Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice.

BACKGROUND: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis. METHODS: On an Apoe-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed. RESULTS: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin ß2-mediated adhesion of monocytes but did not impair integrin α4ß1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn2+- or chemokine-induced activation of integrin α4ß1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4ß1 but was not affected by talin1 deletion or antibodies to integrin ß2. Furthermore, quantitative polymerase chain reaction and ELISA analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin ß3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin ß3. CONCLUSIONS: Integrin α4ß1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4ß1 activation to the high-affinity state and integrin α4ß1-mediated monocyte recruitment. Yet, talin1 is required for integrin ß3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Acetaminophen Overdose Reveals Protease-Activated Receptor 4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium.

Background & Aims: Hepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting. Methods: We generated mice with conditional deletion(s) of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq). Results: We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial Par1 and Par4 had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Conclusions: Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Endothelial Chromatin-Remodeling Enzymes Regulate the Production of Critical ECM Components During Murine Lung Development.

BACKGROUND: The chromatin-remodeling enzymes BRG1 (brahma-related gene 1) and CHD4 (chromodomain helicase DNA-binding protein 4) independently regulate the transcription of genes critical for vascular development, but their coordinated impact on vessels in late-stage embryos has not been explored. METHODS: In this study, we genetically deleted endothelial Brg1 and Chd4 in mixed background mice (Brg1fl/fl;Chd4fl/fl;VE-Cadherin-Cre+), and littermates that were negative for Cre recombinase were used as controls. Tissues were analyzed by immunostaining, immunoblot, and flow cytometry. Quantitative reverse transcription polymerase chain reaction was used to determine gene expression, and chromatin immunoprecipitation revealed gene targets of BRG1 and CHD4 in cultured endothelial cells. RESULTS: We found Brg1/Chd4 double mutants grew normally but died soon after birth with small and compact lungs. Despite having normal cellular composition, distal air sacs of the mutant lungs displayed diminished ECM (extracellular matrix) components and TGFß (transforming growth factor-ß) signaling, which typically promotes ECM synthesis. Transcripts for collagen- and elastin-related genes and the TGFß ligand Tgfb1 were decreased in mutant lung endothelial cells, but genetic deletion of endothelial Tgfb1 failed to recapitulate the small lungs and ECM defects seen in Brg1/Chd4 mutants. We instead found several ECM genes to be direct targets of BRG1 and CHD4 in cultured endothelial cells. CONCLUSIONS: Collectively, our data highlight essential roles for endothelial chromatin-remodeling enzymes in promoting ECM deposition in the distal lung tissue during the saccular stage of embryonic lung development.

Comparative mechanistic study of RPE cell death induced by different oxidative stresses.

Oxidative stress is hypothesized to drive the progression of age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cell layer is important for supporting the function of retina and is particularly susceptible to oxidative stress-induced cell death. How RPE cells die in AMD, especially in geographic atrophy (GA), a late stage of dry AMD, is still controversial. The goal of this study is to compare the features and mechanisms of RPE cell death induced by different oxidative stresses, to identify potential universal therapeutic targets for GA. RPE cell death was induced both in vitro and ex vivo by 4-Hydroxynonenal (4-HNE), a major product of lipid peroxidation, sodium iodate (NaIO3) that has been widely used to model RPE cell death in dry AMD, a ferroptosis inducer RAS-selective lethal 3 (RSL3) or a necroptosis inducer shikonin. We found that RPE necroptosis and ferroptosis show common and distinct features. Common features include receptor-interacting protein kinase (RIPK)1/RIPK3 activation and lipid reactive oxygen species (ROS) accumulation, although lipid ROS accumulation is much milder during necroptosis. This supports cross talk between RPE ferroptosis and necroptosis pathways and is consistent with the rescue of RPE necroptosis and ferroptosis by RIPK1 inhibitor Necrostatin-1 (Nec-1) or in Ripk3-/- RPE explants. Distinct feature includes activated mixed lineage kinase domain like pseudokinase (MLKL) that is translocated to the cell membrane during necroptosis, which is not happening in ferroptosis. This is consistent with the failure to rescue RPE ferroptosis by MLKL inhibitor necrosulfonamide (NSA) or in Mlkl-/- RPE explants. Using this framework, we found that 4-HNE and NaIO3 induced RPE cell death likely through necroptosis based on the molecular features and the rescuing effect by multiple inhibitors. Our studies suggest that multiple markers and inhibitors are required to distinguish RPE necroptosis and ferroptosis, and that necroptosis inhibitor Nec-1 could be a potential therapeutic compound for GA since it inhibits RIPK1/RIPK3 activation and lipid ROS accumulation occurred in both necroptosis and ferroptosis pathways.

