Increased RhoA/Rho-kinase signaling mediates spontaneous tone in aorta from angiotensin II-induced hypertensive rats

Spontaneous tone in large arteries may contribute to the pathogenesis of hypertension. Reactive oxygen species and Ca2+ influx have been shown to stimulate the development of spontaneous tone in isolated aortic rings in several models of hypertensive rats. The aim of this study was to investigate the role of the RhoA/Rho-kinase signaling pathway in the development of spontaneous tone in angiotensin II-induced hypertension and to explore the underlying mechanisms of RhoA/Rho-kinase activation. Our results showed that spontaneous tone was greatly enhanced in endothelium-denuded aortic rings from angiotensin II-induced hypertensive rats compared with their normotensive counterparts (73+/-5 versus 7+/-3% of phenylephrine-induced maximal contraction, respectively). The Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) (0.1-10 microM) concentration dependently inhibited spontaneous tone in aortic rings from angiotensin II-treated rats. NADPH oxidase inhibitors diphenylene iodonium and apocynin also significantly reduced spontaneous tone. Chronic angiotensin II treatment markedly increased RhoA protein expression (57%) but had no effect on Rho guanine nucleotide exchange factor mRNA or Rho-kinase protein expression levels. In endothelium-denuded rings from normotensive rats, angiotensin II (100 nM) increased RhoA membrane translocation and phosphorylation of the myosin light chain phosphatase target subunit, which were both blocked by the NADPH oxidase inhibitor diphenylene iodonium (10 microM). In conclusion, these data suggest that chronic treatment with angiotensin II leads to up-regulation of the RhoA/Rho-kinase pathway, contributing to spontaneous tone development in rat aorta. Increased NADPH oxidase-dependent reactive oxygen species may be one of the mechanisms mediating the RhoA/Rho-kinase activation.     

WNT signaling in stem cell differentiation and tumor formation

Embryonic stem cells (ESCs) hold great therapeutic promise for the regeneration of functional cell types and clinical applications. However, tumorigenic potential of stem cells in a transplanted host remains a major obstacle. In this issue of the JCI, Cui and colleagues identified TCF7-mediated canonical WNT signaling as a critical determinant of both the tumorigenicity and therapeutic function of ESC-derived retinal progenitor cells (ESC-RPCs). Their findings suggested that addressing key extracellular signaling and related intrinsic factors will be essential for the successful use of ESC-derived progenitor transplantation. Photoreceptor degeneration underlies major causes of blindness, including macular degeneration and retinitis pigmentosa, affecting tens of millions of people worldwide. In theory, vision of affected patients may improve if the diseased cells (rods and cones) are replaced with new, healthy cells that form appropriate connections with the host retina. Embryonic stem cells (ESCs) possess unlimited self-renewal capabilities and the ability to differentiate into any adult cell type (1). These unique features make ESC-based therapy appealing for the treatment of various degenerative disorders but may also cause unwanted serious side effects. It has been previously demonstrated that ESCs or ESC-derived progenitors spontaneously form tumors upon transplantation in vivo, even when the cells are predifferentiated or presorted (2). Despite this issue, successful cell replacement of human ESC–derived (hESC-derived) retinal cells for vision restoration in animal models of photoreceptor degeneration has been reported recently. Photoreceptor precursors derived from hESCs have been shown to migrate into Crx-deficient mouse retina following intraocular injection, express appropriate markers for both rod and cone photoreceptors, and subsequently restore some light responses (3). In another study, subretinal transplantation of hESC-derived retinal pigment epithelium in patients with Stargardt’s macular dystrophy and dry age-related macular degeneration improved visual acuity, with no signs of hyperproliferation or tumorigenicity after 4 months (4). These examples illustrate purity, stability, and proper localization of transplanted cells in vivo and prompted the development of numerous differentiation protocols. Still, the risk of tumor formation remains a barrier, because there are no parameters to quantify safety factors and because the appropriate stage of differentiation at which ESC-derived progenitors should be used has not been well evaluated. In this issue, Cui and colleagues attempted to address the above concerns by identifying major extracellular signaling and intrinsic factors controlling tumorigenicity and therapeutic potential of ESC-derived retinal progenitor cells (ESC-RPCs) (5).     

