Objectives: Allergic rhinitis (AR) is an IgE mediated disease which is released by activation of mast cells and basophils, and often leads to sinus headache. Histamine which is the key mediator in the pathogenesis of AR, also plays an important role in the pathogenesis of migraine with nitric oxide (NO). Aim of our study was to investigate the frequency of migraine in patients with AR.Methodology: Headache assessment and neurological examination was performed on patients diagnosed as AR in the outpatient ear nose and throat clinic with age-matched controls. Participants with headache were classified according to the International Headache Society criteria, and migraine frequency was investigated in the patients with AR and control groups.Results: Migraine headache was detected in 50% of the patients with AR. Among these, 95% were migraine without aura, and 5% were migraine with aura. Migraine frequency in the control group was 18.75% in the control group, and all was migraine without aura. Migraine frequency in the patients with AR was four times higher when compared with the control group.Conclusion: While a histamine and IgE associated common mechanism is responsible in the pathogenesis of AR and migraine, not only sinus headache but also migraine headache should be kept in mind. Headache assessment of the patients with AR, and in case of headache existence, referral of these patients to neurology outpatient clinics for differential diagnosis and, to maintain appropriate therapy should not be forgotten. As soon as the Ig E binds to mast cells and basophils containing histamine and other chemicals, the release of these inflammatory mediators cause acute symptoms as sneezing, itchy and watery eyes, swelling and inflammation of the nasal passages, and an increase in mucus production. Late phase occurs 4-8 hours after the exposure to allergen and is characterized with additional symptoms as fatigue, irritability, and headache.3-5Allergic rhinitis is a chronic disease of seasonal and/or perennial course, characterized with antibody immunoglobulin E (IgE) mediated inflammation of nasal mucosa triggered by allergens including pollens, dust, foods, mites, animal dander, occupational allergens and drugs.6 Nitric oxide (NO) is another inflammatory mediator seen in AR, and is considered to play a role in migraine attacks due to its’ vasodilator effects.7,8 Migraine is a combination of autonomic, gastrointestinal, and neurological symptoms which is characterized with severe headaches. Reported prevalence of migraine in Turkey is 16.1%.9 Migraine is considered as a result of neurovascular process triggered by endogenous and/or exogenous factors in genetically predisposed individuals. However, pathogenesis and molecular mechanisms of migraine is still unclear.10Most common form of headache in patients with AR is sinus headache, which is caused by the activation of mast cells and basophils.11 Additionally, it is a well-known fact that plasma histamine levels increase during both the headache and remission phase of migraine.5,12 While local histamine release in AR may trigger migraine attacks in these patients, we aimed to assess the frequency of migraine in patients with AR in our study.Underlying pathophysiological mechanisms include neurogenic inflammation, defects in the metabolism of arachidonic acid and serotonin, cyclic changes in the ovarian steroid concentrations, food allergy, and atopy. These mechanisms are important in assessing the relationship of migraine with allergic diseases.Patients with the diagnoses of AR were included from the Ear Nose and Throat outpatient clinic of Duzce University School of Medicine between July and October 2007. Control group consisted of age-matched healthy individuals. All participants were informed about the content of the study and gave their approval before enrollment.13Exclusion criteria were sinusitis, upper respiratory infection, stroke, cerebral palsy, trigeminal neuralgia, epilepsy and allergic diseases other than AR. Clinical diagnosis was confirmed with skin tests and serum IgE levels in patients with AR. Skin test (Prick test-Allergopharma- Germany) was performed on the forearm of the patients with 16 solutions. Skin tests included solutions with histamine (positive control), physiological saline (negative control), and 14 allergenic substances. Diameter of the induration which existed in the upper layer of reaction was measured 15 minutes after the application of solutions and scored.14All patients and controls underwent a detailed neurological and headache assessment following neurological examination. Participants with headache were re-evaluated and classified according to the criteria of International Headache Society (IHS), and the frequency of migraine was investigated.Data were organized in an SPSS version 11.0  database. Values were given as mean ± standard deviations, and p values < 0.05 were considered to indicate statistical significance. χ2 test was used in the analysis of categorical variables, and numeric variables were compared with student’ s t test.Eighty AR patients with positive skin test were included in the study. Among these, three patients were under desensitization therapy, 77 were newly diagnosed patients without any treatment. Study group consisted of 50 female (62.5%) and 30 male (37.5 %) patients with AR. There were 46 female (57.5%) and 34 male (42.5%) participants in the control group. Mean age of the patients were 32.27 ± 12.50 years (range=12-62), and 34.06 ± 12.79 years (range=15-65) in the control group. There were no statistically significant difference between the age and gender of the study groups . Socio-demographic features and migraine characteristics and frequencies of the study groups were summarized in Skin tests of the patients with AR revealed allergy to acar 1-2 in 68 of the patients (85%), to fungi 1-2 in 5 (6.25%), to mix grass in 4 (5%) and to grass and grain in three of the patients (3.75%). Among these, 40 patients were diagnosed as migraine (50%). Thirty eight of them were diagnosed as classical migraine without aura (95%), and two had migraine with aura (5%). Fifteen of the participants were diagnosed as migraine in the control group, and all was (were) classical migraine without aura (18.75%).Migraine frequency according to the gender in both groups is summarized in 15,16Migraine is a primary episodic headache disorder with various neurological complaints. Previous reports revealed that endogenous molecules such as calcitonin gene related peptide, NO, and histamine play an important role in the pathophysiology of migraine. However, the exact underlying mechanism is still unclear.5 Thus, immune response characterized with histamine and NO mediated inflammation is considered as the fundamental factor of AR pathogenesis.1,7In AR, histamine is considered to contribute the development of migraine headaches via increasing the release of NO. Additionally, it facilitates the evolution of local neurogenic inflammation by increasing vasodilatation and vascular permeability via H1 and H2 receptors.5,15 But differing from the first study evaluating migraine frequency in patients with AR,5 we also investigated the triggering allergens in our study. Min Ku et al, reported that Migraine frequency was 34% in the patients with AR, while this rate was 4% in the control group.5 Similarly, we also found an increased frequency of migraine in patients with AR, when compared with the control group , and this difference between the groups was statistically significant (p<0.05). When we evaluated the migraine frequency according to the gender difference between the groups, we found an increased rate of migraine frequency in women and men in the AR group.With respect to this common pathogenic mechanism in AR and migraine, we aimed to evaluate the migraine frequency in patients with AR. Our literature review revealed limited number of reports demonstrating the association between AR and migraine.11,16,17 Davey at al. emphasized the association between migraine and bronchial asthma,11 while Mortimer et al. reported a higher prevalence of migraine in children with atopy, when compared with children without atophy(atopy).16 A previous study evaluating the possible effects of migraine on allergy investigated the serum IgE and histamine levels during migraine attacks and remission periods in migraine patients with allergy and without allergy. Serum IgE and histamine levels were found to be increased in migraine patients with and without allergy, both when compared with healthy controls. Moreover, there was a statistically significant difference between migraine patients with and without allergy, in which migraine patients with allergy had significantly higher serum IgE and histamine levels. Similar to our results, Gazerani et al. suggested a possible IgE mediated common mechanism in the pathophysiology of migraine and allergy.18There are several reports revealing the relationship of migraine with allergic diseases. However, the number of studies assessing migraine in AR is limited.In conclusion, results of our study were compatible with the hypothesis of IgE and histamine mediated common mechanism in migraine and AR. Thus, it is important to keep in mind that migraine headaches can be seen in patients with AR, as well as sinus headaches. As such AR patients with the complaint of headache should be referred to neurology outpatient clinics for differential diagnosis and appropriate treatment of headache. In such patients, future studies evaluating the effects of AR treatment in migraine headache may be beneficial in enhancing the treatment options, as well.


