Bridging Smart Nanosystems with Clinically Relevant Models and Advanced Imaging for Precision Drug Delivery.

Intracellular delivery of nano-drug-carriers (NDC) to specific cells, diseased regions, or solid tumors has entered the era of precision medicine that requires systematic knowledge of nano-biological interactions from multidisciplinary perspectives. To this end, this review first provides an overview of membrane-disruption methods such as electroporation, sonoporation, photoporation, microfluidic delivery, and microinjection with the merits of high-throughput and enhanced efficiency for in vitro NDC delivery. The impact of NDC characteristics including particle size, shape, charge, hydrophobicity, and elasticity on cellular uptake are elaborated and several types of NDC systems aiming for hierarchical targeting and delivery in vivo are reviewed. Emerging in vitro or ex vivo human/animal-derived pathophysiological models are further explored and highly recommended for use in NDC studies since they might mimic in vivo delivery features and fill the translational gaps from animals to humans. The exploration of modern microscopy techniques for precise nanoparticle (NP) tracking at the cellular, organ, and organismal levels informs the tailored development of NDCs for in vivo application and clinical translation. Overall, the review integrates the latest insights into smart nanosystem engineering, physiological models, imaging-based validation tools, all directed towards enhancing the precise and efficient intracellular delivery of NDCs.

Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Real-World Sarilumab Use and Rule Testing to Predict Treatment Response in Patients with Rheumatoid Arthritis: Findings from the RISE Registry.

INTRODUCTION: Clinical trial findings may not be generalizable to routine practice. This study evaluated sarilumab effectiveness in patients with rheumatoid arthritis (RA) and tested the real-world applicability of a response prediction rule, derived from trial data using machine learning (based on C-reactive protein [CRP] > 12.3 mg/l and seropositivity [anticyclic citrullinated peptide antibodies, ACPA +]). METHODS: Sarilumab initiators from the ACR-RISE Registry, with ≥ 1 prescription on/after its FDA approval (2017-2020), were divided into three cohorts based on progressively restrictive criteria: Cohort A (had active disease), Cohort B (met eligibility criteria of a phase 3 trial in RA patients with inadequate response/intolerance to tumor necrosis factor inhibitors [TNFi]), and Cohort C (characteristics matched to the phase 3 trial baseline). Mean changes in Clinical Disease Activity Index (CDAI) and Routine Assessment of Patient Index Data 3 (RAPID3) were evaluated at 6 and 12 months. In a separate cohort, predictive rule was tested based on CRP levels and seropositive status (ACPA and/or rheumatoid factor); patients were categorized into rule-positive (seropositive with CRP > 12.3 mg/l) and rule-negative groups to compare the odds of achieving CDAI low disease activity (LDA)/remission and minimal clinically important difference (MCID) over 24 weeks. RESULTS: Among sarilumab initiators (N = 2949), treatment effectiveness was noted across cohorts, with greater improvement noted for Cohort C at 6 and 12 months. Among the predictive rule cohort (N = 205), rule-positive (vs. rule-negative) patients were more likely to reach LDA (odds ratio: 1.5 [0.7, 3.2]) and MCID (1.1 [0.5, 2.4]). Sensitivity analyses (CRP > 5 mg/l) showed better response to sarilumab in rule-positive patients. CONCLUSIONS: In real-world setting, sarilumab demonstrated treatment effectiveness, with greater improvements in the most selective population, mirroring phase 3 TNFi-refractory and rule-positive RA patients. Seropositivity appeared a stronger driver for treatment response than CRP, although optimization of the rule in routine practice requires further data.

