Heterogeneity in Kv7 channel function in the cerebral and coronary circulation.

OBJECTIVES: Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels. METHODS AND RESULTS: PCR confirmed that basilar, Circle of Willis and LAD arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50 µM) was significantly greater in the basilar artery compared to the LAD artery. Similarly, the Kv7 channel inhibitor, linopirdine (10 µM) caused a stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than in the coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs. CONCLUSIONS: This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction.

Protein expression levels in the medullary visceral zone of rats with subarachnoid hemorrhage.

We investigated protein expression in the medullary visceral zone (MVZ) of rats with multiple-organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH) to discuss the possible regulatory mechanism of the MVZ in the course of SAH-induced MODS. A SAH-induced MODS model was established in rats by injecting arterial blood into the Willis' circle. Protein expression in the MVZ was analyzed by immunohistochemistry assay. Protein expression in the MVZ peaked 24-36 h after SAH, and was significantly higher than in the control and sham operation groups. Organs at each time point exhibited inflammatory injuries to varying degrees after SAH, which reached a maximum at 24-36 h. Incidences of systemic inflammatory response syndrome and MODS were 100 and 71.67%, respectively, after SAH. There is a consistency between MVZ protein expression and inflammatory changes in each organ after SAH. This prompts the suggestion that the MVZ may be one of the direct regulative centers in SAH-induced MODS, and may be involved in the functional regulation of the surrounding organs after SAH.

[Structural peculiarities of the wall of the vessels of the cerebral arterial circle in the area of bifurcations in people of different age].

The present study was conducted to determine the morphological and morphometric features of the arterial wall structure at the bifurcation of blood vessels of the cerebral arterial circle (CAC) of Willis in people of different age (from birth till 65 years). Material obtained from 80 people was stained with hematoxylin-eosin, Van Gieson stain, with orcein by Unna-Taenzer's method and with sudan. The proliferative activity of the cells in tunica intima and tunica media at the site of bifurcation of the internal carotid and basilar arteries was studied immunohistochemically using monoclonal antibodies against Ki-67. It was found that intimal thickenings (cushions) appeared immediately after birth, initially only in a few places of CAC vessel branching; by 8-10 years they were detected in all the bifurcations of the circle. With aging, the thickening of intimal cushions with a thinning of the underlying tunica media was found. Ki-67 protein expression was noted in both the intimal cushions and underlying tunica media, indicating the activity of atherosclerosis process.

Moyamoya disease: a review of histopathology, biochemistry, and genetics.

OBJECT: Moyamoya disease (MMD) is a rare cerebrovascular disorder involving stenosis of the major vessels of the circle of Willis and proximal portions of its principal branches. Despite concerted investigation, the pathophysiology of the disorder has not been fully elucidated. Currently, the major proteins believed to play an active role in the pathogenesis include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-ß1 (TGFß1), and granulocyte colony-stimulating factor (G-CSF). In terms of the genetics, recent literature suggests a low penetrance autosomal dominant or polygenic mode of transmission involving chromosomes 3, 6, 8, 12, and 17 for familial MMD. This review summarizes the current knowledge on the histopathology, pathophysiology and genetics of MMD. METHODS: A PubMed/Medline systematic study of the literature was performed, from which 45 articles regarding MMD pathophysiology were identified and analyzed. CONCLUSIONS: Moyamoya disease is characterized by the intimal thickening and media attenuation of the proximal vessels of the circle of Willis as well as the development of an aberrant distal vascular network. The primary proteins that are currently implicated in the pathophysiology of MMD include VEGF, bFGF, HGF, TGFß1, and G-CSF. Furthermore, the current literature on familial MMD has pointed to a low penetrance autosomal dominant or polygenic mode of transmittance at loci on chromosomes 3, 6, 8, 12, and 17.

Embolus trajectory through a physical replica of the major cerebral arteries.

