Rigid and remodelled: cerebrovascular structure and function after experimental high-thoracic spinal cord transection.

High-thoracic or cervical spinal cord injury (SCI) is associated with several critical clinical conditions related to impaired cerebrovascular health, including: 300-400% increased risk of stroke, cognitive decline and diminished cerebral blood flow regulation. The purpose of this study was to examine the influence of high-thoracic (T3 spinal segment) SCI on cerebrovascular structure and function, as well as molecular markers of profibrosis. Seven weeks after complete T3 spinal cord transection (T3-SCI, n = 15) or sham injury (Sham, n = 10), rats were sacrificed for either middle cerebral artery (MCA) structure and function assessments via ex vivo pressure myography, or immunohistochemical analyses. Myogenic tone was unchanged, but over a range of transmural pressures, inward remodelling occurred after T3-SCI with a 40% reduction in distensibility (both P < 0.05), and a 33% reduction in vasoconstrictive reactivity to 5-HT trending toward significance (P = 0.09). After T3-SCI, the MCA had more collagen I (42%), collagen III (24%), transforming growth factor ß (47%) and angiotensin II receptor type 2 (132%), 27% less elastin as well as concurrent increased wall thickness and reduced lumen diameter (all P < 0.05). Sympathetic innervation (tyrosine hydroxylase-positive axon density) and endothelium-dependent dilatation (carbachol) of the MCA were not different between groups. This study demonstrates profibrosis and hypertrophic inward remodelling within the largest cerebral artery after high-thoracic SCI, leading to increased stiffness and possibly impaired reactivity. These deleterious adaptations would substantially undermine the capacity for regulation of cerebral blood flow and probably underlie several cerebrovascular clinical conditions in the SCI population.

Targeting leukemia stem cells in vivo with antagomiR-126 nanoparticles in acute myeloid leukemia.

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to 'stemness' and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (⩾60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, that is, miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

Captopril treatment reverses erectile dysfunction in male stroke prone spontaneously hypertensive rats.

The involvement of antihypertensive therapy in the pathology of hypertension associated male erectile dysfunction is unclear. Stroke prone spontaneously hypertensive rats (SHRSP) were treated chronically with the angiotensin converting enzyme (ACE) inhibitor captopril or placebo, normotensive rats served as controls. Mean arterial and intracavernosal pressure were measured during the induction of erection by autonomic ganglion stimulation. SHRSP-placebo treated rats were hypertensive and had a blunted erectile response. Captopril treatment returned both the blood pressure and erectile response to control levels. Therefore, ACE inhibitor therapy may not be responsible for the erectile dysfunction observed in treated hypertensive subjects.

NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat.

We previously reported increased aortic reactive oxygen species (ROS) production in mineralocorticoid (deoxycorticosterone acetate [DOCA]-salt) hypertensive rats. In the present study, we tested the hypothesis that NADH/NADPH oxidase is responsible for increased ROS production, namely superoxide (O(2-)), in aorta from the DOCA-salt rat. Treatment of aortic rings from DOCA-salt rats with the NO synthase inhibitor N-nitro-L-arginine and the xanthine oxidase inhibitor allopurinol did not significantly change O(2-) production. Furthermore, de-endothelialization of aorta from DOCA-salt rats did not affect O(2-) production compared with that of sham-operated rats. Thus, xanthine oxidase and uncoupled endothelial NO synthase were not responsible for increased O(2-) production in the DOCA-salt rats. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased O(2-) production in aortic rings from DOCA-salt rats compared with sham-operated rats. Moreover, long-term administration of apocynin (in drinking water, 1.5 mmol/L, 28 days) to DOCA-salt rats significantly decreased systolic blood pressure compared with that of rats treated with DOCA-salt alone. Furthermore, O(2-) production in aortic rings from DOCA-salt rats treated with apocynin for 28 days was reduced compared with that of untreated DOCA-salt rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that DOCA-salt rats have significantly greater mRNA levels of the NADPH oxidase subunit p22phox than do sham-operated rats. These findings suggest that NADPH oxidase is increased and is responsible for increased O(2-) production and possibly contributes to increased blood pressure in the DOCA-salt hypertensive rat.

