Anna Tjärnlund-Wolf

Fibrinolytic Gene Polymorphism and Ischemic Stroke

Background and Purpose— The tissue-type plasminogen activator (tPA) −7351C>T and the plasminogen activator inhibitor type 1 (PAI-1) -675 4G >5G polymorphisms influence transcriptional activity. Both variants have been associated with myocardial infarction, with increased risk for the T and 4G allele, respectively. In this study we investigated the possible association between these polymorphisms, the respective plasma protein levels, and ischemic stroke. Methods— In the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS), 600 patients with acute ischemic stroke aged 18 to 69 years and 600 matched community controls were recruited. Stroke subtype was determined using Trial of Org 10172 in Acute Treatment criteria. Results— There were no associations between individual genetic variants and ischemic stroke. The multivariate-adjusted odds ratio for overall ischemic stroke was 1.11 (95% CI 0.87 to 1.43) for tPA T allele carriers, and 0.84 (95% CI, 0.64 to 1.11) for subjects homozygous for the PAI-1 4G allele. When genotypes were combined, a protective effect for the tPA CC/PAI-1 4G4G genotype combination was observed (odds ratio 0.65, 95% CI 0.43 to 0.98; P<0.05). Plasma levels of tPA and PAI-1 antigen at follow-up were independently associated with overall ischemic stroke. tPA-antigen differed by stroke subtype and was highest among those with large-vessel disease and cardioembolic stroke. Conclusions— Neither the tPA −7351C>T nor the PAI-1 to 675 4G >5G polymorphism showed significant association with ischemic stroke. For the tPA CC/PAI-1 4G4G genotype combination, a protective effect was observed. Collectively, these results are consistent with a more complex role for tPA and PAI-1 in the brain as compared with the heart.

Plasminogen Activator Inhibitor-1 and Thrombotic Cerebrovascular Diseases

Alterations in thrombosis and fibrinolysis comprise important parts of stroke pathophysiology. A key step in the fibrinolytic process includes the tissue-type plasminogen activator (tPA)-mediated conversion of the proenzyme plasminogen into the active protease plasmin, which in turn degrades the fibrin structure of intravascular thrombi. There are a number of review articles well summarizing molecular mechanisms of the fibrinolytic system.1,2 Inhibition of the fibrinolytic system may occur at the level of plasminogen activation, mainly by a direct inhibition of tPA by plasminogen activator inhibitor 1 (PAI-1) or indirectly by thrombin-activatable fibrinolysis inhibitor and, at the level of plasmin, by α2-antiplasmin. The roles of these 3 inhibitors are complementary in thrombolysis.3 PAI-1 has become recognized as a central molecule linking pathogenesis and progression of thrombotic vascular events including stroke. As a main endogenous inhibitor of tPA, PAI-1 might be related to reperfusion efficacy and hemorrhagic risk of tPA thrombolytic therapy. Moreover, a clear association has been observed between elevated PAI-1 plasma levels and prothrombotic disease conditions such as hypertension, obesity, insulin resistance, and diabetes.4–7 Clinical and experimental studies show that PAI-1 deficiencies cause accelerated fibrinolysis and bleeding, whereas elevated PAI-1 plasma levels are associated with vascular thrombosis.8 Concentrations of active PAI-1 in newly formed thrombi can be several thousandfold greater than active PAI-1 concentrations in normal plasma9 and thus high enough to inhibit doses of tPA used for clinical thrombolysis. Furthermore, beyond its effects in modulating the activity of tPA and the functionally related urokinase-type plasminogen activator, which predominantly catalyzes plasmin formation in the extravascular space, PAI-1 also plays diverse roles in metabolic and vascular disease and may participate in the evolution of brain damage and recovery after stroke.4 …

Expression of plasminogen activator inhibitor-1 and protease nexin-1 in human astrocytes: Response to injury-related factors

