Matrix metalloproteinases as target genes for gene regulatory networks driving molecular and cellular pathways related to a multistep pathogenesis of cerebrovascular disease.

The present study investigated a joint contribution of matrix metalloproteinases (MMPs) genes to ischemic stroke (IS) development and analyzed interactions between MMP genes and genome-wide associated loci for IS. A total of 1288 unrelated Russians (600 IS patients and 688 healthy individuals) from Central Russia were recruited for the study. Genotyping of seven single nucleotide polymorphisms (SNPs) of MMP genes (rs1799750, rs243865, rs3025058, rs11225395, rs17576, rs486055, and rs2276109) and eight genome-wide associated loci for IS were done using Taq-Man-based assays and MALDI-TOF mass spectrometry iPLEX platform, respectively. Allele - 799T at rs11225395 of the MMP8 gene was significantly associated with a decreased risk of IS after adjustment for sex and age (OR = 0.82; 95%CI, 0.70-0.96; P = 0.016). The model-based multifactor dimensionality reduction method has revealed 21 two-order, 124 three-order, and 474 four-order gene-gene (G×G) interactions models meaningfully (Pperm < 0.05) associated with the IS risk. The bioinformatic analysis enabled establishing the studied MMP gene polymorphisms possess a clear regulatory potential and may be targeted by gene regulatory networks driving molecular and cellular pathways related to the pathogenesis of IS. In conclusion, the present study was the first to identify an association between polymorphism rs11225395 of the MMP8 gene and IS risk. The study findings also indicate that MMPs deserve special attention as a potential class of genes influencing the multistep mechanisms of cerebrovascular disease including atherosclerosis in cerebral arteries, acute cerebral artery occlusion as well as the ischemic injury of the brain and its recovery.

Omega-3 fatty acid supplement reduces activation of NADPH oxidase in intracranial atherosclerosis stenosis.

Objectives Intracranial atherosclerotic stenosis (ICAS) is one of the most common causes of stroke worldwide. We adapted a rat model of atherosclerosis to study brain intracranial atherosclerosis, and further investigated how omega-3 fatty acids (O3FA) attenuated the development of ICAS by reducing the generation of reactive oxygen species (ROS) and the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity. Methods Adult male Sprague-Dawley rats were divided into control normal-cholesterol or high-cholesterol diet groups with or without O3FA for up to 6 weeks. NG-nitro-L-arginine methyl ester (L-NAME, 3 mg/mL), a nitric oxide synthase inhibitor, was added to the drinking water of the high-cholesterol groups during the first 2 weeks. The rats received supplementation with O3FA (5 mg/kg/day) by gavage. At 3 and 6 weeks, we measured blood lipid levels, including low-density lipoprotein (LDL), cholesterol (CHO), triglycerides (TG), and high-density lipoprotein (HDL) as atherosclerotic blood markers. The lumen of middle cerebral artery (MCA) and the thickness of the vessel wall were assessed histologically. ROS production was measured. NOX activity and mRNA and protein expression of NOX subunits (p47phox, gp91phox, p22phox, and p67phox) were measured. Results A high-cholesterol diet exhibited a significant increase in the classic blood markers (LDL, CHO, and TG) for atherosclerosis, as well as a decrease in HDL. These markers were found to be progressively more severe with time. Additionally, increased lumen stenosis and intimal thickening were observed in the MCA for this group. Rats given O3FA demonstrated attenuation of blood lipid levels with an absence of morphological changes.O3FA significantly reduced ROS production and NOX activity in the brain. Moreover, O3FA decreased the mRNA and protein expression of the NOX subunits p47phox, gp91phox, and p67phox. Conclusions Long-term O3FA dietary supplementation prevents the development of intracranial atherosclerosis. This O3FA effect appears to be mediated by its attenuation of NOX subunit expression and NOX activity, therefore reducing ROS production. O3FA dietary supplement shows promising results in the prevention of ICAS.

Paraoxonase 1 (PON1) and stroke; the dilemma of genetic variation.

