Beneficial Effects of Small-Molecule Oligopeptides Isolated from Panax Ginseng C. A. Meyer on Cellular Fates in Oxidative Stress-Induced Damaged Human Umbilical Vein Endothelial Cells and PC-12.

Cell fate instability is a crucial characteristic of aging and appears to contribute to various age-related pathologies. Exploring the connection between bioactive substances and cell fate stability may offer valuable insights into longevity. Therefore, the objective of this study was to investigate the potential beneficial effects of ginseng oligopeptides (GOPs) isolated from Panax ginseng C. A. Meyer at the cellular level. Disruption of homeostasis of human umbilical vein endothelial cells (HUVECs) and PC-12 was achieved by culturing them in the growth medium supplemented with 200 µM of H2O2, and 25, 50, and 100 µg/mL GOPs for 4 h. Then, they were cultured in a H2O2-free growth medium containing different concentration of GOPs. We found that GOP administration retards the oxidative stress-induced cell instability in HUVECs by increasing cell viability, inhibiting the cell cycle arrest, enhancing telomerase (TE) activity, suppressing oxidative stress and an inflammatory attack, and protecting mitochondrial function. Furthermore, we hypothesized that GOPs may promote mitochondrial biosynthesis by upregulating PGC-1α expression. Similarly, GOPs positively regulated cell stability in PC-12; notably, the protective effect of GOPs on PC-12 mainly occurred through the inhibition of autophagic cell death of neuronal cells, while the protective effect on mitochondria was weak. In conclusion, it is evident that GOPs demonstrate potential beneficial effects in maintaining cell fate stability, thereby potentially contributing to an enhanced health span and overall well-being.

Flavonoid Compounds in Hippophae rhamnoides L. Protect Endothelial Cells from Oxidative Damage Through the PI3K/AKT-eNOS Pathway.

Sea buckthorn, a traditional medicinal plant, has been used for several years in China for the prevention and treatment of various diseases, a practice closely associated with its significant antioxidant activity. The aim of this study was to investigate the protective effects of sea buckthorn flavonoids on vascular endothelial cells in an oxidative stress environment. We isolated and extracted active compounds from sea buckthorn and investigated their impact on endothelial nitric oxide synthase (eNOS) activity through the PI3K/AKT-eNOS signaling pathway through a combination of network pharmacology and cellular experiments, elucidating the regulatory effects of these compounds on endothelial cell functions. Three flavonoids, named Fr.4-2-1, Fr.4-2-2 and Fr.4-2-3, were obtained from sea buckthorn. The results of network pharmacology indicated that they might exert their effects by regulating the PI3K-AKT signaling pathway. In vitro results showed that all three flavonoids were effective in alleviating the degree of oxidative stress in cells, among which Fr.4-2-1 exerted its antioxidant effects by modulating the PI3K/AKT-eNOS pathway. Flavonoids in sea buckthorn can effectively inhibit oxidative stress-induced cellular damage, preserving the integrity and functionality of endothelial cells, which is crucial for maintaining vascular health and function.

Modified Hongyu Decoction promotes wound healing by activating the VEGF/PI3K/Akt signaling pathway.

Wound healing is a considerable problem for clinicians. Ever greater attention has been paid to the role of Chinese herbal monomers and compounds on wound healing. This study aims to elucidate the wound healing mechanism of Modified Hongyu Decoction (MHD) in vivo and in vitro. MHD wound healing activity in vivo was evaluated using an excision rat model. H and E staining, Masson's staining and immunofluorescence of wound tissue on days 7 and 14 were performed to evaluate the efficacy of MHD on wound healing. Subsequently, human umbilical vein endothelial cells (HUVECs) were used to evaluate wound healing characteristics in vitro. Cell Counting Kit-8 (CCK-8) and scratch assays were conducted to assess the effects of MHD on the proliferation and migration of HUVECs. The involvement of the VEGF/PI3K/Akt signaling pathway was assessed by western blotting. The rats in the MHD group displayed more neovascularization and collagen fibers. Western blotting of wound tissue showed that VEGF, PI3K, p-Akt and p-eNOS expression were significantly increased (p<0.05) in the MHD group. Cell Counting Kit-8 and scratch assays demonstrated that MHD promoted HUVECs proliferation and migration. MHD treatment significantly increased VEGF, PI3K, p-Akt and p-eNOS expression in HUVECs (p<0.05), which was inhibited by LY294002. Both in vivo and in vitro data indicated that MHD promotes wound healing by regulating the VEGF/PI3K/Akt signaling pathway.

