INTEGRINS: A BEDSIDE TO BENCH TO BEDSIDE STORY.

I provide a narrative of the path I took to discover the membrane receptors that mediate leukocyte adhesion, now known as ß2 integrins or CD11/CD18. We followed this discovery with the first determination of the 3-D structures of integrins. The latter advance provided the foundation for understanding the unique features of integrins as divalent cation-dependent signaling receptors and as mechanosensitive conduits between the extracellular matrix and the intracellular cytoskeleton. Our structural studies are now opening new paths for taming overactive integrins in disease while minimizing the collateral damage associated with the faulty pharmacodynamics of current integrin inhibitory drugs.

Cryo-EM structures of full-length integrin αIIbß3 in native lipids.

Platelet integrin αIIbß3 is maintained in a bent inactive state (low affinity to physiologic ligand), but can rapidly switch to a ligand-competent (high-affinity) state in response to intracellular signals ("inside-out" activation). Once bound, ligands drive proadhesive "outside-in" signaling. Anti-αIIbß3 drugs like eptifibatide can engage the inactive integrin directly, inhibiting thrombosis but inadvertently impairing αIIbß3 hemostatic functions. Bidirectional αIIbß3 signaling is mediated by reorganization of the associated αIIb and ß3 transmembrane α-helices, but the underlying changes remain poorly defined absent the structure of the full-length receptor. We now report the cryo-EM structures of full-length αIIbß3 in its apo and eptifibatide-bound states in native cell-membrane nanoparticles at near-atomic resolution. The apo form adopts the bent inactive state but with separated transmembrane α-helices, and a fully accessible ligand-binding site that challenges the model that this site is occluded by the plasma membrane. Bound eptifibatide triggers dramatic conformational changes that may account for impaired hemostasis. These results advance our understanding of integrin structure and function and may guide development of safer inhibitors.

Association between benzodiazepine receptor agonist use and mortality in patients hospitalised for COVID-19: a multicentre observational study.

AIMS: To examine the association between benzodiazepine receptor agonist (BZRA) use and mortality in patients hospitalised for coronavirus disease 2019 (COVID-19). METHODS: A multicentre observational study was performed at Greater Paris University hospitals. The sample involved 14 381 patients hospitalised for COVID-19. A total of 686 (4.8%) inpatients received a BZRA at hospital admission at a mean daily diazepam-equivalent dose of 19.7 mg (standard deviation (s.d.) = 25.4). The study baseline was the date of admission, and the primary endpoint was death. We compared this endpoint between patients who received BZRAs and those who did not in time-to-event analyses adjusted for sociodemographic characteristics, medical comorbidities and other medications. The primary analysis was a Cox regression model with inverse probability weighting (IPW). RESULTS: Over a mean follow-up of 14.5 days (s.d. = 18.1), the primary endpoint occurred in 186 patients (27.1%) who received BZRAs and in 1134 patients (8.3%) who did not. There was a significant association between BZRA use and increased mortality both in the crude analysis (hazard ratio (HR) = 3.20; 95% confidence interval (CI) = 2.74-3.74; p < 0.01) and in the IPW analysis (HR = 1.61; 95% CI = 1.31-1.98, p < 0.01), with a significant dose-dependent relationship (HR = 1.55; 95% CI = 1.08-2.22; p = 0.02). This association remained significant in sensitivity analyses. Exploratory analyses indicate that most BZRAs may be associated with an increased mortality among patients hospitalised for COVID-19, except for diazepam, which may be associated with a reduced mortality compared with any other BZRA treatment. CONCLUSIONS: BZRA use may be associated with an increased mortality among patients hospitalised for COVID-19, suggesting the potential benefit of decreasing dose or tapering off gradually these medications when possible.

Conformational Dynamics in Extended RGD-Containing Peptides.

RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

Structure-guided design of pure orthosteric inhibitors of αIIbß3 that prevent thrombosis but preserve hemostasis.

A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbß3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbß3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as "pure" antagonists of αIIbß3, i.e., they no longer induce the conformational changes in αIIbß3. Both agents inhibit human platelet aggregation but preserve clot retraction. Hr10 and modified tirofiban are as effective as partial agonist drugs in inhibiting vascular thrombosis in humanized mice, but neither causes serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbß3 may thus provide safer alternatives for human therapy, and valuable tools to probe structure-activity relationships in integrins.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVß3 and α5ß1.

