Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca2+]i, forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca2+]i, increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca2+. In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca2+ entry and ceramide formation.Shikonin, the most important component of  Lithospermum erythrorhizon, a traditional Chinese herbal medicine i), e.g., due to stimulation of Ca2+ entry [2+]i leads to the activation of Ca2+-sensitive K+ channels with subsequent cell shrinkage due to K+ exit, hyperpolarization, Cl− exit and thus to cellular loss of KCl and osmotically obliged water [2+]i is further followed by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface i, cell volume and phosphatidylserine exposure were determined in the absence and presence of Shikonin.The present study explored, whether and, if so, how Shikonin stimulates eryptosis. To this end, [Ca2+ activity ([Ca2+]i), the effect of Shikonin on [Ca2+]i was tested in a first series of experiments. To this end, human erythrocytes were loaded with Fluo3-AM and the Fluo 3 fluorescence determined by flow cytometry. Prior to the determination of Fluo 3 fluorescence, the erythrocytes were incubated for 48 h in Ringer solution without or with Shikonin (0.1–1 µM). As illustrated in 2+ concentration. The present study explored whether the naphthoquinone Shikonin triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine translocation. As both hallmarks of eryptosis are stimulated by the increase of cytosolic Ca2+]i may trigger cell shrinkage due to the activation of Ca2+-sensitive K+ channels and the subsequent exit of KCl and osmotically obliged water. Thus, cell volume was estimated from forward scatter in flow cytometry. As illustrated in An increase of [Ca2+]i may further trigger cell membrane phospholipid scrambling with phosphatidylserine translocation to the erythrocyte surface. Phosphatidylserine exposing erythrocytes were identified utilizing annexin V binding as determined in flow cytometry. As illustrated in An increased i, which activates Ca2+-sensitive K+ channels [+ exit, cell membrane hyperpolarization, Cl− exit and, thus, cellular loss of KCl with osmotically obliged water [The Shikonin-induced cell shrinkage is presumably due to the increase of [Cachannels , leadinged water . The celed water .2+ entry. Accordingly, the effect of Shikonin was significantly blunted in the nominal absence of Ca2+. Removal of Ca2+ decreased phosphatidylserine translocation even in the absence of Shikonin, indicating that spontaneous eryptosis following the exposure of erythrocytes to Ringer without Shikonin was similarly, in part, caused by cytosolic Ca2+. However, significant phosphatidylserine exposure was observed in the presence of Shikonin even in the nominal absence of Ca2+, indicating that Shikonin was effective by an additional mechanism. As a matter of fact, Shikonin triggered the formation of ceramide, which, in turn, fosters phosphatidylserine translocation [Stimulation of phosphatidylserine translocation following Shikonin treatment is similarly a result of Calocation . Ceramidlocation . Ceramidlocation . It is elocation . Shikonilocation .− channel, a Ca2+-regulated nonselective Ca2+ permeable cation channel, a scramblase mediating phosphatidylserine translocation upon the increase of cytosolic Ca2+ and a regulator of cell membrane blebbing and microparticle shedding [The molecular mechanism underlying phosphatidylserine translocation has not yet been identified . Recentlshedding . Howevershedding , which ishedding . Thus, t2+-permeable erythrocyte cation channels [+ and Ca2+, and they mediate the disposal of intracellular waste products [2+ entry through the Ca2+-permeable cation channels triggers eryptosis [2+ entry and Ca2+-induced eryptosis thus lead to elimination, not only of the infected erythrocyte, but also of the pathogen [Stimulation of eryptosis may be favorable, as it allows the elimination of defective erythrocytes prior to hemolysis . The phochannels ,78. Actichannels ,78, as tryptosis , which iryptosis . Ca2+ enpathogen .Plasmodia [Accordingly, genetic disorders predisposing to accelerated eryptosis, such as the sickle-cell trait, the beta-thalassemia trait, homozygous Hb-C and G6PD deficiency , lead tolasmodia ,80,81. Mlasmodia , and erylasmodia , chlorprlasmodia  or NO sylasmodia , have beExcessive stimulation of eryptosis may lead to anemia. Phosphatidylserine at the surface of eryptotic cells triggers the phagocytosis of the cells and, thus, leads to the rapid removal of the suicidal erythrocytes from circulating blood . Anemia Anemia and compromised microcirculation may, at least in theory, limit the use of Shikonin. The substance has been proposed to counteract oxidative stress , inflammin vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37°C for 48 h. Where indicated, erythrocytes were exposed to Shikonin  at the indicated concentrations. In Ca2+-free Ringer solution, 1-mM CaCl2 was substituted by 1-mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated 2 and then stained with annexin V FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and the annexin V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation under the respective experimental condition, a 50 µL cell suspension was washed in Ringer solution containing 5-mM CaCl2 and 5-µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5-mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL of Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5-mM CaClFor the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 hour at 37 °C with 1 µg/mL of anti-ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 minutes with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM-specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. 4 (5%). After centrifugation, an aliquot of the supernatant (400 µL) was adjusted to pH 7.7 by the addition of saturated KHCO3 solution. All manipulations were performed at 4 °C to avoid ATP degradation. After dilution of the supernatant, the ATP concentration of the aliquots was determined utilizing the luciferin-luciferase assay kit  and a luminometer  according to the manufacturer´s protocol.For the determination of the intracellular ATP concentration, erythrocytes were lysed in distilled water and proteins were precipitated by HClOt-test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to the triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as the post-test and the 2+ entry and triggers ceramide formation, which, in turn, leads to shrinkage and phosphatidylserine translocation of erythrocytes. Accordingly, Shikonin triggers eryptosis, the suicidal erythrocyte death.Shikonin stimulates Ca


Renal angiomyolipomas (AMLs) are frequent in tuberous sclerosis and are responsible for a significant proportion of the morbidity in adulthood, mainly from bleeding complications, which are correlated to the size of the AMLs. We describe the case of a 19-year-old female with multiple bilateral renal angiomyolipomas.The renal AMLs measured up to 6 cm in size. She was first treated with a low dose of the mammalian target of rapamycin (mTOR) inhibitor sirolimus (up to 3 mg/day over a 12-month period) and following significant AML size reduction, percutaneous cryoablation was performed. No side-effects of either treatment were reported. At 12 months post-cryoablation, no recurrence of the AML was noted.This is the first report of this treatment strategy and the case study reveals that combining a low dose of an mTOR inhibitor with percutaneous cryoablation to treat small tumors mitigates the side-effects while providing a good clinical outcome. This therapeutic approach is a novel tool for the clinician involved in the management of patients with tuberous sclerosis. TSC1 gene (hamartin) or TSC2 gene (tuberin) abnormality affecting one in 6000 births [Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease resulting from a TSC2 and PKD1 (encodes polycystin-1) genes [Renal angiomyolipomas (AML) are common in patients with TSC, affecting nearly 80% of patients, and are responsible for a significant proportion of patient morbidity in adulthood . The mai1) genes .Classic therapeutic options for AML include partial nephrectomy, embolisation, radiofrequency and cryotherapy. In recent years, several studies have shown that mTOR inhibitors (mTORis) can significantly reduce the size of renal AML, with relatively tolerable side-effects ,6. The rWe report the case of a patient with TSC and a large renal AML treated by percutaneous cryotherapy after tumoral mass reduction induced by an mTORi. This is the first report of this novel treatment strategy.2). She had multiple renal AML, including one exophytic AML sized 6 x 5 x 4.5 cm at the upper pole of the right kidney , the AML size was reduced to 4.5 x 4 x 3.5 cm  guidance Figure . CryoablRenal function remained normal and stable throughout follow up, although a mild proteinuria (0.13 g/g of creatinine) occured. Hypercholesterolemia present pre-treatment increased slightly from 2.64 to 2.77 g/l, while triglycerides remained normal. Blood count was normal as well as the level of gammaglobulins. No side-effects of the treatment were reported.The efficacy of mTORis in reducing the size of AML associated with TSC is now well documented. The decrease in size averages from 30 to 47% and has been shown to persist for up to 2 years ,6. HowevIn the current case study, the therapeutic response to sirolimus was a 25% reduction in size of the largest AML. This was considered insufficient to reduce the risk of bleeding and remaining therapeutic options were to increase mTORi dosage, perform a partial nephrectomy, or attempt embolisation or percutaneous ablative therapies.In studies demonstrating the benefits of mTORis, initial target plasma levels were 1–6 ng/ml with potential titration to 9–15 ng/ml dependent on the response .Partial nephrectomy is a definitive therapeutic option but is major surgery with a 5-23% rate of perioperative complications and a 16% conversion rate to radical nephrectomy ,10. RenaStaehler et al. recently published their results in three patients treated with 6 months of neoadjuvant sirolimus treatment, with plasma target level of 4–8 ng/ml, prior