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In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

Purposes The establishment of microcirculation in tissue engineering plays an important role in biomaterials implantation, in which the migration of vascular endothelial cells is the main factor affecting rapid angiogenesis. Tissue and organ injury often leads to inflammation, and high mobility group box 1 (HMGB1) is the initiating factor of inflammation, while its effect on the migration of endothelial cells remains unclear. Methods Human umbilical vein endothelial cells (HUVECs) were extracted and cultured in vitro, and their phenotypes were identified. The concentration gradient of HMGB1 in culture medium was constructed, and the proliferation and migration of HUVECs were detected by CCK-8, scratch test, and Transwell chamber test. The expression of genes and proteins of cell proliferation factor and migration factor in HUVECs was detected by RT-qPCR and Western Blot. Findings Different concentrations of HMGB1 had no significant effect on proliferation of HUVECs, but was concentration-dependent on cell migration. With the increase of HMGB1 concentration, the migration ability of cells was enhanced. The expression of FGF-2 was up-regulated with the increase of HMGB1 concentration. The expression of VEGFA decreased significantly after HUVECs was treated with high HMGB1 concentration. Conclusions HMGB1 can promote the migration of HUVECs through FGF-2, which provides a basis for the study of the effect of HMGB1 on the migration ability of endothelial cells in the future.

The objective of this work was to investigate the protective effect and underlying mechanism of a bioactive peptide from coix seeds (Coix lacryma-jobi) on high glucose-injured human umbilical vein endothelial cells (HUVEC). HUVEC injured by 30 mmol/L glucose were treated with different concentrations of the peptide and were evaluated for the secretion of nitric oxide (NO) by the nitrate reductase method. The content of reactive oxygen species (ROS) was detected by flow cytometry. Cell apoptosis was detected by Annexin V-FITC/PI double staining. Quantitative real-time polymerase chain reaction (qPCR) was applied to detect the expression of inflammation-related genes. The results showed that NO secretion was decreased by 28.72% (P < 0.05), ROS secretion was increased by 96.19% (P < 0.05), and the expression of the ICAM-1, IL-6, MCP-1 and VCAM genes was significantly increased in HUVEC induced by high glucose compared with the normal control group. The prolongation of high glucose treatment time significantly increased the apoptosis rate of HUVECs, and after 14 days, the apoptosis rate of HUVECs was 1.61 times higher than that in the normal control group. Treatment with the peptide at all concentrations increased NO secretion, decreased ROS content and the expression of inflammatory genes and reduced cell apoptosis. In summary, the coix seed biopeptide possesses a protective effect on high glucose-injured HUVEC.

Objective To investigate the effect of cobalt (Co) and calcium-phosphate (Ca/P) doped coating on titanium surfaces and their angiogenic effect. Methods Microarc oxidation (MAO) was used to prepare Co-Ca/P-doped and Co-doped coatings. Titanium (Ti) sheet without MAO treatment was used as control. Scanning electron microscopy (SEM) was used to observe the surface micromorphology of the coatings. Energy dispersive spectrometry (EDS) was also applied to detect the doped chemicals and their contents. Standard soaking solutions of these coatings were prepared using an endothelial cell medium (ECM) solution for subsequent angiogenesis experiments. Human umbilical vein endothelial cells (HUVECs) were cultured on Matrigel with ECM soaking solutions for 4 h and 8 h. The microvessels were observed under a microscope, and the number of microtubules and their interconnecting nodes were analyzed with Image J software. Results Co doped and Co-Ca/P-doped coatings were successfully prepared by MAO, which was demonstrated by both SEM observation and EDS analysis. SEM observation showed that irregular crystals of the above chemicals were present on both Co and Co-Ca/P-doped coatings, commonly with a diameter

Background: Advancing age is one of the independent risk factors for cardiovascular disorders. The Compendium of Materia Medica, a classic book on traditional Chinese medicine, states that ginseng “harmonizes the five internal organs, calming the spirit and prolonging the years of life.'' Considered one of the primary bioactive compounds derived from Panax ginseng, ginsenoside Rb1 (g-Rb1) has been scientifically suggested to possess anti-senescence efficacy. More research is needed to explore the vascular pharmacological activity and potential clinical application value of g-Rb1. Aims of the study: Our previous study demonstrated that g-Rb1 could mitigate cellular senescence via the SIRT1/eNOS pathway. This study was performed to explore the exact mechanisms by which g-Rb1 modulates the SIRT1/eNOS pathway. Materials and methods: We used human primary umbilical vein endothelial cells (HUVECs) to establish a replicative ageing model. Real-time (RT‒PCR), western blotting, small interfering RNA (siRNA), and immunoprecipitation were conducted to detect the effect of g-Rb1 on the SIRT1/caveolin-1/eNOS axis. Results: G-Rb1 increased NO production and alleviated replicative senescence of HUVECs. The application of g-Rb1 elevated the mRNA and protein abundance of both SIRT1 and eNOS while concomitantly suppressing the expression of caveolin-1. Inhibition of SIRT1 and eNOS by siRNAs suppressed the anti-senescence function of g-Rb1, while caveolin-1 siRNA could enhance it. G-Rb1 decreased the acetylation level of caveolin-1 and increased NO production, which was suppressed by SIRT1 siRNA. Both g-Rb1 and caveolin-1 siRNA could reduce the acetylation level of eNOS and increase NO production. Conclusion: G-Rb1 prevents age-related endothelial senescence by modulating the SIRT1/caveolin-1/eNOS signaling pathway.

Previous studies have highlighted significant therapeutic effects of Qiqilian (QQL) capsule on hypertension in spontaneously hypertensive rats (SHRs); however, its underlying molecular mechanism remains unclear. We investigated the potential mechanism by which QQL improves hypertension-induced vascular endothelial dysfunction (VED). In vivo, SHRs were divided into four groups (20 per group) and were administered gradient doses of QQL (0, 0.3, 0.6, and 1.2 g/kg) for 8 weeks, while Wistar Kyoto rats were used as normal control. The vascular injury extent, IL-1β and IL-18 levels, NLRP3, ASC and caspase-1 contents were examined. In vitro, the effects of QQL-medicated serum on angiotensin II (AngII)-induced inflammatory and autophagy in human umbilical vein endothelial cells (HUVECs) were assessed. Compared with the SHR group, QQL significantly decreased thickness (125.50 to 105.45 μm) and collagen density (8.61 to 3.20%) of arterial vessels, and reduced serum IL-1β (96.25 to 46.13 pg/mL) and IL-18 (345.01 to 162.63 pg/mL) levels. The NLRP3 and ACS expression in arterial vessels were downregulated (0.21- and 0.16-fold, respectively) in the QQL-HD group compared with the SHR group. In vitro, QQL treatment restored NLRP3 and ASC expression, which was downregulated approximately 2-fold compared with that of AngII-induced HUVECs. Furthermore, QQL decreased LC3II and increased p62 contents (p < 0.05), indicating a reduction in autophagosome accumulation. These effects were inhibited by the autophagy agonist rapamycin and enhanced by the autophagy inhibitor chloroquine. QQL effectively attenuated endothelial injury and inflammation by inhibiting AngII-induced excessive autophagy, which serves as a potential therapeutic strategy for hypertension. [ABSTRACT FROM AUTHOR]