Targeting both integrins αVß3 and α5ß1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVß3 and α5ß1, whereas knottin 2.5D is αVß3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVß3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVß3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

High-Affinity Bent ß2-Integrin Molecules in Arresting Neutrophils Face Each Other through Binding to ICAMs In cis.

Leukocyte adhesion requires ß2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e., not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation.

Novel Pure αVß3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVß3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVß3-mediated cell adhesion to αVß3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVß3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

Major traumatic complications after out-of-hospital cardiac arrest: Insights from the Parisian registry.

AIM: Due to collapse and cardiopulmonary resuscitation (CPR) maneuvers, major traumatic injuries may complicate the course of resuscitation for out-of-hospital cardiac arrest patients (OHCA). Our goals were to assess the prevalence of these injuries, to describe their characteristics and to identify predictive factors. METHODS: We conducted an observational study over a 9-year period (2007-2015) in a French cardiac arrest (CA) center. All non-traumatic OHCA patients admitted alive in the ICU were studied. Major injuries identified were ranked using a functional two-level scale of severity (life-threatening or consequential) and were classified as CPR-related injuries or collapse-related injuries, depending of the predominant mechanism. Factors associated with occurrence of a CPR-related injury and ICU survival were identified using multivariable logistic regression. RESULTS: A major traumatic injury following OHCA was observed in 91/1310 patients (6.9%, 95%CI: 5.6, 8.3%), and was classified as a life-threatening injury in 36% of cases. The traumatic injury was considered as contributing to the death in 19 (21%) cases. Injuries were related to CPR maneuvers in 65 patients (5.0%, (95%CI: 3.8, 6.1%)). In multivariable analysis, age [OR 1.02; 95%CI (1.00, 1.04); p = 0.01], male gender [OR 0.53; 95%CI (0.31, 0.91); p = 0.02] and CA occurring at home [OR 0.54; 95%CI (0.31, 0.92); p = 0.02] were significantly associated with the occurrence of a CPR-related injury. CPR-related injuries were not associated with the ICU survival [OR 0.69; 95%CI (0.36, 1.33); p = 0.27]. CONCLUSIONS: Major traumatic injuries are common after cardiopulmonary resuscitation. Further studies are necessary to evaluate the interest of a systematic traumatic check-up in resuscitated OHCA patients in order to detect these injuries.

Diagnostic value of early postoperative MRI and diffusion-weighted imaging following trans-sphenoidal resection of non-functioning pituitary macroadenomas.

AIM: To establish the value of early contrast-enhanced magnetic resonance imaging (MRI) and diffusion-weighted imaging (DWI) in differentiating residual pituitary adenoma from postoperative surgical changes. MATERIALS AND METHODS: Thirty patients with non-functioning pituitary macroadenomas, who were undergoing trans-sphenoidal adenomectomy, were prospectively studied. Patients were imaged with both MRI and DWI in the early postoperative period, as well as 6-months post-surgery. Patterns of postoperative contrast enhancement were described (non-enhancement, peripheral enhancement, and nodular enhancement). Apparent diffusion coefficient (ADC) maps were utilised to select the region of interest (ROI) for ADC calculations. RESULTS: Seventeen patients had postoperative surgical granulation tissue and 13 had residual adenoma based on the 6 months follow-up imaging. Mean ADC values of postoperative granulation tissue and residual adenoma were 1.476±0.476×10-3 mm2/s and 0.855±0.190×10-3 mm2/s, respectively, in the early postoperative period, and 1.357±0.416×10-3 mm2/s and 0.829±0.201×10-3 mm2/s, respectively, at the 6-month follow-up. ADC values of granulation tissue were significantly different from that of residual adenoma at both time points (p<0.001). Sensitivity, specificity, positive and negative predictive values of early MRI were 84.6%, 94.1%, 91.7%, and 88.9% respectively, and of early DWI were 91%, 97%, 94.3%, and 93%, respectively. CONCLUSION: Early postoperative DWI after trans-sphenoidal resection of pituitary macroadenomas may be more helpful than early MRI in differentiating residual adenoma from post-surgical changes.

A microfluidic renal proximal tubule with active reabsorptive function.

In the kidney, the renal proximal tubule (PT) reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity.

Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys.

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans.

Biology and structure of leukocyte ß 2 integrins and their role in inflammation.