BACKGROUND AND PURPOSE: The observed distribution of cerebral infarcts varies markedly from expectations based on blood-flow volume or Doppler embolus detection. In this study, we used an in vitro model of the cerebral arteries to test whether embolus microspheres encountering the circle of Willis are carried proportionally to volume flow or express a preferred trajectory related to arterial morphology or embolus size. METHODS: Our model consisted of a patient-specific silicone replica of the cerebral macrocirculation featuring physiologically realistic pulsatile flow of a blood-mimicking fluid at approximately 1000 mL/min and an input pressure of approximately 150/70 mm Hg. Particles of 200, 500, and 1000 microm diameter with equivalent density to thrombus were introduced to the carotid arteries and counted on exiting the model outlets. RESULTS: The middle cerebral arteries (MCAs) of the replica attracted a disproportionate number of emboli compared with the anterior cerebral arteries; 98%+/-3% of 1000 microm and 93%+/-2% of 500 microm emboli entered the MCA compared with 82%+/-5% of the flow. The observed distribution of large emboli was consistent with the ratio of MCA:anterior cerebral artery infarcts, approximately 95% of which occur in territories supplied by the MCA. With decreasing embolus size, the distribution of emboli approaches that of the flow (approximately 89% of 200 microm emboli took the MCA). CONCLUSIONS: Embolus trajectory through the cerebral arteries is dependent on embolus size and strongly favors the MCA for large emboli. The 70:30 ratio of MCA:anterior cerebral artery emboli observed by Doppler ultrasound is consistent with the trajectories of small emboli that tend to be asymptomatic.

Chromatin Conformation Links Putative Enhancers in Intracranial Aneurysm-Associated Regions to Potential Candidate Genes.

Background We previously showed that intracranial aneurysm ( IA )-associated single-nucleotide polymorphisms are enriched in promoters and putative enhancers identified in the human circle of Willis, on which IA s develop, suggesting a role for promoters and enhancers in IAs . We further investigated the role of putative enhancers in the pathogenesis of IA by identifying their potential target genes and validating their regulatory activity. Methods and Results Using our previously published circle of Willis chromatin immunoprecipitation and sequencing data, we selected 34 putative enhancers in IA -associated regions from genome-wide association studies. We then used a chromatin conformation capture technique to prioritize target genes and found that 15 putative enhancers interact with the promoters of 6 target genes: SOX 17, CDKN 2B, MTAP , CNNM 2, RPEL 1, and GATA 6. Subsequently, we assessed the activity of these putative enhancers in vivo in zebrafish embryos and confirmed activity for 8 putative enhancers. Last, we found that all 6 target genes are expressed in the circle of Willis, on the basis of RNA sequencing data and in situ hybridization. Furthermore, in situ hybridization showed that these genes are expressed in multiple cell types in the circle of Willis. Conclusions In 4 of 6 IA -associated genome-wide association study regions, we identified 8 putative enhancers that are active in vivo and interact with 6 nearby genes, suggesting that these genes are regulated by the identified putative enhancers. These genes, SOX 17, CDKN 2B, MTAP , CNNM 2, RPEL 1, and GATA 6, are therefore potential candidate genes involved in IA pathogenesis and should be studied using animal models in the future.

Association of the glutathione S-transferase omega-1 Ala140Asp polymorphism with cerebrovascular atherosclerosis and plaque-associated interleukin-1 alpha expression.

BACKGROUND AND PURPOSE: Glutathione S-transferase omega-1 is a multifunctional enzyme. The Asp/Asp genotype of the Ala140Asp polymorphism of the GSTO1 gene has been alleged to increase the risk of vascular dementia. The objective of this study is to address the question of whether common vessel disorders known to cause vascular dementia are modified in their severity by this polymorphism. METHODS: The severity and expansion of atherosclerosis in the circle of Willis vessels, cerebral small vessel disease, and cerebral amyloid angiopathy were studied in a sample of 79 autopsy cases. Genotyping of the GSTO1 Ala140Asp polymorphism as well as immunohistochemistry for glutathione S-transferase omega-1 was performed. RESULTS: Carriers of the GSTO1 Asp/Asp genotype presented with more severe and widespread atherosclerosis than noncarriers. However, there was no effect on small vessel disease expansion and cerebral amyloid angiopathy severity. Immunohistochemically, we detected interleukin-1 alpha expressing macrophages in the lipid core of atherosclerosis plaques exhibiting glutathione S-transferase omega-1-positive material. GSTO1 Asp/Asp carriers showed larger areas of atherosclerosis plaques containing interleukin-1 alpha-positive material than carriers of the GSTO1 Ala-allele. CONCLUSIONS: The GSTO1 Asp/Asp genotype presumably modulates the severity and expansion of atherosclerosis in the circle of Willis. The cellular colocalization of glutathione S-transferase omega-1 and interleukin-1 alpha suggests a functional interaction between both proteins which in part might explain the function of glutathione S-transferase omega-1 in the pathogenesis of cerebral atherosclerosis.