Telomeres protect the ends of linear chromosomes from being recognized as damaged DNA, and telomere stability is required for genome stability. Here we demonstrate that telomere stability in androgen receptor (AR)-positive LNCaP human prostate cancer cells is dependent on AR and androgen, as AR inactivation by AR antagonist bicalutamide (Casodex), AR-knockdown, or androgen-depletion caused telomere dysfunction, and the effect of androgen-depletion or Casodex was blocked by the addition of androgen. Notably, neither actinomycin D nor cycloheximide blocked the DNA damage response to Casodex, indicating that the role of AR in telomere stability is independent of its role in transcription. We also demonstrate that AR is a component of telomeres, as AR-bound chromatin contains telomeric DNA, and telomeric chromatin contains AR. Importantly, AR inactivation by Casodex caused telomere aberrations, including multiple abnormal telomere signals, remindful of a fragile telomere phenotype that has been described previously to result from defective telomere DNA replication. We suggest that AR plays an important role in telomere stability and replication of telomere DNA in prostate cancer cells, and that AR inactivation-mediated telomere dysfunction may contribute to genomic instability and progression of prostate cancer cells. Telomere DNA at the ends of chromosomes is double-stranded (DS) except for the single-stranded (SS) 3’-overhang . This ovFunctional telomeres are essential for maintaining genome integrity; telomere shortening resulting from telomere dysfunction leads to genomic instability, which is a common cause and hallmark of cancer -31. The in situ hybridization (FISH) analysis. These latter events are reminiscent of those that occur in cells with defective shelterin and non-shelterin proteins at telomeres , or to a biotinylated scrambled DNA probe (desthiobiotin-108 carbons-5’ GaTgTgTgGaTgTggAt-GtGgAtgTgg-3’). Hybridization conditions were 25°C for 3 min, 70°C for 6 min (3 cycles), 38°C for 60 min, 60°C for 2 min, 38°C for 60 min, 60°C for 2 min, 38°C for 120 min, and 25°C final temperature, using an MJ PTC-200 PCR machine. The probes contain a long 108-carbon spacer between the immobilization tag (desthiobiotin) and the LNA probe; the spacer was designed to minimize steric hindrance [The PICh protocol was performed essentially as described by Dejardin and Kingston { and httpindrance . These pindrance , 44}. Hiin situ hybridization (FISH) on metaphase spreads using a telomeric protein nucleic acid (PNA) probe, as described [g (8 min). After hypotonic swelling in 0.075 mM KCl for 25 min at 37o C, cells were fixed in methanol:acetic acid (3:1). Telomere FISH was performed as described [3-peptide nucleic acid (PNA) probe (Panagene).Telomeres were visualized by fluorescence escribed . LNCaP cescribed , using a


The tilt illusion is a well-studied visual phenomenon, whereby the perceived angle of a center stimulus is misjudged in the presence of a differently aligned surround stimulus (e.g. ). The deFirst, we examine is the effect of the tuning curve width on the tilt illusion. Tuning curves widths vary widely in vivo . AlthougSecondly, we consider the consequences of recent experimental findings on the tuning of surround modulation. Most models assume that V1 neurons experience most suppression when the surround stimulus is aligned with the neuron's preferred orientation. However, a recent study showed that for the majority of V1 neurons, the suppression effect depends much more on the relation between center and surround orientation, being strongest when they are co-aligned, regardless of the preferred orientation . We use


IPK) plays an important role in preventing endoplasmic reticulum (ER) stress. It is an interferon-induced kinase that targets the eukaryotic translation initiation factor eukaryotic initiation factor 2 alpha. The aim of this study was to determine the roles of P58IPK in protecting against diabetic retinopathy (DR) by inhibiting ER stress-signaling mediators.58-kilodalton inhibitor of protein kinase -P58IPK or transfection into rat retinal capillary endothelial cells. Retinal vascular permeability was determined by assessing the Evans Blue retinal leakage. To downregulate the P58IPK level in cultured rat retinal capillary endothelial cells, pGIPZ-P58IPK RNA interference (P58IPKRNAi) was introduced in these cells. Real time reverse transcription (RT)–PCR and western blot analyses were performed to evaluate the mRNA and protein levels of Core/emopamil binding protein (C/EBP) homologous protein (CHOP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α).A rat diabetic model was established by intraperitoneal injection of streptozotocin. Overexpression of P58IPK-transfected diabetic rats compared with the control diabetic rats. Both mRNA and protein levels of CHOP, TNF-α, and VEGF in the retina of diabetic rats were remarkably reduced in P58IPK-transfected rats. In vitro study further demonstrated that overexpression of P58IPK downregulated the expression of CHOP, TNF-α, and VEGF under high glucose conditions, whereas introduction of P58IPKRNAi enhanced the expression of CHOP, TNF-α, and VEGF.Retinal blood vessel leakage was significantly decreased in rAAV2-P58IPK against ER stress-mediated DR in diabetic rats, suggesting that P58IPK may act as a DR-resistant gene during diabetes.These results revealed the protecting role of P58 Diabetic retinopathy (DR) is a common complication of diabetes. Owing to the increased number of people with diabetes worldwide, DR has become the most frequent cause of postnatal blindness in people of working age in industrialized countries . AlthougIPK, a member of the heat shock protein 40 (Hsp40) family, phosphorylates eukaryotic initiation factor 2α (elF-2α) and sequesters tis sequesters its guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor /weight of the dry retina (mg).Retinal vascular permeability was quantified by measuring the Evans Blue (EB) retinal leakage . Briefly2. To investigate the expression of P58IPK, cultured rat retinal capillary endothelial cells (RRCECs) were transfected with pGIPZ-P58IPK RNAi (group A), pGIPZ-GFP , irrelevant small interfering RNA , and rAAV2-P58IPK (group D). Real-time PCR and western blot were used to evaluate the expression of P58IPK in RRCECs.Rat eyes were cut circumferentially 1.5 mm posterior to the limbus, and the retinas were harvested and homogenized by two gentle up-and-down strokes in a 15-ml homogenizer . The homogenate was filtered, and the remaining retentate was digested in 0.066% collagenase  at 37 °C for 45 min. The homogenate was centrifuged , and the pellet was resuspended in human serum-free endothelial basal growth medium , supplemented with 20% fetal bovine serum and 50 U/ml endothelial cell growth factor . Cells were cultured in fibronectin-coated dishes and incubated at 37 °C in a humidified atmosphere containing 5% CO9 μg/ml pGIPZ-P58IPK RNAi was added to group 1, and cells were maintained in culture medium supplemented with 30 mM/ml glucose (RNAi/high-glu); 1×109 μg/ml rAAV2-P58IPK was added to group 2, and cells were maintained in culture medium supplemented with 30 mM/ml glucose (P58IPK/high-glu); 1×109 μg/ml pGIPZ-P58IPK RNAi was added to group 3, and cells were cultured with normal human vascular endothelial cell culture media ; 1×109 μg/ml rAAV2-P58IPK was added to group 4, and cells were cultured with normal human vascular endothelial cell culture media .Cells were plated in 24-well culture plates and incubated overnight to 70%–80% confluence. Cells were then washed twice with human endothelial serum-free basal growth medium. Cells were randomly divided into four groups: 1, 2, 3, and 4. Then, 1×10Taq polymerase kit  using SYBR Green technology . β-actin was used as an internal control for the PCRs. Primers for PCRs are presented in IPK was performed in a final reaction volume of 50 μl in the following conditions: a preheating cycle at 94 °C for 3 min, followed by 30 cycles at 94 °C for 1 min, 56 °C for 30 s, and 72 °C for 45 s, and finally elongated at 72 °C for 8 min. The PCR conditions for VEGF, TNF-α, and CHOP were similar, except for the annealing temperature; VEGF was 58 °C, while TNF-α and CHOP were 56 °C. A melting curve analysis was performed by monitoring FAM/SYBR fluorescence.Total RNA was isolated using RNeasy mini Kit  from 30 mg of homogenized tissues with a kit  according to the manufacturer’s protocol. cDNA was then transcribed using a Super Script II RT kit (Invitrogen). Real-time real-time PCR -PCR was performed with a Total protein extracted from the rat retinas or RRCECs was quantified using bicinchoninic acid (Sigma Aldrich). The protein was mixed with the loading buffer, denatured for 6 min at 60 °C, cooled, centrifuged 12,000× g for 5 min, and then separated by sodium-dodecyl sulfate PAGE (PAGE). Monoclonal anti-TNF-α , polyclonal anti-CHOP , and anti-VEGF  antibodies were used to probe the proteins. Some of the same polyvinylidene fluoride membranes were reused to detect β-actin  by incubating them with the mouse antihuman actin antibody . The bands observed on the films were analyzed by automatic image analysis Odyssey scanner , and the integrated optical density of each protein band was normalized to the integrated optical density value of the corresponding β-actin band from the same sample.t test or one-way ANOVA where appropriate. When a significant difference was detected between groups, multiple comparisons of means were performed using the Bonferroni procedure, with the type I error rate at a maximum of 0.017 (0.05/3) adjustment. Statistical analyses were performed using SPSS 15.0 statistical software . Data were presented as the mean±standard deviation (SD). A p value <0.05 was considered to be significant.Normally distributed data were compared by independent two-sample IPK compared with the diabetic rats without P58IPK (rAAV2-GFP). Quantified data revealed that the area of blood vessel leakage was markedly reduced: 15.8±1.8% in the GFP/dia group (n=6); 3.6±0.9% in the GFP/no-dia group (n=6); 6.6±1.2% in the P58IPK/dia group (n=6); and 3.4±1.2% in the P58/no-dia group , pGIPZ-GFP , irrelevant siRNA (group C), and rAAV2-P58IPK (group D). No difference was detected between group C compared with group B. As shown in IPK in group D demonstrated enhanced P58IPK mRNA expression, while P58IPK RNAi decreased the P58IPK mRNA expression significantly. Similar results were obtained in western blot analysis  , have ben kinase . The inttivation , suggestulinemia , suggeste damage . Our datol group . In vitrIPK may relieve ER stress by maintaining ER functions, thus decreasing the levels of inflammatory factors and subsequently reducing the risks for DR development.A growing body of evidence has revealed that CHOP contributes to ER stress-induced retinal cell death ,18. MoreIPK protective retinal blood vessels in high glucose, we did notdemonstrate that overexpressed P58IPK affects the levels of PERK-signaling stress markers, such as PERK, phosphorylated eukaryotic initiation factor 2 (eIF2) and activating transcription factor 4 (ATF4). Future study will be focused on exploring the intracellular signaling pathway regulated by P58IPK. We found significant differences in the mRNA levels of CHOP and TNF between P58IPK/no-dia and GFP/no-dia groups; however, no significant differences were detected in the protein levels of CHOP and TNF between these two groups, suggesting that the effect of P58IPK on CHOP and TNF may be limited to the transcriptional level, although the mechanisms need further study.As the purpose of this paper is to verify the effect of P58IPK reduces the level of inflammatory factors, such as TNF-α. Our previous study confirmed that P58IPK can reduce the expression of VEGFof retinal vascular endothelial cells [IPK protects against ER stress-mediated apoptosis in vascular endothelial cells under high glucose conditions [IPK may act as a DR-resistant gene in diabetic patients.In summary, P58