Integrins comprise a large family of αß heterodimeric cell adhesion receptors that are expressed on all cells except red blood cells and that play essential roles in the regulation of cell growth and function. The leukocyte integrins, which include members of the ß 1, ß 2, ß 3, and ß 7 integrin family, are critical for innate and adaptive immune responses but also can contribute to many inflammatory and autoimmune diseases when dysregulated. This review focuses on the ß 2 integrins, the principal integrins expressed on leukocytes. We review their discovery and role in host defense, the structural basis for their ligand recognition and activation, and their potential as therapeutic targets.

Hypoxic hepatitis after out-of-hospital cardiac arrest: Incidence, determinants and prognosis.

AIM: Hypoxic hepatitis (HH) may complicate the course of resuscitated out-of-hospital cardiac arrest (OHCA) patients admitted in intensive care unit (ICU). Aims of this study were to assess the prevalence of HH, and to describe the factors associated with HH occurrence and outcome. METHODS: We conducted an observational study over a 6-year period (2009-2014) in a cardiac arrest center. All non-traumatic OHCA patients admitted in the ICU after return of spontaneous circulation (ROSC) and who survived more than 24h were included. HH was defined as an elevation of alanine aminotransferase over 20 times the upper limit of normal during the first 72h after OHCA. Factors associated with HH and ICU mortality were picked up by multivariate logistic regression. RESULTS: Among the 632 OHCA patients included in the study, HH was observed in 72 patients (11.4% (95% CI: 9.0%, 14.1%)). In multivariate analysis, time from collapse to ROSC [OR 1.02 per additional minute; 95% CI (1.00, 1.04); p=0.01], male gender [OR 0.53; 95% CI (0.29, 0.95); p=0.03] and initial shockable rhythm [OR 0.35; 95% CI (0.19, 0.65); p<0.01] were associated with HH occurrence. After adjustment for confounding factors, HH was associated with ICU mortality [OR 4.39; 95% CI (1.71, 11.26); p<0.01] and this association persisted even if occurrence of a post-CA shock was considered in the statistical model [OR 3.63; 95% CI (1.39, 9.48); p=0.01]. CONCLUSIONS: HH is not a rare complication after OHCA. This complication is mainly triggered by the duration of resuscitation and is associated with increased ICU mortality.

Guidelines for the Use of Echocardiography in the Evaluation of a Cardiac Source of Embolism.

Embolism from the heart or the thoracic aorta often leads to clinically significant morbidity and mortality due to transient ischemic attack, stroke or occlusion of peripheral arteries. Transthoracic and transesophageal echocardiography are the key diagnostic modalities for evaluation, diagnosis, and management of stroke, systemic and pulmonary embolism. This document provides comprehensive American Society of Echocardiography guidelines on the use of echocardiography for evaluation of cardiac sources of embolism. It describes general mechanisms of stroke and systemic embolism; the specific role of cardiac and aortic sources in stroke, and systemic and pulmonary embolism; the role of echocardiography in evaluation, diagnosis, and management of cardiac and aortic sources of emboli including the incremental value of contrast and 3D echocardiography; and a brief description of alternative imaging techniques and their role in the evaluation of cardiac sources of emboli. Specific guidelines are provided for each category of embolic sources including the left atrium and left atrial appendage, left ventricle, heart valves, cardiac tumors, and thoracic aorta. In addition, there are recommendation regarding pulmonary embolism, and embolism related to cardiovascular surgery and percutaneous procedures. The guidelines also include a dedicated section on cardiac sources of embolism in pediatric populations.

A case of a 42-year-old patient with anomalous origin of the left main coronary artery from the pulmonary artery who delivered three times with no complications: presentation, diagnosis, and review.

An anomalous origin of the left coronary artery (LCA) from the pulmonary artery or Bland-White-Garland (BWG) syndrome is a rare congenital cardiac anomaly, which is unusual to survive to adulthood if left uncorrected. The authors report an unusual case of a multiparity patient who delivered several times without any complications presenting at the age of 42 years with recurrent dyspnea on exertion in which echocardiographic findings of diastolic flow near the origin of the pulmonary valve were suggestive of anomalous origin of the LCA. A CT angiography confirmed that the left main coronary artery arises from the undersurface of the pulmonary flow close :o its origin and gives rise to a left anterior descending (LAD) and left circumflex arteries.

The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish.

The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.

Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Talin (tln) binds and activates integrins to couple extracellular matrix-bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1(fl02k) mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin ß1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgß1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.