Impact of enhanced recovery program implementation on postoperative outcomes after liver surgery: a monocentric retrospective study.

INTRODUCTION: It is still unclear whether enhanced recovery programs (ERPs) reduce postoperative morbidity after liver surgery. This study investigated the effect on liver surgery outcomes of labeling as a reference center for ERP. MATERIALS AND METHODS: Perioperative data from 75 consecutive patients who underwent hepatectomy in our institution after implementation and labeling of our ERP were retrospectively compared to 75 patients managed before ERP. Length of hospital stay, postoperative complications, and adherence to protocol were examined. RESULTS: Patient demographics, comorbidities, and intraoperative data were similar in the two groups. Our ERP resulted in shorter length of stay (3 days [1-6] vs. 4 days [2-7.5], p = 0.03) and fewer postoperative complications (24% vs. 45.3%, p = 0.0067). This reduction in postoperative morbidity can be attributed exclusively to a lower rate of minor complications (Clavien-dindo grade < IIIa), and in particular to a lower rate of postoperative ileus, after labeling. (5.3% vs. 25.3%, p = 0.0019). Other medical and surgical complications were not significantly reduced. Adherence to protocol improved after labeling (17 [16-18] vs. 14 [13-16] items, p < 0.001). CONCLUSIONS: The application of a labeled enhanced recovery program for liver surgery was associated with a significant shortening of hospital stay and a halving of postoperative morbidity, mainly ileus.

Impact of preoperative treatment with antidepressants and/or anxiolytics on outcomes after colorectal surgery with an enhanced recovery programme: a monocentric retrospective study.

BACKGROUND: Preoperative use of antidepressants and anxiolytics was reported to increase length of hospital stay (LOS) and worsen surgical outcomes. However, the surgical procedures studied were seldom performed with an enhanced recovery programme (ERP). This study investigated whether these medications impaired postoperative recovery after colorectal surgery with an ERP. METHODS: The data of all patients scheduled for colorectal surgery between November 2015 and December 2019 prospectively included in our database were analysed. All the patients were managed with the same ERP. Demographic data, risk factors, incidence of postoperative complications, LOS, and adherence to the ERP were compared between patients with and without preoperative antidepressant and/or anxiolytic treatment. RESULTS: Of the 502 patients, 157 (31.3%) were treated with antidepressants and/or anxiolytics. They were older (65.7 vs. 59.5 years, p < 0.001), sicker (higher ASA physical status score, p = 0.001), and underwent surgery more frequently for cancer (73.9 vs. 56.8%, p < 0.001). Overall adherence to ERP (p = 0.99) and adherence to the postoperative items of ERP (p = 0.29), incidence of postoperative complications (35.7 vs. 33.2%, p = 0.61), and LOS (4 [2-7] vs. 4 [2-7], p = 0.99) were similar in the two groups. CONCLUSIONS: Our findings suggest that preoperative treatment with antidepressants and/or anxiolytics does not worsen outcome after elective colorectal surgery with an ERP, does not impact adherence to ERP, and does not prolong LOS. ERP seems efficacious in patients treated with these medications, who should therefore not be excluded from this programme.

Lymphocytic microparticles suppress retinal angiogenesis via targeting Müller cells in the ischemic retinopathy mouse model.

Retinopathy of prematurity (ROP) is the primary cause of visual impairment and vision loss in premature infants, which results from the formation of aberrant retinal neovascularization (NV). An emerging body of evidence has shown that Müller cells are the predominant source of vascular endothelial growth factor (VEGF), which also serves as a chemoattractant for monocyte/macrophage lineage. The recruitment of macrophages is increased during retinal NV, and they exert a pro-angiogenic role in ROP. We have shown that lymphocytic microparticles (microvesicles; LMPs) derived from apoptotic human T lymphocytes possess strong angiogenesis-inhibiting properties. Here, we investigated the effect of LMPs on the chemotactic capacity of Müller cells in vitro using rat Müller cell rMC-1 and mouse macrophage RAW 264.7. In addition, the impact of LMPs was determined in vivo using a mouse model of oxygen-induced ischemic retinopathy (OIR). The results revealed that LMPs were internalized by rMC-1 and reduced their cell proliferation dose-dependently without inducing cell apoptosis. LMPs inhibited the chemotactic capacity of rMC-1 on RAW 264.7 via reducing the expression of VEGF. Moreover, LMPs attenuated pathological retinal NV and the infiltration of macrophages in vivo. LMPs downregulated ERK1/2 and HIF-1α both in vitro and in vivo. These findings expand our understanding of the effects of LMPs, providing evidence of LMPs as a promising therapeutic approach for the treatment of retinal NV diseases.