Early after the turn of the century, much excitement was generated by the reports of Tillerson et al. , 2 that  Although results from studies of the neuroprotective effects of exercise are mixed, one consistent finding from animal models and human trials is the lack of adverse effects of exercise and physical activity on anatomic and behavioral outcomes. The adverse side-effect profile of exercise as an intervention for those with PD appears to be minimal. As such, we think there is no reason to wait for confirmation of neuroprotection. Rather, evidence is accumulating that exercise and physical activity should be utilized as key tools in the management of PD across the spectrum of disease. Evidence-based approaches to rehabilitation are known to improve physical functioning, strength, balance, gait, and health-related quality of life among people with PD –13, but The presently limited scope of rehabilitation in the management of PD, with utilization of rehabilitation as mainly a tertiary prevention measure, reflects a missed opportunity on the part of healthcare providers, patient advocacy groups, and patients themselves. All stakeholders in this situation should be advocates for higher expectations and should work together to develop targets for the future of rehabilitation in PD to include primary and secondary prevention and to improve our current provision of tertiary prevention interventions.From the Spectrum of Rehabilitation Options to Atypical Parkinsonism. In this special issue are several articles that we hope advance the field and move us closer to these future targets. The issue opens with a series of three review articles. The first paper summarizes and synthesizes the nature and features of previous randomized, controlled trials of exercise or motor training in PD, important for the long-term provision of increasing levels of physical activity. The second paper provides a meta-analysis focused on motor learning in upper extremity tasks. The third paper provides an integrated overview of the Lee Silverman Voice Treatment approach to voice and movement therapy, discussing the rationale for the approach as well as the data regarding efficacy. These opening three articles highlight the important roles of speech, occupational, and physical therapy approaches in the rehabilitation of individuals with PD as well as address areas for future research.Several papers in this special issue relate to gait, balance, and falls, examining the relationship between gait economy and six-minute walk distance, reviewing the literature on the costs of dual-task walking, and providing a new theoretical framework for considering freezing of gait (FOG) including methods to improve overall locomotor performance and methods to target the triggers of FOG. Also included are two randomized, controlled trials designed to improve walking. One compares visually cued walking training on a treadmill to overground walking; the other examines use of rotating treadmill training as a means of improving turning, thereby targeting a known trigger of FOG. Turning is also examined in a paper that describes turning impairments in early PD. This is followed by a paper examining the effects of medications on gait-related mobility and postural control, showing that although pharmacologic intervention enhanced some aspects of mobility, reactive postural responses did not improve. This highlights the need for awareness of postural control deficits and the need to be able to measure these deficits, as is addressed by the next two papers in the issue that present the relative merits of different balance measures across different levels of PD severity and examine the relative effectiveness of different balance measures for prospective fall prediction.The inextricable link between posture and gait is addressed in a paper that focuses on the coupling of posture and locomotion and suggests this coupling as a specific target for rehabilitation. This link is also highlighted in two papers demonstrating that walking ability is a major contributor to fear of falling in PD, as is knee strength. The latter leads to the suggestion that resistance training may therefore be warranted as an approach to reduce fear of falling. The rationale for progressive resistance training as well as its potential mechanisms are addressed in a review paper, followed by an article examining the reliability of one repetition maximum strength testing in PD.We conclude the issue with a set of papers addressing the delivery of evidence-based rehabilitation in different settings. These papers include two that are randomized, controlled trials of physiotherapy in outpatient and home-based settings, respectively. These are followed by a paper examining facilitators and barriers to community-based walking exercise among those with PD. Community-based healthcare for PD is also addressed in a paper describing the steps taken to improve the Dutch model of multidisciplinary care. The final paper of the issue demonstrates the effectiveness of multidisciplinary care in an inpatient setting in persons with atypical parkinsonism. All papers in this final section draw attention to the need for a collaborative, cooperative approach to rehabilitation across disciplines, across settings, and with PD and other related disorders.Ultimately, we believe that there is a need to redefine the role of rehabilitation in PD to include the provision of primary, secondary, and tertiary prevention approaches. Across this spectrum from primary through tertiary care, the application of multidisciplinary approaches is needed to optimize the health, function, and quality of life of individuals at risk for, or who already have, PD. Only then will the full potential of rehabilitation in the management of PD be realized.Gammon M. EarhartGammon M. EarhartTerry EllisTerry EllisAlice NieuwboerAlice NieuwboerLeland E. DibbleLeland E. Dibble


Aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences on brain function including profound effects on communication between neurons and the vasculature leading to cerebral ischemia. Physiologically, neurovascular coupling represents a focal increase in cerebral blood flow to meet increased metabolic demand of neurons within active regions of the brain. Neurovascular coupling is an ongoing process involving coordinated activity of the neurovascular unit—neurons, astrocytes, and parenchymal arterioles. Neuronal activity can also influence cerebral blood flow on a larger scale. Spreading depolarizations (SD) are self-propagating waves of neuronal depolarization and are observed during migraine, traumatic brain injury, and stroke. Typically, SD is associated with increased cerebral blood flow. Emerging evidence indicates that SAH causes inversion of neurovascular communication on both the local and global level. In contrast to other events causing SD, SAH-induced SD decreases rather than increases cerebral blood flow. Further, at the level of the neurovascular unit, SAH causes an inversion of neurovascular coupling from vasodilation to vasoconstriction. Global ischemia can also adversely affect the neurovascular response. Here, we summarize current knowledge regarding the impact of SAH and global ischemia on neurovascular communication. A mechanistic understanding of these events should provide novel strategies to treat these neurovascular disorders. Aneurysmal subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality with limited therapeutic options . The maj+ efflux that increases extracellular K+ concentration to >40 mM. Massive influx of Ca2+, Na+, and water accompanies the K+ efflux and triggers uncontrolled release of neurotransmitters, most importantly the excitatory amino acid glutamate. Released K+ and glutamate are believed to depolarize other neurons in the vicinity, and SD slowly propagates in grey matter by way of contiguity. Therefore, extracellular medium, including the perivascular space, is flooded with K+ and neurotransmitters that are vasoactive. Because complete membrane depolarization precludes action potentials and synaptic transmission, SD is associated with suppression of all spontaneous or evoked electrical activity. Consequently, the normal neuronal influence on the vasculature is absent at least until the ability of neurons to generate action potentials returns, which can take several minutes. Moreover, there is ample evidence suggesting that physiological neurovascular coupling is impaired not only during the depolarization but for hours after the SD event e) is artificially elevated e or low glucose, mimicking subarachnoid hemorrhage, SD is associated with severe vasoconstriction , leading to inversion of neurovascular coupling. In addition to responding to neurally released signals, astrocytes exhibit spontaneous Ca2+ oscillations [2+ oscillations occur in both soma and endfeet and have been observed in isolated brain slices [+ channel blocker tetrodotoxin to inhibit neuronal action potentials and represent intracellular Ca2+ release events from the endoplasmic reticulum [2+ events in mouse models of Alzheimer's disease has been linked to vascular instability in vivo [2+ oscillations in astrocyte endfeet was ~490 nM compared to a mean peak amplitude ~320 nM in brain slices from control animals. In comparison, neurally-evoked increases in astrocytic Ca2+ were ~350 nM in both control and SAH animals. Considering that EFS-induced increases in astrocytic endfeet Ca2+ have been shown to induce K+ efflux through endfoot BK channels, spontaneous Ca2+ events are also likely capable of activating endfoot BK channels. Based on these observations, it is conceivable that higher amplitude spontaneous Ca2+ events following SAH enhance BK channel activity contributing to increased basal K+ in restricted perivascular space  toxicity, cortical spreading depression, inflammation, and apoptosis . Emerginpocampus , 102. Global cerebral ischemia may also contribute to brain pathologies associated with SAH. Immediately following cerebral aneurysm rupture, increased intracranial pressure caused by the release of blood into the subarachnoid space can lead to transient global ischemia and contribute to a cascade of events referred to as “early brain injury” , 10. Fur+ due to the enhanced amplitude of spontaneous Ca2+ signaling events in astrocytic endfeet may underlie this inversion of neurovascular coupling, consistent with a bimodal effect of extracellular K+ to cause vasodilation at concentrations below 20 mM and constriction when this threshold of 20 mM is exceeded. Presently mechanisms associated with inversion of the neurovascular response caused by SAH-induced SD have not completely been resolved. However, inversion of SD-induced neurovascular response likely reflects a combination of increased extracellular K+ and the impact of SAH on the relative balance of vasoconstrictor and vasodilator influences. Development of agents and approaches to prevent SAH-induced inversion of neurovascular coupling may provide a much needed additional therapeutic option for SAH patients. Subarachnoid hemorrhage is a multifaceted pathology exhibiting both acute and long-term injury to the brain. It is now clear that SAH profoundly impacts neuronal influences on the vasculature leading to decreased cerebral blood flow that can exacerbate the extent of brain damage. One type of SAH-induced impaired neurovascular signaling arises in the context of SD that can impact large areas of cortical and subcortical grey matter. In the absence of SAH, SD is most frequently associated with a hyperemic response, that is, an increase in cerebral blood flow. However, SAH causes an inversion of the SD-induced neurovascular response leading to vasoconstriction and decreased blood flow to tissue during a time of high metabolic demand. Recently, it has also been shown that SAH can cause inversion of neurovascular coupling at the level of the individual neurovascular unit. Physiologically, coordinated activity of neurons, astrocytes, and parenchymal arterioles ensures increase local blood flow to active neurons in specific regions of the brain engaged in task-dependent processes. After SAH the neurovascular response to neuronal activation switches from vasodilation to vasoconstriction; this also promotes a pathological decrease in the flow of oxygen and nutrients to metabolically active neurons. Evidence suggests that elevated perivascular K


AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19). FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that combined targeting of FGF19 and AR/EGFR may enhance therapeutic efficacy.Hepatocellular carcinoma (HCC) is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR) signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR) in the proliferation, survival and drug resistance of HCC cells.  TP53, common denominator to most HCCs were alterations in intracellular signal transduction pathways triggered by growth factors Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer and a deadly disease The EGF receptor (EGFR) is perhaps one of the signaling systems that undergo most extensive cross-talk in cancer cells, including HCC cells, being considered as a “signaling hub” where different growth and survival signals converge The human HCC cell lines HepG2, Hep3B and PLC/PRF5 were obtained from the ATCC and grown as reported AR gene 5′ region, immediately upstream of the 5′ end of the mRNA start site, nucleotides −210 to −1464 from the ATG AR 5′ DNA fragment was cloned upstream from the luciferase reporter gene in the pGL3-Basic vector . Site-directed mutagenesis of the Tcf-binding elements (TBEs) was performed on this construct using the Quick Change Site-Directed Mutagenesis Kit  according to the protocol supplied by the manufacturer. Mutations were as follows: TBE1: CTTTGTA → CTTTGCC; TBE2: CTTTGAA → CTTTGGC; TBE3: TACAAAC → GCCAAAG. The primers used for site-directed mutagenesis are described in A 1255 bp fragment of human Total RNA was extracted using the TRI Reagent (Sigma). qPCR was performed using an iCycler  and the iQ SYBR Green Supermix (BioRad) as reported AR 5′ regions −1459 to −1287, −987 to −811 and −600 to −364, which include three putative Tcf/Lef binding sites previously identified by DNA sequence analysis The ChIP assay was performed in Huh7 cells essentially as described Cells were homogenized and analyzed by Western blotting as described The sequences of the siRNAs targeting different regions of the human AR gene (siAR1 and siAR2), and the control siRNA (siGL2), along with their specificity have been described Soluble AR was measured in cells’ conditioned medium by ELISA. Cells were cultured in the absence of serum as indicated, conditioned medium was collected and after addition of phenylmethylsulfonyl fluoride (1 mM) it was pre-cleared by centrifugation and lyophilized. The concentration of AR was determined by a sandwich ELISA from R&D Systems, using a monoclonal anti-AR capture antibody (#MAB262) and a biotinylated polyclonal detection antibody (BAF262). A standard curve using recombinant human AR (Sigma) was used to calculate AR concentrations in the conditioned medium.Cells were cultured on coverslips. After treatments cells were fixed with 4% paraformaldehyde (15 min) and subsequently permeabilized with 0.1% Triton X-100. After blocking in Supper blocking Buffer  (30 min) fixed cells were incubated with anti-β-catenin antibodies   overnight at 4°C. Slides were washed with saline and incubated with Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (1∶500 in 1% abumin)  for 1½ h at room temperature, and then washed and mounted.Data mining was performed using public high-throughput gene expression datasets from human HCC tissues obtained from Gene Expression Omnibus (GEO) database. AR and FGF19 expression values were obtained from the following sets of data: GSE1898 (90 HCC tissue samples) t test. The Spearman’s correlation coefficient was used to analyse the correlation between AR and FGF19 gene expression values obtained from the above-mentioned datasets. A P value of <0.05 was considered significant.Statistical analysis was performed with the Graph Pad Prism version 5.00 software . Experiments were performed at least twice in triplicates. Data are means ± SEM. Data were compared among groups using the Student AR gene expression through the activation of the β-catenin signaling pathway we first used two well-known inhibitors of glycogen synthase kinase 3β (GSK3β), LiCl and SB-415286 To examine the regulation of in silico study on the potential regulation of the expression of EGF family members by the Wnt/β-catenin pathway had identified three putative consensus TBE sites in the 5′ region of the human AR gene in vivo binding of β-catenin and Tcf4 to these TBEs . As shown in AR 5′ region was further confirmed by quantitative analysis of immunoprecipitated chromatin (qChIP)  family of transcription factors, leading to the formation of regulatory complexes that bind Tcf-binding elements (TBEs) on the promoters of target genes  (qChIP) . These fTbx3AR promoter luciferase reporter plasmid, as described in Materials and Methods. Transfection of the wild type AR promoter construct in Huh7 cells expressing the active T41A β-catenin mutant resulted in a significant stimulation of AR promoter activity compared to cells transfected with the control vector pcDNA3.1  or the corresponding empty vector. This β-catenin T41A mutant protein has increased stability, and has been previously shown to significantly up-regulate the expression of β-catenin target genes pcDNA3.1 . HoweverpcDNA3.1 . AltogetAR gene expression.Signaling through the β-catenin pathway has been thoroughly demonstrated to contribute to HCC cell growth CTNNB1 gene as well as by altered expression of ligands, receptors and inhibitors of the Wnt pathway Tbx3AR gene expression in HCC cells. As shown in Aberrant activation of the Wnt/β-catenin pathway in HCC can be accounted for by mutations in the Tbx3 and also in decreased AR mRNA levels, confirming the biological activity of this dominant negative Tcf4 mutant and the regulation of AR gene expression in response to the manipulation of the β-catenin pathway. Next we examined the response of ΔNTcf4 expressing HCC cells to FGF19 treatment. We observed that the up-regulation of AR expression in Huh7 cells treated with FGF19 was attenuated in cells expressing ΔNTcf4 compared to cells transfected with control plasmid, and a similar response was observed regarding Tbx3 expression  database for 90 and 239 HCC tumor tissues respectively AR gene expression can be activated in human HCC cells by β-catenin signaling, a key signaling pathway in hepatocarcinogenesis AR promoter region upon β-catenin pathway activation. Endogenous AR expression and secretion to the extracellular medium was induced in HCC cells in response to the ectopic expression of the dominant stable β-catenin mutant T41A Tbx3AR promoter-reporter activity triggered by the expression of this active β-catenin variant was reduced upon mutation of the TBE sites within this AR 5′ region. Together, these observations strongly suggest that AR is a target of β-catenin signaling in human HCC cells. Nevertheless, β-catenin signaling activates a wide variety of target genes that putatively could be involved in HCC tumorigenesis and malignant behaviour The EGFR ligand AR is increasingly recognized as a potent oncogenic factor over-expressed in a variety of human cancers including HCC AR by β-catenin signaling.Functional cross-talk between Wnt-triggered signaling and the EGFR system has been previously identified in other cancer cells of epithelial origin such as breast cancer cells CTNNB1) or in components of the β-catenin degradation complex (AXIN1), as well as dysregulation in the expression of Wnt/Fizzled signaling elements AR expression in epithelial cells AR could be a target of FGF19/FGFR4 signaling. Here we first demonstrate that FGF19 is a novel inducer of AR gene expression in human HCC cells. Moreover, the inhibitory effect of dominant negative ΔNTcf4 on FGF19 induced AR expression, as well as the attenuated response to FGF19 of AR promoter-reporter constructs with mutated TBE sites, strongly suggest that β-catenin signaling is involved in this response. FGF19 has been recently identified as a driver gene in human hepatocarcinogenesis AR and FGF19 gene expression showed a positive correlation in human HCC tissues suggests that to a certain extent AR gene expression could be modulated by FGF19 in liver tumors, and supports the clinical relevance for our experimental observations. Moreover, the increased expression of these two genes in a subclass of tumors corresponding to patients with poorer prognosis As previously mentioned several mechanisms account for β-catenin pathway activation in HCC cells, and these include mutations in β-catenin gene  or the GSK3β inhibitor SB-415286  (24 h) and AR mRNA levels were determined. *P<0.05 vs control (C), §P<0.05 vs cells treated with LiCl 20 mM. AU: arbitrary units.(TIF)Click here for additional data file.Figure S2Knockdown of AR gene expression reduces the proliferation of Huh7 cells expressing mutant β-catenin. T41A β-catenin expressing cells and control pcDNA3.1 transfected cells were transfected with AR specific siRNA (siAR) or control siRNA (siGL). Cells were counted at the indicated time-points 24 h after transfections. *P<0.05 vs siAR transfected cells. At 72 h knockdown of AR expression in T41A β-catenin cells reduced cell proliferation by 45%, while in control pcDNA3.1 transfected cells proliferation was reduced by 26%.(TIF)Click here for additional data file.Figure S3Comparative analysis of AR, FGF19, FGFR4 and β-klotho (KLB) mRNA levels in Huh-7, Hep3B and HepG2 cells. Gene expression was analyzed by qPCR. AU: arbitrary units.(TIF)Click here for additional data file.Table S1Primers used for amplification of human AR gene 5′ region genomic DNA.(DOC)Click here for additional data file.Table S2Primers used in this study for qPCR analysis of gene expression.(DOC)Click here for additional data file.Table S3Primers used for qPCR analysis of the AR gene promoter region encompassing the three studied TBE sites.(DOC)Click here for additional data file.


