Variation in Carotid Artery Stenosis Measurements Among Facilities Seeking Carotid Stenting Facility Accreditation.

BACKGROUND: Based on the inclusion criteria of clinical trials, the degree of cervical carotid artery stenosis is often used as an indication for stent placement in the setting of extracranial carotid atherosclerotic disease. However, the rigor and consistency with which stenosis is measured outside of clinical trials are unclear. In an agreement study using a cross-sectional sample, we compared the percent stenosis as measured by real-world physician operators to that measured by independent expert reviewers. METHODS: As part of the carotid stenting facility accreditation review, images were obtained from 68 cases of patients who underwent carotid stent placement. Data collected included demographics, stroke severity measures, and the documented degree of stenosis, termed operator-reported stenosis (ORS), by 34 operators from 14 clinical sites. The ORS was compared with reviewer-measured stenosis (RMS) as assessed by 5 clinicians experienced in treating carotid artery disease. RESULTS: The median ORS was 90.0% (interquartile range, 80.0%-90.0%) versus a median RMS of 61.1% (interquartile range, 49.8%-73.6%), with a median difference of 21.8% (interquartile range, 13.7%-34.4%), P<0.001. The median difference in ORS and RMS for asymptomatic versus symptomatic patients was not statistically different (24.6% versus 19.6%; P=0.406). The median difference between ORS and RMS for facilities granted initial accreditation was smaller compared with facilities whose accreditation was delayed (17.9% versus 25.5%, P=0.035). The intraclass correlation between ORS and RMS was 0.16, indicating poor agreement. If RMS measurements were used, 72% of symptomatic patients and 10% of asymptomatic patients in the population examined would meet the Centers for Medicare and Medicaid Services criteria for stent placement. CONCLUSIONS: Real-world operators tend to overestimate carotid artery stenosis compared with external expert reviewers. Measurements from facilities granted initial accreditation were closer to expert measurements than those from facilities whose accreditation was delayed. Since decisions regarding carotid revascularization are often based on percent stenosis, such measuring discrepancies likely lead to increased procedural utilization.

Common Mutation in the HFE Gene Modifies Recovery After Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is characterized by bleeding into the brain parenchyma. During an ICH, iron released from the breakdown of hemoglobin creates a cytotoxic environment in the brain through increased oxidative stress. Interestingly, the loss of iron homeostasis is associated with the pathological process of other neurological diseases. However, we have previously shown that the H63D mutation in the homeostatic iron regulatory (HFE) gene, prevalent in 28% of the White population in the United States, acts as a disease modifier by limiting oxidative stress. The following study aims to examine the effects of the murine homolog, H67D HFE, on ICH. METHODS: An autologous blood infusion model was utilized to create an ICH in the right striatum of H67D and wild-type mice. The motor recovery of each animal was assessed by rotarod. Neurodegeneration was measured using fluorojade-B and mitochondrial damage was assessed by immunofluorescent numbers of CytC+ (cytochrome C) neurons and CytC+ astrocytes. Finally, the molecular antioxidant response to ICH was quantified by measuring Nrf2 (nuclear factor-erythroid 2 related factor), GPX4 (glutathione peroxidase 4), and FTH1 (H-ferritin) levels in the ICH-affected and nonaffected hemispheres via immunoblotting. RESULTS: At 3 days post-ICH, H67D mice demonstrated enhanced performance on rotarod compared with wild-type animals despite no differences in lesion size. Additionally, H67D mice displayed higher levels of Nrf2, GPX4, and FTH1 in the ICH-affected hemisphere; however, these levels were not different in the contralateral, non-ICH-affected hemisphere. Furthermore, H67D mice showed decreased degenerated neurons, CytC+ Neurons, and CytC+ astrocytes in the perihematomal area. CONCLUSIONS: Our data suggest that the H67D mutation induces a robust antioxidant response 3 days following ICH through Nrf2, GPX4, and FTH1 activation. This activation could explain the decrease in degenerated neurons, CytC+ neurons, and CytC+ astrocytes in the perihematomal region, leading to the improved motor recovery. Based on this study, further investigation into the mechanisms of this neuroprotective response and the effects of the H63D HFE mutation in a population of patients with ICH is warranted.

