Extracellular Calcium Influx Pathways in Astrocyte Calcium Microdomain Physiology.

Astrocytes are complex glial cells that play many essential roles in the brain, including the fine-tuning of synaptic activity and blood flow. These roles are linked to fluctuations in intracellular Ca2+ within astrocytes. Recent advances in imaging techniques have identified localized Ca2+ transients within the fine processes of the astrocytic structure, which we term microdomain Ca2+ events. These Ca2+ transients are very diverse and occur under different conditions, including in the presence or absence of surrounding circuit activity. This complexity suggests that different signalling mechanisms mediate microdomain events which may then encode specific astrocyte functions from the modulation of synapses up to brain circuits and behaviour. Several recent studies have shown that a subset of astrocyte microdomain Ca2+ events occur rapidly following local neuronal circuit activity. In this review, we consider the physiological relevance of microdomain astrocyte Ca2+ signalling within brain circuits and outline possible pathways of extracellular Ca2+ influx through ionotropic receptors and other Ca2+ ion channels, which may contribute to astrocyte microdomain events with potentially fast dynamics.

MiR-124 and miR-506 are involved in the decline of protein C in children with extra-hepatic portal vein obstruction.

The deficiency of protein C (PROC) can be partly rescued by Rex shunt through restoring portal blood flow in children with extra-hepatic portal venous obstruction (EHPVO). However, the decline of PROC is still found in some patients with a normal portal blood flow after Rex shunt. The aim of this study was to identify the candidate miRNAs involving in the decline of PROC and their mechanism. The protein level of PROC was detected by the ELISA assay, and was compared between sick and healthy groups. The expressions of miRNAs and PROC mRNA were measured using qRT-PCR, and were compared between sick and healthy groups. The correlation between PROC and candidate miRNAs was analysed by a Pearson correlation analysis to identify the most significant miRNAs. The expression of PROC mRNA was detected by qRT-PCR in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors and negative control (NC) mimics, which was compared among the different groups. The rates of liver cells' proliferation and apoptosis were detected in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors or with overexpressing PROC and negative control mimics by CKK8 assay and flow cytometry, which were compared among the different groups. The expressions of COX-2 and VEGF were measured by qRT-PCR, and were compared between the miRNAs groups and NC group. Western blot was assayed for detecting the protein levels of PROC, COX-2, VEGF, Bcl-2 and Bax, which were compared between the miRNAs groups and NC group. The expression of PROC mRNA was lower, and the expressions of miR-506-3p and miR-124-3p were higher in children with EHPVO than healthy group. PROC mRNA was negatively correlated with the expression of miR-506-3p and miR-124-3p. Compared to the NC group, the transcription activity of PROC was lower after exposure of miR-506 and miR-124 mimics in HL-7702 and LX-2 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. The rate of cell proliferation was lower after exposure of miR-506 and miR-124 than the NC group, which was increased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The apoptotic rate was higher after exposure of miR-506 and miR-124 than the NC group, which was decreased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The mRNA levels of COX-2 and VEGF were significantly higher after exposure of miR-506 and miR-124 mimics than those in the NC group. The protein levels of PROC and Bcl-2 were down-regulated, and the levels of COX-2, Bax and VEGF were up-regulated after exposure of miR-506 and miR-124 in HL-7702 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. MiR-506-3p and miR-124-3p may involve in the decline of PROC in protein and transcriptional level, in which the anti-proliferation and pro-apoptosis role of miR-506-3p and miR-124-3p for liver cells may involve in this mechanism.

Loss of m6A demethylase ALKBH5 promotes post-ischemic angiogenesis via post-transcriptional stabilization of WNT5A.