Emerging roles of microRNAs and their implications in uveal melanoma.

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults with an extremely high mortality rate. Genetic and epigenetic dysregulation contribute to the development of UM. Recent discoveries have revealed dysregulation of the expression levels of microRNAs (miRNAs) as one of the epigenetic mechanisms underlying UM tumorigenesis. Based on their roles, miRNAs are characterized as either oncogenic or tumor suppressive. This review focuses on the roles of miRNAs in UM tumorigenesis, diagnosis, and prognosis, as well as their therapeutic potentials. Particularly, the actions of collective miRNAs are summarized with respect to their involvement in major, aberrant signaling pathways that are implicated in the development and progression of UM. Elucidation of the underlying functional mechanisms and biological aspects of miRNA dysregulation in UM is invaluable in the development of miRNA-based therapeutics, which may be used in combination with conventional treatments to improve therapeutic outcomes. In addition, the expression levels of some miRNAs are correlated with UM initiation and progression and, therefore, may be used as biomarkers for diagnosis and prognosis.

The impact of orthostatic intolerance on early ambulation following abdominal surgery in an enhanced recovery programme.

BACKGROUND: The prevalence of orthostatic intolerance on the day of surgery is more than 50% after abdominal surgery. The impact of orthostatic intolerance on ambulation on the day of surgery has been little studied. We investigated orthostatic intolerance and walking ability after colorectal and bariatric surgery in an enhanced recovery programme. METHODS: Eighty-two patients (colorectal: n = 46, bariatric n = 36) were included and analysed in this prospective study. Walk tests for 2 min (2-MWT) and 6 min (6-MWT) were performed before and 24 h after surgery, and 3 h after surgery for 2-MWT. Orthostatic intolerance characterised by presyncopal symptoms when rising was recorded at the same time points. Multivariate binary logistic regressions modelling the probability of orthostatic intolerance and walking inability were performed taking into account potential risk factors. RESULTS: Prevalence of orthostatic intolerance and walking inability was, respectively, 65% and 18% 3-hour after surgery. The day after surgery, patients' performance had greatly improved: approximately 20% of the patients experienced orthostatic intolerance, whilst only 5% of the patients were unable to walk. Adjusted binary logistic regressions demonstrated that age (p = .37), sex (p = .39), BMI (p = .74), duration of anaesthesia (p = .71) and type of surgery (p = .71) did not significantly influence walking ability. CONCLUSION: Our study confirms that orthostatic intolerance was frequent (~ 60%) 3-hour after abdominal surgery but prevented a 2-MWT only in ~20% of patients. No risk factors for orthostatic intolerance and walking inability were evidenced.

Comparison of outcome after right colectomy with an enhanced recovery programme in patients with inflammatory bowel disease and patients operated on for other conditions: a monocentric retrospective study.

PURPOSE: Enhanced recovery programmes (ERPs) after surgery reduce postoperative complications and hospital stay. Patients with inflammatory bowel disease (IBD) often present risk factors for postoperative complications. This accounts for reluctance to include them in ERPs. We compared outcome after right colectomy with an ERP in IBD and non-IBD patients. METHODS: In our GRACE colorectal surgery database comprising 508 patients, we analysed patients scheduled for right colectomy (n = 160). Adherence to the protocol, postoperative complications and length of hospital stay of IBD patients (n = 45) were compared with those of non-IBD patients (n = 115). Data (mean ± SD, median [IQR], count (%)) were compared by Student's t, Mann-Whitney U and chi-square tests when appropriate; p < 0.05 taken as statistically significant. RESULTS: IBD patients were significantly younger (38.9 ± 13.8 vs. 58.9 ± 18.5 years, p < 0.001) and had lower BMI (23.0 ± 5.0 vs. 25.1 ± 5.0 kg m-2, p < 0.01). Adherence to ERP was similar in the two groups. Resumption of eating on the day of the operation was less well tolerated (73.3% vs. 85.2%, p < 0.05) and postoperative pain (p < 0.001) was greater in IBD patients. The incidence of postoperative complications (13.3% vs. 17.3%) and the length of hospital stay (3 [3-4.5] vs. 3 [2-5] days) were comparable in IBD and non-IBD patients, respectively. CONCLUSION: The management of IBD patients in an ERP is not only feasible but also indicated. These patients benefit as much from ERP as non-IBD patients.