Ischaemic stroke, which is mostly caused by atherosclerosis, is one of the most widely causes of mortality and morbidity. One of the most important enzymatic activities of paraoxonase-1 (PON1) in association with high-density lipoprotein (HDL) is the prevention of low-density lipoprotein (LDL) oxidation, which gives it an anti-atherogenic activity. PON1 expression and activity is influenced by various factors; the most important of which is genetic polymorphism, mainly single nucleotide polymorphisms (SNPs). The results of various studies in different populations indicate that some SNPs of the PON1 gene are associated with stroke. Therefore, in this review we studied various researches, in which the association of PON1 SNPs with stroke had been investigated in different populations.

The elevated lipoprotein-associated phospholipase A2 activity is associated with the occurrence and recurrence of acute cerebral infarction.

There is a strong association between lipoprotein-associated phospholipase A2 (Lp-PLA2) levels and atherosclerosis-related diseases. The aim of this study was to investigate the role of Lp-PLA2 in the ischemic stroke and further offer clinical evidence that measuring Lp-PLA2 helps predict the risk of stroke occurrence and recurrence. A total of 328 hospitalized patients were recruited, including 179 cases of acute cerebral infarction (ACI) and 149 non-ACI controls. The serum level of Lp-PLA2 in ACI was significantly higher than non-ACI. The serum level of Lp-PLA2 in the recurrence of ACI was significantly higher than the nonrecurrence. The serum levels of Lp-PLA2 in large-artery atherosclerosis subtype were the highest among the subtypes of the Trial of Org 10172 in Acute Stroke Treatment and non-ACI controls. The level of Lp-PLA2 in large-artery atherosclerosis and the cardioembolism group was statistically significantly higher than that of the control cases. There was no statistically significant difference between the small-vessel occlusion group and the control cases. The present study confirmed that the elevated Lp-PLA2 level can be a risk factor for ischemic stroke in the Chinese population. The serum level of Lp-PLA2 may be a predictive factor for the recurrence of ACI.

Atherosclerosis and arteriosclerosis parameters in stroke patients associate with paraoxonase polymorphism and esterase activities.

BACKGROUND AND PURPOSE: Polymorphic paraoxonase (PON1) variants can variably prevent low- and high-density lipoprotein oxidation, but their role in provoking atherosclerosis remained unclear. We addressed this issue by profiling PON1 polymorphisms and enzymatic activities, and assessing atherosclerosis and cerebral arteriosclerosis severity in post-stroke patients. METHODS: Carotid artery intima-media-thickness (IMT), cerebral white matter lesions (WML), serum PON1 -108C/T, Q192R and L55M polymorphisms, and PON and acetylcholinesterase (AChE) enzyme activities were determined in 237 patients. RESULTS: Genetic variation at the PON1 locus showed a strong influence on PON1 activity in ischaemic stroke patients, but lacked direct influence on IMT. Stroke patients with PON1 QQ192 or MM55 genotypes demonstrated lower PON and arylesterase activities at both Day 1 and 12 months post-stroke than patients with either RQ/RR192 or LM/LL55 genotypes (P < 0.001). Furthermore, patients with carotid atherosclerosis and/or cerebral arteriosclerosis expressed as IMT, carotid plaques and WML had lower 12 months PON1 activity than patients without (P = 0.02, P = 0.027 and P = 0.001, respectively), and PON and AChE hydrolysis rates were more tightly correlated in patients carrying the PON1 192R compared with the 192QQ allele, in a gene dose-dependent manner (P < 0.001). CONCLUSION: Our findings show inverse PON1 activity-carotid atherosclerosis and -cerebral arteriosclerosis association in stroke patients: the lower the PON1 activity the more progressed is the atherosclerotic process and the weaker is the association with AChE activity. Extending previous PON1 genetic studies in stroke populations, our study emphasizes the PON1 activity as a potential anti-atherogenic element and proposes involvement of cholinesterase activities in its effects.

Biomarkers and location of atherosclerosis: matrix metalloproteinase-2 may be related to intracranial atherosclerosis.