to partial nephrectomy . A 38-95Embolization is a common therapeutic option for TSC-associated AML with a primary 90-100% technical success rate; however, there is a 43% rate of recanalization and regrowth and a persisting bleeding risk warranting multiple embolizations ,12. The in situ ablative technique used for many years with good results and no recurrence for up to 4 years [Percutaneous radiofrequency ablation is an  4 years ,18. Howe 4 years ,18. The  4 years ,18. Comp 4 years ,18. The  4 years .Cryoablation of AML was first described using a laparoscopic approach with good results ,21. Subs2 after 24 months of follow-up [Despite severe comorbidities and some minimal collateral normal tissue damage, renal function after cryotherapy has generally been well preserved with a decrease in eGFR of less than 10 ml/min/1.73 mollow-up . The priollow-up . Due to ollow-up . AlthougIn comparison with ablative and surgical treatments of TSC-associated AML, the use of mTORis remain poorly-defined. We suggest a strategy combining an mTORi with ablative treatment, in order to reduce AML size and allow safer radical cryotherapy. This strategy is also likely to avoid an unnecessarily high dosage of the mTORi and prevent medication related side-effects. Cryotherapy appears to have a long-term curative effect with minimal risk on AML smaller than 4 cm although its use in this indication and in combination with mTORi warrants further study in larger series.Written informed consent was obtained from the patient’s parents for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.AML: Angiomyolipoma; eGFR: Estimated glomerular filtration rate.; mTOR: Mammalian target of rapamycin; mTORi: Mammalian target of rapamycin inhibitor; TSC: Tuberous sclerosis complex; PKD1: Polycystic kidney disease gene 1.The authors declare that they have no competing interests.KT wrote the manuscript and performed the literature search. GJ performed the cryoablation and chose the images that illustrate the case. LH managed the patient in the Urology Department; he corrected the manuscript from a urological point of view. GA is the chief of the Interventional Radiology Department; he corrected the manuscript from a radiological point of view. HT is the chief of the Nephrology Department; he corrected the manuscript from a nephrology point of view. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/14/77/prepub


Endometrial adenocarcinoma (EC) usually occurs after menopause, whereas in 2-14% ofcases, it occurs in young patients (less than 40 years old) who may desire to keep their fertility.It is of importance to evaluate women for EC when they develop polycystic ovarian syndromeand abnormal uterine bleeding. Its treatment includes hysterectomy, bilateral salpingo-oo-phorectomy and pelvic lymphadenectomy and in some cases, radiation therapy. We report acase of EC in a 31-year-old woman who presented to Royan Institute. She complained aboutoligomenorrhea with a 10-year history of primary infertility.  Endometrial adenocarcinoma (EC) is the mostcommon gynecologic malignancy in the UnitedStates, predominantly among postmenopausalwomen, at the average age of 59 years. The majorityof cases are diagnosed when the carcinoma isconfined to the uterus, leading to less than 1.5% ofcancer deaths .Although 20-25% of EC are diagnosed before themenopause, 2-14% occur among younger women(less than 40), most of whom wish to preserve theirfertility (3-6). This complication is more common indeveloped countries than the developing countries (7).We report a case of a 31-year-old patient with anendometrial cancer diagnosed at stage II according tothe International Federation of Gynecology and Obstetrics(FIGO), 2000 classification of endometrialcancer (8). This case study will provide a useful guidein diagnosis of EC for the sonographer.The aim of study is to provide opportunity for sonographiststo learn the broad spectrum of findingsthat may be seen at sonohysterography (SHG) inboth benign and malignant processes to raise clinician’sawareness toward the appropriate diagnosisand treatment.A 31-year-old woman who was nulliparous andoverweight [body mass index (BMI)=35.5] with a10-year history of primary infertility presented to theImaging Department of Royan Institute in 2012. Shehad a history of laparoscopy with ovarian cotter, septumand polyp resection by hysteroscopy (HSC) in2010 at different infertility center. In addition, due tomale factor infertility, she underwent ovarian stimulationin IVF cycle and ten embryos were obtained andfrozen in 2010. Her chief complaint was oligomenorrhea.Since she was overweight and had abnormaluterine bleeding (AUB), transvaginal sonography(TVS) and SHG were done for patient. TVS showedthickened endometrium with smooth counter ,while tThis is an interesting case because she was at reproductiveage and adenocarcinoma is uncommon in thisage group. Also obtained findings from transvaginalultra sonography (TVUS) and SHG are not specificfor adenocarcinoma.The sonographic appearance of the normal endometriumhighly depends on the age of the patientand the stage of menstrual cycle for woman of reproductiveage.A carcinoma generally originates from theepithelial cells. EC is a type of uterine cancerthat involves the endometrium. Adenocarcinoma(adeno=gland) refers to a carcinoma featuringmicroscopic glandular-related tissue. Prior to the1980s, EC was broadly characterized as a singledisease. However, observations by Lauchlan (9),Hendrickson et al. (10) have led to the description of two distinct types based on histologic andmolecular characteristics. Type I EC is commonlyreferred to endometrioid adenocarcinoma whichincludes 80-90% of all ECs. Type II EC, nonendometrioidtumors, encompasses the remaining 10-20% of endometrial tumors (11). The etiology andsurvival of these two subtypes are vastly different.The risk factors for EC which are related directlyor indirectly to estrogen exposure including earlymenarche, late menopause, nullparity, poly cycticovarian syndrome (PCO), diabetes and obesity.Besides, adenomatous polyps, breast cancer andthe use of estrogen therapy, are associated withhigher incidence of endometrial cancer .In our case, she had obesity, PCO, and adenomatouspolyp, while she was considered nulliparous.History of estrogen use is more frequently seen inyoung patients. Also hormone-related disorderssuch as ovarian dysfunction, chronic anovulation,infertility, obesity and PCO detected in these patients(18). In this case, it should be mentioned thatshe had one stimulation cycle of assisted reproductiontreatment (ART) in 2010, and subsequently,had 10 frozen embryos.Physicians mostly prefer to perform endometrialbiopsy if there is abnormal uterine bleeding whichis considered as indication of early symptom ofEC . Although postmenopausal bleeding isa common sign of EC, recent studies have shownthat only 4-5% of women with postmenopausalbleeding have endometrial cancer .SHG and TVUS provide a good predictive valuefor endometrial disease in patients with AUB (20).SHG which is instillation of sterile saline solutionby means of a catheter was initially described byNannini et al. (21). SHG produces better images,whereas TVUS provides more accurate measurementof the endometrial thickness allowing moreclear evaluation of the heterogeneity. SHG hasgreater accuracy in the identification of focal lesionsthan in the diagnosis of diffuse lesions particularlymore associated to malignant lesions.Adding 3D imaging to SHG can help getting optimalresults because of allowing real time visualizationof the cavity and more accurate assessingthan conventional 2D. Other advantages are theability to observe the coronal plane of the uterusand saving 3D volumes for later study, which leadsto reduce the duration of examination and to causeless discomfort for the patients. In premenopausalpatients, SHG is preferably performed during theearly proliferative phase of the patient’s menstrualcycle, when the endometrium is a very thin tissue.Although there is no limitation for normal premenopausalendometrial thickness, the endometriumshould be uniform in thickness, homogeneous inechotexture, and not to be displaced by any submucosal,myometrial abnormality (22).In postmenopausal patients, the normal atrophicendometrium should measure be less than 4 mmin double-layer thickness as seen at TVUS andless than 2.5 mm in single-layer thickness as seenat SHG. In addition, the atrophic endometriumshould be smooth and uniform in echotexture andnot to be displaced by any submucosal, myometrialabnormalities .The most common appearance of EC at TVUS isnonspecific thickening of the endometrium. Evenat SHG, endometrial cancer can be difficult to distinguishfrom endometrial hyperplasia and polyps.This diagnosis should be suspected when thesingle layer of the endometrium is thicker than 8mm, irregular, broad based, or poorly marginatedor when the endometrial-myometrial interface isdisrupted. One of interest finding in our case is thatshe had smooth endometrial-myometrial interface.Endometrial thickness measurements often overlapin benign and malignant conditions (24).At SHG, EC is typically a more diffuse process,while early cases can appear as a polypoid mass(19). An intact subendometrium shows localizeddisease, whereas extension of heterogeneity andincreased echogenicity in the myometrium proposeadvanced invasive endometrial carcinoma(25). Sonographic findings related to EC in ourpatient included a thickened, heterogeneous endometrium.Final diagnosis achieved through HSC findingand pathology. Advantages of HSC in the evaluationof abnormal bleeding or abnormal lesion arenotable and the ability to see lesions and to evaluateendometrial cavity is precious. The panoramicHSC, especially with directed biopsy is superior toD&C in patient with abnormal findings (22).SHG make obvious differentiation between focaland diffuse endometrial lesions, so it becomesreliable test in the imaging evaluation of dysfunctional uterine bleeding and postmenopausal bleeding.It is essential for the radiologist to be familiarwith the broad spectrum of findings that may beseen at SHG in both benign and malignant processesin order to direct the clinician toward theappropriate means of diagnostic biopsy or surgery.