Activation of NLRP3 inflammasome by lymphocytic microparticles via TLR4 pathway contributes to airway inflammation.

The presence of elevated T lymphocytic microparticles (TLMPs) during respiratory illness is associated with airway and lung inflammation and epithelial injuries. Although inflammasome and IL-1ß signaling are crucial in airway inflammation, little was known about their regulatory mechanism. We hypothesized that TLMPs trigger inflammasome activation and IL-1ß production in bronchial and alveolar epithelial cells to induce airway and lung inflammation. In this study, TLMPs induced IL-1ß and IL-18 secretion through NLRP3 inflammasome activation and upregulated TLR4 mRNA and protein expression in alveolar (A549) and human airway epithelial (16HBE) cells. Pretreatment with CLI-095, a specific inhibitor of TLR4 signaling, dramatically diminished the TLMP-induced release of IL-1ß and IL-18 by inhibiting the formation of NLRP3/ASC/pro-caspase-1 inflammasome in a dose-dependent manner. The TLMP-induced autophagy inhibition in epithelial cells was dependent on the PI3K/Akt signaling pathway, which significantly increased NLRP3 expression and enhanced TLMP-induced inflammation. TLR4, IL-1ß, and IL-18 proteins harbored in TLMPs were nonessential for the pro-inflammatory effect. In conclusion, TLMPs induce bronchial and alveolar epithelial cell secretion of IL-1ß and IL-18 cytokines by activating the TLR4 and PI3K/Akt signaling pathways and inhibiting autophagy. These effects lead to NLRP3 inflammasome formation and accumulation. TLMPs may be regarded as deleterious markers of airway and lung damage in respiratory diseases.

Extracellular microparticles exacerbate oxidative damage to retinal pigment epithelial cells.

Oxidative stress-induced retinal pigment epithelial cell (RPE) dysfunction is a primary contributing factor to early dry age-related macular degeneration (AMD). Oxidative injury to the retina may promote extracellular vesicles (EVs) released from RPE. In this study, we investigated the effects of oxidative-induced RPE cell-derived microparticles (RMPs) on RPE cell functions. The oxidative stress induced more RMPs released from RPE cells in vitro and in vivo, and significant more RMPs were released from aged RPE cells than that from younger RPE cells. RMPs were taken up by RPE cells in a time-dependent manner; however, blockage of CD36 attenuated the uptake process. Furthermore, the decrease of RPE cell viability by RMPs treatment was associated with an increased expression of cyclin-dependent kinase inhibitors p15 and p21. RMPs enhanced senescence and interrupted phagocytic activity of RPE cells as well. The present study demonstrated that RMPs produce a strong effect of inducing RPE cell degeneration. This finding further supports the postulate that RMPs exacerbate oxidative stress damage to RPE cells, which may uncover a potentially relevant process in the genesis of dry AMD.

Impact of Preoperative Anemia on Outcomes of Enhanced Recovery Program After Colorectal Surgery: A Monocentric Retrospective Study.

BACKGROUND: Anemia is common before major abdominal surgery (35%). It is an independent factor for postoperative complications and longer length of stay (LOS). The aim of this study was to evaluate the extent to which preoperative anemia impacts on enhanced recovery programs (ERP) outcomes. MATERIALS AND METHODS: The data for patients scheduled for colorectal surgery between 2015 and 2019, were analyzed (n = 494). All patients were managed with the same ERP. Demographic data, preoperative risk factors, postoperative complications, LOS and adherence to ERP were compared between anemic and non-anemic patients. Anemia was defined by a hemoglobin concentration < 13 g dL-1 in men and < 12 g dL-1 in women. RESULTS AND DISCUSSION: In total, 173 patients had preoperative anemia. They were older (p < 0.001) and more often male (p = 0.02). The following risk factors were significantly more frequent in the anemic group: renal failure (p = 0.04), malnutrition (p < 0.001), cardiac arrhythmia (p < 0.001), coronaropathy (p = 0.02) and anticoagulant treatment (p < 0.001). Despite more risk factors, anemic patients did not experience more postoperative complications (38.2% vs. 31.2%, p = 0.12). Overall adherence to ERP was similar (18 [16-19] vs. 18 [17-19], p = 0.06). LOS was 4 [3-7] and 3 [2-6.25] days in the anemic and the non-anemic groups, respectively (p < 0.002). Multivariate analysis showed that anemia did not affect LOS (p = 0.27). CONCLUSION: Our study suggests that preoperative anemia does not detract from the benefits of ERP after elective colorectal surgery.

