Search

Background The endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury. Methods Human umbilical vein endothelial cells (HUVECs) were exposed to 1 μg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 μg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence. Results Exposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02). Conclusions Interleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.

Ischemia/reperfusion injury (IRI) has been shown to cause endothelial glycocalyx (EG) damage.Whether the hypoxic/ischemic insult or the oxidative and inflammatory stress of reperfusion plays a greater part in glycocalyx damage is not known. Furthermore, the mechanisms by which IRI causes EG damage have not been fully elucidated. The aims of this study were to determine if hypoxia alone or hypoxia/reoxygenation (H/R) caused greater damage to the glycocalyx, and if this damage was mediated by reactive oxygen species (ROS) and Ca signaling.Human umbilical vein endothelial cells were cultured to confluence and exposed to either normoxia (30 minutes), hypoxia (2% O2 for 30 minutes), or H/R (30 minutes hypoxia followed by 30 minutes normoxia). Some cells were pretreated with ROS scavengers TEMPOL, MitoTEMPOL, Febuxostat, or Apocynin, or with the Ca chelator BAPTA or Ca channel blockers 2-aminoethoxydiphenyl borate, A967079, Pyr3, or ML204. Intracellular ROS was quantified for all groups. Endothelial glycocalyx was measured using fluorescently tagged wheat germ agglutinin and imaged with fluorescence microscopy.Glycocalyx thickness was decreased in both hypoxia and H/R groups, with the decrease being greater in the H/R group. TEMPOL, MitoTEMPOL, BAPTA, and 2-aminoethoxydiphenyl borate prevented loss of glycocalyx in H/R. The ROS levels were likewise elevated compared with normoxia in both groups, but were increased in the H/R group compared with hypoxia alone. BAPTA did not prevent ROS production in either group.In our cellular model for shock, we demonstrate that although hypoxia alone is sufficient to produce glycocalyx loss, H/R causes a greater decrease in glycocalyx thickness. Under both conditions damage is dependent on ROS and Ca signaling. Notably, we found that ROS are generated upstream of Ca, but that ROS-mediated damage to the glycocalyx is dependent on Ca.

The endothelial glycocalyx forms an anti-thrombotic layer on the apical surface of endothelial cells and maintains the selective permeability barrier of blood vessels. Ischemia reperfusion injury like hemorrhagic shock is shown to cause glycocalyx damage. We have previously shown that mitochondrial reactive oxygen species (mitoROS) mediate glycocalyx damage in cultured endothelial cells. Of note, angiotensin II elevates endothelial mitoROS, suggesting a possible exacerbation of glycocalyx damage in hypertensive patients. It is unknown, however, whether mitoROS mediate glycocalyx damage in vivo . We hypothesize that mitoROS mediate glycocalyx damage in a rat model of hemorrhagic shock.We investigated the effect of mitochondrial ROS on the endothelial glycocalyx in vivo , in a rat model of hemorrhagic shock. In anesthetized rats, mean arterial pressure was reduced to 40 mmHg by withdrawing blood from the femoral artery and kept at 40 mmHg for 30 minutes. The rats were then resuscitated with IV (jugular vein) Ringer’s lactate solution for 30 minutes more. Sham rats received the vascular lines only. Syndecan-1 in the plasma was increased after 30 minutes of resuscitation with LR in hemorrhage/resuscitation (H/R) rats (p=0.02) but was not elevated in Sham rats (p=0.52). Resuscitation with IV mitoROS scavenger mitoTEMPOL significantly blunted this increase (5.9 pg/ml vs. 8.7 pg/ml, p=0.03). At the organ level, the glycocalyx was decreased in the endothelium of muscle (p=0.0444) and intestine (P=5.47 * 10 -7 ) vascular beds in H/R rats vs. Sham rats. Our findings show that mitoROS mediate the glycocalyx damage after H/R. This mechanism suggests possible therapies that target mitoROS generation. Future work will investigate whether preexisting hypertension increases glycocalyx damage during H/R due to exacerbated mitoROS levels.