Background: Endothelial cell (EC) inflammation plays a crucial role in the development of several cardiovascular disorders (CD), including atherosclerosis and sepsis. Ligustrazine (Lig), a bioactive constituent derived from Traditional Chinese Medicine Ligusticum chuanxiong Hort, has exhibited in vivo anti-inflammatory properties. Despite the observed positive outcomes, the exact mechanisms underlying these beneficial effects remain unidentified. Aim: The goal of this research is to investigate the influence and potential mechanism of Lig on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). Introduction: These experiments investigate the effectiveness of Lig in preventing LPS-induced damage in HUVECs, with the goal of elucidating the underlying processes at work. Materials and Methods: To evaluate HUVECs' viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted. Enzyme-linked immunosorbent assay (ELISA) was employed to measure changes in ICAM-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein (MCP-1) levels. Real-time polymerase chain reaction (RT-PCR) was used to determine the levels of ICAM-1, IL-6, TNF-α, MCP-1, and toll-like receptors (TLR4) mRNA. Additionally, we performed WB analysis to assess the levels of nuclear factor-κB (NF-κB) p65, TLR4, IκBα, and p-IκBα. Results: The findings demonstrated significantly suppressed cell viability due to LPS treatment, while Lig treatment increased cell viability in a concentration-dependent manner. Lig also effectively reduced the mRNA levels of ICAM-1, TNF-α, IL-6, and MCP-1. Furthermore, Lig pretreatment led to downregulation of TLR4, p-IκBα, and NF-κB p65 in HUVECs. Discussion: The findings indicate that Lig reduces LPS-induced inflammation in HUVECs, and that the TLR4/NF-κB pathway is critical in increasing cell survival and minimizing inflammatory damage. This provides possible anti-inflammatory techniques for treating CD. Conclusion: In conclusion, our work demonstrates Lig's anti-inflammatory actions on LPS-stimulated HUVECs. The data suggest that Lig lowers inflammation via regulating the TLR4/NF-B pathway, boosting cell survival, and decreasing inflammatory responses. [ABSTRACT FROM AUTHOR]

The intricate nature of the tendon–bone interface poses significant challenges for current surgical methods aimed at repairing tendon–bone interface injuries. Despite notable progress in surgical techniques, these methods continue to grapple with hurdles such as complications and suboptimal healing effects. In this study, we prepared a three‐dimensional‐printed composite scaffold by incorporating bioactive ceramic zinc silicate (Zn2SiO4) into sodium alginate (SA) hydrogel. The physicochemical properties and mechanical strength of the SA hydrogel scaffold composited with Zn2SiO4 (ZnSi/SA) were investigated in vitro. Impressively, the 0.5‐ZnSi/SA scaffold exhibited a sustained release of Zn and Si ions, while exhibiting mechanical properties compatible with tendon–bone interface repair. Moreover, cell viability, cell migration, and osteogenic differentiation assay results showed that 0.5‐ZnSi/SA scaffold facilitated the viability, mobility, and osteogenic differentiation of bone marrow stromal cells. In parallel, assessments of cell viability, cell migration, and tendon differentiation indicated that 0.5‐ZnSi/SA scaffold promoted the viability, migration, and tenogenic differentiation of tendon stem/progenitor cells. Moreover, cell viability, cell migration, and tube formation assay results demonstrated that 0.5‐ZnSi/SA scaffold enhanced the viability, migration rate, and angiogenic performance of human umbilical vein endothelial cells. Collectively, our findings suggest a promising therapeutic avenue employing ZnSi/SA scaffold for tendon–bone interface healing. [ABSTRACT FROM AUTHOR]

Background/aim: Heavy-ion irradiation seriously perturbs cellular homeostasis and thus damages cells. Vascular endothelial cells (ECs) play an important role in the pathological process of radiation damage. Protecting ECs from heavy-ion radiation is of great significance in the radioprotection of normal tissues. In this study, the radioprotective effect of ß-D-glucan (BG) derived from Saccharomyces cerevisiae on human umbilical vein endothelial cell (EA.hy926) cytotoxicity produced by carbon-ion irradiation was examined and the probable mechanism was established. Materials and methods: EA.hy926 cells were divided into seven groups: a control group; 1, 2, or 4 Gy radiation; and 10 µg/mL BG pretreatment for 24 h before 1, 2, or 4 Gy irradiation. Cell survival was assessed by colony formation assay. Cell cycles, apoptosis, DNA damage, and reactive oxygen species (ROS) levels were measured through flow cytometry. The level of malondialdehyde and antioxidant enzyme activities were analyzed using assay kits. The activation of NF-κB was analyzed using western blotting and a transcription factor assay kit. The expression of downstream target genes was detected by western blotting. Results: BG pretreatment significantly increased the survival of irradiated cells, improved cell cycle progression, and decreased DNA damage and apoptosis. The levels of ROS and malondialdehyde were also decreased by BG. Further study indicated that BG increased the antioxidant enzyme activities, activated Src, and promoted NF-κB activation, especially for the p65, p50, and RelB subunits. The activated NF-κB upregulated the expression of antioxidant protein MnSOD, DNA damage-response and repair-related proteins BRCA2 and Hsp90a, and antiapoptotic protein Bcl-2. Conclusion: Our results demonstrated that BG protects EA.hy926 cells from high linear-energy-transfer carbon-ion irradiation damage through the upregulation of prosurvival signaling triggered by the interaction of BG with its receptor. This confirms that BG is a promising radioprotective agent for heavy-ion exposure. [ABSTRACT FROM AUTHOR]