One common factor to these conditions is an age-related decline in vascular beta-adrenergic receptor-mediated function and subsequent cAMP generation. Presently, there is no single cellular factor that can explain this age-related decline, and thus, the primary cause of this homeostatic imbalance is yet to be identified. However, the etiology is clearly associated with an age-related change in the ability of beta-adrenergic receptor to respond to agonist at the cellular level in the vasculature. This paper will review what is presently understood regarding the molecular and biochemical basis of age-impaired beta-adrenergic receptor-mediated signaling. A fundamental understanding of why  Life expectancy has increased during the past century, and this has led to a dramatic increase in the aging population. The number of Americans over 65 is expected to double from the years 2000 to 2030: in 2000, there were 34.8 million over the age of 65 (12% of the population), and in 2030, it is predicted that there will be 70.3 million in this age group (representing 20% of the population) [β-AR) subtypes, β1, β2, and β3, are found in vascular smooth muscle cells, the β2-AR subtype is by far the most highly expressed [β2-AR exhibits an age-related decline in signaling with advancing age that leads to impaired vasorelaxation. In contrast, the intrinsic ability for vascular muscle contraction is generally maintained throughout the aging process [β-AR-stimulated cAMP production. Therefore, conditions associated with altered cAMP production are likely affected. To this end, the β-AR is a target for many medications prescribed to the elderly [Vascular tone is regulated by both the intimal  and medial (vascular smooth muscle) layers as well as through interlayer interactions. Age-related changes in the structure and function of each layer is well documented , 5. Smooxpressed . Of spec process . This ch elderly  and are  elderly .β-AR signal cascade has been identified to fully explain the impaired β-AR vascular function observed with aging. Instead, the cause of the change is likely multifactorial. This notion is supported due to intricate nature of β-AR signaling and two biochemical findings: first is that expression of β2-AR does not change with age [β-ARs do not elicit complete vasorelaxation with advancing age, drugs that act on proteins postreceptor in the signaling cascade do [Beta-AR-mediated signal transduction pathways are well described, but new discoveries continue to impart complexity  function through their cognate receptors localized at vascular smooth muscle, and/or endothelial cells and influence an elaborate network of signal transduction pathways that yields homeostatic control . The molβ-AR signal transduction cascade mediates smooth muscle vasorelaxation. Activation of the β-AR stimulates the dissociation of the G protein, Gαs, from the βγ subunit. The G protein βγ subunit can also affect various membrane and/or organelle channels whose action can rapidly alter the ionic milieu of the cell. After uncoupling from the β-AR, Gαs becomes activated by exchanging GDP for GTP. The activated form of Gαs triggers adenylyl cyclase to convert ATP into cAMP [β-AR activation also initiates G protein receptor kinase (GRK) function. GRKs are a family of kinases that phosphorylate, β-ARs [β-ARs are targets for still another group of proteins, the β-arrestins that desensitize β-ARs and mediate internalization, which leads to receptor recycling and/or degradation. In addition, β-arrestins can serve as scaffolds and adaptors for other kinases such as extracellular signal-regulated kinase (ERK), Src, and Raf that regulate several cellular pathways that result in the activation of MAP kinases [In blood vessels, the nto cAMP . Two molnto cAMP . β-AR ace, β-ARs . Phospho kinases .β-AR function has proven elusive due to the complexity of this cascade. Three subtypes of the β-AR  are found in vascular smooth muscle cells [αs-modifying proteins are also known to alter β-AR signaling [β-AR function with age, recent research has focused on proteins that interact with the β-AR, directly or indirectly, that may be critical for optimal receptor signaling. Investigations on age-related changes in various modifications to the β-AR, such as phosphorylation-mediated desensitization, scaffolding proteins that form postreceptor signalosomes, or proteins that directly interact and affect β-AR function may provide insight to explain the change.Identifying a possible locus for the age-related decline in le cells . Also, ale cells , 22, alole cells , three ale cells , and mulle cells  isoformsignaling . Therefoβ-AR itself, rather than a “downstream” change specific to adenylyl cyclase [β-AR function with age. As described above, the β-AR signaling cascade is multifaceted, including numerous protein factors, each of which exists with multiple subtypes. The following is a discussion of what is currently known regarding the mechanism of the age-related change in β-AR function, organized in a sequential manner, starting upstream with the β-AR itself and continuing through the cascade as we currently understand it.The initial observation of an aging effect on vasorelaxation was made in the early 1970s involving analysis of vascular smooth muscle pharmacodynamics in general. Blood vessels from 6-month-old animals relaxed 90% less to isoproterenol as compared to blood vessels from 1-month-old animals , 28. To  cyclase . From thβ-AR have been identified in mammals,  to block the coupling of activated β2-AR to Gαi in aortae isolated from Fischer 344 rats of increasing age, it was found that this treatment did not alter the age-related decline in relaxation. However, a population of β2-AR coupled to Gαi was found, as pertussis toxin treatment improved β2-AR-mediated vasorelaxation in aortae for all ages in equal proportion. Changes in vascular β3-AR function with aging are unknown. However, left ventricular function and age-related heart failure were highly correlated with β3-AR expression in rats [β3-AR function throughout the vasculature.We have evaluated whether an agonist-mediated change in laxation . Using pβ2-AR is the most highly expressed subtype in the vasculature, much interest is the finding that β2-AR sensitivity substantially declines with age [β-ARs were in the high affinity state. This compared to 40%, and 0% high affinity β-ARs for 6- and 24-month-old animals, respectively. To explain these results, age-related changes in the content of β2-AR bound to Gαs was examined: β2 AR : Gαs complexes were found only in aortic preparations from 1-month-old animals. These data strongly suggest that there is a substantial decline in high-affinity β2-AR with advancing age. This change did not appear to be related to a decline in the presence of β-ARs at the membrane (further confirmed [Because the with age . Resultsonfirmed ) or causonfirmed .β2-AR signaling is the possibility that β-AR can form hetero- and homodimers, and this could alter signaling fidelity. Mercier et al. [β-ARs likely exist as either β1-AR :  β1-AR homodimers, or β1-AR : β2-AR heterodimers, and they suggested that changes in the overall cellular configuration of monomers, heterodimers, and homodimers could be altered by agonist as well as disease state, and this finding was supported by Lavoie et al. [β1-AR : β2-AR heterodimers have been shown to exhibit distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation and optimizing β-adrenergic modulation of contractility in cardiomyocytes [β-AR dimerization occurs in cardiovascular tissue. A report by Ianoul et al. [β1-AR and β2-AR may be localized in two different populations of microdomains in cardiomyocytes, an observation inconsistent with the existence dimers. Of additional interest is a recent report by LaRocca et al. [β2-AR form heterodimers with the chemokine receptor type 4 (CXCR4) and this interaction negatively regulated isoproterenol-mediated β2-AR signaling by altering β2-AR sensitivity. Following from these studies, we evaluated whether CXCR4 activity modulation could alter β-AR-meditated vasorelaxation. Using aortae isolated from 2-month-old Fischer 344 rats, it appeared that CXCR4 activation inhibited, and CXCR4 blockade improved the vasorelaxant effect of isoproterenol  into aorta from 6-month-old Fisher 344 rats. In that study [αs-Q227L exhibited enhanced isoproterenol-stimulated vasorelaxation, and both basal- and isoproterenol-stimulated cAMP production was increased. Therefore, Gαs may undergo some age-related change that inhibits its ability to become activated via the action of an agonist-occupied receptor. In support of a functional change in Gαs with age is the identification of a regulator of G protein signaling (RGS)/GTPase activating protein (GAP) that functions on Gαs [β-AR signaling without changes in β-AR or Gαs expression as agonist exposure would not initiate vasorelaxation as Gαs signaling would be quenched due to the high RGS/GAP activity of RGS-PX1. At present, RGS-PX1 has not been evaluated within an aging paradigm. In total, the decline in β-AR-mediated vasorelaxation and cAMP accumulation observed in old vessels could be caused by a change in the function but not expression of Gαs.The age-related change in nchanged . Howeverold rats . Our labpression , suggestpression . Furtherat study , aorta ts on Gαs  called Rβ-AR-mediated signaling could be an increase in Gαi function, as this G protein subunit inhibits adenylyl cyclase activity and thus cAMP production. Also, the β2-AR, the predominant receptor species in vascular smooth muscle, has been shown in cardiomyocytes to rapidly link to Gαi after agonist activation, and it is PKA-phosphorylated [αi with age [αi1&2 protein content between 6- and 24-month-old aortic preparations has been documented [βγ could also affect β-AR-mediated signaling. Gβγ has been shown to either stimulate or inhibit adenylyl cyclase activity in the presence of activated Gαs [βγ also affects numerous plasma and organelle membrane-localized ion channels, thereby affecting the net polarity and potential for tonal changes of vascular smooth muscle [βγ subunit [Another possible explanation for the decline in orylated . We actuwith age . Also, acumented . Age-related Gαs . Gβγ alsh muscle . However subunit .