Dual Delivery of Tetramethylpyrazine and miR-194-5p Using Soft Mesoporous Organosilica Nanoparticles for Acute Lung Injury Therapy.

Background: The respiratory system is intensely damaged by acute lung injury (ALI). The anti-inflammatory effects of tetramethylpyrazine (TMP) against ALI have been confirmed, but it exhibits a short half-life. miR-194-5p could directly target Rac1, but the internalization rate of miRNA cells was low. Purpose: To explore the potential of the soft mesoporous organic silica nanoplatform (NPs) as carriers for delivery of TMP and miR-194-5p through the tail vein. Methods: NPs@TMP and NPs@PEI@miR-194-5p were added to the HUVEC cell-lines, in vitro, to observe the cell uptake and cytotoxic effects. In vivo experiments were conducted by injecting fluorescently labeled NPs through the tail vein and tracking distribution. Therapeutic and toxic side-effects were analyzed systemically. Results: In vitro study exhibited that NPs have no toxic effect on HUVECs within the experimental parameters and have excellent cellular uptake. The IVIS Spectrum Imaging System shows that NPs accumulate mainly in the lungs. NPs@TMP treatment can improved oxidative stress and inflammation levels in ALI mice and inhibited the TLR4/NLRP3/caspase 1 pathway. NPs@PEI@miR-194-5p can inhibit the Rac1/ZO-1/occludin pathway and improved endothelial cell permeability in ALI mice. The co-treatment of NPs@TMP and NPs@PEI@miR-194-5p can significantly improved the survival rates of the mice, reduced pulmonary capillary permeability and improved pathological injury in ALI mice. Innovation: This study combined traditional Chinese medicine, bioinformatics, cellular molecular biology and nanobiomedicine to study the pathogenesis and treatment of ALI. The rate of cellular internalization was improved by changing the shape and hardness of nanoparticles. NPs@TMP and NPs@PEI@miR-194-5p combined application can significantly improve the survival condition and pathological injury of mice. Conclusion: NPs loaded with TMP and miR-194-5p showed a greater therapeutic effect in ALI mice.

Tartary buckwheat protein-derived peptide AFYRW alleviates H2O2-induced vascular injury via the PI3K/AKT/NF-κB pathway.

Tartary buckwheat protein-derived peptide (Ala-Phe-Tyr-Arg-Trp, AFYRW) is a natural active peptide that hampers the atherosclerosis process, but the underlying role of AFYRW in angiogenesis remains unknown. Here, we present a system-based study to evaluate the effects of AFYRW on H2O2-induced vascular injury in human umbilical vein endothelial cells (HUVECs). HUVECs were co-incubated with H2O2 for 2 h in the vascular injury model, and AFYRW was added 24 h in advance to investigate the protective mechanism of vascular injury. We identified that AFYRW inhibits oxidative stress, cell migration, cell invasion, and angiogenesis in H2O2-treated HUVECs. In addition, we found H2O2-induced upregulation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), phosphorylation of nuclear factor-κB (NF-κB) p65 and nuclear translocation of NF-κB decreased by AFYRW. Taken together, AFYRW attenuated H2O2-induced vascular injury through the PI3K/AKT/NF-κB pathway. Thereby, AFYRW may serve as a therapeutic option for vascular injuries.

Schisanhenol Attenuates OxLDL-Induced Endothelial Dysfunction via an AMPK-Dependent Mechanism.