BACKGROUND: Post-ischemic angiogenesis is critical for blood flow recovery and ischemic tissue repair. N6-methyladenosine (m6A) plays essential roles in numerous biological processes. However, the impact and connected mechanism of m6A on post-ischemic angiogenesis are not fully understood. METHODS: AlkB homolog 5 (ALKBH5) was screened out among several methyltransferases and demethylases involved in dynamic m6A regulation. Cardiac microvascular endothelial cells (CMECs) angiogenesis and WNT family member 5A (WNT5A) stability were analyzed upon ALKBH5 overexpression with adenovirus or knockdown with small interfering RNAs in vitro. The blood flow recovery, capillary, and small artery densities were evaluated in adeno-associated virus (AAV)-ALKBH5 overexpression or ALKBH5 knockout (KO) mice in a hind-limb ischemia model. The same experiments were conducted to explore the translational value of transient silencing of ALKBH5 with adenovirus. RESULTS: ALKBH5 was significantly upregulated in hypoxic CMECs and led to a global decrease of m6A level. ALKBH5 overexpression further reduced m6A level in normoxic and hypoxic CMECs, impaired proliferation, migration, and tube formation only in hypoxic CMECs. Conversely, ALKBH5 knockdown preserved m6A levels and promoted angiogenic phenotypes in hypoxic but not in normoxic CMECs. Mechanistically, ALKBH5 regulated WNT5A expression through post-transcriptional mRNA modulation in an m6A-dependent manner, which decreased its stability and subsequently impeded angiogenesis in hypoxic CMECs. Furthermore, ALKBH5 overexpression hindered blood flow recovery and reduced CD31 and alpha-smooth muscle actin expression in hind-limb ischemia mice. As expected, ALKBH5-KO mice exhibited improved blood flow recovery, increased capillary, and small artery densities after hind-limb ischemia, and similar beneficial effects were observed in mice with transient adenoviral ALKBH5 gene silencing. CONCLUSION: We demonstrate that ALKBH5 is a negative regulator of post-ischemic angiogenesis via post-transcriptional modulation and destabilization of WNT5A mRNA in an m6A-dependent manner. Targeting ALKBH5 may be a potential therapeutic option for ischemic diseases, including peripheral artery disease.

Late gestation predictors of a postnatal biventricular circulation after fetal aortic valvuloplasty.

OBJECTIVES: Fetal aortic valvuloplasty (FAV) for severe aortic stenosis (AS) has shown promise in averting progression to hypoplastic left heart syndrome. After FAV, predicting which fetuses will achieve a biventricular (BiV) circulation after birth remains challenging. Identifying predictors of postnatal circulation on late gestation echocardiography will improve parental counseling. METHODS: Liveborn patients who underwent FAV and had late gestation echocardiography available were included (2000-2017, n = 96). Multivariable logistic regression and classification and regression tree analysis were utilized to identify independent predictors of BiV circulation. RESULTS: Among 96 fetuses, 50 (52.1%) had BiV circulation at the time of neonatal discharge. In multivariable analysis, independent predictors of biventricular circulation included left ventricular (LV) long axis z-score (OR 3.2, 95% CI 1.8-5.7, p < 0.001), LV ejection fraction (OR 1.3, 95% CI 1.0-1.8, p = 0.023), anterograde aortic arch flow (OR 5.0, 95% CI 1.2-20.4, p = 0.024), and bidirectional or right-to-left foramen ovale flow (OR 4.6, 95% CI 1.4-15.8, p = 0.015). CONCLUSION: Several anatomic and physiologic parameters in late gestation were found to be independent predictors of BiV circulation after FAV. Identifying these predictors adds to our understanding of LV growth and hemodynamics after FAV and may improve parental counseling.

[William Harvey reinterpreted in the light of species evolution (I) - How and why circulation phylogenesis integrates itself within species evolution]. / William Harvey réinterprété à la lumière de l'évolution des espèces (I) - Comment et pourquoi la phylogenèse circulatoire s'intègre dans l'évolution des espèces.

In this first part we describe the different steps of the evolution of species and the circulatory modifications, which accompany it. From the open circulation in invertebrates, via the in-series closed circulation in fish, to the in-parallel closed circulation in mammals, the local ability to vasodilate in relation to the specific metabolic demand of territorial activity was the driving force for circulatory evolution. This capacity was achieved by the progressive muscularization of small arteries, generating frictional forces, which systemically determine high arterial pressure, and locally determine active vasodilation via the inhibition of vasomotor tone. This determinism differentially impacts the small resistance arteries, which generate blood pressure, and the large conductance arteries, which support the tensional stress generated by the blood pressure.