Increased folate catabolism in mice with ascitic tumours

Folate deficiency is reported in association with certain malignant tumours, and it has been suggested that this arises from increased folate turnover and catabolism in such circumstances. Using an experimental animal model to determine the rate of catabolism of [3H]pteroylglutamate (folic acid) by the quantitative estimation of the two urinary catabolites p-[3H]aminobenzoylglutamate and [3H]acetamidobenzoylglutamate, we have measured the rate of folate catabolism in mice with ascitic tumours. There was a significant increase in the rate of catabolism in the mice with tumours compared with controls over a 10 day period. This was associated with the accumulation of ascitic fluid and an increase in the number of tumour cells in the treated animals. The increase in catabolism appeared to be due to increased cell turnover of the tumour rather than an increase in cell mass, as the increase in mass of the tumour was negligible.     

WNT1-inducible signaling protein–1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/β-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein–1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.     

Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts

Mice deficient in Schnurri-3 (SHN3; also known as HIVEP3) display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts. Here we identified SHN3 as a dampener of ERK activity that functions in part downstream of WNT signaling in osteoblasts. A D-domain motif within SHN3 mediated the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction resulted in aberrant ERK activation and consequent osteoblast hyperactivity in vivo. Additionally, in vivo genetic interaction studies demonstrated that crossing to Lrp5^–/– mice partially rescued the osteosclerotic phenotype of Shn3^–/– mice; mechanistically, this corresponded to the ability of SHN3 to inhibit ERK-mediated suppression of GSK3β. Inducible knockdown of Shn3 in adult mice resulted in a high–bone mass phenotype, providing evidence that transient blockade of these pathways in adults holds promise as a therapy for osteoporosis.     

IL-20RB mediates tumoral response to osteoclastic niches and promotes bone metastasis of lung cancer

Bone is a common site of metastasis in lung cancer, but the regulatory mechanism remains incompletely understood. Osteoclasts are known to play crucial roles in osteolytic bone metastasis by digesting bone matrix and indirectly enhancing tumor colonization. In this study, we found that IL receptor 20 subunit β (IL-20RB) mediated a direct tumoral response to osteoclasts. Tumoral expression of IL-20RB was associated with bone metastasis of lung cancer, and functionally, IL-20RB promoted metastatic growth of lung cancer cells in bone. Mechanistically, tumor cells induced osteoclasts to secrete the IL-20RB ligand IL-19, and IL-19 stimulated IL-20RB–expressing tumor cells to activate downstream JAK1/STAT3 signaling, leading to enhanced proliferation of tumor cells in bone. Importantly, blocking IL-20RB with a neutralizing antibody significantly suppressed bone metastasis of lung cancer. Overall, our data revealed a direct protumor role of osteoclastic niche in bone metastasis and supported IL-20RB–targeting approaches for metastasis treatment.     

Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure

Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of beta-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.     

