ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

Raftlin is recruited by neuropilin-1 to the activated VEGFR2 complex to control proangiogenic signaling.

BACKGROUND: VEGFR2 (vascular endothelial growth factor receptor 2) is the major pro-angiogenic receptor in endothelial cells. Compared to other members of the receptor tyrosine kinase family, we know relatively few VEGFR2 signaling partners. Our objective was to use mass spectrometry-based proteomics to identify novel binding partners of activated VEGFR2. METHODS: We created an endothelial cell line stably expressing GFP-tagged VEGFR2 and isolated activated receptor complexes. Analysis by mass spectrometry identified raftlin as a novel binding partner of VEGFR2. RESULTS: We found that raftlin is recruited to the activated VEGFR2 complex via the co-receptor Nrp1 (neuropilin-1). We show that raftlin regulates the surface levels of Nrp1 in endothelial cells, controlling the availability of Nrp1 for VEGFR2 interaction. Raftlin stabilizes active VEGFR2 at the cell surface by inhibiting endocytosis of the activated receptor. Raftlin also promotes recycling of internalized VEGFR2 to the cell surface. Raftlin alters the signaling outcomes of VEGFR2 activation, inhibiting the activation of p38 and FAK (focal adhesion kinases) specifically. Both pathways are linked to cell migration in endothelial cells, and raftlin inhibits endothelial cell migration in response to VEGF. CONCLUSION: Nrp1 is an important co-receptor for VEGFR2; however, its functions are still only partially understood. We show that raftlin works with Nrp1 in endothelial cells to control intracellular trafficking of the activated VEGFR2. This modulates the response to VEGF and controls endothelial cell migration.

Characterization of the polarized endothelial secretome.

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.

The tumor suppressor RhoBTB1 controls Golgi integrity and breast cancer cell invasion through METTL7B.

BACKGROUND: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. METHODS: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RESULTS: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. CONCLUSION: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.

Platelet Rho GTPases-a focus on novel players, roles and relationships.

Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG-/- platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease.

RhoG protein regulates platelet granule secretion and thrombus formation in mice.

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

The formin FMNL3 is a cytoskeletal regulator of angiogenesis.

The process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.

The small GTPase Rif is an alternative trigger for the formation of actin stress fibers in epithelial cells.

Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation--the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.

The small Rho GTPase Rif and actin cytoskeletal remodelling.

The Rif GTPase is a recent addition to small Rho GTPase family; it shares low homology with other members in the family and evolutionarily parallels with the development of vertebrates. Rif has the conserved Rho GTPase domain structures and cycles between a GDP-bound inactive form and a GTP-bound active form. In its active form, Rif signals through multiple downstream effectors. In the present review, our aim is to summarize the current information about the Rif effectors and how Rif remodels actin cytoskeleton in many aspects.

Site recognition and substrate screens for PKN family proteins.

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr654 in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr654 is shown to be modulated by PKN in vivo.

srGAP2 deactivates RhoA to control the duration of thrombin-mediated endothelial permeability.

The endothelial barrier is a tightly regulated gateway in the transport of material between circulation and the tissues. Inflammatory mediators such as thrombin are able to open paracellular spaces in the endothelial monolayer to allow the extravasation of plasma proteins and leukocytes. Here we show that the protein SLIT-ROBO Rho GTPase-activating protein 2 (srGAP2) plays a critical role in regulating the extent of thrombin-mediated opening. We show that srGAP2 is not required for normal barrier function in resting endothelial cells, but that depletion of srGAP2 significantly increases the magnitude and duration of junctional opening in response to thrombin. We show that srGAP2 acts to switch off RhoA signaling after the contraction phase of thrombin-induced permeability, allowing respreading of cells and reformation of the barrier. srGAP2 is also required for effective restoration of the barrier after treatment with two other vasoactive agents that active RhoA - TNFα and angiotensin II. Taken together, we show that srGAP2 has a general function in controlling RhoA signaling in endothelial permeability, acting to limit the degree and duration of opening, by triggering the switch from endothelial cell contraction to respreading.

Cyclic-AMP Increases Nuclear Actin Monomer Which Promotes Proteasomal Degradation of RelA/p65 Leading to Anti-Inflammatory Effects.

