Morphogenetic Roles of Hydrostatic Pressure in Animal Development.

During organismal development, organs and systems are built following a genetic blueprint that produces structures capable of performing specific physiological functions. Interestingly, we have learned that the physiological activities of developing tissues also contribute to their own morphogenesis. Specifically, physiological activities such as fluid secretion and cell contractility generate hydrostatic pressure that can act as a morphogenetic force. Here, we first review the role of hydrostatic pressure in tube formation during animal development and discuss mathematical models of lumen formation. We then illustrate specific roles of the notochord as a hydrostatic scaffold in anterior-posterior axis development in chordates. Finally, we cover some examples of how fluid flows influence morphogenetic processes in other developmental contexts. Understanding how fluid forces act during development will be key for uncovering the self-organizing principles that control morphogenesis.

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.

Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype.

BACKGROUND: During human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation. METHODS: Primary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy. RESULTS: Our results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-ß type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos. CONCLUSIONS: Our study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.

The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells.

Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.

A 1, 4-benzoquinone derivative isolated from Ardisia crispa (Thunb.) A. DC. root suppresses angiogenesis via its angiogenic signaling cascades.

The root hexane extract of Ardisia crispa (ACRH), which belongs to the Primulaceae family, has been reported to possess anti-inflammatory, chemopreventive, anti-arthritic, and antiangiogenic activities. In this study, we isolated a p-benzoquinone derivative, 2-methoxy-6-undecyl-1,4-benzoquinone (AC2), from ACRH and investigated its potential antiangiogenic activity in human umbilical vein endothelial cells (HUVECs) and zebrafish embryo models. Prior to this study, AC2 was characterized using 1H NMR spectroscopy and MS. AC2 significantly suppressed HUVEC proliferation in a time-independent manner, with an IC50 value of 1.35 ± 0.05, 1.15 ± 0.02, and 1.00 ± 0.01 µg/mL at 24, 48, and 72 h, respectively. AC2 also induced apoptosis in HUVECs and significantly suppressed their migration, invasion, and tube formation in a concentration-dependent manner. Additionally, AC2 significantly attenuated most of the analyzed protein markers, including pro-MMP-2, VEGF-C, VEGF-D, angiopoietin-2, endothelin-1, fibroblast growth factor (FGF)-1, FGF-2, follistatin, heparin-binding epidermal growth factor-like growth factor (HB-EGF), and hepatocyte growth factor (HGF) at all tested concentrations. Furthermore, AC2 significantly inhibited zebrafish embryo intersegmental vessels (ISVs), confirming its antiangiogenic role. In conclusion, AC2 exhibits a potential anti-angiogenic effect by suppressing several proangiogenic and growth factors. Further studies are needed to investigate their effects on other excessive angiogenic diseases.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

Exosomal miR-133a-3p promotes the growth and metastasis of lung cancer cells following incomplete microwave ablation.

PURPOSE: Exosomal miRNAs play key roles in various biological processes such as cell proliferation, angiogenesis, migration and invasion. We explored whether exosomal miRNAs can promote local recurrence (LR) of lung tumors following incomplete microwave ablation (MWA) therapy. METHODS: Exosomal miRNA profiles before and after incomplete MWA in lung cancer (LC) patients with LR (n = 3) were sequenced and compared. The differentially expressed miRNAs of interest were validated in clinical samples (n = 10) and MWA-treated cells using RT-qPCR analysis. Target genes of the miRNAs were predicted and validated. The biological functions of miRNAs in proliferation, angiogenesis and metastasis of A549 cells were evaluated in vitro and in vivo. RESULTS: A total of 270 miRNAs (243 upregulated and 27 downregulated) were differentially expressed after incomplete MWA in patients with local recurrence. Upregulation of miR-133a-3p after MWA was validated in the cells and clinical samples. Cell functional experiments suggested that miR-133a-3p overexpression derived from serum exosomes increased cell viability, migration and invasion ability, tube formation activity and proliferation of A549 cells. Sirtuin 1 (SIRT1) was identified as a target gene for miR-133a-3p. Moreover, miR-133a-3p delivered by exosomes significantly promoted tumor growth, paralleled by reduced SIRT1 expression in a subcutaneous tumorigenesis animal model and increased the number of lung nodules by tail vein metastasis in vivo. CONCLUSION: Exosomal miR-133a-3p overexpression promoted tumor growth and metastasis following MWA and could be a promising biomarker for LC recurrence after incomplete MWA.

