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Objective— Cyclooxygenases 1 and 2 are expressed in atherosclerotic arteries, and local generation of prostacyclin and prostaglandin E 2 (PGE 2 ) occurs. However, the role of cyclooxygenases and individual prostaglandins during plaque progression is currently uncertain. The present study characterizes the effect of vasodilatory prostaglandins on morphology, focal adhesion (FA) function, and migration in human aortic smooth muscle cells (SMCs). Methods and Results— The stable prostacyclin analog iloprost transiently induced: (1) disassembly of FA and stress fibers, (2) partial retraction and rounding of SMCs, (3) hypophosphorylation of FA kinase (FAK) and paxillin, and (4) inhibition of platelet-derived growth factor-BB–induced migration. Inhibition of FAK phosphorylation and morphological changes were mimicked by forskolin, inhibited by H89, and prevented by the protein tyrosine phosphatase inhibitor vanadate and by calpeptin. PGE 2 was by far less efficient with respect to all parameters investigated. This difference correlated with the respective cAMP induction in response to iloprost and PGE 2 . Conclusion— Inhibition of FAK phosphorylation and FA function is a new target of vasodilatory prostaglandins, which might be causally involved in the antimigratory effects of prostaglandins. Importantly, prostacyclin analogs and PGE 2 differ dramatically with respect to dephosphorylation of FAK and inhibition of migration, which might be of relevance for their respective functions in atherosclerosis.

Zusammenfassung Sowohl die Ausrichtung der medizinischen Forschung und Klinik in Richtung “molekulare Medizin” verbunden mit der Entschlusselung des menschlichen Genoms als auch die hieraus resultierende Entwicklung und Nutzung von transgenen Tiermodellen menschlicher Erkrankungen in der medizinischen Forschung erfordern neuartige diagnostische Modalitaten zur Untersuchung von Mensch und Maus. Auserordentlich interessant sind in diesem Zusammenhang bildgebende Verfahren, die direkt molekulare Prozesse im lebenden Organismus (in vivo) untersuchen konnen; diese Verfahren werden unter dem Begriff “Molekulare Bildgebung” subsumiert. Diese Ubersicht fokussiert sich auf die klinisch und “mauseklinisch” verfugbaren szintigraphischen molekularen Bildgebungsverfahren SPECT und PET, deren derzeitiger und potentieller Stellenwert dargestellt wird.

Background— Matrix metalloproteinases (MMPs) are enzymes involved in the proteolytic degradation of extracellular matrix. They play an important role in several disease processes, such as inflammation, cancer, and atherosclerosis. Methods and Results— In this study, we have used the broad-spectrum MMP inhibitor CGS 27023A to develop the radioligand [ 123 I]I-HO-CGS 27023A for in vivo imaging of MMP activity. Using this radioligand, we were able to specifically image MMP activity by scintigraphy in vivo in the MMP-rich vascular lesions that develop after carotid artery ligation and cholesterol-rich diet in apolipoprotein E-deficient mice. These results were confirmed by gamma counting of lesional tissue (counts per minute per milligram). Conclusions— Imaging of MMP activity in vivo is feasible using radiolabeled MMP inhibitors. Additional studies are needed to test the potential of this approach as a novel noninvasive clinical diagnostic tool for the management of human MMP-related diseases.

Non-invasive measurement of matrix metalloproteinase (MMP) activity in vivo is a clinical challenge in many disease processes such as inflammation, tumor metastasis and atherosclerosis. Therefore, radioiodinated analogues of the non-peptidyl broad-spectrum MMP inhibitor (MMPI) CGS 27023A 1a were synthesized for non-invasive detection of MMP activity in vivo using single photon emission computed tomography (SPECT). The compounds Br-CGS 27023A 1b and HO-CGS 27023A 1d were synthesized from the amino acid D-valine and used as precursors for radioiodinated derivatives of CGS 27023A and their non-radioactive references I-CGS 27023A 1c and HO-I-CGS 27023A 1e . Radioiodination of the precursors with [ 123 I]NaI or [ 125 I]NaI produced the no-carrier-added MMP inhibitors [ 123 I]I-CGS 27023A 1f , [ 125 I]I-CGS 27023A 1g , HO-[ 123 I]I-CGS27023A 1h , and HO-[ 125 I]I-CGS 27023A 1i . In vitro studies showed that the non-radioactive analogues of the MMP inhibitors exhibited affinities against gelatinase A (MMP-2) and gelatinase B (MMP-9) in the nanomolar range, comparable to the parent compound CGS 27023A. In vivo biodistribution using HO-[ 125 I]I-CGS 27023A 1i in CL57 Bl6 mice showed rapid blood and plasma clearance and low retention in normal tissues. The preliminary biological evaluation warrant further studies of these radioiodinated MMP inhibitors as potential new radiotracers for imaging MMP activity in vivo.

Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P

Linksventrikulare Unterstutzungssysteme (LVAD) werden hauptsachlich bei terminaler Herzinsuffizienz eingesetzt, um die Wartezeit bis zur Transplantation zu uberbrucken. Unter LVAD verbessert sich oftmals die native Herzfunktion, sodass bei einzelnen Patienten das System entfernt werden kann und eine Herztransplantation nicht mehr notwendig ist. Dieser Prozess wird als "reverse remodeling" bezeichnet. Die ursachlichen molekularen Mechanismen hierfur sind noch weitgehend unklar. Verschiedene molekulare Systeme spielen bei der Entwicklung der Herzinsuffizienz eine Rolle. Unter LVAD-Therapie werden einzelne dieser System reversibel reguliert. Die Verminderung von Volumen- und Druckbelastung mit einer Abnahme der ventrikularen Wandspannung durch die Entlastungstherapie fuhrt neben einer Reduktion der Myozytenhypertrophie auch zu einer verbesserten myokardialen Perfusion und damit zu einer Abnahme der chronischen Ischamie bei Herzinsuffizienz. Diese Verbesserung der myokardialen Durchblutung und Verringerung der ventrikularen Wandspannung waren eine mogliche Erklarung fur die reversible Regulation der beteiligten molekularen Systeme.

