The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G₁₃, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A^−/− mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.     

A multi-domain fragment of Nogo-A protein is a potent inhibitor of cortical axon regeneration via Nogo receptor 1

Nogo-A limits axon regeneration and functional recovery after central nervous system injury in adult mammals. Three regions of Nogo-A (Nogo-A-24, Nogo-66, and Nogo-C39) interact with the neuronal Nogo-66 receptor 1 (NgR1). Nogo-66 also interacts with a structurally unrelated cell surface receptor, paired immunoglobulin-like receptor (PirB). We show here that the other two NgR1-interacting domains, Nogo-A-24 and Nogo-C39, also bind to PirB with high affinity. A purified 22-kDa protein containing all three NgR1- and PirB-interacting domains (Nogo-22) is a substantially more potent growth cone-collapsing molecule than Nogo-66 for chick dorsal root ganglion neurons and mature cortical neurons. Moreover, Nogo-22 inhibits axon regeneration of mature cortical neurons in vitro more potently than does Nogo-66. Although all three NgR1-interacting domains of Nogo-A also interact with PirB, expression of PirB in mature cortical cultures is nearly undetectable. Consistent with a relatively minor role for PirB in mature cortical neurons, Nogo-22 inhibition of axon regeneration is abolished by genetic deletion of NgR1. Thus, NgR1 is the predominant receptor for Nogo-22 in regenerating cortical neurons.     

Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway

Background

Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/Principal Findings

In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/Significance

These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A–specific fragment NogoΔ20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoΔ20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoΔ20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoΔ20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.     

An Open Source Based High Content Screening Method for Cell Biology Laboratories Investigating Cell Spreading and Adhesion

Background

Adhesion dependent mechanisms are increasingly recognized to be important for a wide range of biological processes, diseases and therapeutics. This has led to a rising demand of pharmaceutical modulators. However, most currently available adhesion assays are time consuming and/or lack sensitivity and reproducibility or depend on specialized and expensive equipment often only available at screening facilities. Thus, rapid and economical high-content screening approaches are urgently needed.

Results

We established a fully open source high-content screening method for identifying modulators of adhesion. We successfully used this method to detect small molecules that are able to influence cell adhesion and cell spreading of Swiss-3T3 fibroblasts in general and/or specifically counteract Nogo-A-Δ20-induced inhibition of adhesion and cell spreading. The tricyclic anti-depressant clomipramine hydrochloride was shown to not only inhibit Nogo-A-Δ20-induced cell spreading inhibition in 3T3 fibroblasts but also to promote growth and counteract neurite outgrowth inhibition in highly purified primary neurons isolated from rat cerebellum.

Conclusions

We have developed and validated a high content screening approach that can be used in any ordinarily equipped cell biology laboratory employing exclusively freely available open-source software in order to find novel modulators of adhesion and cell spreading. The versatility and adjustability of the whole screening method will enable not only centers specialized in high-throughput screens but most importantly also labs not routinely employing screens in their daily work routine to investigate the effects of a wide range of different compounds or siRNAs on adhesion and adhesion-modulating molecules.     

Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination

Background

p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.

Methods and Findings

In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.

Conclusions

The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.     

Dopamine D2 Receptor Stimulation Potentiates PolyQ-Huntingtin-Induced Mouse Striatal Neuron Dysfunctions via Rho/ROCK-II Activation

Background

Huntington''s disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.

Methodology/Principal Findings

Using primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.

Conclusions/Significance

Our data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease.     

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive,Actin-Binding Deficient Splice Variant of Vinculin

Background

The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models.

Methodology/Principal Findings

Here, we investigate vinculin-ΔEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-ΔEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-ΔIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-ΔIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-ΔEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-ΔEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-ΔEx20 variant unveils vinculin''s dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites.

Conclusions/Significance

Vinculin-ΔEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.     

Variant RONΔ160 of the RON receptor tyrosine kinase promotes the growth and invasion in vitro and in vivo in gastric cancer cell lines

Background

Recepteur d’origine nantais (RON) is a receptor tyrosine kinase whose overexpression has been observed in human gastric cancers. This study aimed to determine whether overexpression of the variant RONΔ160 could induce tumorigenicity of gastric cancer cells in vitro or in vivo, and whether its specific small molecule inhibitor (Compound I) could inhibit the effect of RONΔ160.