TITLE: William Harvey réinterprété à la lumière de l'évolution des espèces (I) - Comment et pourquoi la phylogenèse circulatoire s'intègre dans l'évolution des espèces. ABSTRACT: Au commencement est la pompe cardiaque qui produit un flux sanguin cyclique (énergie cinétique, Ek). En 1619, William Harvey (1578-1657) décrit expérimentalement, en utilisant des garrots veineux ou artériels, l'anatomie fonctionnelle de la circulation sanguine chez l'homme, à l'exception de la circulation capillaire. Pour la première fois est décrite la circulation sanguine en deux circuits fermés parallèles, l'un à haute pression, l'autre à basse pression. Marcello Malpighi (1628-1694) la complète par l'observation en microscopie du réseau capillaire. Un siècle plus tard, apparaissent les premières hypothèses sur l'évolution des espèces. Jean-Baptiste Lamarck (1744-1829) propose en 1809 une théorie de transmission évolutive des caractères phénotypiques par adaptation aux contraintes environnementales. En 1859, Charles Darwin (1809-1882) élabore une théorie de la sélection naturelle. L'interprétation qui prévaut actuellement intègre à la fois la génétique et l'épigénétique dans la transmission intergénérationnelle, et dans la dynamique de développement des caractères phénotypiques individuels, en particulier chez l'homme.

Comparative transcriptomic and proteomic analyses provide insights into functional genes for hypoxic adaptation in embryos of Tibetan chickens.

The Tibetan chicken is a unique breed that has adapted to the high-altitude hypoxic conditions of the Tibetan plateau. A number of positively selected genes have been reported in these chickens; however, the mechanisms of gene expression for hypoxia adaptation are not fully understood. In the present study, eggs from Tibetan and Chahua chickens were incubated under hypoxic and normoxic conditions, and vascularization in the chorioallantoic membrane (CAM) of embryos was observed. We found that the vessel density index in the CAM of Tibetan chickens was lower than in Chahua chickens under hypoxia conditions. Transcriptomic and proteomic analyses of CAM tissues were performed in Tibetan and Chahua chicken embryos under hypoxic incubation using RNA-Seq and iTRAQ. We obtained 160 differentially expressed genes and 387 differentially expressed proteins that were mainly enriched in angiogenesis, vasculature development, blood vessel morphogenesis, blood circulation, renin-angiotensin system, and HIF-1 and VEGF signaling pathways. Twenty-six genes involved in angiogenesis and blood circulation, two genes involved in ion transport, and six genes that regulated energy metabolism were identified as candidate functional genes in regulating hypoxic adaptation of chicken embryos. This research provided insights into the molecular mechanism of hypoxia adaptation in Tibetan chickens.

Adamts18 deficiency in zebrafish embryo causes defective trunk angiogenesis and caudal vein plexus formation.

ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes play an important role in various morphogenesis processes. To determine the functions of Adamts18 in the early stages of organogenesis, we created Adamts18 deficient zebrafish using morpholino antisense oligonucleotides (MO) to generate exon 3 skipped adamts18 mRNA transcripts. Results showed that Adamts18 deficiency in zebrafish embryos caused developmental defects, including expanded brain ventricle and hindbrain edema, eye defects, and accumulation of blood in the caudal vein. Adamts18 deficiency also led to impaired trunk angiogenesis and formation of the caudal vein plexus (CVP). Consequently, Adamts18 deficient zebrafish embryos exhibited incomplete formation of intersegment vessels (ISVs), disruption of the honeycomb structure of CVP, and reduced CVP area and loop number. Furthermore, Adamts18 deficiency resulted in impaired blood circulation in major trunk, caudal vein (CV), and common cardinal vein (CCV). These aberrant vascular phenotypes in mutant zebrafish embryos were shown to be associated with a decreased expression of multiple angiogenesis-related signaling genes, including slit/robo, dll4/Notch, cox2, and fgfr. These findings indicate the critical role of Adamts18 in the early stages of vascular network development.

Downregulation of AC061961.2, LING01-AS1, and RP11-13E1.5 is associated with dilated cardiomyopathy progression.