Cryopreservation of human cerebral microvascular endothelial cells with glycerol.

The cryopreservation of human cerebral microvascular endothelial cells (hCMEC) has facilitated their commercial availability for research studying the blood-brain barrier. The currently employed cryopreservation protocol uses 10% dimethyl sulfoxide (Me2SO) in cell medium, or 5% Me2SO in 95% fetal bovine serum (FBS) as cryoprotective agents (CPAs). However, Me2SO is toxic to cells and FBS is animal-derived and not chemically defined, so reducing the concentrations of these components is desirable. Recently, we showed that cryopreserving hCMEC in cell medium with 5% Me2SO and 6% hydroxyethyl starch (HES) results in over 90% post-thaw cell viability. This previous work was performed using an interrupted slow cooling (graded freezing) approach followed by SYTO13/GelRed staining to assay for membrane integrity. In this paper, we repeated graded freezing of hCMEC in cell medium containing 5% Me2SO and 6% HES, but this time using Calcein AM/propidium iodide staining to ensure that the stain is an equivalent alternative to SYTO13/GelRed for assessment of cell viability, and that results are comparable to those previously published. Next, using graded freezing experiments and Calcein AM/propidium iodide staining, we examined the effectiveness of non-toxic glycerol as a CPA at different concentrations, loading times, and cooling rates. The cryobiological response of hCMEC was used to develop a protocol that optimizes both the permeating and non-permeating capabilities of glycerol. HCMEC in cell medium loaded with 10% glycerol for 1 h at room temperature, ice nucleated at -5 °C and held for 3 min, and then cooled at -1 °C/min to -30 °C before plunging into liquid nitrogen had post-thaw viability of 87.7% ± 1.8%. Matrigel tube formation assay and immunocytochemical staining of junction protein ZO-1 were carried out on post-thaw hCMEC to ensure that the cryopreserved cells were viable and functional, in addition to being membrane-intact.

A SIRT6 Inhibitor, Marine-Derived Pyrrole-Pyridinimidazole Derivative 8a, Suppresses Angiogenesis.

Angiogenesis refers to the process of growing new blood vessels from pre-existing capillaries or post-capillary veins. This process plays a critical role in promoting tumorigenesis and metastasis. As a result, developing antiangiogenic agents has become an attractive strategy for tumor treatment. Sirtuin6 (SIRT6), a member of nicotinamide adenine (NAD+)-dependent histone deacetylases, regulates various biological processes, including metabolism, oxidative stress, angiogenesis, and DNA damage and repair. Some SIRT6 inhibitors have been identified, but the effects of SIRT6 inhibitors on anti-angiogenesis have not been reported. We have identified a pyrrole-pyridinimidazole derivative 8a as a highly effective inhibitor of SIRT6 and clarified its anti-pancreatic-cancer roles. This study investigated the antiangiogenic roles of 8a. We found that 8a was able to inhibit the migration and tube formation of HUVECs and downregulate the expression of angiogenesis-related proteins, including VEGF, HIF-1α, p-VEGFR2, and N-cadherin, and suppress the activation of AKT and ERK pathways. Additionally, 8a significantly blocked angiogenesis in intersegmental vessels in zebrafish embryos. Notably, in a pancreatic cancer xenograft mouse model, 8a down-regulated the expression of CD31, a marker protein of angiogenesis. These findings suggest that 8a could be a promising antiangiogenic and cancer therapeutic agent.