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge.

BACKGROUND: During infectious diseases, proinflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung, the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG (erythroblast transformation-specific-related gene) as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG were analyzed in cultured HUVECs (human umbilical vein ECs). Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5[PAC]-CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or lipopolysaccharide resulted in a rapid and substantial degradation of ERG within lung ECs but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek-a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. CONCLUSIONS: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge.

Background: During infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. Methods: Cytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNFα or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5(PAC)Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. Results: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek , a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. Conclusions: Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

Proinflammatory mediators, TNFα, IFNγ, and thrombin, directly induce lymphatic capillary tube regression.

In this work, we sought to investigate the direct effects of proinflammatory mediators on lymphatic endothelial cell (LEC) capillaries and whether they might induce regression. Our laboratory has developed novel in-vitro, serum-free, lymphatic tubulogenesis assay models whereby human LEC tube networks readily form in either three-dimensional collagen or fibrin matrices. These systems were initially conceptualized in the hopes of better understanding the influence of proinflammatory mediators on LEC capillaries. In this work, we have screened and identified proinflammatory mediators that cause regression of LEC tube networks, the most potent of which is TNFα (tumor necrosis factor alpha), followed by IFNγ (interferon gamma) and thrombin. When these mediators were combined, even greater and more rapid lymphatic capillary regression occurred. Surprisingly, IL-1ß (interleukin-1 beta), one of the most potent and pathologic cytokines known, had no regressive effect on these tube networks. Finally, we identified new pharmacological drug combinations capable of rescuing LEC capillaries from regression in response to the potent combination of TNFα, IFNγ, and thrombin. We speculate that protecting lymphatic capillaries from regression may be an important step toward mitigating a wide variety of acute and chronic disease states, as lymphatics are believed to clear both proinflammatory cells and mediators from inflamed and damaged tissue beds. Overall, these studies identify key proinflammatory mediators, including TNFα, IFNγ, and thrombin, that induce regression of LEC tube networks, as well as identify potential therapeutic agents to diminish LEC capillary regression responses.

Mouse Chd4-NURD is required for neonatal spermatogonia survival and normal gonad development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

IL-1ß Impacts Vascular Integrity and Lymphatic Function in the Embryonic Omentum.

BACKGROUND: The chromatin-remodeling enzyme BRG1 (brahma-related gene 1) regulates gene expression in a variety of rapidly differentiating cells during embryonic development. However, the critical genes that BRG1 regulates during lymphatic vascular development are unknown. METHODS: We used genetic and imaging techniques to define the role of BRG1 in murine embryonic lymphatic development, although this approach inadvertently expanded our study to multiple interacting cell types. RESULTS: We found that omental macrophages fine-tune an unexpected developmental process by which erythrocytes escaping from naturally discontinuous omental blood vessels are collected by nearby lymphatic vessels. Our data indicate that circulating fibrin(ogen) leaking from gaps in omental blood vessels can trigger inflammasome-mediated IL-1ß (interleukin-1ß) production and secretion from nearby macrophages. IL-1ß destabilizes adherens junctions in omental blood and lymphatic vessels, contributing to both extravasation of erythrocytes and their uptake by lymphatics. BRG1 regulates IL-1ß production in omental macrophages by transcriptionally suppressing the inflammasome trigger RIPK3 (receptor interacting protein kinase 3). CONCLUSIONS: Genetic deletion of Brg1 in embryonic macrophages leads to excessive IL-1ß production, erythrocyte leakage from blood vessels, and blood-filled lymphatics in the developing omentum. Altogether, these results highlight a novel context for epigenetically regulated crosstalk between macrophages, blood vessels, and lymphatics.

