Mesenchymal stem cell-derived exosomes decrease Hyperplasia in Psoriasis by inducing transforming growth factor ß2 (TGF-ß2).

BACKGROUND: Psoriasis, a chronic inflammatory skin disease, is increasingly effectively managed with the targeted immunotherapy; however, long-term immunotherapy carries health risks, and loss of response. Therefore, we need to develop the alternative treatment strategies. Mesenchymal stem/stromal cell (M.S.C.) exosomes stand out for their remarkable immunomodulatory properties, gaining widespread recognition. This study investigated whether M.S.C. exosomes can reduce psoriasis-induced hyperplasia by inducing Transforming Growth Factor beta 2 (TGF-beta2) signaling. METHODOLOGY: Exosomes were isolated from M.S.C.s by ultracentrifugation. Then, scanning electron microscopy was used for the morphology of exosomes. To ascertain the exosome concentration, the Bradford test was used. To ascertain the cellular toxicity of exosomes in Human Umbilical Vein Endothelial Cells ( H.U.V.E.C), an MTT experiment was then conducted. Real-time PCR was used to quantify TGF beta2 expression levels, whereas an ELISA immunosorbent assay was used to determine the protein concentration of TGF beta2. RESULTS: In this study, the exosomes of 15-30 nm in size that were uniform, and cup-shaped were isolated. Moreover, the IC50 value for this Treatment was calculated to be 181.750 µg/ml. The concentration of TGF-ß2 gene in the target cells significantly increased following Treatment with the exosomes. Furthermore, the expression level of the studied gene significantly increased due to the Treatment. CONCLUSION: Upregulating the expression of TGF-ß2 in psoriatic cells via TGF-ß2 signaling is one way exosomes can help reduce hyperplasia.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

[α2-macroglobulin alleviates glucocorticoid-induced avascular necrosis of the femoral head in mice by promoting proliferation, migration and angiogenesis of vascular endothelial cells].

OBJECTIVE: To explore the mechanism underlying the protective effect of α2-macroglobulin (A2M) against glucocorticoid-induced femoral head necrosis. METHODS: In a human umbilical vein endothelial cell (HUVEC) model with injuries induced by gradient concentrations of dexamethasone (DEX; 10-8-10-5 mol/L), the protective effects of A2M at 0.05 and 0.1 mg/mL were assessed by examining the changes in cell viability, migration, and capacity of angiogenesis using CCK-8 assay, Transwell and scratch healing assays and angiogenesis assay. The expressions of CD31 and VEGF-A proteins in the treated cells were detected using Western blotting. In BALB/c mouse models of avascular necrosis of the femoral head induced by intramuscular injections of methylprednisolone, the effects of intervention with A2M on femoral trabecular structure, histopathological characteristics, and CD31 expression were examined with Micro-CT, HE staining and immunohistochemical staining. RESULTS: In cultured HUVECs, DEX treatment significantly reduced cell viability, migration and angiogenic ability in a concentration- and time-dependent manner (P<0.05), and these changes were obviously reversed by treatment with A2M in positive correlation with A2M concentration (P<0.05). DEX significantly reduced the expression of CD31 and VEGF-A proteins in HUVECs, while treatment with A2M restored CD31 and VEGF-A expressions in the cells (P<0.05). The mouse models of femoral head necrosis showed obvious trabecular damages in the femoral head, where a large number of empty lacunae and hypertrophic fat cells could be seen and CD31 expression was significantly decreased (P<0.05). A2M treatment of the mouse models significantly improved trabecular damages, maintained normal bone tissue structures, and increased CD31 expression in the femoral head (P<0.05). CONCLUSION: A2M promotes proliferation, migration, and angiogenesis of DEX-treated HUVECs and alleviates methylprednisolone-induced femoral head necrosis by improving microcirculation damages and maintaining microcirculation stability in the femoral head.

SPAG5 deficiency activates autophagy to reduce atherosclerotic plaque formation in ApoE-/- mice.