Anchorage of cells to the extracellular matrix and integrin-mediated signals play crucial roles in cell survival. We have previously shown that during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (HUVECs), key molecules in focal adhesions and adherens junctions are cleaved by caspases. In this study we provide evidence for a selective upregulation of cell-associated matrix metalloproteinases (MMPs). We observe a physical association of MMP2 with beta1 and alphav integrins, which increased three- to fourfold during apoptosis and is dependent upon integrin beta1 levels and activation state. Both enforced activation of beta1 integrin by a specific antibody and inhibition of MMPs protect HUVECs from apoptosis. We hypothesize that, prior to the commitment to apoptosis, 'inside-out' signals initiated by the apoptotic stimulus alter cell shape together with the activation states and/or the availability of integrins, which promote matrix-degrading activity around dying cells. This 'auxiliary' apoptotic pathway may interrupt ECM-mediated survival signaling, and thus accelerate the efficient execution of the cell death program.

Inherited ventricular arrhythmias such as the long QT syndrome (LQTS), Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation (VF), and arrhythmogenic right ventricular cardiomyopathy (ARVC) account for a relevant proportion of sudden cardiac death cases in young patients cohorts. The detailed pathogenetic mechanisms of inherited ventricular arrhythmias are still poorly understood because systematic investigations are difficult to perform due to low patient numbers and the lack of appropriate experimental models. However, recent advances in research and science have identified a genetic background for many of these diseases. Present Knowledge: In LQTS, various mutations in different genes encoding for cardiac potassium and sodium channel proteins have been identified (“channelopathy”), and initial progress in genotype-phenotype correlation is made. Mutations in the cardiac sodium channel gene have also been identified in a subset of patients with Brugada syndrome, whereas a genetic background has not yet been demonstrated in idiopathic VF and right ventricular outflow-tract tachycardia (RVO-VT). Very recently, mutations in the cardiac ryanodine receptor gene have been identified in CPVT and in a subgroup of patients with ARVC. Although several chromosomal loci were suggested, no other responsible genes or mutations have been found in autosomal dominant forms of ARVC. However, in Naxos disease, a recessive form of ARVC with coexpression of palmoplantar keratoderma and woolly hair, a mutation in the plakoglobin gene has recently been discovered, thus underscoring the potential role of genetic alterations in cytoskeletal proteins in ARVC. Future Perspectives: In the next years, significant progress in the genetic diagnosis pathophysiologic understanding of disease mechanisms, genotype-phenotype correlation, and the development of gene- or target-directed treatment strategies can be expected in the field of inherited ventricular arrhythmias. Conclusion: This review summarizes the current knowledge of the molecular mechanisms, including aspects of pathoanatomy, autonomic innervation, genetics, and genotype-phenotype correlations with their potential implications for diagnosis and treatment of inherited ventricular arrhythmias.

Decorin, a small multifunctional proteoglycan, has been shown to be causally involved in the formation of capillary-like structures and a decrease in apoptosis. Here we investigated signal transduction pathways mediating effects of decorin on endothelial cells (ECs). Addition of decorin led to a fourfold increase in phosphorylation of Akt/protein kinase B on Thr307 and a l.4-fold increase on Ser473 after 10 min, but this phosphorylation could not be blocked by preincubation with Ly29400 (10 micro M). Six hours after the addition of decorin, the synthesis of p21 and p27, two inhibitors of cyclin-dependent kinases, started and increased up to 18 h, while synthesis of cyclin A peaked at 12 h and decreased after 24 h below base level. Induction of dominan-negative Akt by a replication-deficient adenovirus blocked p21 and cyclin A synthesis, but had no effect on p27. Dominant-negative Akt also blocked the antiapoptotic effect of decorin on ECs, but induction of dominant-positive Akt could not rescue the cells from apoptosis. Thus, the matrix proteoglycan decorin is a signaling molecule in ECs that affects cell survival by Akt-dependent and -independent pathways.

ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the β1-integrin. Furthermore, ADAM15 accumulation in cell–cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15–GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15–GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.

Objective: Left ventricular assist devices (LVAD) have been used to ‘bridge’ patients with end-stage heart failure to transplantation. Although several reports have suggested that the native ventricular function recovers after long-term LVAD support, a process called ‘reverse remodeling’, the underlying biological mechanisms are still unknown. As the transcription factor nuclear factor-κB (NF-κB) has been shown to be active in the failing human heart, we examined whether its activity is altered under LVAD support, and may thus contribute to the dynamic process of ‘reverse remodeling’. Methods: The activity of NF-κB was studied in 16 patients with end-stage heart failure (eight with dilated cardiomyopathy, six with ischemic heart disease, one with myocarditis, and one with congenital heart disease) before and after LVAD support by immunohistochemistry using an antibody against active NF-κB. Gel-shifts for NF-κB DNA-binding activity were performed with paired human myocardial tissue from four patients. The mean cardiomyocyte diameter before and after mechanical unloading was measured with an image analyzer system. Results: 15 patients out of 16 showed a significant decrease in the number of NF-κB positive cardiomyocyte nuclei after LVAD support in the left ventricular myocardium. The NF-κB DNA-binding activity also decreased after LVAD support as measured by gel-shift analysis. While the number of positive cardiomyocytes was significantly higher in the subendocardium than in the subepicardium at the time of LVAD implantation, this difference was no longer present at the time of LVAD explantation. The diameter of cardiomyocytes in the left ventricle decreased significantly as a parameter of structural reverse remodeling. Conclusion: LVAD support decreases the extent of NF-κB activation in failing human hearts, suggesting that NF-κB may be involved in the process of ‘reverse remodeling’.

Sphingosine-1-phosphate (S1P) receptors play major roles in cardiovascular, immunological and neurological diseases. The recent approval of the sphingolipid drug Fingolimod (Gilenya®), a sphingosine-1-phosphate agonist for relapsing multiple sclerosis, in 2010 exemplifies the potential for targeting sphingolipids for the treatment of human disorders. Moreover, non-invasive in vivo imaging of S1P receptors that are not available till now would contribute to the understanding of their role in specific pathologies and is therefore of preclinical interest. Based on fluorinated analogues of the S1P1 receptor antagonist W146 showing practically equal in vitro potency as the lead structure, the first S1P receptor antagonist [18F]-radiotracer has been synthesized and tested for in vivo imaging of the S1P1 receptor using positron emission tomography (PET). Though the tracer is serum stable, initial in vivo images show fast metabolism and subsequent accumulation of free [18F]fluoride in the bones.

The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts., The regulatory role of calcitonin in bone homeostasis is well studied, yet its molecular activity is poorly understood. The authors show that calcitonin regulates bone cells function by inhibiting the osteoclast secretion of sphingosine 1-phosphate, a lipid mediator of osteoclast–osteoblast crosstalk.

Endothelial cells undergoing angiogenesis express decorin, a small multifunctional proteoglycan. We have shown that decorin is causally involved in the formation of capillary-like structures and a decrease in apoptosis in endothelial cells cultured in a collagen lattice. Here we investigate signal transduction pathways mediating the effects of decorin. Reverse transcription-polymerase chain reaction demonstrated that p21 and p27, two inhibitors of cyclin-dependent kinases, were up-regulated by decorin induction. Decorin also increased protein levels of p21 and caused its translocation into the nucleus. p21 synthesis started 6 h after decorin addition and reached a plateau after 18 h, while cyclin A, which was also induced, peaked at 12 h and declined below basal levels within 24 h. These effects were mediated by the Akt/protein kinase B pathway. Akt phosphorylation at Thr-308 increased 4-fold and at Ser-473 1.4-fold within 10 min after decorin addition. Overexpression of dominant negative Akt inhibited the decorin-mediated induction of p21 and cyclin A, but had no effect on p27. These results show that decorin is a signaling molecule in sprouting endothelial cells where it acts via different pathways, one of them involving Akt/protein kinase B.

Abstract —Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by arterial smooth muscle cells (ASMCs). This proteoglycan accumulates in topographically defined regions of atherosclerotic lesions and may play a role in the development of this disease. However, little is known about whether decorin has specific effects on the cellular events that contribute to atherosclerotic lesion formation. In the present study, rat ASMCs were transduced with a retroviral vector (LDSN) that carries the bovine decorin gene. Compared with vector control cells (LXSN), these cells constitutively overexpress decorin, as verified by Northern and Western analysis and by metabolic labeling. Experiments were performed to examine the responsiveness of decorin-overexpressing rat ASMCs to platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), 2 growth factors that affect cell proliferation and extracellular matrix production in atherosclerosis. Decorin-overexpressing cells had decreased [ 3 H]thymidine incorporation into DNA and increased the levels of the cyclin-dependent kinase inhibitors p21 and p27 in the first 24 hours of response to serum and PDGF-BB. However, these effects of decorin were not apparent at 48 or 72 hours after plating and did not result in reduced growth of decorin-overexpressing cells in response to serum and PDGF-BB. In contrast, the growth response of decorin-overexpressing ASMCs to TGF-β1, as well as the expression of TGF-β1–responsive genes, such as plasminogen activator inhibitor-1 and versican (an extracellular matrix proteoglycan), was diminished. These results indicate that decorin selectively inhibits the responsiveness of rat ASMCs to TGF-β1 and suggests that the induction of constitutive decorin overexpression by ASMCs in vivo may have therapeutic value in the inhibition of TGF-β1–mediated effects on the development of atherosclerotic lesions.

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A–associated cyclin-dependent kinase 2 (cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21 Cip1/Waf1 and p27 Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21 Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21 Cip1/Waf1 and p27 Kip1 results in a substantial reduction in their association with nuclear cyclin–cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21 Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC50: 30 μM). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.

Sphingosine-1-phosphate (S1P) regulates important functions in cardiac and vascular homeostasis. It has been implied to play causal roles in the pathogenesis of many cardiovascular disorders such as coronary artery disease, atherosclerosis, myocardial infarction, and heart failure. The majority of S1P in plasma is associated with high-density lipoproteins (HDL), and their S1P content has been shown to be responsible, at least in part, for several of the beneficial effects of HDL on cardiovascular risk. The attractiveness of S1P-based drugs for potential cardiovascular applications is increasing in the wake of the clinical approval of FTY720, but answers to important questions on the effects of S1P in cardiovascular biology and medicine must still be found. This chapter focuses on the current understanding of the role of S1P and its receptors in cardiovascular physiology, pathology, and disease.