A Designed Host Defense Peptide for the Topical Treatment of MRSA-Infected Diabetic Wounds.

Diabetes mellitus is a chronic disease characterized by metabolic dysregulation which is frequently associated with diabetic foot ulcers that result from a severely compromised innate immune system. The high levels of blood glucose characteristic of diabetes cause an increase in circulating inflammatory mediators, which accelerate cellular senescence and dampen antimicrobial activity within dermal tissue. In diabetic wounds, bacteria and fungi proliferate in a protective biofilm forming a structure that a compromised host defense system cannot easily penetrate, often resulting in chronic infections that require antimicrobial intervention to promote the healing process. The designed host defense peptide (dHDP) RP557 is a synthesized peptide whose sequence has been derived from naturally occurring antimicrobial peptides (AMPs) that provide the first line of defense against invading pathogens. AMPs possess an amphipathic α-helix or ß-sheet structure and a net positive charge that enables them to incorporate into pathogen membranes and perturb the barrier function of Gram-positive and Gram-negative bacteria along with fungi. The capacity of skin to resist infections is largely dependent upon the activity of endogenous AMPs that provided the basis for the design and testing of RP557 for the resolution of wound infections. In the current study, the topical application of RP557 stopped bacterial growth in the biofilm of methicillin-resistant Staphylococcus aureus (MRSA) USA300 infected wounds on the flanks of clinically relevant diabetic TALLYHO mice. Topical application of RP557 reduced bacterial load and accelerated wound closure, while wound size in control diabetic mice continued to expand. These studies demonstrate that RP557 reduces or eliminates an infection in its biofilm and restores wound-healing capacity.

Hemorrhage and Recurrence of Obliterated Brain Arteriovenous Malformations Treated With Stereotactic Radiosurgery.

BACKGROUND: Although complete nidal obliteration of brain arteriovenous malformations (AVM) is generally presumed to represent durable cure, postobliteration hemorrhage, and AVM recurrence have become increasingly recognized phenomena. The goal of the study was to define hemorrhage and nidal recurrence risks of obliterated AVMs treated with stereotactic radiosurgery (SRS). METHODS: This is a retrospective cohort study from the International Radiosurgery Research Foundation comprising AVM patients treated between 1987 and 2020. Patients with AVM obliteration on digital subtraction angiography (DSA) were included. Outcomes were (1) hemorrhage and (2) AVM recurrence. Follow-up duration began at the time of AVM obliteration and was censored at subsequent hemorrhage, AVM recurrence, additional AVM treatment, or loss to follow-up. Annualized risk and survival analyses were performed. A sensitivity analysis comprising patients with AVM obliteration on magnetic resonance imaging or DSA was also performed for postobliteration hemorrhage. RESULTS: The study cohort comprised 1632 SRS-treated patients with AVM obliteration on DSA. Pediatric patients comprised 15% of the cohort, and 42% of AVMs were previously ruptured. The mean imaging follow-up after AVM obliteration was 22 months. Among 1607 patients with DSA-confirmed AVM obliteration, 16 hemorrhages (1.0%) occurred over 2223 patient-years of follow-up (0.72%/y). Of the 1543 patients with DSA-confirmed AVM obliteration, 5 AVM recurrences (0.32%) occurred over 2071 patient-years of follow-up (0.24%/y). Of the 16 patients with postobliteration hemorrhage, AVM recurrence was identified in 2 (12.5%). In the sensitivity analysis comprising 1939 patients with post-SRS AVM obliteration on magnetic resonance imaging or DSA, 16 hemorrhages (0.83%) occurred over 2560 patient-years of follow-up (0.63%/y). CONCLUSIONS: Intracranial hemorrhage and recurrent arteriovenous shunting after complete nidal obliteration are rare in AVM patients treated with SRS, and each phenomenon harbors an annual risk of <1%. Although routine postobliteration DSA cannot be recommended to SRS-treated AVM patients, long-term neuroimaging may be advisable in these patients.