Kim and Ziff examine the molecular mechanism of synaptic scaling, showing that inhibition of neuronal excitability reduces calcium influx into neurons, resulting in decreased calcineurin activity. This leads to increased surface expression of calcium-permeable AMPA receptors as a homeostatic response. 2+ levels caused by chronic inhibition of neuronal activity are believed to be an important trigger of synaptic scaling, the mechanism of Ca2+-mediated AMPAR-dependent synaptic scaling is not yet understood. Here, we use dissociated mouse cortical neurons and employ Ca2+ imaging, electrophysiological, cell biological, and biochemical approaches to describe a novel mechanism in which homeostasis of Ca2+ signaling modulates activity deprivation-induced synaptic scaling by three steps: (1) suppression of neuronal activity decreases somatic Ca2+ signals; (2) reduced activity of calcineurin, a Ca2+-dependent serine/threonine phosphatase, increases synaptic expression of Ca2+-permeable AMPARs (CPARs) by stabilizing GluA1 phosphorylation; and (3) Ca2+ influx via CPARs restores CREB phosphorylation as a homeostatic response by Ca2+-induced Ca2+ release from the ER. Therefore, we suggest that synaptic scaling not only maintains neuronal stability by increasing postsynaptic strength but also maintains nuclear Ca2+ signaling by synaptic expression of CPARs and ER Ca2+ propagation.Homeostatic synaptic plasticity is a negative-feedback mechanism for compensating excessive excitation or inhibition of neuronal activity. When neuronal activity is chronically suppressed, neurons increase synaptic strength across all affected synapses via synaptic scaling. One mechanism for this change is alteration of synaptic AMPA receptor (AMPAR) accumulation. Although decreased intracellular Ca Synaptic scaling is a form of homeostatic plasticity that normalizes the strength of synapses  and is triggered by chronic inhibition of neuronal activity. Although extensive studies have been conducted, the molecular mechanism of this synaptic adaptation is not understood. Using cultured cortical neurons, we show that chronic inhibition of neuronal activity reduces calcium influx into neurons, which, in turn, decreases the activity of the calcium-dependent phosphatase calcineurin. These changes lead to an increase in GluA1-containing, calcium-permeable AMPA receptors, which mediate communication at the synapse. Newly inserted calcium-permeable AMPA receptors restore calcium currents, which enhance synaptic strength and recover calcium signaling. We also show that inhibition or activation of calcineurin activity is sufficient to induce or block synaptic scaling, respectively, suggesting that calcineurin is an important mediator of homeostatic synaptic plasticity. Taken together, our findings show that synaptic scaling is a homeostatic process that not only enhances synaptic transmission but also maintains calcium signaling in neurons under activity deprivation. Synaptic scaling, a form of homeostatic synaptic plasticity, is a negative feedback process that stabilizes neuronal activity in response to changes in synaptic strength by altering various aspects of neuronal function 2+-impermeable GluA2-containing and Ca2+-permeable, GluA2-lacking/GluA1-containing receptors 2+-permeable AMPARs (CPARs) are generally sensitive to polyamine block, although there is a third class of AMPARs that are Ca2+-permeable but insensitive to polyamines 2+/calmodulin-dependent protein phosphatase, calcineurin, dephosphorylates pGluA1(S845), which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during long-term depression AMPARs are the major excitatory postsynaptic glutamate receptor in the central nervous system and consist of four subunits (GluA1–4) 2+ influx and inhibits activation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), which promotes synaptic scaling 2+-dependent synaptic plasticity 2+ influx is sufficient to induce AMPAR-mediated synaptic scaling 2+ influx can play a critical role in AMPAR-dependent homeostatic scaling, yet there is no complete molecular mechanism linking activity-dependent Ca2+ signals and homeostatic regulation of AMPARs.The most studied experimental system for synaptic scaling is the inhibition of neuronal activity by TTX (tetrodotoxin), which blocks sodium channels and thereby inhibits action potentials. TTX-dependent chronic inhibition of action potentials results in an increase in the strength of synaptic transmission as a compensatory process that can be measured by increases in AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs) 2+ and calcineurin in synaptic scaling. We show that activity suppression reduces Ca2+ influx in neurons, which in turn decreases the activity of calcineurin. This stabilizes pGluA1(S845), which increases synaptic CPARs. This increases synaptic strength as a compensatory response to activity deprivation and restores synapse-to-nucleus Ca2+ signaling via ER Ca2+ wave propagation. Thus, we conclude that synaptic scaling via calcineurin and CPARs provides a means to maintain not only synaptic activity but also Ca2+ signaling as a homeostatic response.Here, we focus on the role of synaptic Cap<.0001) (p = .0008)     consistep<.0001) . Importap<.0001) . There w= .0008) . Because= .0008) . Consistp<.0001) , but frep<.0001) . Naspm a= .0453)  as found= .0453) . Thus, Tp = .024), whereas total GluA1 and GluA2/3 levels were not changed (p = .0127) after TTX treatment, whereas surface GluA2/3 was not altered (Because pGluA1(S845) is required not only for homeostatic scaling in the visual cortex  changed . We furt altered . We next altered . This su2+ influx. We found that calcineurin protein levels were significantly decreased (p = .0414) in synaptosomes following TTX treatment -based calcineurin activity sensor that utilizes a calcineurin activity-dependent molecular switch based on the N-terminal regulatory domain of nuclear factor of activated T cells (NFAT) as a specific substrate, which was inserted between CFP and YFP p<.0001), which confirmed that the reporter detected calcineurin activity  (Increasing pGluA1(S845) can be achieved either by enhancing kinase activity or by decreasing phosphatase activity. A-kinase anchoring protein (AKAP) and SAP97 form a protein complex with GluA1 that tethers PKA and calcineurin, which regulate channel functions, respectively, through GluA1 phosphorylation and dephosphorylation reatment . To measactivity . Calcinep<.0001) . This su2+ lowers calcineurin activity, followed by enhancement of GluA1-containing AMPAR-mediated transmission p<.0001) (p<.0001) (p = .0047), suggesting that CPARs mediated the scaling induced by FK506 , whereas no effect was observed following naspm or PhTX treatment of DMSO-treated neurons  (p = .0474) in synaptosomes, whereas total GluA2/3 and GluA1 levels were not altered (p<.0001) (p = .0299)  . Consistp<.0001) . Furtherby FK506 . Moreoveby FK506 . We confby FK506 , which c neurons . There w neurons . Moreove= .0003) . Similar altered . Surfacep<.0001) . Moreove= .0299) . These r2+-independent, constitutive phosphatase activity, by deleting the calcineurin autoinhibitory domain (CaN-ΔAI) p = .0198) than in cells transfected with GluA1 alone (Ctrl) (p<.0001) (p = .0072)  . Althouge (Ctrl) . When Cae (Ctrl) . Howevere (Ctrl)  as seen p<.0001)  and decr= .0072) , whereas= .0072) . This su2+ signals are thought to be important for synaptic scaling, which suggests that a reduction of Ca2+ influx may be a critical trigger for synaptic scaling 2+ levels has been reported to enhance GluA1-containing AMPAR-mediated transmission 2+ activity in cultured neurons transfected with GCaMP5, a genetically encoded Ca2+ indicator 2+ transients in neurons without TTX treatment  , and this restored activity observed in the presence of TTX was significantly reduced by naspm (p = .0079) (2+ activity that was dependent in part upon the synaptic expression of CPARs.Careatment . To detep<.0001) . Furtheransients , suggest= .0079) . This su2+ signals   , indicating it was from CPARs   . Neuronsreatment . Convers = .005) . To deteecording . Naspm som CPARs . Furtherp<.0001) , consistp<.0001) . This su2+ are used by neurons 2+ influx from extracellular sources is mediated by various Ca2+ channels including NMDA receptors (NMDARs) at synapses and voltage-gated Ca2+ channels in the plasma membrane, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the ER are responsible for intracellular Ca2+ release 2+ sources were responsible for GCaMP5-positive Ca2+ signals , suggesting that GCaMP5 detected Ca2+ signals including those from NMDARs but not from L-type Ca2+ channels (2+ from the ER by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase using 1 µM thapsigargin and found that thapsigargin treatment completely inhibited Ca2+ activity (p<.0001) , suggesting that GCaMP5 detected Ca2+ released from the ER, possibly dependent on the activity of NMDARs  . Moreovef NMDARs . This fu2+ influx evokes Ca2+ signals in the nucleus via Ca2+ wave propagation through the ER 2+ signaling is essential for synaptic plasticity and regulates gene expression through CREB in addition to local signaling in synapses 2+ release 2+ signaling via the ER Ca2+ release when neuronal activity is chronically suppressed by TTX. Consistent with previous findings p = .0004) or 1 µM thapsigargin (p = .0019), confirming that CREB activity was dependent on both neuronal activity and ER Ca2+ , suggesting CPARs were responsible for homeostasis of CREB phosphorylation , providing a balance between GluA1 insertion by kinases and removal by phosphatases in synapses , thereby enhancing synaptic strength and promoting synaptic Ca2+ influx via CPARs instead of NMDARs  TTX inhibited somatic Ca2+ signals, (2) TTX treatment reduced CREB phosphorylation, and (3) synaptic scaling restored Ca2+ signaling and CREB activation.CaMKs are important for Ca2+ signaling