BACKGROUND: MiR-126-3p from adipose-derived mesenchymal stem cells released exosomes (AMSC-Exos) has been used as a potential biomarker of diabetes, but few studies of glucolipotoxicity and mechanism of miR-126-3p on human umbilical vein endothelial cells are found. OBJECTIVE: To explore the effect and mechanism of miR-126-3p from AMSC-Exos on glucolipotoxicity of human umbilical vein endothelial cells. METHODS: (1) Glucolipotoxicity model of human umbilical vein endothelial cells was induced by 30 mmol/L glucose and 100 μmol/L palmitic acid in vitro. (2) The supernatant of passage 4 AMSC-Exos was collected. The morphology of AMSC-Exos was observed by transmission electron microscope. The size distribution of AMSC-Exos was determined by nanoparticle tracking analysis. (3) Human umbilical vein endothelial cells with glucolipotoxicity were treated with AMSC-Exos. The content of reactive oxygen species and apoptosis rate were detected by flow cytometry. ELISA was implemented to detect the expression of inflammatory factors. (4) RT-qPCR was used to detect the expression levels of miR-126-3p mRNA in AMSC-Exos and normal human umbilical vein endothelial cells co-cultured with AMSC. (5) miR-126-3p overexpression was transfected into human umbilical vein endothelial cells. Cell proliferation was detected by CCK- 8 assay. Western blot assay was used to detect the expression levels of apoptotic proteins. (6) The target genes of miR-126-3p were predicted and verified by bioinformatics software. Dual luciferase reporter gene assay was applied to verify and predict signaling pathway. (7) CRK overexpression was transfected into human umbilical vein endothelial cells with glucolipotoxicity. The expression levels of apoptotic protein and p-AKT were detected by western blot assay. (8) Human umbilical vein endothelial cell glucolipotoxicity model was co-treated with AKT pathway inhibitor RG7440 and miR-126-3p overexpression/AMSC-Exos. Apoptotic protein expression was detected by western blot assay and apoptosis rate was detected by flow cytometry. RESULTS AND CONCLUSION: (1) AMSC-Exos were round or oval membranous vesicles, and the particle size ranged from 30 to 200 nm. (2) Compared with the model group, AMSC-Exos could remarkably reduce the expression levels of interleukin-1β, interleukin-6 and interleukin-8 (P < 0.05, P < 0.001), and obviously reduced the reactive oxygen species and cell apoptosis rate (P < 0.05). (3) Compared with adipose-derived mesenchymal stem cells, mRNA expression level of miR-126- 3p from AMSC-Exos was significantly increased (P < 0.001). Compared with human umbilical vein endothelial cells, co-culture with adipose-derived mesenchymal stem cells could significantly increase the expression level of miR-126-3p of human umbilical vein endothelial cells (P < 0.001). (4) miR-126-3p overexpression could significantly promote cell proliferation, reduce cell apoptotic rate and expression levels of cleaved caspase 3, 9 (P < 0.01, P < 0.001). (5) Bioinformatics analysis and dual luciferase reporter gene assay proved that CRK was the target gene of miR-126-3p. The AKT pathway was as the object of next experimental research. (6) CRK overexpression could activate AKT pathway and up-regulate expression level of cleaved caspase 3, 9. Compared with the Exos group, the cell apoptosis rate was significantly higher in the Exos + AKT inhibitor group (P < 0.001). (7) The results showed that miR-126-3p from AMSC-Exos could improve the glucolipotoxicity of human umbilical vein endothelial cells. It is supposed that the mechanism maybe work by miR-126-3p regulating the AKT pathway via targeting CRK gene. [ABSTRACT FROM AUTHOR]

The design of physiologically relevant blood vessel in vitro models has been impaired by the difficulty to reproduce the complex architecture of native blood vessels and the mechanisms mediating key cellular functions within miniaturized perfusable systems. Aiming to simulate blood vessel walls, in this work innovative 2D platforms are designed and patterned with magnetic‐responsive gelatin for enabling in situ co‐culture of mesenchymal stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs) within confined compartments. The performance of the 2D chips is evaluated based on HUVECs migration, adherence, and angiogenic behavior (proliferation and sprouting), as well as production of Endothelin‐1 (endothelium marker), and compared with the results of 3D single channel models, designed to mimic the morphology of native arteries and veins. The 2D chips obtain better cell adhesion and angiogenic performance, which is attributed to flow profiles and VEGF concentration gradients. Magnetic stimulation is then used as a novel strategy to increase cell sprouting and endothelization ≈1.5 times above the control condition. These bio‐inspired devices advance the exploration of magnetism for a finer convergence to the native vascular conditions in vitro and improved modulation of angiogenesis, showing promising contributions to the development of sophisticated therapeutics for vascular ischemia‐related diseases. [ABSTRACT FROM AUTHOR]

Objective To explore the effect of lncRNA SNHG10 on lipopolysaccharide （LPS)-induced oxidative stress injury in vascular endothelial cells. Methods The optimal concentration of LPS was screened out to be 500 ng/ mL. The in vitro models were constructed by inducing human umbilical vein endothelial cells （HUVEC) with LPS. HU⁃ VEC in the logarithmic growth phase were taken, si-NC and si-SNHG10 plasmids were transfected into HUVEC, and then the cells were treated with LPS （500 ng/mL)（recorded as LPS + si-NC group and LPS+si-SNHG10 group, respectively). HUVEC without any treatment were selected as the Control group, and only 500 ng/mL LPS was added. CCK-8 and flow cytometry were used to detect cell viability and apoptosis level； RT-qPCR was used to detect the relative expression of small nucleolar RNA host gene 10（SNHG10)； Western blotting was used to detect Ki-67, Bcl-2, and Bax； ELISA was used to detect tumor necrosis factor-α（TNF-α), interleukin 6（IL-6), interleukin 1β（IL-1β), MDA content, and SOD activity. Results The cell viability, Ki-67 protein, and Bcl-2 protein expression of HUVEC in the LPS + si-SNHG10 group were 75. 6% ± 4. 1%, 0. 74 ± 0. 06, 0. 65 ± 0. 05, and they were 45. 4 ± 2. 8%, 0. 52% ± 0. 04, and 0. 42 ± 0. 03, respectively, in the LPS+si-NC group； SNHG10 expression, apoptosis rate, Bax protein expression, TNF-α, 1L-6 and 1L-1β expression in the LPS+si-SNHG10 group were 1. 88 ± 0. 11, 16. 8% ± 0. 9%, 1. 32 ± 0. 07(289. 2 ± 25. 1) pg/mL(363. 3 ± 22. 4) pg/mL, and （402. 6 ± 21. 3) pg/mL, which were 3. 24 ± 0. 19, 23. 1% ± 1. 2%, 1. 68 ± 0. 11(536. 6 ± 33. 0) pg/mL(634. 5 ± 33. 1) pg/mL, and （664. 5 ± 38. 1) pg/mL, respectively, in the LPS + si-NC group. SNHG10 expression, apoptosis rate, Bax protein expression, TNF-α, 1L-6, and 1L-1β expression levels were lower in the LPS + si-SNHG10 group than in the LPS + si-NC group （all P<0. 05). Compared with the Control group, the expression of MDA increased in the LPS group （P<0. 05), and the SOD activity decreased （P<0. 05)； compared with the LPS + si-NC group, MDA in HUVEC cells of the LPS + si-SNHG10 group decreased （P<0. 05), while the SOD activity increased （P< 0. 05). Conclusion Interfering with the expression of SNHG10 can alleviate the apoptosis, inflammation and oxidative stress of LPS-induced HUVEC, and promote cell proliferation. [ABSTRACT FROM AUTHOR]

