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.

Variants in COX-2, PTGIS, and TBXAS1 Are Associated with Carotid Artery or Intracranial Arterial Stenosis and Neurologic Deterioration in Ischemic Stroke Patients.

BACKGROUND: Eicosanoids may play a role in ischemic stroke (IS). However, the association of variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and neurologic deterioration (ND) is not fully understood. The aim of the present study was to investigate the association of 11 variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and ND. METHODS: Eleven variants in eicosanoid genes were examined using mass spectrometry method in 297 IS patients. The symptomatic carotid artery or intracranial arterial stenosis was assessed by computed tomographic angiography. Platelet aggregation and platelet-leukocyte aggregates were measured. The primary outcome was ND within 10 days of admission. ND was defined as an increase of 2 or more points in National Institutes of Health Stroke Scale score. RESULTS: Among 297 IS patients, 182 (61.3%) cases had symptomatic carotid artery or intracranial arterial stenosis, and 88 (29.6%) patients experienced ND within 10 days after admission. Symptomatic carotid artery or intracranial arterial stenosis was significantly associated with higher ND (P < .001). Rs20417CC, rs41708TT, and rs5629CC were independent risk factors for symptomatic carotid artery or intracranial arterial stenosis and ND, and associated with higher platelet aggregation and platelet-leukocyte aggregates. CONCLUSIONS: Symptomatic carotid artery or intracranial arterial stenosis was associated with higher ND. Rs20417CC, rs41708TT, and rs5629CC were not only independent risk factors for symptomatic carotid artery or intracranial arterial stenosis, but also independent risk predictors for ND.

MTHFR C677T gene mutation as a risk factor for arterial stroke: a hospital based study.

Elevated homocysteine level is an independent risk factor for ischemic stroke, thrombotic and cardiovascular diseases. The enzyme methylenetetrahydrofolate reductase (MTHFR) plays a crucial role in regulating the levels of homocysteine. A C677T mutation in this gene results in reduced activity. Sixty-nine patients with arterial stroke, six patients with venous stroke (confirmed by computed tomography and/or magnetic resonance imaging) with hyperhomocysteinemia were selected for the study. Forty-nine subjects with no past history of stroke served as controls. MTHFR genotypes were determined by PCR using specific primers, followed by restriction digestion and gel analysis. The prevalence of the mutated homozygous and heterozygous C677T MTHFR genotype in the patients with arterial stroke was 1.4% (one of 69) and 31.88% (21 of 69), respectively. There frequency was 16.6% (one of six) and 33.3% (two of six) in venous stroke. The genotyping results from controls showed that there was only one heterozygote out of the 49 studied (2.08%). There was a significant difference between the control and the patient groups. Odds ratio for the probability of the C677T MTHFR gene mutation in the patients versus control group was 22.29 (95% CI 4.89-98.8). This indicates that C677T MTHFR mutation is strongly associated with arterial stroke especially in young adults. MTHFR allele evaluation will help in preventing/reducing morbidity caused by stroke.

Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice.

Heme oxygenase-1 (HO-1, HSP32) is an early gene that is responsive to an array of pathological conditions including, but not limited to, hypoxia and cerebral ischemia. HO-1 cleaves the heme molecule and produces carbon monoxide (CO) and biliverdin (an antioxidant) and is essential for iron homeostasis. The purpose of this study was to investigate, using transgenic (Tg) mice, whether overexpression of HO-1 in the brain augments or attenuates cellular injury caused by ischemic stroke. Homozygous HO-1 Tg mice that overexpress HO-1 under the control of the neuron-specific enolase promoter (characterized previously) were used. Under halothane anesthesia and normothermic conditions, wild-type nontransgenic (nTg; n = 22) and HO-1 Tg (n = 24) mice were subjected to middle cerebral artery occlusion (MCAo). Six hours after induction of ischemia, Tg and nTg mice developed infarcts that were 39 +/- 6 and 63 +/- 9 mm3, respectively (p < 0.01). No significant difference between the two strains was observed in the values of brain edema (11.3 +/- 4% in Tg vs. 14.6 +/- 5% in nTg; p < 0.1). At 24 h after MCAo, Tg mice exhibited significant neuroprotection as determined by the stroke volumes (41 +/- 2 mm3 in Tg vs. 74 +/- 5 mm3 in nTg; p < 0.01) and values of ischemic cerebral edema (21 +/- 6% in Tg vs. 35 +/- 11% in nTg; p < 0.01). Data suggest that neuroprotection in Tg mice was, at least in part, related to the following findings: (a) constitutively up-regulated cyclic GMP and bcl-2 levels in neurons; (b) inhibition of nuclear localization of p53 protein; and (c) antioxidant action of HO-1, as detected by postischemic neuronal expression of ferritin, and decreases in iron staining and tissue lipid peroxidation. We suggest that pharmacological stimulation of HO-1 activity may constitute a novel therapeutic approach in the amelioration of ischemic injury during the acute period of stroke.

[Lack of evidence that inducible nitric oxide synthase participates in the development of ischemic brain damage].

We have already reported that the concentration of nitric oxide (NO) increases during and after cerebral ischemia and a selective inhibitor of neuronal NO synthase (nNOS) suppresses this increase and subsequently mitigates brain damage in rats. Although the selective inhibition of nNOS is a promising pharmacological strategy for the treatment of stroke, the role of inducible NOS (iNOS) remains to be clarified. Toward this end, we investigated temporal alterations in iNOS mRNA by the RT-PCR method in a rat model of middle cerebral artery (MCA) occlusion. We found that iNOS mRNA in the ischemic hemisphere began to increase at 3 hr and reached the maximum level at 24 hr of reperfusion following 3 hr of MCA occlusion. However, quantitative analysis revealed that no significant difference existed between 6 hr or 24 hr reperfusion group and their respective time-matched sham operation group. In addition, neither Western blotting nor immunocytochemical study disclosed an apparent induction of iNOS at any time points examined. Similar results were obtained at 24 hr of permanent MCA occlusion. Taken together, these data indicate that iNOS induction during and after MCA occlusion may be not a critical event for the development of infarction caused by ischemia itself.

Elevated immunoreactivity for glutamic acid decarboxylase in the rat cerebral cortex following transient middle cerebral artery occlusion.

We investigated the expression of glutamic acid decarboxylase (molecular weight 67,000; GAD67) immunohistochemically in the rat cerebral cortex following transient middle cerebral artery occlusion (MCAO) capable of producing slowly progressive neuronal damage. An increase in GAD67 immunoreactivity was observed in the cerebral cortex ipsilateral to the ischemic insult, most prominent in lamina IV, 3 to 14 days after MCAO. At this stage, light microscopy showed GAD67-positive puncta to be larger and more strongly immunoreactive in the ipsilateral cortex than those in the contralateral side. The elevated expression of GAD67 in the insulted cortex may reflect part of the adaptive functional changes in GABA transmission with slowly progressive cortical ischemic damage.

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20.

Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a full-length cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.