The Dual Regulatory Role of MiR-181a in Breast Cancer.

MicroRNAs (miRNAs) are a family of highly conserved noncoding single-stranded RNA molecules of 21 to 25 nucleotides. miRNAs silence their cognate target genes at the post-transcriptional level and have been shown to have important roles in oncogenesis, invasion, and metastasis via epigenetic post-transcriptional gene regulation. Recent evidence indicates that the expression of miR-181a is altered in breast tumor tissue and in the serum of patients with breast cancer. However, there are several contradicting findings that challenge the biological significance of miR-181a in tumor development and metastasis. In fact, some studies have implicated miR-181a in regulating breast cancer gene expression. Here we summarize the current literature demonstrating established links between miR-181a and human breast cancer with a focus on recently identified mechanisms of action. This review also aims to explore the potential of miR-181a as a diagnostic and/or prognostic biomarker for breast cancer and to discuss the contradicting data regarding its targeting therapeutics and the associated challenges.

Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.

CXCR6-GFP(+) cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60-70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.

Retinal neurons curb inflammation and enhance revascularization in ischemic retinopathies via proteinase-activated receptor-2.

Ischemic retinopathies are characterized by sequential vaso-obliteration followed by abnormal intravitreal neovascularization predisposing patients to retinal detachment and blindness. Ischemic retinopathies are associated with robust inflammation that leads to generation of IL-1ß, which causes vascular degeneration and impairs retinal revascularization in part through the liberation of repulsive guidance cue semaphorin 3A (Sema3A). However, retinal revascularization begins as inflammation culminates in ischemic retinopathies. Because inflammation leads to activation of proteases involved in the formation of vasculature, we hypothesized that proteinase-activated receptor (Par)-2 (official name F2rl1) may modulate deleterious effects of IL-1ß. Par2, detected mostly in retinal ganglion cells, was up-regulated in oxygen-induced retinopathy. Surprisingly, oxygen-induced retinopathy-induced vaso-obliteration and neovascularization were unaltered in Par2 knockout mice, suggesting compensatory mechanisms. We therefore conditionally knocked down retinal Par2 with shRNA-Par2-encoded lentivirus. Par2 knockdown interfered with normal revascularization, resulting in pronounced intravitreal neovascularization; conversely, the Par2 agonist peptide (SLIGRL) accelerated normal revascularization. In vitro and in vivo exploration of mechanisms revealed that IL-1ß induced Par2 expression, which in turn down-regulated sequentially IL-1 receptor type I and Sema3A expression through Erk/Jnk-dependent processes. Collectively, our findings unveil an important mechanism by which IL-1ß regulates its own endothelial cytotoxic actions by augmenting neuronal Par2 expression to repress sequentially IL-1 receptor type I and Sema3A expression. Timely activation of Par2 may be a promising therapeutic avenue in ischemic retinopathies.

SYK is a target of lymphocyte-derived microparticles in the induction of apoptosis of human retinoblastoma cells.

Retinoblastoma (Rb) is an aggressive childhood cancer of the developing retina. This disease is associated with epigenetic deregulation of several cancer pathways including upregulation of the proto-oncogene spleen tyrosine kinase (SYK). We have previously demonstrated that lymphocyte-derived microparticles (LMPs) possess strong cytotoxic effect on cancer cells. This report demonstrated that LMPs have potent pro-apoptotic properties on human Rb cells, which was associated with a strong reduction of SYK expression. Perturbing SYK activity in Rb cells induced cell apoptosis and upregulated expression of p53 and p21. Interestingly, inhibition of p53 or knockdown of p21, abolished LMP-induced caspase-3 activity and cell death. Blocking oxidized phospholipid-rich LMPs with a specific antibody significantly prevented LMP-induced Rb apoptosis and reversed the expression patterns of SYK, p53, p21. In summary, our results suggest that LMPs are important pro-apoptotic regulators for Rb cells through reduction of SYK expression and upregulation of the p53-p21 pathway which ultimately activates caspase-3. These data may open unexpected avenues for the development of LMPs as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of Rb.