Site-specific elevation of interleukin-1ß and matrix metalloproteinase-9 in the Willis circle by hemodynamic changes is associated with rupture in a novel rat cerebral aneurysm model.

The pathogenesis of subarachnoid hemorrhage remains unclear. No models of cerebral aneurysms elicited solely by surgical procedures and diet have been established. Elsewhere we reported that only few rats in our original rat aneurysm model manifested rupture at the anterior and posterior Willis circle and that many harbored unruptured aneurysms at the anterior cerebral artery-olfactory artery bifurcation. This suggests that rupture was site-specific. To test our hypothesis that a site-specific response to hemodynamic changes is associated with aneurysmal rupture, we modified our original aneurysm model by altering the hemodynamics. During 90-day observation, the incidence of ruptured aneurysms at the anterior and posterior Willis circle was significantly increased and the high incidence of unruptured aneurysms at the anterior cerebral artery-olfactory artery persisted. This phenomenon was associated with an increase in the blood flow volume. Notably, the level of matrix metalloproteinase-9 associated with interleukin-1ß was augmented by the increase in the blood flow volume, suggesting that these molecules exacerbated the vulnerability of the aneurysmal wall. The current study first demonstrates that a site-specific increase in interleukin-1ß and matrix metalloproteinase-9 elicited by hemodynamic changes is associated with rupture. Our novel rat model of rupture may help to develop pharmaceutical approaches to prevent rupture.

Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite.

Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that SAGE does not require prior knowledge of the genes of interest and provides quantitative and qualitative data of potentially every transcribed sequence in a particular tissue or cell type. Furthermore, SAGE can quantify low-abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations. However, SAGE demands high input levels of mRNA which are often unavailable, particularly when studying human disease. To overcome this limitation, we have developed a modification of SAGE that allows detailed global analysis of gene expression in extremely small quantities of tissue or cultured cells. We have called this approach 'SAGE-Lite'. This technique was used for the global analysis of transcription in samples of normal and pathological human cerebrovasculature to study the molecular pathology of intracranial aneurysms. These samples, which are obtained during operative surgical repair, are typically no bigger than 1 or 2 mm and yield <100 ng of total RNA. In addition, we show that SAGE-Lite allows simple and rapid isolation of long cDNAs from short (15 bp) SAGE sequence tags.

Characterization of melatonin receptors and signal transduction system in rat arteries forming the circle of Willis.

The aims of this study were to characterize the melatonin receptors in rat brain arteries forming the circle of Willis. Saturation studies performed using in vitro autoradiography and [125I]iodomelatonin revealed the presence of two binding sites: one with a Kd of 13 pM, and the second characterized by a Kd of 832 pM. Coincubation with a nonhydrolyzable guanine nucleotide analog [guanosine-5'-O-(3-thiotriphosphate)] inhibited 2-[125I]iodomelatonin binding in a concentration-dependent manner, whereas adenine nucleotide adenosine-5'-O-(3-thiotriphosphate) was ineffective. In saturation studies performed in the presence of guanosine-5'-O-(3-thiotriphosphate), the high affinity site was no longer detectable, and the affinity of the receptor was decreased to the high picomolar range. Melatonin, at nanomolar concentrations, was able to inhibit forskolin-stimulated cAMP production in rat circle of Willis arteries. Preincubation with pertussis toxin counteracted the effect of melatonin. Our results demonstrate that melatonin receptors in rat cerebral arteries are linked to their second messenger through a pertussis toxin-sensitive G-protein, similar to what has been described for melatonin receptors in different areas of vertebrate brain.

Acetylcholine levels and choline acetyltransferase activity in rat cerebrovascular bed after uni- or bilateral sphenopalatine ganglionectomy.

Endogenous acetylcholine (ACh) levels and choline acetyltransferase (ChAT) activity were measured in several vascular segments (major cerebral arteries, cortical pial vessels, and peripheral arteries) and nervous tissues [including the sphenopalatine ganglion (SPG)] in the rat. The effects of uni- or bilateral surgical ablation of the SPG, a putative origin of the cholinergic cerebrovascular innervation, were investigated on these two specific cholinergic markers at various postoperative times. ChAT activity and ACh levels were enriched in the cerebral as compared to the peripheral arteries. Among the cerebrovascular tissues tested, ACh levels were particularly high in the circle of Willis and the vertebrobasilar segments and, to a lesser extent, in the middle cerebral artery. Lower levels were found in the small pial vessels and choroid plexus. Overall, ChAT activity measured in different arterial beds paralleled the distribution of ACh. Following uni- or bilateral removal of the SPG, slight reductions (18-36%, statistically not significant) were observed in ChAT activity in rostral cerebral arteries and pial vessels overlying the frontal cortex. Similarly, bilateral ganglionectomy resulted in minor decreases (11-22%, not significant) in the cerebrovascular contents of ACh in these same vascular segments. These results clearly show that the SPG does not or only partly contributes to the cholinergic fibers that supply the cerebrovascular bed.

