Response Of Endothelial Cells Research Articles

The BCR::ABL1 tyrosine kinase inhibitors ponatinib and nilotinib differentially affect endothelial angiogenesis and signalling.

BCR::ABL1 inhibitors, the treatment of choice for the majority of patients with chronic myeloid leukaemia (CML), can cause vascular side effects that vary between agents. The exact underlying mechanisms are still poorly understood, but the vascular endothelium has been proposed as a site of origin. The present study investigates the effects of three BCR::ABL1 inhibitors, ponatinib, nilotinib and imatinib, on angiogenesis and signalling in human endothelial cells in response to vascular endothelial growth factor (VEGF). The experiments were performed in endothelial cells isolated from human umbilical veins. After exposure to imatinib, ponatinib and nilotinib, the angiogenic capacity of endothelial cells was assessed in spheroid assays. VEGF-induced signalling pathways were examined in Western blotting experiments using different specific antibodies. RNAi technology was used to downregulate proteins of interest. Intracellular cGMP levels were measured by ELISA. Imatinib had no effect on endothelial function. Ponatinib inhibited VEGF-induced sprouting, while nilotinib increased spontaneous and VEGF-stimulated angiogenesis. These effects did not involve wild-type ABL1 or ABL2, as siRNA-mediated knockdown of these kinases did not affect angiogenesis and VEGF signalling. Consistent with their effects on sprouting, ponatinib and nilotinib affected angiogenic pathways in opposite directions. While ponatinib inhibited VEGF-induced signalling and cGMP formation, nilotinib activated angiogenic signalling, in particular phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). The latter occurred in an epidermal growth factor receptor (EGFR)-dependent manner possibly via suppressing Fyn-related kinase (FRK), a negative regulator of EGFR signalling. Both, pharmacological inhibition of Erk1/2 or EGFR suppressed nilotinib-induced angiogenic sprouting. These results support the notion that the vascular endothelium is a site of action of BCR::ABL1 inhibitors from which side effects may arise, and that the different vascular toxicity profiles of BCR::ABL1 inhibitors may be due to their different actions at the molecular level. In addition, the as yet unknown pro-angiogenic effect of nilotinib should be considered in the treatment of patients with comorbidities associated with pathological angiogenesis, such as ocular disease, arthritis or obesity.

Influence of Substrate Structure and Associated Properties on Endothelial Cell Behavior in the Context of Behaviors Associated with Laminar Flow Conditions.

In order to treat most vascular diseases, arterial grafts are commonly employed for replacing small-diameter vessels, yet they often cause thrombosis. The growth of endothelial cells along the interior surfaces of these grafts (substrates) is critical to mitigate thrombosis. Typically, endothelial cells are cultured inside these grafts under laminar flow conditions to emulate the native environment of blood vessels and produce an endothelium. Alternatively, the substrate structure could have a similar influence on endothelial cell behavior as laminar flow conditions. In this study, we investigated whether substrates with aligned fiber structures could induce responses in human umbilical vein endothelial cells (HUVECs) akin to those elicited by laminar flow. Our observations revealed that HUVECs on aligned substrates displayed significant morphological changes, aligning parallel to the fibers, similar to effects reported under laminar flow conditions. Conversely, HUVECs on random substrates maintained their characteristic cobblestone appearance. Notably, cell migration was more significant on aligned substrates. Also, we observed that while vWF expression was similar between both substrates, the HUVECs on aligned substrates showed more expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31), laminin, and collagen IV. Additionally, these cells exhibited increased gene expression related to critical functions such as proliferation, extracellular matrix production, cytoskeletal reorganization, autophagy, and antithrombotic activity. These findings indicated that aligned substrates enhanced endothelial growth and behavior compared to random substrates. These improvements are similar to the beneficial effects of laminar flow on endothelial cells, which are well-documented compared to static or turbulent flow conditions.

ANOTHER NOTCH IN THE BELT OF RHEUMATOID ARTHRITIS.