Astrocytes play a diverse role in central nervous system (CNS) injury. Production of the serine protease inhibitors (serpins) plasminogen activator inhibitor‐1 (PAI‐1) and protease nexin‐1 (PN‐1) by astrocytes may counterbalance excessive serine protease activity associated with CNS pathologies such as ischemic stroke. Knowledge regarding the regulation of these genes in the brain is limited, so the objective of the present study was to characterize the effects of injury‐related factors on serpin expression in human astrocytes. Native human astrocytes were exposed to hypoxia or cytokines, including interleukin‐6 (IL‐6), IL‐1β, tumor necrosis factor‐α (TNF‐α), IL‐10, transforming growth factor‐α (TGF‐α), and TGF‐β for 0–20 hr. Serpin mRNA expression and protein secretion were determined by real‐time RT‐PCR and ELISA, respectively. Localization of PAI‐1 and PN‐1 in human brain tissue was examined by immunohistochemistry. Hypoxia and all assayed cytokines induced a significant increase in PAI‐1 expression, whereas prolonged treatment with IL‐1β or TNF‐α resulted in a significant down‐regulation. The most pronounced induction of both PAI‐1 and PN‐1 was observed following early treatment with TGF‐α. In contrast to PAI‐1, the PN‐1 gene did not respond to hypoxia. Positive immunoreactivity for PAI‐1 in human brain tissue was demonstrated in reactive astrocytes within gliotic areas of temporal cortex. We show here that human astrocytes express PAI‐1 and PN‐1 and demonstrate that this astrocytic expression is regulated in a dynamic manner by injury‐related factors.

Genetic variation at the BDNF locus: evidence for association with long-term outcome after ischemic stroke.

Background and Purpose Rates and extent of recovery after stroke vary considerably between individuals and genetic factors are thought to contribute to post-stroke outcome. Brain-derived neurotrophic factor (BDNF) plays important roles in brain plasticity and repair and has been shown to be involved in stroke severity, recovery, and outcome in animal models. Few clinical studies on BDNF genotypes in relation to ischemic stroke have been performed. The aims of the present study are therefore to investigate whether genetic variation at the BDNF locus is associated with initial stroke severity, recovery and/or short-term and long-term functional outcome after ischemic stroke. Methods Four BDNF tagSNPs were analyzed in the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS; 600 patients and 600 controls, all aged 18–70 years). Stroke severity was assessed using the NIH Stroke Scale (NIHSS). Stroke recovery was defined as the change in NIHSS over a 3-month period. Short- and long-term functional outcome post-stroke was assessed using the modified Rankin Scale at 3 months and at 2 and 7 years after stroke, respectively. Results No SNP was associated with stroke severity or recovery at 3 months and no SNP had an impact on short-term outcome. However, rs11030119 was independently associated with poor functional outcome 7-years after stroke (OR 0.66, 95% CI 0.46–0.92; P =  0.006). Conclusions BDNF gene variants were not major contributors to ischemic stroke severity, recovery, or short-term functional outcome. However, this study suggests that variants in the BDNF gene may contribute to poor long-term functional outcome after ischemic stroke.

Two conserved regions within the tissue-type plasminogen activator gene promoter mediate regulation by brain-derived neurotrophic factor

Tissue‐type plasminogen activator (t‐PA) has recently been identified as a modulator of neuronal plasticity and can initiate conversion of the pro‐form of brain‐derived neurotrophic factor (BDNF) into its mature form. BDNF also increases t‐PA gene expression implicating t‐PA as a downstream effector of BDNF function. Here we demonstrate that BDNF‐mediated induction of t‐PA mRNA requires an increase in t‐PA gene transcription. Reporter constructs harboring 9.5 kb of the human t‐PA promoter conferred BDNF‐responsiveness in transfected mouse primary cortical neurons. This regulation was recapitulated in HEK 293 cells coexpressing the TrkB neurotrophin receptor. t‐PA promoter‐deletion analysis revealed the presence of two BDNF‐responsive domains, one located between −3.07 and −2.5 kb and the other within the proximal promoter. The upstream region was shown to confer BDNF responsiveness in a TrkB‐dependent manner when attached to a heterologous promoter. We also identify homologous regions within the murine and bovine t‐PA gene promoters and demonstrate that the equivalent upstream murine sequence functions as a BDNF‐responsive enhancer when inserted 5′ of the human proximal t‐PA promoter. Hence, BDNF‐mediated induction of t‐PA transcription relies on conserved modular promoter elements including a novel upstream BDNF‐responsive domain and the proximal t‐PA gene promoter.