USP14 Regulates ATF2/PIK3CD Axis to Promote Microvascular Endothelial Cell Proliferation, Migration, and Angiogenesis in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness in diabetic patients. However, the pathogenesis of DR is complex, and no firm conclusions have been drawn so far. It has become a hot spot in ophthalmology research to deeply study the mechanism of DR pathological changes and find effective treatment options. Human retinal microvascular endothelial cells (HRMECs) were induced by high glucose (HG) to construct DR cell model. CCK-8 assay was used to detect the viability of HRMECs. Transwell assay was used to detect the migration ability of HRMECs. Tube formation assay was used to identify the tube formation ability of HRMECs. The expressions of USP14, ATF2 and PIK3CD were detected by Western blot analysis and qRT-PCR assay. Immunoprecipitation (IP) was used to ascertain the relationship of USP14 and ATF2. To explore the regulatory relationship between ATF2 and PIK3CD by dual-luciferase reporter gene assay and Chromatin immunoprecipitation (ChIP) assay. High glucose treatment promoted the proliferation, migration, and tube formation of HRMEC, and the expressions of USP14, ATF2 and PIK3CD were significantly up-regulated. USP14 or ATF2 knockdown inhibited HG-induced HRMECs proliferation, migration, and tube formation. USP14 regulated the expression of ATF2, and ATF2 promoted PIK3CD expression. PIK3CD overexpression attenuated the inhibitory effectiveness of USP14 knockdown on proliferation, migration and tube formation of DR cell model. Here, we revealed that USP14 regulated the ATF2/PIK3CD axis to promote proliferation, migration, and tube formation in HG-induced HRMECs.

Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM2.5 exposure.

Cooking oil fume-derived PM2.5 (COF-PM2.5) is a major source of indoor air contamination in China, which has been demonstrated to be a hazard factor of cardiovascular and cerebrovascular diseases. This study aimed to investigate the role of ROS-mediated PERK/ATF4 signaling activation in COF-PM2.5-inhibited extracorporeal tube formation in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 100 µg/mL COF-PM2.5 at different times, with or without 100 nM PERK activity inhibitor GSK2606414 (GSK) or 200 µM antioxidant N-acetylcysteine (NAC) pretreatment. Our results showed that COF-PM2.5 exposure can inhibit extracorporeal tube formation and down-regulate VEGFR2 expression in HUVECs. Furthermore, our data indicated that COF-PM2.5 exposure can activate the PERK/ATF4 signaling in HUVECs. Mechanistically, pretreatment with GSK interdicted PERK/ATF4 signaling, thereby reversing COF-PM2.5-downregulated VEGFR2 protein expression in HUVECs. Furthermore, NAC reversed VEGFR2 expression downregulated induced by COF-PM2.5 by inhibiting the upregulation of intracellular ROS levels and PERK/ATF4 signaling in HUVECs. As above, COF-PM2.5 exposure could induce ROS release from HUVECs, which in turn activate the endoplasmic reticulum PERK/ATF4 signaling and inhibit tube formation of HUVECs.

The proliferation and angiogenesis in hemangioma-derived endothelial cells is affected by STC2 medicated VEGFR2/Akt/eNOS pathway.

Objective: Stanniocalcin-2 (STC2), a secreted glycoprotein that is involved in the regulation of angiogenesis, was proposed as one of the mechanisms of neovascularization in hemangioma (HA). We aimed to investigate the effect of STC2 on proliferation and angiogenesis in hemangioma-derived endothelial cells. Methods: The hemangioma samples from HA patients with the median age of six months were surgically collected in the Affiliated Hospital of Weifang Medical University from October 2019 to June 2021, and divided into normal skin tissues (n=10), involuting-phase HAs (n=10) and proliferating-phase HAs (n=10) according to the Mulliken classification. The expression of STC2 was detected in involuting-phase HAs and proliferating-phase HAs. Hemangioma endothelial cells (HemEC) were transfected with small interfering RNA (siRNA) specific for STC2, and cell survival and tube formation were analyzed. Results: STC2 expression in proliferating-phase HAs was markedly higher than in the normal skin tissues and involving-phase HAs. Similarly, STC2 expression was higher in HemEC compared to the control human umbilical vein endothelial cells (HUVEC). Knockdown of STC2 slowed the proliferation of HemEC and decreased the expression of proliferating cell nuclear antigen (PCNA) in HemEC. Moreover, knockdown of STC2 in HemEC inhibited vascular endothelial cell angiogenesis and regulated the expression and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). Mechanistically, STC2 knockdown attenuated the activation of Akt/eNOS signaling, which was abolished by insulin growth factor-1 (IGF-1), the activator of Akt signaling, accompanying by increased proliferation and tube formation of HemEC. Conclusion: Inhibition of STC2 suppresses HemEC proliferation and angiogenesis by VEGFR2/Akt/eNOS pathway, which warrants further development of STC2-based strategies for HA treatment.

Alpha-crystallin B chains in trastuzumab-resistant breast cancer cells promote endothelial cell tube formation through activating mTOR.