as a homeostatic response to suppression of neuronal activity.In summary, we conclude that synaptic scaling not only maintains neuronal stability by increasing CPAR-dependent postsynaptic strength but also maintains basal CREB transcriptional activity through nuclear Ca2+ imaging. Cells were grown in Neurobasal medium with B27 and 0.5 mM Glutamax (Life Technologies). For neuronal transfection, DIV4 neurons were transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol, and analysis was performed at DIV14–17.Cortical primary neurons were prepared by a modification of the previously described method 2+-independent, constitutive phosphatase activity Constitutively active calcineurin mutant (CaN-ΔAI) was generated according to the previous study 2, 1.5 MgCl2, 30 glucose 20 HEPES (Life Technologies), and 0.001 glycine (Sigma), pH 7.4. Patch electrodes (5–8 MΩ) were filled with (in mM) 120 K-gluconate (Sigma), 9 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA (Sigma), 2 Mg-ATP (Sigma), and 0.2 GTP (Sigma). We added 1 µM TTX (Tocris Biosciences) and 10 µM bicuculline (Tocris Biosciences) to the bath to inhibit action potentials and miniature inhibitory postsynaptic currents, respectively. mEPSCs were recorded at −60 mV with a Warner amplifier (PC-501A) and filtered at 1 kHz. Recordings were digitized  and analyzed using the Mini Analysis software (Synaptosoft). The access resistance (Ra<25 MΩ) was monitored during recording to eliminate artifacts. Events whose amplitude was less than 7.5 pA were rejected. To induce synaptic scaling, neurons were pretreated with 2 µM TTX for 48 h or 5 µM FK506 or DMSO for 12 hrs. We added 20 µM naspm  or 5 µM philanthotoxin-74 (Tocris Biosciences) to suppress CPAR-mediated transmission in the bath solution. For the CaN-ΔAI experiment, GFP was cotransfected with CaN-ΔAI to visualize transfected neurons, and mEPSCs were analyzed at DIV14–17.Miniature EPSCs were measured in cortical neurons cultured from C57Bl6 or GluA1 S845A embryos at DIV14–17 as described previously Synaptosomal fractions from DIV14 primary cortical neurons were prepared as described previously Neurons were transfected with the calcineurin activity biosensor, and FRET activity was measured at DIV14 according to a modification of the previously described method 2 using a Zeiss stage incubator model S with CTI, digital temperature, and humidity controller. The imaging was captured for periods of 0.5 to 1.0 s depending on the intensity of the fluorescence signal using a 63× oil-immersion objective. One hundred images were obtained with a 1-s interval, and Ca2+ activity in the cell body (excluding dendrites) was analyzed using the ImageJ software. F0 was determined as the minimum value during the imaging. Total Ca2+ activity was obtained by combining 100 values of ΔF/F0 = (Ft−F0)/F0 in each image, and values of ΔF/F0<0.3 were rejected due to bleaching.DIV4 neurons were transfected with GCaMP5 . Neurons were grown for 10–12 d after transfection in Neurobasal medium without phenol red and supplemented with B27 and 0.5 mM Glutamax. Glass-bottom dishes were mounted on a temperature-controlled stage on Zeiss Axiovert 200M and maintained at 37°C and 5% CO2, 10 mM KCl, and 0.25% NP40). Nuclear fraction was collected by centrifugation, resuspended in a nuclear preparation buffer B , and analyzed by immunoblots.Neurons pretreated with 2 µM TTX for 6 or 48 h or 1 µM thapsigargin for 6 h were lysed with a nuclear preparation buffer A  followed by Fisher's Least Significant Difference (LSD) test to determine statistical significance. The Kolmogorov-Smirnov (K-S) test (http://www.physics.csbsju.edu/stats/KS-test.html) was used for comparisons of cumulative probabilities. Results were represented as mean ± s.e.m., and a p value<.05 was considered statistically significant.Most statistical comparisons were analyzed with the GraphPad Prism6 software. Unpaired two-tailed Student's Figure S12+ activity. (a) Representative images of GluA1 surface labeling (green) and mCherry were used as markers for transfection (red) (n = number of cells). Scale bar is 2 µm. A summary graph of surface GluA1 levels in each condition. (b) Example bar graphs of Ca2+ activity in each condition. Each bar represents the GCaMP5 fluorescence intensity detected in a single exposure frame. Scale bars are 50 s. Normalized average of total Ca2+ activity in each condition .Effects of CaN-ΔAI on surface GluA1 and Ca(TIF)Click here for additional data file.


RSK4) is a factor downstream of the MAPK/ERK pathway and an important tumor suppressor which inhibits ERK trafficking. Decrease in RSK4 expression has been reportedin some malignancies, which leads to an increase in growth and proliferation and eventually poor prognosis. In this study we measured RSK4 expression rate in acute myeloidleukemia (AML). Signaling pathways such as extracellular regulated kinase/mitogen activated protein kinase (ERK/MAPK) have increased activity in leukemia. Ribosomal 6 kinase and employing the ΔΔCT method. Data were analyzed using Mann-Whitneyand Spearman tests using SPSS (version 11.5).RSK4 was significantly decreased in the AML group in comparison with the non-AML group (P<0.001). There was also a significant decrease inRSK4 expression in AML with t in comparison to other translocations (P=0.004). Expression rate of RSK4 in AML patients. This may lead toan increase in the activity of the ERK/MPAK pathway and exacerbate leukemogenesis orthe prognosis of the patients.We detected a down-regulation of  A proportion of acute myeloid leukemia (AML) cases exhibit irregularities in the expression of genes including transcription factors, oncogenes, tumor suppressors, and abnormal activities in tyrosine kinase receptors which regulate blood production . TyrosinRSK4 (known as RPS6KA6) has the ability to inhibit the ERK signaling pathway (RSK4 (RSK4 has an inhibitory role in embryo development (c-MYC (a cell cycle regulator) (RSK4 is able to phosphorylate and activate check point kinase one (CK1) and halt the cell cycle progression  which is also the most important substrate of ERK . The RSK pathway  and is a pathway . Also itay (RSK4 . In addielopment  and has gulator) . When DNgression . In coloobserved . In fibrobserved . In breaor cells . RSK4 to act as a tumor suppressor and also ERK/MPAK pathway exhibiting increased activity in AML, we analyzed RSK4 expression changes in blood and bone marrow samples of AML patients by quantitative teal-time polymerase change reaction (PCR). Given that previous studies have shown RSK4 expression, initially RNA was extracted from mononuclear cells of the bone marrow samples  and cDNA was synthesised . Then real-time qPCR was undertaken to quantitate the expression level of RSK4 using a SYBR Green master mix kit  on an ABI thermocycler . Differential expression was analyzed by the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as the reference gene (a housekeeping gene). Prior to differential expression analyzes, efficiency of both GADPH and RSK4 was determined using dilution series. The real-time qPCR procedure was performed according to the instruction of the kit used , however, due to low RSK4 expression levels in normal tissues and the possibility of expression reduction in our sample cells (based on our hypothesis), the annealing temperature was reduced, whilst the number of the cycles was increased . Primers used are shown in This cross-sectional study was performed in the Cancer Molecular Pathology Research Center, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran, in 2013-2014. The local Ethical Committee approved the research protocol and written informed consents were taken from all participants. Peripheral blood and bone marrow samples of diagnosed AML patients [according to World Health Organization (WHO) criteria] and those of non-AML individuals (as the control group) were collected in anticoagulant-containing blood sampling tubes Ethylene Diamine Tetra Acetic acid-K2 (EDTA-K2). For all patients, complete blood count (CBC) was analyzed and peripheral blood and bone marrow smears were prepared and observed after staining. AML was diagnosed as existence of at least 20% blasts in peripheral blood or bone marrow smears that were positive for myeloperoxidase or Sudan Black B staining and myeloid markers CD33, CD13, CD117, CD64 and CD14. According to morphologic features of the smears, other staining including periodic acid schiff and none-specific esterase were also performed. In the next step, for AML patients only, among the recurrent cytogenetic abnormalities determined by the WHO classification, three translocations including t, t and inv were anaData were analyzed using SPSS (Version11.5). Initially, descriptive and then comparative analyses of parameters were performed.Due to the non-normal distribution of expression values, sample means were compared usingMann-Whitney test and correlation analysis wasundertaken by Spearman’s test. P≤0.05 was considered to be significant.After exclusion of five samples, 40 patientsdiagnosed with AML and 10 individuals as thecontrol group were analyzed. In the patient group,24 (60%) were male and 16 (40%) were female,while in the control group, 5 (50%) were male and5 (50%) were female. The average age ± SD was 31± 17 and 29 ± 5.9 years in the patient and the con-trol groups respectively. The mean age differencebetween two groups was not significant (P=0.544).Differences between AML patients and the controlgroup in red blood cell (RBC) and Platelet (PLT)count, Haematocrit (HCT) and Hemoglobin (Hb)were significant (P<0.05), however, white bloodcell (WBC) count was not significantly different.RSK4 in thepatient group compared with the control group was0.0041 ± 0.0048 (about 250-fold down-regulation)that shows a significant reduction (P<0.001). The expression level change was also analyzed in differentmorphologic subtypes. Among these, the