This study aimed to explore long noncoding RNAs (lncRNAs) implicated in dilated cardiomyopathy (DCM). Ten samples of failing hearts collected from the left ventricles of patients with DCM undergoing heart transplants, and ten control samples obtained from normal heart donors were included in this study. After sequencing, differentially expressed genes (DEGs) and lncRNAs between DCM and controls were screened, followed with functional enrichment analysis and weighted gene coexpression network analysis (WGCNA). Five key lncNRAs were validated through real-time polymerase chain reaction (PCR). Total 1,398 DEGs were identified, including 267 lncRNAs. WGCNA identified seven modules that were significantly correlated with DCM. The top 50 genes in the three modules (black, dark-green, and green-yellow) were significantly correlated with DCM disease state. Four core enrichment lncRNAs, such as AC061961.2, LING01-AS1, and RP11-557H15.4, in the green-yellow module were associated with neurotransmitter secretion. Five core enrichment lncRNAs, such as KB-1299A7.2 and RP11-13E1.5, in the black module were associated with the functions of blood circulation and heart contraction. AC061961.2, LING01-AS1, and RP11-13E1.5 were confirmed to be downregulated in DCM tissues by real-time PCR. The current study suggests that downregulation of AC061961.2, LING01-AS1, and RP11-13E1.5 may be associated with DCM progression, which may serve as key diagnostic biomarkers and therapeutic targets for DCM.

The expression dynamics of mechanosensitive genes in extra-embryonic vasculature after heart starts to beat in chick embryo.

BACKGROUND: Fluid flow plays an important role in vascular development. However, the detailed mechanisms, particularly the link between flow and modulation of gene expression during vascular development, remain unexplored. In chick embryo, the key events of vascular development from initiation of heart beat to establishment of effective blood flow occur between the stages HH10 and HH13. Therefore, we propose a novel in vivo model to study the flow experienced by developing endothelium. OBJECTIVE: Using this model, we aimed to capture the transcriptome dynamics of the pre- and post-flow conditions. METHODS: RNA was isolated from extra embryonic area vasculosa (EE-AV) pooled from three chick embryos between HH10-HH13 and RNA sequencing was performed. RESULTS: The whole transcriptome sequencing of chick identified up-regulation of some of the previously well-known mechanosensitive genes including NFR2, HAND1, CTGF and KDR. GO analyses of the up-regulated genes revealed enrichment of several biological processes including heart development, extracellular matrix organization, cell-matrix adhesion, cell migration, blood vessel development, patterning of blood vessels, collagen fibril organization. Genes encoding for gap junctions proteins which are involved in vascular remodeling and arterial-venous differentiation, and genes involved in cell-cell adhesion, and ECM interactions were significantly up-regulated. Validation of selected genes through semi quantitative PCR was performed. CONCLUSION: The study indicates that shear stress plays a major role in development. Through appropriate validation, this platform can serve as an in vivo model to study conditions of disturbed flow in pathology as well as normal flow during development.

Brief Exercises Affect Gene Expression in Circulating Monocytes.

We aimed to give a systematic hypothesis on the functions of exercise on circulating monocytes by identifying a discrete set of genes in circulating monocytes that were altered by exercise. The microarray expression profile of GSE51835 was downloaded from gene expression omnibus (GEO) database for the identification of differentially expressed genes (DEGs) using limma and affy packages in R language. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for DEGs, followed by the construction of co-expression network and protein-protein interaction (PPI) network. The top 10 nodes in PPI network were screened, and subnetwork was constructed for the key genes identification. Totally, 35 DEGs, including 2 upregulated genes and 33 downregulated genes, were identified. The enriched GO terms were mainly linked to immune response and defence response, and the enriched KEGG pathways were mainly associated with natural killer cell-mediated cytotoxicity and graft-versus-host disease. Dual-specificity phosphatase 2 (DUSP2) was identified as a key node in the co-expression network. In the PPI network, CD247 module (CD247), chemokine (C-X-C motif) receptor 4 (CXCR4), granzyme B (GZMB) and perforin 1 (PRF1) were identified as key nodes. An important interaction, GZMB/PRF1, was detected. Five key genes, including DUSP2, CD247, CXCR4, GZMB and PRF1, and an interaction of GZMB/PRF1, were significant factors in the immune processes of circulating monocytes, which might be regulated by brief exercises, leading to the enhancement of immune function.

Low levels of insulin-like growth factor-1 contribute to alveolar macrophage dysfunction in cystic fibrosis.