The specific human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibody trastuzumab shows considerable clinical efficacy in patients with HER2-overexpressing breast cancer. However, about 20% of patients who receive trastuzumab in the adjuvant setting relapse, and approximately half of patients with metastatic HER2-positive breast cancer develop resistance to trastuzumab within 1 year. Although the mechanism of trastuzumab resistance has been explored broadly, whether and how angiogenesis participates in trastuzumab resistance is unclear. Here, we examined the association between angiogenesis and trastuzumab resistance by using a trastuzumab-resistant cell line (SKBR3-TR). Compared with that from the parental trastuzumab-sensitive SKBR3 cells, the culture supernatant from SKBR3-TR cells significantly increased the sprouting of endothelial cells. To identify intercellular features that contribute to the induction of endothelial tube formation, proteomics revealed that α-crystallin B chain (αB-crystallin) was upregulated in SKBR3-TR cells. Moreover, silencing of αB-crystallin significantly repressed SKBR3-TR-induced tube formation, and knockdown of αB-crystallin in SKBR3-TR cells suppressed the activation of mechanistic target of rapamycin (mTOR) in endothelial cells. In addition, treatment with rapamycin, an inhibitor of mTOR, reversed the SKBR3-TR-induced promotion of tube formation. In summary, αB-crystallin enhanced the ability of SKBR3-TR cells to activate mTOR in endothelial cells and thus promote angiogenesis.

Improvement of tube formation model of cell: Application for acute hypoxia in in vitro study of angiogenesis.

Angiogenesis caused by acute vascular occlusion occurs in various ischemic diseases. The in vitro tube formation assay by endothelial cells is a rapid, quantitative method for drug discovery on angiogenesis. Tube formation assay on Matrigel has been widely used to identify the angiogenesis, however, there are some problems to limit its application. In this study, we found for the first time that sodium dithionite (SD) could induce endothelial cell tube formation without Matrigel under hypoxia condition. To further verify our findings, the angiogenesis related proteins and mRNA at different time points after tube formation were measured both in primary human large-vessel endothelial cell (HUVECs) and murine microvascular endothelial cell line (Bend.3). In conclusion, compared with traditional tube formation on Matrigel, the novel model exhibits the following advantages: (1) Combination oxygen glucose deprivation with sodium dithionite (OGD-SD) model is operated more easily than traditional tube formation. (2) OGD-SD can be used for not only cell imaging, but also immunofluorescence, protein extraction and gene analysis. (3) OGD-SD is more applicable to acute hypoxia model of endothelial cell in vitro. (4) OGD-SD may be more suitable to identify molecular mechanism of compound that intervenes processes of pro-tube formation, tube formation and tube disconnection.

SMAD-specific E3 ubiquitin ligase 2 promotes angiogenesis by facilitating PTX3 degradation in MSCs from patients with ankylosing spondylitis.

Dysregulated angiogenesis of mesenchymal stem cells (MSCs) is closely related to inflammation and disrupted bone metabolism in patients with various autoimmune diseases. However, the role of MSCs in the development of abnormal angiogenesis in patients with ankylosing spondylitis (AS) remains unclear. In this study, we cultured human umbilical vein endothelial cells (HUVECs) with bone marrow-derived MSCs from patients with AS (ASMSCs) or healthy donors (HDMSCs) in vitro. Then, the cocultured HUVECs were assayed using a cell counting kit-8 (CCK-8) to evaluate the cell proliferation. A wound healing assay was performed to investigate cell migration, and a tube formation assay was conducted to determine the angiogenesis efficiency. ASMSCs exhibited increased angiogenesis, and increased expression of SMAD-specific E3 ubiquitin ligase 2 (Smurf2) in MSCs was the main cause of abnormal angiogenesis in patients with AS. Downregulation of Smurf2 in ASMSCs blocked angiogenesis, whereas overexpression of Smurf2 in HDMSCs promoted angiogenesis. The pro-angiogenic effect of Smurf2 was confirmed by the results of a Matrigel plug assay in vivo. By functioning as an E3 ubiquitin ligase in MSCs, Smurf2 regulated the levels of pentraxin 3 (PTX3), which has been shown to suppress angiogenesis through the PTX3-fibroblast growth factor 2 pathway. Moreover, Smurf2 transcription was regulated by activating transcription factor 4-induced endoplasmic reticulum stress. In conclusion, these results identify novel roles of Smurf2 in negatively regulating PTX3 stability and promoting angiogenesis in ASMSCs.