Increased oxidant activity mediates vascular dysfunction in vibration injury

Occupational exposure to hand-operated vibrating tools causes a spectrum of pathological changes in the vascular, neurological, and musculoskeletal systems described as the hand-arm vibration syndrome (HAVS). Experiments were performed to determine the effects of acute vibration on the function of digital arteries. Rats paws were exposed to a vibrating platform (4 h, 125 Hz, constant acceleration of 49 m/s(2) root mean squared), and digital artery function was assessed subsequently in vitro using a pressure myograph system. Constriction to phenylephrine or 5-hydroxytryptamine was reduced in digital arteries from vibrated paws. However, after endothelium denudation, constriction to the agonists was no longer impaired in vibrated arteries. Inhibition of nitric-oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. However, nitric oxide (NO) activity, determined using the NO-sensitive probe 4-amino-5-methylamino-2', 7'-difluorofluorescein, was reduced in vibrated compared with control arteries. Endogenous levels of reactive oxygen species (ROS), determined using the ROS-sensitive probe 5-(and 6)-chloromethyl-2',7'-dichlorodihydro-fluorescein, were increased in vibrated compared with control arteries. The increased ROS levels were abolished by L-NAME or by catalase, which degrades extracellular hydrogen peroxide. Catalase also increased constriction to phenylephrine or 5-hydroxytryptamine in vibrated but not control arteries and abolished the vibration-induced depression in constrictor responses. The results suggest that acute vibration causes vascular dysfunction in digital arteries by increasing ROS levels, which is probably mediated by uncoupling of endothelial NOS. Therefore, therapeutic strategies to inhibit ROS or augment NO activity may be beneficial in HAVS.     

Increased running response to morphine in morphine-pretreated mice.

The running response of B6AF1/J mice to 25 mg/kg of morphine sulfate was increased up to 3-fold when this dose was administered either twice daily for 5 days or once a week for 2 or 3 weeks. The effect of weekly pretreatment was proportional to the dose of morphine and lasted as long as 1 month after pretreatment was stopped. There was no sensitization when the mice were less than 15 days old at the time of pretreatment. Of the parental strains, untreated C57Bl/6J mice showed a good running response to morphine, while A/J mice showed little response. Pretreatment of either of these strains produced only slight sensitization. Pretreatment of the hybrids with levorphanol increased the response to morphine. Dextrorphan and naloxone were ineffective. Sensitization by morphine was blocked by naloxone. Increased morphine running was not associated with analgesic tolerance as measured by the tail-flick assay. Morphine pretreatment produced some increase in the running response to amphetamine and to cocaine. Pretreatment with amphetamine or cocaine did not increase the response to morphine.     

CD36 mediates the innate host response to beta-amyloid

Accumulation of inflammatory microglia in Alzheimer's senile plaques is a hallmark of the innate response to beta-amyloid fibrils and can initiate and propagate neurodegeneration characteristic of Alzheimer's disease (AD). The molecular mechanism whereby fibrillar beta-amyloid activates the inflammatory response has not been elucidated. CD36, a class B scavenger receptor, is expressed on microglia in normal and AD brains and binds to beta-amyloid fibrils in vitro. We report here that microglia and macrophages, isolated from CD36 null mice, had marked reductions in fibrillar beta-amyloid-induced secretion of cytokines, chemokines, and reactive oxygen species. Intraperitoneal and stereotaxic intracerebral injection of fibrillar beta-amyloid in CD36 null mice induced significantly less macrophage and microglial recruitment into the peritoneum and brain, respectively, than in wild-type mice. Our data reveal that CD36, a major pattern recognition receptor, mediates microglial and macrophage response to beta-amyloid, and imply that CD36 plays a key role in the proinflammatory events associated with AD.     

Decreased atherogenic response to dietary cholesterol in pigeons after stimulation of cholesterol catabolism in early life

Cholesterol catabolism was stimulated in 6-wk-old White Carneau pigeons using a laboratory stock diet containing 1.3% cholestyramine resin. After 8 wk on this diet the animals were returned to control stock diet (no resin) for another 8-wk period. When subsequently challenged with a diet containing 0.5% cholesterol, cholestyramine-pretreated pigeons exhibited significantly lower serum cholesterol level when compared with controls and this "hyporesponder" behavior persisted throughout the study period. Furthermore, the aorta of cholestyramine-treated animals exhibited significantly (a) lower prevalence and severity of atherosclerosis and (b) lower cholesterol content. These studies demonstrate for the first time that enhancement of cholesterol catabolism in early life improves resistance to diet-induced atherosclerosis in later life in this avian model.     