Concentrations of putative neurovascular transmitters in major cerebral arteries and small pial vessels of various species.

The levels of noradrenaline, neuropeptide Y, 5-hydroxytryptamine, and substance P were measured and compared between the large arteries of the circle of Willis and the small cerebral vessels of the pia mater in the rat, rabbit, cat, and monkey. In all species, noradrenaline and neuropeptide Y concentrations were greater in the larger arteries than in small pial vessels. Noradrenaline concentrations were dramatically reduced following cervical sympathectomy, with the extent of diminution differing greatly in the various species; the effects of cervical ganglionectomy on neuropeptide Y concentrations were less pronounced. 5-Hydroxytryptamine concentrations in rats, cats, and rabbits were significantly greater in the small pial vessels, although measurable concentrations existed in the circle of Willis. In cats and monkeys, substance P was found in major arteries, but was not detectable at the level of the small pial vessels. The differences in the regional distribution of the various neurotransmitter candidates in the cerebrovascular bed may reflect their physiological significance.

L-Citrulline recycle for synthesis of NO in cerebral perivascular nerves and endothelial cells.

Recycle of L-citrulline to form L-arginine in cerebral perivascular nerves has been well described, providing direct evidence that nitric oxide (NO) is synthesized and released from these nerves to act as the transmitter for vasodilation. NO is also synthesized and released from cerebral endothelial cells, involving L-citrulline conversion to L-arginine. Evidence for the presence of enzymes involved in the conversion, however, has not been shown. The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL), and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in endothelial cells of middle cerebral arteries and the circle of Willis of the pig, therefore, were examined using combined immunohistochemical and histochemical techniques. NOS-, ASS-, and ASL-immunoreactivities were found in almost all endothelial cells of all cerebral arteries examined. All ASS-, ASL-, and NOS-immunoreactive (I) endothelial cells also stained positively for NADPHd, suggesting that ASS, ASL, and NOS were colocalized in endothelial cells of middle cerebral arteries and the circle of Willis. These results provide morphological evidence that cerebral vascular endothelial cells like cerebral perivascular nerves contain enzymes necessary for recycling L-citrulline to L-arginine to synthesize NO via an argininosuccinate (AS) pathway.

Autoradiographic localization of the GABAA receptor agonist [3H]muscimol in rat cerebral vessels.

By the use of combined in vitro radioreceptor binding and autoradiographic techniques with [3H]muscimol as a ligand, we analyzed the distribution of GABAA receptor sites in the arteries of the circle of Willis as well as in the arteries and arterioles of the pial-arachnoid membrane in the rat. [3H]Muscimol was bound by sections of rat cerebral vessels in a manner consistent with the existence of GABAA receptors, with Kd and Bmax values of 46 nM and 0.60 pmol/mg tissue respectively. [3H]Muscimol was bound by the medial layer of cerebral arteries, while no specific binding was observed in the intima, the adventitia and the adventitial-medial border. These findings suggest that the vasodilatory action of GABA on in vitro preparations of cerebral vessels is mediated by muscular receptor sites. The posterior cerebral arteries are richer in [3H]muscimol binding sites than the anterior ones. Pial-arachnoid arterioles, which are of critical importance in controlling local cerebral blood flow, did not exhibit any significant binding of [3H]muscimol. These results may explain the difficulty in manipulating pharmacologically the cerebral tissue perfusion in intact animals using GABAergic agonists.

Substance P-like immunoreactivity in rat forebrain leptomeninges and cerebral vessels originates from the trigeminal but not sympathetic ganglia.

