Genetic Determinants of Cerebral Arterial Adaptation to Flow-loading.

BACKGROUND: In animal models, flow-loading is a necessary and sufficient hemodynamic factor to express the Cerebral Aneurysm (CA) phenotype. Using a rat model, this study characterizes the molecular events that comprise the cerebral arterial response to flow-loading and reveals their significance relating to the CA phenotype. OBJECTIVE: To characterize the molecular events that underlie expansive remodeling of cerebral arteries in two genetically distinct inbred rat strains with differential susceptibility to flow-dependent cerebrovascular pathology. METHODS: Thirty-two rats underwent bilateral common carotid artery ligation (BCL) (n=16) or Sham Surgery (SS) (n=16). Nineteen days later, vertebrobasilar arteries were harvested, histologically examined and analyzed for mRNA and protein expression. Flow-induced changes in histology, mRNA and protein expression were compared between BCL and SS rats. Differences between aneurysm-prone (Long Evans, LE) and resistant (Brown Norway, BN) strains were evaluated. RESULTS: Basilar Artery (BA) medial thickness/luminal diameter ratio was significantly reduced in BCL rats, without significant differences between LE (2.02 fold) and BN (1.94 fold) rats. BCL significantly altered BA expression of mRNA and protein but did not affect blood pressure. Eight genes showed similarly large flow-induced expression changes in LE and BN rats. Twenty-six flow responsive genes showed differences in flow-induced expression between LE and BN rats. The Cthrc1, Gsta3, Tgfb3, Ldha, Myo1d, Ermn, PTHrp, Rgs16 and TRCCP genes showed the strongest flow responsive expression, with the largest difference between LE and BN rats. CONCLUSIONS: Our study reveals specific molecular biological responses involved in flow-induced expansive remodeling of cerebral arteries that may influence differential expression of flowdependent cerebrovascular pathology.

Variable MR and pathologic patterns of hemorrhage after iodinated contrast infusion in MCA occlusion/reperfusion model.

OBJECTIVE: To examine the hypothesis that IA reperfusion with iso-osmolar iodixanol, low-osmolar iopamidol, or saline causes different effects on MR signal changes and pathologic cut-brain section related to hemorrhagic transformation (HT) or iodinated radiographic contrast media (IRCM) deposition. METHODS: Infarct was induced in 30 rats by middle cerebral artery suture occlusion. Reperfusion was performed after 5 hours with iso-osmolar iodixanol (n=9), low-osmolar iopamidol (n=12) or saline (n=9). MR images were obtained immediately after reperfusion and rats were sacrificed at 24 hours. Hypointense areas within the infarction on T2-weighted (T2-WI) or gradient echo (GRE) images were recorded and compared with HT on pathology. Fisher's exact test was used for proportions, and receiver operator curve analysis to evaluate MRI discrimination of hemorrhage. RESULTS: Two types of HT were noted on pathology: confluent >0.2 mm petechial hemorrhage (PeH, 78%) or well-defined ≤0.2 mm hemorrhagic focus (HF, 22%). PeH was least common in the iodixanol subgroup (p<0.02). HF was more common in the IRCM group. Hypointense areas on T2-WI but not on GRE were significantly more common in the IRCM group (p<0.05). Hypointense areas on T2-WI and GRE discriminated HT (area under the curve: 0.714, p<0.002). CONCLUSIONS: IRCM and saline induced different MRI signal and pathologic patterns in our sample. We postulate that T2 hypointensity with no GRE hypointensity might be associated with IRCM deposition; and decreased frequency of PeH after iodixanol infusion and the presence of HF almost exclusively in the IRCM group might represent a direct/indirect effect of contrast infusion/deposition in the brain parenchyma after reperfusion. Our results support previous observations in IMS III and are hypothesis generating.

The Effect of Creatine Kinase Inhibition on Contractile Properties of Human Resistance Arteries.

BACKGROUND: Creatine kinase (CK) is a main predictor of blood pressure, and this is thought to largely depend on high resistance artery contractility. We previously reported an association between vascular contractility and CK in normotensive pregnancy, but pregnancy is a strong CK inducer, and data on human hypertension are lacking. Therefore, we further explored CK-dependency of vascular contractility outside the context of pregnancy in normotensive and hypertensive women. METHODS AND RESULTS: Nineteen consecutive women, mean age 42 years (SE 1.3), mean systolic/diastolic blood pressure respectively 142.6 (SE 5.9)/85.6 (3.4) mm Hg (9 hypertensive), donated an omental fat sample during abdominal surgery. We compared vasodilation after the specific CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB; 10(-6) mol/l) to sodium nitroprusside (10(-6) mol/l) in isolated resistance arteries using a wire myograph. Additionally, we assessed predictors of vasoconstrictive force. DNFB reduced vascular contractility to 24.3% (SE 4.4), P < 0.001, compared to baseline. Sodium nitroprusside reduced contractility to 89.8% (SE 2.3). Maximum contractile force correlated with DNFB effect as a measure of CK (r = 0.8), and with vessel diameter (r = 0.7). The increase in contractile force was 16.5 mN [9.1-23.9] per unit DNFB effect in univariable and 10.35 mN [2.10-18.60] in multivariable regression analysis. CONCLUSION: This study extends on our previous findings in pregnant normotensive women of CK-dependent microvascular contractility, indicating that CK contributes significantly to resistance artery contractility across human normotension and primary hypertension outside the context of pregnancy. Further studies should explore the effect of CK inhibitors on clinical blood pressure.