Methods

We constructed human gastric cancer cell line MGC-803 that was stably transfected with a recombinant plasmid expressing RONΔ160, and the effect of RONΔ160 overexpression and macrophage-stimulating protein (MSP) activation on proliferation, migration and invasion abilities of MGC-803 cells were evaluated. Tumor-bearing mice with gastric cancer cells were used to analyze the effects of RONΔ160 overexpression and Compound I on implanted tumor growth.

Results

In vitro, overexpression of RONΔ160 in MGC-803 cells resulted changes to their cell morphology, and promoted cell proliferation, migration and invasion. In addition, overexpression of RONΔ160 increased the proportion of cells in the S phase. The effect of RONΔ160 was significantly enhanced by induction of MSP inducing (p < 0.05). In vivo, RONΔ160 promoted the growth of MGC-803 cells in nude mice, including increased tumor size and weight, and lower tumor incubation period. The Compound I inhibited the tumorigenic abilities of RONΔ160 (p <0.05).

Conclusions

The results indicate that overexpression of the variant RONΔ160 altered the phenotype and tumorigenicity of MGC-803 cells. Its specific small molecule inhibitor could inhibit the effect of RONΔ160. Therefore, the variant RONΔ160 may become a potential therapeutic target for gastric cancer.     

Anti-Cancer Effect and the Underlying Mechanisms of Gypenosides on Human Colorectal Cancer SW-480 Cells

Background

Gypenosides (Gyp), the main components from Gynostemma pentaphyllum Makino, are widely used in traditional Chinese medicine. The present study aimed to investigate the anti-cancer effect and the underlying mechanisms of Gyp on human colorectal cancer cells SW-480.

Materials and Methods

The inhibitory effect of Gyp on SW-480 cells was evaluated by MTT assay. Apoptotic cell death was detected by nuclear Hoechst 33342 staining and DNA fragmentation analysis. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining. Cell membrane integrity was evaluated with flow cytometry following PI staining. Changes of mitochondrial membrane potential (Δψ _m) were detected through flow cytometry analysis of rhodamine 123 (Rh123). The role of reactive oxygen species (ROS) in Gyp induced cell death was investigated by intracellular ROS generation and general ROS scavenger. Wound-healing assay was carried out to investigate Gyp-inhibited migration of SW-480 cells in vitro. Additionally, the alterations in F-actin microfilaments were analyzed by FITC-labeled phalloidin toxin staining and the morphological changes were evaluated under scanning electron microscope (SEM).

Results

After the Gyp treatment, the plasma membrane permeability of SW-480 cell was increased, Δψ _m was decreased significantly, the level of intracellular ROS level was increased, DNA fragmentation and apoptotic morphology were observed. Cells treated with Gyp exert serious microfilament network collapse as well as the significant decrease in the number of microvilli. Gyp induced the changes of cell viability, cell migration, intracellular ROS generation and nuclear morphology were alleviated obviously by NAC.

Conclusion

The results in this study implied that ROS play an important role in Gyp induced cell toxicity and apoptosis, and the mitochondria damage may be upstream of ROS generation post Gyp treatment. The findings of the present study provide new evidences for anti-tumor mechanisms by which Gyp induces apoptosis in vitro.     

Structural characterization of the human Nogo-A functional domains. Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration.

The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury. Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration. So far, no structural information is available for Nogo-A or any of its structural domains. We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli. CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble. Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration. Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment. The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol. The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands. Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration.     

Interaction between Genetic Predisposition to Adiposity and Dietary Protein in Relation to Subsequent Change in Body Weight and Waist Circumference

Background

Genetic predisposition to adiposity may interact with dietary protein in relation to changes of anthropometry.

Objective

To investigate the interaction between genetic predisposition to higher body mass index (BMI), waist circumference (WC) or waist-hip ratio adjusted for BMI (WHR_BMI) and dietary protein in relation to subsequent change in body weight (ΔBW) or change in WC (ΔWC).