Rheumatoid arthritis (RA) is a condition that may cause joint damage, if untreated. Sarilumab is an advanced medication, approved for treating moderate-to-severe RA in patients not responding to initial standard medicines. Clinical trials have shown that sarilumab improves RA symptoms; however, some people do not respond. This is a common problem in RA treatment. Physicians measure proteins in people's blood (called biomarkers; e.g., anticyclic citrullinated peptide antibodies [ACPA], C-reactive protein [CRP], and rheumatoid factor [RF]) to predict a medicine's response. A previous study showed that people with positive blood tests for ACPA and CRP (> 12.3 mg/l) responded well to sarilumab; this study was based on machine learning (a branch of science using computers) and identified factors that could be linked to treatment benefits. The present study analyzed routine data of 2949 people from the ACR-RISE Registry and showed an improvement in RA symptoms after 6 and 12 months of sarilumab, with a greater improvement noted in patients previously treated with other medicines. Biomarkers were tested in 205 people to check whether they could predict treatment response in day-to-day life. People were called rule-positive if they tested positive for RF and/or ACPA with CRP > 12.3 mg/l, and otherwise rule-negative. After 24 weeks of treatment, rule-positive people had a greater chance of disease improvement than rule-negative people. These results showed the benefits of sarilumab in RA in routine care and suggested the usefulness of machine learning in identifying biomarkers that physicians can use to make treatment decisions.

Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.

The arginine methyltransferase PRMT7 promotes extravasation of monocytes resulting in tissue injury in COPD.

Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.

The voltage-gated potassium channel KV1.3 regulates neutrophil recruitment during inflammation.

AIMS: Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling. METHODS AND RESULTS: Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3. CONCLUSION: We show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.

Identification of a Rule to Predict Response to Sarilumab in Patients with Rheumatoid Arthritis Using Machine Learning and Clinical Trial Data.

INTRODUCTION: In rheumatoid arthritis, time spent using ineffective medications may lead to irreversible disease progression. Despite availability of targeted treatments, only a minority of patients achieve sustained remission, and little evidence exists to direct the choice of biologic disease-modifying antirheumatic drugs in individual patients. Machine learning was used to identify a rule to predict the response to sarilumab and discriminate between responses to sarilumab versus adalimumab, with a focus on clinically feasible blood biomarkers. METHODS: The decision tree model GUIDE was trained using a data subset from the sarilumab trial with the most biomarker data, MOBILITY, to identify a rule to predict disease activity after sarilumab 200 mg. The training set comprised 18 categorical and 24 continuous baseline variables; some data were omitted from training and used for validation by the algorithm (cross-validation). The rule was tested using full datasets from four trials (MOBILITY, MONARCH, TARGET, and ASCERTAIN), focusing on the recommended sarilumab dose of 200 mg. RESULTS: In the training set, the presence of anti-cyclic citrullinated peptide antibodies, combined with C-reactive protein > 12.3 mg/l, was identified as the "rule" that predicts American College of Rheumatology 20% response (ACR20) to sarilumab. In testing, the rule reliably predicted response to sarilumab in MOBILITY, MONARCH, and ASCERTAIN for many efficacy parameters (e.g., ACR70 and the 28-joint disease activity score using CRP [DAS28-CRP] remission). The rule applied less to TARGET, which recruited individuals refractory to tumor necrosis factor inhibitors. The potential clinical benefit of the rule was highlighted in a clinical scenario based on MONARCH data, which found that increased ACR70 rates could be achieved by treating either rule-positive patients with sarilumab or rule-negative patients with adalimumab. CONCLUSIONS: Well-established and clinically feasible blood biomarkers can guide individual treatment choice. Real-world validation of the rule identified in this post hoc analysis is merited. CLINICAL TRIAL REGISTRATION: NCT01061736, NCT02332590, NCT01709578, NCT01768572.

Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins.

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. KEY POINTS: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells.

A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions.

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic I n VI tro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 µm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o-) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.

Applications and Immunological Effects of Quantum Dots on Respiratory System.

Quantum dots (QDs), are one kind of nanoscale semiconductor crystals with specific electronic and optical properties, offering near-infrared mission and chemically active surfaces. Increasing interest for QDs exists in developing theranostics platforms for bioapplications such as imaging, drug delivery and therapy. Here we summarized QDs' biomedical applications, toxicity, and immunological effects on the respiratory system. Bioapplications of QDs in lung include biomedical imaging, drug delivery, bio-sensing or diagnosis and therapy. Generically, toxic effects of nanoparticles are related to the generation of oxidative stresses with subsequent DNA damage and decreased lung cells viability in vitro and in vivo because of release of toxic metal ions or the features of QDs like its surface charge. Lastly, pulmonary immunological effects of QDs mainly include proinflammatory cytokines release and recruiting innate leukocytes or adaptive T cells.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

The surface chemistry determines the spatio-temporal interaction dynamics of quantum dots in atherosclerotic lesions.