The endothelial glycocalyx is a proteoglycan and glycoprotein matrix that coats the luminal surface of endothelial cells. It is an important component of the permeability barrier function of endothelial cells, including in the glomerular capillaries. Furthermore, it is a sensor of shear stress, and in vessels treated with enzymes to remove the glycocalyx do not exhibit flow-mediated vasodilation. Sparse previous studies have implicated a reduced glycocalyx thickness in hypertensive patients, but the causative signaling mechanisms have not been investigated. We and others have found that the endothelial glycocalyx can be studied in cultured endothelial cells if the medium contains significant soluble protein. We hypothesized that elevated catecholamines cause shedding of the endothelial glycocalyx in vitro . We cultured human umbilical vein endothelial cells (HUVECs) in M200 medium supplemented with 1% bovine serum albumin to permit development of a glycocalyx. We exposed these cells to concentrations of epinephrine, norepinephrine, phenylephrine, or isoproterenol from 0.01 to 10 μM for 30 minutes. We then imaged the glycocalyx using fluorescently labelled wheat germ agglutinin. We found that norepinephrine, epinephrine, and isoproterenol all caused a significant decrease in the endothelial glycocalyx staining intensity (5.82 ± 0.28 vs. 4.82 ± 0.21, 6.82 ± 0.28 vs. 5.09 ± 0.25, 7.46 ± 0.33 vs. 6.51 ± 0.28, at 1 μM, 1 μM, and 0.1 μM, respectively). However, phenylephrine caused an increase in glycocalyx staining intensity (6.27 ± 0.25 vs. 7.72 ± 0.32 at 1 μM). These results suggest that catecholamines acutely cause degradation of the glycocalyx in endothelial cells, but only when β adrenergic receptors are involved, since phenylephrine actually acted to increase glycocalyx. Future work will be needed to investigate the pathophysiological role of the reduced glycocalyx in hypertension. Furthermore, in vivo experiments are needed to determine whether these results are applicable to endothelial cells in the complex environment of intact blood vessels. Future work will also investigate the signaling mechanisms leading from adrenergic receptor activation to enzymatic degradation of the glycocalyx.

Rationale: Epicardial epithelial-to-mesenchymal trasition (EMT) is a vital process in embryonic heart development. During EMT, epicardial cells acquire migratory and invasive properties, and differentiate into new cell types, including cardiac fibroblasts and coronary smooth muscle cells. Non-histone protein methylation is an emerging modulator of cell signaling. We have recently established a role for protein arginine methyltransferase-1 (PRMT1) in TGF-β-induced EMT in cultured cells. Objective: To determine the role of PRMT1 in epicardial EMT. Methods and Results: We investigated the role of PRMT1 in epicardial EMT in mouse epicardial cells. Embryonic day 9.5 (E9.5) tamoxifen administration of WT1-Cre ERT ;PRMT1 fl/fl ;ROSA-YFP fl/fl mouse embryos was used to delete PRMT1 in the epicardium. Epicardial PRMT1 deletion led to reduced epicardial migration into the myocardium, a thinner compact myocardial layer, and dilated coronary blood vessels at E15.5. Using the epicardial cell line MEC1, we found that PRMT1 siRNA prevented the increase in mesenchymal proteins Slug and Fibronectin and the decrease in epithelial protein E-Cadherin during TGF-β treatment-induced EMT. PRMT1 siRNA also reduced the migration and invasion of MEC1 cells. We further identified that PRMT1 siRNA also increased the expression of p53, a key regulator of the Slug degradation pathway. PRMT1 siRNA increases p53 expression by decreasing p53 degradation, and shifted p53 localization to the cytoplasm. In vitro methylation assays further demonstrated that PRMT1 methylates p53. Knockdown of p53 increased Slug levels and enhanced EMT, establishing p53 as a regulator of epicardial EMT through controlling Slug expression. Furthermore, RNAseq experiments in MEC1 cells demonstrated that 40% (545/1,351) of TGF-β-induced transcriptional changes were prevented by PRMT1 siRNA. Furthermore, when p53 and PRMT1 were simultaneously knocked down, TGF-β induced transcriptional control of 37% (201/545) of these PRMT1-dependent genes was restored. Conclusions: The PRMT1-p53-Slug pathway is necessary for epicardial EMT in cultured MEC1 cells as well as in the epicardium in vivo . Epicardial PRMT1 is required for the development of compact myocardium and coronary blood vessels.

