Signaling In Endothelial Cells Research Articles

FGD5 in basal cells induces CXCL14 secretion that initiates a feedback loop to promote murine mammary epithelial growth and differentiation

The interactions of environmental compartments with epithelial cells are essential for mammary gland development and homeostasis. Currently, the direct crosstalk between the endothelial niche and mammary epithelial cells remains poorly understood. Here, we show that faciogenital dysplasia 5 (FGD5) is enriched in mammary basal cells (BCs) and mediates critical interactions between basal and endothelial cells (ECs) in the mammary gland. Conditional deletion of Fgd5 reduced, whereas conditional knockin of Fgd5 increased, the engraftment and expansion of BCs, regulating ductal morphogenesis in the mammary gland. Mechanistically, murine mammary BC-expressed FGD5 inhibited the transcriptional activity of activating transcription factor 3 (ATF3), leading to subsequent transcriptional activation and secretion of CXCL14. Furthermore, activation of CXCL14/CXCR4/ERK signaling in primary murine mammary stromal ECs enhanced the expression of HIF-1α-regulated hedgehog ligands, which initiated a positive feedback loop to promote the function of BCs. Collectively, these findings identify functionally important interactions between BCs and the endothelial niche that occur through the FGD5/CXCL14/hedgehog axis.

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.

SEMA3G regulates BMP9 inhibition of VEGF-mediated migration and network formation in pulmonary endothelial cells

AimsBone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence. Methods and resultsWe identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays. ConclusionsThis study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.

Vascular endothelial cell morphology and alignment regulate VEGF-induced endothelial nitric oxide synthase activation.

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) inhibits platelet and leukocyte adhesion while promoting vasorelaxation in smooth muscle cells. Dysfunctional regulation of eNOS is a hallmark of various vascular pathologies, notably atherosclerosis, often associated with areas of low shear stress on endothelial cells (ECs). While the link between EC morphology and local hemodynamics is acknowledged, the specific impact of EC morphology on eNOS regulation remains unclear. Morphological differences between elongated, aligned ECs and polygonal, randomly oriented ECs correspond to variations in focal adhesion and cytoskeletal organization, suggesting differing levels of cytoskeletal prestress. However, the functional outcomes of cytoskeletal prestress, particularly in the absence of shear stress, are not extensively studied in ECs. Some evidence suggests that elongated ECs exhibit decreased immunogenicity and enhanced NO production. This study aims to elucidate the signaling pathways governing VEGF-stimulated eNOS regulation in the aligned EC phenotype characterized by elongated and aligned cells within a monolayer. Using anisotropic topographic cues, bovine aortic endothelial cells (BAECs) were elongated and aligned, followed by VEGF treatment in the presence or absence of cytoskeletal tension inhibitors. Phosphorylation of eNOS ser1179, AKT ser437 and FAK Tyr397 in response to VEGF challenge were significantly heightened in aligned ECs compared to unaligned ECs. Moreover this response proved to be robustly tied to cytoskeletal tension as evinced by the abrogation of responses in the presence of the myosin II ATPase inhibitor, blebbistatin. Notably, this work demonstrates for the first time the reliance on FAK phosphorylation in VEGF-mediated eNOS activation and the comparatively greater contribution of the cytoskeletal machinery in propagating VEGF-eNOS signaling in aligned and elongated ECs. This research underscores the importance of utilizing appropriate vascular models in drug development and sheds light on potential mechanisms underlying vascular function and pathology that can help inform vascular graft design.