Selective and Marked Blockade of Endothelial Sprouting Behavior Using Paclitaxel and Related Pharmacologic Agents.

Whether alterations in the microtubule cytoskeleton affect the ability of endothelial cells (ECs) to sprout and form branching networks of tubes was investigated in this study. Bioassays of human EC tubulogenesis, where both sprouting behavior and lumen formation can be rigorously evaluated, were used to demonstrate that addition of the microtubule-stabilizing drugs, paclitaxel, docetaxel, ixabepilone, and epothilone B, completely interferes with EC tip cells and sprouting behavior, while allowing for EC lumen formation. In bioassays mimicking vasculogenesis using single or aggregated ECs, these drugs induce ring-like lumens from single cells or cyst-like spherical lumens from multicellular aggregates with no evidence of EC sprouting behavior. Remarkably, treatment of these cultures with a low dose of the microtubule-destabilizing drug, vinblastine, led to an identical result, with complete blockade of EC sprouting, but allowing for EC lumen formation. Administration of paclitaxel in vivo markedly interfered with angiogenic sprouting behavior in developing mouse retina, providing corroboration. These findings reveal novel biological activities for pharmacologic agents that are widely utilized in multidrug chemotherapeutic regimens for the treatment of human malignant cancers. Overall, this work demonstrates that manipulation of microtubule stability selectively interferes with the ability of ECs to sprout, a necessary step to initiate and form branched capillary tube networks.

Genomic locus proteomic screening identifies the NF-κB signaling pathway components NFκB1 and IKBKG as transcriptional regulators of Ripk3 in endothelial cells.

The receptor-interacting protein kinase 3 (RIPK3) is a multi-functional protein best known for facilitating cellular necroptosis and inflammation. Recent evidence from our lab indicates that RIPK3 expression must be tightly regulated in endothelial cells to promote angiogenesis, to maintain vascular integrity during embryogenesis, and to provide protection from postnatal atherosclerosis. RIPK3 activity and stability are regulated by post-translational modifications and caspase-dependent cleavage. However, less is known about the transcriptional regulation of Ripk3. Here we utilized an unbiased CRISPR-based technology called genomic locus proteomics (GLoPro) to screen transcription factors and coregulatory proteins associated with the Ripk3 locus in a murine endothelial cell line. We found that 41 nuclear proteins are specifically enriched at the Ripk3 locus, including the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway components NFκB1 and IKBKG. We further verified that NFκB1 and IKBKG directly bind the Ripk3 promoter and prevent TNFα-induced Ripk3 transcription in cultured human primary endothelial cells. Moreover, NFκB1 prevents RIPK3-mediated death of primary endothelial cells. These data provide new insights into NF-κB signaling and Ripk3 transcriptional regulation in endothelial cells.

RIPK3 modulates growth factor receptor expression in endothelial cells to support angiogenesis.