Notch ligands and receptors, including JAG1/2, DLL1/4, and Notch1/3, are enriched on macrophages (MΦs), fibroblast-like synoviocytes (FLS), and/or endothelial cells in rheumatoid arthritis (RA) compared to normal synovial tissues (ST). Power Doppler ultrasound-guided ST studies reveal that the Notch family is highly involved in early active RA, especially during neovascularization. In contrast, the Notch family is not implicated during the erosive stage, evidenced by their lack of correlation with radiographic damage in RA STs. TLRs and TNF are the common inducers of Notch expression in RA MΦs, FLS, and endothelial cells. Among Notch ligands, JAG1 and/or DLL4 are most inducible by inflammatory responses in RA MΦs or endothelial cells and trans-activate their receptors on RA FLS. TNF plays a central role on Notch ligands, as anti-TNF good responders display JAG1/2 and DLL1/4 transcriptional downregulation in RA ST myeloid cells. In in vitro studies, TNF increases Notch3 expression in MΦs, which is further amplified by RA FLS addition. Specific disease-modifying anti-rheumatic drugs (DMARDs) reduced JAG1 and Notch3 expression in MΦ and RA FLS cocultures. Organoids containing FLS and endothelial cells have increased expression of JAG1 and Notch3. Nonetheless, Methotrexate, IL-6R antibodies, and B cell blockers are mostly ineffective at decreasing Notch family expression. NF-κB, MAPK, and AKT pathways are involved in Notch signaling, whereas JAK/STATs are not. Although Notch blockade has been effective in RA preclinical studies, its small molecule inhibitors have failed in phase I and II studies, suggesting that alternative strategies may be required to intercept their function.

Quercetin ameliorates atherosclerosis by inhibiting inflammation of vascular endothelial cells via Piezo1 channels

BackgroundNatural antioxidants, exemplified by quercetin (Qu), have been shown to exert a protective effect against atherosclerosis (AS). However, the precise pharmacological mechanisms of Qu also remain elusive. PurposeHere, we aimed to uncover the anti-atherosclerotic mechanisms of Qu. Methods/Study designsThe inflammatory cytokine expression, activity of NLRP3 inflammasome and NF-κB, as well as mechanically activated currents and intracellular calcium levels were measured in endothelial cells (ECs). In addition, to explore whether Qu inhibited atherosclerotic plaque formation via Piezo1 channels, Ldlr−/- and Piezo1 endothelial-specific knockout mice (Piezo1△EC) were established. ResultsOur findings revealed that Qu significantly inhibited Yoda1-evoked calcium response in human umbilical vein endothelial cells (HUVECs), underscoring its role as a selective modulator of Piezo1 channels. Additionally, Qu effectively reduced mechanically activated currents in HUVECs. Moreover, Qu exhibited a substantial inhibitory effect on inflammatory cytokine expression and reduced the activity of NF-κB/NLRP3 in ECs exposed to ox-LDL or mechanical stretch, and these effects remained unaffected after Piezo1 genetic depletion. Furthermore, our study demonstrated that Qu substantially reduced the formation of atherosclerotic plaques, and this effect remained consistent even after Piezo1 genetic depletion. ConclusionThese results collectively provide compelling evidence that Qu ameliorates atherosclerosis by inhibiting the inflammatory response in ECs by targeting Piezo1 channels. In addition, Qu modulated atherosclerosis via inhibiting Piezo1 mediated NFκB/IL-1β and NLRP3/caspase1/ IL-1β axis to suppress the inflammation. Overall, this study reveals the potential mechanisms by which natural antioxidants, such as Qu, protect against atherosclerosis.

Impact of Uniaxial Cyclic Stretching on Matrix-Associated Endothelial Cell Responses

Uniaxial cyclic stretching plays a pivotal role in the fields of tissue engineering and regenerative medicine influencing cell behaviors and functionality based on physical properties, including matrix morphology and mechanical stimuli. This study delves into the response of endothelial cells to uniaxial cyclic strain within the geometric constraints of micro-nano fibers. Various structural scaffolds forms of poly(L-lactide-co-caprolactone) (PLCL), such as flat membranes, randomly oriented fiber membranes, and aligned fiber membranes, were fabricated through solvent casting and electrospinning methods. Our investigation focuses on the morphological variation of endothelial cells under diverse geometric constraints, and the mechanical-dependent release of nitric oxide (NO) on oriented fibrous membranes. Our results indicate that while uniaxial cyclic stretching promotes endothelial cell spreading, the anisotropy of the matrix morphology remains the primary driving factor for cell alignment. Additionally, uniaxial cyclic stretching significantly enhances NO release, with a notably strengthening effect correlated to the increasing strain amplitude. Importantly, this study reveals that uniaxial cyclic stretching enhances the mRNA expression of key proteins, including talin, vinculin, rac, and nitric oxide synthase (eNOS).