An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes.

Intermediate monocytes (iMo; CD14+CD16+) increase in number in the circulation of patients with unstable coronary artery disease (CAD), and their recruitment to inflamed arteries is implicated in events leading to mortality following MI. Monocyte recruitment to inflamed coronary arteries is initiated by high affinity ß2-integrin (CD11c/CD18) that activates ß1-integrin (VLA-4) to bind endothelial VCAM-1. How integrin binding under shear stress mechanosignals a functional shift in iMo toward an inflammatory phenotype associated with CAD progression is unknown. Whole blood samples from patients treated for symptomatic CAD including non-ST elevation MI, along with healthy age-matched subjects, were collected to assess chemokine and integrin receptor levels on monocytes. Recruitment on inflamed human aortic endothelium or rVCAM-1 under fluid shear stress was assessed using a microfluidic-based artery on a chip (A-Chip). Membrane upregulation of high affinity CD11c correlated with concomitant activation of VLA-4 within focal adhesive contacts was required for arrest and diapedesis across inflamed arterial endothelium to a greater extent in non-ST elevation MI compared with stable CAD patients. The subsequent conversion of CD11c from a high to low affinity state under fluid shear activated phospho-Syk- and ADAM17-mediated proteolytic cleavage of CD16. This marked the conversion of iMo to an inflammatory phenotype associated with nuclear translocation of NF-κB and production of IL-1ß+ We conclude that CD11c functions as a mechanoregulator that activates an inflammatory state preferentially in a majority of iMo from cardiac patients but not healthy patients.

Clonal Vγ6+Vδ4+ T cells promote IL-17-mediated immunity against Staphylococcus aureus skin infection.

T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1ß, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.

Risk Factors Associated with ICU-Specific Care in Patients Undergoing Endovascular Treatment of Unruptured Intracranial Aneurysms.

BACKGROUND: Multiple studies suggest routine postoperative intensive care unit (ICUs) stays in presumed high-risk neurosurgical procedures may be unnecessary. Our objective was to evaluate the risk factors associated with ICU-specific needs in patients undergoing elective endovascular treatment of unruptured intracranial aneurysms. METHODS: A retrospective review of consecutive patients undergoing elective endovascular treatment of unruptured aneurysms was performed between January 2010 and January 2020 in a single academic medical center. Patient demographic information, aneurysm and treatment characteristics, intraoperative and postoperative complications, as well as ICU-specific needs, were abstracted. The primary outcome was ICU-specific needs. RESULTS: A total of 382 patient encounters in 344 unique patients were abstracted. 13.6% (52 of 382) of patient encounters had an ICU-specific need. Multivariate analysis revealed that age [adjusted odds ratio (OR) 1.04, 95% confidence interval (CI) 1.01-1.07, p = 0.03], procedure duration greater 200 min (adjusted OR 2.75, 95% CI 1.34-5.88, p = 0.007), and any intraoperative complication (adjusted OR 20.41, CI 7.97-56.57, p < 0.001) were independent predictors of postoperative ICU-specific needs. The majority of ICU-specific needs (94%, 49 of 52) occurred within 6 h of surgery. CONCLUSIONS: Our results show that age, procedure duration greater than or equal to 200 min, and intraoperative complication were independent predictors of postoperative ICU-specific needs in patients presenting for elective endovascular treatment of unruptured intracranial aneurysms. The majority of ICU-specific needs and associated complications occurred in the immediate postoperative period. This data can be used to help decide the appropriate postoperative level of care in this patient population.