Atherosclerotic cardiovascular diseases, commonly known as the formation of fibrofatty lesions in the artery wall, are the leading causes of death globally. Oxidized low-density lipoprotein (oxLDL) is one of the major components of atherosclerotic plaques. It is evident that dietary supplementation containing sources of antioxidants can prevent atherogenic diseases. Schisanhenol (SAL), a dibenzocyclooctene lignin, has been shown to attenuate oxLDL-induced apoptosis and the generation of reactive oxygen species (ROS) in endothelial cells. However, the underlying molecular mechanisms are still largely unknown. In this study, human umbilical vein endothelial cells (HUVECs) were pre-treated with SAL and oxLDL. Our results showed that adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was enhanced in cells pre-treated with SAL in time-dependent and dose-dependent manners. Subsequently, oxLDL-induced AMPK dephosphorylation and protein kinase C (PKC) phosphorylation were significantly reversed in the presence of SAL. In addition, SAL treatment led to an inhibiting effect on the oxLDL-induced membrane assembly of NADPH oxidase subunits, and a similar effect was observed in ROS generation. This effect was further confirmed using knockdown AMPK with small interfering RNA (siRNA) and pharmaceutical reagents, such as the AMPK activator (AICAR), PKC inhibitor (Gö 6983), and ROS inhibitor (DPI). Furthermore, the oxLDL-induced intracellular calcium rise and the potential collapse of the mitochondrial membrane reduced the Bcl-2/Bax ratio, and released cytochrome c from the mitochondria, leading to the subsequent activation of caspase-3 in HUVECs, which were also markedly suppressed by SAL pretreatment. The results mentioned above may provide additional insights into the possible molecular mechanisms underlying the cardiovascular protective effects of SAL.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells.

BACKGROUND: Breast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HYPOTHESIS: Earlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. METHODS: Co-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. RESULTS: Data revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. CONCLUSION: In silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200 µg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Saikosaponin b2 inhibits tumor angiogenesis in liver cancer via down­regulation of VEGF/ERK/HIF­1α signaling.

Saikosaponin b2 (SSb2) is an active component of Radix Bupleuri, which is commonly used in traditional Chinese medicine for defervescence and liver protection. In the present study, it was demonstrated that SSb2 exhibited potent antitumor activity by inhibiting tumor angiogenesis in vivo and in vitro. As measured by tumor weight and measures of immune function such as thymus index, spleen index and white blood cell count, SSb2 inhibited tumor growth, with low immunotoxicity, in H22 tumor­bearing mice. Furthermore, proliferation and migration of HepG2 liver cancer cells was inhibited following SSb2 treatment, which demonstrated SSb2's antitumor effect. The angiogenesis marker CD34 was downregulated in the SSb2­treated tumor samples, which suggested the antiangiogenic activity of SSb2. Furthermore, the chick chorioallantoic membrane assay demonstrated the potent inhibitory effect of SSb2 on basic fibroblast growth factor­induced angiogenesis. In vitro, SSb2 significantly inhibited numerous stages of angiogenesis, including the proliferation, migration and invasion of human umbilical vein endothelial cells. Further mechanistic studies demonstrated that SSb2 treatment reduced the levels of key proteins involved in angiogenesis, including vascular endothelial growth factor (VEGF), phosphorylated ERK1/2, hypoxia­inducible factor (HIF)­1α, MMP2 and MMP9 in H22 tumor­bearing mice, which supported the HepG2 liver cancer cell results. Overall, SSb2 effectively inhibited angiogenesis via the VEGF/ERK/HIF­1α signal pathway and may serve as a promising natural agent for liver cancer treatment.

Homoplantaginin attenuates high glucose-induced vascular endothelial cell apoptosis through promoting autophagy via the AMPK/TFEB pathway.

Vascular endothelial cell (VEC) injury is a key factor in the development of diabetic vascular complications. Homoplantaginin (Hom), one of the main flavonoids from Salvia plebeia R. Br. has been reported to protect VEC. However, its effects and mechanisms against diabetic vascular endothelium remain unclear. Here, the effect of Hom on VEC was assessed using high glucose (HG)-treated human umbilical vein endothelial cells and db/db mice. In vitro, Hom significantly inhibited apoptosis and promoted autophagosome formation and lysosomal function such as lysosomal membrane permeability and the expression of LAMP1 and cathepsin B. The antiapoptosis effect of Hom was reversed by autophagy inhibitor chloroquine phosphate or bafilomycin A1. Furthermore, Hom promoted gene expression and nuclear translocation of transcription factor EB (TFEB). TFEB gene knockdown attenuated the effect of Hom on upregulating lysosomal function and autophagy. Moreover, Hom activated adenosine monophosphate-dependent protein kinase (AMPK) and inhibited the phosphorylation of mTOR, p70S6K, and TFEB. These effects were attenuated by AMPK inhibitor Compound C. Molecular docking showed a good interaction between Hom and AMPK protein. Animal studies indicated that Hom effectively upregulated the protein expression of p-AMPK and TFEB, enhanced autophagy, reduced apoptosis, and alleviated vascular injury. These findings revealed that Hom ameliorated HG-mediated VEC apoptosis by enhancing autophagy via the AMPK/mTORC1/TFEB pathway.