Resistance artery creatine kinase mRNA and blood pressure in humans.

Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

Down-regulation of interleukin 7 mRNA by hypoxia is calcium dependent.

OBJECTIVE: The discovery of IL-7R(alpha) polymorphisms implicated in the pathogenesis of multiple sclerosis has highlighted the importance of interleukin 7 (IL-7) in central nervous system diseases. Hypoxia affects neurological disease states in part by modulating expression of many early and late response genes. The present work used cultured PC12 cells to investigate the effect of hypoxia on IL-7 expression. METHOD: PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium. RNA was isolated and reverse transcriptase-polymerase chain reaction (RT-PCR) was run to quantify messenger RNA (mRNA) change. Western blots were used to assess IL-7 protein change in the medium. Extracellular free Ca(2+) was removed by using Ca(2+)-free DMEM/F12 with 1 mM ethylene glycol tetraacetic acid for 45 minutes before the start of hypoxia. RESULTS: Exposure of PC12 cells to 1% oxygen for 6 hours decreased IL-7 mRNA by 77% using RT-PCR (p<0.01). Exposure to 1% oxygen for 24 hours decreased IL-7 protein in the medium by 21% (p<0.05). As hypoxia duration increased (2, 4, 6 and 24 hours) or oxygen concentrations decreased (10%, 5% and 1%), IL-7 mRNA expression progressively decreased. Removal of extracellular free Ca(2+) completely prevented these hypoxia-induced decreases of IL-7 mRNA. DISCUSSION: Since IL-7 exhibits trophic properties in developing brain, down-regulation of IL-7 by hypoxia may contribute to hypoxia-induced injury to neural cells.

Hypertension risk in idiopathic hyperCKemia.

Patients with idiopathic hyperCKemia are usually reassured and discharged. However, these subjects may have increased hypertension risk, based on data from our population study, which showed that the population tertile with the highest serum creatine kinase activities had the highest systolic and diastolic blood pressure levels. Therefore, we assessed whether subjects with idiopathic hyperCKemia have greater occurrence of hypertension than controls. We included 46 participants aged 18 to 67 years, diagnosed with idiopathic hyperCKemia at the departments of Neurology of the Universities of Amsterdam and Utrecht, The Netherlands. We found that 48% of the subjects with idiopathic hyperCKemia were hypertensive, as compared to 19% of the random population controls (n = 22,612, aged 20 to 65 years), an odds ratio of 3.9 (95 % CI, 2.2 to 6.9) before, and 2.0 (1.1 to 3.8) after adjustment for sex, age, and body mass index. In accord with our previous finding of an association between creatine kinase and blood pressure in the general population, the data reported here suggest that subjects with idiopathic hyperCKemia have greater hypertension risk than controls. This may be due to relatively high tissue creatine kinase activity, resulting in greater ATP buffer capacity to create and sustain high blood pressure levels.Larger, prospective studies are needed to further assess this association, but as active case finding is important in the diagnosis of hypertension, subjects with idiopathic hyperCKemia should be screened and monitored for the presence of hypertension.

Creatine kinase activity is associated with blood pressure.

BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular contraction, and antagonizes nitric oxide-mediated functions. Relatively high activity of the enzyme, particularly in resistance arteries, is thought to enhance pressor responses and increase blood pressure. Tissue creatine kinase activity is reported to be high in black people, a population subgroup with greater hypertension risk; the proposed effects of high creatine kinase activity, however, are not "race dependent." We therefore assessed whether creatine kinase is associated with blood pressure in a multiethnic population. METHODS AND RESULTS: We analyzed a stratified random sample of the population of Amsterdam, The Netherlands, consisting of 1444 citizens (503 white European, 292 South Asian, 580 black, and 69 of other ethnicity) aged 34 to 60 years. We used linear regression analysis to investigate the association between blood pressure and normal serum creatine kinase after rest, as a substitute measure of tissue activity. Creatine kinase was independently associated with blood pressure, with an increase in systolic and diastolic pressure, respectively, of 8.0 (95% CI, 3.3 to 12.7) and 4.7 (95% CI, 1.9 to 7.5) mm Hg per log creatine kinase increase after adjustment for age, sex, body mass index, and ethnicity. CONCLUSIONS: Creatine kinase is associated with blood pressure. Further studies are needed to explore the nature of this association, including how variation in cardiovascular creatine kinase activity may affect pressor responses.