Allele-specific transcription of the PAI-1 gene in human astrocytes

The 4G allele of the PAI-1 -675(4G/5G) insertion/deletion promoter polymorphism has been associated with elevated plasma levels of PAI-1 and an increased risk of myocardial infarction. However, this allele has also been associated with a reduced risk of ischaemic stroke. In the brain, PAI-1 is mainly produced by astrocytes, and can reduce the neurotoxic effects exerted by tissue-type plasminogen activator during pathophysiologic conditions. The aim of the present study was to investigate whether the PAI-1 -675(4G/5G) polymorphism and the linked -844A/G polymorphism affect transcriptional activity of the PAI-1 gene in human astrocytes. Haplotype chromatin immunoprecipitation (haploChIP) was used in order to quantify allele-specific promoter activity in heterozygous cells. Protein-DNA interactions were investigated by electrophoretic mobility shift assay (EMSA). A clear allele-specific difference in PAI-1 gene expression was observed in astrocytes, where the haplotype containing the 4G and the -844A alleles was associated with higher transcriptional activity compared to the 5G and -844G-containing haplotype. EMSA revealed an allele-specific binding of nuclear proteins to the 4G/5G site as well as to the -844A/G site. Supershift experiments identified specific binding of the transcription factors Elf-1 and Elk-1 to the -844G allele. The relative impact of the different sites on allele-specific PAI-1 promoter activity remains to be determined. We demonstrate that common polymorphisms within the PAI-1 promoter affect transcriptional activity of the PAI-1 gene in human astrocytes, thus providing a possible molecular genetic mechanism behind the association between PAI-1 promoter variants and ischaemic stroke.

Retinoids and activation of PKC induce tissue‐type plasminogen activator expression and storage in human astrocytes

Summary.  Background: Emerging data demonstrate important roles for tissue‐type plasminogen activator (t‐PA) in the central nervous system (CNS). In contrast to endothelial cells, little is known about the regulation of t‐PA gene expression and secretion in astrocytes. Objectives: The aims of the present study were to investigate whether t‐PA gene expression is regulated by retinoids and the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) in human astrocytes, and to study whether t‐PA is stored and subject to regulated release from these cells, as with endothelial cells. Methods: Native human astrocytes were treated with RA and/or PMA. mRNA was quantified by real‐time RT‐PCR and protein secretion determined by ELISA. Intracellular t‐PA immunoreactivity in astrocytes was examined by immunocyto‐ and histochemistry. Results: RA and/or PMA induced a time‐dependent increase in t‐PA mRNA and protein levels in astrocytes, reaching 10‐fold after combined treatment. This was associated with increased amounts of t‐PA storage in intracellular granular structures. Both forskolin and histamine induced regulated release of t‐PA. The presence of t‐PA in reactive astrocytes was confirmed in human brain tissue. Conclusions: These data show that RA and PKC activation induce a strong up‐regulation of t‐PA expression in astrocytes, and increased intracellular storage pools. Moreover, a regulated release of t‐PA can be induced from these cells. This raises the possibility that astrocytes contribute to the regulation of extracellular t‐PA levels in the CNS.