Streptococcal arginine deiminase regulates endothelial inflammation, mTOR pathway and autophagy.

Endothelial cells (EC) are active participants in the inflammation process. During the infection, the change in endothelium properties provides the leukocyte infiltrate formation and restrains pathogen dissemination due to coagulation control. Pathogenic microbes are able to change the endothelium properties and functions in order to invade the bloodstream and disseminate in the host organism. Arginine deiminase (ADI), a bacterial arginine-hydrolyzing enzyme, which causes the amino acid deficiency, important for endothelium biology. Previous research implicates altered metabolism of arginine in the development of endothelial dysfunction and inflammation. It was shown that arginine deficiency, as well as overabundance affects the balance of mechanical target of rapamycin (mTOR)/S6 kinase (S6K) pathway, arginase and endothelial nitric oxide synthase (eNOS) resulted in reactive oxygen species (ROS) production and EC activation. ADI creating a deficiency of arginine can interfere cellular arginine-dependent processes. Thus, this study was aimed at investigation of the influence of streptococcal ADI on the metabolism and inflammations of human umbilical vein endothelial cells (HUVEC). The action of ADI was studied by comparing the effect Streptococcus pyogenes M49-16 paternal strain expressing ADI and its isogenic mutant M49-16delArcA with the inactivated gene ArcA. Based on comparison of the parental and mutant strain effects, it can be concluded, that ADI suppressed mTOR signaling pathway and enhanced autophagy. The processes failed to return to the basic level with arginine supplement. Our study also demonstrates that ADI suppressed endothelial proliferation, disrupted actin cytoskeleton structure, increased phospho-NF-κB p65, CD62P, CD106, CD54, CD142 inflammatory molecules expression, IL-6 production and lymphocytes-endothelial adhesion. In spite of the ADI-mediated decrease in arginine concentration in the cell-conditioned medium, the enzyme enhanced the production of nitric oxide in endothelial cells. Arginine supplementation rescued proliferation, actin cytoskeleton structure, brought NO production to baseline and prevented EC activation. Additional evidence for the important role of arginine bioavailability in the EC biology was obtained. The results allow us to consider bacterial ADI as a pathogenicity factor that can potentially affect the functions of endothelium.

Differential effects of oleate on vascular endothelial and liver sinusoidal endothelial cells reveal its toxic features in vitro.

Several fatty acids, in particular saturated fatty acids like palmitic acid, cause lipotoxicity in the context of non-alcoholic fatty liver disease . Unsaturated fatty acids (e.g. oleic acid) protect against lipotoxicity in hepatocytes. However, the effect of oleic acid on other liver cell types, in particular liver sinusoidal endothelial cells (LSECs), is unknown. Human umbilical vein endothelial cells (HUVECs) are often used as a substitute for LSECs, however, because of the unique phenotype of LSECs, HUVECs cannot represent the same biological features as LSECs. In this study, we investigate the effects of oleate and palmitate (the sodium salts of oleic acid and palmitic acid) on primary rat LSECs in comparison to their effects on HUVECs. Oleate induces necrotic cell death in LSECs, but not in HUVECs. Necrotic cell death of LSECs can be prevented by supplementation of 2-stearoylglycerol, which promotes cellular triglyceride (TG) synthesis. Repressing TG synthesis, by knocking down DGAT1 renders HUVECs sensitive to oleate-induced necrotic death. Mechanistically, oleate causes a sharp drop of intracellular ATP level and impairs mitochondrial respiration in LSECs. The combination of oleate and palmitate reverses the toxic effect of oleate in both LSECs and HUVECs. These results indicate that oleate is toxic and its toxicity can be attenuated by stimulating TG synthesis. The toxicity of oleate is characterized by mitochondrial dysfunction and necrotic cell death. Moreover, HUVECs are not suitable as a substitute model for LSECs.

Tongluo Shenggu capsule promotes angiogenesis to ameliorate glucocorticoid-induced femoral head necrosis via upregulating VEGF signaling pathway.

BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined. PURPOSE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH. METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a "drug targets-disease genes" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs). RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro. CONCLUSION: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.