Impact of uniaxial cyclic stretching on matrix-associated endothelial cell responses

Uniaxial cyclic stretching plays a pivotal role in the fields of tissue engineering and regenerative medicine, influencing cell behaviors and functionality based on physical properties, including matrix morphology and mechanical stimuli. This study delves into the response of endothelial cells to uniaxial cyclic strain within the geometric constraints of micro-nano fibers. Various structural scaffold forms of poly(l-lactide-co-caprolactone) (PLCL), such as flat membranes, randomly oriented fiber membranes, and aligned fiber membranes, were fabricated through solvent casting and electrospinning methods. Our investigation focuses on the morphological variation of endothelial cells under diverse geometric constraints and the mechanical-dependent release of nitric oxide (NO) on oriented fibrous membranes. Our results indicate that while uniaxial cyclic stretching promotes endothelial cell spreading, the anisotropy of the matrix morphology remains the primary driving factor for cell alignment. Additionally, uniaxial cyclic stretching significantly enhances NO release, with a notably stronger effect correlated to the increasing strain amplitude. Importantly, this study reveals that uniaxial cyclic stretching enhances the mRNA expression of key proteins, including talin, vinculin, rac, and nitric oxide synthase (eNOS).

A feedback loop driven by H3K9 lactylation and HDAC2 in endothelial cells regulates VEGF-induced angiogenesis.

Vascular endothelial growth factor (VEGF) is one of the most powerful proangiogenic factors and plays an important role in multiple diseases. Increased glycolytic rates and lactate accumulation are associated with pathological angiogenesis. Here, we show that a feedback loop between H3K9 lactylation (H3K9la) and histone deacetylase 2 (HDAC2) in endothelial cells drives VEGF-induced angiogenesis. We find that the H3K9la levels are upregulated in endothelial cells in response to VEGF stimulation. Pharmacological inhibition of glycolysis decreases H3K9 lactylation and attenuates neovascularization. CUT& Tag analysis reveals that H3K9la is enriched at the promoters of a set of angiogenic genes and promotes their transcription. Interestingly, we find that hyperlactylation of H3K9 inhibits expression of the lactylation eraser HDAC2, whereas overexpression of HDAC2 decreases H3K9 lactylation and suppresses angiogenesis. Collectively, our study illustrates that H3K9la is important for VEGF-induced angiogenesis, and interruption of the H3K9la/HDAC2 feedback loop may represent a novel therapeutic method for treating pathological neovascularization.

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions.

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.

Inositol-requiring enzyme 1 pathway and autophagy drive sequential response of endothelial cells to febrile range hyperthermia

Inositol-requiring enzyme 1 pathway and autophagy drive sequential response of endothelial cells to febrile range hyperthermia

Exploring the underlying molecular mechanisms of acute myocardial infarction after SARS-CoV-2 infection

Exploring the underlying molecular mechanisms of acute myocardial infarction after SARS-CoV-2 infection

Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Correction to: ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

Correction to: ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

An essential role for EROS in redox-dependent endothelial signal transduction

The chaperone protein EROS (“Essential for Reactive Oxygen Species”) was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

Shear stress and pathophysiological PI3K involvement in vascular malformations.

