Biology subjects

Fleming, I.

Publications and source records attributed to Fleming, I..

At least 19 recordsLinked to original sources

bioRxiv2025

Pericyte contact alters endothelial cell metabolism by promoting exchange of lactate through SLC16A3

Intimate crosstalk between endothelial cells and pericytes is fundamental for vascular development and stability, but the metabolic dimension of this interaction remains poorly defined. Using a filter-based co-culture system that mimics the shared basement membrane of the microcirculation, we performed an integrated multi-omics analysis to investigate how direct contact reprograms both cell types. We found that initial contact does not induce immediate quiescence but rather triggers a transient, low-level activation of an endothelial-to-mesenchymal transition (EndMT)-like state in endothelial cells, characterized by specific upregulation of genes involved in extracellular matrix production (e.g., collagen isoforms) and PDGFR signaling, without a full loss of endothelial identity. Concomitantly, pericytes shifted endothelial cell metabolism, increasing glycolysis and elevating intracellular pyruvate and lactate. Proteomic analysis of the contact interface revealed enrichment of solute carriers, most notably the lactate transporter SLC16A3. Functional studies demonstrated that pericytes act as a glycolytic partner, actively shuttling lactate to endothelial cells via SLC16A3. This lactate did not fuel the TCA cycle but instead served as a signaling metabolite, driving widespread alterations in the endothelial acetylome and lactylome, particularly affecting proteins involved in glycolytic metabolism. In vitro, exogenous lactate potentiated cytokine-induced EndMT and upregulated lactylation-associated genes. The physiological relevance of this lactate shuttle was confirmed in vivo, where endothelial-specific deletion of Slc16a3 in mice impaired postnatal retinal angiogenesis, leading to reduced vessel density and diminished endothelial-pericyte overlap without affecting endothelial cell proliferation. Our findings establish that vessel maturation is orchestrated by a metabolically gated phase of plasticity initiated upon first contact, wherein a targeted EndMT-like program and a pericyte-driven lactate signaling axis are integrated to coordinate vascular network assembly.

physiology↗

bioRxiv2025

SMOC1 Regulates Endothelial-to-Mesenchymal Transition During Cardiac Repair After Myocardial Infarction

Endothelial-to-mesenchymal transition (EndMT) is a crucial, dual-phase process in cardiac repair after myocardial infarction (MI), driving both initial scar stabilization and subsequent pathological fibrosis. Therapeutic targeting requires precise temporal control rather than complete inhibition. This study identifies the matricellular protein SPARC-related modular calcium-binding protein 1 (SMOC1) as a novel regulator of EndMT. Analysis of single-cell RNA sequencing data from post-MI mouse hearts revealed that SMOC1 is highly enriched in a subpopulation of endothelial cells undergoing late EndMT. In vitro, SMOC1 expression was upregulated during cytokine-induced EndMT in human endothelial cells. Its siRNA-mediated knockdown exacerbated the EndMT phenotype, increasing mesenchymal marker expression and cell morphology changes, effects rescued by recombinant SMOC1 (rSMOC1). Mechanistically, SMOC1 deficiency enhanced TGF-{beta}2-induced SMAD2 phosphorylation, while rSMOC1 attenuated this pathway and promoted a shift from the short to the long, signaling-competent isoform of endoglin. In vivo, endothelial-specific SMOC1 deficiency (SMOC1{Delta}EC) in mice promoted age-associated EndMT and profoundly worsened post-MI outcomes. After MI, SMOC1{Delta}EC mice exhibited exacerbated cardiac dysfunction, ventricular dilation, pathological fibrosis, increased inflammatory cell infiltration, reduced survival, and a higher incidence of cardiac rupture compared to controls. Collectively, these findings establish SMOC1 as a critical endogenous modulator of EndMT that restrains its pathological progression. SMOC1 coordinates endothelial cell phenotype, in part by fine-tuning TGF-{beta}/endoglin signaling, and its loss accelerates maladaptive remodeling post-MI. Thus, SMOC1 represents a potential therapeutic target for spatially and temporally controlling EndMT to improve cardiac repair

pathology↗

bioRxiv2025

Soluble epoxide hydrolase upregulation in Alzheimer's disease promotes blood-brain barrier dysfunction