Acute Liver Failure (ALF) is a life-threatening illness characterized by the rapid onset of abnormal liver biochemistries, coagulopathy, and the development of hepatic encephalopathy. Extracorporeal bioengineered liver (BEL) grafts could offer a bridge therapy to transplant or recovery. The present study describes the manufacture of clinical scale BELs created from decellularized porcine-derived liver extracellular matrix seeded entirely with human cells: human umbilical vein endothelial cells (HUVECs) and primary human liver cells (PHLCs). Decellularized scaffolds seeded entirely with human cells were shown to adhere to stringent sterility and safety guidelines and demonstrated increased functionality when compared to grafts seeded with primary porcine liver cells (PPLCs). BELs with PHLCs were able to clear more ammonia than PPLCs and demonstrated lower perfusion pressures during patency testing. Additionally, to determine the full therapeutic potential of BELs seeded with PHLCs, longer culture periods were assessed to address the logistical constraints associated with manufacturing and transporting a product to a patient. The fully humanized BELs were able to retain their function after cold storage simulating a product transport period. Therefore, this study demonstrates the manufacture of bioengineered liver grafts and their potential in the clinical setting as a treatment for ALF. [ABSTRACT FROM AUTHOR]

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2+/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2-/-). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

A critical component of tissue engineering is the ability to functionally replace native tissue stroma. Electrospinning is a technique capable of forming fibrous constructs with a high surface area for increased cell-material interaction and enhanced biocompatibility. However, physical and biological properties of electrospun scaffolds are limited by design controllability on a macroscale. We developed a methodology for generating electrospun scaffolds with defined patterns and topographic features to influence physical properties and biological interactions. Five unique design electrospinning target collectors were fabricated to allow for generation of defined polymeric scaffold patterns including lines, sinusoids, squares, zigzags, and solid. Poly(lactic-co-glycolic) acid was electrospun under identical conditions utilizing these varied targets, and constructs generated were examined as to their physical configuration, mechanical and chemical properties, and their ability to foster vascular smooth muscle cell adhesion and retention at 24 h. Modifying collector designs led to significant differences in fiber target coverage ranging from 300 mm2 for solid (100% of the target area) to 217.8 mm2 for lines (72.6% of the target area). Measured fiber excess, residual open area, and contact angle (hydrophobicity) followed the same trend as fiber target coverage with respect to the collector pattern: lines > sinusoids > squares > zigzags > solid. Similarly, the line design allowed for the greatest cell adhesion and retention (258 ± 31 cells), whereas solid exhibited the lowest (150 ± 15 cells); p < 0.05. There was a strong direct correlation of cell adhesion to construct residual open area (R2 = 0.94), normalized fiber excess (R2 = 0.99), and fiber grammage (R2 = 0.72), with an inverse relationship to fiber target coverage (R2 = 0.94). Our results demonstrate the ability to utilize patterned collectors for modifying macroscopic and microscopic electrospun scaffold features, which directly impact cell adhesion and retention, offering translational utility for designing specific tissue constructs.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 μM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 μM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

Objective To explore the anti-atherogenic activity and mechanism involved of ginsenoside derivative CK201 by ApoE-/- mouse and human umbilical vein endothelial cells (HUVECs). Methods In vivo experiment: 6-week-old male ApoE-/- mice(n=30) were randomly divided into 5 groups: the solvent control group, the model group, the ginsenoside compound K(CK) group, the atorvastatin calcium group and the CK201 group. Except the solvent control group, mice in other groups were fed high-fat diet. At the same time, mice in the CK group, the atorvastatin calcium group and the CK201 group were administrated intragastrically 3 mg/kg compound, respectively. The effect of CK201 and atorvastatin calcium on the formation of AS plaque was evaluated by aorta oil red O staining. The plasma levels of TC and TG were detected, and the effects of CK201 and atorvastatin calcium on blood lipid in mice were investigated. Western blot assay was used to analyze the effects of CK201 and atorvastatin calcium on the expression of inflammatory factors IL-6 and TNF-α of the aorta. The effects of CK201 and atorvastatin calcium on liver lipid accumulation were evaluated by HE and oil red O staining in liver sections. Liver and kidney function tests and blood routine tests of mice were performed to evaluate the effect of CK201 on related indicators. In vitro experiments: Western blot assay was used to analyze the effects of CK201 on the levels of IL-6 and TNF-α in HUVECs cells stimulated by palmitic acid. The effects of CK201 on the migration and recruitment of monocytes of HUVECs were analyzed by the scratch healing experiment of HUVECs cells and the adhesion experiment between monocytes (THP-1) and HUVECs cells. Results The CK201 group significantly inhibited the formation of atherosclerotic aortic plaques in ApoE-/- mice fed with high-fat diet, compared with the atorvastatin calcium group (plaque inhibition rate: 62.4% vs 26.6%, P < 0.05). The CK201 group had significantly reduced hepatic lipid accumulation, which had significantly better effect than those in the atorvastatin calcium group(P < 0.05). In the CK201 group, TC content in plasma was significantly decreased, the expressions of IL-6 and TNF-α in aorta were inhibited(P < 0.05), the expression of palmitic acid-stimulated inflammatory cytokines IL-6 and TNF-α in HUVECs cells was inhibited, the adhesion of THP-1 cells to HUVECs cells was reduced, and the migration of HUVECs cells was promoted(P < 0.05). In addition, the experimental dose of CK201 had no significant effect on the liver function and blood routine tests. Conclusion CK201, a ginsenoside derivative, has a significant anti-atherosclerosis effect, and the mechanism involved may be related to the reduction of plasma TC level, inhibition of aortic inflammation, inhibition of cell adhesion of monocyte and endothelial cells.

