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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.

INTRODUCTION Metropolitan cities in the United States suffer from higher rates of gun violence. However, the specific structural factors associated with increased gun violence are poorly defined. We hypothesized that firearm homicide in metropolitan cities would be impacted by Black-White segregation index. METHODS This cross-sectional analysis evaluated 51 US metropolitan statistical areas (MSAs) using data from 2013 to 2017. Several measures of structural racism were examined, including the Brooking Institute's Black-White segregation index. Demographic data were derived from the US Census Bureau, US Department of Education, and US Department of Labor. Crime data and firearm homicide mortality rates were obtained from the Federal Bureau of Investigation and the Centers for Disease Control. Spearman ρ and linear regression were performed. RESULTS Firearm mortality was associated with multiple measures of structural racism and racial disparity, including White-Black segregation index, unemployment rate, poverty rate, single parent household, percent Black population, and crime rates. In regression analysis, percentage Black population exhibited the strongest association with firearm homicide mortality (β = 0.42, p < 0.001). Black-White segregation index (β = 0.41, p = 0.001) and percent children living in single-parent households (β = 0.41, p = 0.002) were also associated with higher firearm homicide mortality. Firearm legislation scores were associated with lower firearm homicide mortality (β = -0.20 p = 0.02). High school and college graduation rates were not associated with firearm homicide mortality and were not included in the final model. CONCLUSION Firearm homicide disproportionately impacts communities of color and is associated with measures of structural racism, such as White-Black segregation index. Public health interventions targeting gun violence must address these systemic inequities. Furthermore, given the association between firearm mortality and single-parent households, intervention programs for at-risk youth may be particularly effective. LEVEL OF EVIDENCE Epidemiological level II.

Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1-Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1-Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses.

Background How the surgical stabilization of rib fractures after trauma affects the development of acute respiratory distress syndrome and impacts survival has yet to be determined in a large database. We hypothesized that surgical stabilization of rib fractures would not decrease the incidence of acute respiratory distress syndrome. Methods The National Trauma Data Bank was queried for all traumatic rib fractures in 2016. Patients were divided into groups with single rib fractures, multiple rib fractures, and flail chest. Nonoperative therapy was compared with stabilization of rib fractures of 1 to 2 ribs or 3+ ribs. Results There were 114,972 total patients with rib fractures meeting inclusion criteria, with 5,106 (4.4%) having flail chest, 24,726 (21.5%) having single rib fractures, and 85,140 (74.1%) having multiple rib fractures. Those with flail chest (15.9%) were most likely to get rib plating in comparison to multiple rib fractures (0.9%) and single rib fractures (0.2%); P < .001. On logistic regression, surgical stabilization of rib fractures 1 to 2 ribs (odds ratio: 0.17, 95% confidence interval: 0.10–0.28) or 3+ ribs (odds ratio: 0.17, 95% confidence interval: 0.11–0.28), with nonoperative therapy as the reference was associated with survival. Variables associated with mortality included increasing age, male sex, increasing injury severity score, decreased Glasgow coma scale, requirement of transfusions, and hypotension on admission. Surgical stabilization of rib fractures 3+ ribs (odds ratio: 2.30, 95% confidence interval: 1.58–3.37) was associated with acute respiratory distress syndrome but not 1 to 2 ribs (odd ratio: 1.55, 95% confidence interval: 0.97–2.48). On logistic regression of only patients with flail chest, stabilization of rib fractures was associated with decreased mortality but not increased risk of acute respiratory distress syndrome. Conclusion The increased risk of acute respiratory distress syndrome should be considered in the preoperative assessment for stabilization of rib fractures.