Functional compartmentation of glycogen phosphorylase with creatine kinase and Ca2+ ATPase in skeletal muscle.

This manuscript discusses aspects of functional compartmentation in the regulation of metabolism. The functional consequences of enzymes coupling between creatine kinase, glycogen phosphorylase and sarcoplasmic reticular Ca2+ ATPase is examined. It is proposed that the coupling of creatine kinase and glycogen phosphorylase classifies as a novel class of diazyme complex with an important regulatory role in the inhibition of glycogenolysis at rest. In addition it is suggested that creatine kinase, glycogen phosphorylase and the sarcoplasmic reticular Ca2+ ATPase may couple to form a three-enzyme complex. From a consideration of the structure and chemical catalysis of the putative three-enzyme complex, a novel net reaction for glycogenolysis in the vicinity of the sarcoplasmic reticulum is suggested (Phosphocreatine+Glycogen+H(+)Creatine+Glycogen(n)(-1)+Glucose-1-Phosphate). The three-enzyme complex may also have an important role in inhibiting glycogenolysis at rest as well as improving the efficiency of high-energy phosphate transfer.

Presence of normal creatine in the muscle of a patient with a mutation in the creatine transporter: a case study.

To date, more than seven families have been reported who carry a mutation in the X-linked creatine-transporter (CrT) gene. The resulting lack of creatine in the brain is associated with mental retardation, severe expressive language disorder, mild epilepsy, and a complete absence of Cr in the brain (measured using MRS). Conversely, these patients had no observable cardiac or musculo-skeletal deficits. In this case study, a 22-year-old patient underwent surgical repair for scoliosis. Proton MRS of this patient's brain demonstrated the near-absence of creatine and phosphocreatine within the cerebral white and deep gray matter structures. Cerebral atrophy was noted with serial MRI examinations. Subsequent genetic and metabolic analysis showed some biochemical anomalies consistent with a CrT deficiency. The mutation in this patient was identified as a deletion at phenylalanine 107 (delF107). Control muscle biopsies were obtained from archived samples, which had been taken with informed consent during routine muscle biopsies for diagnostic purposes. We determined that the total Cr concentration in the skeletal muscle biopsy was 39.3 +/- 2.94 mmol/kg wet wt., which is not significantly different from non-CrT controls, n = 3 (43.3 +/- 3.57 mmol/kg wet wt.). We conclude that the brain appears to lack the ability to transport creatine when there is a mutation in the CrT gene. However, the muscle utilizes another mechanism for maintaining normal creatine levels. Identifying this alternative creatine-transport mechanism may be useful in treating the neurologic and cognitive impairments of patients with creatine-transporter deficiency.

Mechanisms for monovalent cation-dependent depletion of intracellular Mg2+:Na(+)-independent Mg2+ pathways in guinea-pig smooth muscle.

It has been suggested that magnesium deficiency is correlated with many diseases. 31P NMR experiments were carried out in order to investigate the effects of Na+ substitution on Mg2+ depletion in smooth muscle under divalent cation-free conditions. In the taenia of guinea-pig caeci, the intracellular free Mg2+ concentration ([Mg2+]i) was estimated from the chemical shifts of (1) the beta-ATP peak alone and (2) beta- and gamma-ATP peaks. Both estimations indicated that [Mg2+]i decreased only very slowly in Mg(2+)-free, Ca(2+)-free solutions in which Na+ was substituted with large cations such as NMDG (N-methyl-D-glucamine) and choline. Furthermore, the measurements of tension development supported the suggestion of preservation of intracellular Mg2+ with NMDG substitution. Substituting extracellular Na+ with the small cation, Li+, also shifted the beta-ATP peak towards a lower frequency, but the frequency shift was significantly less than that seen upon Na+ substitution with K+. The estimated [Mg2+]i depletion was, however, comparable with that seen after Na+ substitution with K+ using the titration curves of metal-free and Mg(2+)-bound ATP obtained in Li(+)-based model solutions. It was concluded that Mg2+ rapidly decreases only when small cations were the major electrolyte of the extracellular medium. Na+ substitutions with NMDG, choline or Li+ had little effect on intracellular ATP concentration after 100 min treatment.