Alveolar macrophages are major contributors to lung innate immunity. Although alveolar macrophages from cystic fibrosis (CF) transmembrane conductance regulator(-/-) mice have impaired function, no study has investigated primary alveolar macrophages in adults with CF. CF patients have low levels of insulin-like growth factor 1 (IGF-1), and our prior studies demonstrate a relationship between IGF-1 and macrophage function. We hypothesize that reduced IGF-1 in CF leads to impaired alveolar macrophage function and chronic infections. Serum and bronchoalveolar lavage (BAL) samples were obtained from eight CF subjects and eight healthy subjects. Macrophages were isolated from BAL fluid. We measured the ability of alveolar macrophages to kill Pseudomonas aeruginosa. Subsequently, macrophages were incubated with IGF-1 prior to inoculation with bacteria to determine the effect of IGF-1 on bacterial killing. We found a significant decrease in bacterial killing by CF alveolar macrophages compared with control subjects. CF subjects had lower serum and BAL IGF-1 levels compared with healthy control subjects. Exposure to IGF-1 enhanced alveolar macrophage macrophages in both groups. Finally, exposing healthy alveolar macrophages to CF BAL fluid decreased bacterial killing, and this was reversed by the addition of IGF-1, whereas IGF-1 blockade worsened bacterial killing. Our studies demonstrate that alveolar macrophage function is impaired in patients with CF. Reductions in IGF-1 levels in CF contribute to the impaired alveolar macrophage function. Exposure to IGF-1 ex vivo results in improved function of CF alveolar macrophages. Further studies are needed to determine whether alveolar macrophage function can be enhanced in vivo with IGF-1 treatment.

Effects of blockade of peripheral interleukin-6 trans-signaling on hippocampus-dependent and independent memory in mice.

Besides functions of the interleukin-6 (IL-6)/gp130 cytokine family in immunology, IL-6 signaling has influence on memory processes. IL-6 acts on target cells via a membrane-bound IL-6 receptor (IL-6R) and subsequent association with the signal-transducing protein gp130. While gp130 is expressed on all cells in the body, IL-6R is expressed in only on few cells such as hepatocytes and some leukocytes. Cells lacking IL-6R were shown not to be responsive to the cytokine. Interestingly, a soluble form of the IL-6R in complex with IL-6 can stimulate cells that do not express the membrane-bound IL-6R. This signaling pathway has been called IL-6 trans-signaling. IL-6 trans-signaling can specifically be blocked by a soluble gp130 protein (sgp130Fc) without affecting IL-6 classic signaling via the membrane-bound IL-6R. Transgenic mice expressing sgp130Fc in the blood, but not in the central nervous system, were analyzed for hippocampus-dependent and independent memory, together with exploratory- and anxiety-related behavior. Transgenic animals did not show impaired hippocampus-dependent or independent learning and memory. However, compared to wild-type animals, they showed reduced exploratory behavior and an increased thermal pain threshold, indicating that these effects depend on IL-6 trans-signaling. These results bear important consequences for the therapeutic blockade of IL-6 activity in autoimmune diseases.

Systems and synthetic biology of the vessel wall.

Atherosclerosis is intimately coupled to blood flow by the presence of predilection sites. The coupling is through mechanotransduction of endothelial cells and approximately 2000 gene are associated with this process. This paper describes a new platform to study and identify new signalling pathways in endothelial cells covering an atherosclerotic plaque. The identified networks are synthesized in primary cells to study their reaction to flow. This synthetic approach might lead to new insights and drug targets.

RTEF-1, an upstream gene of hypoxia-inducible factor-1α, accelerates recovery from ischemia.

The amount of available hypoxia-inducible factor (HIF)-1α has been considered to be largely a consequence of post-translational modification by multiple ubiquitin-proteasome pathways. However, the role of transcriptional regulation of HIF-1α is less certain, and the mechanisms of transcriptional regulation of HIF-1α require further investigation. Here we report that related transcriptional enhancer factor-1 (RTEF-1), a member of the TEF transcriptional factor family, transcriptionally regulates the HIF-1α gene under normoxic and hypoxic conditions. The expression of HIF-1α mRNA was decreased in endothelial cells in which RTEF-1 was knocked down with siRNA. Sequential deletional analysis of the HIF-1α promoter revealed that the MCAT-like element in the HIF-1α promoter was essential for HIF-1α transcription. Binding of RTEF-1 to the MCAT-like element was confirmed by ChIP. Treatment of endothelial cells with a HIF-1 inhibitor resulted in retardation of RTEF-1-induced proliferation and tube formation. Moreover, increased HIF-1α expression was observed in transgenic mice expressing RTEF-1 under the VE-cadherin promoter (VE-Cad/RTEF-1). VE-Cad/RTEF-1 mice subjected to hindlimb ischemia demonstrated increased levels of HIF-1α, accelerated recovery of blood flow, and increased capillary density compared with littermate controls. These results identify RTEF-1 as a regulator of HIF-1α transcription, which results in up-regulation of HIF-1α and acceleration of recovery from ischemia.