Bone-morphogenetic-protein (BMP)/Smads signaling pathway plays crucial role during heart development and vessel angiogenesis. BMP signaling is induced by the binding of BMP ligands (eg. BMP4) to their receptors, which recruit and phosphorylate receptor-Smads (R-Smads, eg. Smad1, Smad5) that form nuclear-transporting complexes with Smad4 for transcriptional regulation. Smad6 is an inhibitory Smad expresses predominantly in atria-ventricular cushion and outflow tract of the developing mouse heart, and expands to valves and great vessels. At the cell surface level, Smad6 binds to BMP type I receptor to block R-Smads recruitment to the receptor. At cytosolic level, Smad6 block Smad1/Smad4 complex formation. In the nucleus, Smad6 represses transcription. How these three levels of regulation are coordinated to inhibit BMP signaling is not known. We previously showed that BMP ligand induces an acute Smad6 methylation at arginine 74 (R74) at the cell surface level by a methyltransferase PRMT1, and methyl-Smad6 dissociates from receptor to allow receptor-induced Smad1/5 phosphorylation and activation. We further identified a delayed methylation on arginine 81 (R81) of Smad6 in the cytosol by PRMT1. We found that R81 methylation is required for BMP signaling-induced recruitment of Smad6 to phosphor-Smad1; it is also required for Smad6 to disrupt phosphor-Smad1/Smad4 complex formation and the following nuclear transportation, as well as for Smad6 to suppress Smad1 targeting gene transactivation. Previous findings indicate that Smad6 binds to type I receptor and Smad1 through its C-terminal region. We examined how arginine methylation in the N-terminal region, regulates the binding properties of C-terminal Smad6. We found that N-terminal Smad6 stabilizes the interaction between C-terminal Smad6 and Smad1 and enhances Smad6 inhibitory function. Disruption of R81 methylation results in loss of inhibitory function because of an increase in binding between N-term and C-term Smad6 that results in a "closed" conformation. In summary, R81 methylation controls Smad6 activity and R81 methylation of Smad6 defines the duration and intensity of BMP-induced Smad1/5 signaling.

Rationale: Epicardial epithelial-to-mesenchymal trasition (EMT) is a vital process in embryonic heart development. During EMT, epicardial cells acquire migratory and invasive properties, and differentiate into new cell types, including cardiac fibroblasts and coronary smooth muscle cells. EMT is characterized by an increase in mesenchymal proteins such as Slug and Fibronectin, and a decrease in cell-junction proteins such as E-Cadherin, and is dependent on TGF-β signaling. We have recently demonstrated that protein arginine methyltransferase-1 (PRMT1) is necessary for TGF-β family signaling and EMT in non-epicardial cell types. Objective: To determine the role of PRMT1 in epicardial EMT. Methods and Results: We investigated the role of PRMT1 in epicardial EMT in mouse epicardial cells. PRMT1 siRNA prevented the increase in Slug and Fibronectin and the decrease in E-Cadherin in TGF-β treatment-induced EMT of mouse epicardial cell line MEC1. PRMT1 siRNA also reduced the migration and invasion of MEC1 cells. These results demonstrate that PRMT1 is required for epicardial EMT. In WT1-Cre ERT ;ROSA-YFP fl/fl mouse embryos, PRMT1 siRNA reduced epicardial EMT in a thorax culture model. Among the key transcription factors that regulate the EMT program, Slug, but not Snail, is specifically regulated by PRMT1. We further identified that PRMT1 siRNA also increased the expression of p53, a key regulator of the Slug degradation pathway. PRMT1 siRNA increases p53 expression by decreasing p53 degradation, and shifted p53 localization to the cytoplasm. In vitro methylation assays further demonstrated that PRMT1 methylates p53. Knockdown of p53 increased Slug levels and enhanced EMT, establishing p53 as a regulator of epicardial EMT through controlling Slug expression. Conclusions: The PRMT1-p53-Slug pathway is necessary for epicardial EMT in cultured MEC1 cells as well as in the epicardium ex vivo .