Recent advances in anti-amyloid therapies for Alzheimers disease have been promising, but they have also highlighted critical challenges, including increased vascular complications, such as amyloid-related imaging abnormalities. Emerging evidence suggests that the soluble epoxide hydrolase may be a promising therapeutic target due to the involvement of sEH-derived diols in inflammation, oxidative stress, and vascular destabilization. APPPS1 mice were crossed with an inducible soluble epoxide hydrolase knock-out mouse line. The knock-out was induced before onset of amyloid deposition, and then the mice were analyzed using histological, molecular, and RNA sequencing techniques. Here, we identify astrocytic soluble epoxide hydrolase as a key mediator of vascular instability in Alzheimers disease. Targeted astrocyte-specific deletion of soluble epoxide hydrolase in APPPS1 mice dramatically mitigated vascular changes, reducing the vascular amyloid burden by 67.95% and preserving VE-cadherin architecture. Importantly, vasomotion was markedly impaired in the Alzheimers disease model and was preserved in soluble epoxide hydrolase-deficient animals. Transcriptomic profiling of vasculature in APPPS1xsEH{Delta}AC mice revealed upregulated expression of genes critical for neurovascular protection. These findings identify soluble epoxide hydrolase as a central regulator of neurovascular dysfunction and underscore its therapeutic potential in increasing vascular stability in Alzheimers disease.

neuroscience↗

bioRxiv2025

A microprotein encoded by FERMT3 modulates endothelial cell protein catabolism and induces p53-mediated cell cycle arrest and senescence

BackgroundHuman endothelial cells express numerous microproteins (miPs) encoded by small open reading frames (smORFs) distributed throughout the genome, yet the biological functions of most remain unknown. This study set out to characterize a novel 69 amino acid miP encoded by a smORF located within the coding sequence of the FERM domain containing kindlin-3 transcript (miP-FERMT3). MethodsConfocal microscopy was used to determine the subcellular localization of miP-FERMT3 in endothelial cells and its interaction partners were determined by mass spectrometry and immunoblotting. RNA sequencing identified transcriptional alterations induced by miP-FERMT3 overexpression. Cell proliferation and cell cycle stages were assessed by live cell imaging, EdU incorporation and flow cytometry, while senescence was examined by senescence-associated {beta}-galactosidase staining, live cell imaging and RT-qPCR-based measurement of telomere length. ResultsIn endothelial cells miP-FERMT3 localized mainly to centriole subdistal appendages, where it interacted with proteins involved in ubiquitin- and proteasome-dependent protein catabolism, including PSMD9, CUL2 and TRIM8. Consistent with these interactions, cells expressing miP-FERMT3 exhibited increased global protein ubiquitination, enhanced centrosomal neddylation and elevated proteasomal activity. MiP-FERMT3 also promoted the nuclear accumulation of p53, which subsequently repressed FOXM1 expression, leading to the downregulation of genes required for cell-cycle progression and upregulation of genes involved in cell cycle inhibition, resulting in cell-cycle arrest. Cells expressing the miP also demonstrated multiple hallmarks of cellular senescence, including enlarged size, DNA damage, increased senescence-associated {beta}-galactosidase activity, telomere shortening and paracrine pro-inflammatory activation of naive endothelial cells. Analyses of independent murine and human transcriptomic and proteomic aging datasets further revealed that FERMT3 expression and protein abundance increase with age. ConclusionsThese findings identify miP-FERMT3 as a novel regulator of protein catabolism and p53-dependent cell cycle arrest and cellular senescence in endothelial cells. Given the aging-associated upregulation of FERMT3 in mouse and human endothelial cells, increased miP-FERMT3 expression may contribute to the onset of vascular senescence as a hallmark of aging.