Genomics of the periinfarction cortex after focal cerebral ischemia.

Understanding transcriptional changes in brain after ischemia may provide therapeutic targets for treating stroke and promoting recovery. To study these changes on a genomic scale, oligonucleotide arrays were used to assess RNA samples from periinfarction cortex of adult Sprague-Dawley rats 24 h after permanent middle cerebral artery occlusions. Of the 328 regulated transcripts in ischemia compared with sham-operated animals, 264 were upregulated, 64 were downregulated, and 163 (49.7%) had not been reported in stroke. Of the functional groups modulated by ischemia: G-protein-related genes were the least reported; and cytokines, chemokines, stress proteins, and cell adhesion and immune molecules were the most highly expressed. Quantitative reverse transcription polymerase chain reaction of 20 selected genes at 2, 4, and 24 h after ischemia showed early upregulated genes (2 h) including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, and DAF. Late upregulated genes (24 h) included Cathepsin C, Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which is involved in mitochondrial reoxidation of glycolysis derived NADH, was regulated more than 60-fold. Plasticity-related transcripts were regulated, including Narp, agrin, and Cpg21. A newly reported lung pathway was also regulated in ischemic brain: C/EBP induction of Egr-1 (NGFI-A) with downstream induction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regulated acutely after stroke may modulate cell survival and death; also, late regulated genes may be related to tissue repair and functional recovery.

Heme and iron metabolism: role in cerebral hemorrhage.

Heme and iron metabolism are of considerable interest and importance in normal brain function as well as in neurodegeneration and neuropathologically following traumatic injury and hemorrhagic stroke. After a cerebral hemorrhage, large numbers of hemoglobin-containing red blood cells are released into the brain's parenchyma and/or subarachnoid space. After hemolysis and the subsequent release of heme from hemoglobin, several pathways are employed to transport and metabolize this heme and its iron moiety to protect the brain from potential oxidative stress. Required for these processes are various extracellular and intracellular transporters and storage proteins, the heme oxygenase isozymes and metabolic proteins with differing localizations in the various brain-cell types. In the past several years, additional new genes and proteins have been discovered that are involved in the transport and metabolism of heme and iron in brain and other tissues. These discoveries may provide new insights into neurodegenerative diseases like Alzheimer's, Parkinson's, and Friedrich's ataxia that are associated with accumulation of iron in specific brain regions or in specific organelles. The present review will examine the uptake and metabolism of heme and iron in the brain and will relate these processes to blood removal and to the potential mechanisms underlying brain injury following cerebral hemorrhage.

Smooth muscle and NMR review: an overview of smooth muscle metabolism.

Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles. It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary. NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg2+ ([Mg2+]i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg2+]i is highly protected in the muscle (within confined range), but also responds to the environment around it. In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases.

Simultaneous estimation of intracellular free Mg2+ and pH by use of a new pH-dependent dissociation constant of MgATP.

In the present technical note for 31P-NMR, we used a new pH-dependent dissociation constant of MgATP, and re-estimated changes in the intracellular free Mg2+ concentration and pH from the chemical shifts of beta- and gamma-ATP during Na+-removal in smooth muscle. We confirmed the role of Na+ - Mg2+ exchange.

Oxidation of bilirubin produces compounds that cause prolonged vasospasm of rat cerebral vessels: a contributor to subarachnoid hemorrhage-induced vasospasm.

The authors have previously shown that bilirubin-oxidation products (BOXes) are present in CSF of subarachnoid hemorrhage patients with vasospasm, and that BOXes cause vasoconstriction in vitro. This study determined whether BOXes cause vasospasm in vivo. Identical volumes of either lysed blood or standardized amounts of BOXes were injected into the cisterna magna of adult rats. BOX injections caused 6 of 10 rats to die within 10 minutes, whereas 12 of 12 rats survived for 24 hours after blood injections. The mechanism for this significant (P < or = 0.01) increase in mortality was unclear. To directly test whether BOXes produced vasospasm, a cranial window technique was used. Application of 20 microL of 10-micromol/L bilirubin had little effect on the vessels. However, application of BOXes produced marked, dose-dependent small artery and arteriole vasospasm that approached a 90% decrease in diameter by 40 minutes after application in some vessels, and persisted for at least 24 hours. To determine if BOX-mediated vasospasm led to cortical injury, histology and immunocytochemistry were performed on animals that survived for 24 hours. There was a BOX-related stress protein response for HSP25 and HSP32 (HO-1) without evidence of infarction. The finding that the BOXes produce vasospasm of cerebral vessels in vivo, in conjunction with BOXes being found in CSF of vasospasm patients, supports our hypothesis that BOXes contribute to or cause cerebral vasospasm after subarachnoid hemorrhage.