AIM: To optimize the design of nanoparticles for diagnosis or therapy of vascular diseases, it is mandatory to characterize the determinants of nano-bio interactions in vascular lesions. MATERIALS & METHODS: Using ex vivo and in vivo microscopy, we analyzed the interactive behavior of quantum dots with different surface functionalizations in atherosclerotic lesions of ApoE-deficient mice. RESULTS: We demonstrate that quantum dots with different surface functionalizations exhibit specific interactive behaviors with distinct molecular and cellular components of the injured vessel wall. Moreover, we show a role for fibrinogen in the regulation of the spatio-temporal interaction dynamics in atherosclerotic lesions. CONCLUSION: Our findings emphasize the relevance of surface chemistry-driven nano-bio interactions on the differential in vivo behavior of nanoparticles in diseased tissue.

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo.

Changes in intra- and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging.

Dexamethasone-conjugated DNA nanotubes as anti-inflammatory agents in vivo.

The biopolymer DNA allows to create nanoscale, biocompatible structures, which can be designed in a target-specific and stimuli-responsive manner. DNA carrier systems with these characteristics hold a great potential for nanomedical applications, such as for the treatment of inflammatory diseases. Here we used a DNA-based drug carrier system for the pH-dependent delivery of the glucocorticoid dexamethasone into macrophages, a cell type with a key role in the regulation of inflammation. Dexamethasone (Dex) nanotubes were internalized within minutes by MH-S macrophages in vitro and by tissue resident macrophages in the mouse cremaster muscle in vivo and localized in their endosomes. Treatment with Dex nanotubes in vitro significantly reduced the LPS-induced TNF secretion by macrophages, as compared to equivalent amounts of free dexamethasone without affecting cell viability. Microinjection of Dex nanotubes into postischemic muscle tissue of anesthetized mice resulted in a marked reduction of ischemia-reperfusion-elicited leukocyte transmigration and diminished vascular expression of the endothelial adhesion molecules VCAM-1 and ICAM-1. Taken together, our results demonstrate that DNA nanotubes can be used as a platform for the targeted delivery of glucocorticoids and could thus foster the development of nanomedical therapeutics with reduced off-target effects.

New View on Endothelial Cell Migration: Switching Modes of Migration Based on Matrix Composition.

OBJECTIVE: Cell-matrix interactions are crucial for regulating cellular activities, such as migration. This is of special importance for morphogenic processes, such as angiogenesis (the development of new blood vessels). Most of our understanding of cell migration relies on 2-dimensional (2D) experiments. However, the awareness that 3D settings might elicit different results has increased. Knowledge about endothelial cell (EC) behavior in 3D environments and the influence of matrix composition on EC migration, in particular, is still limited. APPROACH AND RESULTS: We characterize the migration of single ECs through 2 structurally different hydrogels: spongy Matrigel and fibrillar collagen I. Our observations reveal an elongated migration phenotype in Matrigel and a rounded phenotype with pronounced cell blebs (blebs >2 µm) in collagen I, which have not previously been described in ECs. Directed migration seems to depend on Rac1 and Cdc42 in collagen, but not in Matrigel (shown using appropriate pharmacological inhibitors). By applying anti-integrin antibodies and supplementing laminin in collagen gels, we identify laminin as the main determinant of the elongated phenotype. Laminin seems to induce a morphological switch between modes of migration. As an in situ proof of principle, we performed live imaging of EC migration during vascular growth in a murine retina in the absence and presence of anti-integrin antibodies. CONCLUSIONS: We show that, surprisingly, ECs can evade the pharmacological inhibition of central signaling pathways involved in migration (contractility, small GTPases, and proteolysis) by shifting gears between modes of migration. This finding indicates an unexpected contextual plasticity of EC behavior.

Perivascular Mast Cells Govern Shear Stress-Induced Arteriogenesis by Orchestrating Leukocyte Function.

The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit(+)/CXCR-4(+) cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.