In this study, the total sugar, protein, 3,6-anhydro galactose and sulfate contents, apparent molecular mass and monosaccharide composition of porphyran isolated from Porphyra yezoensis were analyzed. The pro-angiogenic activity and underlying mechanism of porphyran were further investigated using human umbilical vein endothelial cells (HUVECs) as an in vitro angiogenesis model. The effect of porphyran on the cell viability of HUVECs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell adhesion and migration capacity were evaluated using cell adhesion and wound healing assays. The number of lumens formed by HUVECs was analyzed using in vitro tube formation assay. The expression levels of E-cadherin and N-cadherin in HUVECs were analyzed by Western Blotting, and the pro-angiogenic mechanism of porphyran in HUVECs was explored using specific inhibitors of mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors. The results showed that porphyran contained (77.48 ± 2.62)% total sugar, (3.24 ± 0.58)% protein, and (20.30 ± 1.06)% 3,6-anhydrogalactose as well as (20.78 ± 3.31)% sulfate groups. The apparent molecular mass of porphyran was 39.81 kDa. Porphyran was mainly composed of galactose, 3,6-anhydrogalactose and sulfate groups, suggesting it to be a sulfated galactan. Furthermore, porphyran treatment in the concentration range of 0–200 μg/mL had no significant toxicity no HUVECs, but instead promote cell proliferation, adhesion, migration, and vascular network formation. Further studies demonstrated that porphyran exerted its pro-angiogenic effect primarily by mediating the down-regulation of E-cadherin expression and the up-regulation of N-cadherin expression through the c-Jun N-terminal kinase (JNK) MAPK and VEGF receptor signaling pathways. In conclusion, porphyran has pro-angiogenic potential, which will provide a scientific basis for the development of porphyran as a novel pro-angiogenic factor and the precision nutrition-guided deep processing and high-value utilization of the red seaweed P. yezoensis.

Background/purpose: Porphyromonas gingivalis (P. gingivalis) could induce the activation of vascular endothelial cells and promote the formation of atherosclerosis. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing (NLRP) 6 could recognize P. gingivalis, but its role in atherosclerosis was unknown. The purpose of this study is to investigate the role of NLRP6 in the activation of inflammation in human umbilical vein endothelial cells (HUVECs) stimulated by P. gingivalis. Materials and methods: The expression level of NLRP6 in HUVECs with or without P. gingivalis-challenge was observed. Down-regulating the expression of NLRP6 in HUVECs, the expression levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein (MCP)-1 were detected. Then, the HUVECs with NLRP6-overexpressed were stimulated by P. gingivalis, the levels of inflammatory cytokines above were examined and compared with those in HUVECs triggered by P. gingivalis only. To evaluate the effect of NLRP6 on bacterial immune escape, the NLRP6 was overexpressed, and the colonies of P. gingivalis that survived in HUVECs were calculated. Results: NLRP6 was expressed in HUVECs and decreased after P. gingivalis stimulation. Downregulation of NLRP6 decreased the expression levels of IL-1β, IL-6, IL-8, TNF-α and MCP-1 in HUVECs. Those cytokines above in NLRP6-overexpressed HUVECs with P. gingivalis-stimulation significantly increased than in the cells with P. gingivalis-stimulation only. Furthermore, over-expression of NLRP6 decreased the colonies of P. gingivalis survival in HUVECs. Conclusion: NLRP6 regulated the activation of inflammation in HUVECs triggered by P. gingivalis and played an important role in P. gingivalis survival in endothelial cells.

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

The two main blood flow patterns, namely, pulsatile shear (PS) prevalent in straight segments of arteries and oscillatory shear (OS) observed at branch points, are associated with atheroprotective (healthy) and atheroprone (unhealthy) vascular phenotypes, respectively. The effects of blood flow-induced shear stress on endothelial cells (ECs) and vascular health have generally been studied using human umbilical vein endothelial cells (HUVECs). While there are a few studies comparing the differential roles of PS and OS across different types of ECs at a single time point, there is a paucity of studies comparing the temporal responses between different EC types. In the current study, we measured OS and PS transcriptomic responses in human aortic endothelial cells (HAECs) over 24 h and compared these temporal responses of HAECs with our previous findings on HUVECs. The measurements were made at 1, 4, and 24 h in order to capture the responses at early, mid, and late time points after shearing. The results indicate that the responses of HAECs and HUVECs are qualitatively similar for endothelial function-relevant genes and several important pathways with a few exceptions, thus demonstrating that HUVECs can be used as a model to investigate the effects of shear on arterial ECs, with consideration of the differences. Our findings show that HAECs exhibit an earlier response or faster kinetics as compared to HUVECs. The comparative analysis of HAECs and HUVECs presented here offers insights into the mechanisms of common and disparate shear stress responses across these two major endothelial cell types.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.

High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets.

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.

Abstract The intricate nature of the tendon–bone interface poses significant challenges for current surgical methods aimed at repairing tendon–bone interface injuries. Despite notable progress in surgical techniques, these methods continue to grapple with hurdles such as complications and suboptimal healing effects. In this study, we prepared a three‐dimensional‐printed composite scaffold by incorporating bioactive ceramic zinc silicate (Zn2SiO4) into sodium alginate (SA) hydrogel. The physicochemical properties and mechanical strength of the SA hydrogel scaffold composited with Zn2SiO4 (ZnSi/SA) were investigated in vitro. Impressively, the 0.5‐ZnSi/SA scaffold exhibited a sustained release of Zn and Si ions, while exhibiting mechanical properties compatible with tendon–bone interface repair. Moreover, cell viability, cell migration, and osteogenic differentiation assay results showed that 0.5‐ZnSi/SA scaffold facilitated the viability, mobility, and osteogenic differentiation of bone marrow stromal cells. In parallel, assessments of cell viability, cell migration, and tendon differentiation indicated that 0.5‐ZnSi/SA scaffold promoted the viability, migration, and tenogenic differentiation of tendon stem/progenitor cells. Moreover, cell viability, cell migration, and tube formation assay results demonstrated that 0.5‐ZnSi/SA scaffold enhanced the viability, migration rate, and angiogenic performance of human umbilical vein endothelial cells. Collectively, our findings suggest a promising therapeutic avenue employing ZnSi/SA scaffold for tendon–bone interface healing.