Phosphoenolpyruvate carboxykinase-1 targeted siRNA promotes wound healing in type 2 diabetic mice by restoring glucose homeostasis

It is well-accepted that the liver plays a vital role in the metabolism of glucose and its homeostasis. Dysregulated hepatic glucose production and utilization, leads to type 2 diabetes (T2DM). In the current study, RNA sequencing and qRT-PCR analysis of nanoformulation-treated T2DM mice (TGthr group) revealed beneficial crosstalk of PCK-1 silencing with other pathways involved in T2DM. The comparison of precise genetic expression profiles of the different experimental groups showed significantly improved hepatic glucose, fatty acid metabolism and several other T2DM-associated crucial markers after the nanoformulation treatment. As a result of these improvements, we observed a significant acceleration in wound healing and improved insulin signaling in vascular endothelial cells in the TGthr group as compared to the T2DM group. Enhanced phosphorylation of PI3K/Akt pathway proteins in the TGthr group resulted in increased angiogenesis as observed by the increased expression of endothelial cell markers (CD31, CD34) thereby improving endothelial dysfunctions in the TGthr group. Additionally, therapeutic nanoformulation has been observed to improve the inflammatory cytokine profile in the TGthr group. Overall, our results demonstrated that the synthesized therapeutic nanoformulation referred to as GPR8:PCK-1siRNA holds the potential in ameliorating hyperglycemia-associated complications such as delayed wound healing in diabetes.

How selective antagonists and genetic modification have helped characterise the expression and functions of vascular P2Y receptors.

Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y1 receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y2 receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y2 receptor knockdown reduced endothelial signalling and endothelial P2Y2 receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y2 receptor knockout were complex. No P2Y4 receptor antagonists are available and P2Y4 knockout did not affect the vascular actions of UTP and UDP. The P2Y6 receptor antagonist, MRS2578, identified endothelial P2Y6 receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y6 knockout abolished, contractions evoked by UDP. P2Y6 receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y11 receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y12/P2Y13 and P2Y14 receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.

Role of endothelial Raptor in abnormal arteriogenesis after lower limb ischaemia in type 2 diabetes.

Proper arteriogenesis after tissue ischaemia is necessary to rebuild stable blood circulation; nevertheless, this process is impaired in type 2 diabetes mellitus (T2DM). Raptor is a scaffold protein and a component of mammalian target of rapamycin complex 1 (mTORC1). However, the role of the endothelial Raptor in arteriogenesis under the conditions of T2DM remains unknown. This study investigated the role of endothelial Raptor in ischaemia-induced arteriogenesis during T2DM. Although endothelial mTORC1 is hyperactive in T2DM, we observed a marked reduction in the expression of endothelial Raptor in two mouse models and in human vessels. Inducible endothelial-specific Raptor knockout severely exacerbated impaired hindlimb perfusion and arteriogenesis after hindlimb ischaemic injury in 12-week high-fat diet fed mice. Additionally, we found that Raptor deficiency dampened vascular endothelial growth factor receptor 2 (VEGFR2) signalling in endothelial cells (ECs) and inhibited VEGF-induced cell migration and tube formation in a PTP1B-dependent manner. Furthermore, mass spectrometry analysis indicated that Raptor interacts with neuropilin 1 (NRP1), the co-receptor of VEGFR2, and mediates VEGFR2 trafficking by facilitating the interaction between NRP1 and Synectin. Finally, we found that EC-specific overexpression of the Raptor mutant (loss of mTOR binding) reversed impaired hindlimb perfusion and arteriogenesis induced by endothelial Raptor knockout in high-fat diet fed mice. Collectively, our study demonstrated the crucial role of endothelial Raptor in promoting ischaemia-induced arteriogenesis in T2DM by mediating VEGFR2 signalling. Thus, endothelial Raptor is a novel therapeutic target for promoting arteriogenesis and ameliorating perfusion in T2DM.

Bone morphogenetic protein signalling in pulmonary arterial hypertension: revisiting the BMPRII connection.

