Shock drives a STAT3 and JunB-mediated coordinated transcriptional and DNA methylation response in the endothelium.

Endothelial dysfunction is a crucial factor in promoting organ failure during septic shock. However, the underlying mechanisms are unknown. Here, we show that kidney injury after lipopolysaccharide (LPS) insult leads to strong endothelial transcriptional and epigenetic responses. Furthermore, SOCS3 loss leads to an aggravation of the responses, demonstrating a causal role for the STAT3-SOCS3 signaling axis in the acute endothelial response to LPS. Experiments in cultured endothelial cells demonstrate that IL-6 mediates this response. Furthermore, bioinformatics analysis of in vivo and in vitro transcriptomics and epigenetics suggests a role for STAT, AP1 and interferon regulatory family (IRF) transcription factors. Knockdown of STAT3 or the AP1 member JunB partially prevents the changes in gene expression, demonstrating a role for these transcription factors. In conclusion, endothelial cells respond with a coordinated response that depends on overactivated IL-6 signaling via STAT3, JunB and possibly other transcription factors. Our findings provide evidence for a critical role of IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction.

Resolvin D2-G-Protein Coupled Receptor 18 Enhances Bone Marrow Function and Limits Steatosis and Hepatic Collagen Accumulation in Aging.

Aging is associated with nonresolving inflammation and tissue dysfunction. Resolvin D2 (RvD2) is a proresolving ligand that acts through the G-protein-coupled receptor called GPR18. Unbiased RNA sequencing revealed increased Gpr18 expression in macrophages from old mice, and in livers from elderly humans, which was associated with increased steatosis and fibrosis in middle-aged (MA) and old mice. MA mice that lacked GPR18 on myeloid cells had exacerbated steatosis and hepatic fibrosis, which was associated with a decline in Mac2+ macrophages. Treatment of MA mice with RvD2 reduced steatosis and decreased hepatic fibrosis, correlating with increased Mac2+ macrophages, increased monocyte-derived macrophages, and elevated numbers of monocytes in the liver, blood, and bone marrow. RvD2 acted directly on the bone marrow to increase monocyte-macrophage progenitors. A transplantation assay further demonstrated that bone marrow from old mice facilitated hepatic collagen accumulation in young mice. Transient RvD2 treatment to mice transplanted with bone marrow from old mice prevented hepatic collagen accumulation. Together, this study demonstrates that RvD2-GPR18 signaling controls steatosis and fibrosis and provides a mechanistic-based therapy for promoting liver repair in aging.

Neutrophil-to-Lymphocyte Ratios Distinguish Idiopathic Orbital Inflammation From Orbital Infectious Disease.

PURPOSE: The neutrophil-to-lymphocyte ratio (NLR) is a relatively novel biomarker to distinguish between acute stresses. This study was performed to determine whether the NLR may discern infectious orbital maladies from idiopathic orbital inflammation (IOI). METHODS: The NLR was calculated by a review of the initial blood draws of adult patients who presented to the emergency department at a single academic medical center. Statistical comparisons were performed to identify the significance of these results. RESULTS: Ten patients with IOI, 12 patients with necrotizing fasciitis (NF), and 12 patients with orbital cellulitis (OC) presented to the emergency department. The groups were not statistically significantly different in terms of age or gender. The mean NLRs were 3.48 (standard deviation = 1.80), 13.5 (standard deviation = 14.5), and 8.15 (standard deviation = 6.56) for IOI, NF, and OC, respectively. Patients with IOI had statistically significantly lower NLRs than patients with NF ( p = 0.037) and OC ( p = 0.034). However, the NLRs of patients with OC were not statistically significantly different from those of patients with NF ( p = 0.27). CONCLUSIONS: The NLR appears to distinguish IOI from infectious etiologies, but does not discern between variants of infection. These results should be juxtaposed against appropriate imaging and clinical evaluations, but elevated NLR values may heighten clinicians' concerns for an infectious process and encourage them to initiate appropriate management steps.

New therapies for unresectable or metastatic cutaneous eyelid and orbital melanoma.