Isolation and identification of angiogenesis-promoting components in Huanglian Jiedu decoction using live cell bio-specific extraction.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu Decoction (HLJDD) is a traditional heat-dissipating and detoxicating prescription used in Chinese medicine and has been extensively applied in the clinical treatment of ischemic stroke. Preliminary research confirmed that HLJDD exerts a neuroprotective effect on brain tissue injury caused by cerebral ischemia by promoting angiogenesis. However, the components of HLJDD responsible for its medicinal activity in ischemic injury remain unclear. AIM OF THE STUDY: The aim of this study was to identify the active components of HLJDD that could promote angiogenesis and investigate its underlying mechanism, as well as Hypoxia-inducible factor-1α (HIF-1α)/Vascular endothelial growth factor (VEGF) signalings in human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: The specific binding components of HLJDD with HUVECs were isolated and identified through a combination of live cell biospecific extraction, solid-phase extraction, and ultra performance liquid chromatography (UPLC)-Orbitrap Fusion Tribrid mass spectrometry (MS). Their pharmacological activity against oxygen-glucose deprivation-reperfusion (OGD/R) injury and in vitro pro-angiogenesis was validated using Cell Counting Kit-8 (CCK-8) and tube formation analysis, respectively. Finally, we explored the effect of active ingredients on the expression levels of HIF-1α and VEGF using enzyme-linked immunosorbent assay. Molecular docking was used to predict the potential binding of six active components to phosphoinositide 3-kinase (PI3K), serine/threonine-specific protein kinase (AKT) and Von Hippel-Lindau (VHL) proteins, which are involved in the regulation of HIF-1α and are highly associated with angiogenesis. RESULTS: A total of 13 HUVECs-specific HLJDD components were identified, and 10 of them were shown to protect against OGD/R injury. We were the first to demonstrate that two of these components have a protective role in OGD/R-induced HUVECs injury. Additionally, seven of these 10 components exhibited angiogenesis-promoting activity, and two of these components were shown, for the first time, to promote angiogenesis in HUVECs. These effects might occur through the HIF-1α/VEGF pathway. Molecular docking results showed that all six active ingredients could stably bind to PI3K and AKT proteins, suggesting that these two proteins may be potential targets for six active ingredients. CONCLUSIONS: The approach employed in this study effectively identified proangiogenic components in HLJDD that might act via PI3K/AKT/HIF-1α/VEGF pathways and other mechanisms involved in angiogenesis. In conclusion, this study was the first to demonstrate four compounds with new bioactivities and could also provide insight into the isolation and discovery of new bioactive compounds existing in Chinese medicine with potential clinical value.

Anti-angiogenic effects of aqueous extract from Agrostemma githago L. seed in human umbilical vein endothelial cells via regulating Notch/VEGF, MMP2/9, ANG2, and VEGFR2.

Abnormal angiogenesis contributes to the pathogenesis of various diseases. The medicinal usage of Agrostemma githago L. seed (A. githago herein) has been stated in traditional medicine. This study aims to investigate the anti-angiogenic potential of aqueous extract of A. githago. In order to test the effect of A. githago extract, its impact on HUVECs, T98G, and HGF2PI2 cells was assessed by looking at cellular viability, changes in the distribution of cells in different phases of the cell cycle, induction of oxidative stress, and apoptosis. In addition, the release of VEGF, ANG2, and MMP2/9 factors, along with the expressions of the critical Notch signaling pathway players and VEGF receptors (VEGFR), was measured. Furthermore, a γ-secretase inhibitor (LY411575) was applied to determine whether Notch inhibition restores A. githago effects. As a further characterization, total phenolic and flavonoid contents of A. githago were estimated, and five triterpene saponin compounds were identified using LC-ESI-MS. In response to A. githago extract, a reduction in total cell viability, along with the induction of ROS and apoptosis, was detected. Exposure to the A. githago extract could modulate the release of VEGF and ANG2 from T98G and HUVECs, respectively. In addition, A. githago reduced the release of MMP2/9. Furthermore, Notch1, DLL4, and HEY2 transcripts and protein expressions were up-regulated, while VEGFR2 was down-regulated in treated HUVEC cells. Treatment with the A. githago extract resulted in a dose-dependent inhibition of AKT phosphorylation. Inhibition of Notch signaling retrieved the viability loss, reduced intracellular ROS, and alleviated the impaired tube formation in A. githago-treated HUVECs. Overall, these data underscore the anti-angiogenic potential of A. githago via inducing apoptosis, modifying the expression levels of VEGF/VEGFR2, and impacting the release of MMP2/9 and ANG2, effects that are most probably modulated through the Notch/VEGF signaling axis.