Pulmonary arterial hypertension (PAH) is a rare and life-threatening vascular disorder, characterised by abnormal remodelling of the pulmonary vessels and elevated pulmonary artery pressure, leading to right ventricular hypertrophy and right-sided heart failure. The importance of bone morphogenetic protein (BMP) signalling in the pathogenesis of PAH is demonstrated by human genetic studies. Many PAH risk genes are involved in the BMP signalling pathway and are highly expressed or preferentially act on vascular endothelial cells. Endothelial dysfunction is recognised as an initial trigger for PAH, and endothelial BMP signalling plays a crucial role in the maintenance of endothelial integrity. BMPR2 is the most prevalent PAH gene, found in over 80% of heritable cases. As BMPRII protein is the major type II receptor for a large family of BMP ligands and expressed ubiquitously in many tissues, dysregulated BMP signalling in other cells may also contribute to PAH pathobiology. Sotatercept, which contains the extracellular domain of another transforming growth factor-β family type II receptor ActRIIA fused to immunoglobin Fc domain, was recently approved by the FDA as a treatment for PAH. Neither its target cells nor its mechanism of action is fully understood. This review will revisit BMPRII function and its extracellular regulation, summarise how dysregulated BMP signalling in endothelial cells and smooth muscle cells may contribute to PAH pathogenesis, and discuss how novel therapeutics targeting the extracellular regulation of BMP signalling, such as BMP9 and Sotatercept, can be related to restoring BMPRII function.

CalDAG-GEFIII controls Ca2+ entry via Rap1 and H-Ras, triggering distinct signaling pathways

Objective: Small GTPase Rap1 plays key functions in endothelium, including controlling Ca2+-dependent endothelial function (NO release). Our recent study suggests the Rap1A isoform restricts Ca2+ entry, to control eNOS activity. The activity of Rap1, a GDP to GTP switch, is controlled by Guanine Nucleotide Exchange Factors (GEFs). However, how Rap1 is activated in the context of Ca2+ signaling and regulation of eNOS activity is unknown. A Ca2+ and diacylglycerol (DAG)-activated CalDAG-GEFIII can activate Ha-Ras, R-Ras, and Rap1, but its functions in endothelial cells (ECs), and the signaling specificity of CalDAG-GEFIII through Rap1 and Ras pathways are not well understood. Our study aims to elucidate the role of CalDAG-GEFIII in regulating Ca2+ signaling in ECs. Approach and Results: To determine relative expression of Rap1 GEFs in ECs in different tissues, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data from eleven publicly available scRNA-seq datasets. We found RasGRP3 (encoding CalDAG-GEFIII) was highly expressed compared to other Rap1-GEFs in endothelial cells from different organs. To examine the involvement of CalDAG-GEFIII Rap1 and Ras activation in heart ECs, we determined the effect of on Vascular Endothelial Growth Factor (VEGF)-induced Rap1-GTP and Ras-GTP loading using pull-down assay. Knockdown of RasGRP3 blocked Rap1 and Ras activation downstream from VEGFR2, suggesting CalDAG-GEFIII acts as a GEF (activator) of both Rap1 and Ras in endothelial cells. To examine the involvement of CalDAG-GEFIII and H-Ras in Ca2+ signaling in ECs, we measured VEGF-induced intracellular Ca2+ using Fura2 dye, an intracellular Ca2+ indicator, in siRasGRP3, siH-Ras (most highly expressed Ras isoform) and siControl HUVECs. We found that Ca2+ release unchanged, but Ca2+ entry was increased in siRasGRP3 and siRas ECs, similarly to what we had found in siRap1A ECs. This suggests that Ras, like Rap1, limits ATP and TG-induced Ca2+ entry in HUVECs and that CalDAG-GEFIII may act upstream from both Rap1 and Ras. The elevated Ca2+ entry in siRap1A, siRasGRP3 ECs was partially normalized by exogenous expression of constitutively activated Rap1A or activation of Rap1 A by cAMP-dependent GEF, Epac. This indicates that Rap1A controls Ca2+ entry both acutely via a signaling mechanism, and at the transcript level. To determine the involvement of CalDAG-GEFIII in signaling by Rap1 and Ras, we determined the impact of siRNA knockdown of siRap1, siRas and siRasGRP3 on Ser1177 eNOS phosphorylation and Thr202/Tyr204 ERK phosphorylation in response to VEGF stimulation. Knockdown of RasGRP3 or Rap1 attenuated VEGF-induced Ser-1177 eNOS phosphorylation, while knockdown of RasGRP3 or Ras, but not Rap1, inhibited ERK phosphorylation in response to VEGF. Conclusions: Rap1 and Ras, activated downstream from CalDAG-GEFIII in response to VEGF activation, limit agonist-induced Ca2+ entry in endothelial cells. CalDAG-GEFIII promotes eNOS phosphorylation through Rap1B and promotes ERK phosphorylation via Ras, independent of Rap1. This suggests that CalDAG-GEFIII acts in two separate pathways, that are Rap1 and Ras dependent. NIH NHLBI: R01HL111582. 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.