Design

Three different Danish cohorts were used. In total 7,054 individuals constituted the study population with information on diet, 50 single-nucleotide polymorphisms (SNPs) associated with BMI, WC or WHR_BMI, as well as potential confounders. Mean follow-up time was ∼5 years. Four genetic predisposition-scores were based on the SNPs; a complete-score including all selected adiposity- associated SNPs, and three scores including BMI, WC or WHR_BMI associated polymorphisms, respectively. The association between protein intake and ΔBW or ΔWC were examined and interactions between SNP-score and protein were investigated. Analyses were based on linear regressions using macronutrient substitution models and meta-analyses.

Results

When protein replaced carbohydrate, meta-analyses showed no associations with ΔBW (41.0 gram/y/5 energy% protein, [95% CI: −32.3; 114.3]) or ΔWC (<−0.1 mm/y/5 energy % protein, [−1.1; 1.1]). Similarly, there were no interactions for any SNP-scores and protein for either ΔBW (complete SNP-score: 1.8 gram/y/5 energy% protein/risk allele, [−7.0; 10.6]) or ΔWC (complete SNP-score: <0.1 mm/y/5 energy% protein/risk allele, [−0.1; 0.1]). Similar results were seen when protein replaced fat.

Conclusion

This study indicates that the genetic predisposition to general and abdominal adiposity, assessed by gene-scores, does not seem to modulate the influence of dietary protein on ΔBW or ΔWC.     

The Thioamides Methimazole and Thiourea Inhibit Growth of M. avium Subspecies paratuberculosis in Culture

Background

Thyrotoxicosis is conceptualized as an “autoimmune” disease with no accepted infectious etiology. There are increasingly compelling data that another “autoimmune” affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition.

Methods

The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec® culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as “cumulative growth index,” (cGI) or “percent decrease in cumulative GI” (%-ΔcGI).

Principal Findings

Methimazole was the most effective thioamide at inhibiting MAP growth. At 128µg/ml: MAP UCF-4; 65%-ΔcGI & MAP ATCC 19698; 90%-ΔcGI. Thiourea inhibited MAP “Ben” maximally; 70%-ΔcGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested.

Significance

We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.     

Hip and Knee Strength Is Not Affected in 12-16 Year Old Adolescents with Patellofemoral Pain - A Cross-Sectional Population-Based Study

Background

One of the rationales behind using strength training in the treatment of adolescents with Patellofemoral Pain (PFP) is that reduced strength of the lower extremity is a risk factor for PFP and a common deficit. This rationale is based on research conducted on adolescents >15 years of age but has never been investigated among young adolescents with PFP.

Objectives

To compare isometric muscle strength of the lower extremity among adolescents with PFP compared to age- and gender-matched pain-free adolescents.

Methods

In 2011 a population-based cohort (APA2011-cohort) consisting of 768 adolescents aged 12–15 years from 8 local schools was formed. In September 2012, all adolescents who reported knee pain in September 2011 were offered a clinical examination if they still had knee pain. From these, 20 adolescents (16 females) were diagnosed with PFP. Pain-free adolescents from the APA2011-cohort (n = 20) were recruited on random basis as age- and gender-matched pairs. Primary outcome was isometric knee extension strength normalized to body weight (%BW) and blinded towards subject information. Secondary outcomes included knee flexion, hip abduction/adduction and hip internal/external rotation strength. Demographic data included Knee Injury and Osteoarthritis Outcome Score (KOOS) and symptom duration.

Results

Adolescents with PFP reported long symptom duration and significantly worse KOOS scores compared to pain-free adolescents. There were no significant differences in isometric knee extension strength (Δ0.3% BW, p = 0.97), isometric knee flexion strength (Δ0.4% BW, p = 0.84) or different measures of hip strength (Δ0.4 to 1.1% BW, p>0.35).

Conclusion

Young symptomatic adolescents with PFP between 12 and 16 years of age did not have decreased isometric muscle strength of the knee and hip. These results question the rationale of targeting strength deficits in the treatment of adolescents with PFP. However, strength training may still be an effective treatment for those individuals with PFP suffering from strength deficits.     