BACKGROUND: Autophagy, as a regulator of cell survival, plays an important role in atherosclerosis (AS). Sperm associated antigen 5 (SPAG5) is closely associated with the classical autophagy pathway, PI3K/Akt/mTOR signaling pathway. This work attempted to investigate whether SPAG5 can affect AS development by regulating autophagy. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized-low density lipoprotein (ox-LDL) to induce cell damage. ApoE-/- mice were fed a Western diet to establish an AS mouse model. Haematoxylin and eosin (H&E) staining and Oil Red O staining evaluated the pathological changes and in lipid deposition in aortic tissues. CCK-8 and flow cytometry detected cell proliferation and apoptosis. Immunohistochemistry, Enzyme linked immunosorbent assay, qRT-PCR and western blotting assessed the levels of mRNA and proteins. RESULTS: Ox-LDL treatment elevated SPAG5 expression and the expression of autophagy-related proteins, LC3-I, LC3-II, Beclin-1, and p62, in HUVECs. GFP-LC3 dots were increased in ox-LDL-treated HUVECs and LPS-treated HUVECs. SPAG5 knockdown reversed both ox-LDL and LPS treatment-mediated inhibition of cell proliferation and promotion of apoptosis in HUVECs. SPAG5 silencing further elevated autophagy and repressed the expression of PI3K, p-Akt/Akt, and p-mTOR/mTOR in ox-LDL-treated HUVECs. 3-MA (autophagy inhibitor) treatment reversed SPAG5 silencing-mediated increase of cell proliferation and decrease of apoptosis in ox-LDL-treated HUVECs. In vivo, SPAG5 knockdown reduced atherosclerotic plaques in AS mice through activating autophagy and inhibiting PI3K/Akt/mTOR signaling pathway. CONCLUSION: This work demonstrated that SPAG5 knockdown alleviated AS development through activating autophagy. Thus, SPAG5 may be a potential target for AS therapy.

Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-ß-MRTF axis inhibited by aspirin.

BACKGROUND: The SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT). METHODS: HUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-ß-neutralizing antibodies, and inhibitors of the TGF-ß-dependent pathway were used to investigate the role of the TGF-ß-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation. RESULTS: Both viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-ß-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-ß2, whose secretion is induced through TLR4-ROS action. TGF-ß2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-ß1 secretion as a result of ACE2 downregulation. TGF-ß1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-ß-MRTF pathway deregulation. CONCLUSION: The reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-ß2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-ß1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.

Hydrogels with Ultrasound-Treated Hyaluronic Acid Regulate CD44-Mediated Angiogenic Potential of Human Vascular Endothelial Cells In Vitro.

The development of hydrogels that allow vascular endothelial cells to form capillary-like networks is critical for advancing tissue engineering and drug discovery. In this study, we developed hydrogels composed of phenolated hyaluronic acid (HA-Ph) with an average molecular weight of 490-159 kDa via sonication in an aqueous solution. These hydrogels were synthesized by the horseradish peroxidase-catalyzed crosslinking of phenol moieties in the presence of hydrogen peroxide and phenolated gelatin. The sonication-degraded HA-Ph (198 kDa) significantly enhanced the migration ability of human umbilical vein endothelial cells (HUVECs) on cell culture plates when added to the medium compared to the original HA-Ph (490 kDa) and less-degraded HA-Ph (312-399 kDa). In addition, HUVECs cultured on these hydrogels formed networks that did not occur on hydrogels made from the original HA-Ph. CD44 expression and PI3K gene expression, both markers related to angiogenesis, were 3.5- and 1.8-fold higher, respectively, in cells cultured on sonication-degraded HA-Ph hydrogels than in those cultured on hydrogels comprising the original HA-Ph. These results highlight the potential of hydrogels containing sonication-degraded HA-Ph for tissue engineering and drug-screening applications involving human vascular endothelial cells.

CCCTC-binding factor suppresses alpha-2-macroglobulin transcription to improve vascular endothelial cell functions in lower extremity arteriosclerosis obliterans.