Cardiac Progenitor Cells and Adipocyte Stem Cells from Same Patients Exhibit In Vitro Functional Differences.

Cardiac progenitor cells (CPCs) and adipocyte stem cells (ASCs) are widely tested for their efficacy in repairing the diseased heart with varying results. However, no study has directly compared the functional efficacy of CPCs and ASCs collected from the same patient. CPCs and ASCs were isolated from the right atrial appendage and epicardial adipose tissue of the same patients, using explant culture. The flow cytometry analysis confirmed that both the cell types express common mesenchymal stem cells markers CD90 and CD105. ASCs, in addition, expressed CD29 and CD73. The wound-healing assay demonstrated that CPCs migrate faster to cover the wound area. Both cell types were resistant to hypoxia-induced cell death when exposed to hypoxia and serum deprivation; however, the ASCs showed increased proliferation. Conditioned medium (CM) collected after culturing serum-deprived CPCs and ASCs showed differential secretion patterns, with ASC CM showing an increased IGF-1 level, while CPC CM showed an increased FGF level. Only CPC CM reduced hypoxia-induced apoptosis in AC-16 human ventricular cardiomyocytes, while vascular network formation by endothelial cells was comparable between CPC and ASC CM. In conclusion, ASCs and CPCs exhibit differential characteristics within the same patient, and in vitro studies showed that CPCs have marginally superior functional efficacy.

Permethrin inhibits tube formation and viability of endothelial cells.

BACKGROUND: Exposure to environmental chemicals has been linked with endothelial dysfunction, which is a leading cause of human diseases, including atherosclerosis. Permethrin is a frequently used synthetic pyrethroid insecticide for which longer exposure may cause toxicity in several types of tissues and the development of metabolic diseases, including atherosclerosis, obesity and diabetes. The present study was designed to evaluate the potential adverse effect of permethrin on the function and activity of human endothelial cells. RESULTS: Permethrin was found to repress migration and tube formation by human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, as well as to significantly repress their viability after 24 and 48 h of treatment. Furthermore, increased reactive oxygen species (ROS) production was observed in cells treated with permethrin, and the permethrin-induced repression of cell viability was ROS-dependent. Permethrin did not influence apoptosis, necrosis or mitochondrial membrane potential in HUVECs. CONCLUSION: The results of the present study suggest that permethrin represses angiogenesis and viability through ROS-dependent and cell growth-, apoptosis- and necrosis-independent means. © 2022 Society of Chemical Industry.

Inhibition of hypoxia-inducible factor-1 by salidroside in an in vitro model of choroidal neovascularization.

PURPOSE: As a characteristic of age-related macular degeneration (AMD), choroidal neovascularization (CNV) causes severe vision loss. The current treatment has limited efficacy. This study was to investigate effects of Salidroside against CNV and explore its underlying mechanisms. METHODS: RF/6A cells were treated with 200 mM cobalt chloride (CoCl2) for 6 hr to mimic hypoxic condition. Cells were then treated with Salidroside at 10, 30, and 100 µM for 24 hr. Cells treated with DMSO were used as negative control. The cell proliferation was assessed using 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid assay. The tube formation was investigated on Matrigel. The cell migration was measured by a Transwell assay. RT-qPCR was used to detect the gene expression. Immuohistochemistry and western blot were used to detect the expression of proteins. RESULTS: Salidroside significantly inhibited the cell migration and tube formation activity of RF/6A cells under hypoxia. Moreover, Salidroside reduced the expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1) in RF/6A cells. CONCLUSIONS: Our data suggested that Salidroside could be a potential novel therapeutic agent against CNV.

PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA positron emission tomography/computed tomography has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. METHODS: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium (CM) derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the CM. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived CM using a tube formation assay that mimics angiogenesis. RESULTS: Immunohistochemistry of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in 4 of 33 prostate cancer patients (12.1%). We also found that the 10,000g pellet fraction of the LNCaP-derived CM containing PSMA-positive membranes, such as microvesicles transformed HUVECs "PSMA-negative" into "PSMA-positive." Furthermore, treatment of HUVECs with the 10,000g pellet fraction of the LNCaP-derived CM significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. CONCLUSIONS: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues.

The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension.

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis.