Amanda M.G. Versteilen

Involvement of RhoA/Rho Kinase Signaling in VEGF-Induced Endothelial Cell Migration and Angiogenesis In Vitro

Objective—Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced signaling pathways, but its role has not been clarified. Methods and Results—VEGF induced the activation of RhoA and recruited RhoA to the cell membrane of human ECs. This increase in RhoA activity is necessary for the VEGF-induced reorganization of the F-actin cytoskeleton, as demonstrated by adenoviral transfection of dominant-negative RhoA. Rho kinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase. Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration. Furthermore, in an in vitro model for angiogenesis, inhibition of either RhoA or Rho kinase attenuated the VEGF-mediated ingrowth of ECs in a 3-dimensional fibrin matrix. Conclusions—VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signaling is involved in the VEGF-induced in vitro EC migration and angiogenesis.

Myofilament dysfunction in cardiac disease from mice to men

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the β-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the β-adrenergic receptors (β-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of β-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Prevention of Myofilament Dysfunction by β-Blocker Therapy in Postinfarct Remodeling

Background—Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether &bgr;-blocker therapy could restore myofilament contractility. Methods and Results—In pigs with a MI induced by ligation of the left circumflex coronary artery, &bgr;-blocker therapy (bisoprolol, MI+&bgr;) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (Fmax) was lower, whereas Ca2+ sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca2+ sensitivity was abolished by treatment of cells with the &bgr;-adrenergic kinase, protein kinase A. &bgr;-blocker therapy partially reversed Fmax and Ca2+ sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca2+ sensitivity in MI+&bgr; compared with untreated myocardium, the protein kinase A induced reduction in Ca2+ sensitivity was largest in cardiomyocytes from myocardium treated with &bgr;-blockers. Phosphorylation of &bgr;-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. &bgr;-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions—&bgr;-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of &bgr;-blockade on global left ventricular function after MI.

Endothelin receptor blockade combined with phosphodiesterase-5 inhibition increases right ventricular mitochondrial capacity in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A (ET(A))/type B (ET(B)) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart.

Enhanced myofilament responsiveness upon β-adrenergic stimulation in post-infarct remodeled myocardium

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed β-adrenergic receptor (β-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F(max)) and higher Ca(2+)-sensitivity in MI compared to sham. F(max) did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca(2+)-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of β-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca(2+)-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.

Mechanisms of the urinary concentration defect and effect of desmopressin during endotoxemia in rats.

Acute renal failure during human sepsis is often nonoliguric. To study the underlying mechanisms, renal function was assessed in endotoxic and control male Wistar rats during and after saline loading and treatment with the selective V2 receptor agonist desmopressin. Escherichia coli endotoxin (dose, 8 mg/kg) was administered from time (t) = 0 to t = 60 min; saline loading (rate, 5 mL/100 g per hour) was administered from t = 0 to t = 120 min. Thereafter, half of each group received desmopressin (dose, 10 &mgr;g) for 1 h. The inner medullary (IM) osmolality, hematocrit, plasma, and urinary concentrations of sodium, potassium, urea, and osmolality were measured; then, aquaporin 2 (AQP2) immunohistochemistry was performed. Plasma vasopressin concentrations were measured at t = 180 min. Saline loading increased urine volume in all rats. In the endotoxic group, mean arterial pressure decreased when saline loading was stopped. Despite increased hematocrit and vasopressin levels (>16 pg/mL), the endotoxin group had a low IM osmolality (mean ± SEM, 412 ± 0.04 mOsm/kg H2O) in comparison with the control group (mean ± SEM, 1,094 ± 0.17 mOsm/kg H2O) and was not able to either decrease urine volume or raise urine osmolality. Desmopressin treatment in endotoxin-treated rats maintained mean arterial pressure, increased sodium reabsorption, IM osmolality, and urine osmolality, and decreased urine flow. The AQP2 intensity decreased in the endotoxin group, and the apical localization disappeared; both were not affected by desmopressin. Our results indicate that endotoxemia in rats acutely diminishes renal urinary concentration capacity and is associated with a decreased IM osmolality and diminished apical AQP2 localization. These findings may help to explain nonoliguric acute renal failure in human septic shock.

