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Conclusion: Proteins containing a Jumonji C (JmjC) domain appear in almost all living organisms and catalyze a variety of oxidation reactions. Therefore, they are important regulators in many biological processes such as proliferation and differentiation. They act either as protein hydroxylases, histone demethylases or by regulate mRNA splicing. Given the fact that some of the JmjC domain-containing proteins are shown to be upregulated in response to hypoxia as well as the dependency of JmjC domain catalytic activity on oxygen led to the assumption of an involvement in angiogenesis. For Jmjd6, a member of the JmjC domain-containing protein family, a regulatory involvement in mRNA splicing has been shown. The Jmjd6-/- mouse dies perinatally due to several severe organ malformations, especially in the heart. Despite the pale appearance, the growth retardation and the cardiac defects, it is unclear whether these mice exhibit defects of cells comprising the vasculature. Therefore, the involvement of Jmjd6 in angiogenesis was examined in vitro using angiogenesis assays as well as in vivo using the Jmjd6+/- mouse. An siRNA-mediated knockdown of Jmjd6 in ECs significantly impaired the formation of capillary-like networks in the tube formation assay as well as sprouting in the spheroid assay. Moreover, after siRNA-mediated knockdown of Jmjd6 in ECs cell migration was significantly reduced. These findings were confirmed in the matrigel plug assay in vivo. Implanted matrigel plugs of Jmjd6+/- mice exhibited significantly less perfused vessels compared to wildtype littermates. Furthermore, cultured lung ECs from Jmjd6+/- mice exhibited impaired network forming activity ex vivo compared to cells isolated from wildtype littermates. To elucidate the mechanisms underlying the requirement of Jmjd6 in angiogenesis, an Affymetrix exon-array was performed, which allows detection of changes in gene expression as well as splicing. The siRNA-mediated knockdown of Jmjd6 altered the expression of genes known to play a role in vascular biology. The bioinformatic assessment of alternative splice variants revealed that Jmjd6 silencing affects the splicing of the VEGF receptor 1 (Flt1). Differential splicing of Flt1 was shown to generate a short and soluble form of Flt1 (sFlt1), which sequestrates VEGF and PlGF, and thereby inhibits angiogenesis. In particular, a significant increase in sFlt1 expression was observed. Jmjd6 was recently reported to hydroxylate the splicing factor U2AF65. Therefore, we investigated whether U2AF65 might mediate Flt1 splicing and binds to Flt1 mRNA. Indeed, U2AF65 co-immunoprecipitated with Jmjd6 in ECs, while an interaction of U2AF65 with sFlt1 was demonstrated. Moreover, inhibition of Jmjd6 catalytic function by reduced oxygen concentration altered splicing of Flt1 resulted in an increase of the sFlt1 splice variant. Finally, saturating concentrations of VEGF or PlGF or neutralizing antibodies against sFlt1 significantly reduced the inhibition of sprouting caused by Jmjd6 knockdown in vitro.
Collectively, our results indicate that Jmjd6 has an essential role in the oxygen-dependent regulation of angiogenesis by controlling the splicing of Flt1 mRNA, thereby adjusting the generation of the anti-angiogenic short splice variant sFlt1. Several publications demonstrated a major importance for sFlt1 as a biomarker for many severe human diseases such as preeclampsia, sepsis, cancer, myocardial infarction as well as chronic heart failure. Therefore, the identification of the molecular mechanism behind the generation of sFlt1 might enable the development of new or more precise clinical markers for the diagnosis of the corresponding diseases. Furthermore, the discovery of the enzymes involved in the generation of sFlt1 provides further possibilities to modulate sFlt1 levels and thereby may potentially gives rise to the development of new therapies.
Background: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.
Methodology/Principal findings: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).
Conclusions/Significance: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.
Improved risk stratification in prevention by use of a panel of selected circulating microRNAs
(2017)
Risk stratification is crucial in prevention. Circulating microRNAs have been proposed as biomarkers in cardiovascular disease. Here a miR panel consisting of miRs related to different cardiovascular pathophysiologies, was evaluated to predict outcome in the context of prevention. MiR-34a, miR-223, miR-378, miR-499 and miR-133 were determined from peripheral blood by qPCR and combined to a risk panel. As derivation cohort, 178 individuals of the DETECT study, and as validation cohort, 129 individuals of the SHIP study were used in a case-control approach. Overall mortality and cardiovascular events were outcome measures. The Framingham Risk Score(FRS) and the SCORE system were applied as risk classification systems. The identified miR panel was significantly associated with mortality given by a hazard ratio(HR) of 3.0 (95% (CI): 1.09–8.43; p = 0.034) and of 2.9 (95% CI: 1.32–6.33; p = 0.008) after adjusting for the FRS in the derivation cohort. In a validation cohort the miR-panel had a HR of 1.31 (95% CI: 1.03–1.66; p = 0.03) and of 1.29 (95% CI: 1.02–1.64; p = 0.03) in a FRS/SCORE adjusted-model. A FRS/SCORE risk model was significantly improved to predict mortality by the miR panel with continuous net reclassification index of 0.42/0.49 (p = 0.014/0.005). The present miR panel of 5 circulating miRs is able to improve risk stratification in prevention with respect to mortality beyond the FRS or SCORE.
Copeptin is the C-terminal end of pre-provasopressin released equimolar to vasopressin into circulation and recently discussed as promising cardiovascular biomarker amendatory to established markers such as troponins. Vasopressin is a cytokine synthesized in the hypothalamus. A direct release of copeptin from the heart into the circulation is implied by data from a rat model showing a cardiac origin in hearts put under cardiovascular wall stress. Therefore, evaluation of a potential release of copeptin from the human heart in acute myocardial infarction (AMI) has been done.
The use of cardiac troponins (cTn) is the gold standard for diagnosing myocardial infarction. Independent of myocardial infarction (MI), however, sex, age and kidney function affect cTn levels. Here we developed a method to adjust cTnI levels for age, sex, and renal function, maintaining a unified cut-off value such as the 99th percentile. A total of 4587 individuals enrolled in a prospective longitudinal study were used to develop a model for adjustment of cTn. cTnI levels correlated with age and estimated glomerular filtration rate (eGFR) in males/females with rage = 0.436/0.518 and with reGFR = −0.142/−0.207. For adjustment, these variables served as covariates in a linear regression model with cTnI as dependent variable. This adjustment model was then applied to a real-world cohort of 1789 patients with suspected acute MI (AMI) (N = 407). Adjusting cTnI showed no relevant loss of diagnostic information, as evidenced by comparable areas under the receiver operator characteristic curves, to identify AMI in males and females for adjusted and unadjusted cTnI. In specific patients groups such as in elderly females, adjusting cTnI improved specificity for AMI compared with unadjusted cTnI. Specificity was also improved in patients with renal dysfunction by using the adjusted cTnI values. Thus, the adjustments improved the diagnostic ability of cTnI to identify AMI in elderly patients and in patients with renal dysfunction. Interpretation of cTnI values in complex emergency cases is facilitated by our method, which maintains a single diagnostic cut-off value in all patients.