β-AR stimulation. This decline is directly correlated to an inability to synthesize appreciable concentrations of cAMP. However, old vessels do maintain the ability to relax entirely, as acetylcholine-, forskolin-, and nitrate-mediated vasorelaxation is complete [αs (as discussed previous) or G protein receptor kinase (see following). The classical effector of Gαs is adenylyl cyclase. Forskolin directly activates adenylyl cyclase, and thus stimulates cAMP production. Because forskolin stimulates blood vessels from young and old animals to relax completely, and to accumulate cAMP equally, it is generally thought that adenylyl cyclase function does not change with advancing age. Our results further support that adenylyl cyclase activity is maintained across aging [As discussed, the fundamental change in blood vessels from older animals is a pronounced inability to relax to complete . Therefoss aging . Howeverss aging . Perhapsss aging . TherefoAnother line of reasoning would be that adenylyl cyclase activity is unchanged across age, but cAMP processing is altered. Therefore, changes in physiology could be due to age-related changes in the processing and degradation of cAMP through phosphodiesterases. We have determined that there are no age-related changes in general phosphodiesterase inhibitor-mediated vasorelaxation using 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase subtype inhibitory agent . Howeverβ-AR desensitization is initiated by phosphorylation of the receptor, which is followed by its uncoupling from its signaling cascade. The kinases PKA, GRK, and others phosphorylate β-ARs [β-ARs are targets for another family of proteins that mediate uncoupling/desensitization, the arrestins [β-AR kinases: β-ARK-1 and β-ARK-2, resp.) target β-ARs and are highly expressed in the cardiovascular system [te β-ARs . Phosphorrestins . Thereforrestins , 67. To r system .β-AR-mediated cAMP production and vasorelaxation. Also, overexpression caused elevations in resting blood pressure and was accompanied by an increase in vascular thickening, suggesting a decline in cAMP generation.Evidence shows that GRKs are important regulators of pathology in humans . Signifiβ-AR mediated vasorelaxation. Only a few studies have been performed to assess age-related changes in GRKs. No changes in GRK activity or GRK-2 and 5 expression were observed in lymphocytes of aged humans [β-arrestin expression increased approximately 1.6-fold. In the membrane fraction, GRK-2 expression increased approximately 1.5-fold, GRK-3 expression increased nearly 2.1-fold, while there was not an age-related change in the expression of GRK-5. These data suggest that a critical feature of age-related impaired β-AR signaling may be imparted through an increase in total pool of GRK that could be explained by either increased expression, or decreased degradation [β-AR. Whether β-ARs from aged vessels have increased phosphorylated residues, and thus enhanced desensitization is yet to be established. Also, the mechanism for the enhanced expression of GRKs with advancing age is likewise yet to be explained but is an active interest of our lab.An age-related change in GRK activity or expression in vascular tissue would implicate GRKs in the age-related decline in d humans . Howeverd humans . We haved humans . In aortradation . This inβ-AR signaling depends on the interaction between numerous proteins. Therefore, β-AR-mediated function requires appropriate localization (cytoplasmic versus membrane) and organization  of these proteins. Therefore, recent research has focused on “scaffolding proteins,” which are intracellular proteins that compartmentalize multiple related signaling molecules to specific intracellular domains. This theory has replaced the classical “random collision-coupling theory” of signal transduction [β-AR signaling cascade is anchored within the plasma membrane by the scaffolding protein caveolin [β2-ARs [αs) [sduction . The β-Acaveolin . β2-ARs[β2-ARs , 78, numARs [αs) , 80, adeARs [αs) , and GRKARs [αs)  all locaβ-AR signaling pocket, and thereby alter signaling. Indeed, Carman et al. [Age-related changes in caveolin have recently been demonstrated revealing tissue-specific changes in expression . Also, on et al.  found thn et al. . Therefoα-AR agonist). Also, acetylcholine-mediated (nitric oxide-dependent) vasorelaxation was similarly altered in null animals. Finally, caveolin-1 null mice displayed hyperproliferation in certain cell types, suggesting (but not documented) a decline in cAMP production. Changes in β-AR-, cAMP-, or age-mediated effects in the caveolin-1-null mice have yet to be evaluated. However, these data from transgenic animals clearly indicate that caveolin is an important modulator of vascular function.Other reports implicate caveolin as a regulator of vascular function . Razani αq-linked receptors and β-ARs have been observed. Activation of PKC by Gαq-linked-agonists directed GRKs to the membrane, enhancing β-AR phosphorylation and desensitization [β-ARs after its activation by PKC [β-AR agonist-induced desensitization rather than the expected attenuation result. In addition, these authors subsequently found that this effect was linked to phosphatase activity [β-AR resensitization through interaction with a phosphatase. Therefore, phosphorylation/dephosphorylation and desensitization/resensitization of β-ARs can be induced from a number of stimuli including angiotensin II.A potentially interesting phenomenon observed in molecular signaling is receptor cross-talk events. Cross-talk between Gtization . Also, in by PKC . Finallyactivity . These fβ-AR being altered by receptor cross-talk are in vitro studies showing that angiotensin II enhanced β-AR-mediated cAMP production in cultured aortic vascular smooth muscle cells [1) [β-AR-mediated vasorelaxation, and angiotensin II [β-AR-mediated signaling are unknown but may involve adenylyl cyclase, Gαs, or calcineurin. Further study may show that aging may effect a factor common to both angiotensin II and β-AR signaling pathways or that aging may impair cross-talk between these two receptor pathways. In terms of vascular le cells , 92, 93 le cells , 95. In ells [1) . We examensin II . Our resβ-AR-mediated signaling is understanding the role of various ion channels; it is well understood that the function of numerous ion channels is responsible for determining membrane potential [ATP). They also determined that the isoproterenol/KATP-mediated hyperpolarization was impaired in smooth muscle cells from older rats. However, the effect of direct activation of KATP was unchanged between young and old groups. Therefore, their data fit well with what has been previously known about the age-related changes in β-AR signaling—the alteration appears to be localized proximal to adenylyl cyclase and may involve changes in the β-AR itself or in its ability to couple to other regulatory molecules.A final interesting aspect of age-related changes in otential . The effotential . Resultsβ-AR function. It is clear that age-related changes in the endothelium occur [β-AR [β-AR ligands [β-AR agonists [β-AR-mediated vasorelaxation appears to be induced via both nitric oxide-mediated pathways , and cAMP-mediated pathways (vascular smooth muscle) [A controversial topic is the endothelium-mediated effect on the age-related change in um occur , and theur [β-AR . It is aur [β-AR . Data sh ligands  and thatagonists . Therefo muscle) .β-AR vasorelaxation is initiated in both endothelial and vascular smooth muscle cells. However, [ATP-mediated hyperpolarization was found to be impaired in vascular smooth muscle cells from older rats [β-AR-mediated vasorelaxation. When comparing the effect of age on isoproterenol-mediated vasorelaxation on phenylephrine- versus KCl-contracted aorta, vessels contracted with phenylephrine relaxed to a substantially higher degree that those contracted with KCl, although the age-effect was maintained [β-AR signaling is vascular smooth muscle-, rather than endothelium-dependent is that other agents that initiate vasorelaxation through vascular smooth muscle localized G protein-coupled receptors  also show impaired vasorelaxation with age [β-AR-stimulated vasorelaxation in general.In terms of aging, However,  found thder rats . Our labintained . One intwith age , 106. Thβ-AR signaling. Impaired β-AR-mediated vasorelaxation with age is observed throughout species and arterial beds, and in aged vascular tissue, β-ARs are desensitized. One cellular process that changes with age that modulates β-AR sensitivity is phosphorylation by GRK-2. But, there are multiple other protein factors that may modulate β-AR function with aging. Similarly, there are multiple factors that may modulate GRK function [β-AR signaling is altered (such as in the kidney and liver [Hypertension, orthostatic hypotension, arterial insufficiency, atherosclerosis, and restenosis are common disorders in the elderly that lead to significant morbidity and mortality. These clinically significant conditions all may have a common feature in that they are associated with and age-related change in function . Therefond liver , heart [nd liver , lung [1nd liver , and brand liver .


