Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole.

Primary cilia from the brain microvascular endothelial cells (ECs) are specialized cell-surface organelles involved in mediating sensory perception, cell signaling, and vascular stability. Immunofluorescence (IF) analysis of human primary brain microvascular ECs reveals two cilia per cell. To confirm the in vitro observation of the two-cilia phenotype in human primary brain ECs, ECs isolated from mouse brain were cultured and stained for cilium. Indeed, brain ECs from a ciliopathic mouse (polycystic kidney disease or Pkd2 -/-) also possess more than one cilium. Primary cilium emerges from the mother centriole. Centriole analysis by IF suggests that in brain ECs, markers for the mother and daughter centrioles stain both cilia, suggesting that the second cilium in brain ECs arises from the daughter centriole. Further quantification of cilia size in brain ECs revealed that cilia arising from the mother centriole are bigger in size compared with cilia from the daughter centriole. Cell cycle analyses using immunoblotting and flow cytometry suggest that the ciliary proteins ARL13B and IFT88 involved in brain EC ciliogenesis are highly expressed only in the G0/G1 and S phases of the cell cycle. The IF analyses of cells arrested at different cell cycle stages indicate that the two-cilia phenotype is highly specific to the G0/G1 phase. Our findings suggest that in addition to the mother centriole, the daughter centriole also plays a role in ciliogenesis in primary cultured ECs.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability.

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1-2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

Cystathione ß-synthase regulates HIF-1α stability through persulfidation of PHD2.

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine ß-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

SNRK: a metabolic regulator with multifaceted role in development and disease.

Sucrose nonfermenting 1-related kinase (SNRK) is a serine/threonine kinase and a member of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) family that is involved in the metabolic regulatory mechanisms in various cell types. SNRK is an important mediator in maintaining cellular metabolic homeostasis. In this review, we discuss the role of SNRK in metabolic tissues where it is expressed, including heart and adipose tissue. We discuss its role in regulating inflammation in these tissues and the pathways associated with regulating inflammation. We also discuss SNRK's role in vascular development and the processes associated with it. Finally, we review SNRK's potential as a target in various metabolic dysfunction-associated diseases such as cardiovascular diseases, diabetes, obesity, and cancer. This comprehensive review on SNRK suggests that it has therapeutic value in the suppression of inflammation in cardiac and adipose tissue.

Hepatic Vein Blood Increases Lung Microvascular Angiogenesis and Endothelial Cell Survival-Toward an Understanding of Univentricular Circulation.

To improve our understanding of pulmonary arteriovenous malformations in univentricular congenital heart disease, our objective was to identify the effects of hepatic vein and superior vena cava constituents on lung microvascular endothelial cells independent of blood flow. Paired blood samples were collected from the hepatic vein and superior vena cava in children 0-10 years old undergoing cardiac catheterization. Isolated serum was subsequently used for in vitro endothelial cell assays. Angiogenic activity was assessed using tube formation and scratch migration. Endothelial cell survival was assessed using proliferation (BrdU incorporation, cell cycle analysis) and apoptosis (caspase 3/7 activity, Annexin-V labeling). Data were analyzed using Wilcoxon signed-rank test and repeated measures analysis. Upon incubating lung microvascular endothelial cells with 10% patient serum, hepatic vein serum increases angiogenic activity (tube formation, P = 0.04, n = 24; migration, P< 0.001, n = 18), increases proliferation (BrdU, P < 0.001, n = 32; S-phase, P = 0.04, n = 13), and decreases apoptosis (caspase 3/7, P < 0.001, n = 32; Annexin-V, P = 0.04, n = 12) compared to superior vena cava serum. Hepatic vein serum regulates lung microvascular endothelial cells by increasing angiogenesis and survival in vitro. Loss of hepatic vein serum signaling in the lung microvasculature may promote maladaptive lung microvascular remodeling and pulmonary arteriovenous malformations.

Neuronal vulnerability to fetal hypoxia-reoxygenation injury and motor deficit development relies on regional brain tetrahydrobiopterin levels.