Contractile pericyte-mediated cerebrovascular deficits in a mouse model of CADASIL

Small vessel disease (SVD) of the brain underlies 25% of stroke cases and is the most common cause of vascular cognitive impairment. The most common monogenic form of SVD is CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), caused by mutations of the NOTCH3 protein which is widely expressed in mural cells (vascular smooth muscle cells [SMCs] and pericytes). Cerebral blood flow (CBF) deficits present early in CADASIL, attributable in part to NOTCH3-mediated extracellular matrix dysregulation, which alters heparin-binding epidermal growth factor-like (HB-EGF) signaling in SMCs and endothelial cells (ECs). Pericytes also robustly express NOTCH3 and are a relatively underexplored cell type in the context of CADASIL. Pericytes proximal to the penetrating arteriole (PA), known as contractile pericytes, express α-smooth muscle actin (αSMA) and can contract to rapidly regulate capillary diameter and thus blood flow in the downstream capillary tree. We conducted two-photon laser scanning microscopy (2PLSM) of blood flow and pericyte contractile responses with CADASIL Notch3R169C transgenic and control mice. Our data indicates that contractile pericyte-mediated control of blood flow is impaired prior to other known CBF deficits, thus raising the tantalizing possibility that these pericytes could be a target for early clinical intervention in CADASIL. While PA responses to stimulation of capillary electrical signaling remain intact, both blood flow as well as capillary diameter of contractile pericyte-covered vessels are diminished. Taken together, this work will establish pericytes as a novel site of dysfunction and provide a new candidate mechanism by which CBF is diminished early in CADASIL pathogenesis for potential therapeutic intervention. 5T32GM008181-33 (AV), 1DP2OD02944801 (TL), 1R01AG066645 (TL), 5R01NS115401 (TL). 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.

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. 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.

In High Glucose Exposed Endothelial Cells BLNK and BTK are Novel Components of the Nuclear Factor-kappa B Pathway

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFκB signaling in ischemic endothelial cells (EC). We have previously shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFκB pathway through activation of protein kinase C β (PKCβ). However, the signaling pathways upstream of PKCβ involved in this high glucose induced desensitization of NFκB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFκB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates from NG, HG, NGI and HGI groups were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spots by the signal intensity of the corresponding non-phosphorylated spots for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling by associating with the scaffold protein BLNK. However, neither of these proteins has been known to be expressed in ECs or play a role in EC function. In this study, we confirmed BLNK and BTK protein and transcript expression in ECs. Inhibition of BTK by a selective inhibitor terreic acid decreased PKCβ-S661 phosphorylation and restored NFκB activation through IκBα in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as potentially new components of the NFκB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. National Heart, Lung, and Blood Institute (R01 HL130399) to AOD. 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.