Blood Pressure Control with a Single-Pill Combination of Indapamide Sustained-Release and Amlodipine in Patients with Hypertension: The EFFICIENT Study

Objective

Despite antihypertensive treatment, most hypertensive patients still have high blood pressure (BP), notably high systolic blood pressure (SBP). The EFFICIENT study examines the efficacy and acceptability of a single-pill combination of sustained-release (SR) indapamide, a thiazide-like diuretic, and amlodipine, a calcium channel blocker (CCB), in the management of hypertension.

Methods

Patients who were previously uncontrolled on CCB monotherapy (BP≥140/90 mm Hg) or were previously untreated with grade 2 or 3 essential hypertension (BP≥160/100 mm Hg) received a single-pill combination tablet containing indapamide SR 1.5 mg and amlodipine 5 mg daily for 45 days, in this multicenter prospective phase 4 study. The primary outcome was mean change in BP from baseline; percentage of patients achieving BP control (BP<140/90 mm Hg) was a secondary endpoint. SBP reduction (ΔSBP) versus diastolic BP reduction (ΔDBP) was evaluated (ΔSBP/ΔDBP) from baseline to day 45. Safety and tolerability were also assessed.

Results

Mean baseline BP of 196 patients (mean age 52.3 years) was 160.2/97.9 mm Hg. After 45 days, mean SBP decreased by 28.5 mm Hg (95% CI, 26.4 to 30.6), while diastolic BP decreased by 15.6 mm Hg (95% CI, 14.5 to 16.7). BP control (<140/90 mm Hg) was achieved in 85% patients. ΔSBP/ΔDBP was 1.82 in the overall population. Few patients (n = 3 [2%]) reported side effects, and most (n = 194 [99%]) adhered to treatment.

Conclusion

In patients who were previously uncontrolled on CCB monotherapy or untreated with grade 2 or 3 hypertension, single-pill combination indapamide SR/amlodipine reduced BP effectively—especially SBP— over 45 days, and was safe and well tolerated.

Trial Registration

Clinical Trial Registry – India CTRI/2010/091/000114     

Glaucomatous and Age-Related Changes in Corneal Pulsation Shape. The Ocular Dicrotism

Purpose

To ascertain whether the incidence of ocular dicrotic pulse (ODP) increases with age, it is more pronounced in glaucomatous than in normal eyes and whether it is related to cardiovascular activity.

Methods

261 subjects aged 47 to 78 years were included in the study and classified into four groups: primary open angle glaucoma (POAG), primary angle-closure glaucoma (PACG), glaucoma suspects with glaucomatous optic disc appearance (GODA) and the controls (CG). Additionally, in each group, subjects with ODP were divided into two age subgroups around the median age. A non-contact ultrasonic method was used to measure corneal indentation pulse (CIP) synchronically with the acquisition of electrocardiography (ECG) and blood pulse signals. ODP was assessed from the acquired signals that were numerically processed in a custom written program.

Results

ODP incidence was about 78%, 66%, 66% and 84% for CG, GODA, POAG, and PACG group, respectively. With advancing age, the ODP incidence increased for all subjects (Δ = 12%), the highest being for the PACG and POAG groups (Δ = 30%). GODA group did not show an age-related increase in the incidence of ODP.

Conclusions

The ocular dicrotism, measured with non-contact ultrasonic method, was found to be a common phenomenon in elderly subjects. The increased ODP incidence in PACG and POAG group may correspond to either higher stiffness of glaucoma eyes, biochemical abnormalities in eye tissues, changes in ocular hemodynamics, may reflect the effect of medications or be a combination of all those factors. The results of GODA group suggest different mechanisms governing their ocular pulse that makes them less susceptible to generating ODP and having decreased predisposition to glaucoma.     

Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia

Aim

Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66^Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H₂O₂. Ablation of p66^Shc protects against IR injury, but it is unknown if and when p66^Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66^Shc activation during IR is associated with cardioprotection.

Methods

Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66^Shc at serine 36 and levels of p66^Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion.

Results

Phosphorylation of p66^Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66^Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66^Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66^Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion.

Conclusion

Our results show that IR activates p66^Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66^Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.