Signaling In Endothelial Cells Research Articles

WNK kinase is a vasoactive chloride sensor in endothelial cells

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl-) is the most abundant anion in ECs and the Cl- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl- concentration ([Cl-]i) due to the activation of TMEM16A, a Cl- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca2+ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca2+ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl-]i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl- signaling and WNK kinase activity in ECs to control arterial contractility.

Homocysteine metabolites inhibit autophagy by upregulating miR-21-5p, miR-155-5p, miR-216-5p, and miR-320c-3p in human vascular endothelial cells

Nutritional and genetic deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis, which is a major cause of cardiovascular disease (CVD). Impaired autophagy causes the accumulation of damaged proteins and organelles and is associated with CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone and N-Hcy-protein. However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of Hcy-thiolactone, N-Hcy-protein, and Hcy on autophagy human umbilical vein endothelial cells. We found that treatments with Hcy-thiolactone, N-Hcy-protein, or Hcy significantly downregulated beclin 1 (BECN1), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 (LC3) mRNA and protein levels. We also found that these changes were mediated by upregulation by Hcy-thiolactone, N-Hcy-protein, and Hcy of autophagy-targeting microRNA (miR): miR-21, miR-155, miR-216, and miR-320c. The effects of these metabolites on levels of miR targeting autophagy as well as on the levels of BECN1, ATG5, ATG7, and LC3 mRNA and protein were abrogated by treatments with inhibitors of miR-21, miR-155, miR-216, and mir320c. Taken together, our findings show that Hcy metabolites can upregulate miR-21, miR-155, miR-216, and mir320c, which then downregulate autophagy in human endothelial cells, important for vascular homeostasis.

RelA-mediated signaling connects adaptation to chronic cardiomyocyte stress with myocardial and systemic inflammation in the ADCY8 model of accelerated aging.

Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. We have previously demonstrated through bioinformatics that constitutive adenylyl cyclase activation in TGAC8 mice is associated with the activation of inflammation-related signaling pathways. However, the immune response associated with chronic myocardial stress in the TGAC8 mouse remains unexplored. Here we demonstrate that chronic activation of adenylyl cyclase in cardiomyocytes of TGAC8 mice results inactivation of cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. All these changes precede the appearance of cardiac fibrosis. We provide evidence indicating that RelA activation in cardiomyocytes with chronic activation of adenylyl cyclase is mediated by calcium-protein Kinase A (PKA) signaling. Using a model of chronic cardiomyocyte stress and accelerated aging, we highlight a novel, calcium/PKA/RelA-dependent connection between cardiomyocyte stress, myocardial inflammation, and systemic inflammation. These findings suggest that RelA-mediated signaling in cardiomyocytes might be an adaptive response to stress that, when chronically activated, ultimately contributes to bothcardiac and systemic aging.

Ca2+ signals in human umbilical endothelial cells derived from pregnancy with fetal growth restriction associated with hypertensive disorder.

Fetal growth restriction associated with hypertensive disorders of pregnancy (FGR-HDP) is a prevalent pathology with a higher risk of perinatal morbimortality. In this condition, placental insufficiency and endothelial dysfunction serve key roles. The present prospective cohort study monitored 11 patients with an FGR-HDP and 15 with full-term normotensive pregnancies and studied post-natal intracellular calcium concentration ([Ca2+]i) signals in human umbilical vein endothelial cells (HUVECs). Small fetuses with placental insufficiency were identified using fetal biometry with Doppler velocimetry. Mean gestational age and birth weight were 31.8±4.1 weeks and 1,260±646 g for FGR-HDP and 39.2±0.8 weeks and 3,320±336 g for normal births, respectively. Abnormal umbilical artery Doppler waveforms were found in 64% of neonates with FGR-HDP. A significant percentage (86%) of FGR newborns were admitted to the neonatal intensive care unit at Gustavo Fricke hospital, Viña del Mar, Chile, with one case of death after birth. [Ca2+]i signals were measured by microfluorimetry in Fluo-3-loaded HUVECs from primary cultures. Altered [Ca2+]i signals were observed in HUVECs from FGR-HDP, where the sustained phase of ATP-induced [Ca2+]i responses was significantly reduced compared with the normotensive group. Also, the [Ca2+]i signals induced with 10 mM Ca2+ after depletion of internal Ca2+ stores were significantly higher. The present study provides a better comprehension of the role of altered cytosolic Ca2+ dynamics in endothelial dysfunction and an in vitro model to assess novel therapeutic approaches for decreasing or preventing complications in FGR-HDP.