The lipid sphingosine-1-phosphate (S1P) is a chemokine for a variety of immune cells including lymphocytes and monocytes. Migration toward S1P is determined by the S1P receptor expression profile, with S1PR1/3 (where S1PR is S1P receptor) stimulating and S1PR2 attenuating migration. However, the impact and physiological significance of S1P-induced migration of macrophages is largely unclear. We observed that alternative activation of human macrophages, by IL-4 or apoptotic cells (ACs), enhanced S1PR1 expression. Moreover, ACs provoked macrophage migration toward S1P in an S1PR1-dependent manner as confirmed by pharmacological receptor inhibition and S1PR1-deficient murine macrophages. In a mouse model of resolving peritoneal inflammation, F4/80-driven deletion of S1PR1 reduced postinflammatory macrophage emigration from inflammatory sites. S1PR1 expression on macrophages might, therefore, be relevant for restoring tissue homeostasis during the resolution of inflammation.

Sphingosine-1-phosphate (S1P) regulates important functions in cardiac and vascular homeostasis. It has been implied to play causal roles in the pathogenesis of many cardiovascular disorders such as coronary artery disease, atherosclerosis, myocardial infarction, and heart failure. The majority of S1P in plasma is associated with high-density lipoproteins (HDL), and their S1P content has been shown to be responsible, at least in part, for several of the beneficial effects of HDL on cardiovascular risk. The attractiveness of S1P-based drugs for potential cardiovascular applications is increasing in the wake of the clinical approval of FTY720, but answers to important questions on the effects of S1P in cardiovascular biology and medicine must still be found. This chapter focuses on the current understanding of the role of S1P and its receptors in cardiovascular physiology, pathology, and disease.

Survivin (Surv) belongs to the inhibitor of apoptosis protein family. Its cardiac-specific deletion results in reduced cardiomyocyte number, increased cardiomyocyte size and ploidy, and development of heart failure. Its impact on cardiac electrophysiology is unknown. In vivo transvenous electrophysiological studies were carried out in adult male mice with a cardiac-specific deletion of survivin (Surv(-/-); n = 12) and wild-type controls (Surv(+/+); n = 12). Epicardial activation mapping (EAM) was performed in Langendorff-perfused hearts of 16 Surv(-/-) and 6 Surv(+/+) mice. Surface-ECG showed lower heart rates in Surv(-/-) mice (326 ± 66 bpm vs. 440.6 ± 39 ms; P = 0.0001), accompanied by significantly prolonged P waves (20.3 ± 5.8 vs. 14.6 ± 2.0 ms; P = 0.009), PQ-(47.4 ± 8.6 vs. 41.1 ± 3.7 ms; P = 0.043), QRS- (19.5 ± 4.8 vs. 14.0 ± 1.0 ms; P = 0.002) and QT-intervals (41.6 ± 4.4 vs. 36.2 ± 3.4 ms; P = 0.003). The HV-interval was prolonged in Surv(-/-) mice (12.1 ± 2.4 vs. 9.3 ± 1.4 ms; P = 0.0045). We found impaired sinus-nodal function (sinus node recovery times: 310.2 ± 76.6 vs. 207.8 ± 68.6 ms; P = 0.003) and AV-nodal conduction (Wenckebach-periodicity: 105.9 ± 15.9 vs. 79.6 ± 8.1 ms; P = 0.0002). EAM showed significant slowing and heterogeneity of conduction in the myocardium of Surv(-/-) mice. All Surv(-/-) mice showed spontaneous supraventricular and ventricular ectopic beats (P0.0001 vs. wildtype). Quantitative immunofluorescence staining for connexin43 (Cx43) revealed a decrease in both per cardiomyocyte and single gap junction. Surv(-/-) mice exhibit severe global conduction attenuations in atrial and ventricular myocardium as well as the specific conduction system, accompanied by lower connexin43 levels. Lack of susceptibility to AF and VT suggests that reduced cardiomyocyte number and increased size constitute determinants of electrical stableness in the heart and counteract potentially proarrhythmogenic connexin43 loss in Surv(-/-).

Linksventrikulare mechanische Unterstutzungssysteme („left ventricular assist devices”; LVAD) werden bei terminaler Herzinsuffizienz entweder als „bridge to transplantation“ oder als „destination therapy“ bei Vorliegen von Kontraindikationen zur Herztransplantation eingesetzt. LVAD sind pulsatile oder nonpulsatile Systeme, die Blut vom linken Ventrikel parallel zur Zirkulation in die Aorta ascendens transportieren und so zu einer profunden Druck- und Volumenentlastung im linken Ventrikel fuhren. Bei einem Teil der Patienten kommt es infolge des Einsatzes des LVAD zu einer deutlichen Abnahme der Herzhypertrophie und -dilatation sowie zu einer signifikanten Verbesserung der kardialen Pumpfunktion. Dieser Prozess wird als „reverse cardiac remodeling” bezeichnet und ist durch Abnahme der Kardiomyozytengrose und reversible Regulation zahlreicher molekularer Systeme des Myokards gekennzeichnet.

