation of ceramide. Pharmacological inhibition of autophagy prevented development of premature senescence but did lead to the enhanced rate of apoptosis in human umbilical vein endothelial cells exposed to GC. Pharmacological induction of autophagy resulted in reciprocal changes. These observations appear to represent a mechanistic molecular cascade whereby advanced glycation end products like GC induce sphingomyelinase activity, accumulation of ceramide, clustering, and later internalization of lipid rafts.

Which patients benefit from hemodialysis therapy in hepatorenal syndrome

Background and Aim:  Hepatorenal syndrome (HRS) occurs in patients with advanced liver cirrhosis and has a poor outcome. The aim of the present study was to investigate which patients with HRS are likely to benefit from hemodialysis.

Carbon Monoxide Stimulates the Ca2+–Activated Big Conductance K Channels in Cultured Human Endothelial Cells

We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca2+-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, NG-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with NG-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

Stress-induced premature senescence of endothelial cells: a perilous state between recovery and point of no return.

Purpose of reviewTo discuss most recently published studies on morphologic patterns and molecular mechanisms of stress-induced premature senescence (SIPS) of vascular endothelial cells. Recent findingsLysosomal dysfunction and impaired autophagy, which have been well established in replicative senescence, were also described in SIPS induced by advanced glycation end products (AGEs). Recently, strides were made to prevent and reverse senescence. The role of lysosomal dysfunction and Lamp-2A deficiency has been demonstrated in aging. Molecular analyses identified the role of sirtuin 1 in preventing cell senescence; shed light on the role of polycomb group (PcG) protein Bmi-1 in senescence. Additionally, intriguing data on the role of caveolin-1 in cell senescence have emerged. SummaryIn aging organisms and chronic diseases properly functioning tissue is replaced by senescent cells. Comparison between replicative senescence and SIPS indicates that replicative senescence is almost exclusively associated with the reduction of telomerase activity and attrition of telomeres, whereas SIPS does not require these events, thus conferring potential reversibility onto this process.

Effects of baroreflex activation therapy on arterial stiffness and central hemodynamics in patients with resistant hypertension.

Background: High central blood pressure, augmentation index and pulse wave velocity are independent cardiovascular risk factors. Little is known of the effect of baroreflex activation therapy on central hemodynamics. Method: In this prospective clinical trial, radial artery applanation tonometry and pulse wave analysis were used to derive central aortic pressure and hemodynamic indices (i.e. augmentation pressure, augmentation index, pulse wave velocity, systolic and diastolic pressure time integral, subendocardial viability index) at baseline and 6 months after starting baroreflex activation therapy in 25 patients with resistant hypertension. Results: Apart from peripheral blood pressure reduction, 6 months of baroreflex activation therapy significantly reduced mean central aortic blood pressure from 109.7 ± 20.5 to 97.4 ± 18.8 mmHg (P < 0.01) and aortic pulse pressure from 62.9 ± 18.6 to 55.2 ± 16.0 mmHg (P < 0.01). Aortic augmentation pressure and augmentation index at a heart rate of 75 b.p.m. were significantly reduced by 4.3 ± 7.9 mmHg (P = 0.01) and 3.5 ± 6.8% (P = 0.02). Additionally, pulse wave velocity decreased from 10.3 ± 2.6 to 8.6 ± 1.3 m/s (P < 0.01) 6 months after starting baroreflex activation therapy. Systolic pressure time integral was significantly reduced (P = 0.03), whereas subendocardial viability index remained unchanged. Conclusion: Apart from peripheral blood pressure, baroreflex activation therapy reduces central blood pressure, augmentation index at a heart rate of 75 b.p.m. and pulse wave velocity in patients with resistant hypertension, suggesting strong potential to reduce cardiovascular risk.

Autophagy: The missing link between non-enzymatically glycated proteins inducing apoptosis and premature senescence of endothelial cells?

In a series of studies into the fate of endothelial cells exposed to non-enzymatically glycated collagen I, a model of cytotoxic molecules relevant to diabetic vasculopathy, we demonstrate that cells either undergo apoptosis or become prematurely senescent despite relatively spared telomeres and telomerase activity. Our most recent work shows that long-lived advanced glycation end product (AGE)-modified proteins induce 1) lysosomal permeabilization leading to the inefficiency of autophagy due to the reduced digestion (early) and non-fusion (later) of lysosomes with phagosomes—a frustrated autophagy; and 2) accumulation of lipid mediators, such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death. Under the experimental conditions described here, the seesaw relations between premature senescence and apoptosis are integrated by autophagy, which plays a novel function of a cellular switch between states of premature senescence and apoptosis. Addendum to: Patschan SA, Chen J, Polotskaia A, Mendelev N, Cheng J, Patschan D, Goligorsky MS. Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells. Am J Physiol Heart Circ Physiol 2008; In press. and Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS. Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells. Am J Physiol Renal Physiol 2008; 294:F100-9.