The second messenger, cAMP has potent immunosuppressive and anti-inflammatory actions. These have been attributed, in part, to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappa B (NF-κB). However, the mechanisms underlying the modulation of NF-κB activity by cAMP remain unclear. Here we demonstrate an important role for cAMP-mediated increase in nuclear actin monomer levels in inhibiting NF-κB activity. Elevated cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-ActinR62D) inhibited basal and TNFα induced mRNA levels of NF-κB-dependent genes and NF-κB-dependent reporter gene activity. Elevated cAMP or NLS-ActinR62D did not affect NF-κB nuclear translocation but did reduce total cellular and nuclear RelA/p65 levels. Preventing the cAMP-induced increase in nuclear actin monomer, either by expressing a nuclear localised active mutant of the actin polymerising protein mDIA, silencing components of the nuclear actin import complex IPO9 and CFL1 or overexpressing the nuclear export complex XPO6, rescued RelA/p65 levels and NF-κB reporter gene activity in forskolin-stimulated cells. Elevated cAMP or NLS-ActinR62D reduced the half-life of RelA/p65, which was reversed by the proteasome inhibitor MG132. Accordingly, forskolin stimulated association of RelA/p65 with ubiquitin affinity beads, indicating increased ubiquitination of RelA/p65 or associated proteins. Taken together, our data demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP and highlight the important role played by nuclear actin in the regulation of inflammation.

VEGFR1 (Flt1) regulates Rab4 recycling to control fibronectin polymerization and endothelial vessel branching.

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear.Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports alphavbeta3 Integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and alphavbeta3 Integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.

The role of formins in filopodia formation.

Filopodia are highly dynamic cell-surface protrusions used by cells to sense their external environment. At the core of the filopodium is a bundle of actin filaments. These give form to the filopodia and also drive the cycle of elongation and retraction. Recent studies have shown that two very different actin nucleating proteins control the formation of filopodial actin filaments - Arp2/3 and Formins. Although the actin filaments produced by these two nucleators have very different structures and properties, recent work has begun to piece together evidence for co-operation between Arp2/3 and formins in filopodia formation, leading to a deeper understanding of these sensory organelles.

Uses of the in vitro endothelial-fibroblast organotypic co-culture assay in angiogenesis research.

Angiogenesis is a complex process that involves multiple cellular events. In addition to receiving inputs from a range of stimulatory and inhibitory factors, endothelial cells undergoing angiogenesis make multiple interactions with the extracellular matrix and with other cell types in the stroma. Recreating angiogenesis in vitro is probably an impossible goal; however, a number of assays have been developed that recapitulate many of the key events of the process. These assays are indispensible tools for investigating the signalling pathways that control the formation of new blood vessels. In the present paper, we review the organotypic co-culture assay of angiogenesis - until recently, a comparatively underemployed assay, but one with a number of powerful advantages for angiogenesis research. We give a set of optimized protocols for its use, including protocols for siRNA (small interfering RNA)-based screens, and we discuss appropriate methods for obtaining quantitative data from the assay.

A functional antagonism between RhoJ and Cdc42 regulates fibronectin remodelling during angiogenesis.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Angiogenesis requires endothelial cells to change shape and polarity, as well as acquire the ability to directionally migrate ‒ processes that are classically regulated by the Rho family of GTPases. RhoJ (previously TCL) is an endothelium enriched Rho GTPase with a 78% amino acid similarity to the ubiquitously expressed Cdc42. In our recent publication, we demonstrate that α5ß1 integrin co-traffics with RhoJ. RhoJ specifically represses the internalization of the active α5ß1 conformer, leading to a reduced ability of endothelial cells to form fibronectin fibrils. Surprisingly, this function of RhoJ is in opposition to the role of Cdc42, a known driver of fibrillogenesis. Intriguingly, we discovered that the competition for limiting amounts of the shared effector, PAK3, could explain the ability of these two Rho GTPases to regulate fibrillogenesis in opposing directions. Consequently, RhoJ null mice show excessive fibronectin deposition around retinal vessels, possibly due to the unopposed action of Cdc42. Our work suggests that the functional antagonism between RhoJ and Cdc42 could restrict fibronectin remodelling to sites of active angiogenesis to form a provisional matrix for vessel growth. One correlate of our findings is that RhoJ dependent repression of fibronectin remodelling could be atheroprotective in quiescent vessels.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.