Substance P-like immunoreactivity (SPLI) is present in the pia mater, arachnoid and pial vessels (leptomeninges) of the rat. Unilateral electrolytic lesions of the trigeminal ganglion decreased levels of SPLI in leptomeninges on the lesioned side. Levels did not change following unilateral or bilateral superior cervical ganglionectomies or after i.c.v. injections of 6-hydroxydopamine, sufficient to deplete norepinephrine in pial arteries by more than 90%. SP levels did not decrease in leptomeninges or in blood vessels within the circle of Willis following treatment of adult or neonatal rats with capsaicin despite the fact that levels were reduced in the cornea and dorsal spinal cord in these same animals. These results suggest that not all substance P-containing sensory fibers are susceptible to the peptide-depleting effects of capsaicin.

Characterization of [3H]5-hydroxytryptamine uptake within rat cerebrovascular tree.

The in vitro uptake of tritiated serotonin ([3H]5HT) was studied in a preparation of rat extracerebral arteries. The uptake of [3H]5HT was time- and temperature-dependent and of high affinity; linear regression analysis gave a Km value of 6.48 X 10(-7) M for the specific uptake. Bilateral superior cervical ganglionectomy was without effect on [3H]5HT uptake while it significantly reduced the uptake of tritiated norepinephrine by the preparation of rat extracerebral arteries. The serotonergic neurotoxin 5,7-dihydroxytryptamine and lesions to both the medial and the dorsal raphe nuclei caused a marked loss of [3H]5HT uptake but did not change the uptake of tritiated norepinephrine. Competition studies with norepinephrine, desimipramine (a noradrenergic uptake blocker), nomifensine (a dopaminergic uptake blocker) and fluoxetine (a 5HT uptake blocker) confirmed the specificity of the [3H]5HT uptake mechanism. Histoautoradiographic studies showed the highest density of silver grains at the level of the adventitial-medial border of the basilar artery. Fluoxetine inhibited the accumulation of silver grains within the adventitial-medial border in the blood vessel studied. The present data further support the view that a neuronal serotonergic system may play a role in the control of blood flow in the cerebrovascular tree.

Abnormalities of collagen cross-linkage in posterior communicating artery aneurysms: a preliminary study.

The collagen of tissues submitted to mechanical load (i.e. arterial wall) is characterised by the presence of intermolecular covalent cross-links (hydroxylysyl pyridinoline or pyridinoline: PYD; and lysyl-pyridinoline or deoxypyridinoline: DPD) which stabilise the molecular structure. In this preliminary study we look for quantitative or qualitative alterations of collagen cross-linkage in intracranial aneurysms of posterior communicating artery (PCoA) which may be considered a site of elective weakening in the intracranial arterial circulation, being one of the most frequent locations of intracranial aneurysms and of so called "infundibular widening'. We analysed the collagen cross-linkage in 6 autopsy samples of intracranial arterial segments of the Circle of Willis which were removed from patients whose cause of death was other than cerebral hemorrhage, and in 6 samples of intracranial PCoA aneurysms obtained at surgery. The analysis of cross-links showed that there was no significant difference in collagen and cross-link content between autopic and aneurysmatic samples except for PCoA. In autoptic nonaneurysmatic samples of PCoA a lower content of PYD than in internal carotid artery (ICA) and a lower content of DPD than in all other arterial segments has been demonstrated; moreover a lower content of cross-links (DPD + PYD/ Collagen) was evident in nonaneurysmatic PCoA samples when compared to other segments (ICA and Anterior Communicating Artery). On the other hand, the mean content of DPD was significantly lower in PCoA aneurysms than in nonaneurysmatic samples of the artery and moreover, a significantly low content of cross-links (DPD + PYD/Collagen) is overemphasised in PCoA aneurysms, suggesting that the peculiar lower content of DPD in PCoA arteries may be considered the expression of minor resistance of the arterial wall at this site, and may be related to the higher incidence of aneurysms or infundibular widening of this arterial segment.

TNF-α induces phenotypic modulation in cerebral vascular smooth muscle cells: implications for cerebral aneurysm pathology.

Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-α) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-α activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-α and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-α inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-α induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.

Genetic endothelial systems biology of sickle stroke risk.

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1beta/tumor necrosis factoralpha. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.

Expression of alpha2-macroglobulin, neutrophil elastase, and interleukin-1alpha differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis.

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor alpha(2)-macroglobulin (A2M), the neutrophil elastase (NE)-an enzyme degrading elastin and collagen fibers-and the proinflammatory protein interleukin-1alpha (IL-1alpha) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61-91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1alpha. In initial AS lesions A2M was found, whereas NE and IL-1alpha were absent. NE and IL-1alpha became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1alpha was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1alpha in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1alpha in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.