BACKGROUND: Various biomarkers are linked with the pathophysiology of atherosclerosis. We hypothesized that these factors may be associated with the location and burden of cerebral atherosclerosis. METHODS: We evaluated 177 consecutive patients with chronic (>6 months) ischemic stroke: 68 with small vessel occlusion (SVO) and 109 with large-artery atherosclerosis (LAA), with the latter further sub-classified into 80 patients with intracranial atherosclerosis (ICAS) and 29 with extracranial atherosclerosis (ECAS). The number of ≥50% steno-occlusions on magnetic resonance angiography was used to assess the burden of atherosclerosis. Serum concentrations of the biomarkers (matrix metalloproteinases (MMP)-2 and -9, homocysteine, interleukin (IL)-6, tumor necrosis factor-α, C-reactive protein, adiponectin, leptin, resistin, free fatty acid, and lipoprotein(a)) and the metabolic syndrome were measured in each study subject. RESULTS: Decreased plasma concentrations of MMP-2 (p = 0.020) and homocysteine (p = 0.038) were more closely associated with ICAS than with ECAS, whereas increased IL-6 concentrations were related to severe (≥4 steno-occlusions) atherosclerosis (p = 0.031). Multiple logistic regression analysis showed that the lowest tertile of MMP-2 was independently associated with ICAS (OR 4.84, 95% CI 1.29-18.19, p = 0.022). CONCLUSION: Low MMP-2 plasma levels are associated with intracranial location of cerebral atherosclerosis, suggesting that MMP-2 may play a role in the development of ICAS.

Changes in plasma matrix metalloproteinase-9 levels in patients with acute ischemic stroke.

Matrix metalloproteinases (MMP) have been thought to be involved in stroke pathogenesis. MMP-9 contributes to tissue destruction. Our aim was to analyze the MMP-9 levels in blood within 24 hours of acute ischemic stroke onset to observe the role of MMP-9 in the pathogenesis of atherothrombotic stroke. In this study we investigated prospectively MMP-9 levels in serum from 106 patients (42 men and 64 women, mean age 71.52 +/- 6.32 years) with acute ischemic stroke in the middle cerebral artery area in the first 24 hours from the onset (mean duration 7.8 +/- 4.5 hours) as compared to 112 controls (48 men and 64 women, mean age 70.36 +/- 6.8 years). Serum samples were collected under sterile conditions and stored in aliquots at -70 degrees C until assay. Serum MMP-9 levels were determined by enzyme-linked immunosorbent assay (ELISA) in blood samples obtained on admission. Statistical analysis was performed by Mann-Whitney and Log-Likeliwood Ratio tests. All values reported are expressed as mean (x) +/- SD. Mean serum MMP-9 concentrations were higher in group with ischemic stroke 172 +/- 32.4 ng/mL, range 139.6-204.4 ng/mL vs. controls 57 +/- 9.6 ng/mL, range 47.4-66.6 ng/mL (95% CI, 3.17 to 14.18; p < 0.014). In conclusion, MMP-9 activity is associated with early acute ischemic stroke. The high levels of MMP-9 in acute ischemic stroke document the involvement of this enzyme in the regulation of inflammation in stroke.

The paraoxonase gene polymorphism in stroke patients and lipid profile.

OBJECTIVES: The paraoxonase (PON) gene can reduce the risk of developing atherosclerosis. We investigated the associations between PON polymorphisms and ischemic stroke. We also investigated the associations between PON polymorphisms and lipid profile in stroke patients. METHODS: A total of 350 patients with ischemic stroke and 242 control subjects in Korean population were genotyped for the PON1M55 L, PON1Q192R, PON2A148 G and PON2S311C polymorphisms using melting point analysis with LightCycler real-time polymerase chain reaction (PCR) technology. RESULTS: There were no significant differences in genotype and allele distribution of the PON polymorphisms between the ischemic stroke patients and control subjects. The concentration of total homocysteine was significantly different in the PON1M55 L polymorphism (P = 0.047), and the apolipoprotein (Apo)B concentration was significantly different in the PON1Q192R polymorphism (P = 0.02) in stroke patients. The concentrations of low-density lipoprotein (LDL) cholesterol and ApoB were significantly different between the PON2A148 G (P = 0.011, P = 0.000, respectively) and PON2S311C polymorphisms (P = 0.046, P = 0.003, respectively) in stroke patients. CONCLUSIONS: This study did not provide association between PON gene polymorphisms and ischemic stroke. However, it confirmed that the PON1L55 L allele is associated with plasma concentration of total homocysteine and that the PON2G148 G and PON2S311S allele is associated with plasma concentrations of LDL cholesterol and ApoB.