Objective: This study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. Methods: We employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. Results: At extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. Conclusion: Hydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

AIM: To investigate the mechanism and the protective effect of matrine against hydrogen peroxide (H2O2) -induced oxidative injury in human umbilical vein endothelial cells (HUVECs) based on the KLF4/Nrf2 pathway. METHODS: The HUVECs were cultured and screened to determine the experimental concentration of matrine using CCK-8 method. The HUVECs were divided into control group, model group, and low-, medium- and high-concentration (0.5, 1 and 2 mmol/L) matrine groups. An oxidative injury model of HUVECs was induced by H2O2 (0.5 mmol/L), and cell viabilty and intracellular reactive oxygen species (ROS), malondialdehyde(MDA), superoxide dismutase (SOD) and glutathione peroxidase(GPX)levels were detected. The mRNA and protein levels of Kriippel-like factor 4(KLF4), nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), quinone oxidoreductase 1(NQO1) and y-glutamyl cysteine synthetase (y-GCS) were detected using RT-qPCR and Western blot. Changes in these indicators were observed after siRNA-mediated knockdown of KLF4 gene expression and cotreatment with matrine. RESULTS: Compared with model group, the cell viability was increased (P<0. 05), ROS and MDA levels were decreased (PVO. 05), SOD and GPX activity was increased (PVO. 05), and the expression levels of KLF4, NRF2, HO-1, NOQI and y-GCS were increased in matrine groups（PV0. 05）. Inhibiting KLF4 expression significantly weakened the protective effect of matrine against H2O2- induced decrease in cell viability, increase in ROS, and expression of oxidative stress-related molecules（PV0. 05）. CONCLUSION: Matrine can alleviate H2O2-induced oxidative injury in HUVECs by activating KLF4/Nrf2 pathway and inhibiting oxidative stress. [ABSTRACT FROM AUTHOR]

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Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation. [ABSTRACT FROM AUTHOR]

Cedrol is a sesquiterpene alcohol isolated from Cedrus atlantica, which has been traditionally used in aromatherapy and has anticancer, antibacterial and antihyperalgesic effects. One characteristic of glioblastoma (GB) is the overexpression of vascular endothelial growth factor (VEGF), which induces a high degree of angiogenesis. Although previous studies have reported that cedrol inhibits GB growth by inducing DNA damage, cell cycle arrest and apoptosis, its role in angiogenesis remains unclear. The aim of the present study was to investigate the effects of cedrol on VEGF-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 0-112 µM cedrol and 20 ng/ml VEGF for 0-24 h, and then anti-angiogenic activation of cedrol was determined by MTT assay, wound healing assay, Boyden chamber assay, tube formation assay, semi-quantitative reverse transcription-PCR and western blotting. These results demonstrated that cedrol treatment inhibited VEGF-induced cell proliferation, migration and invasion in HUVECs. Furthermore, cedrol prevented VEGF and DBTRG-05MG GB cells from inducing capillary-like tube formation in HUVECs and decreased the number of branch points formed. Moreover, cedrol downregulated the phosphorylation of VEGF receptor 2 (VEGFR2) and the expression levels of its downstream mediators AKT, ERK, VCAM-1, ICAM-1 and MMP-9 in HUVECs and DBTRG-05MG cells. Taken together, these results demonstrated that cedrol exerts anti-angiogenic effects by blocking VEGFR2 signaling, and thus could be developed into health products or therapeutic agents for the prevention or treatment of cancer and angiogenesis-related diseases in the future. [ABSTRACT FROM AUTHOR]

Objective We aimed to explore the protective effect of Qingre Zhixue Formula and tripterygium wilfordii multiglucoside on the endothelial damage induced by serum-derived poly IgA in patients with Henoch-Schönlein purpura nephritis and its effect on the expression of key proteins of the NF-κB signaling pathway. Methods A total of 80 patients with Henoch-Schönlein purpura nephritis from The First Affliated Hospital of Henan University of Chinese Medicine and 38 healthy controls were enrolled in this study. Serum IgA was purified from all participants in order to prepare serum-derived poly IgA. Moreover, 60 Sprague-Dawley rats were divided into the following five groups (n = 12 rats per group): the Qingre Zhixue Formula group (31. 25 mg/ kg), the tripterygium wilfordii multiglucoside group (46. 88 mg/ kg), the combined group (31. 25 mg/ kg + 46. 88 mg/ kg), the prednisone acetate group (23. 44 mg/ kg) and the saline group. Rats in each group were gavaged twice a day for 7 days. Additionally, human umbilical vein endothelial cells (HUVECs) were stimulated with serum-derived poly IgA (200 µg/ mL) derived from patients with Henoch-Schönlein purpura nephritis for 24 h to construct HUVECs injury model. The cells were divided into the following seven groups: the blank group, the healthy group, the model group, the Qingre Zhixue Formula group, the tripterygium wilfordii multiglucoside group, the combined group and the prednisone acetate group, and provide corresponding intervention. siRNA transfection was used to inhibit the NF-κB signaling pathway, and divide the transfected cells into the blank group, the model group, the model+normal control group, the model+ transfection group, the Qingre Zhixue Formula group, the tripterygium wilfordii multiglucoside group, the combined group and the prednisone acetate group. Cell viability was detected by the MTT assay, the apoptosis rate was detected by flow cytometry, and the protein expression levels of p-P65, p-P50, IKKβ, and IκBα were detected by Western blotting before and after transfection. Results Compared with the blank group, the cell viability of the model group was decreased (P<0. 05). Compared with the model group, the cell viability of each treatment group was increased (P<0. 05). Compared with the combined group, the cell viability of the Qingre Zhixue Formula group, the tripterygium wilfordii multiglucoside group, and the prednisone acetate group was decreased (P<0. 05). Compared with the blank group, the protein expression levels of p-P65, p-P50 and IKKβ in the model group were increased (P<0. 05), and the protein expression level of IκBα was decreased (P <0. 05). Compared with the model group, the protein expression levels of p-P65, p-P50 and IKKβ were decreased in each group (P<0. 05), and the protein expression level of IκBα was increased (P <0. 05). Compared with the combined group, the protein expression levels of p-P65, p-P50, and IKKβ in the Qingre Zhixue Formula group, the tripterygium wilfordii multiglucoside group and the prednisone acetate group were increased (P<0. 05), while the protein expression level of IκBα was decreased ( P < 0. 05). After siRNA transfection, compared with the blank group, the protein expression levels of p-P65, p-P50 and IKKβ in the model group and the model+normal control group were increased (P<0. 05), and the protein expression level of IκBα was decreased (P<0. 05). Compared with the model group, the protein expression levels of p-P65, p-P50 and IKKβ were decreased (P<0. 05), and the protein expression level of IκBα was increased (P< 0. 05) in the cells of the model+transfection group, the Qingre Zhixue Formula group, the tripterygium wilfordii multiglucoside group, the combined group and the prednisone acetate group. Conclusion Qingre Zhixue Formula combined with tripterygium wilfordii multiglucoside may inhibit the activation of the NF-κB pathway to improve the cell viability of HUVECs induced by serum-derived poly IgA in children with Henoch-Schönlein purpura nephritis, so it plays a role in protecting endothelial cells. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