PURPOSE: Newer treatment options offer the promise of improved outcomes for metastatic and unresectable melanoma. This investigation was performed to review these modalities for cutaneous eyelid and orbital disease. METHODS: A search for articles that were related to this subject was performed in the PubMed database, and the bibliographies of these manuscripts were reviewed to ensure capture of the appropriate literature. Data was abstracted and analyzed. RESULTS: Historically, patients who suffer from melanoma of the ocular adnexa have fared poorly. Approaches that employ BRAF and mitogen-associated protein kinase inhibitors, immunotherapy, and novel cellular therapies improve outcomes and survival rates, although the side effect profiles of these agents are problematic. Most of the existing strategies have not explored ocular adnexal disease specifically, and treatment plans are generally adapted from the general cutaneous oncology literature. CONCLUSIONS: Thanks to advances in our comprehension of the cellular biology of the disease, the management of unresectable and metastatic melanoma has evolved considerably over the past several years. Newer modalities will likely continue to improve survival and reduce adverse events.

Emerging therapeutic options for periorbital and orbital cutaneous basal and squamous cell carcinomas.

PURPOSE: Recently, several new therapies have emerged to address locally advanced cutaneous basal cell and squamous cell carcinomas. Given the constraints of the ocular adnexa and orbit, this review was designed to discuss the role of these modalities in this region. METHODS: A PubMed search was carried out to analyze the utility of United States Food and Drug Administration-approved therapies to address these malignancies. The data presented in the identified investigations were analyzed and abstracted. RESULTS: Multiple novel interventions may be useful in the management of periocular cutaneous basal cell and squamous cell carcinomas, including imiquimod, hedgehog inhibitors, and immunotherapy. While many of these treatments have not been specifically explored in the orbit and ocular adnexa, the literature generally shows favorable response rates. However, adverse events were common in these studies. CONCLUSIONS: Several novel treatments may address periorbital cutaneous malignancies, and these therapies may be particularly useful in patients with unresectable disease and those who are poor surgical candidates.

SOCS3 limits TNF and endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin-6 signal in human endothelial cells.

Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein and dermal blood microvascular endothelial cells. We show that cotreatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This cotreatment led to strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, whereas SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-κB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

CaMKIIδ is upregulated by pro-inflammatory cytokine IL-6 in a JAK/STAT3-dependent manner to promote angiogenesis.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine threonine kinase with established roles in physiological and pathophysiological vascular remodeling. Based on our previous study demonstrating that CaMKIIδ promotes thrombin-induced endothelial permeability and recent reports that CaMKII may contribute to inflammatory remodeling in the heart, we investigated CaMKIIδ-dependent regulation of endothelial function downstream of an interleukin-6 (IL-6)/JAK/STAT3 signaling axis. Upon treatment with IL-6 and its soluble receptor (sIL-6r), CaMKIIδ expression is significantly induced in HUVEC. Using pharmacological inhibitors of JAK and siRNA targeting STAT3, we demonstrated that activation of STAT3 is sufficient to induce CaMKIIδ expression. Under these conditions, rather than promoting IL-6-induced permeability, we found that CaMKIIδ promotes endothelial cell migration as measured by live cell imaging of scratch wound closure and single-cell motility analysis. In a similar manner, endothelial cell proliferation was attenuated upon knockdown of CaMKIIδ as determined by growth curves, cell cycle analysis, and capacitance of cell-covered electrodes as measured by ECIS. Using inducible endothelial-specific STAT3 knockout mice, we demonstrate that STAT3 signaling promotes developmental angiogenesis in the neonatal mouse retina assessed at postnatal day 6. CaMKIIδ expression in retinal endothelium was attenuated in these animals as measured by qPCR. STAT3's effects on angiogenesis were phenocopied by the endothelial-specific knockout of CaMKIIδ, with significantly reduced vascular outgrowth and number of junctions in the developing P6 retina. For the first time, we demonstrate that transcriptional regulation of CaMKIIδ by STAT3 promotes endothelial motility, proliferation, and in vivo angiogenesis.

MEF2 (Myocyte Enhancer Factor 2) Is Essential for Endothelial Homeostasis and the Atheroprotective Gene Expression Program.

OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.

Resolvin D1 Enhances Necroptotic Cell Clearance Through Promoting Macrophage Fatty Acid Oxidation and Oxidative Phosphorylation.