HrpA, an RNA helicase involved in RNA processing, is required for mouse infectivity and tick transmission of the Lyme disease spirochete.

The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 [p66], bba74, bb0241 [glpK], bb0242 and bb0243 [glpA]) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.

Lymphocyte-derived microparticles induce apoptosis of airway epithelial cells through activation of p38 MAPK and production of arachidonic acid.

The airway epithelium is critical for the normal integrity and function of the respiratory system. Excessive epithelial cell apoptosis contributes to cell damage and airway inflammation. We previously demonstrated that lymphocyte-derived microparticles (LMPs) induce apoptosis of human bronchial epithelial cells. However, the underlying mechanisms contributing to LMPs-evoked epithelial cell death are largely unknown. Here we used bronchial and lung tissue cultures to confirm the pro-apoptotic effects of LMPs. In cell culture experiments, we found that LMPs induced human airway epithelial cell apoptosis with associated increases in caspase-3 activity. In addition, LMPs treatment triggered oxidative stress in epithelial cells by enhancing production of malondialdehyde, superoxide, and reactive oxygen species (ROS), and by inhibiting production of the antioxidant glutathione. Moreover, decreasing cellular ROS with the antioxidant N-acetylcysteine rescued epithelial cell viability. Together, these results demonstrate an important role for oxidative stress in LMPs-induced cell death. In epithelial cells, LMPs treatment induced phosphorylation of p38 MAPK and arachidonic acid accumulation. Moreover, arachidonic acid was significantly cytotoxic towards LMPs-treated epithelial cells, whereas inhibition of p38 MAPK was protective against these cytotoxic effects. Similarly, inhibition of arachidonic acid production led to decreased caspase-3 activity, thus rescuing airway epithelial cells from LMPs-induced cell death. In conclusion, our results show that LMPs induce airway epithelial cell apoptosis by activating p38 MAPK signaling and stimulating production of arachidonic acid, with consequent increases in oxidative stress and caspase-3 activity. As such, LMPs may be regarded as deleterious markers of epithelial cell damage in respiratory diseases.

Fatty acid receptor Gpr40 mediates neuromicrovascular degeneration induced by transarachidonic acids in rodents.

OBJECTIVE: Nitro-oxidative stress exerts a significant role in the genesis of hypoxic-ischemic (HI) brain injury. We previously reported that the ω-6 long chain fatty acids, transarachidonic acids (TAAs), which are nitrative stress-induced nonenzymatically generated arachidonic acid derivatives, trigger selective microvascular endothelial cell death in neonatal neural tissue. The primary molecular target of TAAs remains unidentified. GPR40 is a G protein-coupled receptor activated by long chain fatty acids, including ω-6; it is highly expressed in brain, but its functions in this tissue are largely unknown. We hypothesized that TAAs play a significant role in neonatal HI-induced cerebral microvascular degeneration through GPR40 activation. APPROACH AND RESULTS: Within 24 hours of a HI insult to postnatal day 7 rat pups, a cerebral infarct and a 40% decrease in cerebrovascular density was observed. These effects were associated with an increase in nitrative stress markers (3-nitrotyrosine immunoreactivity and TAA levels) and were reduced by treatment with nitric oxide synthase inhibitor. GPR40 was expressed in rat pup brain microvasculature. In vitro, in GPR40-expressing human embryonic kidney (HEK)-293 cells, [(14)C]-14E-AA (radiolabeled TAA) bound specifically, and TAA induced calcium transients, extracellular signal-regulated kinase 1/2 phosphorylation, and proapoptotic thrombospondin-1 expression. In vivo, intracerebroventricular injection of TAAs triggered thrombospondin-1 expression and cerebral microvascular degeneration in wild-type mice, but not in GPR40-null congeners. Additionally, HI-induced neurovascular degeneration and cerebral infarct were decreased in GPR40-null mice. CONCLUSIONS: GPR40 emerges as the first identified G protein-coupled receptor conveying actions of nonenzymatically generated nitro-oxidative products, specifically TAAs, and is involved in (neonatal) HI encephalopathy.

Microglia and interleukin-1ß in ischemic retinopathy elicit microvascular degeneration through neuronal semaphorin-3A.