Endothelial POFUT1 controls injury-induced liver fibrosis by repressing fibrinogen synthesis

NOTCH signaling in liver sinusoidal endothelial cells (LSECs) regulates liver fibrosis, a pathological feature of chronic liver diseases. POFUT1 is an essential regulator of NOTCH signaling. Here, we investigated the role of LSEC-expressed POFUT1 in liver fibrosis. Endothelial-specific Pofut1 knockout mice were generated and experimental liver fibrosis was induced by chronic carbon tetrachloride exposure or common bile duct ligation. Liver samples were assessed by ELISA, histology, electron microscopy, immunostaining and RNA in situ hybridization. LSECs and hepatic stellate cells (HSCs) were isolated for gene expression analysis by RNA sequencing, qPCR, and western blotting. Signaling crosstalk between LSECs and HSCs was investigated by treating HSCs with supernatant from LSEC cultures. Liver single-cell RNA sequencing datasets from patients with cirrhosis and healthy individuals were analyzed to evaluate the clinical relevance of gene expression changes observed in mouse studies. POFUT1 loss promoted injury-induced LSEC capillarization and HSC activation, leading to aggravated liver fibrosis. RNA sequencing analysis revealed that POFUT1 deficiency upregulated fibrinogen expression in LSECs. Consistently, fibrinogen was elevated in LSECs of patients with cirrhosis. HSCs treated with supernatant from LSECs of Pofut1 null mice showed exacerbated activation compared to those treated with supernatant from control LSECs, and this effect was attenuated by knockdown of fibrinogen or by pharmacological inhibition of fibrinogen receptor signaling, altogether suggesting that LSEC-derived fibrinogen induced the activation of HSCs. Mechanistically, POFUT1 loss augmented fibrinogen expression by enhancing NOTCH/HES1/STAT3 signaling. Endothelial POFUT1 prevents injury-induced liver fibrosis by repressing the expression of fibrinogen, which functions as a profibrotic paracrine signal to activate HSCs. Therapies targeting the POFUT1/fibrinogen axis offer a promising strategy for the prevention and treatment of fibrotic liver diseases. Paracrine signals produced by liver vasculature play a major role in the development of liver fibrosis, which is a pathological hallmark of most liver diseases. Identifying those paracrine signals is clinically relevant in that they may serve as therapeutic targets. In this study, we discovered that genetic deletion of Pofut1 aggravated experimental liver fibrosis in mouse models. Moreover, fibrinogen was identified as a downstream target repressed by Pofut1 in liver endothelial cells and functioned as a novel paracrine signal that drove liver fibrosis. In addition, fibrinogen was found to be relevant to cirrhosis and may serve as a potential therapeutic target for this devastating human disease.

Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-β signaling in primary human dermal microvascular endothelial cells.

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-β secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-β secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-β production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-β production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-β response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-β secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-β response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

Single-Cell Transcriptomic Analysis of Salivary Gland Endothelial Cells

Vascular endothelial cells have important tissue-specific functions in fibrosis and regeneration. In the salivary gland, endothelial cells are required for proper development, but their roles within adult glands are largely unknown. To identify ligand–receptor interactions between endothelial cells and other cell types that may be important during fibrosis and regeneration, we used a reversible ductal ligation injury. To induce injury, a clip was applied to the primary ducts for 14 d, and to induce a regenerative response, the clip was subsequently removed for 5 d. To identify endothelial cell-produced factors, we used single-cell RNA sequencing of stromal-enriched cells from adult female submandibular and sublingual salivary glands. Transcriptional profiles of homeostatic salivary gland endothelial cells were compared to endothelial cells of other organs. Salivary gland endothelial cells expressed many unique genes and displayed the highest overlap in gene expression with other fenestrated endothelial cells from the colon, small intestine, and kidney. Comparison of the 14-d ligated, mock-ligated, and 5-d deligated stromal-enriched transcripts and lineage tracing revealed that endothelial cells retain their identity following ligation and recovery from injury. CellChat and NATMI were used to predict changes in ligand–receptor interactions from endothelial cells to other cells in response to ligation and deligation. CellChat and NATMI predicted that after ligation, interactions with fibroblasts, epithelial cells, and glial cells were increased, and following deligation, interactions with pericyte, glia, fibroblasts, and immune cells were increased. Some of the highest-ranked interactions predicted in ligated compared to mock endothelial cells were between glial cells via Col4a2-Cd93 and Jag2-Notch1, as well as epithelial cells via Pecam1-Cd38, while in deligated compared to ligated endothelial cells, the top interactions were between fibroblasts via Ntf3-Ntrk2, glial cells via Hspg2-Itgb1, and pericytes via Jam2-F11r. Understanding salivary gland endothelial cell signaling will inform future endothelial cell-based regenerative therapies.