OBJECTIVE: Plaque necrosis is a key feature of defective resolution in atherosclerosis. Recent evidence suggests that necroptosis promotes plaque necrosis; therefore, we sought to determine how necroptotic cells (NCs) impact resolution programs in plaques. Approach and Results: To investigate the role(s) of necroptosis in advanced atherosclerosis, we used mice deficient of Mlkl, an effector of necroptosis. Mlkl-/- mice that were injected with a gain-of-function mutant PCSK9 (AAV8-gof-PCSK9) and fed a Western diet for 16 weeks, showed significantly less plaque necrosis, increased fibrous caps and improved efferocytosis compared with AAV8-gof-PCSK9 injected wt controls. Additionally, hypercholesterolemic Mlkl-/- mice had a significant increase in proresolving mediators including resolvin D1 (RvD1) and a decrease in prostanoids including thromboxane in plaques and in vitro. We found that exuberant thromboxane released by NCs impaired the clearance of both apoptotic cells and NCs through disruption of oxidative phosphorylation in macrophages. Moreover, we found that NCs did not readily synthesize RvD1 and that exogenous administration of RvD1 to macrophages rescued NC-induced defective efferocytosis. RvD1 also enhanced the uptake of NCs via the activation of p-AMPK (AMP-activated protein kinase), increased fatty acid oxidation, and enhanced oxidative phosphorylation in macrophages. CONCLUSIONS: These results suggest that NCs derange resolution by limiting key SPMs and impairing the efferocytic repertoire of macrophages. Moreover, these findings provide a molecular mechanism for RvD1 in directing proresolving metabolic programs in macrophages and further suggests RvD1 as a potential therapeutic strategy to limit NCs in tissues. Graphic Abstract: A graphic abstract is available for this article.

Myeloid differentiation factor 88 expression in eyelid specimens of rosacea.

PURPOSE: Rosacea is a common cause of ocular surface disease. Specific immunologic features have been implicated in its pathogenesis, including toll-like receptors, mitogen-associated kinase, and nuclear factor kappa-B. Myeolid differentiation factor 88 (MYD88) has been associated with these elements, suggesting a role for this protein in rosacea. This study was designed to compare the expression of MYD88 in the eyelids of patients with and without this disease. METHODS: Western blotting for MYD88 was performed in 14 control patients and 15 patients with rosacea. Bands were quantified and normalized to actin. Immunohistochemical staining for MYD88 was performed in a different cohort of 12 patients with rosacea and 12 controls, and positively-staining cells were counted across five consecutive 40x fields. Statistical analyses compared the differences between the two groups via a dedicated software package. RESULTS: On western blotting, the mean ratios of MYD88 to actin were 13.8 (standard deviation = 14.1) and 44.3 (standard deviation = 39.6) in control and rosacea patients, respectively (p = .002). On immunohistochemistry, the mean numbers of positively-staining cells were 12.1/40x field (standard deviation = 9.61/40x field) and 27.4/40x (standard deviation = 18.7/40x field) in control and rosacea patients, respectively (p = .0438). CONCLUSIONS: MYD88 is enriched in eyelid specimens of rosacea. This finding further implicates the innate immune system in the pathogenesis of rosacea, and is consistent with previous reports regarding the role of this protein in ocular surface disease and the previously-implicated cellular features of the disease. Inhibition of MYD88 may be a successful treatment strategy to manage rosacea.

Unique inflammatory profile is associated with higher SARS-CoV-2 acute respiratory distress syndrome (ARDS) mortality.

The COVID19 pandemic has caused more than a million of deaths worldwide, primarily due to complications from COVID19-associated acute respiratory distress syndrome (ARDS). Controversy surrounds the circulating cytokine/chemokine profile of COVID19-associated ARDS, with some groups suggesting that it is similar to patients without COVID19 ARDS and others observing substantial differences. Moreover, although a hyperinflammatory phenotype associates with higher mortality in non-COVID19 ARDS, there is little information on the inflammatory landscape's association with mortality in patients with COVID19 ARDS. Even though the circulating leukocytes' transcriptomic signature has been associated with distinct phenotypes and outcomes in critical illness including ARDS, it is unclear whether the mortality-associated inflammatory mediators from patients with COVID19 are transcriptionally regulated in the leukocyte compartment. Here, we conducted a prospective cohort study of 41 mechanically ventilated patients with COVID19 infection using highly calibrated methods to define the levels of plasma cytokines/chemokines and their gene expressions in circulating leukocytes. Plasma IL1RA and IL8 were found positively associated with mortality, whereas RANTES and EGF negatively associated with that outcome. However, the leukocyte gene expression of these proteins had no statistically significant correlation with mortality. These data suggest a unique inflammatory signature associated with severe COVID19.

Cytotoxic T-Lymphocyte-Associated Protein-4 and Lymphocyte Activation Gene-3 Expression in Orbitally-Invasive Versus Nodular Basal Cell Carcinoma.