The epithelial-to-mesenchymal transition (EMT) is an important cellular mechanism in a diverse range of biological processes such as development, wound healing, cancer metastasis, and organ fibrosis. Epicardial cells are mesothelial cells lining the outer surface of the heart that are an important progenitor population and a source of growth factors during development. Epicardial cells undergo EMT and invade the myocardium, differentiating into cardiac fibroblasts and coronary smooth muscle cells. Of note, resident fibroblasts of epicardial origin are a major cellular mediator of cardiac fibrosis. Our recent work has established an important function for the protein arginine methyltransferase PRMT1 in EMT. We hypothesized that PRMT1 is required for EMT in a mouse epicardial cell line (MEC-1). We found that PRMT1 is required for a subset of the EMT marker changes in epicardial EMT, such as upregulation of vimentin, fibronectin, and slug, as well as loss of E-cadherin. Furthermore, PRMT1 knockdown reduced MEC-1 migration and invasion, suggesting that PRMT1 is critical for epicardial progenitor function. Epicardial cells secrete a variety of signaling factors that affect cardiomyocyte proliferation and structure. This is a critical component of heart development, and may also affect heart diseases such as hypertophic heart failure. Therefore we assessed whether EMT and PRMT1 affect the paracrine functions of epicardial cells. Co-culture of MEC-1 cells with rat neonatal cardiomyocytes caused cardiomyocyte hypertrophy, and this was enhanced when MEC-1 cells were pre-treated with TGF-β to induce EMT. Interestingly, MEC-1 cells treated with PRMT1 siRNA also induced cardiomyocyte hypertrophy, but TGF-β pre-treatment of these cells did not enhance this effect. In conclusion, epicardial EMT is largely dependent on PRMT1. Epicardial cells also promote cardiomyocyte hypertrophy, which is enhanced in epicardial cells that have undergone PRMT1-mediated EMT towards a mesenchymal fate. These studies establish a role for protein methylation in the EMT process, and could lead to novel treatments for heart failure and other diseases affected by EMT.

Hydrogen sulfide (H 2 S), produced by the enzyme cystathionine-γ lyase (CSE), dilates arteries by hyperpolarizing and relaxing vascular smooth muscle cells (VSMC) and CSE knock-out causes hypertension and endothelial dysfunction showing the importance of this system. However, it is not clear if H 2 S-induced VSMC depolarization and relaxation is mediated by direct effects on VSMC or indirectly through actions on endothelial cells (EC). We reported previously that disrupting EC prevents H 2 S-induced vasodilation suggesting H 2 S might act directly on EC. Because inhibiting large-conductance Ca 2+ -activated K + (BK Ca ) channels also inhibits H 2 S-induced dilation, we hypothesized that H 2 S activates EC BK Ca channels to hyperpolarize EC and increase EC Ca 2+ which stimulates release of a secondary hyperpolarizing factor. Small mesenteric arteries from male Sprague-Dawley rats were used for all experiments. We found that EC disruption prevented H 2 S-induced VSMC membrane potential ( E m ) hyperpolarization. Blocking EC BK Ca channels with luminal application of the BK Ca inhibitor, iberiotoxin (IbTx, 100 nM), also prevented NaHS-induced dilation and VSMC hyperpolarization but did not affect resting VSMC E m showing EC specific actions. Sharp electrode recordings in arteries cut open to expose EC demonstrated H 2 S-induced hyperpolarization of EC while Ca 2+ imaging studies in fluor-4 loaded EC showed that H 2 S increases EC Ca 2+ event frequency. Thus H 2 S can act directly on EC. Inhibiting the EC enzyme cytochrome P 450 2C (Cyp2C) with sulfaphenazole also prevented VSMC depolarization and vasodilation. Finally, inhibiting TRPV4 channels to block the target of the Cyp2C product 11,12-EET inhibited NaHS-induced dilation. Combined with our previous report that CSE inhibition decreases BK Ca currents in EC, these results suggest that H 2 S stimulates EC BK Ca channels and activates Cyp2C upstream of VSMC hyperpolarization and vasodilation.