Liquid crystal elastomer foams with elastic properties specifically engineered as biodegradable brain tissue scaffolds.

Tissue regeneration requires 3-dimensional (3D) smart materials as scaffolds to promote transport of nutrients. To mimic mechanical properties of extracellular matrices, biocompatible polymers have been widely studied and a diverse range of 3D scaffolds have been produced. We propose the use of responsive polymeric materials to create dynamic substrates for cell culture, which goes beyond designing only a physical static 3D scaffold. Here, we demonstrated that lactone- and lactide-based star block-copolymers (SBCs), where a liquid crystal (LC) moiety has been attached as a side-group, can be crosslinked to obtain Liquid Crystal Elastomers (LCEs) with a porous architecture using a salt-leaching method to promote cell infiltration. The obtained SmA LCE-based fully interconnected-porous foams exhibit a Young modulus of 0.23 ± 0.07 MPa and a biodegradability rate of around 20% after 15 weeks both of which are optimized to mimic native environments. We present cell culture results showing growth and proliferation of neurons on the scaffold after four weeks. This research provides a new platform to analyse LCE scaffold-cell interactions where the presence of liquid crystal moieties promotes cell alignment paving the way for a stimulated brain-like tissue.

Non-Fermi-liquid behavior within the ferromagnetic phase in URu2-xRexSi2.

The URu2-xRexSi2 system exhibits ferromagnetic order for Re concentrations 0.3 < x < or =1.0. Non-Fermi-liquid (NFL) behavior is observed in the specific heat for 0.15< or = x< or =0.6 [C/T proportional to, -lnT (or T(-0.1))], and also in the power-law T dependence of the electrical resistivity [rhoT proportional to, Tn] with n<2 for 0.15< or = x <0.8, at low T, providing strong evidence that the NFL behavior persists within the ferromagnetic phase. Furthermore, the deviation of the physical properties of URu2-xRexSi2 from Fermi-liquid behavior is most pronounced at the ferromagnetic quantum critical point, and the NFL behavior found in the ferromagnetic phase may be consistent with the Griffiths-McCoy phase model.

The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism.

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.

Interrater reliability of the National Institutes of Health Stroke Scale: rating by neurologists and nurses in a community-based stroke incidence study.

The reliability of the National Institutes of Health Stroke Scale (NIHSS) for use by trained neurologists in clinical trials of acute stroke has been established in several hospital-based studies. However, it also has the potential for application in community-based settings and to be used by nonneurologists: issues which have not been explored before. Hence, we aimed to determine the reliability of the NIHSS when administered by research nurses within the existing North Eastern Melbourne Stroke Incidence Study. Using the NIHSS, thirty-one consecutively registered stroke patients were assessed by 2 neurologists and 1 of 2 trained research nurses. The interrater reliability of observations was compared using weighted and unweighted kappa statistics and intraclass correlation coefficients (ICC). There was a high level of agreement for total scores between the 2 neurologists (ICC = 0.95) and between each neurologist and research nurse (ICC = 0.92 and 0.96). While there was moderate to excellent agreement among neurologists and research nurse (weighted kappa > 0.4) for the majority of the NIHSS items, there was poor agreement for the component 'limb ataxia'. Overall, agreement between nurse and neurologist for individual items was not significantly different from agreement between neurologists. It appears that in both hospital and community settings, trained research nurses can administer the NIHSS with a reliability similar to stroke-trained neurologists. This ability could be used to advantage in large community-based trials and epidemiological studies.

The role of AT1 angiotensin receptor activation in the pathogenesis of preeclampsia.