Receptor-interacting protein kinase 3 (RIPK3) is a multifunctional intracellular protein that was first recognized as an important component of the necroptosis programmed cell death pathway. RIPK3 is also highly expressed in non-necroptotic murine embryonic endothelial cells (ECs) during vascular development, indicating its potential contribution to angiogenesis. To test this hypothesis, we generated mice lacking endothelial RIPK3 and found non-lethal embryonic and perinatal angiogenesis defects in multiple vascular beds. Our in vitro data indicate that RIPK3 supports angiogenesis by regulating growth factor receptor degradation in ECs. We found that RIPK3 interacted with the membrane trafficking protein myoferlin to sustain expression of vascular endothelial growth factor receptor 2 (VEGFR2) in cultured ECs following vascular endothelial growth factor A (VEGFA) stimulation. Restoration of myoferlin, which was diminished after RIPK3 knockdown, rescued decreased VEGFR2 expression and vascular sprouting in RIPK3-deficient ECs after VEGFA treatment. In addition, we found that RIPK3 modulated expression of genes involved in endothelial identity by inhibiting ERK signaling independently of growth factor receptor turnover. Altogether, our data reveal unexpected non-necroptotic roles for RIPK3 in ECs and evidence that RIPK3 promotes developmental angiogenesis in vivo.

An inhibitor of endothelial ETS transcription factors promotes physiologic and therapeutic vessel regression.

During the progression of ocular diseases such as retinopathy of prematurity and diabetic retinopathy, overgrowth of retinal blood vessels results in the formation of pathological neovascular tufts that impair vision. Current therapeutic options for treating these diseases include antiangiogenic strategies that can lead to the undesirable inhibition of normal vascular development. Therefore, strategies that eliminate pathological neovascular tufts while sparing normal blood vessels are needed. In this study we exploited the hyaloid vascular network in murine eyes, which naturally undergoes regression after birth, to gain mechanistic insights that could be therapeutically adapted for driving neovessel regression in ocular diseases. We found that endothelial cells of regressing hyaloid vessels underwent down-regulation of two structurally related E-26 transformation-specific (ETS) transcription factors, ETS-related gene (ERG) and Friend leukemia integration 1 (FLI1), prior to apoptosis. Moreover, the small molecule YK-4-279, which inhibits the transcriptional and biological activity of ETS factors, enhanced hyaloid regression in vivo and drove Human Umbilical Vein Endothelial Cells (HUVEC) tube regression and apoptosis in vitro. Importantly, exposure of HUVECs to sheer stress inhibited YK-4-279-induced apoptosis, indicating that low-flow vessels may be uniquely susceptible to YK-4-279-mediated regression. We tested this hypothesis by administering YK-4-279 to mice in an oxygen-induced retinopathy model that generates disorganized and poorly perfused neovascular tufts that mimic human ocular diseases. YK-4-279 treatment significantly reduced neovascular tufts while sparing healthy retinal vessels, thereby demonstrating the therapeutic potential of this inhibitor.

The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development.

ATP-dependent chromatin-remodeling complexes epigenetically modulate transcription of target genes to impact a variety of developmental processes. Our lab previously demonstrated that CHD4-a central ATPase and catalytic enzyme of the NuRD chromatin-remodeling complex-plays an important role in murine embryonic endothelial cells by transcriptionally regulating vascular integrity at midgestation. Since NuRD complexes can incorporate the ATPase CHD3 as an alternative to CHD4, we questioned whether the CHD3 enzyme likewise modulates vascular development or integrity. We generated a floxed allele of Chd3 but saw no evidence of lethality or vascular anomalies when we deleted it in embryonic endothelial cells in vivo (Chd3ECKO). Furthermore, double-deletion of Chd3 and Chd4 in embryonic endothelial cells (Chd3/4ECKO) did not dramatically alter the timing and severity of embryonic phenotypes seen in Chd4ECKO mutants, indicating that CHD3 does not play a cooperative role with CHD4 in early vascular development. However, excision of Chd3 at the epiblast stage of development with a Sox2-Cre line allowed us to generate global heterozygous Chd3 mice (Chd3Δ/+), which were subsequently intercrossed and revealed partial lethality of Chd3Δ/Δ mutants prior to weaning. Tissues from surviving Chd3Δ/Δ mutants helped us confirm that CHD3 was efficiently deleted in these animals and that CHD3 is highly expressed in the gonads and brains of adult wildtype mice. Therefore, Chd3-flox mice will be beneficial for future studies about roles for this chromatin-remodeling enzyme in viable embryonic development and in gonadal and brain physiology.

Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis.

Receptor-interacting protein kinase 3 (RIPK3) was recently implicated in promoting atherosclerosis progression through a proposed role in macrophage necroptosis. However, RIPK3 has been connected to numerous other cellular pathways, which raises questions about its actual role in atherosclerosis. Furthermore, RIPK3 is expressed in a multitude of cell types, suggesting that it may be physiologically relevant to more than just macrophages in atherosclerosis. In this study, Ripk3 was deleted in macrophages, endothelial cells, vascular smooth muscle cells or globally on the Apoe-/- background using Cre-lox technology. To induce atherosclerosis progression, male and female mice were fed a Western diet for three months before tissue collection and analysis. Surprisingly, necroptosis markers were nearly undetectable in atherosclerotic aortas. Furthermore, en face lesion area was increased in macrophage- and endothelial-specific deletions of Ripk3 in the descending and abdominal regions of the aorta. Analysis of bone-marrow-derived macrophages and cultured endothelial cells revealed that Ripk3 deletion promotes expression of monocyte chemoattractant protein 1 (MCP-1) and E-selectin in these cell types, respectively. Western blot analysis showed upregulation of MCP-1 in aortas with Ripk3-deficient macrophages. Altogether, these data suggest that RIPK3 in macrophages and endothelial cells protects against atherosclerosis through a mechanism that likely does not involve necroptosis. This protection may be due to RIPK3-mediated suppression of pro-inflammatory MCP-1 expression in macrophages and E-selectin expression in endothelial cells. These findings suggest a novel and unexpected cell-type specific and athero-protective function for RIPK3.This article has an associated First Person interview with the first author of the paper.

The NuRD chromatin-remodeling complex enzyme CHD4 prevents hypoxia-induced endothelial Ripk3 transcription and murine embryonic vascular rupture.

Physiological hypoxia can trigger transcriptional events that influence many developmental processes during mammalian embryogenesis. One way that hypoxia affects transcription is by engaging chromatin-remodeling complexes. We now report that chromodomain helicase DNA binding protein 4 (CHD4), an enzyme belonging to the nucleosome remodeling and deacetylase (NuRD) chromatin-remodeling complex, is required for transcriptional repression of the receptor-interacting protein kinase 3 (Ripk3)-a critical executor of the necroptosis cell death program-in hypoxic murine embryonic endothelial cells. Genetic deletion of Chd4 in murine embryonic endothelial cells in vivo results in upregulation of Ripk3 transcripts and protein prior to vascular rupture and lethality at midgestation, and concomitant deletion of Ripk3 partially rescues these phenotypes. In addition, CHD4 binds to and prevents acetylation of the Ripk3 promoter in cultured endothelial cells grown under hypoxic conditions to prevent excessive Ripk3 transcription. These data demonstrate that excessive RIPK3 is detrimental to embryonic vascular integrity and indicate that CHD4 suppresses Ripk3 transcription when the embryonic environment is particularly hypoxic prior to the establishment of fetal-placental circulation at midgestation. Altogether, this research provides new insights into regulators of Ripk3 transcription and encourages future studies into the mechanism by which excessive RIPK3 damages embryonic blood vessels.

Proinflammatory Mediators, IL (Interleukin)-1ß, TNF (Tumor Necrosis Factor) α, and Thrombin Directly Induce Capillary Tube Regression.