Rationale: The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. Objective: We hypothesized that the S1P receptor 3 (S1P 3 ) plays a causal role in the pathogenesis of atherosclerosis. Methods and Results: We examined atherosclerotic lesion development in mice deficient for S1P 3 and apolipoprotein (Apo)E. Although S1P 3 deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P 3 −/− /ApoE −/− double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P 3 -deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow–derived S1P 3 -deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P 3 -deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P 3 in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P 3 deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P 3 in the arterial injury response. Conclusions: Our data suggest that S1P 3 mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

Rationale: Stent implantation into atherosclerotic plaques releases, apart from particulate debris, soluble substances that contribute to impaired microvascular perfusion. Objective: To quantify the release of vasoconstrictors and to determine the efficacy of coronary dilators to attenuate their action. Methods and Results: Using a distal protection/aspiration device, coronary arterial blood was retrieved before and during stenting in 22 patients with severe saphenous vein aorto-coronary bypass stenoses. The release of catecholamines, endothelin, serotonin, thromboxane B 2 , and tumor necrosis factor (TNF)α was measured. The response of rat mesenteric arteries with intact (+E) and denuded (−E) endothelium to aspirate plasma was normalized to that by KCl. Responses to selective receptor blockade, adenosine, nitroprusside, and verapamil against the aspirate-induced constriction were determined. The coronary arterial plasma withdrawn before stenting induced 21±5% and the aspirate plasma after stenting induced 95±8% of maximum KCl-induced vasoconstriction. Serotonin, thromboxane B 2 , and TNFα release into aspirate plasma increased by 1.9±0.2 μmol/L, 25.6±3.1 pg/mL, and 19.7±6.1 pg/mL, respectively, during stenting. The aspirate-induced vasoconstriction was largely antagonized by selective serotonin receptor blockade, with little further antagonism by additional thromboxane receptor blockade. TNFα did not induce constriction per se but potentiated the constriction with serotonin and the thromboxane-analog U-46619 in arteries +E. The concentrations to induce half-maximal vasodilation were comparable for nitroprusside (+E, 3.3×10 −8 ; −E, 1.9×10 −8 mol/L) and verapamil (+E, 8.3×10 −8 ; −E, 7.8×10 −8 mol/L), and the vasoconstriction was eventually eliminated. The vasodilator response to adenosine was dependent on functional endothelium and weaker. Conclusion: Serotonin is the main coronary vasoconstrictor after stenting, and thromboxane and TNFα somewhat potentiate the serotonin response. Nitroprusside and verapamil are more potent than adenosine to attenuate the aspirate plasma-induced vasoconstriction, and they are not dependent on functional endothelium.

Background— Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. Methods and Results— Four-week-old apolipoprotein E–deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. Conclusions— The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.

The biologically active sphingolipid sphingosine-1-phosphate (S1P) plays key functions in the immune, inflammatory, and cardiovascular systems. In the vasculature, S1P and its receptors are involved in vessel morphogenesis and angiogenesis during embryonic development and in the adult organism both under normal and pathological conditions. Via its actions on endothelial and smooth muscle cells, S1P regulates arterial tone, vascular permeability, and tissue perfusion. Elevated local S1P levels during inflammation induce endothelial adhesion molecules, recruit inflammatory cells, and activate dendritic cells. At the same time, S1P activates a negative feedback loop that consecutively seals endothelial cell-cell contacts, decreases vascular leakage, and inhibits cytokine-induced leukocyte adhesion. Thus S1P determines not only the build-up, magnitude, and duration of an inflammatory reaction but also the pace of its resolution. This review focuses on the role S1P plays in endothelial function, its receptors and signalling pathways, and the role its major carrier high-density lipoproteins (HDL) play in its bioavailability and transport. We will also discuss the potential of interfering with S1P-S1P receptor interactions for the treatment of endothelial disorders and vascular pathologies.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that regulates immunity, inflammation, and cardiovascular function. S1P gradients between plasma and the interstitium, and dynamically regulated S1P receptors in hematopoietic and endothelial cells, orchestrate immune and inflammatory cell trafficking. S1P generating enzymes are activated in inflammation, and high local S1P levels boost inflammatory responses by inducing adhesion molecules, recruiting and retaining inflammatory cells and lymphocytes, and activating lymphatic dendritic cells. At the same time, S1P has clear anti-inflammatory effects: it strengthens endothelial cell-cell contacts, decreases permeability, and inhibits cytokine-induced leukocyte adhesion. Thus, S1P activates both positive and negative feedback mechanisms in inflammation and determines not only the build-up, magnitude, and duration of inflammation but also its restraint and resolution. This review elucidates these mechanisms and the important issue of S1P bioavailability and delivery by high-density lipoproteins as its major plasma carrier. Finally, the potential application of S1P receptor analogues as new treatment strategies in inflammation is discussed.

Background The ubiquitin–proteasome system (UPS) breaks down misfolded and normal proteins, including cell cycle regulatory proteins involved in cardiac hypertrophy. Because congestive heart failure (CHF) increases cardiomyocyte cellular mass, indicative of increased protein synthesis and/or impaired breakdown, and ventricular unloading decreases cardiac hypertrophy and changes regulation of multiple molecular systems (“reverse cardiac remodeling”), we tested the hypothesis that ventricular unloading alters myocardial UPS. Methods In 23 paired myocardial specimens (before and after unloading) ubiquitin, 20S proteasome, and cyclin D1 were investigated immunohistochemically and morphometrically quantified in relation to cardiomyocyte hypertrophy, DNA content, nuclear profile area and perimeter, and cyclin D1 protein expression. Moreover, 20S proteasome plasma concentrations were measured by enzyme-linked immunoassay (ELISA). Results In CHF, sarcoplasmic 20S proteasome protein expression was significantly decreased compared with controls, but significantly increased after unloading. In contrast, sarcoplasmic ubiquitin protein was increased in CHF but significantly decreased after unloading, and both variables were inversely correlated. Cardiomyocyte 20S proteasome expression correlated inversely with cell size, mean DNA content, and cyclin D1, whereas ubiquitin protein expression was positively correlated with these parameters. The 20S proteasome plasma concentration was significantly increased after unloading. Conclusions Our data indicate that: (1) the UPS is depressed in CHF; and (2) this is reversed by ventricular unloading and associated with decreased cardiomyocyte hypertrophy, mean DNA content, and cell cycle regulatory proteins. The findings support the view that the UPS is involved in both the pathogenesis of cardiac hypertrophy and “reverse cardiac remodeling” after ventricular unloading.

