Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways.

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

Evidence for Pro-angiogenic Functions of VEGF-Ax.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Cell-Type-Specific Interleukin 1 Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities.

Interleukin-1 (IL-1) signaling is important for multiple potentially pathogenic processes in the central nervous system (CNS), but the cell-type-specific roles of IL-1 signaling are unclear. We used a genetic knockin reporter system in mice to track and reciprocally delete or express IL-1 receptor 1 (IL-1R1) in specific cell types, including endothelial cells, ventricular cells, peripheral myeloid cells, microglia, astrocytes, and neurons. We found that endothelial IL-1R1 was necessary and sufficient for mediating sickness behavior and drove leukocyte recruitment to the CNS and impaired neurogenesis, whereas ventricular IL-1R1 was critical for monocyte recruitment to the CNS. Although microglia did not express IL-1R1, IL-1 stimulation of endothelial cells led to the induction of IL-1 in microglia. Together, these findings describe the structure and functions of the brain's IL-1R1-expressing system and lay a foundation for the dissection and identification of IL-1R1 signaling pathways in the pathogenesis of CNS diseases.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Basic and therapeutic aspects of angiogenesis.

Blood vessels form extensive networks that nurture all tissues in the body. Abnormal vessel growth and function are hallmarks of cancer and ischemic and inflammatory diseases, and they contribute to disease progression. Therapeutic approaches to block vascular supply have reached the clinic, but limited efficacy and resistance pose unresolved challenges. Recent insights establish how endothelial cells communicate with each other and with their environment to form a branched vascular network. The emerging principles of vascular growth provide exciting new perspectives, the translation of which might overcome the current limitations of pro- and antiangiogenic medicine.

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

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

Human blood vessel organoids as a model of diabetic vasculopathy.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

Leptin treatment has vasculo-protective effects in lipodystrophic mice.

Lipodystrophy syndromes (LDs) are characterized by loss of adipose tissue, metabolic complications such as dyslipidemia, insulin resistance, and fatty liver disease, as well as accelerated atherosclerosis. As a result of adipose tissue deficiency, the systemic concentration of the adipokine leptin is reduced. A current promising therapeutic option for patients with LD is treatment with recombinant leptin (metreleptin), resulting in reduced risk of mortality. Here, we investigate the effects of leptin on endothelial to mesenchymal transition (EndMT), which impair the functional properties of endothelial cells and promotes atherogenesis in LD. Leptin treatment reduced inflammation and TGF-ß2-induced expression of mesenchymal genes and prevented impairment of endothelial barrier function. Treatment of lipodystrophic- and atherosclerosis-prone animals (Ldlr-/-; aP2-nSrebp1c-Tg) with leptin reduced macrophage accumulation in atherosclerotic lesions, vascular plaque protrusion, and the number of endothelial cells with mesenchymal gene expression, confirming a reduction in EndMT in LD after leptin treatment. Treatment with leptin inhibited LD-mediated induction of the proatherosclerotic cytokine growth/differentiation factor 15 (GDF15). Inhibition of GDF15 reduced EndMT induction triggered by plasma from patients with LD. Our study reveals that in addition to the effects on adipose tissue function, leptin treatment exerts beneficial effects protecting endothelial function and identity in LD by reducing GDF15.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Role of KCNK3 Dysfunction in Dasatinib-associated Pulmonary Arterial Hypertension and Endothelial Cell Dysfunction.

Pulmonary arterial (PA) hypertension (PAH) is a severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (Potassium channel subfamily K member 3; coding for outward K+ channel) variant in a patient with dasatinib-associated PAH and investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control human PA smooth muscle cells (hPASMCs) and human pulmonary endothelial cells (hPECs), we evaluated the consequences of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp experiments revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to PA constriction by decreasing KCNK3 function and expression. In control hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that a loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

GPR30 alleviated subarachnoid hemorrhage-induced blood-brain barrier dysfunction by activating the PI3K/Akt and Nrf2/HO-1 pathways.