Differential effect of basal vitamin D status in monocrotaline induced pulmonary arterial hypertension in normal and vitamin D deficient rats: Possible involvement of eNOS/TGF-ß/α-SMA signaling pathways.

Vitamin D deficiency is common and linked to poor prognosis in pulmonary arterial hypertension (PAH). We investigated the differential effect of basal vitamin D levels in monocrotaline (MCT) induced PAH in normal and vitamin D deficient (VDD) rats. Rats were fed a VDD diet and exposed to filtered fluorescent light to deplete vitamin D. Normal rats were pretreated with vitamin D 100 IU/d and treated with vitamin D 100 and 200 IU/d, while VDD rats received vitamin D 100 IU/d. Vitamin D receptor (VDR) silencing was done in human umbilical vein endothelial cells (HUVECs) using VDR siRNA. Calcitriol (50 nM/mL) was added to human pulmonary artery smooth muscle cells (HPASMCs) and HUVECs before and after the exposure to TGF-ß (10 ng/mL). Vitamin D 100 IU/d pretreatment in normal rats up-regulated the expression of eNOS and inhibited endothelial to mesenchymal transition significantly and maximally. Vitamin D 100 IU/d treatment in VDD rats was comparable to vitamin D 200 IU/d treated normal rats. These effects were significantly attenuated by L-NAME (20 mg/kg), a potent eNOS inhibitor. Exposure to TGF- ß significantly reduced the expression of eNOS and increased the mesenchymal marker expression in normal and VDR-silenced HUVECs and HPASMCs, which were averted by treatment and maximally inhibited by pretreatment with calcitriol (50 nM). To conclude, this study provided novel evidence suggesting the beneficial role of higher basal vitamin D levels, which are inversely linked with PAH severity.

Jatrorrhizine Improves Endothelial Function in Diabetes and Obesity through Suppression of Endoplasmic Reticulum Stress.

Jatrorrhizine (JAT) is one of the major bioactive protoberberine alkaloids found in rhizoma coptidis, which has hypoglycemic and hypolipidemic potential. This study aimed to evaluate the vasoprotective effects of JAT in diabetes and obesity and the underlying mechanism involved. Mouse aortas, carotid arteries and human umbilical cord vein endothelial cells (HUVECs) were treated with risk factors (high glucose or tunicamycin) with and without JAT ex vivo and in vitro. Furthermore, aortas were obtained from mice with chronic treatment: (1) control; (2) diet-induced obese (DIO) mice fed a high-fat diet (45% kcal% fat) for 15 weeks; and (3) DIO mice orally administered JAT at 50 mg/kg/day for the last 5 weeks. High glucose or endoplasmic reticulum (ER) stress inducer tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations (EDRs) in mouse aortas, induced oxidative stress in carotid arteries and HUVECs, downregulated phosphorylations of Akt at Ser473 and eNOS at Ser1177 and enhanced ER stress in mouse aortas and HUVECs, and these impairments were reversed by cotreatment with JAT. JAT increased NO release in high-glucose-treated mouse aortas and HUVECs. In addition, chronic JAT treatment restored endothelial function with EDRs comparable to the control, increased Akt/eNOS phosphorylation, and attenuated ER stress and oxidative stress in aortas from DIO mice. Blood pressure, glucose sensitivity, fatty liver and its morphological change, as well as plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and plasma lipid profile, were also normalized by JAT treatment. Collectively, our data may be the first to reveal the vasoprotective effect of JAT that ameliorates endothelial dysfunction in diabetes and obesity through enhancement of the Akt/eNOS pathway and NO bioavailability, as well as suppression of ER stress and oxidative stress.

Extracellular vesicle-mediated transfer of lncRNA CLDN10-AS1 aggravates low-density lipoprotein-induced vascular endothelial injury.