HDL, through sphingosine-1-phosphate (S1P), exerts direct cardioprotective effects on ischemic myocardium. It remains unclear whether other HDL-associated sphingophospholipids have similar effects. We therefore examined if HDL-associated sphingosylphosphorylcholine (SPC) reduces infarct size in a mouse model of transient myocardial ischemia/reperfusion. Intravenously administered SPC dose-dependently reduced infarct size after 30 minutes of myocardial ischemia and 24 hours reperfusion compared to controls. Infarct size was also reduced by postischemic, therapeutical administration of SPC. Immunohistochemistry revealed reduced polymorphonuclear neutrophil recruitment to the infarcted area after SPC treatment, and apoptosis was attenuated as measured by TUNEL.In vitro, SPC inhibited leukocyte adhesion to TNFα-activated endothelial cells and protected rat neonatal cardiomyocytes from apoptosis. S1P3was identified as the lysophospholipid receptor mediating the cardioprotection by SPC, since its effect was completely absent in S1P3-deficient mice. We conclude that HDL-associated SPC directly protects against myocardial reperfusion injuryin vivovia the S1P3receptor.

Coronary microembolization from the erosion or rupture of a vulnerable atherosclerotic plaque occurs spontaneously in acute coronary syndromes and iatrogenically during percutaneous coronary interventions. Typical consequences of coronary microembolization are microinfarcts with an inflammatory response, contractile dysfunction, and reduced coronary reserve. Apart from transient elevations of creatine kinase and troponin, microemboli can be visualized by intracoronary Doppler and the resulting microinfarcts by late-enhancement nuclear magnetic resonance. Statins, antiplatelet agents, and coronary vasodilators protect against microembolization and microinfarction when started before percutaneous coronary interventions. Distal protection devices can retrieve atherothrombotic debris and prevent its embolization into the microcirculation, but their effect on clinical outcome has been disappointing so far, except for saphenous vein bypass grafts. Devices for aspiration of thrombi and thrombus-derived vasoconstrictor, thrombogenic, and inflammatory substances, however, reduce thrombus burden, improve perfusion, and provide protection in patients with acute myocardial infarction.

Aims The study tested whether high-density lipoprotein-cholesterol (HDL-C) has an effect on percutaneous coronary intervention (PCI)-induced myocardial infarction and its prognosis. Elevation of cardiac troponin I (cTnI) > 3× upper normal limit after PCI is defined as PCI-related myocardial infarction (PMI) and is associated with a negative prognosis. No data exist on the relationship of HDL-C to PMI and PMI-related outcome. Methods and results Pre-procedural HDL-C levels and post-procedural peak cTnI levels were collected in 350 patients undergoing PCI. Data were analysed for PMI and for acute myocardial infarction (AMI) during follow-up. Patients with PMI ( n = 115) had lower HDL-C levels than patients without PMI [ n = 235; 1.17 mmol/L (0.75–2.51) vs. 1.27 mmol/L (0.70–2.87), P < 0.001]. Pre-procedural HDL-C levels were inversely related to the occurrence of PMI [odds ratio for PMI: 0.884, 95% CI: 0.80, 0.98; P = 0.02 for an HDL-C-increment of 5 mg/dL (0.13 mmol/L)] and to AMI during follow-up [hazard ratio (HR): 0.697, 95% CI: 0.54, 0.90; P = 0.005]. The occurrence of PMI was associated with an elevated HR for AMI (4.702, 95% CI: 1.79, 12.37; P = 0.002). Low-risk levels of pre-procedural HDL-C [men ≥40 mg/dL (≥1.03 mmol/L), women ≥45 mg/dL (≥1.16 mmol/L)] did not influence the negative effects of PMI on outcome (HR: 5.510, 95% CI: 1.43, 21.31; P = 0.013) and reduction of AMI-free survival [mean AMI-free survival time with PMI: 1167.5 days (95% CI: 1098.27, 1236.67) vs. 1240.7 days (95% CI: 1220.94, 1290.49) without PMI; log-rank P = 0.005]. Conclusion Small increases in HDL-C in patients undergoing elective PCI convert into a substantial reduction of risk for PMI, which has adverse effects on the long-term prognosis. Patients with PMI are at a high risk for AMI at any HDL-C level and therefore should receive particular monitoring by the treating physician over a long period after PCI.

Survivin regulates cell division and inhibits apoptosis. Liver regeneration is a complex process involving both proliferation and apoptosis. The role of survivin is not well elucidated and no data exist in humans.Seventy per cent liver resection was used to investigate liver regeneration in rats. Survivin was identified by means of reverse transcriptase polymerase chain reaction, Western blotting and immunohistochemistry. Proliferation and apoptosis were quantified. Liver biopsies from 33 patients who underwent living donor liver transplantation were used to study survivin immuno-expression, proliferation and apoptosis within the first 17 days after transplantation. Seven healthy donors served as controls.Survivin transcript and protein were significantly upregulated in rat hepatocytes after 24-72 h during regeneration and showed a significant correlation with proliferation but not with apoptosis. In humans, survivin was nearly absent in donor and reperfused liver tissue but increased significantly 5-7 days after transplantation and correlated with proliferation but not with apoptosis.Survivin is upregulated in human and rodent liver regeneration and correlates with proliferation, suggesting an association of survivin and cell division.