Vascular endothelial zinc finger 1 (VEZF1) plays important roles in endothelial lineage definition and angiogenesis. Vasohibins 1 and 2 (VASH1 and VASH2) can form heterodimers with small vasohibin-binding protein (SVBP) and were recently shown to regulate angiogenesis by acting as tubulin detyrosinases. Here, we showed that VEZF1 binds directly with DNA guanine quadruplex (G quadruplex, G4) structures in vitro and in cells, which modulates the levels of the two isoforms of VASH1 mRNA. Disruption of this interaction, through genetic depletion of VEZF1 or treatment of cells with G4-stabilizing small molecules, led to increased production of the long over short isoform of VASH1 (i.e. VASH1A and VASH1B, respectively) mRNA and elevated tubulin detyrosinase activity in cells. Moreover, disruption of VEZF1-G4 interactions in human umbilical vein endothelial cells resulted in diminished angiogenesis. These results suggest that the interaction between VEZF1 and G4 structures assumes a crucial role in angiogenesis, which occurs through regulating the relative levels of the two isoforms of VASH1 mRNA and the detyrosinase activity of the VASH1-SVBP complex. Together, our work revealed VEZF1 as a G4-binding protein, identified a novel regulatory mechanism for tubulin detyrosinase, and illustrated that the VEZF1- and VASH1-mediated angiogenesis pathways are functionally connected.

Angiogenesis is a complex process that is required for development and tissue regeneration and it may be affected by many pathological conditions. Chemicals and drugs can impact formation and maintenance of the vascular networks; these effects may be both desirable (e.g., anti-cancer drugs) or unwanted (e.g., side effects of drugs). A number of in vivo and in vitro models exist for studies of angiogenesis and endothelial cell function, including organ-on-a-chip microphysiological systems. An arrayed organ-on-a-chip platform on a 96-well plate footprint that incorporates perfused microvessels, with and without tumors, was recently developed and it was shown that survival of the surrounding tissue was dependent on delivery of nutrients through the vessels. Here we describe a technology transfer of this complex microphysiological model between laboratories and demonstrate that reproducibility and robustness of these tissue chip-enabled experiments depend primarily on the source of the endothelial cells. The model was highly reproducible between laboratories and was used to demonstrate the advantages of the perfusable vascular networks for drug safety evaluation. As a proof-of-concept, we tested Fluorouracil (1-1,000 μM), Vincristine (1-1,000 nM), and Sorafenib (0.1-100 μM), in the perfusable and non-perfusable micro-organs, and in a colon cancer-containing micro-tumor model. Tissue chip experiments were compared to the traditional monolayer cultures of endothelial or tumor cells. These studies showed that human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs. The data from the 3D models confirmed advantages of the physiological environment as compared to 2D cell cultures. We demonstrated how these models can be translated into practice by verifying that the endothelial cell source and passage are critical elements for establishing a perfusable model.

Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1, a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.

Periodontitis is a local and systemic inflammatory condition and a risk factor of atherosclerosis, but no studies investigated the effect of a statin on atherogenesis affected by severe periodontitis. In this study, we investigated the effect of rosuvastatin (RSV) on atherogenesis in Apolipoprotein E-deficient mice receiving silk ligature placement around the maxillary second molars. Mice with the ligature placement developed severe periodontitis and vascular inflammation. RSV significantly inhibited the development of periodontitis and vascular inflammation and remarkably blocked the increased lipid deposition and the atherogenic gene expression in the arterial wall and aortic sinus induced by severe periodontitis. To understand the mechanistic effect of RSV on periodontitis-associated atherogenesis, we investigated the in vitro effect of RSV on various effect of TNF-α, a major proinflammatory cytokine for periodontitis and atherogenesis. We found that RSV notably inhibited the TNF-α-induced osteoclast formation, endothelial cell phenotypic changes, foam cell formation, and the expression of CD47 and other oncogenes in arterial smooth muscle cells. Taken together, our study indicates that RSV prevents the exacerbation of atherosclerosis induced periodontitis by inhibiting local, systemic and vascular inflammation, as well as the expression of CD47 from arterial smooth muscle cells in mice.

Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy. SIGNIFICANCE: Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1498/F1.large.jpg.

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumors own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.

The accumulation of senescent cells (SnCs) is a causal factor of various age-related diseases as well as some of the side effects of chemotherapy. Pharmacological elimination of SnCs (senolysis) has the potential to be developed into novel therapeutic strategies to treat these diseases and pathological conditions. Here we show that ubiquitin-specific peptidase 7 (USP7) is a novel target for senolysis because inhibition of USP7 with an inhibitor or genetic depletion of USP7 by RNA interference induces apoptosis selectively in SnCs. The senolytic activity of USP7 inhibitors is likely attributable in part to the promotion of the human homolog of mouse double minute 2 (MDM2) ubiquitination and degradation by the ubiquitin-proteasome system. This degradation increases the levels of p53, which in turn induces the pro-apoptotic proteins PUMA, NOXA, and FAS and inhibits the interaction of BCL-XL and BAK to selectively induce apoptosis in SnCs. Further, we show that treatment with a USP7 inhibitor can effectively eliminate SnCs and suppress the senescence-associated secretory phenotype (SASP) induced by doxorubicin in mice. These findings suggest that small molecule USP7 inhibitors are novel senolytics that can be exploited to reduce chemotherapy-induced toxicities and treat age-related diseases.