Mutations in the acid alpha-glucosidase gene (M. Pompe) in a patient with an unusual phenotype.

Glycogenosis type II (Pompe disease) is a lysosomal storage disease caused by deficiency of acid alpha-glucosidase (acid maltase). The disease is autosomal recessive inherited and is clinically and genetically heterogenous. The authors describe a 30-year-old woman affected by late-onset Pompe disease with vascular affection resembling atherosclerotic angiopathy of the elderly. Genetic analysis revealed two novel mutations (Ala237Val and Gly293Arg) in the acid alpha-glucosidase gene in this patient.

Age-related effects on atherogenesis and scavenger enzymes of intracranial and extracranial arteries in men without classic risk factors for atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis occurs later and is less extensive in intracranial arteries than in extracranial arteries. However, the mechanisms responsible are poorly understood. A previous study has suggested a better antioxidant protection of intracranial arteries. METHODS: To assess the influence of age on arterial activity of antioxidant enzymes and atherogenesis, we compared intracranial and extracranial arteries of humans of different ages who retrospectively lacked confounding classic risk factors (48 premature fetuses aged 6.4+/-0.8 months [mean+/-SD], 58 children aged 7.9+/-3.8 years, 42 adults aged 42.5+/-5.1 years, and 40 elderly subjects aged 71.8+/-3.4 years; all males). Lesions were quantified by computer-assisted imaging analysis of sections of the middle cerebral and basilar arteries, the left anterior descending coronary artery, the common carotid artery, and the abdominal aorta. Macrophages, apolipoprotein B, oxidized LDL, and matrix metalloproteinase-9 in lesions were determined by immunocytochemistry. The effect of aging on atherogenesis was then compared with that on the activity of 4 antioxidant enzymes in the arterial wall. RESULTS: Atherosclerosis was 6- to 19-fold greater (P<0.01) in extracranial arteries than in intracranial arteries, and it increased linearly with age. Intracranial arteries showed significantly greater antioxidant enzyme activities than did extracranial arteries. However, the antioxidant protection of intracranial arteries decreased significantly in older age, coinciding with a marked acceleration of atherogenesis. An increase in matrix metalloproteinase-9 protein expression and in gelatinolytic activity consistent with the degree of intracranial atherosclerosis was also observed. CONCLUSIONS: These results suggest that a greater activity of antioxidant enzymes in intracranial arteries may contribute to their greater resistance to atherogenesis and that with increasing age intracranial arteries respond with accelerated atherogenesis when their antioxidant protection decreases relatively more than that of extracranial arteries.

Elastase-type enzymes and their relation to blood lipids in atherosclerotic patients.

Serum elastase-type activity, elastase inhibitory capacity and their relation to lipids were examined in 140 male patients with ischemic vascular disease (coronary, cerebral, peripheral) and in 60 control subjects. In a further 24 patients with acute myocardial infarction elastase activity, inhibitory capacity and lipids during the course of the illness have also been investigated. Serum elastase-type activity was found to be significantly lower and inhibitory capacity significantly higher in the groups of patients than in the controls. HDL- and HDL2-cholesterol as well as apo A concentration showed significant negative correlation with elastase inhibitory capacity both in atherosclerotic and in control subjects. During the course of myocardial infarction a significant elevation of serum elastase-type activity could be observed at the end of the first week; serum triglyceride levels increased, HDL- and HDL2-concentrations decreased significantly in the first 3 weeks, than gradually approached the initial values. In the patients with an elevation of serum elastase-like activity by more than 30% in the first week, there was a significantly higher elevation of serum GOT and LDH1 and a greater occurrence of transmural (Q) infarction than in those with a smaller variation of elastase-like activity.

[Changes in the neural apparatus of the cerebral arteries in atherosclerosis]. / Izmeneniia nervnogo apparata arterii golovnogo mozga pri ateroskleroze.