OBJECTIVES: Preeclampsia is characterized by an increase in vascular tone associated with reduced uteroplacental flow. The nature of hypertension arising in pregnancy suggests that the abnormal increase in blood pressure is dependent on some humoral factor that mediates vasospasm. There is evidence that preeclampsia results from a breakdown in the balance between vasodilators such as prostacyclin and prostaglandin E2 and nitric oxide and the vasoconstrictors angiotensin II, thromboxane A2, serotonin, and endothelin. Furthermore, vascular reactivity to angiotensin II is greatly enhanced in preeclampsia as opposed to normal pregnancies. The increased vascular tone and the enhanced thromboxane production noted in preeclampsia may be mediated by the increased sensitivity to angiotensin II because angiotensin II coupled to an AT1 receptor is a potent vasoconstrictor and stimulates the accumulation of free arachidonic acid, the precursor of thromboxane and the prostaglandins. STUDY DESIGN: We used a rat model that has been shown to express the relevant clinical features of human preeclampsia to investigate the involvement of the AT1 angiotensin receptor in this pathologic condition. Pregnant rats were divided into three groups that were either infused with saline or endotoxin on the 14th day of pregnancy. One of the endotoxin-infused groups was further treated with the AT1-selective antagonist losartan from day 11 until day 19 of pregnancy. RESULTS: Perinatal outcome, blood pressure, and urine protein were monitored for each group. We observed that endotoxin infusion resulted in a decrease in pup weight and number of pups and caused an increase in mean arterial pressure as well as increased proteinuria when compared with saline solution-infused animals. In contrast, endotoxin-infused rats receiving losartan exhibited no change in number or weight of pups when compared with control, and losartan tended to diminish the rise in mean arterial pressure. In addition, the increase in urinary protein excretion was completely blocked by losartan. CONCLUSIONS: Endotoxin infusion in pregnant rats appears to be a suitable model for the study of preeclampsia. Moreover, the angiotensin II-dependent activation of an AT1 receptor appears to mediate a portion of the pathophysiologic features associated with preeclampsia.

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells.

The accumulation of radiolabeled arachidonic acid (AA), immunoblot analysis of subcellular fractions, and immunofluorescence tagging of proteins in intact cells were used to examine the coupling of ANG II receptors with the activity and location of a cytosolic phospholipase A2 (cPLA2) in vascular smooth muscle cells (VSMC). ANG II induced the accumulation of AA, which peaked by 10 min and was downregulated by 20 min. A large proportion of the AA released in response to ANG II was due to the activation of a Ca(2+)-dependent lipasc coupled to an AT1 receptor. However, regulation of Ca2+ availability failed to completely block AA release, and a small but significant reduction in ANG II-mediated AA release was observed in the presence of an AT2 antagonist. These findings, coupled with a 25% reduction in the ANG II-induced AA release by an inhibitor specific for a Ca(2+)-independent PLA2, are consistent with the presence and activation of a Ca(2+)-independent PLA2. In contrast, immunoblot analysis and immunofluorescence detection showed that the ANG II-mediated translocation of cPLA2 to a membrane fraction was exclusively AT1 dependent and regulated by Ca2+ availability. Furthermore, the nucleus was the membrane target. We conclude that ANG II regulates the Ca(2+)-dependent activation and translocation of cPLA2 through an AT1 receptor and that this event is targeted at the nucleus in VSMC.

Angiotensin-(1-7) inhibits vascular smooth muscle cell growth.

Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.

Angiotensins differentially activate phospholipase D in vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats.

We previously showed that angiotensin (Ang) II activates phospholipase D (PLD) through AT1 receptors in vascular smooth muscle cells (VSMC) isolated from Sprague-Dawley rats [Freeman and Tallant, Biochem J. 304:543-548, (1994)]. In the present study, we compared activation of PLD by angiotensin peptides in VSMC from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats. Ang II caused a dose-dependent increase in PLD activity in VSMC from both rat strains. However, the response to Ang II in VSMC from hypertensive rats was approximately three times higher than that observed in VSMC from normotensive controls. Furthermore, Ang II-induced activation of PLD in VSMC from hypertensive rats was significant within 1 min, whereas significant increases in PLD activity in cells from normotensive rats were not seen until 10 min after exposure to Ang II. Ang-(2-8) caused a similar increase in PLD activity which was three times higher in SHR VSMC than in WKY controls. In contrast, Ang-(1-7) did not affect PLD activity in either smooth muscle cell population. The Ang II-mediated increases in PLD activity in VMSC from both rat strains were completely blocked by AT1 receptor antagonists (EXP 3174 or L-158,809). Conversely, the AT2 receptor antagonist PD 123177 (1 mumol/L) was ineffective. Thus Ang II stimulation of PLD in VSMC derived from both the hypertensive and normotensive rat aorta and the accumulation of its metabolites (e.g., phosphatidic acid and diacylglycerol) is coupled to activation of AT1 receptors predominantly and occurs in response to Ang II or Ang-(2-8) but not Ang-(1-7). Moreover, activation of PLD by angiotensins in VMSC from the SHR is significantly more robust than that observed in VSMC from the normotensive WKY rat. We conclude that increased activation of PLD by Ang II in genetically-induced hypertension may reflect an additional mechanism linking enhanced contractile responses to enhanced growth.