M3 varianthad the lowest and the M6 variant had the highestexpression levels but these differences were not significant. Among the AML with chromosomal abnormalities, a significant decrease of RSK4 expressionwas observed in AML with t (P=0.004). Wealso analyzed correlation between the rates of RSK4expression in the AML group and their CBC parameters and observed only a correlation between RBCand gene expression rate .Among the AML patients, the most common subtype (according to the FAB classification) was theAML-M3 and alsoRSK4 may play a role inAML leukemogenesis. As a result, we analyzedpossible expression changes of RSK4 in AML.The expression level of RSK4 decreased inAML patients and this decrease was significantin AML with the t chromosomal disorder. According to some studies, it is possiblethat decrease in RSK4 expression results in overactivating the ERK/MPAK signaling pathwayin AML cells and finally myeloid proliferation(RSK4expression (~250-fold down-regulation) may beeither the low expression of the gene in normaltissues or down regulation of RSK4 in AML asa tumor suppressor. The reason for reductionof RSK4 expression may relate to the missensemutation in the N-terminal of RSK4 kinase thathas also been observed in primary malignantcells in lung cancer . However, we observed decrease in expression of RSK4 inAML with t. It is possible that this difference may be due to the double-sided effectsof c-MYC on cellular growth and proliferation in AML patients. Given that theERK/MPAK pathway exhibits increased activityin AML progenitors and the suppressive effect ofRSK4 on this pathway, it would be interesting toinvestigate the effect of RSK4 on AML further.We observed down-regulation of


Mutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies.7 receptor and the transient receptor potential channel, TRPV2, another Ca2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.Human deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins . The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2Xde novo expression Cx HC causes a dysregulation of intracellular free Ca2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.The data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the The online version of this article (doi:10.1186/s12860-016-0096-6) contains supplementary material, which is available to authorized users. In addition, an altered permeability to dyes such as Evans blue has been reported in skeletal muscles from Dysf-/- mice  [de novo expression of Cx HCs has been observed in similar pathologies, where they mediate myofiber atrophy induced by denervation [2+ and Na+) and small compounds, including signaling molecules such as ATP and NAD+ and dyes including ethidium (Etd+) and Evans blue [de novo expression of Cx HCs promotes the increase of oxidative stress in pathological conditions such as muscle denervation [Dysferlinopathies are muscular dystrophies caused by mutations in dysferlin, a 230 kDa membrane protein, mainly localized in the sarcolemma  and the nopathy) , and prenopathy) . Hence, ervation . Intereservation . Since Cans blue , 13, theervation  and theyervation , 12, 14.-/- mice) results in the recovery of membrane resealing function. However, the progressive degeneration, ascertained from muscle histology studies, remains unabated [2+ in these cells.To date there is no effective treatment to arrest or even reduce the symptomatology of the patients affected with dysferlinopathies. Nevertheless, the introduction of a mini-dysferlin in animal models of the disease , which have been previously associated with muscular atrophy [7 receptors were detected in one of the two patients evaluated, whereas TRPV2 was found in the biopsies of both patients , the absence of dysferlin was confirmed by immunohistochemistry assays (data not shown). As shown in Fig.  atrophy . P2X7 rents Fig. . Conversnts Fig. .Fig. 1Co2+ influx is reportedly increased in human muscles bearing dysferlin mutations [2+ channels; connexin-based hemichannels, transient potential receptor TRPV2 and P2X7, all of which have been previously linked in muscular pathologies [7 receptors were only present in 107 and 379  derived myotubes . In the absence of dysferlin, RCMH cells express connexins 40.1, 43 and 45 , which were in control myotubes (RCMH) that express dysferlin  uptake assays [+ uptake compared with control myotubes , was enough to reduce the elevated basal Ca2+ concentration to levels similar to those of control cells. The mechanisms regulating Ca2+ in myotubes, such as the Na+/Ca2+ exchanger, Ca2+ binding proteins and most importantly the sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA), are extremely efficient in controlling the cytosolic levels of this cation [2+ concentration by ~8 %, which may be enough to alter cellular homeostasis of myocytes. This increment in Ca2+ levels could be enough to alter the cellular homeostasis of the myocytes. Indeed, an altered cytosolic Ca2+ level could induce several changes in the myotubes, including an increased proteolytic activity [2+ levels in human DMD myotubes were increased compared with normal myotubes [In the present report, we have demonstrated that muscles from patients diagnosed with dysferlinopathy express Cxs 40.1, 43 and 45 at the plasma membrane. Additionally, four immortalized muscle cell lines generated from muscle samples of dysferlinopathy patients also expressed these Cxs, which in turn, formed functional Cx-based hemichannels. This phenomenon was also induced in control myotubes , after decreasing dysferlin expression through the transfection (in myoblast stage) of a specific small hairpin RNA against dysferlin. Moreover, human dysferlin-mutated 107 and 379 myotubes expressed two additionals Cas cation . Even soactivity , which oactivity . The latactivity . These fmyotubes .de novo expression of functional connexin-based hemichannels suggests a direct relation between the absence of dysferlin and the presence of connexin. However, it has been demonstrated that some conditions such as inflammation induces the expression of functional connexin-based hemichannels in skeletal muscles through proinflammatory cytokines and the transcription factor NFκB [Therefore, in addition to the deficit in membrane repair mechanism found in dysferlin-mutated myofibers , we proptor NFκB , 12. Inftor NFκB . Whether2+ levels in human myotubes. The presence of these channels may represent a novel pathological mechanism in dysferlinopathies, as well as a most attractive therapeutical target, considering their localization at the membrane level.In this study, we have demonstrated the presence of connexins 40.1, 43 and 45 in muscular biopsies of patients with different dysferlin mutations. This was confirmed in myotubes derived from cell lines of human dysferlinopathy patients, as well as in normal myotubes where dysferlin was knocked down. The Cxs expressed formed functional connexin based hemichannels, that are responsible of elevated basal intracellular Ca+) bromide was from GIBCO/BRL , fluoromount-G was from Electron Microscopy Science , Previously described polyclonal anti-Cx40.1, -43, and -Cx45 antibodies were used [Anti-rabbit or anti-mouse IgG antibodies-conjugated to Cy2 (green) or Cy3 (red) were purchased from Jackson immunoresearch laboratories . Ethidium  HTZ; ER, G1628R(c.4882G > A)HMZ.Four different dysferlin-mutated myoblast lines were generated and called 107, 379, AB320 and ER, each one bearing a different dysferlin mutation as follow: 2+ signals were evaluated in myotubes by using the ratiometric dye FURA 2-AM. The myotubes were incubated in Krebs-Ringer buffer  containing FURA2-AM dye (2 μM) during 55 min at room temperature. Then, the Ca2+ signal was evaluated in a Nikon Eclipse Ti microscope equipped with epifluorescence illumination, and images were obtained by using a Clara camera (Andor), at 2 wavelength (λ) 340 and 380 nm, calculating the ratio 340 vs 380.Basal intracellular Ca+) was evaluated by using time lapse measurements. Human myotubes plated onto glass cover slips were washed twice with Krebs-buffer solution. For time lapse measurements myotubes were incubated in Krebs buffer solution containing 5 μM Etd+ bromide. Etd+ fluorescence was recorded in regions of interest located in nuclei of the myofibers. An epifluorescence Nikon Ti microscope was used. Images were captured with a Clara camera (Andor) every 30 s. Image processing was performed off-line with ImageJ software .The uptake of ethidium (Etd7 (1:100) antibodies followed by 4 washes with PBS 1X and then, incubated with an appropriate dilution of alexa488-conjugated goat anti-rabbit or anti-mouse IgG antibodies. Samples were rinsed with PBS 1X, mounted with fluoromount G containing DAPI on glass slides and representative images were acquired in an epifluorescence microscope Nikon Ti .To detect different proteins in myotubes or cross sections (10 μm) of muscles (fast frozen with iso-methyl-butane cooled in liquid nitrogen) samples were obtained and processed as described previously . BrieflyCommercial small hairpin RNA plasmid against dysferlin , with puromycin resistance, was used to transfect RCMH myoblast through lypofectamine method . The silFollowing ethical guidelines, healthy volunteers and dysferlinopathy patients who participated in this study signed an informed consent approved by the local Ethics Committee in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Diagnosis of dysferlinopathy was achieved based on clinical examination, muscular biopsy and mutation analysis. The mutations of the patients are published in Woudt et al. .P < 0.05 was considered statistically significant.Results are presented as mean ± standard error (SE). For multiple comparisons with a single control, a non-parametric one-way ANOVA followed by the Dunn’s multiple comparison test was used. Analyses were carried out using GRAPHPAD software.