A small scale expression screen identifies tissue specific markers in the Dugesia japonica strain Pek-1.

Freshwater planaria has tremendous capacity to reform the missing part of the body and therefore is considered as one of the most important model organism for regeneration study. At present, Schmidtea mediterranea and Dugesia japonica are the two major species utilized for laboratory manipulations. Dugesia japonica flatworms are widely distributed in the Far East including Cherry Valley region in the north-west area of Beijing, China. We reported here the establishment of an asexual Dugesia japonica strain Pek-1, as a suitable system for regeneration study. Using morphological, karyotypical as well as phylogenetic analyses, we confirmed that these flatworms indeed belonged to Dugesia japonica. We went on to show that the commonly used in situ probes and immunohistochemistry reagents and protocols were applicable to the Pek-1 strain. Using this strain, we carried out small scale analysis on EST, RNAi and gene expression. We identified 193 unique EST sequences and 65 of them had not been reported in planarian. By RNAi analysis, we showed that 48 genes, when down-regulated individually, had no effect on regeneration. Furthermore, we identified 3 groups of tissue specific expressing genes that were useful for cell lineage analysis. We concluded that the Dugesia japonica Pek-1 strain could be another suitable animal model to regeneration research.

Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart.

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis.

Zebrafish Jak2a plays a crucial role in definitive hematopoiesis and blood vessel formation.

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2(-/-) transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.

The CCR2 promoter polymorphism T-960A, but not the serum MCP-1 level, is associated with endothelial function in prediabetic individuals.

Monocyte-chemoattractant-protein (MCP)-1 and its receptor CCR2 have been shown to play a pivotal role in vascular inflammation and atherosclerotic plaque formation. However, it is currently unclear whether MCP-1/CCR2 triggered inflammation affects nitric oxide (NO)-bioavailability, hence influencing vascular function, a sign of early atherosclerosis. Therefore, we sought to investigate the association between serum levels of MCP-1 and NO-bioavailability, expressed as flow mediated dilation (FMD) in vivo, and the impact of CCR2 gene variations on FMD. We studied a German population of 242 prediabetic individuals (144 women, 98 men; mean age 45+/-0.8 years) via FMD by high-resolution ultrasound (13MHz). In order to replicate our findings, a second, independent population (n=115; 44 women, 77 men; mean age 48+/-1.0 years) (total=357 individuals) from Italy was studied. Vascular function in the Italian population was studied via intra-arterial application of acetylcholine. MCP-1 serum-levels were assessed by ELISA and CCR2 polymorphisms were determined by sequencing. MCP-1 serum levels showed no association with FMD (p=0.90), whereas the CCR2 promoter polymorphism was associated with elevated FMD (T/T: 5.6+/-0.3%; T/A: 6.7+/-0.4%; A/A: 8.3+/-0.8%; p=0.01) after adjusting for possible confounders. These results were confirmed in the independent Italian population (A/A: 97.1+/-20.3 vs. T/T: 60.5+/-5.6% forearm blood-flow increase; p<0.05). When testing for the functional relevance of the T-960A (rs3918359) polymorphism, we found that the A/A-genotype was associated with moderately increased protein binding in EMSA, increased promoter activity in luciferase assays and reduced transendothelial monocyte migration. In conclusion, MCP-1 serum levels do not reflect endothelial function in vivo in prediabetic individuals. However, the functionally relevant CCR2 promoter polymorphism T-960A (rs3918359) is associated with elevated vascular function. This might be due to reduced subendothelial inflammation, mediated by reduced transendothelial monocyte-migration ability.