Endothelial cells signaling and patterning under hypoxia: a mechanistic integrative computational model including the Notch-Dll4 pathway.

Introduction: Several signaling pathways are activated during hypoxia to promote angiogenesis, leading to endothelial cell patterning, interaction, and downstream signaling. Understanding the mechanistic signaling differences between endothelial cells under normoxia and hypoxia and their response to different stimuli can guide therapies to modulate angiogenesis. We present a novel mechanistic model of interacting endothelial cells, including the main pathways involved in angiogenesis. Methods: We calibrate and fit the model parameters based on well-established modeling techniques that include structural and practical parameter identifiability, uncertainty quantification, and global sensitivity. Results: Our results indicate that the main pathways involved in patterning tip and stalk endothelial cells under hypoxia differ, and the time under hypoxia interferes with how different stimuli affect patterning. Additionally, our simulations indicate that Notch signaling might regulate vascular permeability and establish different Nitric Oxide release patterns for tip/stalk cells. Following simulations with various stimuli, our model suggests that factors such as time under hypoxia and oxygen availability must be considered for EC pattern control. Discussion: This project provides insights into the signaling and patterning of endothelial cells under various oxygen levels and stimulation by VEGFA and is our first integrative approach toward achieving EC control as a method for improving angiogenesis. Overall, our model provides a computational framework that can be built on to test angiogenesis-related therapies by modulation of different pathways, such as the Notch pathway.

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Critical Role of CD55 in Controlling Wound Healing.

How reparative processes are coordinated following injury is incompletely understood. In recent studies, we showed that autocrine C3a and C5a receptor (C3ar1 and C5ar1) G protein-coupled receptor signaling plays an obligate role in vascular endothelial growth factor receptor 2 growth signaling in vascular endothelial cells. We documented the same interconnection for platelet-derived growth factor receptor growth signaling in smooth muscle cells, epidermal growth factor receptor growth signaling in epidermal cells, and fibroblast growth factor receptor signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. In this study, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 (also known as decay accelerating factor), which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is heightened C3ar1/C5ar1 signaling resulting from the loss of CD55's restraint jointly potentiating growth factor production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury (burn, corneal denudation, ear lobe puncture, and reengraftment of autologous skin) showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In wild-type mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking Ab (against CD55's active site) to wounds accelerated the healing rate by 40-70%. To our knowledge, these results provide new insights into mechanisms that underlie wound repair and open up a new tool for accelerating healing.

Hyaluronic acid turnover controls the severity of cerebral cavernous malformations in bioengineered human micro-vessels.

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell-matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

Calcium signaling in endothelial and vascular smooth muscle cells: sex differences and the influence of estrogens and androgens.

Calcium signaling in vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) is essential for the regulation of vascular tone. However, the changes to intracellular Ca2+ concentrations are often influenced by sex differences. Furthermore, a large body of evidence shows that sex hormone imbalance leads to dysregulation of Ca2+ signaling and this is a key factor in the pathogenesis of cardiovascular diseases. In this review, the effects of estrogens and androgens on vascular calcium-handling proteins are discussed, with emphasis on the associated genomic or nongenomic molecular mechanisms. The experimental models from which data were collected were also considered. The review highlights 1) in female ECs, transient receptor potential vanilloid 4 (TRPV4) and mitochondrial Ca2+ uniporter (MCU) enhance Ca2+-dependent nitric oxide (NO) generation. In males, only transient receptor potential canonical 3 (TRPC3) plays a fundamental role in this effect. 2) Female VSMCs have lower cytosolic Ca2+ levels than males due to differences in the activity and expression of stromal interaction molecule 1 (STIM1), calcium release-activated calcium modulator 1 (Orai1), calcium voltage-gated channel subunit-α1C (CaV1.2), Na+-K+-2Cl- symporter (NKCC1), and the Na+/K+-ATPase. 3) When compared with androgens, the influence of estrogens on Ca2+ homeostasis, vascular tone, and incidence of vascular disease is better documented. 4) Many studies use supraphysiological concentrations of sex hormones, which may limit the physiological relevance of outcomes. 5) Sex-dependent differences in Ca2+ signaling mean both sexes ought to be included in experimental design.

Polyethylenimine Triggers Dll4 Degradation to Regulate Angiogenesis In Vitro.