ADGRD1 (GPR133) locus are linked with differences in metabolism, human height and heart frequency. ADGRD1 is a Gs protein-coupled receptor belonging to the class of adhesion GPCRs.A large number of human inherited and acquired diseases and phenotypes are caused by mutations in G protein-coupled receptors (GPCR). Genome-wide association studies (GWAS) have shown that variations in the ADGRD1 as listed in public data bases. Approximately 2.4 % of these SNPs are located in exons resulting in 129 non-synonymous SNPs (nsSNPs) at 119 positions of ADGRD1. However, the functional relevance of those variants is unknown. In-depth characterization of these amino acid changes revealed several nsSNPs  causing full or partial loss of receptor function, while one nsSNP (F383S) significantly increased basal activity of ADGRD1.Analysis of more than 1000 sequenced human genomes revealed approximately 9000 single nucleotide polymorphisms (SNPs) in the human ADGRD1 variants is present in the human population which may cause clinically relevant phenotypes, while being compatible with life when heterozygous.Our results show that a broad spectrum of functionally relevant The online version of this article (doi:10.1186/s12864-016-2937-2) contains supplementary material, which is available to authorized users. More than three dozen human inherited and acquired diseases are caused by mutations in G protein-coupled receptors (GPCRs) –4. At thADGRD1 with the postprandial levels of very low density lipoproteins (VLDL)  and G141D , in the spacer region, led to significant increase in activity, which cannot be explained by an associated elevated membrane expression Fig. . E78K waa tissue . G141D wThere were two mutations, Y85C  and F110L , in the spacer region that led to significant decrease in activity Fig. . Y85C apADGRD1 orthologs whereas the position E178 is substituted by aspartate in 12 % of orthologous sequences but not by lysine , E178K  and G195R  are located within two alpha helical structures of the PTX-like domain, as predicted via hydrophobicity plot according to Kyte- Doolittle with Protean 7.1.0 . The latter two SNPs have also been identified by Samuels’ laboratory (NHGRI/NIH) as being found in samples from malignant melanoma. Amino acid changes at these positions significantly decrease cell surface expression, which results in an even more pronounced reduction of CRE levels Fig. . W174 anADGRD1 has been associated with changes in heart frequency [The GAIN domain is considered an important structural and functional element of aGPCRs . Nine ourequency  it is strequency  that doe12-CTF chimera [Several nsSNPs within the highly conserved 7TM core  in ICL2, A761E (rs369201469 C > A) and V764M  in TM6 and N795K rs369853823, C > A) in TM7) led to a reduction in receptor-function Fig. . N795K c, C > A i76, C > TThe importance of cleavage for aGPCR function is still a matter of debate, yet GPR133-cleavage deficient mutants have been shown to display similar cell surface and cAMP levels compared to the wt receptor . In lineOur analysis includes data from three studies with a pathophysiological focus: (1) coronary artery disease , (2) maADGRD1 allele is compatible with life and does not result in a clinically relevant phenotype in humans. However, association of nsSNPs in ADGRD1 with common human diseases cannot be excluded. Of note, the nsSNP A446V, which was previously identified by a study searching for genes influencing human height [Since all variants were found in adults our data indicate that inactivation or constitutive activation of only one n height  displayeTo evaluate the capability of these receptor variants to be activated through the tethered peptide agonist of ADGRD1, we stimulated selected receptor mutants with 500 μM of the synthetic peptide agonist p13  Fig. 2d2d. As a The constitutively active F383S-mutant, as a part of the second β-strand in subdomain B of the GAIN domain , exhibitThe natural variant Q600stop  leads to a premature truncation of ADGRD1 in the first intracellular loop Fig. . NonetheThere is a natural variation of ADGRD1 that causes an extension of the reading frame Fig. . In C632cis-signaling’ while the NTF resolves the ‘trans-signaling’ to other cells. Since there is no report on ADGRD1-deficient animal models available the individual functions and the physiological relevance of NTF vs. CTF are unknown yet.In sum, there are at least two naturally occurring nsSNPs that lead to non-functional ADGRD1 due to gross alteration of the receptor protein chain. In contrast to classical rhodopsin-like GPCR, where truncation after the first transmembrane domain would be considered dysfunctional, aGPCRs are thought to have bimodal functions –37. HereADGRD1 gene showed that this receptor displays mutation frequencies that are similar to a prototypic GPCR. While functional consequences of the nsSNPs with respect to intracellular signal transduction are highly variable, cleavage does not seem to be affected. It seems that a range between complete abolishment of up to a 3-fold increase in ADGRD1 function is compatible with human life and development at least with only one allele being affected. Gene-deficient animal models will shed light on the physiologic relevance of this evolutionarily conserved GPCR. However, the existence of clinically healthy carriers for functionally relevant ADGRD1 variants will eventually lead to the identification of homozygous / compound heterozygous individuals exposing the phenotypic relevance also in humans.Our analysis of naturally occurring SNPs within the Thermo Fisher Scientific Inc., .If not stated otherwise, all standard substances were purchased from Sigma-Aldrich , Merck , and C. Roth GmbH . Cell culture material and primers were obtained from ADGRD1 were extracted (latest update 21-04-2015) from the NCBI database of Single Nucleotide Polymorphisms (dbSNP) [Non-synonymous SNPs (nsSNPs) found in human  (dbSNP) , which c (dbSNP) , exome s (dbSNP) , ClinSeq (dbSNP) ) as well (dbSNP)  and was ADGRD1 mRNA 3′UTR region (NM_198827.3) from human leucocytes  and insert a Flag tag into the new frame in front of the new stop codon to evaluate its expression levels. One of the nsSNPs, (Q600stop), results in a premature stop codon central of the intracellular loop 1 (ICL1). To generate this mutant it was necessary to clone the FLAG-epitope upstream of the stop signal .The human ADGRD1 was N-terminally tagged with a haemagglutinin (HA) epitope and C-terminally tagged with a FLAG epitope based on . nsSNPs All plasmids were introduced into the eukaryotic expression vector pcDps. All generated constructs were confirmed by sequencing.2 in a humidified atmosphere. One day prior to transfection, COS-7 cells were split into 48-well plates (3 × 104 cells/well for cell surface ELISA), or 6-well plates (3 × 105 cells/well for sandwich ELISA and Western Blot). For secreted alkaline phosphatase (SeAP) and luciferase assays HEK293T cells were split into 96-well plates (3 × 104 cells/well). Transfection of cells was done using Lipofectamine™ 2000  according to the manufacture’s protocol.COS-7 and HEK293T cells were cultured in Dulbecco’s minimum essential medium (DMEM) containing 10 % fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5 % COs-coupled receptor function was measured with cAMP response element (CRE) secreted alkaline phosphatase (SeAP) and CRE binding protein (CREB) luciferase assays. SeAP plasmid  and wt or mutant ADGRD1 constructs were co-transfected with each 75 ng of DNA/well. Basal and peptide agonist induced luciferase as well as SeAP levels were measured after 5 h of incubation. For SeAP experiments endogenously expressed alkaline phosphatases were heat inactivated for 2 h right before the measurement. 10 μM forskolin was used as positive control for CRE and CREB activities. As an additional readout for cAMP formation we measured luciferase activity under the control of CREB. Thus, co-transfection of 50 ng receptor plasmid and 50 ng luciferase plasmid (PathDetect trans-reporting System) was performed using HEK293T cells. Luciferase activity was detected using steadylite plus .Because ADGRD1 is a GTo detect cell surface and whole cell expression in transfected COS-7 cells ELISAs were used as described previously .Measurements were performed at least three times in triplicates. After normalization to mock-transfection (empty vector (EV)) data were compared to wt function and expression in percentage of each trial. All amino acid positions are based on wt ADGRD1 variant NP_942122.2. Graphical and statistical analyses were done using Prism version 5.01 . Statistical methods are described in the figure legends.aGPCR, adhesion G protein-coupled receptor; CRE, cAMP response element; CREB, CRE binding protein; CTF, C-terminal fragment; dbSNP, NCBI database of Single Nucleotide Polymorphisms; DMEM, Dulbecco’s minimum essential medium; ELISA, enzyme-linked immunosorbent assay; EV, empty vector; GAIN, GPCR autoproteolysis inducing; GPS, GPCR proteolysis site; GWAS, Genome-wide association study; HA, haemagglutinin; ICL1, intracellular loop 1; LamG3, Laminin G3; nsSNP, non-synonymous SNP; NTF, N-terminal fragment; PTX, pentraxin; SD, standard deviation; SeAP, secretion of alkaline phosphatase; SNP, single nucleotide polymorphism; UTR, untranslated regions; VLDL, very low density lipoproteins; wt, wildtype


The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function. The smooth muscle cells of blood vessels display considerable plasticity in their phenotype. In healthy, young blood vessels, the phenotype is largely contractile and blood pressure is well autoregulated. However, during the life span of an individual, vascular cells can switch to a synthetic, largely noncontractile phenotype in response to proinflammatory stimuli, diet or other factors that result in the development of atherosclerosis or vessel remodeling. We will not focus on these processes here but refer the reader to several recent reviews on this topic .Here we will focus on the contractile phenotype, which also can display plasticity of function through a range of more subtle adaptations to aging, biomechanical stress, and vasoactive physiologic and pathophysiologic molecules. The current review will focus on these responses and especially focus, as a prototype disease of contractile vascular smooth muscle, on the complex role of this cell type in hypertension and where many opportunities exist for the exploration of untapped potential therapeutic targets.In humans, the diagnosis of hypertension is widespread, but typically asymptomatic; 20–50% of the world’s population has hypertension and in the United States ∼30% of the population is hypertensive . Further and therefore blood pressure  result in severe hypertension, whereas mutations that decrease Na+ resorption (volume contraction) produce hypotension. We will not discuss the well-accepted role of renal fluid balance in regulation blood pressure, because this topic has been the subject of a number of reviews  inhibitors, angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs)], until recently, there was little experimental evidence consistent with the regulation of vascular tone being an important factor for the molecular mechanism that produces hypertension (2+ channel β1 subunit knockout (KO)  , estrogeO)      is more likely to be effective  trial demonstrated that treatment of black patients with heart failure with the combination of hydralazine and isosorbide dinitrate reduced mortality, whereas this regimen did not change mortality compared with placebo for white patients . In contThese racial and regional differences in the response to