cell biology↗

bioRxiv2025

Ferroptosis governs lymphatic vessel growth and regression

Whether, when, and how lymphatic vessels undergo cell death remains poorly understood. Here we identify ferroptosis as a physiological, cell-intrinsic regulator of the lymphatic endothelial cell survival during development and following injury, in stark contrast to the resilient organotypic blood endothelial cells. The lymphatic susceptibility to ferroptosis stems from tampered cystine/ hydropersulfide metabolism, alongside reduced glutathione availability triggered by an SH3RF3 E3 ligase mediated GPX4 degradation, and enhanced integration of polyunsaturated fatty acid enriched membrane phospholipids. Inducing ferroptosis genetically or pharmacologically elevated lymphatic lipid peroxidation, halted embryonic lymphangiogenesis and prevented post-injury lymphatic overgrowth while simultaneously shaped immune responses. Conversely, ferroptosis inhibition through saturated fatty acid supplementation led to pathological lymphatic hyperplasia. Targeting lymphatic ferroptotic mechanisms holds promise against pathological lymphatic growth in response to injury.

cell biology↗

bioRxiv2025

Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

BackgroundA large number of microproteins (miPs) encoded by small open reading frames (smORFs) are expressed in endothelial cells, yet their function remains largely unknown. In this study, we characterized a novel 46-amino-acid miP encoded by a smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript that was upregulated under inflammatory conditions and we refer to as miP-PSTPIP2. MethodsImmunoprecipitation coupled with mass spectrometry-based proteomics, immunoblotting, immunofluorescence and proximity ligation assays were used to identify and validate miP-PSTPIP2 interacting proteins in human endothelial cells. The impact of adenovirus-mediated overexpression of miP-PSTPIP2 on endocytosis, cytoskeleton dynamics and abundance of proteins involved in these processes was investigated by confocal microscopy and immunoblotting. Live cell imaging was used to assess endothelial cell migration and vascular permeability. ResultsmiP-PSTPIP2 physically associated with caveolar proteins, proteins involved in the regulation of cytoskeleton dynamics, intracellular transport, clathrin adaptor activity, as well as nuclear proteins. Human endothelial cells overexpressing miP-PSTPIP2 demonstrated enhanced endocytosis and transcytosis of transferrin as well as low-density lipoprotein. Mechanistically, miP-PSTPIP2 modulated Arp2/3-mediated actin nucleation and branching, which are required for dynamic cytoskeleton rearrangements. Moreover, altered cytoskeleton dynamics in miP-PSTPIP2-expressing endothelial cells resulted in impaired cell migration as well as increased permeability and monocyte trans-endothelial migration. ConclusionsmiP-PSTPIP2 is an inflammation-induced endothelial miP that regulates Arp2/3-dependent actin dynamics, thereby enhancing lipid uptake and leukocyte permeability. Its upregulation under inflammatory conditions suggests a contributory role in endothelial dysfunction and vascular inflammation.

cell biology↗

bioRxiv2025

Morphogen-guided neocortical organoids recapitulate regional areal identity and model neurodevelopmental disorder pathology

The human neocortex exhibits characteristic regional patterning (arealization) critical for higher-order cognitive function. Disrupted arealization is strongly implicated in neurodevelopmental disorders (NDDs), but current neocortical organoid models largely fail to recapitulate this patterning, limiting mechanistic understanding. Here, we establish a straightforward method for generating arealized organoids through short-term early exposure to anterior (FGF8) or posterior (BMP4/CHIR-99021) morphogens. These treatments created distinct anterior and posterior signaling centers, supporting long-lasting polarization, which we validated with single-cell RNA sequencing that revealed area-specific molecular signatures matching prenatal human cortex. To demonstrate the utility of this platform, we modeled Fragile X Syndrome (FXS) in organoids with distinct anterior and posterior regional identities. FXS organoids showed highly disrupted SOX4/SOX11 expression gradients along the anterior-posterior axis, consistent with alterations found in autism spectrum disorder (ASD) and demonstrate how regional patterning defects may contribute to NDD pathology. Together, our study provides a robust platform for generating neocortical organoids with anterior-posterior molecular signatures and highlights the importance of modeling NDDs using experimental platforms with neuroanatomic specificity.