PURPOSE: To compare the expression of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in nodular and orbitally-invasive forms of basal cell carcinoma (BCC). METHODS: Immunohistochemical staining for CTLA-4 and LAG-3 was performed on the pathology specimens of BCC from orbital exenteration and nodular forms. The numbers of positively-staining cells/×40 field were counted across 5 consecutive fields of each specimen and statistical analysis was performed to calculate the difference in expression between the 2 groups. RESULTS: Nine cases of orbitally-invasive BCC and 6 cases of nodular BCC were studied. The mean numbers of CTLA-4-positively staining cells were 11.51 cells/×40 field (median = 6.60 cells/×40 field, range = 0.4-31.8 cells/×40 field) in invasive BCC and 0.90 cells/×40 field (median = 0.60 cells/×40 field, range = 0.0-2.8 cells/×40 field) in nodular specimens. The difference between the 2 groups was statistically significant (p = 0.0030). The mean number of LAG-3-positively staining cells was 0.58 cells/×40 field (median = 0.0, range = 0.0-2.8 cells/×40 field) in invasive BCC and 3.13 cells/×40 field (median = 0.0, range = 0.0-18.18 cells/×40 field). There was no significant difference in LAG-3 positivity between tumor groups (p = 0.5564). CONCLUSIONS: CTLA-4 expression was enriched in orbitally invasive BCC compared with nodular forms of BCC, whereas LAG-3 expression did not differ between these entities. CTLA-4 mediated immune suppression may facilitate the development of orbitally invasive BCC. Treatment strategies that use existing medications to target CTLA-4 may decrease the requirement for orbital exenteration and provide enhanced survival outcomes.

High CO2 Downregulates Skeletal Muscle Protein Anabolism via AMP-activated Protein Kinase α2-mediated Depressed Ribosomal Biogenesis.

High CO2 retention, or hypercapnia, is associated with worse outcomes in patients with chronic pulmonary diseases. Skeletal muscle wasting is also an independent predictor of poor outcomes in patients with acute and chronic pulmonary diseases. Although previous evidence indicates that high CO2 accelerates skeletal muscle catabolism via AMPK (AMP-activated protein kinase)-FoxO3a-MuRF1 (E3-ubiquitin ligase muscle RING finger protein 1), little is known about the role of high CO2 in regulating skeletal muscle anabolism. In the present study, we investigated the potential role of high CO2 in attenuating skeletal muscle protein synthesis. We found that locomotor muscles from patients with chronic CO2 retention demonstrated depressed ribosomal gene expression in comparison with locomotor muscles from non-CO2-retaining individuals, and analysis of the muscle proteome of normo- and hypercapnic mice indicates reduction of important components of ribosomal structure and function. Indeed, mice chronically kept under a high-CO2 environment show evidence of skeletal muscle downregulation of ribosomal biogenesis and decreased protein synthesis as measured by the incorporation of puromycin into skeletal muscle. Hypercapnia did not regulate the mTOR pathway, and rapamycin-induced deactivation of mTOR did not cause a decrease in ribosomal gene expression. Loss-of-function studies in cultured myotubes showed that AMPKα2 regulates CO2-mediated reductions in ribosomal gene expression and protein synthesis. Although previous evidence has implicated TIF1A (transcription initiation factor-1α) and KDM2A (lysine-specific demethylase 2A) in AMPK-driven regulation of ribosomal gene expression, we found that these mediators were not required in the high CO2-induced depressed protein anabolism. Our research supports future studies targeting ribosomal biogenesis and protein synthesis to alleviate the effects of high CO2 on skeletal muscle turnover.

CD73 Is Enriched in Cutaneous Carcinomas That Invade the Orbit.

PURPOSE: To compare the expression of CD73 in indolent nodular basal cell carcinomas and cutaneous lesions that invade the orbit. METHODS: Immunohistochemical staining for CD73 was performed on nodular basal cell carcinomas and invasive basal cell carcinomas. The numbers of positively staining cells were counted across 5 consecutive ×40 microscopic fields, and a statistical comparison was performed via a dedicated computerized software package. RESULTS: Ten control specimens and 11 invasive tumors were analyzed. In nodular basal cell carcinomas, the mean number of positively staining cells was 0.72/×40 field (standard deviation = 0.80 cells/×40 field), whereas invasive tumors demonstrated a mean of 6.53 positive staining cells/×40 field (standard deviation = 3.33 cells/×40 field). This difference was statistically significant (p = 0.0002). CONCLUSIONS: CD73 is enriched in orbital invasion of cutaneous carcinomas. This checkpoint may represent an important target for therapeutic management of these lesions. Immunotherapeutic agents that have been designed to treat other neoplasms may decrease the requirement for orbital exenteration in this clinical scenario.