Increased membrane sphingomyelin and arachidonic acid in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Cell membrane composition and fluidity are altered in hypertension. Previous reports suggest arachidonic acid, a metabolically active fatty acid, is increased in the membranes of hypertensive animals compared to control. This increase in unsaturated fatty acids does not explain the observed reduction in fluidity in hypertensive rats, suggesting some other factors affecting fluidity may be present. It has been suggested that the metabolism of sphingomyelin is altered in genetic hypertension. We hypothesized that membrane sphingomyelin content is increased in hypertensive animals. PROCEDURES: Stroke-prone spontaneously hypertensive rats (n = 8) were compared with Wistar-Kyoto rats (n = 8). Erythrocyte membranes were prepared and the lipids extracted and separated. Fatty acid methyl esters were produced, identified, and quantified by gas chromatography-mass spectrometry; membrane lipid content was also assessed. RESULTS: The concentration of sphingomyelin was higher in the membrane of the hypertensive rats (45.7+/-6 v 22.4+/-2 microg/mg of protein) compared to control. The previously observed increase in membrane arachidonic acid content was observed in hypertensive animals when compared to control (130+/-32 v 40+/-3 microg/mg of protein). However, this difference was confined to the phosphatidylinositol (18+/-4 v 6.5+/-1.5 microg/mg of protein) and free fatty acid (2.1+/-0.4 v 0.6+/-0.1 microg/mg of protein) fractions. CONCLUSION: We hypothesize that reports of reduced membrane fluidity observed in hypertension may be due to an increase in the proportion of sphingomyelin in the cell membrane.

Spironolactone reduces cerebral infarct size and EGF-receptor mRNA in stroke-prone rats.

Remodeling of the cerebral vasculature contributes to the pathogenesis of cerebral ischemia. Remodeling is caused by increased smooth muscle proliferation and may be due to an increase in the responsiveness of vascular cells to epidermal growth factor (EGF). Aldosterone is a risk factor for stroke, and the literature suggests it may play a role in increasing the expression of the receptor for EGF (EGFR). We hypothesized that mRNA for the EGF-stimulated pathway would be elevated in the vasculature of stroke-prone spontaneously hypertensive rats (SHRSP) and that this and experimental ischemic cerebral infract size would be reduced by aldosterone inhibition with spironolactone. We found that spironolactone treatment reduced the size of cerebral infarcts after middle cerebral artery occlusion in SHRSP (51.69 +/- 3.60 vs. 22.00 +/- 6.69% of hemisphere-infarcted SHRSP vs. SHRSP + spironolactone P < 0.05). Expression of EGF and EGFR mRNA was higher in cerebral vessels and aorta from adult SHRSP compared with Wistar-Kyoto rats. Only the expression of EGFR mRNA was elevated in the young SHRSP. Spironolactone reduced the EGFR mRNA expression in the aorta (1.09 +/- 0.25 vs. 0.56 +/- 0.11 phosphorimage units SHRSP vs. SHRSP + spironolactone P < 0.05) but had no effect on EGF mRNA. In vitro incubation of aorta with aldosterone +/- spironolactone produced similar results, suggesting a direct effect of aldosterone. Thus spironolactone may reduce the size of cerebral infarcts via a reduction in the expression of the EGFR mRNA, leading to reduced remodeling.

Glucocorticoids inhibit tetrahydrobiopterin-dependent endothelial function.

Tetrahydrobiopterin (BH4) acts as an important co-factor for endothelial nitric oxide synthase (eNOS). Glucocorticoids have been shown to inhibit expression of the rate-limiting enzyme for tetrahydrobiopterin synthesis, GTP cyclohydrolase, in other cell types. We hypothesized that endothelium-dependent vasodilator responses would be blunted in rats made hypertensive with dexamethasone. Further, we hypothesized that treatment of rat vascular segments with dexamethasone would result in attenuation of endothelial function accompanied by decreased GTP cyclohydrolase expression. We report that endothelium-dependent relaxation responses to the calcium ionophore A23187 are reduced in aortic rings from dexamethasone-hypertensive rats compared with sham values. Dexamethasone incubation abolishes contraction to Nomega-nitro-L-arginine (L-NNA, 10(-5) M) in endothelium-intact aortic rings, and inhibits expression of GTP cyclohydrolase. We conclude that inhibition of BH4 synthesis by glucocorticoid regulation of GTP cyclohydrolase expression may contribute to reduced endothelium-dependent vasodilation characteristic of glucocorticoid-induced hypertension.

Decreased penile erection in DOCA-salt and stroke prone-spontaneously hypertensive rats.

Numerous etiological studies have established a positive clinical association between hypertension and erectile dysfunction. However, to date, the mechanism underlying this dysfunction remains to be established. In this study, we demonstrate the presence of erectile dysfunction in two rat models of hypertension, and hypothesize that increased vasoconstrictor signaling via Rho-kinase contributes to the decreased erectile response. We found deoxycorticosterone-salt and stroke prone-spontaneously hypertensive rats to exhibit a decreased erectile response, recorded as intracavernosal pressure/mean arterial pressure (ICP/MAP) upon electrical stimulation of the major pelvic ganglion. As previously shown, inhibition of Rho-kinase activity by intracavernosal injection of the selective inhibitor, Y-27632, resulted in an increase in ICP/MAP. However, Y-27632 was significantly less effective at increasing ICP/MAP in the hypertensive as compared to normotensive rats. Additionally, intracavernosal injection of Y-27632 potentiated the voltage-stimulated increase in ICP/MAP in both hypertensive and normotensive rats, but was less effective at potentiating the voltage-mediated erectile response in the hypertensive rats. Altogether, our data demonstrate a decreased erectile response in a mineralocorticoid and genetic model of hypertension, and suggest the role of increased cell signaling by Rho-kinase in the vasoconstrictor activity of erectile dysfunction associated with hypertension.