Mangiferin has been shown to have the effect of improving dyslipidemia. Plasma free fatty acids (FFA) are closely associated with blood lipid metabolism as well as many diseases including metabolic syndrome. This study is to investigate whether mangiferin has effects on FFA metabolism in hyperlipidemic rats. Wistar rats were fed a high-fat diet and administered mangiferin simultaneously for 6 weeks. Mangiferin  decreased dose-dependently FFA and triglycerides (TG) levels in plasma, and their accumulations in liver, but increased the β-hydroxybutyrate levels in both plasma and liver of hyperlipidemic rats. HepG2 cells were treated with oleic acid  to simulate the condition of high level of plasma FFA in vitro, and were treated with different concentrations of mangiferin simultaneously for 24 h. We found that mangiferin significantly increased FFA uptake, significantly decreased intracellular FFA and TG accumulations in HepG2 cells. Mangiferin significantly increased AMP-activated protein kinase (AMPK) phosphorylation and its downstream proteins involved in fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT1), but significantly decreased acyl-CoA: diacylgycerol acyltransferase 2 (DGAT2) expression and acetyl-CoA carboxylase (ACC) activity by increasing its phosphorylation level in both in vivo and in vitro studies. Furthermore, these effects were reversed by Compound C, an AMPK inhibitor in HepG2 cells. For upstream of AMPK, mangiferin increased AMP/ATP ratio, but had no effect on LKB1 phosphorylation. In conclusion, mangiferin decreased plasma FFA levels through promoting FFA uptake and oxidation, inhibiting FFA and TG accumulations by regulating the key enzymes expression in liver through AMPK pathway. Therefore, mangiferin is a possible beneficial natural compound for metabolic syndrome by improving FFA metabolism. A large body of evidence suggests that elevated plasma free fatty acids (FFA) are a risk factor for metabolic syndrome, including insulin resistance, atherogenic dyslipidemia and type 2 diabetes The liver is an important site of FFA removal from the blood Anemarrhena asphodeloides rhizomes. Mangiferin has many beneficial biological activities, including anti-inflammatory, anti-oxidant and anti-diabetic effects in vivo and in vitro models, we found mangiferin was able to decrease the levels of plasma FFA, promote FFA catabolism, and inhibit the synthesis of FFA and TG in liver through AMPK signaling pathway.Mangiferin is a xanthone glucoside and exists in many kinds of food and folk medicines such as mangoes and Rats were fed with different concentrations of mangiferin  for 30 days. There were no significant effects on body weight, blood cell counting, hemoglobin, urea nitrogen, creatinine, blood glucose, total cholesterol, triglycerides, aspartate transaminase and alanine transaminase activity . This inP<0.05), TG (P<0.01) and FFA (P<0.01) in both plasma and liver tissue compared with rats in normal control (P<0.05), and fenofibrate and mangiferin at 100 or 150 mg/kg BW significantly decreased plasma TG level (P<0.01), and liver FFA (P<0.01) and TG (P<0.01) levels in hyperlipidemic rats. In addition, fenofibrate and high dose of mangiferin may decrease the weights of abdominal and epididymal fat pads in hyperlipidemic rats  in order to simulate the condition of high level of plasma FFA in animal experiment. Mangiferin significantly decreased the OA concentration of cell culture medium in a dose-dependent manner, and fenofibrate (positive control) at 100 µmol/L also significantly decreased the OA concentrations P<0.01, . OA uptain vitro, we determined the protein levels of AMPK, CD36, CPT1, ACC and DGAT2 in HepG2 cells by mangiferin supplementation. Mangiferin significantly increased the phosphorylation levels of AMPK and ACC  to simulate the condition of high plasma levels of FFA in animal experiment. As a result, 0.2 mmol/L OA increased significantly the intracellular TG content in liver cells, a condition that is observed often when plasma FFA is elevated in human and animal studies. Fenofibrate, a hypotriglyceride drug as positive control agent, can reduce plasma FFA and hepatic TG in vivo and in vitro. Our study showed for the first time that mangiferin decreased significantly plasma FFA levels in hyperlipidemic rats and FFA levels in culture medium of HepG2 cells. It is well known that plasma FFA is mainly transported into the liver at rest. CD36 is the rate-limiting enzyme in high-affinity peripheral FFA uptake in the liver We have confirmed that the doses of mangiferin used in this study are safe based on the toxicity experiment of 30-day feeding test and MTT assay both AMPK, an upstream regulator of CD36, CPT1, ACC and DGAT2, is a critical enzyme involved in FFA metabolism In conclusion, we investigated systematically the effect of mangiferin on FFA levels and FFA metabolism, and its possible mechanism for the first time. Mangiferin decreases the levels of FFA in both plasma and liver tissue in hyperlipidemic rats, promotes FFA catabolism by regulating the key enzymes of FFA uptake and utilization and inhibiting intracellular TG synthesis in liver. The effect of mangiferin on decreasing plasma FFA levels and increasing FFA catabolism is possibly exerted through the AMPK pathway by enhancing the ratio of AMP to ATP. Therefore, mangiferin results in beneficial effects on FFA metabolism, which may improve metabolic syndrome.All rats were purchased from Shanghai SLAC Laboratory. Animals were housed in cages individually in an environmentally controlled room at 21±2.0°C and 50±5% humidity. Animals were subject to a 12∶12 h light∶dark cycle and had access to food and water ad libitum. After an acclimatization period of one week, the animals were used in the following experiments.Forty male rats and forty female rats (100±20 g) were divided into 4 groups respectively, 10 rats in each group, treated with different concentrations of mangiferin  by oral gavage for 30 days. The blood chemistry results, hematologic measures, and liver enzyme values were determined by using SYSMEX-SF-3000 Automatic Hematology Analyzer .Sixty male rats (220±20 g) were divided randomly into six groups (n = 10 in each group): control group, fed an AIN-93G diet; hyperlipidemia group, fed a high-fat diet ; positive drug group, fed a high-fat diet and fenofibrate at dose of 100 mg/kg BW/d; mangiferin-supplemented groups, fed the high-fat diet and different doses of mangiferin . Mangiferin  and fenofibrate was given by oral gavage in 1% carboxymethyl cellulose buffer solution. Rats in the control and hyperlipidemia groups were given 1% carboxymethyl cellulose buffer only. After 6 weeks of mangiferin supplementation, all the animals were fasted for 12 h until sacrifice. Rats were anesthetized by pentobarbital sodium and blood samples were taken from abdominal aorta into EDTA-tubes. Abdominal and epididymal fat pads were removed and weighted separately. Liver was collected and stored at −80°C for analysis. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Harbin Medical University, and conducted in compliance with the animal-use guidelines (SYXK (Hei) 2006-010).Dulbecco's Modified Eagle Medium (DMEM) was purchased from GIBCO . Fetal bovine serum (FBS) was purchased from Sijiqing Co . Mangiferin for cell experiment, oleic acid (OA), fenofibrate and compound C were obtained from Sigma-Aldrich . Mangiferin  for animal experiment were obtained from Zhongxin Innova Laboratories . Mangiferin, fenofibrate and compound C were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium. Antibodies against AMP-activated protein kinase α (AMPKα), phosphor-AMPKα (p-AMPKα), acetyl CoA carboxylase (ACC) and phosphor-ACC (p-ACC) were purchased from Cell Signaling . Antibody against fatty acid translocase (CD36) was purchased from Cayman Chemical . Antibodies against LKB1, phosphor-LKB1, carnitine palmitoyltransferase 1 (CPT-1), acyl-CoA: diacylglycerol acyltransferase-2 (DGAT2), GAPDH and β-actin were purchased from Santa Cruz Biotechnology .2. The cytotoxicity of mangiferin was determined by the 3--2, 5-diphenyltetrazolium bromide (MTT) assay. When 70–80% confluence was reached, the cells were treated with different concentrations of mangiferin  or 100 µmol/L fenofibrate (positive control) in serum-free medium with 1% FFA-free bovine serum albumin (BSA) in the presence of OA (0.2 mmol/L) for 24 h. In parallel, HepG2 cells treated with an equivalent volume of DMSO (0.1%) without OA were used as the normal control. HepG2 cells were treated with OA (0.2 mmol/L) in order to simulate the condition of high level of plasma FFA in liver cells. Compound C (25 µmol/L) was added to HepG2 cells 1 h before treatment with mangiferin to verify the AMPK signal pathway.The HepG2 cell line was obtained from the Chinese Academy of Science . The cells were maintained in DMEM containing 10% (v/v) FBS and 1% antibiotic/antimycotic at 37°C in an atmosphere containing 95% air and 5% COA portion of liver was homogenized and the lipids were extracted with Chloroform and methanol (1∶2 v/v) 2 times as described by Bligh et al HepG2 cells were incubated with 0.2 mmol/L OA alone, or with different concentrations of mangiferin  or 100 µmol/L fenofibrate simultaneously in serum-free medium with 1% FFA-free BSA for 24 h. The cellular lipid was extracted using a previously described method The concentration of OA remaining in the medium after treatment was determined to assess the uptake of OA by HepG2 cells. OA was measured by GC-MS . Aliquots (1 mL) of cell culture medium, or 200 µL of cell lysates in each group were spiked with an internal standard (IS) working solution , and the following method of sample pretreatment and GC-MS conditions was used, as described previously by our laboratory β-actin. For each study, western blot analysis was conducted three times and representative blots were shown.The proteins CD36, CPT-1, ACC, DGAT2, AMPK and LKB1 were determined by western blot analysis. Proteins from the cultured cells or liver tissues were extracted with a RIPA lysis buffer . Protein concentrations were determined by the Bradford method. Equal amounts of protein were separated by SDS-PAGE, and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 1% BSA and TBS-T  for 0.5 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies in TBS-T. The membranes were washed three times with TBS-T (6 minutes each), and incubated with appropriate secondary antibodies for 1 h at room temperature. The signal was amplified by color development using the ProtoBlot II AP System with a stabilized substrate . Data were presented as the ratios of target protein to After the treatment as described above, cells were lysed in 300 µ L media with 20 µ L of 1 mol/L HClO4. HClO4 was removed by mixed phase extraction employing tri-n-octylamine and Freon 11  P<0.05 was considered to be statistically significant.All data were analyzed for statistical significance with SPSS 13.0 software. Data were presented as means ± standard deviation. Statistical analysis used one-way ANOVA. Table S1Effects of mangiferin on blood chemistry results, hematologic measures and liver enzyme values in 30-day feeding test.(DOC)Click here for additional data file.Table S2List of all actual numbers to generate the relative results of .(DOC)Click here for additional data file.