The Ras signaling pathway mediates cetuximab resistance in nasopharyngeal carcinoma

This work aimed to investigate the role of the Ras signaling pathway in cetuximab resistance in human nasopharyngeal carcinoma (hNPC). An hNPC 5-8F cell line was induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect protein levels. Our results are as follows: cetuximab-resistant hNPC 5-8F/Erbitux cell lines were successfully developed. After treatment with cetuximab for 3 and 5 d, the RI was 1.2 and 1.1, respectively. Compared with the 5-8F cells, the protein expression levels of H-ras, JNK/P-JNK, P-ERK1/2, p38/P-p38, P-AKT, NF-κB p65/P-NF-κB p65 and c-fos were significantly increased in the 5-8F/Erbitux cells (P = 0.000 for all); however, the protein expression levels of ERK1/2 and c-jun/P-c-jun were significantly decreased (P = 0.000 for all) and AKT protein expression showed no significant change (P = 0.176). After the 5-8F/Erbitux cells were transfected with H-ras shRNA, H-ras protein expression was significantly decreased (P = 0.000) and cetuximab sensitivity improved. In contrast, in the 5-8F/Erbitux + siH-ras cells, protein expression levels of P-ERK1/2, P-JNK, P-AKT and NF-κB p65/P-NF-κB p65 were significantly decreased (P = 0.000 for all). Additionally, protein expression levels of JNK, ERK1/2, p38/P-p38 and c-jun/P-c-jun were significantly increased (P = 0.000 for all), but protein expression levels of AKT and c-fos did not change significantly (P = 0.061 and P = 0.068, respectively). In conclusion, the activation of the H-ras/ERK1/2, H-ras/JNK and PI3K-AKT signaling pathways is closely associated with cetuximab resistance in 5-8F/Erbitux cells. NF-κB is activated in 5-8F/Erbitux cells without activation of c-jun.     

间隙连接通讯功能对小剂量甲状旁腺激素成骨效应调控的机制

INTRODUCTIONBoneformativeprocessesinducedbyintermittenttreatmentoflow－doseparathyroidhormones（PTH）possessnovelandhopefullytherapeuticeffectsonosteoporosis[１－２]．Theimportantsignaltrans－ductionsandmechanismsmediatedbygapjunctionalintercellularcommunication（GJIC）viagapjunctions（GJ）composedofConnex－in４３（Cx４３）betweenadjacentosteoblastsisconceivedtoplaycru－cialrolesintheseprocesses[３－４]．Whereas，theessentialcontributionsofGJICtolow－dosePTHassociatedboneformationa…     

Study on roles of gap junctional intercellular communication in low dose parathyroid hormones-induced bone anabolism in vitro

AIM:To investigate mediating and reguatory effects of osteoblastic gap junctional interecllular communication(GJIC)on low-dose parathyroid hor-mones(PTH)-stimulated bone formation activties in vitro.METHODS:Rat calvarial osteoblasts(ROBs) in cultures wer divided into three groups ac-cording to the different mode of exposure.Group A:vehicle (sodium acetate,SA)-treated group;GroupB:1&;#215;10^-8mol/L hPTH(1-34)intermittent exposure group;GroupC:1&;#215;10^-8mol/LhPTH(1-34)+1&;#215;10^-7mol/L TPA exposure group.48h incubation cycles in three groups were repeated for eight times.GJIC and mineralized bone nodules formation in three groups were detected using Lucifer Yellow(LY) scrape loading dye transfer(SLDT) and mineralized nodule staining together with nodule index,respectively.RESULTS:At various measuring time points of SA&;#215;6h+TPA&;#215;1h in group C,LY(+) cell numbers were 6.8&;#177;2.5,19.5&;#177;6.5,14.0&;#177;3.6 and 5.7&;#177;2.4,respectively.Diffusion and transfer of LY fluorescent probe was much more noticeably discerned in group B than in group A and C(P&;lt;0.01).Mineralized bone nodule indices were 45.2&;#177;12.5,88.0&;#177;15.3and 38.5&;#177;17.9 in group A,B and C respectively.Bone formation activity was much better revealed in group B than in group A and C(P&;lt;0.01）,whereas no statistically significant difference of bone formation activities were found in group A compared with group C(P=0.465).CONCLUSION:Mediations and regulations of the coordinating signals in osteoblastic network via GJIC essentially contribute to PTH-stimulated bone anabolism.However,disruption of GJIC not only hinders osteoblastic intercellular coordination but also frustrated PTH-induced bone formation activities in vitro.Therefor,GJIC may evidently play imprtant roles in regulations on low-dose PTH-induced bone formation.     