Fresh frozen plasma (FFP) contains proinflammatory mediators released from cellular debris during frozen storage. In addition, recent studies have shown that transfusion of never-frozen plasma (NFP), instead of FFP, may be superior in trauma patients. We hypothesized that FFP would have higher levels of inflammatory mediators when compared to NFP.FFP (n = 8) and NFP (n = 8) samples were obtained from an urban, level 1 trauma center blood bank. The cytokines in these samples were compared using a Milliplex (Milliplex Sigma) human cytokine magnetic bead panel multiplex assay for 41 different biomarkers.Growth factors that were higher in NFP included platelet-derived growth factor-AA (PDGF-AA; 8.09 versus 108.00 pg/mL, P0.001) and PDGF-AB (0.00 versus 215.20, P= 0.004). Soluble CD40-ligand (sCD40L), a platelet activator and pro-coagulant, was higher in NFP (31.81 versus 80.45 pg/mL, P0.001). RANTES, a leukocyte chemotactic cytokine was higher in NFP (26.19 versus 1418.00 pg/mL, P0.001). Interleukin-4 (5.70 versus 0.00 pg/mL, P= 0.03) and IL-8 (2.20 versus 0.52 pg/ml, P= 0.03) levels were higher in were higher in FFP.Frozen storage of plasma may result in decrease of several growth factors and/or pro-coagulants found in NFP. In addition, the freezing and thawing process may induce release of pro-inflammatory chemokines. Further studies are needed to determine if these cytokines result in improved outcomes with NFP over FFP in transfusion of trauma patients.

The epithelial-to-mesenchymal transdifferentiation (EMT) is crucial for tissue differentiation in development and drives essential steps in cancer and fibrosis. EMT is accompanied by reprogramming of gene expression and has been associated with the epithelial stem-cell state in normal and carcinoma cells. The cytokine transforming growth factor β (TGF-β) drives this program in cooperation with other signaling pathways and through TGF-β–activated SMAD3 as the major effector. TGF-β–induced SMAD3 activation is inhibited by SMAD7 and to a lesser extent by SMAD6, and SMAD6 and SMAD7 both inhibit SMAD1 and SMAD5 activation in response to the TGF-β–related bone morphogenetic proteins (BMPs). We previously reported that, in response to BMP, protein arginine methyltransferase 1 (PRMT1) methylates SMAD6 at the BMP receptor complex, thereby promoting its dissociation from the receptors and enabling BMP-induced SMAD1 and SMAD5 activation. We now provide evidence that PRMT1 also facilitates TGF-β signaling by methylating SMAD7, which complements SMAD6 methylation. We found that PRMT1 is required for TGF-β–induced SMAD3 activation, through a mechanism similar to that of BMP-induced SMAD6 methylation, and thus promotes the TGF-β–induced EMT and epithelial stem-cell generation. This critical mechanism positions PRMT1 as an essential mediator of TGF-β signaling that controls the EMT and epithelial cell stemness through SMAD7 methylation.

The balance between pro- and anti-inflammatory signals maintains tissue homeostasis and defines the outcome of chronic inflammatory diseases such as periodontitis, a condition that afflicts the tooth-supporting tissues and exerts an impact on systemic health. The induction of tissue inflammation relies heavily on Toll-like receptor (TLR) signaling, which drives a proinflammatory pathway through recruiting myeloid differentiation primary response gene 88 (MyD88) and activating nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). TLR-induced production of proinflammatory cytokines and chemokines is reined in by anti-inflammatory cytokines, including the transforming growth factor β (TGFβ) family of cytokines. Although Smad6 is a key mediator of TGFβ-induced anti-inflammatory signaling, the exact mechanism by which TGFβ regulates TLR proinflammatory signaling in the periodontal tissue has not been addressed to date. In this study, we demonstrate for the first time that the ability of TGFβ to inhibit TLR-NFκB signaling is mediated by protein arginine methyltransferase 1 (PRMT1)–induced Smad6 methylation. Upon methylation, Smad6 recruited MyD88 and promoted MyD88 degradation, thereby inhibiting NFκB activation. Most important, Smad6 is expressed and methylated in the gingival epithelium, and PRMT1-Smad6 signaling promotes tissue homeostasis by limiting inflammation. Consistent with this, disturbance of Smad6 methylation exacerbates inflammation and bone loss in experimental periodontitis. The dissected mechanism is therapeutically important, as it highlights the manipulation of PRMT1-Smad6 signaling as a novel promising strategy to modulate the host immune response in periodontitis.

