Anja Meissner

Sphingosine-1-Phosphate–Dependent Activation of p38 MAPK Maintains Elevated Peripheral Resistance in Heart Failure Through Increased Myogenic Vasoconstriction

Rationale: Mechanisms underlying vasomotor abnormalities and increased peripheral resistance exacerbating heart failure (HF) are poorly understood. Objective: To explore the role and molecular basis of myogenic responses in HF. Methods and Results: 10 weeks old C57Bl6 mice underwent experimental myocardial infarction (MI) or sham surgery. At 1 to 12 weeks postoperative, mice underwent hemodynamic studies, mesenteric, cerebral, and cremaster artery perfusion myography and Western blot. Organ weights and hemodynamics confirmed HF and increased peripheral resistance after MI. Myogenic responses, ie, pressure-induced vasoconstriction, were increased as early as 1 week after MI and remained elevated. Vasoconstrictor responses to phenylephrine were decreased 1 week after MI, but not at 2 to 6 weeks after MI, whereas those to endothelin (ET)-1 and sphingosine-1-phosphate (S1P) were increased at all time points after MI. An antagonist (JTE-013) for the most abundant S1P receptor detected in mesenteric arteries (S1P2R) abolished the enhanced myogenic responses of HF, with significantly less effect on controls. Mice with genetic absence of sphingosine-kinases or S1P2R (Sphk1−/−; Sphk1−/−/Sphk2+/−; S1P2R−/−) did not manifest enhanced myogenic responses after MI. Mesenteric arteries from HF mice exhibited increased phosphorylation of myosin light chain, with deactivation of its phosphatase (MLCP). Among known S1P-responsive regulators of MLCP, GTP-Rho levels were unexpectedly reduced in HF, whereas levels of activated p38 MAPK and ERK1/2 (extracellular signal-regulated kinase 1/2) were increased. Inhibiting p38 MAPK abolished the myogenic responses of animals with HF, with little effect on controls. Conclusions: Rho-independent p38 MAPK-mediated deactivation of MLCP underlies S1P/S1P2R-regulated increases in myogenic vasoconstriction observed in HF. Therapeutic targeting of these findings in HF models deserves study.

Tumor Necrosis Factor-α–Mediated Downregulation of the Cystic Fibrosis Transmembrane Conductance Regulator Drives Pathological Sphingosine-1-Phosphate Signaling in a Mouse Model of Heart Failure

Background— Sphingosine-1-phosphate (S1P) signaling is a central regulator of resistance artery tone. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzyme sphingosine kinase 1 and its functional antagonist S1P phosphohydrolase-1. The intracellular localization of S1P phosphohydrolase-1 necessitates the import of extracellular S1P into the intracellular compartment before its degradation. The present investigation proposes that the cystic fibrosis transmembrane conductance regulator transports extracellular S1P and hence modulates microvascular S1P signaling in health and disease. Methods and Results— In cultured murine vascular smooth muscle cells in vitro and isolated murine mesenteric and posterior cerebral resistance arteries ex vivo, the cystic fibrosis transmembrane conductance regulator (1) is critical for S1P uptake; (2) modulates S1P-dependent responses; and (3) is downregulated in vitro and in vivo by tumor necrosis factor-&agr;, with significant functional consequences for S1P signaling and vascular tone. In heart failure, tumor necrosis factor-&agr; downregulates the cystic fibrosis transmembrane conductance regulator across several organs, including the heart, lung, and brain, suggesting that it is a fundamental mechanism with implications for systemic S1P effects. Conclusions— We identify the cystic fibrosis transmembrane conductance regulator as a critical regulatory site for S1P signaling; its tumor necrosis factor-&agr;–dependent downregulation in heart failure underlies an enhancement in microvascular tone. This molecular mechanism potentially represents a novel and highly strategic therapeutic target for cardiovascular conditions involving inflammation.