LDL lipid apheresis rapidly increases peripheral endothelial progenitor cell competence.

Endothelial progenitor cells (EPCs) have been shown to promote neovascularization under physiologic and pathologic conditions. Statins have been documented to increase the total number of circulating EPCs in long‐term treated patients. Lipid apheresis is used to treat patient with refractory hyperlipidemia. The aim of our study was to evaluate whether lipid apheresis is associated with EPC mobilization.

Endothelial Progenitor Cells in Acute Ischemic Kidney Injury: Strategies for Increasing the Cells' Renoprotective Competence

Acute ischemic kidney injury is the most frequent cause of acute renal failure in daily clinical practice. It has become increasingly recognized that microvascular endothelial cell dysfunction (ED) in peritubular capillaries inhibits the process of postischemic renal reperfusion. ED can serve as therapeutic target in the management of acute ischemic kidney injury. Postischemic reflow can be restored by systemic administration of either mature endothelial cells or of endothelial progenitor cells. Endothelial progenitor cells EPCs can be cultured from the peripheral circulation of humans and different animals. The cells act vasoprotectively by direct and indirect mechanisms. The protective effects of EPCs in acute ischemic kidney injury can be stimulated by preincubating the cells with different agonistic mediators. This paper summarizes the currently available data on strategies to improve the renoprotective activity of EPCs in acute ischemic kidney injury.

Angiopoietin-2 modulates eEOC-mediated renoprotection in AKI in a dose-dependent manner.

BACKGROUND Early endothelial outgrowth cells (eEOCs) significantly protect mice from acute kidney injury (AKI). Angiopoietin-2 (Ang-2) has been shown to be critically involved in vascular repair and homeostasis. The aim of this study was to investigate consequences of Ang-2 treatment of syngeneic murine eEOCs in a cell-based therapeutic approach for AKI. METHODS Male 8- to 12-week-old C57/Bl6N mice, subjected to unilateral renal ischemia (40 minutes) postuninephrectomy were systemically injected with 0.5 × 10(6) untreated or Ang-2-pretreated syngeneic murine eEOCs. Renal function and morphology were analyzed 48 hours later. Cellular consequences of eEOC treatment with Ang-2 were evaluated using different in vitro assays (direct and indirect migration, apoptosis/necrosis, ELISA studies). RESULTS Administration of untreated eEOCs did not protect mice from AKI. Ang-2 dose-dependently modulated cell effects in AKI. While incubating the cells at a concentration of 200 ng/mL (1 hour) did not have any effect on renal function, doubling the concentration (400 ng/mL) resulted in significant renoprotection of cell-injected mice. With 800 ng/mL, cell injection dramatically worsened renal function of treated animals. In vitro analysis showed significantly accelerated migration of cultured mature endothelial cells after incubation with supernatant from Ang-2-treated eEOCs (200 and 400 ng/mL). These effects were most pronounced with 400 mg/mL. In addition, Ang-2 promoted survival of eEOCs. Cellular releases of VEGF and IL-6 were decreased by Ang-2, while TGF-β levels in the medium of Ang-2-stimulated eEOCs were not different from those in untreated cells. CONCLUSION Ang-2 acts as modulator of eEOCs in AKI. The migration analysis indicates that the Ang-2 significantly alters indirect (paracrine) activity of eEOCs, thus promoting renoprotection in a dose-dependent manner.

Regulation of Arginine Methylation in Endothelial Cells : Role in Premature Senescence and Apoptosis

With the recent characterization of enzymes responsible for protein arginine methylation and demonstration that catabolic products of arginine methylation, such as asymmetric dimethylarginine (ADMA), are among the most powerful mechanisms of atherogenesis, developing endothelial dysfunction and cardiovascular complications in a variety of pathologic processes, the need for functional characterization of the methylation-demethylation processes becomes ever more urgent. Therefore, the aims of the present study were to refine the feedback regulation of protein arginine methylation using one of the heavily methylated proteins, an RNA-binding protein Sam68, as a prototype, to elucidate the relations between Sam68 methylation and tyrosine phosphorylation and the role of methylation in RNA binding and subcellular distribution, as well as the cellular consequences of reduced protein methylation. Screening pro-atherogenic substances known to induce endothelial dysfunction showed that ADMA did not affect the level of arginine methylation of Sam68, whereas peroxynitrite was a strong inhibitor of methylation. Adavanced glycation-modified collagen I, which accumulats in diabetes and induces formation of peroxynitrite and premature endothelial cell senescence, also inhibited arginine methylation of Sam68. When the level of arginine methylation of Sam68 was pharmacologically reduced, this did not affect its RNA binding or degree of tyrosine phosphorylation, but resulted in the predominantly nuclear hypomethylation pattern. Furthermore, protein hypomethylation resulted in the increased rate of apoptosis and premature senescence. This data may offer an additional explanation for the pro-apoptotic and senescence-accelerating action of peroxynitrite, a potent inhibitor of protein methylation.