The blood-brain barrier (BBB) plays a critical role in the development and outcome of subarachnoid hemorrhage (SAH). This study focuses on the potential mechanism by which G-protein-coupled estrogen receptor 30 (GPR30) affects the BBB after SAH. A rat SAH model was established using an intravascular perforation approach. G1 (GPR30 agonist) was administered to investigate the mechanism of BBB damage after SAH. Brain water content, Western blotting, Evans blue leakage, and immunofluorescence staining were performed. Brain microvascular endothelial cells were induced by hemin to establish SAH model in vitro. By adding LY294002 [a phosphatidylinositol 3-kinase (PI3K) blocker] and zinc protoporphyrin IX (ZnPP IX) [a heme oxygenase 1 (HO-1) antagonist], the mechanism of improving BBB integrity through the activation of GPR30 was studied. In vivo, GPR30 activation improved BBB disruption, as evidenced by decreased cerebral edema, downregulated albumin expression, and reduced extravasation of Evans blue and IgG after G1 administration in SAH rats. Moreover, SAH downregulated the levels of tight junction (TJ) proteins, whereas treatment with G1 reversed the effect of SAH. The protective effect of G1 on BBB integrity in vitro was consistent with that in vivo, as evidenced by G1 reducing the impact of hemin on transendothelial electrical resistance (TEER) value, dextran diffusivity, and TJ protein levels in brain microvascular endothelial cells. In addition, G1 activated the PI3K/ protein kinase B (Akt) and nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 pathways both in vivo and in vitro. Furthermore, the administration of LY294002 and ZnPP IX partially reversed the protective effect of G1 on BBB integrity in hemin-stimulated cells. We demonstrated that the activation of GPR30, at least partly through the PI3K/Akt and Nrf2/HO-1 pathways, alleviated BBB damage both in vivo and in vitro. This study introduced a novel therapeutic approach for protecting the BBB after SAH.NEW & NOTEWORTHY The PI3K/Akt and Nrf2/HO-1 pathways might be potential mechanisms by which GPR30 protected the integrity of the BBB in SAH models. Therefore, treatment of SAH with GPR30 activator might be a promising therapeutic strategy.

Puerarin mitigated LPS-ATP or HG-primed endothelial cells damage and diabetes-associated cardiovascular disease via ROS-NLRP3 signalling.

The occurrence and development of diabetic vascular diseases are closely linked to inflammation-induced endothelial dysfunction. Puerarin (Pue), the primary component of Pueraria lobata, possesses potent anti-inflammatory properties. However, its vasoprotective role remains elusive. Therefore, we investigated whether Pue can effectively protect against vascular damage induced by diabetes. In the study, Pue ameliorated lipopolysaccharide-adenosine triphosphate (LPS-ATP) or HG-primed cytotoxicity and apoptosis, while inhibited reactive oxygen species (ROS)-mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome in HUVECs, as evidenced by significantly decreased ROS level, NOX4, Caspase-1 activity and expression of NLRP3, GSDMD, cleaved caspase-1, IL-1ß and IL-18. Meanwhile, ROS inducer CoCI2 efficiently weakened the effects of Pue against LPS-ATP-primed pyroptosis. In addition, NLRP3 knockdown notably enhanced Pue's ability to suppress pyroptosis in LPS-ATP-primed HUVECs, whereas overexpression of NLRP3 reversed the inhibitory effects of Pue. Furthermore, Pue inhibited the expression of ROS and NLRP3 inflammasome-associated proteins on the aorta in type 2 diabetes mellitus rats. Our findings indicated that Pue might ameliorate LPS-ATP or HG-primed damage in HUVECs by inactivating the ROS-NLRP3 signalling pathway.

Naringin activates semaphorin 3A to ameliorate TGF-ß-induced endothelial-to-mesenchymal transition related to atrial fibrillation.