Vascular endothelial cell functions affect lower extremity arteriosclerosis obliterans (LEASO), while alpha-2-macroglobulin (A2M) and CCCTC-binding factor (CTCF) are closely related to the function of such cells. This paper aims to identify the influences of CTCF on vascular endothelial cells in LEASO by regulating A2M. A rat model of LEASO was established to measure intima-media ratio, blood lipid, and inflammatory factor levels. By constructing LEASO cell models, cell viability and apoptosis were assayed, while autophagy-related proteins, CTCF and A2M levels in femoral artery tissues and HUVECs were determined. The transcriptional regulation of CTCF on A2M was verified. In LEASO rat models, femoral artery lumen was narrowed and endothelial cells were disordered; levels of total cholesterol, IL-1, and TNF-α enhanced, and HDL-C decreased, with strong expression of A2M and low expression of CTCF. The viability of ox-LDL-treated HUVECs was decreased, together with higher apoptosis, lower LC3II/I expression, and higher p62 expression, which were reversed by sh-A2M transfection. Overexpression of CTCF inhibited A2M transcription, promoted the viability and autophagy of HUVECs, and decreased apoptosis. Collectively, CTCF improves the function of vascular endothelial cells in LEASO by inhibiting A2M transcription.

Dapagliflozin Improves Angiogenesis after Hindlimb Ischemia through the PI3K-Akt-eNOS Pathway.

Recently, the vascular protective effect of anti-diabetic agents has been receiving much attention. Sodium glucose cotransporter 2 (SGLT2) inhibitors had demonstrated reductions in cardiovascular (CV) events. However, the therapeutic effect of dapagliflozin on angiogenesis in peripheral arterial disease was unclear. This study aimed to explore the effect and mechanism of dapagliflozin on angiogenesis after hindlimb ischemia. We first evaluated the effect of dapagliflozin on post-ischemic angiogenesis in the hindlimbs of rats. Laser doppler imaging was used to detect the hindlimb blood perfusion. In addition, we used immunohistochemistry to detect the density of new capillaries after ischemia. The relevant signaling pathways of dapagliflozin affecting post-ischemic angiogenesis were screened through phosphoproteomic detection, and then the mechanism of dapagliflozin affecting post-ischemic angiogenesis was verified at the level of human umbilical vein endothelial cells (HUVECs). After subjection to excision of the left femoral artery, all rats were randomly distributed into two groups: the dapagliflozin group (left femoral artery resection, receiving intragastric feeding with dapagliflozin (1 mg/kg/d), for 21 consecutive days) and the model group, that is, the positive control group (left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days). In addition, the control group, that is the negative control group (without left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days) was added. At day 21 post-surgery, the dapagliflozin-treatment group had the greatest blood perfusion, accompanied by elevated capillary density. The results showed that dapagliflozin could promote angiogenesis after hindlimb ischemia. Then, the ischemic hindlimb adductor-muscle tissue samples from three rats of model group and dapagliflozin group were taken for phosphoproteomic testing. The results showed that the PI3K-Akt-eNOS signaling pathway was closely related to the effect of dapagliflozin on post-ischemic angiogenesis. Our study intended to verify this mechanism from the perspective of endothelial cells. In vitro, dapagliflozin enhanced the tube formation, migration, and proliferation of HUVECs under ischemic and hypoxic conditions. Additionally, the dapagliflozin administration upregulated the expression of angiogenic factors phosphorylated Akt (p-Akt) and phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor A (VEGFA), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. dapagliflozin could promote angiogenesis after ischemia. This effect might be achieved by promoting the activation of the PI3K-Akt-eNOS signaling pathway. This study provided a new perspective, new ideas, and a theoretical basis for the treatment of peripheral arterial disease.

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

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

rESWT promoted angiogenesis via Bach1/Wnt/ß-catenin signaling pathway.

Previous reports have established that rESWT fosters angiogenesis, yet the mechanism by which rESWT promotes cerebral angiogenesis remains elusive. rESWT stimulated HUVECs proliferation as evidenced by the CCK-8 test, with an optimal dosage of 2.0 Bar, 200 impulses, and 2 Hz. The tube formation assay of HUVECs revealed that tube formation peaked at 36 h post-rESWT treatment, concurrent with the lowest expression level of Bach1, as detected by both Western blot and immunofluorescence. The expression level of Wnt3a, ß-catenin, and VEGF also peaked at 36 h. A Bach1 overexpression plasmid was transfected into HUVECs, resulting in a decreased expression level of Wnt3a, ß-catenin, and VEGF. Upon treatment with rESWT, the down-regulation of Wnt3a, ß-catenin, and VEGF expression in the transfected cells was reversed. The Wnt/ß-catenin inhibitor DKK-1 was utilized to suppress Wnt3a and ß-catenin expression, which led to a concurrent decrease in VEGF expression. However, rESWT treatment could restore the expression of these three proteins, even in the presence of DKK-1. Moreover, in the established OGD model, it was observed that rESWT could inhibit the overexpression of Bach1 and enhance VEGF and VEGFR-2 expression under the OGD environment.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation.