Medical events, such as stroke, limb fractures, joint replacements and spinal injuries, can lead to acute functional disability at all ages and to chronic disability, especially among the elderly. Rehabilitation is, therefore, essential for the prevention of permanent disability among older individuals. There are international practice guidelines for stroke and hip fracture management, including recommendations that rehabilitation services be an integral part of the provision of treatment in either an inpatient setting or in the community. There are no organized data on provision of rehabilitation services in Israel or on the distribution of these services throughout the country. Such information would be of great assistance in designing these services where they are needed and in making changes in the existing ones where necessary.Patients aged 65 years or older with stroke or hip fracture were identified through one-day surveys conducted in 2009–2010 in all 26 acute care hospitals in Israel. Data on inpatient and ambulatory rehabilitation services were collected from discharge medical summaries, telephone interviews with the patients or their relatives and reports from the healthcare provider. The extent of rehabilitation services was described and the association between receipt of inpatient rehabilitation and the geographic district based on the patients’ listed address was examined in a multivariate analysis.A total of 570 patients with stroke and 768 patients with hip fracture were identified and interviews were conducted in regards to 421 and 672 respectively. Out of the stroke patients 238(56.5%) received inpatient rehabilitation, 46(10.9%) received ambulatory rehabilitation treatment without inpatient phase and 137 (32.5%) received no rehabilitation. In fracture these rates were 494(73.5%), 96(14.3%) and 82(12.2%) respectively. Patients living in districts with lower availability of rehabilitation beds were less likely to receive inpatient rehabilitation after controlling for patient characteristics.Regional disparities in the provision of inpatient rehabilitation care for elderly after an acute episode of stroke or hip fracture were identified and could be partially attributed to the distribution of rehabilitation beds. These findings highlight the need to plan the rehabilitation resources based on the population needs and to routinely monitor the provision of these services. Rehabilitation is essential following medical events that lead to an acute functional disability, such as stroke, limb fractures, joint replacements and spinal injuries . The incIsrael has a limited number of rehabilitation day-centers that offer multidisciplinary treatment in an outpatient setting. Therefore, post-acute care usually starts in a specialized rehabilitation inpatient facility. Currently, there are 18 licensed wards for geriatric rehabilitation and ten for general rehabilitation, with a total of about 1500 beds for general and geriatric rehabilitation. This translates into about 2 beds per 1000 residents 65 year or older. In 2009, the Ministry of Health in collaboration with the Ministry for Senior Citizens launched a multidisciplinary initiative to improve and promote the medical rehabilitation services delivered to the elderly in Israel. The initiative encompassed the dissemination of practice guidelines to the acute care wards regarding the referral process for rehabilitation treatment [The study population included patients aged 65 years or older who were hospitalized for acute stroke or hip fracture. The patients were identified during a series of 6 single-day surveys conducted in the neurology, internal medicine, orthopedic and geriatric wards in all of the 26 general hospitals in Israel. Data regarding rehabilitation was collected only for those who were discharged from the acute care after exclusion of in-hospital mortality.Data on each study patient were collected from three sources. The first was the medical discharge letter which was used to extract data on demographic variables, the patient’s functional status prior to the hospitalization, co-morbidities, course of the acute event, in-hospital complications, and recommendations for rehabilitation treatment. Three variables were used to describe the patient’s functional status prior to the event: indoor mobility was dichotomized to mobile (independent or with assistance) versus wheel-chair dependent, general function was dichotomized to fully dependent versus independent or need some assistance and cognitive function was dichotomized to with or without dementia. The severity of stroke was assessed according to the NIH stroke scale (NIHSS) which has been noted to be a god predictor of patient outcomes ,17. NIHSThe second source was a telephone interview with the patient, or with a close family member, which was conducted 4–6 months after the patient was discharged in order to collect information on any inpatient or ambulatory rehabilitation care that had been received. The contact details of a family member were taken from the hospital medical record. A caretaker was contacted only if the patient could not be interviewed. The interview started by asking the family member if he was familiar with the patients’ care, and if not he was asked to provide contact details for an appropriate caretaker. The third source was the administrative data on any inpatient rehabilitation that the patient received within the first four months after hospitalization for the acute event as recoded in the patient’s health maintenance organization (HMO) file.The study was conducted as part of the routine monitoring activities of the Ministry of Health and therefore did not require formal ethical approval.Statistical analyses were performed using SPSS software for Windows version 19. Each patient was assigned to a group according to the initial rehabilitation services provided . Comparisons between the characteristics of patients were done by the chi- square test and t-test as appropriate. We conducted a multivariate logistic regression to explore the association between receipt of inpatient rehabilitation and the patient’s district of residence. Patients who were not mobile at baseline were excluded from the fracture analysis (9 patients).The models included in addition to age the factors that were found to be linked to inpatient rehabilitation in a univariate analysis (p < 0.05): presence of dementia, dependency status and stroke severity.For the purpose of sensitivity analysis we ran the regression models twice: once with dependent variable defined by the self reported data and once defined by the health provider's data.p <0.001).Six one-day surveys were conducted between March, 2009 and October, 2010. 570 patients with stroke and 768 with hip fracture were identified, out of them 81 and 19 died in hospital and 489 and 749 were discharged and included in this study. The mean age of all the stroke patients was 78.8 years (range 65–100) and the mean age of all the hip fracture patients was 82.6 years (range 65–107). In-hospital mortality was much higher among stroke patients compared to fracture patients  discharged stroke cases and 672/749 (89.9%) discharged fracture cases with the patients themselves (16.1%) or with their caretakers 83.9%). The distribution of rehabilitation setting by diagnosis is given in Table .9%. The Ambulatory rehabilitation treatments were given to 177/421 (42%) of the stroke patients and 432/672 (64%) of the fracture patients. Ambulatory treatments were given either as a sole treatment or mostly, following an episode of in-hospital rehabilitation both in stroke  and in fracture . However, a total of 107 (25%) stroke patients and 158 (24%) fracture patients did not receive any rehabilitation treatments in the community after discharge from inpatient rehabilitation.In a multivariate analysis stroke patients residing in Jerusalem were two times less likely to receive inpatient rehabilitation compared to patients in the Centre District  . These rates are similar to the 52.2% inpatient rehabilitation rate reported from a stroke registry in Germany  but highIn the present study, almost one-third of the stroke patients and 12.2% of the fracture patients received no rehabilitation treatment in any setting. As might be expected, these patients were, on the average, older and more likely to be dependent and bed-bound before the event. Yet, the majority of them lived in the community prior to the event. In addition, 50% of the stroke patients and 30% of the fracture patients that did not get any rehabilitation were discharged to their homes and not to a geriatric nursing facility, suggesting that this group might include individuals that could have benefited from a trial of rehabilitation.Selecting people for rehabilitation poses a difficult dilemma in trying to balance between the objective to provide proper treatment for all the patients that may benefit from it, and the need to avoid wasting scarce resources on candidates who are expected to fail ,26. AvaiThe geographic disparities in rehabilitation rates that were observed in this study lend support to the premise that non-patient-related factors play a role in the selection process. Geographic disparities in rehabilitation care were reported in the USA both between states and between regions in the same state . FreburgSupply could be one reason for geographic variation, since the availability of post-acute care services was found to strongly limit their appropriate use following an acute event of hip fracture, stroke or joint replacement . We deteThe availability of beds is a pre-condition for the provision of rehabilitation services, but it does not guarantee an equal access to those services. In practice, the provision of inpatient rehabilitation care is a multistage process that involves the interaction of many factors involved in the patient‘s care. The acute care ward needs to assess the need and potential for rehabilitation and refer the patient for further treatment, the healthcare provider must agree to pay for the additional treatments, the rehabilitation unit needs to accept the patient and, finally, the patient and his/her family need to agree to the suggested rehabilitation program. In order to streamline the referral process and ensure the patient’s right to receive proper rehabilitation care, the Ministry of Health Geriatric Department issued a circular with practice guidelines for rehabilitation therapy for the elderly in 2009 . These gIn our data we did not detect differences in the rates of inpatient rehabilitation between types of wards but the frequency of including treatment recommendations regarding rehabilitation in the discharge letter differed: The issue of rehabilitation was not mentioned in the discharge letter of 35.6% of the fracture patients discharged from the orthopedic ward compared to only 12.5% of those discharged from the geriatric ward (p < 0.001).The present study is the first national survey that assessed the extent of rehabilitation services provided to the elderly in Israel. By including all 26 acute care hospitals in Israel, it provides a representative national overview. The usage of different resources increased the validity and completeness of the collected data. We are aware that there was a great variability in the quality of discharge letters from different hospitals, resulting in some lack of information on the functional status of the patient before the event and at discharge. Decisions with regard to rehabilitation therapy were not always well documented in the discharge letters so we could not always distinguish between patients who were assessed clinically to be not fit for rehabilitation versus patients who did not receive rehabilitation for other reasons. This limitation was valid for all districts and therefore could not explain the regional gaps that were documented in our study. The use of proxy respondents of different relatedness to the patient could theoretically impair the validity of the data. But as the respondents were closely involved with the patient care we believe the validity was quite high as demonstrated by the good correlation with the health-provider’s data.On the national level, policy makers in the Ministry of Health should initiate appropriate planning of rehabilitation services based on the current and future needs of the growing population. Such planning should consider the growth and aging of the population in each district over the next 10 years, the incidence of medical conditions that require rehabilitation and the estimated mean number of inpatient rehabilitation days per event. If the planned reform to integrate nursing services with primary care services goes through, this may encourage the Health Maintenance Organizations to provide rehabilitation services. In addition, there should be ongoing supervision of the implementation of the guidelines set by the Ministry of Health.In conclusion, distinct regional differences were found in the provision of rehabilitation care after an acute episode of stroke or hip fracture, and these differences seemed to be related to the availability of rehabilitation beds. An ongoing follow-up survey will examine whether this trend continues and attempt to better characterize its causes.The authors declare that they have no competing interests.All the authors were actively involved in designing the study. IZ - Data analysis and manuscript writing. TS – Data analysis and revision of the manuscript. IL, IR, SH and AC - revision of the manuscript. All authors read and approved the final manuscript.