The loss of semaphorin 3A (Sema3A), which is related to endothelial-to-mesenchymal transition (EndMT) in atrial fibrosis, is implicated in the pathogenesis of atrial fibrillation (AF). To explore the mechanisms by which EndMT affects atrial fibrosis and assess the potential of a Sema3A activator (naringin) to prevent atrial fibrosis by targeting transforming growth factor-beta (TGF-ß)-induced EndMT, we used human atria, isolated human atrial endocardial endothelial cells (AEECs), and used transgenic mice expressing TGF-ß specifically in cardiac tissues (TGF-ß transgenic mice). We evaluated an EndMT marker (Twist), a proliferation marker (proliferating cell nuclear antigen; PCNA), and an endothelial cell (EC) marker (CD31) through triple immunohistochemistry and confirmed that both EndMT and EC proliferation contribute to atrial endocardial fibrosis during AF in TGF-ß transgenic mice and AF patient tissue sections. Additionally, we investigated the impact of naringin on EndMT and EC proliferation in AEECs and atrial fibroblasts. Naringin exhibited an antiproliferative effect, to which AEECs were more responsive. Subsequently, we downregulated Sema3A in AEECs using small interfering RNA to clarify a correlation between the reduction in Sema3A and the elevation of EndMT markers. Naringin treatment induced the expression of Sema3A and a concurrent decrease in EndMT markers. Furthermore, naringin administration ameliorated AF and endocardial fibrosis in TGF-ß transgenic mice by stimulating Sema3A expression, inhibiting EndMT markers, reducing atrial fibrosis, and lowering AF vulnerability. This suggests therapeutic potential for naringin in AF treatment.

Establishment of the deuterium oxide dilution method as a new possibility for determining the transendothelial water permeability.

Increase in transendothelial water permeability is an essential etiological factor in a variety of diseases like edema and shock. Despite the high clinical relevance, there has been no precise method to detect transendothelial water flow until now. The deuterium oxide (D2O) dilution method, already established for measuring transepithelial water transport, was used to precisely determine the transendothelial water permeability. It detected appropriate transendothelial water flow induced by different hydrostatic forces. This was shown in four different endothelial cell types. The general experimental setup was verified by gravimetry and absorbance spectroscopy. Determination of transendothelial electrical resistance (TEER) and immunocytochemical staining for proteins of the cell-cell contacts were performed to ensure that no damage to the endothelium occurred because of the measurements. Furthermore, endothelial barrier function was modulated. Measurement of transendothelial water flux was verified by measuring the TEER, the apparent permeability coefficient and the electrical capacity. The barrier-promoting substances cyclic adenosine monophosphate and iloprost reduced TEER and electrical capacity and increased permeability. This was accompanied by a reduced transendothelial water flux. In contrast, the barrier-damaging substances thrombin, histamine and bradykinin reduced TEER and electrical capacity, but increased permeability. Here, an increased water flow was shown. This newly established in vitro method for direct measurement of transendothelial water permeability was verified as a highly precise technique in various assays. The use of patient-specific endothelial cells enables individualized precision medicine in the context of basic edema research, for example regarding the development of barrier-protective pharmaceuticals.

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration.

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

Lysophosphatidic acid (LPA) receptor-mediated signaling regulates hypoxia-induced biological functions of lymphatic endothelial cells.

The tumor microenvironment is an extremely complex composed of cancer cells and various non-cancer cells, including lymphatic endothelial cells. Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) activate a variety of malignant properties in human malignancies. In the present study, we examined the roles of LPA receptor-mediated signaling in biological responses of lymphatic endothelial SVEC4-10 cells induced by hypoxia. Lpar1, Lpar2 and Lpar3 expressions were decreased in SVEC4-10 cells cultured at hypoxic conditions (1 % O2). LPA had no impact on the cell growth activity of SVEC4-10 cells in 21 % O2 culture conditions. Conversely, the cell growth activity of SVEC4-10 cells in 1 % O2 culture conditions was reduced by LPA. The cell motile activity of SVEC4-10 cells was elevated by 1 % O2 culture conditions. GRI-977143 (LPA2 agonist) and (2S)-OMPT (LPA3 agonist) stimulated SVEC4-10 cell motility as well as AM966 (LPA1 antagonist). In tube formation assay, the tube formation of SVEC4-10 cells in 1 % O2 culture conditions was markedly increased, in comparison with 21 % O2. GRI-977143 and (2S)-OMPT elevated the tube formation of SVEC4-10 cells. Furthermore, the tube formation of SVEC4-10 cells was increased by AM966. These results suggest that LPA receptor-mediated signaling contributes to the modulation of hypoxic-induced biological functions of lymphatic endothelial cells.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone.

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.