developmental biology↗

bioRxiv2025

Immunomodulatory endothelial cells contribute to T cell recruitment and activation through antigen presentation on MHC class II

AimsA subset of endothelial cells referred to as immunomodulatory endothelial cells (IMEC) has been proposed to regulate T cell responses in atherosclerosis, but their phenotype and function remain poorly understood. Here, we characterized the inflammation-induced emergence of IMEC and their crosstalk with T cells. Methods and ResultsAn in vitro model to study IMEC was established and characterized using flow cytometry and proteomics. Single-cell transcriptome data from human atherosclerotic arteries as well as single cell transcriptome and endothelial cell-specific translatome data from a murine atherogenesis model were used to determine pathophysiological relevance. Immunopeptidomics was performed to detect antigen presentation. T cell chemotaxis, adhesion and activation were assessed through flow cytometry and microscopy. IMEC were induced by treating human endothelial cells with interleukin-1{beta}, interferon-{gamma}, and transforming growth factor-{beta}2. These cells expressed lower levels of classical endothelial cell markers but expressed major histocompatibility complex (MHC) class II, proteins involved in antigen processing and presentation (CD83, CD80 and CD86) and pro-inflammatory cytokines as well as chemokines, including CXCL9. An endothelial cell subpopulation with similar immunomodulatory features was identified in a mouse model of accelerated atherogenesis as well as in human atheromas. Conditioned medium from IMEC enhanced the migration of peripheral blood mononuclear cells and induced T cell chemotaxis, the latter being partially inhibited by antagonizing CXCL9. Proteins related to glycosaminoglycan degradation were significantly downregulated in IMEC which was relevant inasmuch as the glycocalyx plays a key role in the establishment of chemokine gradients. Indeed, the accumulation of heparan sulfates in IMEC contributed to the adhesion of T cells. Notably, IMEC that had been exposed to monocyte lysates presented 627 peptide antigens on MHC class II and induced T cell activation. ConclusionOur data demonstrate the role of IMEC as non-professional antigen-presenting cells that potentially contribute to T cell-mediated immune responses in cardiovascular disease. Translational PerspectiveThis study characterizes immunomodulatory endothelial cells (IMEC) as critical mediators of vascular inflammation through their capacity to process and present exogenous antigens and activate T cells. Induced by pro-atherogenic cytokines (IFN-{gamma}, IL-1{beta}, TGF-{beta}2), IMEC upregulate MHC class II and costimulatory molecules, promote leukocyte chemotaxis, and enhance T cell adhesion through surface heparan sulfate. The identification of IMEC-like populations in both murine models and human atherosclerotic plaques indicates a conserved immunological function in atherogenesis. These findings position IMEC as novel, non-professional antigen-presenting cells and potential therapeutic targets to modulate vascular immune responses in atherosclerotic cardiovascular disease.

immunology↗

bioRxiv2024

The transaminase-omega-amidase pathway is a redox switch in glutamine metabolism that generates alpha-ketoglutarate