Increased renal catabolism of 1,25-dihydroxyvitamin D3 in murine X-linked hypophosphatemic rickets.

The hypophosphatemic (Hyp) mouse, a murine homologue of human X-linked hypophosphatemic rickets, is characterized by renal defects in brush border membrane phosphate transport and vitamin D3 metabolism. The present study was undertaken to examine whether elevated renal 25-hydroxyvitamin D3-24-hydroxylase activity in Hyp mice is associated with increased degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by side chain oxidation. Metabolites of 1,25(OH)2D3 were separated by HPLC on Zorbax SIL and identified by comparison with standards authenticated by mass spectrometry. Production of 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, and 24-oxo-1,23,25-trihydroxyvitamin D3 was twofold greater in mitochondria from mutant Hyp/Y mice than from normal +/Y littermates. Enzyme activities, estimated by the sum of the three products synthesized per milligram mitochondrial protein under initial rate conditions, were used to estimate kinetic parameters. The apparent Vmax was significantly greater for mitochondria from Hyp/Y mice than from +/Y mice (0.607 +/- 0.064 vs. 0.290 +/- 0.011 pmol/mg per protein per min, mean +/- SEM, P less than 0.001), whereas the apparent Michaelis-Menten constant (Km) was similar in both genotypes (23 +/- 2 vs. 17 +/- 5 nM). The Km for 1,25(OH)2D3 was approximately 10-fold lower than that for 25-hydroxyvitamin D3 [25(OH)D3], indicating that 1,25(OH)2D3 is perhaps the preferred substrate under physiological conditions. In both genotypes, apparent Vmax for 25(OH)D3 was fourfold greater than that for 1,25(OH)2D3, suggesting that side chain oxidation of 25(OH)D3 may operate at pharmacological concentrations of substrate. The present results demonstrate that Hyp mice exhibit increased renal catabolism of 1,25(OH)2D3 and suggest that elevated degradation of vitamin D3 hormone may contribute significantly to the clinical phenotype in this disorder.     

Increased dilator response to nitrate and decreased flow-mediated dilatation in migraineurs

Background.—It has been known that in a migraine attack intracranial and extracranial arteries on the headache side dilate and when the migraine attack has subsided, the intracranial arteries show segmental narrowing. We hypothesized that patients with migraine had an underlying systemic vasomotion abnormality and there might be an increased nitrate-mediated vasodilatory response in the brachial artery of migraineurs. Accordingly we aimed to measure endothelium dependent and independent functions of brachial artery in migraineurs and healthy subjects.
Materials and Methods.—Twenty-four patients who fulfilled the diagnostic criteria of migraine were enrolled in the study. Twenty-six age- and sex-matched healthy control subjects comprised the control group. Flow-mediated dilatation and nitrate-mediated dilatation were measured in all patients and control subjects by means of brachial artery ultrasonography.
Results.—Flow-mediated dilatation of patients with migraine was significantly lower than that of control subjects (7.6 ± 3.7% vs 10.4 ± 3.5%, respectively, P = .008). However, nitrate-mediated dilatation in migraineurs was significantly higher than that of nonmigraineurs (25% vs 14%, respectively, P < .001).
Conclusion.—We have shown that migraineurs have decreased endothelium dependent function whereas increased nitrate-mediated response in their brachial artery. It can be suggested that the mechanism underlying migraine may be a diffuse vascular vasomotion abnormalities and migraine may be a local manifestation of systemic vascular abnormality rather than a primary cerebral phenomenon.