antihypertensive regimens could be due to polymorphisms. A number of genome wide-association studies (GWAS) have investigated this question  Ca2+ channels to produce a large repertoire of Ca2+ signals, which differ in their spatial and temporal distribution . Extracellular Ca2+ influx is mainly mediated by the opening of voltage dependent L-type Ca2+ channels (LTCC), but there are a number of other channels that modulate intracellular Ca2+, including transient receptor potential (TRP) cation channels. Because of their high single-channel conductance and expression in VSMCs, LTCCs have the largest influence on global [Ca2+]i, and their activity largely determines the VSMC’s contractile state and ultimately vessel diameter , like all other muscle cells, depend on Cadiameter . In resp2+ compartmentalization was introduced when it was demonstrated that local increases in Ca2+ could activate the contractile apparatus without influencing other Ca2+-dependent signaling pathways 2+ compar2+ resulting from influx through single or small clusters of LTCCs are called Ca2+ sparklets  activity and is counteracted by the serine phosphatase calcineurin. In pathologic conditions such as diabetes, however, protein kinase A (PKA) becomes a mediator of enhanced sparklet activity . In a strtension . In thisrtension . Althougrtension .It should be noted, that in some studies, there was significant PKC activation in agonist-mediated vasoconstriction, but not in pressure-mediated vasoconstriction , suggest2+-permeable, nonselective TRPV4 channels. These channels can produce Ca2+ sparklets with 100-fold higher Ca2+ flux compared with LTCCs, yet they have been linked to VSMC relaxation  found in VSMCs.In a recent study , investilaxation . This as2+ release from the SR, but overall LTCC sparklet activity is higher and hence LTCCs have a much greater effect on global Ca2+. Through the effect on global Ca2+, LTCC sparklet activity determines the rate at which the SR can refill its Ca2+ stores. However, neither the SR Ca2+ content nor the number and amplitude of SR Ca2+ release events appear to be directly linked to LTCC sparklet activity  that oppose vasoconstriction by allowing hyperpolarizing outward K+ currents (2+ gated Cl− channels (CaCCs) depolarize the plasmalemma and thereby enhance Ca2+ influx through LTCCs  inhibitors and IP3R blockers, which led to a decrease in [Ca2+i] and vasodilation. Therefore, IP3Rs contribute to Ca2+ waves in both tissues as part of a positive feedback loop for myogenic tone. In contrast, despite the inhibition of Ca2+ sparks and waves in feed arteries, inhibition of RyRs caused an increase in [Ca2+i] and led to vasoconstriction. This was abolished in the presence of BK-channel blocker paxilline, which supports the hypothesis that RyRs, which are involved in Ca2+ waves, are also coupled to BK channels and part of a negative feedback regulation of myogenic tone  and are inactivated as the [Ca2+] rises further. The IP3R’s Ca2+-dependent activation/inactivation properties are reflected in the wave-like pattern of IP3R-mediated Ca2+ release  relocates to the SR-plasmalemmal junction and physically interacts with and activates the selective Ca2+ channel Orai i in hypertension  containing transcription factors directly  cells and coordinated by a multitude of scaffolding proteins. However, these pathways are often overlapping, multilayered, and tissue specific. The tissue-specific nature of these pathways, even between different vessels or sizes of vessels, has led to much controversy on the relative importance of one pathway versus another. Ultimately, however, the possibility of multiple pathways that could be activated or inhibited in various disease states or as functional compensation to physiologic stress gives the system considerable functional plasticity.Pathways #1 & #2, Pathway #3, Pathway #4, At the simplest level, it is well established that vascular tone can be increased either by increasing activation of myosin . 2+i]) levels  decreases its activity, and conversely, inhibition of MP will increase its activity. A large number of pathways, such as those activated by PGF2a and lysophosphatidic acid   and delt2+ levels the G-2 variant translocates to the cortical focal adhesions  release, generated by the activation of GPCRs in vascular smooth muscle, or by myometrial stretch, RU486, and labor in the rat and human myometrium (2+ (Cae), phenylephrine (PE) will activate pathways 1, 3, and 4 but in the absence of Cae and in the absence of changes in 20kda light chain phosphorylation, a contraction persists in aorta of the ferret . In contractile smooth muscle, these downstream effects include phosphorylation of the actin binding protein caldesmon. Caldesmon has been described as being functionally analogous to the troponin complex in striated muscle in that it blocks the access of myosin to actin and hence impairs crossbridge cycling. The C-terminal end of caldesmon is responsible for the direct inhibition of myosin ATPase activity  in dVSM with the photoprotein aequorin, we noticed that agonists often cause tonic contractions with only transient increases in [Ca2+]i or differing magnitudes of [Ca2+]i, reflecting apparent changes in “Ca2+ sensitivity” of the contractile apparatus     is an ERK-binding and actin-binding protein that has been extensively studied in nonmuscle systems but little studied in smooth muscle systems. In cultured vascular smooth muscle cells, knockdown of IQGAP prevents the phosphorylation and activation of an actin-associated pool of ERK in response to PKC activation. Proximity ligation assays demonstrated direct tethering of ERK1/2 to actin by IQGAP. Interestingly caveolin is also required for activation of this pathway unless ERK is already associated with actin. Caveolin appears to be required specifically for upstream C-raf activation .Pathway #2, RIP, is thought to link active Rho/ROCK to the inhibition of MP , and hence, myosin activity  signaling pathways . These pY27632, the first ROCK inhibitor described, decreases blood pressure in 11-Deoxycorticosterone acetate (DOCA)-salt rat model of hypertension. A similar effect was obtained with the newer ROCK inhibitors fasudil, SAR07899, in other animal models of hypertension, including the spontaneously hypertensive rat (SHR), angiotensin II-induced hypertension in several animals, and L-NG-Nitroarginine Methyl Ester (L-NAME)-induced hypertension . Of noteThe endothelin pathway, linked to PLC and ERK signaling, has been identified as an effective antihypertensive target .Of interest is the fact that beta receptor mediated relaxation of vascular smooth muscle has been reported to decline with age in both the human and animal models. In aortas from Fischer 344 rats, an increase in the level of G-protein receptor kinase-2, which desensitizes the beta adrenergic receptor by phosphorylation of the receptor has been reported to increase with age , and thuPathway #1; The crossbridge cycle describes the development of force through a series of complexes between actin (A), myosin (M), ATP, and its hydrolysis products, ADP and Pi   and MLC phosphatase , i.e., Rlmodulin , whereaslmodulin . At a cotization , whereastization .MLC phosphatase is a holoenzyme  consistiPathway #2, 2+ sensitization has been the subject of a number of reviews  activation but increased the force produced by stimulation with both prostaglandin F2α and thromboxane (U46619). These results suggest that Rac1 signaling is agonist dependent and can result in either an enhancement of the Ca2+ transient and force or an inhibition of Ca2+ sensitization.Although the role of RhoA in the regulation of Cah muscle . Howeverh muscle , and Pakh muscle . Furtherh muscle . Recentlh muscle . Using a2+ sensitization  are produced by alternative splicing of a single gene. Alternative splicing of a 5′-site produces two isoforms that express a unique aa sequence of either 43  or 9  residues at the carboxy terminus of the SM MHC tail , whereas2+-activated skinned bladder strips to elevated Pi and ADP suggested that the lower shortening velocity of the SMA isoform is due to a slower rate of ADP dissociation or an additional force producing isomerization of the AM-ADP state in the crossbridge cycle , alternative splicing of exon six, which encodes 44 bp, also produces two isoforms, which differ in the expression of 9 aa at the carboxy terminus of the ELC17 . Exon 6 2+ sensitization (2+ desensitization (2+ desensitization) and/or nitric oxide (NO)-mediated vasodilatation  and carboxy-terminal leucine zipper (LZ+/−). The expression of the CI (residues 512–552)  is both tization , a role tization . The aa tization , which ctization . Subsequtization  as well tization , but thelatation . PKG pholatation , but MYPlatation . The mect Ser668 , 2014, at Ser668 . MYPT1 Lt Ser668 , and modt Ser668 , pre-eclt Ser668 , portal t Ser668 , pulmonat Ser668 , nitratet Ser668 , and sept Ser668 . Furthert Ser668 , 2014. TThe importance of the LZ domain of MYPT1 for the regulation of vascular tone has been established using two different transgenic mice. Because NO-mediated vasodilation is a fundamental property of the vasculature , preservβ, an atypical member of RNA binding proteins, are higher in fast (phasic) compared with slow (tonic) smooth muscle, and in an animal model of portal hypertension, Tra-2β is downregulated coincident with the decrease in LZ+ MYPT1 expression (E23 exon exclusion) and the shift to the expression of LZ− MYPT1 isoforms (β binds to E23 and transactivates E23 splicing (exon inclusion) to generate a LZ− MYPT1 isoform and deletion or mutation of the Tra2β binding site abolished E23 splicing (exon exclusion) to produce a LZ+ MYPT1. These investigators also showed that an siRNA-induced decrease of Tra-2β decreased E23 splicing to produce an increase in LZ+ MYPT1 expression, which is consistent with Tra-2β expression regulating E23 splicing and LZ+/− MYPT1 expression  of the resistance vessels; until recently, investigation of the signaling pathways regulating the myogenic response of the small resistance arteries was not possible. But, recent improvements in the sensitivity of protein phosphorylation levels have allowed for direct investigation of signaling pathways that regulate the myogenic response  myosin , NMIIA, The kinetics of the individual steps of the AMATPase of NMIIB  and NMIIμM ADP , which would decrease the rate of ATP usage to <0.01 ATP per head per second during force maintenance  characteristic of wild-type bladder smooth muscle , and thus, SVR is predominantly regulated at the level of the small resistance arteries with a diameter of 50–300 μm. Because of their small size, the molecular contractile phenotype of the resistance vessels have not been fully characterized, but the small resistance vessels express a mixture of fast and slow contractile proteins , and Ca2+ activation of these channels results in a membrane hyperpolarizing current that opposes vasoconstriction  was responsible for the increase in blood pressure in RGS-2 KO mice compared with WT littermates , but notably not FAK, undergo Src-dependent endosomal processing and regulate, through processes not yet fully understood, vascular contractility and stiffness . In contZyxin and vasodilator-stimulated phosphoprotein (VASP) are FA proteins associated with regulation of actin polymerization. This is consistent with a model whereby in VSMCs, only proteins at the edge of the focal adhesion, furthest from the plasmalemma, are capable of breaking off and undergoing remodeling in smooth muscle. This model is consistent with recent nanoscale super-resolution imaging of cultured fibroblast focal adhesion