Hypertonia is pathognomonic of cerebral palsy (CP), often caused by brain injury before birth. To understand the early driving events of hypertonia, we utilized magnetic resonance imaging (MRI) assessment of early critical brain injury in rabbit fetuses (79% term) that will predict hypertonia after birth following antenatal hypoxia-ischemia. We examined if individual variations in the tetrahydrobiopterin cofactor in the parts of the brain controlling motor function could indicate a role in specific damage to motor regions and disruption of circuit integration as an underlying mechanism for acquiring motor disorders, which has not been considered before. The rabbit model mimicked acute placental insufficiency and used uterine ischemia at a premature gestation. MRI during the time of hypoxia-ischemia was used to differentiate which individual fetal brains would become hypertonic. Four brain regions collected immediately after hypoxia-ischemia or 48 h later were analyzed in a blinded fashion. Age-matched sham-operated animals were used as controls. Changes in the reactive nitrogen species and gene expression of the tetrahydrobiopterin biosynthetic enzymes in brain regions were also studied. We found that a combination of low tetrahydrobiopterin content in the cortex, basal ganglia, cerebellum, and thalamus brain regions, but not a unique low threshold of tetrahydrobiopterin, contributed etiologically to hypertonia. The biggest contribution was from the thalamus. Evidence for increased reactive nitrogen species was found in the cortex. By 48 h, tetrahydrobiopterin and gene expression levels in the different parts of the brain were not different between MRI stratified hypertonia and non-hypertonia groups. Sepiapterin treatment given to pregnant dams immediately after hypoxia-ischemia ameliorated hypertonia and death. We conclude that a developmental tetrahydrobiopterin variation is necessary with fetal hypoxia-ischemia and is critical for disrupting normal motor circuits that develop into hypertonia. The possible mechanistic pathway involves reactive nitrogen species.

Cardiomyocyte-Specific Snrk Prevents Inflammation in the Heart.

Background The SNRK (sucrose-nonfermenting-related kinase) enzyme is critical for cardiac function. However, the underlying cause for heart failure observed in Snrk cardiac conditional knockout mouse is unknown. Methods and Results Previously, 6-month adult mice knocked out for Snrk in cardiomyocytes (CMs) displayed left ventricular dysfunction. Here, 4-month adult mice, on angiotensin II (Ang II) infusion, show rapid decline in cardiac systolic function, which leads to heart failure and death in 2 weeks. These mice showed increased expression of nuclear factor κ light chain enhancer of activated B cells (NF-κB), inflammatory signaling proteins, proinflammatory proteins in the heart, and fibrosis. Interestingly, under Ang II infusion, mice knocked out for Snrk in endothelial cells did not show significant systolic or diastolic dysfunction. Although an NF-κB inflammation signaling pathway was increased in Snrk knockout endothelial cells, this did not lead to fibrosis or mortality. In hearts of adult mice knocked out for Snrk in CMs, we also observed NF-κB pathway activation in CMs, and an increased presence of Mac2+ macrophages was observed in basal and Ang II-infused states. In vitro analysis of Snrk knockdown HL-1 CMs revealed similar upregulation of the NF-κB signaling proteins and proinflammatory proteins that was exacerbated on Ang II treatment. The Ang II-induced NF-κB pathway-mediated proinflammatory effects were mediated in part through protein kinase B or AKT, wherein AKT inhibition restored the proinflammatory signaling protein levels to baseline in Snrk knockdown HL-1 CMs. Conclusions During heart failure, SNRK acts as a cardiomyocyte-specific repressor of cardiac inflammation and fibrosis.

Tetrahydrobiopterin in antenatal brain hypoxia-ischemia-induced motor impairments and cerebral palsy.

Antenatal brain hypoxia-ischemia, which occurs in cerebral palsy, is considered a significant cause of motor impairments in children. The mechanisms by which antenatal hypoxia-ischemia causes brain injury and motor deficits still need to be elucidated. Tetrahydrobiopterin is an important enzyme cofactor that is necessary to produce neurotransmitters and to maintain the redox status of the brain. A genetic deficiency of this cofactor from mutations of biosynthetic or recycling enzymes is a well-recognized factor in the development of childhood neurological disorders characterized by motor impairments, developmental delay, and encephalopathy. Experimental hypoxia-ischemia causes a decline in the availability of tetrahydrobiopterin in the immature brain. This decline coincides with the loss of brain function, suggesting this occurrence contributes to neuronal dysfunction and motor impairments. One possible mechanism linking tetrahydrobiopterin deficiency, hypoxia-ischemia, and neuronal injury is oxidative injury. Evidence of the central role of the developmental biology of tetrahydrobiopterin in response to hypoxic ischemic brain injury, especially the development of motor deficits, is discussed.