Regulation of endogenous tissue-type plasminogen activator expression is modulated by the −7351C>T enhancer polymorphism

biologic risk factors for thrombosis include splenectomy, thrombocytosis, and plasma coagulation factor abnormalities [4]. The improved lifespan and clinical status of the thalassemia population has allowed successful term pregnancies in some patients. During pregnancy, a hypercoagulable state, the risk of developing thrombosis is further increased. It is possible that our patient’s thrombotic events are multifactorial, pregnancy and TI being two contributing factors. Other risk factors include age (>20 years), anemia with no prior transfusions, previous thrombosis, thrombocytosis, and splenectomy. Her molecular studies revealed heterozygosity for the MTHFR mutation, which is rather frequent in our population [5]. The MTHFR mutation does not increase the risk of thrombosis during pregnancy probably due to pregnancy-related physiologic reduction in homocysteine levels, and/or the effects of folic acid supplements that are nowwidely used [6]. In addition, despite the high incidence of inherited thrombophilias in Lebanon, FVL, MTHFR C677T, and prothrombin G20210A mutations were not shown to play a role in increasing the risk of thromboembolism in Lebanese TI patients [5]. This excludes inherited thrombophilias as a contributing factor for our patient’s recurrent thrombosis. The current experience with need for thromboprophylaxis during pregnancy in patients with TI is scant to define recommendations. The use of prophylactic heparin seems to be reasonable particularly in patients, like ours, who previously had thrombosis. What is not clear is whether pregnant TI patients with additional risk factors, such as splenectomy, thrombocytosis, previous deep vein thrombosis, anemia, all of which were present in our patient who developed recurrent thrombosis, would require therapeutic anticoagulation. It will be critical to study the biologic risk factors, thrombosis risk, and prophylaxis in prospective clinical trials with precise data ascertainment regarding transfusion regimens, phenotype, genotype, and other thrombosis risk factors. Only after performing such large-scale controlled trials could evidencebased regimens for thromboprophylaxis in patients with TI be recommended.

Allelic imbalance of tissue-type plasminogen activator (t-PA) gene expression in human brain tissue

We have identified a single-nucleotide polymorphism (SNP) in the t-PA enhancer (-7351C>T), which is associated with endothelial t-PA release in vivo. In vitro studies demonstrated that this SNP is functional at the level of transcription. In the brain, t-PA has been implicated in both physiologic and pathophysiologic processes. The aim of the present study was to examine the effect of the t-PA -7351C>T SNP on t-PA gene expression in human brain tissue. Allelic mRNA expression was measured in heterozygous post-mortem brain tissues using quantitative TaqMan genotyping assay. Protein-DNA interactions were assessed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Significantly higher levels of t-PA mRNA were generated from chromosomes that harboured the wild-type -7351C allele, as compared to those generated from the mutant T allele (for the hippocampus, C to T allelic ratio of ~1.3, p=0.010, n=12; and for the cortex, C to T allelic ratio of ~1.2, p=0.017, n=12). EMSA showed reduced neuronal and astrocytic nuclear protein binding affinity to the T allele, and identified Sp1 and Sp3 as the major transcription factors that bound to the -7351 site. ChIP analyses confirmed that Sp1 recognises this site in intact cells. In conclusion, the t-PA -7351C>T SNP affects t-PA gene expression in human brain tissue. This finding might have clinical implications for neurological conditions associated with enhanced t-PA levels, such as in the acute phase of cerebral ischaemia, and also for stroke recovery.

Species-Specific Regulation of t-PA and PAI-1 Gene Expression in Human and Rat Astrocytes.

In recent years, the role and physiological regulation of the serine protease tissue-type plasminogen activator (t-PA) and its inhibitors, including plasminogen activator inhibitor type-1 (PAI-1), in the brain have received much attention. However, as studies focusing these issues are difficult to perform in humans, a great majority of the studies conducted to date have utilized rodent in vivo and/or in vitro models. In view of the species-specific structural differences present in both the t-PA and the PAI-1 promoters, we have compared the response of these genes in astrocytes of rat and human origin. We reveal marked quantitative and qualitative species-specific differences in gene induction following treatment with various physiological and pathological stimuli. Thus, our findings are of importance for the interpretation of previous and future results related to t-PA and PAI-1 expression.