Oxidized low-density lipoprotein (ox-LDL) stimulation impairs the oxidation-reduction equilibrium in vascular endothelial cells (VECs) and contributes to atherosclerosis (AS). This study probed the mechanisms of extracellular vesicle (EV)-mediated transfer of lncRNA CLDN10 antisense RNA 1 (CLDN10-AS1) in ox-LDL-induced VEC injury. Initially, VEC injury models were established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL. EVs were isolated from HUVECs (HUVECs-EVs) and identified. CLDN10-AS1, microRNA (miR)-186, and Yin Yang 1 (YY1) expressions in ox-LDL-treated HUVECs and EVs derived from these cells (ox-EVs) were measured. HUVECs were incubated with EVs, after which the cell viability, apoptosis, and concentrations of proinflammatory cytokines and oxidative stress markers were measured. We discovered that CLDN10-AS1 and YY1 were upregulated in ox-LDL-treated HUVECs, whereas miR-186 was downregulated. ox-EVs treatment elevated CLDN10-AS1 expression in HUVECs and ox-EVs overexpressing CLDN10-AS1 promoted VEC injury. Besides, CLDN10-AS1 is competitively bound to miR-186 and promoted YY1 expression. Rescue experiments revealed that miR-186 overexpression or YY1 suppression partially reversed the roles of ox-EVs overexpressing CLDN10-AS1 in ox-LDL-induced VEC injury. Lastly, clinical serum samples were collected for verification. Overall, CLDN10-AS1 carried by HUVECs-EVs into HUVECs competitively bound to miR-186 to elevate YY1 expression, thereby aggravating ox-LDL-induced VEC injury.

Flavonoids from Lycium barbarum Leaves Exhibit Anti-Aging Effects through the Redox-Modulation.

Lycium barbarum leaves are a kind of vegetable, and modern nutrition studies have found that they have an anti-aging function. Our study aims to investigate the anti-aging effects of Lycium barbarum leaf flavonoid (LBLF) extracts and its underlying molecular mechanism. LBLFs were purified using D101 and polyamide resin, characterized by ultraperformance liquid chromatography coupled with mass spectrometry, and administered to hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. Appropriate enrichment conditions were optimized through dynamic adsorption and desorption experiments, the content of flavonoids reached 909.84 mg/g, rutin and kaempferol being the main ones. LBLFs attenuated H2O2-induced HUVEC apoptosis, decreased reactive oxygen species and malondialdehyde production levels, increased superoxide dismutase, glutathione peroxidase and catalase activities. Furthermore, pre-treatment with LBLF increased mRNA expression of erythropoietin (EPO) and heme oxygenase-1 (HO-1) via the mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. Compared with 100 µM rutin monomer, LBLF prolonged the lifespan of Caenorhabditis elegans, enhanced their mobility in middle life stages and upregulated expression of sod-2, gcs-1 and skn-1 genes, which indicated that the anti-aging effects of LBLF were due to its redox-modulation.

Disturbance of Fatty Acid Metabolism Promoted Vascular Endothelial Cell Senescence via Acetyl-CoA-Induced Protein Acetylation Modification.

Endothelial cell senescence is the main risk factor contributing to vascular dysfunction and the progression of aging-related cardiovascular diseases. However, the relationship between endothelial cell metabolism and endothelial senescence remains unclear. The present study provides novel insight into fatty acid metabolism in the regulation of endothelial senescence. In the replicative senescence model and H2O2-induced premature senescence model of primary cultured human umbilical vein endothelial cells (HUVECs), fatty acid oxidation (FAO) was suppressed and fatty acid profile was disturbed, accompanied by downregulation of proteins associated with fatty acid uptake and mitochondrial entry, in particular the FAO rate-limiting enzyme carnitine palmitoyl transferase 1A (CPT1A). Impairment of fatty acid metabolism by silencing CPT1A or CPT1A inhibitor etomoxir facilitated the development of endothelial senescence, as implied by the increase of p53, p21, and senescence-associated ß-galactosidase, as well as the decrease of EdU-positive proliferating cells. In the contrary, rescue of FAO by overexpression of CPT1A or supplement of short chain fatty acids (SCFAs) acetate and propionate ameliorated endothelial senescence. In vivo, treatment of acetate for 4 weeks lowered the blood pressure and alleviated the senescence-related phenotypes in aortas of Ang II-infused mice. Mechanistically, fatty acid metabolism regulates endothelial senescence via acetyl-coenzyme A (acetyl-CoA), as implied by the observations that suppression of acetyl-CoA production using the inhibitor of ATP citrate lyase NDI-091143 accelerated senescence of HUVECs and that supplementation of acetyl-CoA prevented H2O2-induced endothelial senescence. Deficiency of acetyl-CoA resulted in alteration of acetylated protein profiles which are associated with cell metabolism and cell cycle. These findings thus suggest that improvement of fatty acid metabolism might ameliorate endothelial senescence-associated cardiovascular diseases.