Nervous apparatus of the brain arteries (450-60 microns in diameter) in 55-64-year-old patients with cerebral atherosclerosis was studied. Alterations in the afferent innervation were manifested in the increased sinuosity of fibers, areas of hyper- and hypoimpregnation, appearance of thickenings in the fibers. Changes in the vasomotor innervation consisted of the disturbances of the nerve network structure, an increase of nerve fibers concentration and varicosity in the cholinergic plexuses and their decrease in the adrenergic ones. Restructurings of the nerve apparatus were more pronounced in the arteries of large caliber.

[Histochemical characteristics of the vascular-capillary bed of the brain during aging and in arteriosclerosis]. / Gistokhimicheskaia kharakteristika sosudisto-kapilliarnogo rusla golovnogo mozga pri starenii i ateroskleroze.

Using the alkaline phosphatase test, the authors studied the capillaries of 41 fields in clinically healthy people aged 22-64 years and animals (dogs, cats, rats) and also in individuals aged 45 to 64 years with mild atherosclerosis in the area of the median cerebral artery trunk. It has been established that unlike "normal" people, atherosclerotic patients of the respective age show an increase in the volume of deposited blood in the presence of a reduction of the working surface and the average indices of the activity of alkaline phosphatase leading to a considerable diminution (21.5-23.5%) in the intensity of metabolic processes.

Effect of cardiac glycosides on the release of adenylate kinase into cerebrospinal fluid of patients with cerebral arteriosclerosis.

48 patients with cerebral arteriosclerosis were found to have a manifest release of adenylate kinase (AK) into cerebrospinal fluid (CSF). This release was most probably due to an increased leak in the brain cells subsequent to a lowered adenylate charge potential followed by a diminished electrochemical potential in these cells suffering from disturbed oxygen supply. A further increase of AK release into CSF was noted for the 22 patients receiving cardiac glycosides compared to the 26 patients not treated with these drugs. The mean AK value of the former group was 0.119 +/- 0.028 U/l compared to that of the latter group, being 0.089 +/- 0.025 U/l, and this difference was significant (p less than 0.001). The effect of cardiac glycosides is most probably explained by an additional lowering of the membrane electrochemical potential in brain cells of these patients due to the direct action of cardiac glycosides on the Na+- and K+-dependent ATPase system in these cells, resulting in an increased leak in the plasma membrane.

[Morphologo-histochemical characteristics of ischemic cerebral infarcts in the acute stage of the stroke in patients with atherosclerosis]. / Morfologo-gistokhimicheskaia kharakteristika ishemicheskikh infarktov mozga v ostroi stadii insul'ta u bol'nykh aterosklerozom.

Morphological and histochemical investigations of the activity of succinate, malate, lactate, isocitrate, and glucoso-6-phosphate dehydrogenases, and NAD-diaphorase in the central, marginal, borderline and perifocal zones of ischemic brain infarctions were carried out. Deep changes in the activity of the enzymes listed, and peculiarities of this activity in various brain structures and tissues were revealed. A connection between the activity of the enzymes, the duration of the infarction, and the structural and biochemical peculiarities of the affected brain part is demonstrated. A suggestion is made on the pathogenesis of the ischemic infarctions and on the possibility of using histochemical data for the purposes of the pathoanatomic diagnosis.

[Alkaline phosphatase and ATPase activity in the walls of the cerebral vessels in hypertension and arteriosclerosis with disorders of the cerebral circulation]. / Aktivnost' shchelochnoi fosfatazy i ATF-azy v stenkakh sosukov golovnogo mozga pri gipertonicheskoi bolezni i ateroskleroze s narusheniiami mozgovogo krovoobrashcheniia.

In hypertensive disease and atherosclerosis without acute disorders of cerebral circulation it was established that in the cerebral vessel walls there was a high activity of alkaline phosphatase and adenosintriphosphtase. In the symmetrical areas of the subcortical nodes differences in the activity of these enzymes were insignificant and not valid. In vessel walls, located in the peripheral zone of the apoplectic hemorrhage and in the adjacent brain tissue the activity of alkaline phosphatase and adenosintriphosphatase drops. The existence of a high activity of enzymes in the vessel walls on the early stages of hemorrhages gives ground to the authors to claim that in the peripheral zone of an extensive hemorrhage a drop in the enzyme activity appears in the process of a development of a stroke.