Balloon-Assisted Roadmap Technique to Enable Flow Diversion of a High-Flow Direct Carotid-Cavernous Fistula.

BACKGROUND: The use of flow diverters as a first-line treatment for direct carotid cavernous fistula (CCF) is a relatively new approach in the neurointerventional field which allows obliteration of the fistula with less mass effect from coils in the cavernous sinus. Safe and successful deployment of a flow diverter requires adequate imaging of the parent vessel, which may be challenging in the setting of high-flow CCF without antegrade flow. OBJECTIVE: To facilitate adequate parent vessel imaging in the setting of high-flow CCF to enable the safe development of a flow diverter device. METHODS: Here we present the case of a patient with delayed presentation of post-traumatic direct CCF after a motor vehicle accident, with no antegrade flow past the fistulous connection. We used temporary balloon occlusion of the fistulous connection to enable road-map imaging of the parent vessel and flow-diverter placement. "Drag and drop" device opening in the middle cerebral artery facilitated better deployment of the flow-diverter against retrograde cavernous flow through the fistula. RESULTS: Temporary balloon occlusion of the fistulous connection was used to acquire a roadmap to facilitate safe deployment of a flow diverter and subsequent treatment of the CCF with transvenous coil embolization, with complete resolution of symptoms. CONCLUSION: Balloon-assisted roadmap use is a novel means of visualizing the parent vessel in direct CCF to facilitate safe flow diverter deployment.

Replacing Saturated Fat With Unsaturated Fat in Western Diet Reduces Foamy Monocytes and Atherosclerosis in Male Ldlr-/- Mice.

OBJECTIVE: A Mediterranean diet supplemented with olive oil and nuts prevents cardiovascular disease in clinical studies, but the underlying mechanisms are incompletely understood. We investigated whether the preventive effect of the diet could be due to inhibition of atherosclerosis and foamy monocyte formation in Ldlr-/- mice fed with a diet in which milkfat in a Western diet (WD) was replaced with extra-virgin olive oil and nuts (EVOND). Approach and Results: Ldlr-/- mice were fed EVOND or a Western diet for 3 (or 6) months. Compared with the Western diet, EVOND decreased triglyceride and cholesterol levels but increased unsaturated fatty acid concentrations in plasma. EVOND also lowered intracellular lipid accumulation in circulating monocytes, indicating less formation of foamy monocytes, compared with the Western diet. In addition, compared with the Western diet, EVOND reduced monocyte expression of inflammatory cytokines, CD36, and CD11c, with decreased monocyte uptake of oxLDL (oxidized LDL [low-density lipoprotein]) ex vivo and reduced CD11c+ foamy monocyte firm arrest on vascular cell adhesion molecule-1 and E-selectin-coated slides in an ex vivo shear flow assay. Along with these changes, EVOND compared with the Western diet reduced the number of CD11c+ macrophages in atherosclerotic lesions and lowered atherosclerotic lesion area of the whole aorta and aortic sinus. CONCLUSIONS: A diet enriched in extra-virgin olive oil and nuts, compared with a Western diet high in saturated fat, lowered plasma cholesterol and triglyceride levels, inhibited foamy monocyte formation, inflammation, and adhesion, and reduced atherosclerosis in Ldlr-/- mice.

IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium.

AIMS: Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. METHODS AND RESULTS: Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1-/- mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. CONCLUSIONS: The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