Shear Stress Induces Morphology Changes in Human Lung Microvascular Endothelial Cells via Calcium Influx

Introduction: The pulmonary vascular endothelium is in contact with laminar blood flow, resulting in a physiologic degree of shear stress exerted on endothelial cells (ECs). In vitro cell culture of ECs is typically performed under static conditions, leading to underappreciation of the impact of shear stress. Calcium signaling is among the most ubiquitous cell signaling mechanisms, and changes in intracellular calcium concentration ([Ca2+]i) have been linked to shear stress. The impact of shear stress on human lung microvascular endothelial cell (hLMVEC) morphology and cell signaling is not well described. We hypothesized that hLMVECs exposed to shear stress would undergo Ca2+-dependent morphologic changes. Methods: The effect of shear stress on [Ca2+]i was first tested using ratiometric measurement. Early passage (p≤8) primary hLMVECs from three unique donor lots were plated in gelatin-coated Ibidi 0.2 uM depth plates for 16 h, then incubated with the calcium indicator Fura-2 AM for 1 h. Measurement of [Ca2+]i was obtained over 15 minutes under static and physiologic shear stress (0 and 12 dyn/cm2, respectively), using validated flow rates from the Ibidi system. Measured [Ca2+]i was compared between cells treated vehicle or an inhibitor of the mechanosensitive Ca2+-permeable channel TRPV4 (HC-067047). HC treatment was for 1 h prior to and throughout the duration of shear exposure. Morphology changes were then assessed in hLMVECs from the same three cell lots grown in standard 6-well plates; one plate kept in static culture, while another was exposed to 24 h of 12 dyn/cm2 shear stress using an orbital shaker. Treatments in each plate were control, vehicle, and HC. Representative photos of each well were taken at 0 h and at 24 h. Average ratios of shortest to longest dimensions from 30 cells/field were measured using ImageJ and compared between treatments and shear conditions. A 2-way ANOVA with post-test was used to test for statistically significant changes in morphology. Results: Shear stress increased [Ca2+]i within 15 minutes, and both baseline [Ca2+]i and observed change in response to shear stress were decreased by TRPV4 inhibition with HC. Morphologic changes were observed after exposure to shear stress on microscopic examination, with cells grown in static culture having typical cobblestone appearance and shear-adapted cells having an elongated, spindle like appearance aligned in the direction of flow. The calculated ratio (shortest to longest dimension) of all wells at 0 h was ~0.70 with no significant differences between groups. 24 h of shear stress caused a nearly 80% decrease in ratio compared to static culture in untreated cells (p<0.05), with a decrease of only 30% in HC-treated cells after 24 h of shear (p<0.05). Conclusions: Exposure to shear stress at physiological levels results in a rapid increase [Ca2+]i through mechanosensitive, Ca2+-permeable TRPV4 channels as well as notable morphologic changes to hLMVECs with prolonged exposure to shear stress that appear to be mediated in large part by TRPV4. Further investigation into the impact of shear stress on hLMVECs is warranted to elucidate biochemical pathways and other downstream functional consequences of increased [Ca2+]i. NIH-T32HL007534. 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.

Remodeled Extracellular Matrix Guides Endothelial Cell Phenotype and Transcriptome to Fibrotic Niche