Oxidative stress is caused by short-lived molecules and metabolic changes belong to the fastest cellular responses. Here we studied how the endothelial cell metabolome reacts to acute oxidative challenges (menadione or H2O2) to identify redox-sensitive metabolic enzymes. H2O2 selectively increased -ketoglutaramate (KGM), a largely uncharacterized metabolite produced by glutamine transamination and a yet unrecognized intermediate of endothelial glutamine catabolism. The enzyme nitrilase-like 2 {omega}-amidase (NIT2) converts KGM to -ketoglutarate (KG). Reversible oxidation of specific cysteine in NIT2 by H2O2 inhibited its catalytic activity. Furthermore, a variant in the NIT2 gene that decreases its expression is associated with high plasma KGM level in humans. Endothelial-specific knockout mice of NIT2 exhibited increased levels of KGM and impaired angiogenesis. Knockout of NIT2 impaired endothelial cell proliferation and sprouting and induced senescence. In conclusion, we show that the glutamine transaminase-{omega}-amidase pathway is a metabolic switch in which NIT2 is the redox-sensitive enzyme. The pathway is modulated in humans and functionally important for endothelial glutamine metabolism.

physiology↗

bioRxiv2024

Metabolic alterations drive inflammatory phenotypes in CHIP-associated heart failure

Mutations in DNA methyltransferase 3 alpha (DNMT3A) are the most frequent driver of clonal hematopoiesis of indeterminate potential (CHIP), and associated with higher risk of cardiovascular disease and pro-inflammatory activation of immune cells. Here, we investigated the mechanisms underlying DNMT3A CHIP-associated inflammatory phenotypes in macrophages. We show that monocytes of DNMT3A CHIP-driver mutation carriers are associated with DNA hypomethylation of succinate dehydrogenase A (SDHA) and an altered tricarboxylic acid cycle metabolite profile. Silencing of DNMT3A in monocytes increased SDHA and elevated mitochondria complex II activity. The secreted complex II product, malate, further increased inflammatory activation in wild type monocytes to further augment inflammation in a paracrine manner. Pharmacological inhibition of SDHA (using dimethyl malonate) in mice harboring DNMT3A mutations in hematopoietic stem cells ameliorated the inflammatory response and improved cardiac function after myocardial infarction. Thus, interfering with the altered metabolic state may provide a new therapeutic option to dampen inflammatory activation in DNMT3A CHIP carrying patients.

molecular biology↗

bioRxiv2024

Nuclear eNOS interacts with and S-nitrosates ADAR1 to modulate type I interferon signaling and endothelial function.

BackgroundNitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. Most eNOS is localized at the cell membrane or in the Golgi apparatus, but the enzyme is also present in the endothelial cell nucleus. Here we assessed the relevance of nuclear eNOS and NO signaling for endothelial cell function. MethodseNOS gain- and loss-of-function approaches were combined with confocal microscopy, biochemical, histological and multi-omics analyses. The pathophysiological relevance of the findings was assessed in murine models of atherogenesis (ApoE-/- and partial carotid ligation) as well as in samples from patients with atherosclerosis. ResultseNOS was present in the nucleus of unstimulated human and murine endothelial cells (in vitro and ex vivo) and stimulation with vascular endothelial growth factor (VEGF) enhanced its nuclear localization. Co-immunoprecipitation studies coupled with proteomics revealed the association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among the latter was double-stranded RNA-specific adenosine deaminase (ADAR1), an enzyme involved in editing double-stranded RNA (dsRNA) via the deamination of adenosine (A) to inosine (I). ADAR1 was S-nitrosated in human endothelial cells, and the knockdown of eNOS resulted in altered ADAR1-mediated A-to-I editing and an increase in dsRNA. Mitochondrial antiviral signaling protein (MAVS) was the transcript most affected by eNOS depletion, and endothelial cells lacking eNOS accumulated dsRNA as well as MAVS protein aggregates. These changes resulted in activation of the type I interferon (IFN) signaling pathway and a marked downregulation of cell cycle-related genes. ADAR1 depletion elicited similar effects on the activation of type I IFN signaling. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as stimulated cell death were increased. Endothelial dysfunction in mice as well as in subjects with atherosclerosis was accompanied by the accumulation of dsRNA and the activation of the type I IFN signaling. Preserving NO bioavailability in vivo in an eNOS gain-of-function mouse model (Tyr657Phe eNOS) prevented these effects. ConclusionsOur findings have uncovered a novel mechanism linking nuclear eNOS-derived NO with the activity of ADAR1 to maintain vascular homeostasis. Reduced NO bioavailability results in the previously unrecognized activation of a type I IFN response in the endothelium, which contributes to atherogenesis.