The Dll4-Notch signaling pathway plays a crucial role in the regulation of angiogenesis and is a promising therapeutic target for diseases associated with abnormal angiogenesis, such as cancer and ophthalmic diseases. Here, we find that polyethylenimine (PEI), a cationic polymer widely used as nucleic acid transfection reagents, can target the Notch ligand Dll4. By immunostaining and immunoblotting, we demonstrate that PEI significantly induces the clearance of cell-surface Dll4 and facilitates its degradation through the lysosomal pathway. As a result, the activation of Notch signaling in endothelial cells is effectively inhibited by PEI, as evidenced by the observed decrease in the generation of the activated form of Notch and expression of Notch target genes Hes1 and Hey1. Furthermore, through blocking Dll4-mediated Notch signaling, PEI treatment enhances angiogenesis in vitro. Together, our study reveals a novel biological effect of PEI and establishes a foundation for the development of a Dll4-targeted biomaterial for the treatment of angiogenesis-related disease.

Desmoglein-2 Affects Vascular Function in Moyamoya Disease by Interacting with MMP-9 and Influencing PI3K Signaling

The pathogenesis and development of Moyamoya disease are still unclear. This study aimed to investigate the effect of desmoglein-2 (DSG2) on Moyamoya disease and determine the inhibitory effect of DSG2 in vascular remodeling in Moyamoya disease.RNA sequencing, immunohistochemistry (IHC), and western blotting were used to detect the expression of DSG2 in the superficial temporal artery (STA) tissues of Moyamoya disease. The association between DSG2 and endothelial cells’ biological activities was investigated by cell counting kit-8 (CCK-8), migration assay, tube formation assay, flow cytometry with Annexin V-FITC/PI staining, and TUNEL apoptotic cell detection kit. Pathways affected by overexpression or knockdown of DSG2 were identified in endothelial cells.The expression of DSG2 in the STA tissues of Moyamoya disease was lower than that in normal controls. Overexpression of DSG2 inhibits the proliferation and migration but promotes apoptosis in endothelial cells, and low DSG2 levels result in impaired angiogenesis. In addition, there was an interaction between DSG2 and MMP-9, and DSG2 acted through the PI3K signaling in endothelial cells.Our results indicate that DSG2 affects PI3K signaling in vascular endothelial cells, and MMP-9 is involved in DSG2-mediated vascular changes in Moyamoya disease.

Shear Stress and Sub-Femtomolar Levels of Ligand Synergize to Activate ALK1 Signaling in Endothelial Cells.

Endothelial cells (ECs) respond to concurrent stimulation by biochemical factors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported synergy between SS and BMP9 to include BMP10 and BMP9/10. Moreover, we demonstrated that this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1's basal activity and response to minimal ligand levels depend on endocytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. However, an in-depth analysis of ALK1 receptor trafficking's molecular mechanisms requires further investigation.

Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes.

Engineered Wnt7a ligands rescue blood-brain barrier and cognitive deficits in a COVID-19 mouse model.

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating cerebrovascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we performed unbiased transcriptional analysis to identify brain endothelial cell signalling pathways dysregulated by mouse adapted SARS-CoV-2 MA10 in aged immunocompetent C57Bl/6 mice in vivo. This analysis revealed significant suppression of Wnt/β-catenin signalling, a critical regulator of blood-brain barrier (BBB) integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/β-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected BBB integrity, reduced T-cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/β-catenin signalling or its downstream effectors could be potential interventional strategies for restoring cognitive health following viral infections.

Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced β-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.

Abstract A044: P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination

Abstract The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neural cell signaling. Here we report the importance of ATP-dependent P2Y2 signaling in breast epithelial cells and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular calcium (Ca2+) in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca2+ responses. The non-tumorigenic cells respond to increased Ca2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. Citation Format: Makenzy Mull, Stephen Pratt, Keyata Thompson, David Annis, Julia Ju, Megan Stemberger, Liron Boyman, Michele Vitolo, W. Jonathan Lederer, Stuart Martin. P2Y2 signaling disruption reduces ATP-dependent calcium elevation and actin localization to promote breast tumor cell dissemination [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A044.

Alcohol and e-cigarette damage alveolar-epithelial barrier by activation of P2X7r and provoke brain endothelial injury via extracellular vesicles

BackgroundUse of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC).MethodsWe used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75 µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca2+) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca2+ levels.ResultsETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca2+ accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca2+ levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function.ConclusionAbusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.