We thank Dr Csont and Dr Ferdinandy for their interest in our work.1 In principle, we agree that it is possible and even likely that more that one factor controls the complex events that accompany myocardial infarction (MI). Nevertheless, we have clearly shown that mortality, frequency of ventricular ruptures, and hemodynamic dysfunction were significantly increased 3 weeks post MI in bgn −/ 0 mice.1 These findings prove that absence of biglycan …

See related article, pages 315–324 Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid that plays an important role in the physiology of the cardiovascular, nervous and immune systems.1 Red blood cells are the major source of plasma S1P, where it mainly associates with High-density lipoproteins (HDL). There is a steep concentration gradient of S1P between plasma and the interstitial compartment, with S1P being present in the low micromolar range in plasma and at least 1 order of magnitude lower in the interstitial fluid. S1P exerts numerous effects on the vascular cells regulating arterial tone—the smooth muscle cell and the endothelial cell —via a set of 5 cognate G protein–coupled receptors (S1P1 to 5) expressed differentially in the different cell types.2 In addition, S1P has negative effects on cardiac rate, contractility, and output. All of this makes S1P a candidate for a bona fide modulator of vascular tone. Direct vasoactive effects of S1P have been studied both in isolated vessel strips ex vivo and different vascular territories in vivo.2 The consensus from all these studies is that S1P induces vasoconstriction in resistance vessels such as mesenteric, cerebral, and coronary arteries but has little or no effect on conduit vessels such as aorta, carotid, and femoral arteries. This implies S1P as a player in the control of blood flow in the periphery. However, these studies have been all performed by using exogenously added S1P, making it difficult to draw conclusions on the role of endogenous S1P sources in the homoeostatic regulation of resistance artery tone. In a previous study, this has been addressed by Bolz et al by overexpressing sphingosine kinase (Sphk)1 in hamster resistance arteries, showing that endogenously produced S1P participates in the regulation of basal tone, as well as that of the myogenic response,3 the …

An approach to the in vivo imaging of locally upregulated and activated matrix metalloproteinases (MMPs) found in many pathological processes is offered by positron emission tomography (PET). Hence, appropriate PET radioligands for MMP imaging are required. Here, we describe the syntheses of novel fluorinated MMP inhibitors (MMPIs) based on lead structures of the broad-spectrum inhibitors N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](benzyl)-amino]-3-methyl-butanamide (CGS 25966) and N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)-amino]-3-methyl-butanamide (CGS 27023A). Additionally, tailor-made precursor compounds for radiolabeling with the positron-emitter 18F were synthesized. All prepared hydroxamate target compounds showed high in vitro MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13. As a consequence, the promising fluorinated hydroxamic acid derivative 1f was resynthesized in its 18F-labeled version via two different procedures yielding the potential PET radioligand [18F]1f. As expected, the biodistribution behavior of this novel compound and that of the more hydrophilic variant [18F]1j, also developed by our group, indicates that there was no tissue specific accumulation in wild-type (WT) mice.

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P(3). Upon activation with S1P, S1P(3) couples to G(i)- and G(q)-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P(3)-mediated and G(i)-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G(q) via S1P(3) in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P(3). The lack of receptor desensitization prevented S1P(3)-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P(1) and S1P(3), and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P(1) and S1P(3). FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.

The question raised by Dr Xia is based on the hypothesis that plasma proteins “buffer” the large amounts of sphingosine 1-phosphate (S1P) present in plasma to prevent it from erroneously activating S1P receptors.1 Although reconstituted high-density lipoprotein (HDL) that does not contain S1P has numerous biological effects when administered in vivo, it is unknown whether and how rapidly it may actually acquire S1P from the plasma once it enters the circulation. Subsequently, such “S1P-charged” HDL may be partially responsible for the effects attributed to …

All stages of atherosclerosis have been identified as a chronic vascular inflammatory disease. In the last few years there is increasing evidence that endogenous lysophospholipids such as sphingosine-1-phosphate (S1P) have potent anti-inflammatory properties. The S1P analogue FTY720 that has been developed as a potent, orally active, immunosuppressant in the field of transplantation and autoimmune disease has interesting effects on inflammatory processes in the arterial vessel wall. S1P targets five specific S1P receptors (S1P(1-5)), which are ubiquitously expressed. S1P(1-3) receptor expression is identified in arterial vessels. S1P and FTY720 show potent silencing effects on some vascular proinflammatory mechanisms in endothelial and vascular smooth muscle cells. In addition, the interaction of monocytes with the vessel wall is inhibited. As shown recently, FTY720 can effectively reduce the progression of atherosclerosis in apolipoprotein E-deficient mice having a high-cholesterol diet. It is not entirely clear which S1P receptor subtype is mainly involved in this process. However, it is currently speculated that the S1P(3) and probably the S1P(1) is involved in the anti-atherosclerotic effects of FTY720. This review summarizes the current knowledge about S1P- and FTY720-effects on mechanisms of vascular inflammatory disease. In addition S1P receptor subtypes are identified which might be interesting for molecular drug targeting.