Epicardial cells are cardiac progenitors that give rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during development. An integral phase of epicardial fate transition is epithelial-to-mesenchymal transition (EMT) that confers motility. We uncover an essential role for the protein arginine methyltransferase 1 (PRMT1) in epicardial invasion and differentiation. Using scRNA-seq, we show that epicardial-specific deletion of Prmt1 reduced matrix and ribosomal gene expression in epicardial-derived cell lineages. PRMT1 regulates splicing of Mdm4, which is a key controller of p53 stability. Loss of PRMT1 leads to accumulation of p53 that enhances Slug degradation and blocks EMT. During heart development, the PRMT1-p53 pathway is required for epicardial invasion and formation of epicardial-derived lineages: cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Consequently, this pathway modulates ventricular morphogenesis and coronary vessel formation. Altogether, our study reveals molecular mechanisms involving the PRMT1-p53 pathway and establish its roles in heart development.

Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGFβ, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.

TGFβ superfamily includes the transforming growth factor βs (TGFβs), bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and Activin/Inhibin families of ligands. Among the 33 members of TGFβ superfamily ligands, many act on multiple types of cells within the heart, including cardiomyocytes, cardiac fibroblasts/myofibroblasts, coronary endothelial cells, smooth muscle cells, and immune cells (e.g. monocytes/macrophages and neutrophils). In this review, we highlight recent discoveries on TGFβs, BMPs, and GDFs in different cardiac residential cellular components, in association with functional impacts in heart development, injury repair, and dysfunction. Specifically, we will review the roles of TGFβs, BMPs, and GDFs in cardiac hypertrophy, fibrosis, contractility, metabolism, angiogenesis, and regeneration.

SummaryThe spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

Background and objective Increased air pollutants correlate with increased incidence of cardiovascular disease potentially due to vascular dysfunction. We have reported that acute diesel engine exhaust (DE) exposure enhances vasoconstriction and diminishes acetylcholine (ACh)-induced dilation in coronary arteries in a nitric oxide synthase (NOS)-dependent manner. We hypothesize that acute DE inhalation leads to endothelial dysfunction by uncoupling NOS. Methods Rats inhaled fresh DE (300 μg particulate matter/m3) or filtered air for 5 hr. After off-gassing, intraseptal coronary arteries were isolated and dilation to ACh recorded using videomicroscopy. Results Arteries from DE-exposed animals dilated less to ACh than arteries from air-exposed animals. NOS inhibition did not affect ACh dilation in control arteries but increased dilation in the DE group, suggesting NOS does not normally contribute to ACh-induced dilation in coronary arteries but does contribute to endothelial dysfunction after DE inhalation. Cyclooxygenase (COX) inhibition did not affect ACh dilation in the DE group, but combined inhibition of NOS and COX diminished dilation in both groups and eliminated intergroup differences, suggesting that the two pathways interact. Superoxide scavenging increased ACh dilation in DE arteries, eliminating differences between groups. Tetrahydrobiopterin (BH4) supplementation with sepiapterin restored ACh-mediated dilation in the DE group in a NOS-dependent manner. Superoxide generation (dihydroethidium staining) was greater in DE arteries, and superoxide scavenging, BH4 supplementation, or NOS inhibition reduced the signal in DE but not air arteries. Conclusion Acute DE exposure appears to uncouple NOS, increasing reactive oxygen species generation and causing endothelial dysfunction, potentially because of depletion of BH4 limiting its bioavailability.

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O2-5% CO2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H2S)-induced vasodilation in rats. The enzyme that produces H2S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca2+ concentration ([Ca2+]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca2+] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P < 0.05), and fewer EC were NFATc3 nuclear positive in IH rat arteries than in sham rat arteries (IH = 13 ± 3, sham = 59 ± 11%, P < 0.05). H2S production was also lower in mesenteric tissue from IH rats vs. sham rats. Endothelium-dependent vasodilation to acetylcholine (ACh) was lower in mesenteric arteries from IH rats than in arteries from sham rats, and inhibiting CSE with β-cyanoalanine diminished ACh-induced vasodilation in arteries from sham but not IH rats but did not affect dilation to the H2S donor NaHS. Thus, IH lowers EC [Ca2+], NFAT activity, CSE expression and activity, and H2S production while inhibiting NFAT activation lowers CSE expression. The observations that IH exposure decreases NFATc3 activation and CSE-dependent vasodilation support a role for NFAT in regulating endothelial H2S production. NEW & NOTEWORTHY This study identifies the calcium-regulated transcription factor nuclear factor of activated T cells as a novel regulator of cystathionine γ-lyase (CSE). This pathway is basally active in mesenteric artery endothelial cells, but, after exposure to intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation.

Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.