physiology↗

bioRxiv2023

Age-dependent RGS5 loss in pericytes induces cardiac dysfunction and fibrosis in the heart

BackgroundPericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although ageing is one of the main risk factors for cardiovascular disease, the consequences of ageing on cardiac pericytes are unknown. MethodsIn this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of ageing on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of Regulator of G protein signalling 5 (RGS5) loss of function and finally have performed pericytes-fibroblasts co-culture studies to understand the effect of RGS5 deletion in pericytes on the neighbouring fibroblasts. ResultsAgeing reduced the pericyte area and capillary coverage in the murine heart. Single nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, fibrosis, and induced morphological changes and a pro-fibrotic gene expression signature in pericytes characterized by the expression of different extracellular matrix components and growth factors e.g. TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5 deficient pericytes induced their activation as evidenced by the increased expression of smooth muscle actin in a TGF{beta}2-dependent mechanism. ConclusionsOur results have identified RGS5 as a crucial regulator of pericyte function during cardiac ageing. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac ageing.

physiology↗

bioRxiv2023

Early cellular and molecular signatures correlate with severity of West Nile Virus infection

Infection with West Nile Virus (WNV) can drive a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To identify cellular and molecular signatures distinguishing WNV severity, we employed systems profiling of peripheral blood from asymptomatic and severely ill individuals infected with WNV. We interrogated immune responses longitudinally from acute infection through convalescence at 3 months and 1 year employing multiplexed single cell protein and transcriptional profiling (CyTOF and Seq-Well) complemented with matched serum proteomics and metabolomics. At the acute time point, we detected both an elevated proportion of pro-inflammatory markers in innate immune cell types and reduced frequency of regulatory T cell activity in participants with severe infection compared to those with asymptomatic infection. Single-cell transcriptomics of paired samples revealed that asymptomatic donors had higher expression of genes associated with innate immune pathways, in particular anti-inflammatory CD16+ monocytes at the acute time point. A multi-omics analysis identified factors--beyond those from individual analyses--that distinguished immune state trajectory between severity groups. Here we highlighted the potential of systems immunology using multiple cell-type and cell-state-specific analyses to identify correlates of infection severity and host cellular activity contributing to an effective anti-viral response.

immunology↗

bioRxiv2023

Inhibition of proline-rich-tyrosine kinase 2 restores cardioprotection by remote ischemic preconditioning in type 2 diabetes mellitus