OBJECTIVE: In this work, we examine the molecular basis for capillary tube regression and identify key proregressive factors, signaling pathways, and pharmacological antagonists of this process. Approach and Results: We demonstrate that the proinflammatory mediators, IL (interleukin)-1ß, TNF (tumor necrosis factor) α, and thrombin, singly and in combination, are potent regulators of capillary tube regression in vitro. These proregressive factors, when added to endothelial cell-pericyte cocultures, led to selective loss of endothelial cell-lined tube networks, with retention and proliferation of pericytes despite the marked destruction of adjacent capillary tubes. Moreover, treatment of macrophages with the TLR (toll-like receptor) agonists Pam3CSK4 and lipopolysaccharide generates conditioned media with marked proregressive activity, that is completely blocked by a combination of neutralizing antibodies directed to IL-1ß and TNFα but not to other factors. The same combination of blocking antibodies, as well as the anti-inflammatory cytokine IL-10, interfere with macrophage-dependent hyaloid vasculature regression in mice suggesting that proinflammatory cytokine signaling regulates capillary regression in vivo. In addition, we identified a capillary regression signaling signature in endothelial cells downstream of these proregressive agents that is characterized by increased levels of ICAM-1 (intercellular adhesion molecule-1), phospho-p38, and phospho-MLC2 (myosin light chain-2) and decreased levels of phospho-Pak2, acetylated tubulin, phospho-cofilin, and pro-caspase3. Finally, we identified combinations of pharmacological agents (ie, FIST and FISTSB) that markedly rescue the proregressive activities of IL-1ß, TNFα, and thrombin, individually and in combination. CONCLUSIONS: Overall, these new studies demonstrate that the major proinflammatory mediators, IL-1ß, TNFα, and thrombin, are key regulators of capillary tube regression-a critical pathological process regulating human disease.

Characterizing Epigenetic Changes in Endothelial Cells.

Chromatin immunoprecipitation (ChIP) is an antibody-based method used to identify protein-DNA interactions and sites of protein modifications to chromatin in living cells. ChIP is a powerful method for identifying genomic sites at which epigenetic changes occur in cell types of interest because many antibodies have been developed that recognize specific epigenetic modifications of histone tails. This chapter provides detailed ChIP and subsequent polymerase chain reaction (ChIP-PCR) protocols for use in cultured endothelial cells. These protocols will allow investigators to make consistent and quantitative discoveries about epigenetic changes that occur in endothelial cells at specific genomic sites under varying treatment conditions.

Excessive Plasmin Compromises Hepatic Sinusoidal Vascular Integrity After Acetaminophen Overdose.

The serine protease plasmin degrades extracellular matrix (ECM) components both directly and indirectly through activation of matrix metalloproteinases. Excessive plasmin activity and subsequent ECM degradation cause hepatic sinusoidal fragility and hemorrhage in developing embryos. We report here that excessive plasmin activity in a murine acetaminophen (APAP) overdose model likewise compromises hepatic sinusoidal vascular integrity in adult animals. We found that hepatic plasmin activity is up-regulated significantly at 6 hours after APAP overdose. This plasmin up-regulation precedes both degradation of the ECM component fibronectin around liver vasculature and bleeding from centrilobular sinusoids. Importantly, administration of the pharmacological plasmin inhibitor tranexamic acid or genetic reduction of plasminogen, the circulating zymogen of plasmin, ameliorates APAP-induced hepatic fibronectin degradation and sinusoidal bleeding. Conclusion: These studies demonstrate that reduction of plasmin stabilizes hepatic sinusoidal vascular integrity after APAP overdose. (Hepatology 2018; 00:1-13).

Endothelial dysfunction and angiogenesis impairment in the ageing vasculature.

Ageing is the main risk factor for the development of cardiovascular diseases. A central mechanism by which ageing promotes vascular pathologies is compromising endothelial health. The age-related attenuation of endothelium-dependent dilator responses (endothelial dysfunction) associated with impairment of angiogenic processes and the subsequent pathological remodelling of the microcirculation contribute to compromised tissue perfusion and exacerbate functional decline in older individuals. This Review focuses on cellular, molecular, and functional changes that occur in the endothelium during ageing. We explore the links between oxidative and nitrative stress and the conserved molecular pathways affecting endothelial dysfunction and impaired angiogenesis during ageing. We also speculate on how these pathological processes could be therapeutically targeted. An improved understanding of endothelial biology in older patients is crucial for all cardiologists because maintenance of a competently functioning endothelium is critical for adequate tissue perfusion and long-term cardiac health.