Proximal Cerebral Arteries Develop Myogenic Responsiveness in Heart Failure via Tumor Necrosis Factor-α–Dependent Activation of Sphingosine-1-Phosphate Signaling

Background— Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Methods and Results— Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-&agr; (TNF&agr;)–dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNF&agr; expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNF&agr; antagonist etanercept. TNF&agr; mediates its effect via a sphingosine-1-phosphate (S1P)–dependent mechanism, requiring sphingosine kinase 1 and the S1P2 receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Conclusions— Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNF&agr;/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Proximal Cerebral Arteries Develop Myogenic Responsiveness in Heart Failure via TNFα-Dependent Activation of S1P Signaling

Background— Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Methods and Results— Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-&agr; (TNF&agr;)–dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNF&agr; expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNF&agr; antagonist etanercept. TNF&agr; mediates its effect via a sphingosine-1-phosphate (S1P)–dependent mechanism, requiring sphingosine kinase 1 and the S1P2 receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Conclusions— Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNF&agr;/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries

Objective—The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results—In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1S225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations, and Ca2+ sensitivity. The lack of function of the Sk1S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca2+ sensitivity and myogenic tone were attenuated, whereas Ca2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca2+ sensitivity) and intracellular effects of S1P (Ca2+ handling). Accordingly, S1P2 receptor inhibition (1&mgr;mol/L JTE013) attenuated myogenic tone without effect on Ca2+. Conclusions—Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.

Activation of the c‐Met receptor complex in fibroblasts drives invasive cell behavior by signaling through transcription factor STAT3

c‐Met is the receptor for hepatocyte growth factor/scatter factor (HGF/SF). It mediates multiple cellular responses in development and adult life, and c‐Met hyperactivity is associated with malignant transformation of cells and the acquisition of metastatic properties. Signal transducer and activator of transcription 3 (STAT3) has been shown to contribute to c‐Met‐mediated cell motility and is, thus, potentially involved in the control of invasive cell behavior. We have functionally reconstituted c‐Met‐dependent signal transduction in fibroblasts with the aim of studying Met‐driven cell invasiveness and the role of STAT3 in this phenomenon. Activation of the system was achieved by means of a hybrid receptor comprising the extracellular domain of the nerve growth factor (NGF) receptor TrkA, the cytoplasmic part of c‐Met and a C‐terminally fused blue fluorescent protein (BFP). In addition, a GFP‐tagged derivative of adaptor protein Gab1 was expressed. NGF‐stimulation of mouse fibroblasts expressing tagged versions of both Trk‐Met and Gab1 with NGF resulted in anchorage‐independent growth and enhanced invasiveness. By freeze‐fracture cytochemistry and electron microscopy, we were able to visualize the ligand‐induced formation of multivalent receptor complex assemblies within the cell membrane. NGF‐stimulation of the heterologous receptor system evoked activation of STAT3 as evidenced by tyrosine phosphorylation and the formation of STAT3 clusters at the cell membrane. siRNA‐mediated ablation of STAT3 expression resulted in a drastic reduction of c‐Met‐driven invasiveness, indicating an important role of STAT3 in the control of this particularly relevant property of transformed cells.

Therapeutically Targeting Tumor Necrosis Factor-α/Sphingosine-1-Phosphate Signaling Corrects Myogenic Reactivity in Subarachnoid Hemorrhage

Background and Purpose— Subarachnoid hemorrhage (SAH) is a complex stroke subtype characterized by an initial brain injury, followed by delayed cerebrovascular constriction and ischemia. Current therapeutic strategies nonselectively curtail exacerbated cerebrovascular constriction, which necessarily disrupts the essential and protective process of cerebral blood flow autoregulation. This study identifies a smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in a murine model of experimental SAH: it links tumor necrosis factor-&agr; (TNF&agr;), the cystic fibrosis transmembrane conductance regulator, and sphingosine-1-phosphate signaling. Methods— Mouse olfactory cerebral resistance arteries were isolated, cannulated, and pressurized for in vitro vascular reactivity assessments. Cerebral blood flow was measured by speckle flowmetry and magnetic resonance imaging. Standard Western blot, immunohistochemical techniques, and neurobehavioral assessments were also used. Results— We demonstrate that targeting TNF&agr; and sphingosine-1-phosphate signaling in vivo has potential therapeutic application in SAH. Both interventions (1) eliminate the SAH-induced myogenic tone enhancement, but otherwise leave vascular reactivity intact; (2) ameliorate SAH-induced neuronal degeneration and apoptosis; and (3) improve neurobehavioral performance in mice with SAH. Furthermore, TNF&agr; sequestration with etanercept normalizes cerebral perfusion in SAH. Conclusions— Vascular smooth muscle cell TNF&agr; and sphingosine-1-phosphate signaling significantly enhance cerebral artery tone in SAH; anti-TNF&agr; and anti–sphingosine-1-phosphate treatment may significantly improve clinical outcome.