Role of calcium and protein kinase C in the activation of phospholipase D by angiotensin II in vascular smooth muscle cells.

We previously showed that cultured rat aortic vascular smooth muscle cells (VSMC) possess an AT1 angiotensin (Ang) receptor coupled to the activation of a phospholipase D (PLD). AT1 receptors in VSMC are also coupled to the activation of a phosphoinositide-specific phospholipase C (PLC), mobilization of intracellular Ca2+, and activation of protein kinase C (PKC). To determine whether PLD stimulation by Ang II is the result of PLC activation and the subsequent elevation of cytosolic free Ca2+ and PKC activation, we investigated the role of Ca2+ and PKC in the activation of PLD. Chelation of extracellular Ca2+ by EGTA, blockade of voltage-sensitive Ca2+ channels, or chelation of intracellular Ca2+ with BAPTA partially attenuated PLD activation and Ca2+ mobilization in response to Ang II. However, the simultaneous chelation of extracellular Ca2+ with EGTA and intracellular Ca2+ with BAPTA completely attenuated both PLD activation and Ca2+ accumulation. Ca2+ ionophores mimicked Ang II and the combined effects of Ang II and ionophore resulted in no further stimulation of PLD activity above that observed in the presence of either agonist alone. Although the putative PLC inhibitor U73122 blocked the activation of PLD by Ang II, it also may inhibit PLD activation directly, since it attenuated both Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA)-mediated increases in PLD activity. PMA also activated PLD in VSMC in a dose-dependent manner; however, Ang II and PMA stimulation were additive. Down-regulation of PKC via exposure to phorbol dibutyrate almost completely blocked PMA-induced stimulation of PLD while it had no effect on Ang II- or Ca(2+)-ionophore-mediated increases in PLD activity. The PKC inhibitor staurosporine augmented basal PLD activity and partially inhibited PMA stimulation of PLD while it had little effect on Ang II-induced increases in PLD activity. Thus, optimal Ang II stimulation of PLD is dependent on the availability of both intracellular and extracellular Ca2+ and independent of PMA-mediated effects. Furthermore, these data suggest that Ang II stimulation of PLD may occur subsequent to activation of PLC, since Ang II activates PLC and PLC is shown to be responsible for increases in intracellular Ca2- in response to Ang II.

Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation.

We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.

Modulation of glutamate release from hippocampal mossy fiber nerve endings by arachidonic acid and eicosanoids.

Arachidonic acid has been implicated in normal synaptic transmission processes, including those related to the development of hippocampal long-term synaptic potentiation. Hippocampal mossy fiber (MF) synaptosomes were used to investigate the role of arachidonate in the evoked accumulation of presynaptic Ca2+ and the release of endogenous glutamate, since these nerve terminals express long-term potentiation and selectively release glutamate as the excitatory transmitter. It was demonstrated that membrane depolarization evoked the accumulation of Ca2+, the release of glutamate, and the production of unesterified arachidonic acid. These events may be functionally related, since exogenous arachidonate and phospholipase A2 activation mimicked the effects of depolarization on Ca2+ availability and glutamate release, while secretion processes were attenuated in the presence of phospholipase A2 inhibitors. In addition, pretreatment of the nerve terminals with arachidonate or melittin allowed for the facilitated release of glutamate in response to a subsequent depolarizing stimulus. Inhibition of cyclooxygenase or lipoxygenase activities also potentiated presynaptic responses to membrane depolarization. In contrast, 12-lipoxygenase products attenuated the depolarization-evoked accumulation of intraterminal free Ca2+ and glutamate release. It is suggested that arachidonic acid acts as a positive modulator of mossy fiber secretion processes, including those involved in the increased glutamate release required for the induction of long-term potentiation, while 12-lipoxygenase metabolites provide negative feedback signals designed to limit neurotransmitter secretion.

12-Lipoxygenase products attenuate the glutamate release and Ca2+ accumulation evoked by depolarization of hippocampal mossy fiber nerve endings.

Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium-dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12-lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5- or 15-lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-10 microM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12-lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid.

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.