Objective— Activation of collagen receptors expressed by smooth muscle cells induces matrix metalloproteinase (MMP) expression. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to interfere with integrin signaling, but their effects on collagen receptor-mediated MMP expression have not been investigated. Methods and Results— In the present study, we show that simvastatin (3 μmol/L) reduces MMP1 expression and secretion in human smooth muscle cells cultured on polymerized type I collagen by 39.9±11.2% and 36.0±2.3%, respectively. Reduced MMP1 protein levels correlate with a similar decrease in MMP1 promoter activity (−33.0±8.9%), MMP1 mRNA levels (−37.8±10.5%), and attenuation of smooth muscle cell collagen degradation (−34.2±6.1%). Mevalonate, and the isoprenoid derivative geranylgeraniol, precursors of geranylgeranylated proteins, completely prevent the inhibitory effect of simvastatin on MMP1. Moreover, the protein geranylgeranyltransferase inhibitor GGTI-286 significantly decreases MMP1 expression. Retroviral overexpression of dominant-negative mutants of geranylgeranylated Rac1 lead to a reduction of MMP1 protein (−50.4±5.4%) and mRNA levels (−97.9±1.0%), and knockdown of Rac1 by small interfering RNA downregulates MMP1 expression. Finally, simvastatin reduces GTP-bound Rac1 expression levels in smooth muscle cells cultured on polymerized collagen. Conclusions— These results demonstrate that simvastatin, by inhibiting Rac1 activity, reduces MMP1 expression and collagen degradation in human smooth muscle cells.

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 S1P3sphingolipid 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.

Objectives Cyclooxygenase-2 is associated with cardiac hypertrophy during chronic heart failure and is regulated through the PI3K/Akt pathway. Cyclooxygenase-2-induced cell growth through Akt phosphorylation was demonstrated in vitro. In chronic heart failure, left ventricular assist devices lead to hypertrophy regression and molecular changes. Therefore, the expression of cyclooxygenase-2, phosphorylated Akt (p-Akt), and p-Erk 1/2, as well as cardiac hypertrophy before and after left ventricular assist device insertion, was investigated. Methods In myocardial tissue before and after left ventricular assist device insertion, the expression of cyclooxygenase-2, p-Akt (Thr308) , p-Akt (Ser473) , and p-Erk 1/2 was demonstrated by immunohistochemistry and quantified by morphometry. Colocalization of cyclooxygenase-2 and p-Akt (Thr308) was investigated by immuno-doublestaining. Results A significant decrease of cyclooxygenase-2, p-Akt (Thr308) , p-Akt (Ser473) , and p-Erk 1/2 protein expression and hypertrophy regression was observed after left ventricular assist device insertion. A significant correlation between cyclooxygenase-2 and p-Akt (Thr308) expression, as well as between cyclooxygenase-2 expression and cardiomyocyte diameter, was observed before, but not after, left ventricular assist device insertion. Only cyclooxygenase-2-positive cardiomyocytes showed significant hypertrophy regression on unloading. Sarcoplasmic colocalization of cyclooxygenase-2 and p-Akt (Thr308) is present before left ventricular assist device insertion and is decreased after unloading, whereas the normal myocardium is completely devoid of it. Conclusions Left ventricular assist device treatment is associated with a significant decrease of cyclooxygenase-2, p-Akt (Thr308) , p-Akt (Ser473) , and p-Erk 1/2, and cardiac hypertrophy regression of cyclooxygenase-2-positive cardiomyocytes. The significant correlation and colocalization in cardiomyocytes of cyclooxygenase-2 and p-Akt (Thr308) before left ventricular assist device insertion suggests a cross-talk between the 2 molecules in the progression of cardiac hypertrophy, which is reversibly regulated by the left ventricular assist device.

Due to their tremendous apoptosis-inducing potential, proteasomal inhibitors (PIs) have recently entered clinical trials. Here we show, however, that various PIs rescued proliferating tumor cells from death receptor-induced apoptosis. This protection correlated with the stabilization of X-linked IAP (XIAP) and c-FLIP and the inhibition of caspase activation. Together with the observation that PIs could not protect cells expressing XIAP or c-FLIP short interfering RNAs (siRNAs) from death receptor-induced apoptosis, our results demonstrate that PIs mediate their protective effect via the stabilization of these antiapoptotic proteins. Furthermore, we show that once these proteins were eliminated, either by long-term treatment with death receptor ligands or by siRNA-mediated suppression, active caspases accumulated to an even larger extent in the presence of PIs. Together, our data support a biphasic role for the proteasome in apoptosis, as they show that its constitutive activity is crucial for the rapid initiation of the death program by eliminating antiapoptotic proteins, whereas at later stages, the proteasome acts in an antiapoptotic manner due to the proteolysis of caspases. Thus, for a successful PI-based tumor therapy, it is crucial to carefully evaluate basal proteasomal activity and the status of antiapoptotic proteins, as their PI-mediated prolonged stability might even cause adverse effects, leading to the survival of a tumor.

Light microscopic alterations reflecting both previous and preservation-induced changes in the donor organ are usually not very distinctive. The ischemia/reperfusion-associated injury depends primarily on the conditions of donor organ preservation. The present study examined human kidney biopsies with special attention paid to the molecular mechanisms of preservation-induced injury preceding reperfusion. Stress-associated proteins hemeoxygenase-1 (HO-1), heat shock protein 70 (HSP 70), and metallothionein (MT) were studied in human zero-hour biopsies of transplanted kidneys prior to reperfusion in 29 patients. Protein expression was evaluated by semiquantitative immunohistochemistry and Western blotting for HO-1 and HSP 70. These findings were correlated with terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphosphate-digoxigenin nick end labeling (TUNEL) staining and follow up. Compared to controls, MT and HSP 70 expression was significantly higher at zero hour. In contrast, HO-1 and the number of TUNEL-positive cells were not elevated. MT and HO-1 immunoexpression were inversely associated with graft function, and hence, were of prognostic relevance. MT and HSP 70 were sensitive to the duration of cold ischemia. MT and HO-1 are suitable indicators for tissue injury during ischemia and may serve as new predictive markers that need to be validated in further independent studies.