Decreased levels of stem cell factor in subjects with incident coronary events.

BACKGROUND: It has been proposed that vascular progenitor cells play an important role in vascular repair, but their possible clinical importance in cardiovascular disease has not been fully characterized. Vascular endothelial growth factor A, placental growth factor and stem cell factor (SCF) are three growth factors that are important in recruiting vascular progenitor cells. In this study, we investigated the association between the plasma levels of these growth factors and incident coronary events (CEs). METHODS: Levels of the three growth factors were measured using the proximity extension assay technique in baseline plasma samples from 384 subjects with a first CE (mean follow-up 14.0 ± 4.3 years) and 409 event-free control subjects matched by sex and age, as well as in homogenates from 201 endarterectomy specimens. RESULTS: After controlling for known cardiovascular disease risk factors in a Cox regression model, subjects in the lowest SCF tertile had a hazard ratio of 1.70 (95% confidence interval 1.14-2.54) compared with subjects in the highest SCF tertile. Lower SCF levels were also associated with more severe carotid disease, less fibrous atherosclerotic plaques and an increased incidence of heart failure. Expression of the SCF receptor c-kit was demonstrated in the subendothelial layer and fibrous cap of human atherosclerotic plaques. Smokers and subjects with diabetes had decreased levels of SCF compared with control subjects. CONCLUSION: To our knowledge, this is the first clinical study to provide evidence to support a key role for SCF and progenitor cells in vascular repair. We suggest that the SCF-c-kit pathway may be a promising biomarker and therapeutic target in cardiovascular disease.

Plasmin deficiency leads to fibrin accumulation and a compromised inflammatory response in the mouse brain.

BACKGROUND: Excess fibrin in blood vessels is cleared by plasmin, the key proteolytic enzyme in fibrinolysis. Neurological disorders and head trauma can result in the disruption of the neurovasculature and the entry of fibrin and other blood components into the brain, which may contribute to further neurological dysfunction. OBJECTIVES: While chronic fibrin deposition is often implicated in neurological disorders, the pathological contributions attributable specifically to fibrin have been difficult to ascertain. An animal model that spontaneously acquires fibrin deposits could allow researchers to better understand the impact of fibrin in neurological disorders. METHODS: Brains of plasminogen (plg)- and tissue plasminogen activator (tPA)-deficient mice were examined and characterized with regard to fibrin accumulation, vascular and neuronal health, and inflammation. Furthermore, the inflammatory response following intrahippocampal lipopolysaccharide (LPS) injection was compared between plg(-/-) and wild type (WT) mice. RESULTS AND CONCLUSIONS: Both plg(-/-) and tPA(-/-) mice exhibited brain parenchymal fibrin deposits that appear to result from reduced neurovascular integrity. Markers of neuronal health and inflammation were not significantly affected by proximity to the vascular lesions. A compromised neuroinflammatory response was also observed in plg(-/-) compared to WT mice following intrahippocampal LPS injection. These results demonstrate that fibrin does not affect neuronal health in the absence of inflammation and suggest that plasmin may be necessary for a normal neuroinflammatory response in the mouse CNS.

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.

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood-brain barrier.

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9-11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.

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

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.