Molecular characterization of vascular anomalies has revealed that affected endothelial cells (ECs) harbor gain-of-function (GOF) mutations in the gene encoding the catalytic α subunit of PI3Kα (PIK3CA). These PIK3CA mutations are known to cause solid cancers when occurring in other tissues. PIK3CA-related vascular anomalies, or "PIKopathies," range from simple, i.e., restricted to a particular form of malformation, to complex, i.e., presenting with a range of hyperplasia phenotypes, including the PIK3CA-related overgrowth spectrum. Interestingly, development of PIKopathies is affected by fluid shear stress (FSS), a physiological stimulus caused by blood or lymph flow. These findings implicate PI3K in mediating physiological EC responses to FSS conditions characteristic of lymphatic and capillary vessel beds. Consistent with this hypothesis, increased PI3K signaling also contributes to cerebral cavernous malformations, a vascular disorder that affects low-perfused brain venous capillaries. Because the GOF activity of PI3K and its signaling partners are excellent drug targets, understanding PIK3CA's role in the development of vascular anomalies may inform therapeutic strategies to normalize EC responses in the diseased state. This Review focuses on PIK3CA's role in mediating EC responses to FSS and discusses current understanding of PIK3CA dysregulation in a range of vascular anomalies that particularly affect low-perfused regions of the vasculature. We also discuss recent surprising findings linking increased PI3K signaling to fast-flow arteriovenous malformations in hereditary hemorrhagic telangiectasias.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

Targeting Neutrophil Extracellular Trap Formation: Exploring Promising Pharmacological Strategies for the Treatment of Preeclampsia.

Neutrophils, which constitute the most abundant leukocytes in human blood, emerge as crucial players in the induction of endothelial cell death and the modulation of endothelial cell responses under both physiological and pathological conditions. The hallmark of preeclampsia is endothelial dysfunction induced by systemic inflammation, in which neutrophils, particularly through the formation of neutrophil extracellular traps (NETs), play a pivotal role in the development and perpetuation of endothelial dysfunction and the hypertensive state. Considering the potential of numerous pharmaceutical agents to attenuate NET formation (NETosis) in preeclampsia, a comprehensive assessment of the extensively studied candidates becomes imperative. This review aims to identify mechanisms associated with the induction and negative regulation of NETs in the context of preeclampsia. We discuss potential drugs to modulate NETosis, such as NF-κβ inhibitors, vitamin D, and aspirin, and their association with mutagenicity and genotoxicity. Strong evidence supports the notion that molecules involved in the activation of NETs could serve as promising targets for the treatment of preeclampsia.

Phototuning of Hyaluronic-Acid-Based Hydrogel Properties to Control Network Formation in Human Vascular Endothelial Cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA-Ph) and phenolated gelatin (Gelatin-Ph) are presented. Visible-light irradiation in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA-Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible-light irradiation for 45min (42W cm-2 at 450nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60min. The irradiation time-dependent degradation of HA-Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and β-actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA-Ph degradation. These findings highlight the potential of tailoring HA-based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.

Endogenous Acetylcholine Contributes to Flow-Mediated Dilation in Humans

Background &amp; Objective. Flow-mediated dilation (FMD) is a crucial function of the vascular endothelium wherein intraluminal shear stress stimulates endothelium-dependent vasodilation mediated primarily by nitric oxide. Brachial artery FMD (baFMD) is widely evaluated in clinical research as an index of endothelial function, and impaired baFMD is an independent predictor of cardiovascular risk. Despite the physiological and clinical relevance of FMD, the mechanisms by which shear stress is transduced to endothelium-dependent vasodilation (i.e., upstream of nitric oxide production) remain unclear. baFMD corresponds closely with sensitivity to the endothelium-dependent vasodilator acetylcholine (ACh), and preclinical models suggest ACh is released from endothelial cells in response to shear stress. Therefore, the present study tested the hypothesis that activation of muscarinic ACh receptors facilitates baFMD in humans. Methods. baFMD was evaluated using Doppler ultrasound in healthy participants (n=11; 5F/6M; 23±2 y; means ± SE) under control conditions and following local inhibition of muscarinic ACh receptors via intra-arterial atropine infused proximal to the measurement site. baFMD was assessed in response to transient and sustained shear stimuli in separate trials: 1) reactive hyperemia following 5 min of arterial cuff occlusion (RH-FMD) and 2) steady-state rhythmic handgrip exercise at graded intensities (10, 15, 20, &amp; 25% maximal voluntary contraction, MVC) to produce step increases in shear rate (EX-FMD). Blockade effcacy was confirmed via a dose-response to intra-arterial ACh infusion pre and post atropine. Results. Baseline brachial artery diameter and forearm blood flow were similar in all trials (all P&gt;0.05). Atropine reduced peak brachial artery dilation following cuff release (RH-FMD, Δ diameter, atropine: 4.6±0.8 vs. control: 6.5±0.8%, P=0.04) and increased the shear stimulus (shear rate area under the curve, SRAUC: atropine, 29±3 vs. control: 21±3 s−1×103, P=0.01) such that the percent change in brachial diameter was diminished by 35±16% when normalized to SRAUC (baFMD:SRAUC ratio, atropine: 1.7±0.3 vs. control: 3.9±1.0, P=0.02). Similarly, in the EX-FMD trial, atropine reduced brachial artery diameter at all exercise intensities (25% MVC, percent change in diameter: atropine, 10.8±1.4 vs. control, 15.5±1.7%, P=0.04) despite a greater shear stimulus (shear rate: atropine, 10.9±1.1 vs. control, 9.3±0.9 s−1×102, P=0.01). Thus, atropine reduced the slope describing the relationship between brachial artery vasodilation (percent change) and the change in shear rate by 38±8% across all exercise intensities (slope, atropine: 1.3±0.1 vs. control: 2.4±0.4% Δ×s×10−2, P=0.007). As expected, atropine reduced brachial artery vasodilation in response to upstream infusion of ACh (1.5 μg ACh/dl forearm volume/min, Δ diameter, atropine: 2±1 vs. control: 23±4%, P=0.001). Conclusions. The results suggest endogenous ACh facilitates baFMD in response to both transient and sustained shear stimuli, which highlights a novel physiological role of endogenous ACh in vascular regulation in humans and provides insight regarding the basic mechanisms of flow-mediated vasodilation. NIH R01 HL119337 and F31 HL142240. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of neuroendothelial cells in aging-related vascular cognitive impairment