Calcitonin Gene-Related Peptide in Blind, Painful Eyes.

PURPOSE: To determine if calcitonin gene-related peptide (CGRP) is overexpressed in blind, painful eyes. METHODS: This was a retrospective cross-sectional randomized control study conducted at a tertiary level hospital. Eight specimens of eyes enucleated by a single surgeon were included in the study. The control group patients underwent exenteration for cutaneous malignancy without intraocular involvement, while the case group was enucleated for management of blind, painful eye. Each eye was stained using immunohistochemistry for the expression of CGRP. Expression of CGRP was examined by counting the number of cells staining positive for CGRP in 5 consecutive ×40 fields in the choroid, iris, and cornea of each specimen. RESULTS: The mean number of cells staining positively for CGRP in the choroid of blind, painful eye group was 45.5 (26.3) versus 5.5 (7.7) in nonpainful enucleated eyes. This difference was found to be statistically significant (p = 0.016). Comparison of iris and cornea did not reach statistical significance. Expression of CGRP was compared using a paired t test. CONCLUSIONS: This study demonstrates that CGRP is overexpressed in the choroid of enucleated blind, painful eyes. Modulation of this protein may represent a meaningful, nonsurgical therapeutic strategy to addressing the blind, painful eye.

Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

Interleukin-6 promotes a sustained loss of endothelial barrier function via Janus kinase-mediated STAT3 phosphorylation and de novo protein synthesis.

Vascular leakage is a hallmark of the inflammatory response. Acute changes in endothelial permeability are due to posttranslational changes in intercellular adhesion and cytoskeleton proteins. However, little is known about the mechanisms leading to long-term changes in vascular permeability. Here, we show that interleukin-6 (IL-6) promotes an increase in endothelial monolayer permeability that lasts over 24 h and demonstrate that activation of Src and MEK/ERK pathways is required only for short-term increases in permeability, being dispensable after 2 h. In contrast, Janus kinase (JAK)-mediated STAT3 phosphorylation at Y705 (but not S727) and de novo synthesis of RNA and proteins are required for the sustained permeability increases. Loss of junctional localization of VE-cadherin and ZO-1 is evident several hours after the maximal IL-6 response, thus suggesting that these events are a consequence of IL-6 signaling, but not a cause of the increased permeability. Understanding the mechanisms involved in sustaining vascular permeability may prove crucial to allow us to directly target vascular leakage and minimize tissue damage, thus reducing the rates of mortality and chronic sequelae of excessive edema. Targeting endothelial-specific mechanisms regulating barrier function could provide a new therapeutic strategy to prevent vascular leakage while maintaining the immune response and other beneficial aspects of the inflammatory response that are required for bacterial clearance and tissue repair.

Endothelial Myocyte Enhancer Factor 2c Inhibits Migration of Smooth Muscle Cells Through Fenestrations in the Internal Elastic Lamina.

OBJECTIVE: Laminar flow activates myocyte enhancer factor 2 (MEF2) transcription factors in vitro to induce expression of atheroprotective genes in the endothelium. Here we sought to establish the role of Mef2c in the vascular endothelium in vivo. APPROACH AND RESULTS: To study endothelial Mef2c, we generated endothelial-specific deletion of Mef2c using Tie2-Cre or Cdh5-Cre-ERT2 and examined aortas and carotid arteries by en face immunofluorescence. We observed enhanced actin stress fiber formation in the Mef2c-deleted thoracic aortic endothelium (laminar flow region), similar to those observed in normal aortic inner curvature (disturbed flow region). Furthermore, Mef2c deletion resulted in the de novo formation of subendothelial intimal cells expressing markers of differentiated smooth muscle in the thoracic aortas and carotids. Lineage tracing showed that these cells were not of endothelial origin. To define early events in intimal development, we induced endothelial deletion of Mef2c and examined aortas at 4 and 12 weeks postinduction. The number of intimal cell clusters increased from 4 to 12 weeks, but the number of cells within a cluster peaked at 2 cells in both cases, suggesting ongoing migration but minimal proliferation. Moreover, we identified cells extending from the media through fenestrations in the internal elastic lamina into the intima, indicating transfenestral smooth muscle migration. Similar transfenestral migration was observed in wild-type carotid arteries ligated to induce neointimal formation. CONCLUSIONS: These results indicate that endothelial Mef2c regulates the endothelial actin cytoskeleton and inhibits smooth muscle cell migration into the intima.

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis.

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.