Impairment of endothelial barrier integrity causes vascular injury leading to acute lung injury (ALI), which, if not resolved, can compromise a patient’s lung functions and mortality. While it is known that both endothelial cells (EC) and their underlying extracellular matrix (ECM) are required for preserving lung homeostasis and functions, much of the efforts to understand mechanisms regulating these functions have focused mainly on EC. A fundamental unresolved question is whether lung-remodeled ECM formed due to leaky endothelium impacts EC functions, and if so, how? Herein, using lung ECM derived from a mouse lacking focal adhesion kinase (FAK), specifically in the endothelium (remodeled ECM), we assessed ECM components and their role in regulating EC functions and their relevance in vivo. Previously, we have shown that conditional deletion of FAK in endothelium increases EC permeability and induces vascular injury at homeostasis. Mass spectrometric analysis showed 805 proteins in control ECM (FAKfl/fl mice lung derived ECM) consisting of glycoproteins, matrisome-associated proteins, and secreted factors. However, the remodeled ECM had 722 proteins and was highly enriched with fibrillin 1, fibrinogen, and collagen I. Moreover, the remodeled ECM was two-fold stiffer than the control ECM. Remodeled ECM with its inherent composition recapitulates the 3D microenvironment, encouraging EC morphological alteration, increased cell stiffness, and enhanced focal adhesion (FA) formation compared to control ECM. In addition, RNA sequencing identified increased expression of genes linked with fibrosis, including integrin b8, TGFb2, GDF15, SMAD7, COL1A1, IL33, ID3, BMPR1A, and ADAMTS4 in EC seeded on remodeled ECM. Interestingly, fibrotic signaling observed in EC plated on remodeled ECM mimicked in vivo in EC-FAK−/− mice as they showed increased hydroxyproline content and collagen deposition in the lungs, indicating loss of EC-FAK as the crucial initiator of fibrotic signaling. In summary, studies for the first time show disrupted EC signaling as the instructor of fibrotic niche in the lungs for targeting pulmonary fibrosis. NIH. 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.

Abstract 1051: Endothelial CEPT1 Impacts Aortic Atherosclerosis And Hepatic Lipoprotein Metabolism

Background: Circulating lipids that contribute to the development of atherosclerosis are derived from very low-density lipoprotein (VLDL), produced in liver by a process requiring microsomal triglyceride transfer protein (MTTP). Since choline ethanolamine phosphotransferase 1 (CEPT1) generates phospholipids critical for membrane composition and signaling, we hypothesized that Cept1 deficiency reduces hepatic MTTP and aortic atherosclerosis. Methods: Conditional endothelial cell (EC)-specific knockdown of Cept1 ( Cept1 fl/fl Cre+ ) was performed in ApoE -/- mice maintained on a 42% high-fat diet for 12 weeks, serum lipids were measured, and aorta, aortic roots, and liver tissue were harvested (Figure, A). In addition, EC-hepatocyte (HepG2 cells) signaling was evaluated using an in vitro co-culture assay, and Mttp gene expression was assessed in HepG2 cells, cultured with and without ECs treated with Cept1 siRNA (Figure, B and C). Results: Compared to ApoE -/- mice, Cept1 fl/fl Cre+ ApoE -/- mice had significantly reduced serum cholesterol (P < .05; Figure, D) and triglyceride (P < .01; Figure, E), and reduced atherosclerotic lesions of the aorta and aortic root (P < .01 and P < .05, respectively; Figure, F-I). Cept1 fl/fl Cre+ ApoE -/- mice also demonstrated more severe hepatic steatosis and numerically greater content of liver lipid droplets (Figure, J and K). Cept1 knockdown in ECs co-cultured with HepG2 cells resulted in a significant decrease in Mttp expression (P < .01; Figure, L). Conclusion: Our findings suggest that deficiency of endothelial Cept1 significantly reduces aortic atheroprogression. This may, in part, be due to a reduction in serum lipid levels via inhibition of hepatocyte MTTP driven by signals from endothelial cells. These findings open a new avenue of exploration regarding the influence of EC signaling on hepatic lipid metabolism and its impact on hyperlipidemia and atherosclerotic cardiovascular diseases.

Emerging Roles of Spatial Transcriptomics in Liver Research.