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Cav channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Cav channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca2+ through Cav channels contributes to the regulation of hLEC proliferation, identifying Cav channel blockers as potential therapeutic substances in ocular diseases.

Sphingosine-1-phosphate signalling-a key player in the pathogenesis of Angiotensin II-induced hypertension.

Aims Hypertension is a complex condition involving functional and structural alterations of the microvasculature and an activation of the immune system. T-lymphocytes play a crucial role during the development of hypertension in experimental models, yet the underlying mechanisms remain elusive. Lymphocyte egress from lymph nodes is controlled by sphingosine-1-phosphate (S1P), a natural lipid mediator regulating immune cell and vascular function in health and disease. We therefore investigated the involvement of S1P signalling in the pathogenesis of hypertension. Methods and results Angiotensin-II (AngII) treatment resulted in high blood pressure (BP) associated to increased plasma S1P and circulating T-cell counts. T-cell egress from lymph nodes was found to be a critical initial step for the onset of hypertension as fingolimod, a S1P-receptor agonist sequestering lymphocytes in the lymph nodes and inducing lymphopenia, blunted BP responses to AngII. Furthermore, activity of S1P-generating enzyme type 2 (SphK2) in haematopoietic cells critically contributed to AngII-induced lymphocyte mobilization from the lymph nodes as SphK2-/- mice and mice where SphK2 was ablated only in the haematopoietic system presented an accumulation of T-cells in mesenteric lymph nodes and a blunted BP response. In addition, deregulation of vascular SphK2 expression associated to a thrombo-inflammatory phenotype of the microvasculature, and to functional alterations of small resistance arteries. Conclusion The presented results point to a critical involvement of S1P and its signalling axis in the pathogenesis of hypertension. Specifically, SphK2 evolves as key player in immune cell trafficking and vascular dysfunction contributing to the development of overt hypertension.

Hypertension and the Brain: A Risk Factor for More Than Heart Disease.

Background: Cerebral small vessel disease (cSVD), a common risk factor for cognitive impairment, involves unspecific arteriopathy characterized by hypertrophy and endothelial dysfunction that alter cerebrovascular function and auto-regulation of cerebral blood flow (CBF). Microbleedings, subcortical lacunar infarctions and diffuse areas of white matter lesions resulting from vascular injury are associated with reduced cognitive function mostly characterized by difficulties in learning and retention, attention deficits, gait disorders or depression. In recent years, it has become evident that vascular risk factors contribute to the development of cSVD and associated vascular cognitive impairment (VCI). Among them, hypertension emerged as such a major modifiable risk factor since the brain presents an early target for organ damage due to changes in blood pressure (BP). Subsequently both high and, especially in the elderly, low BP have been linked to cognitive decline, which initiated controversial discussions about BP control as a potential therapeutic strategy to achieve optimal brain perfusion and thus, reduce the occurrence of cSVD and cognitive dysfunction. Yet, recent randomized controlled trials examined the impact of anti-hypertensive therapy on cognitive performance with conflicting results. Summary: In light of the current knowledge, it becomes apparent that there is an urgent need to understand the mechanisms underlying hypertension-induced cerebrovascular complications in order to identify effective therapeutic targets to prevent and most importantly also reverse cognitive decline mediated through hypertension. Key Message: This review summarizes the current knowledge of cSVD pathogenesis as well as possible links to hypertension-mediated cerebrovascular complications. By pointing out knowledge gaps, it aims to spur future studies in search of specific targets helping to prevent therapy failures and decelerate the rapidly progressing neuro-degeneration of patients suffering from cerebrovascular diseases emanating from hypertension.