function described by An interesting scenario occurs immediately after birth when there is a drastic demand for a decrease in pulmonary vascular resistance. This triggers a transition from the intrauterine pulmonary vascular requirement for only 8–10% of cardiac output, whereas the placenta performs the function of the primary site of gas exchange to the independent support of the newborn’s lung function. As a result, in the first days to weeks of the newborn’s life, dramatic vascular remodeling is required. If this fails it is associated with the disorder of persistent pulmonary hypertension of the newborn. Interestingly, the remodeling is not just of the cells of the vascular tissue but also a subcellular remodeling of the actin isoforms within the vascular cells with a persistence of excessive gamma actin being a marker of persistent pulmonary hypertension of the newborn . Cortica2+ entry to the cell, activation of myosin light chain kinase, as well as increased activation of ROCK and protein kinase C pathways that regulate actin polymerization , especially in the context of aging, as an independent biomarker and predictor of hypertension and atherosclerosis, which are associated with increased mortality rates and severe damage to organ systems via stroke, renal failure, and heart disease . Given tAssessment of arterial stiffness exists on multiple scale levels. Macroscale techniques that characterize the hemodynamics of pulsatile blood flow are available clinically in humans and more easily interpretable, whereas microscale techniques better elucidate the underlying mechanisms causative of changes in the physical properties of blood vessels . In studIn vivo arterial stiffness can be measured representatively and noninvasively as pulse wave velocity (PWV). This measurement of the pulse wave generated by cardiac systole is the ratio of the distance it travels along the vascular wall to the time delay between its arrivals at different points along the circulatory pathway. Most commonly, PWV is measured via ultrasound between the carotid and femoral arteries as representative of aortic stiffness. As the current “gold standard” of arterial stiffness measurements, increased PWV is strongly correlated to both aging and CVD .E is the incremental Young’s modulus of the vessel, h and a are the thickness and radius of the aortic wall, respectively, and ρ is blood density. The value of E represents a material stiffness of the wall that is more precisely descriptive of its biomechanical properties. The Moens-Korteweg equation is itself dependent upon key assumptions: that the aortic wall is isotropic and homogeneous and that the aorta undergoes small isovolumetric changes in response to pulse wave propagation. Unfortunately, these assumptions do not hold, as demonstrated by previous studies  produced by endothelial cells induces vasodilation and relaxation in VSM . NO bioaRecently, attempts to quantify the constituency of total arterial stiffness attributable to VSMC activity have become more prevalent. Ex vivo studies have shown that VSMCs contribute significantly to total stiffness, potentially in conjunction with recruitment of collagen . Under oTherefore, it is of great interest to probe the cellular mechanisms that produce this effect to identify underlying origins of dysfunction and degeneration. A series of studies highlighting stimulated VSMCs have found that focal adhesions connecting the cortical cytoskeleton to the matrix play a critical role in the regulation of signaling cascades triggered by smooth muscle activation, independently of actomyosin crossbridges . In partIt has become increasingly clear that VSM may play a major role in the regulation of arterial stiffness.Epigenetic changes refer to the heritable alterations in gene expression that occur without changes in genome sequence . The conDNA methylation involves the covalent addition of a methyl group to the 5′-position of the pyrimidine ring of cytosine residues to form 5-methylcytosine . This prVariable methylation patterns are observed in the genes of VSMCs originating from different tissues . Abnormaα-actin and SM MHC in VSMC gene loci -dependent deacetylase activity  residues of a wide range of nonhistone proteins and are therefore also denoted as KATs and KDACs, respectively . Nonhistα-actin , tropomyα-actin . KDAC6 iα-actin . KDAC inα-actin . In a seα-actin .Epigenetic mechanisms influence the changes in vascular structure that occur during hypertension . Angiote+-dependent deacetylation. Currently, there are seven known mammalian sirtuins (SIRT1-7), located in the nucleus   , cytopla, and 7) . Becausend ISDH2 . Extensind ISDH2 ,α, which modulates vascular tone and enhances cell survival through induction of antioxidant enzymes to promote angiogenesis -2ogenesis . The deaogenesis . Overexpogenesis .Interestingly, recent studies implicated SIRT1 in blood pressure regulation through targeting VSMCs. Overexpression of SIRT1 in VSMCs has been found to modulate blood pressure by downregulation of the Ang II type 1a receptor gene . MoreoveNoncoding RNAs comprise many functional RNA transcripts that are not transcribed into proteins, but regulate the transcription, stability, or translation of protein-encoding genes . NoncodiCaenorhabditis elegans larval development more than 20 years ago (MicroRNAs (miRs) are a family of short (21–25 nucleotide) RNAs, which typically negatively regulate protein translation by direct binding to the 3′ untranslated (3′ UTR) region of mRNA targets. The first miR, lin-4, was discovered to have a role in ears ago . To dateears ago . It is nMicroRNAs are transcribed in the nucleus by RNA polymerase II to long primary transcripts  Fig. 10Fig. 10. There are several excellent reviews that discuss the role of miRs in vascular structure, function, and disease . The curThe Dicer KO model has also demonstrated an important role for miRs in the regulation of small arterial myogenic tone, therefore modulating peripheral arterial resistance, which is raised in hypertension. Myogenic tone of mesenteric arteries was abolished from Dicer KO mice, which was associated with a loss of calcium influx through the L-type calcium channel . It is tα, through the targeted downregulation of KLF4 and 5 , arbitrarily classified as longer than 200 nucleotides, have a role in regulating the expression of neighboring genes. LncRNAs have a much more widespread mode of action than miRs, and their function cannot currently be inferred from sequence or structure . LncRNAsAlthough the field is newer than that for miRs, LncRNAs are emerging as potentially important players in cardiovascular disease , thus aiding the design of preclinical studies to determine their efficacy and safety. In addition, miRs have the potential to target many members of the same molecular pathway and as a result having a greater combined effect than siRNAs, which typically target only a single gene. For example, the miR-200 family has been implicated in regulating many targets that control actin filament organization and dynamics . FurtherThere are two main techniques to modulate miR activity in vivo . For example, miR-130a promotes PASMC proliferation through the negative regulation of its targets CDKN1A and growth arrest-specific homeobox . Interestriction .2α receptor that was more prevalent in hypertensive individuals ), anti-miR-92 , and anti-miR-145  have already reached preclinical trials. This has clearly been a rapidly expanding field and we expect to see many further developments, including approaches to target LncRNAs, in the coming years.2+, whereas ACE inhibitors and ARBs will decrease the activation of the RhoA/Rho kinase signaling pathway to reduce Ca2+ sensitization. K+ channel openers produce vasodilatation; nicorandil acts on BK channels and hyperpolarizes smooth muscle cells  all target the smooth muscle cell to decrease vascular tone and blood pressure. CCBs decrease the activation of SM myosin by decreasing intracellular Cale cells , whereasle cells . The mecothelium .In addition to essential hypertension, there are a number of other diseases of the vasculature and diseases with associated abnormal vascular reactivity. These defects in vascular reactivity contribute to morbidity and mortality. In this section, we will outline these diseases, their mechanism, current therapies, and suggest novel targets for both the treatment of essential hypertension as well as these other diseases of the vasculature .Considerable evidence from both human subjects and animal models have shown that HF is associated with an impaired vasodilatory response to acetylcholine, via an endothelium-dependent mechanism, as well as to nitroglycerin, via an endothelial-independent mechanism . Both MYβ (κB to produce a proinflammatory state by inducing expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α -α  and also (TNF)-α . In hear (TNF)-α . Further (TNF)-α , which sα  is a rare, incurable disease with a poor prognosis . In patiThere are two components that produce PAH: the extent of the structural changes  downregulation of AKAP150, 2) increasing the expression of the β1 subunit of the BK channel, 3) novel BK channel openers, 4) inhibiting or decreasing the expression of STIM/Orai (Ca2+ release-activated Ca2+ channels), and 5) increasing cleavage of the C terminal of LTCC or increasing CCt expression. One could also consider inhibition of MLCK as a novel target; the soybean isoform of calmodulin (SCaM-4) has been demonstrated to bind Ca2+ but not activate MLCK and thus inhibit the activation of smooth muscle  and NO unresponsive to a lower force (phasic) and NO responsive and hence produce a reduction in vascular tone and blood pressure.There is no question that essential hypertension is due, in part, to changes at the level of vascular smooth muscle that lead to excess vasoconstriction and a concomitant increase in SVR and blood pressure. In this review, we suggested have several novel targets for the treatment of essential hypertension. Details and the strategy for the each target can be found in the individual sections. These targets include those that affect Cah muscle . Thus, tThese novel targets could form the basis for rationale drug design, and could be exploited for development of therapeutic agents that are effective for the treatment of hypertension. For some targets, small molecule inhibitors/activators would appear to be a reasonable choice, whereas for other targets, i.e., to reverse the decreases in MYPT1 expression, adenoviral delivery of MYPT1 to the vasculature could also be used as a strategy to increase the sensitivity to NO or flow-mediated vasodilation, which would decrease in vascular tone and blood pressure.2+ signaling, regulation of the cytoskeleton and contractility, and biomechanics to the pathogenesis of essential hypertension may vary among ethnic groups and/or individuals. Currently, antihypertensive therapy is generally approached by a method that could be best summarized as trial and error: an agent is selected, usually without consideration of the patient’s ethnic background, and if control is not adequate, either the dose is increased or another agent is added.The contribution of alteration in the vascular phenotype from CaIn this review, we discussed the importance of the vascular phenotype in the mechanism that leads to essential hypertension. Therefore, mechanisms to identify the etiology of the increase in blood pressure in each patient are important for ultimately selecting an individualized and effective therapeutic agent. These future studies will hopefully lead to a more personalized approach to antihypertensive therapy.