Spatial transcriptomics, leveraging sequencing- and imaging-based techniques, has emerged as a groundbreaking technology for mapping gene expression within the complex architectures of tissues. This approach provides an in-depth understanding of cellular and molecular dynamics across various states of healthy and diseased livers. Through the integration of sophisticated bioinformatics strategies, it enables detailed exploration of cellular heterogeneity, transitions in cell states, and intricate cell-cell interactions with remarkable precision. In liver research, spatial transcriptomics has been particularly revelatory, identifying distinct zonated functions of hepatocytes that are crucial for understanding the metabolic and detoxification processes of the liver. Moreover, this technology has unveiled new insights into the pathogenesis of liver diseases, such as the role of lipid-associated macrophages in steatosis and endothelial cell signals in liver regeneration and repair. In the domain of liver cancer, spatial transcriptomics has proven instrumental in delineating intratumor heterogeneity, identifying supportive microenvironmental niches and revealing the complex interplay between tumor cells and the immune system as well as susceptibility to immune checkpoint inhibitors. In conclusion, spatial transcriptomics represents a significant advance in hepatology, promising to enhance our understanding and treatment of liver diseases.

Betaglycan sustains HGF/Met signaling in lung cancer and endothelial cells promoting cell migration and tumor growth

Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFβ receptor or TGFBR3), a multi-faceted proteoglycan TGFβ co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan.

A Review: The Significance of Toll-Like Receptors 2 and 4, and NF-κB Signaling in Endothelial Cells during Atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that begins with endothelial activation followed by a series of inflammatory responses, plaque formation, and finally rupture. An early event in endothelial dysfunction is activation of the nuclear factor-κB (NF-κB) signaling axis. Toll-like receptors (TLRs) in endothelial cells (ECs) play an essential role in recognizing pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and lifestyle-associated molecular patterns (LAMPs). Activation of the canonical NF-κB pathway stimulates the expression of cytokines, chemokines, and an array of additional genes which activate and amplify AS-associated inflammatory responses. In this review, we discuss the involvement of TLR2/4 and NF-κB signaling in ECs during AS initiation, as well as regulation of the inflammatory response during AS by noncoding RNAs, especially microRNA (miRNA) and circular RNA (circRNA).

Single-cell RNA sequencing reveals cellular and molecular landscape of fetal cystic hygroma

BackgroundThe molecular mechanism of fetal cystic hygroma (CH) is still unclear, and no study has previously reported the transcriptome changes of single cells in CH. In this study, single-cell transcriptome sequencing (scRNA-seq) was used to investigate the characteristics of cell subsets in the lesion tissues of CH patients.MethodsLymphoid tissue collected from CH patients and control donors for scRNA-seq analysis. Differentially expressed gene enrichment in major cell subpopulations as well as cell-cell communication were analyzed. At the same time, the expression and interactions of important VEGF signaling pathway molecules were analyzed, and potential transcription factors that could bind to KDR (VEGFR2) were predicted.ResultsThe results of scRNA-seq showed that fibroblasts accounted for the largest proportion in the lymphatic lesions of CH patients. There was a significant increase in the proportion of lymphatic endothelial cell subsets between the cases and controls. The VEGF signaling pathway is enriched in lymphatic endothelial cells and participates in the regulation of cell-cell communication between lymphatic endothelial cells and other cells. The key regulatory gene KDR in the VEGF signaling pathway is highly expressed in CH patients and interacts with other differentially expressed EDN1, TAGLN, and CLDN5 Finally, we found that STAT1 could bind to the KDR promoter region, which may play an important role in promoting KDR up-regulation.ConclusionOur comprehensive delineation of the cellular composition in tumor tissues of CH patients using single-cell RNA-sequencing identified the enrichment of lymphatic endothelial cells in CH and highlighted the activation of the VEGF signaling pathway in lymphoid endothelial cells as a potential modulator.Simple summaryThe molecular and cellular pathogenesis of fetal cystic hygroma (CH) remains largely unknown. This study examined the distribution and gene expression signature of each cell subpopulation and the possible role of VEGF signaling in lymphatic endothelial cells in regulating the progression of CH by single-cell transcriptome sequencing. The enrichment of lymphatic endothelial cells in CH and the activation of the VEGF signaling pathway in lymphatic endothelial cells provide some clues to the pathogenesis of CH from the perspective of cell subpopulations.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.