Endothelial up-regulation of VCAM-1 at susceptible sites in arteries modulates the recruitment efficiency of inflammatory monocytes that initiates atherosclerotic lesion formation. We reported that hydrodynamic shear stress (SS) mechanoregulates inflammation in human aortic endothelial cells through endoplasmic reticulum (ER) stress via activation of the transcription factor x-box binding protein 1 (XBP1). Here, a microfluidic flow channel that produces a linear gradient of SS along a continuous monolayer of endothelium was used to delve the mechanisms underlying transcriptional regulation of TNF-α-stimulated VCAM-1 expression. High-resolution immunofluorescence imaging enabled continuous detection of platelet endothelial cell adhesion molecule 1 (PECAM-1)-dependent, outside-in signaling as a function of SS magnitude. Differential expression of VCAM-1 and intercellular adhesion molecule 1 (ICAM-1) was regulated by the spatiotemporal activation of MAPKs, ER stress markers, and transcription factors, which was dependent on the mechanosensing of SS through PECAM-1 and PI3K. Inhibition of p38 specifically abrogated the rise to peak VCAM-1 at low SS (2 dyn/cm2), whereas inhibition of ERK1/2 attenuated peak ICAM-1 at high SS (12 dyn/cm2). A shear stress-regulated temporal rise in p38 phosphorylation activated the nuclear translocation of XBP1, which together with the transcription factor IFN regulatory factor 1, promoted maximum VCAM-1 expression. These data reveal a mechanism by which SS sensitizes the endothelium to a cytokine-induced ER stress response to spatially regulate inflammation promoting atherosclerosis.-Bailey, K. A., Moreno, E., Haj, F. G., Simon, S. I., Passerini, A. G. Mechanoregulation of p38 activity enhances endoplasmic reticulum stress-mediated inflammation by arterial endothelium.

Temporal augmentation with poly methyl methacrylate at the time of autologous cranioplasty.

Patients undergoing decompressive hemicraniectomy often have temporal wasting. We present a technique to manage temporalis wasting at the time of cranioplasty. Using poly methyl methacrylate, a small temporal implant is placed during cranioplasty. This is both a simple and cost effective technique for temporal augmentation at the time of cranioplasty.

Downregulation of GATA6 in mTOR-inhibited human aortic endothelial cells: effects on TNF-α-induced VCAM-1 expression and monocytic cell adhesion.

Increased expression of vascular cell adhesion molecule 1 (VCAM-1) on the aortic endothelium is an early marker of atherogenesis, promoted in part by elevated levels of inflammatory cytokines such as TNF-α. Mammalian target of rapamycin (mTOR) is a ubiquitous signaling molecule that has been considered to contribute to diverse cellular processes through mTOR complex 1 (mTORC1) or complex 2 (mTORC2). This study aimed to elucidate the role of mTOR signaling in TNF-α-induced VCAM-1 expression by the arterial endothelium. Primary human aortic endothelial cells (HAECs) were treated with low-dose (0.1 ng/ml) TNF-α, and VCAM-1 expression was measured by real-time quantitative PCR, Western blot analysis, and flow cytometry. Inhibition of mTOR through siRNA-mediated depletion or treatment with chemical inhibitors rapamycin or torin 1 suppressed VCAM1 transcription, which translated to inhibition of VCAM-1 surface expression by HAECs and concomitant decreased adhesion of monocytes. A promoter luciferase assay and chromatin immunoprecipitation indicated that mTOR regulated VCAM1 transcription through a mechanism involving transcription factor GATA6. Activation of PKC-α and an increase in miR-200a-3p expression, caused by mTOR inhibition but not disruption of mTORC1 or mTORC2 singly or together, decreased TNF-α-induced GATA6 expression and its enrichment at the VCAM1 promoter. In conclusion, mTOR inhibition activates PKC-α independently of disruption of mTORC1 and/or mTORC2, which challenges the conventional wisdom regarding mTOR signaling. Moreover, mTOR signals through transcriptional and posttranscriptional mechanisms to elicit maximal cytokine-induced endothelial inflammation that precedes atherosclerosis. NEW & NOTEWORTHY Both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 contribute to PKC-α activation in the human aortic endothelium. Inhibition of mTOR is not equivalent to disruption of mTORC1 and/or mTORC2 in affecting human aortic endothelial cell signaling. Specifically, inhibition of mTOR causes PKC-α activation and miR-200a-3p upregulation, which independently suppresses TNF-α-induced transcription factor GATA6 expression and subsequently inhibits VCAM-1 expression and monocytic cell adhesion onto the aortic endothelium.

Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow.

E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewisx (sLex), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity ß2-integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLex expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of ß2-integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLex, resulting in focal clusters that deliver a distinct signal to upshift ß2-integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended ß2-integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1ß secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1ß promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Gnb isoforms control a signaling pathway comprising Rac1, Plcß2, and Plcß3 leading to LFA-1 activation and neutrophil arrest in vivo.

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and ß subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C ß2 (Plcß2) and Plcß3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcß2-, Plcß3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcß2, and Plcß3 are critically involved in integrin activation and leukocyte arrest.

Calcium signalling and related ion channels in neutrophil recruitment and function.

The recruitment of neutrophils to sites of inflammation, their battle against invading microorganisms through phagocytosis and the release of antimicrobial agents is a highly coordinated and tightly regulated process that involves the interplay of many different receptors, ion channels and signalling pathways. Changes in intracellular calcium levels, caused by cytosolic Ca2+ store depletion and the influx of extracellular Ca2+ via ion channels, play a critical role in synchronizing neutrophil activation and function. In this review, we provide an overview of how Ca2+ signalling is initiated in neutrophils and how changes in intracellular Ca2+ levels modulate neutrophil function.

Multifactorial Experimental Design to Optimize the Anti-Inflammatory and Proangiogenic Potential of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E2 (PGE2 ) and vascular endothelial growth factor (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE2 and VEGF production versus those formed with 10,000 cells per spheroid in 21% oxygen with no inflammatory stimulus (Spheroid 2). Compared to Spheroid 2, Spheroid 1 produced fivefold more PGE2 and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these factors from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and keratinocyte migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells 2017;35:1493-1504.

You've got to be kindlin!

In this issue of Blood, Liao et al report that kindlin-2 is necessary for angiogenic sprouting in vitro and for developmental and tumor angiogenesis in vivo. The process of blood vessel sprouting is known to involve the αVß3 vitronectin integrin on endothelium. Kindlin-2 linkage to the C-terminal tail of ß3 completed the outside-in circuit necessary for integrin signaling that is critical for navigation of new vessel sprouts.

CD11c/CD18 Signals Very Late Antigen-4 Activation To Initiate Foamy Monocyte Recruitment during the Onset of Hypercholesterolemia.

Recruitment of foamy monocytes to inflamed endothelium expressing VCAM-1 contributes to the development of plaque during atherogenesis. Foamy CD11c(+) monocytes arise in the circulation during the onset of hypercholesterolemia and recruit to nascent plaque, but the mechanism of CD11c/CD18 and very late Ag-4 (VLA-4) activation and cooperation in shear-resistant cell arrest on VCAM-1 are ill defined. Within 1 wk of the onset of a Western high-fat diet (WD) in apolipoprotein E-deficient mice, an inflammatory subset of foamy monocytes emerged that made up one fourth of the circulating population. These cells expressed ∼3-fold more CD11c/CD18 and 50% higher chemokine receptors than nonfoamy monocytes. Recruitment from blood to a VCAM-1 substrate under shear stress was assessed ex vivo using a unique artery-on-a-chip microfluidic assay. It revealed that foamy monocytes from mice on a WD increased their adhesiveness over 5 wk, rising to twice that of mice on a normal diet or CD11c(-/-) mice fed a WD. Shear-resistant capture of foamy human or mouse monocytes was initiated by high-affinity CD11c, which directly activated VLA-4 adhesion via phosphorylated spleen tyrosine kinase and paxillin within focal adhesion complexes. Lipid uptake and activation of CD11c are early and critical events in signaling VLA-4 adhesive function on foamy monocytes competent to recruit to VCAM-1 on inflamed arterial endothelium.