Vascular cognitive impairment (VCI) is a growing health problem in many developed countries due to aging of its population. Aging-induced impairment of neurovascular coupling (NVC) responses and brain microvascular rarefaction have been implicated as major driving mechanisms in age-related cognitive decline. One of the cellular hallmarks of NVC involve the release of vasodilator nitric oxide (NO) by endothelial cells in response to mediators released from activated neurons. Recent studies from our group identified and characterized a novel, minority subtype of scattered ECs with endothelial and neuronal characteristics, hence the name neuroendothelial cell (NECs). NECs present a well-defined arteriovenous zonal localization exclusively in small resistance arterioles with the highest density in the brain followed by the kidney. Intravital multiphoton microscopy (MPM) of intact brain arterioles in vivo suggest that NECs play an important role in regulating blood flow to the brain and able to detect changes in systemic blood pressure, hypoxia and perhaps other metabolic changes in the local microenvironment. Our central hypothesis is that NECs are novel key players in blood flow autoregulation, vascular control of organ functions in both short-term (hemodynamics) and long-term (angiogenesis, tissue regeneration). We further hypothesize that the decrease in NEC density and function in aged subjects is a major player in the development of age-related cerebral microvascular pathologies and that augmenting NEC number and function in aged animals will provide therapeutic benefit. This study used a NEC specific comprehensive research toolbox including NEC-GFP transgenic mouse models and high-resolution single-cell based transcriptome analysis to establish the NEC specific molecular atlas in young adult (3 month of age) and aged (2 years of age) animals. Furthermore, transgenic animal models combined with serial intravital imaging of the same tissue volume over consecutive days was used to study changes in vascular endothelial cell composition in response to NEC stimuli in young adult and aged mice. Cell fate tracking studies of the Nos1-lineage combined with Nos1 immunostaining revealed that NECs represent a permanent cell type with terminal neuronal differentiation. Importantly, NEC density is reduced with aging in all organs including the brain. High resolution single-cell based transcriptome analysis of 100K endothelial cells isolated from mouse brain demonstrated that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Angpt1, Egfl8) and novel (e.g., Cytl1, MGP, Gkn3) tissue trophic factors that are known to play important roles in acute hemodynamic responses including NO mediated functional hyperemia, and in chronic settings in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Furthermore, the expression of many of these factors were significantly reduced in NECs isolated from aged animals. In conclusion, the newly discovered NEC-specific molecular and signaling mechanisms may play a role in the age-related decrease in NVC capacity and microvascular rarefaction and may be targeted for therapeutic benefits in age related VCI. American Society of Nephrology Carl W. Gottschalk Research Scholar Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.