Rho-Kinase Inhibition Reduces Early Microvascular Leukocyte Accumulation in the Rat Kidney following Ischemia-Reperfusion Injury: Roles of Nitric Oxide and Blood Flow

Aim: To study whether microvascular leukocyte accumulation after rat renal ischemia and reperfusion (IR) is decreased by Rho kinase inhibition, independently of effects upon nitric oxide (NO) and renal blood flow. Methods: Male Wistar rats were subjected to 60 min of ischemia by bilateral clamping and 60 min of reperfusion of the renal arteries, or a sham procedure. Haemodynamics were monitored and microsphere blood flow to the kidneys was measured. The infusion of the Rho kinase inhibitor (Y27632) was commenced before clamping and IR. The NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), was administered after the start of reperfusion whilst the dopamine-1 receptor agonist fenoldopam, a renal vasodilator, was infused during the reperfusion period. Digital imaging microscopy analysis of cryosections was done to determine leukocyte accumulation and vasodilator-stimulated phosphoprotein serine 239 phosphorylation (p-VASP ser 239), a marker of endothelial NO. Results: Leukocytes (60–70% neutrophils) accumulated within blood vessels in the corticomedullary junction and medulla of the kidney. Leukocyte accumulation was markedly reduced by the Rho kinase inhibitor but not by fenoldopam. However, both drugs improved renal blood flow and microvascular expression of p-VASP ser 239 in the corticomedullary junction and medulla, which were decreased following IR. L-NAME treatment of IR animals pretreated with the Rho kinase inhibitor reduced blood flow and p-VASP ser 239 expression and increased leukocyte accumulation. Conclusion: Early microvascular leukocyte accumulation in the corticomedullary junction and medulla of the rat kidney after IR is ameliorated by Rho kinase inhibition. This effect is partly independent upon attenuation of decreased NO and renal blood flow.

Role of cyclooxygenase and derived reactive oxygen species in rho-kinase-mediated impairment of endothelium-dependent vasodilation and blood flow after ischemia-reperfusion of the rat kidney

Background/Aims: Decreased endothelium-dependent vasodilation and blood flow in renal ischemia-reperfusion (IR) may result in part from ρ-kinase activation, and cyclooxygenase (COX) activation, and resultant reactive oxygen species (ROS) may be involved. Methods: We tested this hypothesis in male Wistar rats, subjected to 60 min of bilateral clamping of the renal arteries and 60 min of reperfusion or a sham procedure, and treated by the ρ-kinase inhibitor Y27632 (1 mg/kg) and/or the nonspecific COX inhibitor diclofenac (10 mg/kg). Renal blood flow was measured by fluorescent microspheres, and ROS in the arterial endothelium was quantified by dihydroethidium staining. Endothelium-dependent vasodilation was determined by an acetylcholine concentration-response curve in the presence or absence of diclofenac (10 µM). Results: Y27632 increased renal blood flow and reduced ROS in vivo, and improved endothelium-dependent vasodilation in vitro, following IR with or without diclofenac. Following IR, diclofenac had no effect on renal blood flow and ROS in vivo, but improved endothelium-dependent vasodilation in vitro. Conclusion: Activation of ρ-kinase impairs endothelium-dependent vasodilation and perfusion following renal IR, independently of COX and resultant ROS. In contrast, the vasodilatory effect of ρ-kinase inhibition may be partly mediated by decreasing ROS, unrelated to COX and resultant vasoconstricting prostanoids.

RHO-KINASE INHIBITION ATTENUATES ENDOTHELIAL CELL APOPTOSIS FOLLOWING SIMULATED ISCHEMIA AND REPERFUSION BY AKT PHOSPHORYLATION

TOWARDS RESOLVING THE CHALLENGE OF SEPSIS DIAGNOSTIC. Thomas Herget* and Thomas Joos . *Merck KGaA, Darmstadt, Germany; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany Biomarkers have proven to be very useful in clinical conditions such as heart attack, stroke and cancer. There are characteristics linked to sepsis like in blood pressure, body temperature and heart rate. Efforts over the last decade to improve diagnosis for infectious inflammation have been unsuccessful in identifying a single and universal biomarker that provides sufficiently high sensitivity and specificity. In gramnegative septicemia and following major abdominal trauma, the determination of endotoxin continues to be a leading candidate which could become adopted into clinical practice. The importance of endotoxin measurement continues to grow as more clinicians recognize the added value of measuring endotoxin in critically ill patients and with the emergence of major pharmaceutical trials directly targeting endotoxin in the bloodstream. However, hundreds of other candidates potentially serving as biomarker for sepsis have been recently described, e.g. cysteinyl-leukotriene (LTC4) generation, procalcitonin (PCT) and C-reactive protein (CRP). However, none of them fulfils the criteria requested by clinicians, namely being specific and sensitive. The presentation will discuss criteria for a sepsis biomarker, will give an overview of obtaining samples from appropriate cell systems and from patients. Furthermore, tools will be described to identify marker candidates on genetic-, proteinand metabolite level. The integration of these data sets covering e.g. signal transduction, protein : protein interaction, gene expression with the help of bioinformatics and systems biology will help to validate such candidates. The final goal is manufacturing a robust diagnostic device for clinical routine work. A solid sepsis diagnostics method will be beneficial for patients, but also for the healthcare systems and will open challenges for the pharmaceutical industry.