OBJECTIVE: Proinflammatory cytokines contribute to the development of retinal vasculopathies. However, the role of these factors and the mechanisms by which they elicit their effects in retina are not known. We investigated whether activated microglia during early stages of ischemic retinopathy produces excessive interleukin-1ß (IL-1ß), which elicits retinal microvascular degeneration not directly but rather by triggering the release of the proapoptotic/repulsive factor semaphorin-3A (Sema3A) from neurons. APPROACH AND RESULTS: Sprague Dawley rats subjected to retinopathy induced by hyperoxia (80% O2; O2-induced retinopathy) exhibited retinal vaso-obliteration associated with microglial activation, NLRP3 upregulation, and IL-1ß and Sema3A release; IL-1ß was mostly generated by microglia. Intraperitoneal administration of IL-1 receptor antagonists (Kineret, or rytvela [101.10]) decreased these effects and enhanced retinal revascularization; knockdown of Sema3A resulted in microvessel preservation and, conversely, administration of IL-1ß caused vaso-obliteration. In vitro, IL-1ß derived from activated primary microglial cells, cultured under hyperoxia, stimulated the release of Sema3A in retinal ganglion cells-5, which in turn induced apoptosis of microvascular endothelium; antagonism of IL-1 receptor decreased microglial activation and on retinal ganglion cells-5 abolished the release of Sema3A inhibiting ensuing endothelial cell apoptosis. IL-1ß was not directly cytotoxic to endothelial cells. CONCLUSIONS: Our findings suggest that in the early stages of O2-induced retinopathy, retinal microglia are activated to produce IL-1ß, which sustains the activation of microglia and induces microvascular injury through the release of Sema3A from adjacent neurons. Interference with IL-1 receptor or Sema3A actions preserves the microvascular bed in ischemic retinopathies and, consequently, decreases ensued pathological preretinal neovascularization.

The microRNA Let-7f Induces Senescence and Exacerbates Oxidative Stress in Retinal Pigment Epithelial Cells.

This study aims to investigate the role of microRNA let-7f in the dysfunction and degeneration of retinal pigment epithelium (RPE) cells through the induction of senescence and oxidative stress. Furthermore, we explore whether let-7f inhibition can protect these cells against sodium iodate (SI)-induced oxidative stress. Oxidative stress and let-7f expression are reciprocally regulated in retinal pigment epithelial cells. Overexpression of let-7f in ARPE-19 cells induced oxidative stress as demonstrated by increased reactive oxygen species (ROS) production as well as senescence. Inhibition of let-7f successfully protected RPE cells from the detrimental effects induced by SI. In addition, let-7f overexpression induced RPE cellular dysfunction by diminishing their migratory capabilities and reducing the phagocytosis of porcine photoreceptor outer segments (POS). Results were further confirmed in vivo by intravitreal injections of SI and let-7f antagomir in C57BL/6 mice. Our results provide strong evidence that let-7f is implicated in the dysfunction of RPE cells through the induction of senescence and oxidative injury. These findings may help to uncover novel and relevant processes in the pathogenesis of dry AMD.

Modulating the Nature of Ionizable Lipids and Number of Layers in Hyaluronan-Decorated Lipid Nanoparticles for In Vitro Delivery of RNAi.

Lipid nanoparticles (LNPs) have established their position as nonviral vectors for gene therapy. Tremendous efforts have been made to modulate the properties of LNPs to unleash their full clinical potential. Among the strategies being pursued, the layer-by-layer (LbL) technique has gained considerable attention in the biomedical field. Illuminated by our previous work, here we investigate if the LbL approach could be used to modify the LNP cores formulated with three different ionizable lipids: DODMA, MC3, and DODAP. Additionally, we wondered if more than three layers could be loaded onto LNPs without disrupting their gene transfection ability. Taking advantage of physicochemical analysis, as well as uptake and gene silencing studies, we demonstrate the feasibility of modifying the surface of LNPs with the LbL assembly. Precisely, we successfully modified three different LNPs using the layer-by-layer strategy which abrogated luciferase activity in vitro. Additionally, we constructed a 5×-layered HA-LNP containing the MC3 ionizable lipid which outperformed the 3×-layered counterpart in transfecting miRNA-181-5p to the pediatric GBM cell line, as a proof-of-concept in vitro experiment. The method used herein has been proven reproducible, of easy modification to adapt to different ionizable lipid-containing LNPs, and holds great potential for the translation of RNA-based therapeutic strategies.