AimsSeveral signaling pathways contribute to endothelial-mesenchymal transitions and vascular calcification, including bone morphogenetic protein (BMP) and transforming growth factor (TGF) β signaling. The transcription factor homeobox D3 (Hoxd3) is known to regulate an invasive endothelial phenotype, and the aim of the study is to determine if HOXD3 modulates BMP and TGFβ signaling in the endothelium.Methods and researchWe report that the endothelium with high BMP activity due to the loss of BMP inhibitor matrix Gla protein (MGP) shows induction of Hoxd3. HOXD3 is part of a BMP-triggered cascade. When activated by BMP9, activin receptor-like kinase (ALK) 1 induces HOXD3 expression. Hoxd3 promoter is a direct target of phosphorylated (p) SMAD1, a mediator of BMP signaling. High BMP activity further results in enhanced TGFβ signaling due to induction of TGFβ1 and its receptor, ALK5. This is mediated by HOXD3, which directly targets the Tgfb1 promoter. Finally, TGFβ1 and BMP9 stimulate the expression of MGP, which limits the enhanced ALK1 induction by counteracting BMP4. The cascade of BMP9-HOXD3-TGFβ also affects Notch signaling and angiogenesis through induction of Notch ligand Jagged 2 and suppression of Notch ligand delta-like 4 (Dll4).ConclusionThe results suggest that HOXD3 is a novel link between BMP9/ALK1 and TGFβ1/ALK5 signaling.Translational perspectiveBMP and TGFβ signaling are instrumental in vascular disease such as vascular calcification and atherosclerosis. This study demonstrated a novel type of cross talk between endothelial BMP and TGFβ signaling as mediated by HOXD3. The results provide a possible therapeutic approach to control dysfunctional BMP and TGFβ signaling by regulating HOXD3.

Toxoplasma gondii can infect and replicate in vascular endothelial cells prior to entering host tissues. However, little is known about the molecular interactions at the parasite-endothelial cell interface. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low-molecular-weight polymers. T. gondii disrupted vascular endothelial cadherin (VE-cadherin) and β-catenin localization to the cell periphery and reduced VE-cadherin protein expression. Notably, T. gondii infection led to reorganization of the host cytoskeleton by reducing filamentous actin (F-actin) stress fiber abundance under static and microfluidic shear stress conditions and by reducing planar cell polarity. RNA sequencing (RNA-Seq) comparing genome-wide transcriptional profiles of infected to uninfected endothelial cells revealed changes in gene expression associated with cell-cell adhesion, extracellular matrix reorganization, and cytokine-mediated signaling. In particular, genes downstream of Hippo signaling and the biomechanical sensor and transcriptional coactivator Yes-associated protein (YAP) were downregulated in infected endothelial cells. Interestingly, T. gondii infection activated Hippo signaling by increasing phosphorylation of LATS1, leading to cytoplasmic retention of YAP, and reducing YAP target gene expression. These findings suggest that T. gondii infection triggers Hippo signaling and YAP nuclear export, leading to an altered transcriptional profile of infected endothelial cells.IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

The delivery of growth factors is often challenging due to their short half-life, low stability, and rapid deactivation. In native tissues, the sulfated residual of glycosaminoglycan (GAG) polymer chains of proteoglycans immobilizes growth factors through the proteoglycans'/proteins' complexation with nanoscale organization. These biological assemblies can influence growth factor-cell surface receptor interactions, cell differentiation, cell-cell signaling, and mechanical properties of the tissues. Here, we introduce a facile procedure to prepare novel biomimetic proteoglycan nanocarriers, based on naturally derived polymers, for the immobilization and controlled release of growth factors. We developed polyelectrolyte complex nanoparticles (PCNs) as growth factor nanocarriers, which mimic the dimensions, chemical composition, and growth factor immobilization of proteoglycans in native tissues. PCNs were prepared by a polymer-polymer pair reaction method and characterized for physicochemical properties. Fourier transform infrared spectroscopy (FTIR) analysis indicated that complexation occurred through electrostatic interactions. Transmission electron microscopy (TEM) results showed that the nanocarriers had a diameter of 60 ± 11 nm and 91 ± 33 nm for dermatan sulfate sodium salt-poly-l-lysine (DS-PLL) and gum tragacanth-poly-l-lysine (GT-PLL) complexes, respectively. The colloidal nanoparticles were stable due to their negative zeta potential, i.e.-25 ± 4 mV for DS-PLL and -18 ± 3.5 mV for GT-PLL. Cytocompatibility of PCNs in contact with human bone marrow stromal cells (HS-5) was confirmed through a live/dead assay and metabolic activity measurement. In addition, vascular endothelial growth factor (VEGF) was used to evaluate the ability of PCNs to stabilize growth factors. The capability of PCNs to preserve VEGF activity for up to 21 days was confirmed by analyzing the metabolic and mitogenic characteristics of human umbilical vein endothelial cells (HUVECs). Our results demonstrated the potential applications of these nanoparticles in therapeutic delivery for tissue regeneration applications.

Interleukin-15 is a pleotropic factor, capable of modulating metabolism, survival, proliferation, and differentiation in many different cell types. The rationale behind this study relates to previous work demonstrating that IL-15 is a major factor present in stem cell extracts, which protects cardiomyocytes subjected to hypoxic stress in vitro. The objective of this current study was to assess whether administration of IL-15 peptide will also show protective effects in vivo. The data indicate that administration of IL-15 reduces cell death, increases vascularity, decreases scar size, and significantly improves left ventricular ejection fraction in a mouse model of myocardial infarction.

Proper storage of excessive dietary fat into subcutaneous adipose tissue (SAT) prevents ectopic lipid deposition-induced insulin resistance, yet the underlying mechanism remains unclear. Here, we identify angiopoietin-2 (Angpt2)-integrin α5β1 signaling as an inducer of fat uptake specifically in SAT. Adipocyte-specific deletion of Angpt2 markedly reduced fatty acid uptake and storage in SAT, leading to ectopic lipid accumulation in glucose-consuming organs including skeletal muscle and liver and to systemic insulin resistance. Mechanistically, Angpt2 activated integrin α5β1 signaling in the endothelium and triggered fatty acid transport via CD36 and FATP3 into SAT. Genetic or pharmacological inhibition of the endothelial integrin α5β1 recapitulated adipocyte-specific Angpt2 knockout phenotypes. Our findings demonstrate the critical roles of Angpt2-integrin α5β1 signaling in SAT endothelium in regulating whole-body fat distribution for metabolic health and highlight adipocyte-endothelial crosstalk as a potential target for prevention of ectopic lipid deposition-induced lipotoxicity and insulin resistance.