BackgroundEndothelial function and cardioprotection through remote ischemic preconditioning (rIPC) are severely impaired in type 2 diabetes mellitus (T2DM). Proline-rich tyrosine kinase 2 (Pyk2), a downstream target of the insulin receptor, reduces endothelial nitric oxide synthase (eNOS) activity. Therapeutic options to rescue cardioprotection in T2DM and improve outcomes after acute myocardial infarction (AMI) are lacking. We hypothesized that vascular endothelium contributes to rIPC, and that inhibition of Pyk2 restores cardioprotection in T2DM through modulation of eNOS, thus limiting infarct size. MethodsNew Zealand Obese (NZO) mice were used as a polygenic model of T2DM. Effects of Pyk2-inhibition on endothelial function, remote ischemic preconditioning (rIPC), and infarct size (IS) after ischemia/reperfusion (I/R) were compared in NZO, eNOS KO, and C57Bl/6 (Bl6) mice. Plasma derived from mice and individuals with or without T2DM at baseline and after rIPC was transferred to isolated hearts and aortic rings to assess the effects of Pyk2-inhibition on remote tissue protection. ResultsTransfer experiments with plasma drawn from non-diabetic humans and mice exposed to rIPC demonstrate that endothelium-dependent signals for remote tissue protection are conveyed by plasma. Key features reflecting the glucometabolic spectrum in T2DM were detected in NZO mice, including hyperinsulinemia, insulin resistance, obesity, and impaired glucose tolerance. Similar to T2DM patients, these mice also revealed endothelial dysfunction with decreased flow-mediated dilation (FMD), reduced circulating nitrite levels, elevated arterial blood pressure, and larger infarct size after I/R. Pyk2 increased the phosphorylation of eNOS on its inhibitory site (Tyr656). Cardioprotective effects by rIPC were lost in NZO mice. Inhibition of Pyk2 restored endothelial function and rescued endothelium-dependent cardioprotection after rIPC displayed by lower IS and improved LV function post I/R. ConclusionEndothelial function contributing to remote tissue protection is severely impaired in diabetes mellitus. Proline-rich tyrosine kinase 2 is a novel target to rescue cardioprotection through endothelium-dependent remote ischemic preconditioning, advocating its role in limiting infarct size in diabetes mellitus. Clinical perspective What is new?O_LIVascular endothelium contributes to remote tissue protection in ischemic preconditioning, which is severely impaired in diabetes C_LIO_LIProline-rich tyrosine kinase 2 reduces eNOS-activity, causes endothelial dysfunction, and impairs cardioprotection through ischemic preconditioning C_LIO_LIInhibition of proline-rich tyrosine kinase 2 restores eNOS activity, endothelial function, and cardioprotective effects of remote ischemic preconditioning limiting infarct size in an experimental model of diabetes. C_LI What are the clinical implications?O_LIProper endothelial function is cirtical to maintain cardiovascular health. Endothelial dysfunction contributes to impaired remote tissue protection in diabetes. C_LIO_LIThese data demonstrate for the first time that endothelium-dependent cardioprotection in myocardial ischemia/reperfusion through remote ischemic preconditioning can be restored in diabetes. C_LIO_LIProline-rich tyrosine kinase 2 is a novel target to restore endothelium-dependent remote cardioprotection to improve the outcome of diabetic patients with acute myocardial infarction. C_LI

physiology↗

bioRxiv2023

Mena and VASP are required for vascular smooth muscle relaxation

Enabled/vasodilator stimulated phosphoproteins (Ena/VASP) proteins are important regulators of the cytoskeleton, linking kinase signaling pathways to actin assembly. In mammals, the Ena/VASP family of proteins consists of mammalian enabled (Mena), VASP, and Ena-VASP-like protein (EVL). The proteins are well known targets of cAMP- and cGMP-dependent protein kinases, PKA and PKG, respectively. Given the importance of cyclic nucleotide signaling in mediating vasodilation, we investigated the role of Ena/VASP protein in vascular smooth muscle relaxation. Whereas VASP and Mena were strongly expressed in vascular smooth muscle cells, EVL was undetectable in the arterial wall and EVL-deficiency had no impact on agonist-induced smooth muscle relaxation. VASP deletion impaired the acetylcholine (ACh)- and nitric oxide (NO)-induced relaxation murine mesenteric arteries ex vivo. Similarly, the ACh-induced and NO-dependent relaxation of aorta from 7-month-old but not 3- month-old VASP-/- mice was also reduced. Aortas from animals lacking VASP and expressing only minimal amounts of Mena displayed significantly impaired relaxations in response to NO, cAMP and cGMP stimulation. These results suggest that Mena and VASP play an important role in agonist induced smooth muscle relaxation and functionally compensate for each other.

physiology↗

bioRxiv2023

Ena/VASP proteins mediate endothelial cell repulsion from ephrin ligands

The interaction of Eph receptor tyrosine kinases with their transmembrane ligands; the ephrins, is important for the regulation of cell-cell communication. Ephrin-Eph signaling is probably best known for the discrimination of arterial and venous territories by repulsion of venous endothelial cells away from those with an arterial fate. Ultimately, cell repulsion is mediated by initiating the collapse of the actin cytoskeleton in membrane protrusions. Here, we investigated the role of the Ena/VASP family of actin binding proteins in endothelial cell repulsion initiated by ephrin ligands. Human endothelial cells dynamically extended sheet-like lamellipodia over ephrin-B2 coated surfaces. While lamellipodia of control siRNA transfected cells rapidly collapsed, resulting in a pronounced cell repulsion from the ephrin-B2 surfaces, the knockdown of Ena/VASP proteins impaired the cytoskeletal collapse of membrane protrusions and the cells no longer avoided the repulsive surfaces. Mechanistically, ephrin-B2 stimulation elicited the EphB-mediated tyrosine phosphorylation of VASP, which abrogated its interaction with the focal adhesion protein Zyxin. Nck2 was identified as a novel VASP binding protein, which only interacted with the tyrosine phosphorylated VASP protein. Nck links Eph-receptors to the actin cytoskeleton. Therefore, we hypothesize that Nck-Ena/VASP complex formation is required for actin reorganization and/or Eph receptor internalization downstream of ephrin-Eph interaction in endothelial cells, with implications for endothelial navigation and pathfinding.

cell biology↗

bioRxiv2023

Impaired H19 lncRNA expression contributes to the compromised developmental angiogenesis in EVL-deficient mice

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. Endothelial-specific deletion of EVL, a member of the mammalian Ena/VASP protein family, reduced the expression of the tip cell marker protein endothelial cell specific molecule-1 (Esm1) and compromised the radial sprouting of the vascular plexus in the postnatal mouse retina. The latter effects could at least partly be attributed to reduced VEGF receptor 2 (VEGFR2) internalization and signaling but the underlying mechanisms(s) are not fully understood. In the present study, we revealed that the expression of the long non-coding RNA H19 was significantly reduced in endothelial cells from postnatal EVL-/- mice and in siRNA-transfected human endothelial cells under hypoxic conditions. H19 was recently shown to promote VEGF expression and bioavailability via Esm1 and hypoxia inducible factor 1 (HIF-1). Similar to EVL-/- mice, the radial outgrowth of the vascular plexus was significantly delayed in the postnatal retina of H19-/- mice. In summary, our data suggests that loss of EVL not only impairs VEGFR2 internalization and downstream signaling, but also impairs VEGF expression and bioavailability in the hypoxic retina via downregulation of lncRNA H19.

cell biology↗

bioRxiv2023

Impaired AMPK activity contributes to the inflammatory phenotype and the reduced phagocytosis capacity of VASP-deficient macrophages

Macrophage polarization plays an important role in tissue regeneration. Numerous factors and signaling molecules affect polarization processes. Here we investigated the consequences of the genetic deletion of vasodilator-stimulated phosphoprotein (VASP), which increases macrophage M1 polarization through augmented signal transducer and activator of transcription 1 (STAT1) signaling, and AMP-activated protein kinase (AMPK), which attenuates inflammation by inhibiting STAT1 expression and signaling. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNF and IL-1{beta} was increased in these cells. Consistent with the role of AMPK in macrophage phagocytosis, VASP-/- macrophage phagocytosis was also significantly impaired. Interestingly, impaired phagocytosis could be rescued by exogenous activation of AMPK. Mechanistically, we found that VASP binds directly to protein phosphatase 1 regulatory subunit 6 (PP1-R6) and we hypothesize that VASP-binding to PP1- R6/PP1 limits the PP1-dependent de-phosphorylation of AMPK in wild-type cells. Conversely, AMPK dephosphorylation by the PP1-R6/PP1 complex is enhanced in the absence of VASP. In summary, we have identified a link between VASP and AMP-activated protein kinase (AMPK) activity, which may contribute to the pro-inflammatory phenotype of VASP-deficient macrophages.

cell biology↗