Novel signaling pathways contributing to vascular changes in hypertension.

In hypertension, increased peripheral resistance maintains elevated levels of arterial blood pressure. The increase in peripheral resistance results, in part, from abnormal constrictor and dilator responses and vascular remodeling. In this review, we consider four cellular signaling pathways as possible explanations for these abnormal vascular responses: (1) augmented signaling via the epidermal growth factor receptor to cause remodeling of the cerebrovasculature; (2) reduced sphingolipid signaling leading to blunted vasodilation and increased smooth muscle proliferation; (3) increased signaling via Rho/Rho kinase leading to enhanced vasoconstriction, and (4) a relative state of microtubular depolymerization favoring vasoconstriction in hypertension. These novel cell signaling pathways provide new pharmacological targets to reduce total peripheral vascular resistance in hypertension.

Identification of a common low density lipoprotein receptor mutation (C163Y) in the west of Scotland.

Familial hypercholesterolaemia (FH) is an autosomal codominant disorder characterised by high levels of LDL cholesterol and a high incidence of coronary artery disease. Our aims were to track the low density lipoprotein receptor (LDLR) gene in individual families with phenotypic FH and to identify and characterise any mutations of the LDLR gene that may be common in the west of Scotland FH population using single strand conformational polymorphism analysis (SSCP). Patient samples consisted of 80 heterozygous probands with FH, 200 subjects who were related to the probands, and a further 50 normal, unrelated control subjects. Tracking of the LDLR gene was accomplished by amplification of a 19 allele tetranucleotide microsatellite that is tightly linked to the LDLR gene locus. Primers specific for exon 4 of the LDLR gene were used to amplify genomic DNA and used for SSCP analysis. Any PCR products with different migration patterns as assessed by SSCP were then sequenced directly. In addition to identifying probands with a common mutation, family members were screened using a forced restriction site assay and analysed using microplate array diagonal gel electrophoresis (MADGE). Microsatellite D19S394 analysis was informative in 20 of 23 families studied. In these families there was no inconsistency with segregation of the FH phenotype with the LDLR locus. Of the FH probands, 15/80 had a mutant allele as assessed by SSCP using three pairs of primers covering the whole of exon 4 of the LDLR gene. Direct DNA sequencing showed that 7/15 of the probands had a C163Y mutation. Using a PCR induced restriction site assay for the enzyme RsaI and MADGE, it was determined that the C163Y mutation cosegregated with the FH phenotype in family members of the FH probands. This mutant allele was not present in any of the control subjects. Microsatellite analysis has proven useful in tracking the LDLR gene and could be used in conjunction with LDL cholesterol levels to diagnose FH, especially in children and young adults where phenotypic diagnosis can be difficult.

The role of thyroidal type-I iodothyronine deiodinase in tri-iodothyronine production by human and sheep thyrocytes in primary culture.

We have studied the origin of tri-iodothyronine (T3) secreted by human and sheep thyrocytes in primary culture and also the expression of type-I thyroidal iodothyronine deiodinase (ID-I) in the thyroid and liver of man and various other animals. Inhibitors of ID-I reduced T3 secretion from human but not sheep thyrocytes. In contrast, inhibitors of de-novo thyroid hormone synthesis reduced both thyroxine (T4) and T3 production in sheep thyrocytes, but had no effect on the T3 secreted by human thyrocytes. Human thyrocytes did not produce T4 under the culture conditions used, although some endogenous T4 was present in the cells following their isolation. Although thyrotrophin (TSH) stimulated T3 production in both human and sheep thyrocytes, iodine in the form of potassium iodide was only essential for T3 and T4 production by the sheep cells. Although 125I from Na125I was incorporated into T3 and T4 in TSH-stimulated sheep thyrocytes, no 125I incorporation into T3 or T4 was detected in TSH-stimulated human thyrocytes. Using activity measurements and affinity labelling, ID-I was present in the livers of all species studied, but ID-I could not be detected in thyroid tissue from cattle, pigs, sheep, goats, rabbits, deer or llamas. In contrast, thyroid tissue from man, mice, guinea-pigs and rats had significant ID-I activity and expressed an affinity-labelled protein with a molecular mass of approximately 28.1 kDa on SDS-PAGE. These data show that under the culture conditions used, sheep thyrocytes produced T3 by de-novo synthesis, whilst human thyrocytes produced T3 by deiodination of endogenous T4. We conclude that thyroidal ID-I shows marked species difference in its expression and that, in those species which express the enzyme (man, mice, guinea-pigs and rats, in this study), it appears that it may make an important contribution to thyroidal T3 production.