Diabetes complications such as nephropathy, retinopathy, or cardiovascular disease arise from vascular dysfunction. In this context, it has been observed that past hyperglycemic events can induce long-lasting alterations, a phenomenon termed "metabolic memory." In this study, we evaluated the genome-wide gene expression and chromatin accessibility alterations caused by transient high-glucose exposure in human endothelial cells (ECs) in vitro. We found that cells exposed to high glucose exhibited substantial gene expression changes in pathways known to be impaired in diabetes, many of which persist after glucose normalization. Chromatin accessibility analysis also revealed that transient hyperglycemia induces persistent alterations, mainly in non-promoter regions identified as enhancers with neighboring genes showing lasting alterations. Notably, activation of the NRF2 pathway through NRF2 overexpression or supplementation with the plant-derived compound sulforaphane, effectively reverses the glucose-induced transcriptional and chromatin accessibility memories in ECs. These findings underscore the enduring impact of transient hyperglycemia on ECs' transcriptomic and chromatin accessibility profiles, emphasizing the potential utility of pharmacological NRF2 pathway activation in mitigating and reversing the high-glucose-induced transcriptional and epigenetic alterations.

[The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms].

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment.

BACKGROUND: Vasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear. METHODS: Vascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation. RESULTS: Our study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1. CONCLUSION: In summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Major endothelial damage markers identified from hemadsorption filters derived from treated patients with septic shock - endoplasmic reticulum stress and bikunin may play a role.

Introduction: In septic patients the damage of the endothelial barrier is decisive leading to circulatory septic shock with disseminated vascular coagulation, edema and multiorgan failure. Hemadsorption therapy leads to rapid resolution of clinical symptoms. We propose that the isolation of proteins adsorbed to hemadsorption devices contributes to the identification of mediators responsible for endothelial barrier dysfunction. Material and methods: Plasma materials enriched to hemadsorption filters (CytoSorb®) after therapy of patients in septic shock were fractionated and functionally characterized for their effect on cell integrity, viability, proliferation and ROS formation by human endothelial cells. Fractions were further studied for their contents of oxidized nucleic acids as well as peptides and proteins by mass spectrometry. Results: Individual fractions exhibited a strong effect on endothelial cell viability, the endothelial layer morphology, and ROS formation. Fractions with high amounts of DNA and oxidized DNA correlated with ROS formation in the target endothelium. In addition, defined proteins such as defensins (HNP-1), SAA1, CXCL7, and the peptide bikunin were linked to the strongest additive effects in endothelial damage. Conclusion: Our results indicate that hemadsorption is efficient to transiently remove strong endothelial damage mediators from the blood of patients with septic shock, which explains a rapid clinical improvement of inflammation and endothelial function. The current work indicates that a combination of stressors leads to the most detrimental effects. Oxidized ssDNA, likely derived from mitochondria, SAA1, the chemokine CXCL7 and the human neutrophil peptide alpha-defensin 1 (HNP-1) were unique for their significant negative effect on endothelial cell viability. However, the strongest damage effect occurred, when, bikunin - cleaved off from alpha-1-microglobulin was present in high relative amounts (>65%) of protein contents in the most active fraction. Thus, a relevant combination of stressors appears to be removed by hemadsorption therapy which results in fulminant and rapid, though only transient, clinical restitution.

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

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

3-Hydroxytanshinone Inhibits the Activity of Hypoxia-Inducible Factor 1-α by Interfering with the Function of α-Enolase in the Glycolytic Pathway.

Tumor cells in hypoxic conditions control cancer metabolism and angiogenesis by expressing HIF-1α. Tanshinone is a traditional Chinese medicine that has been shown to possess antitumor properties and exerts a therapeutic impact on angiogenesis. However, the precise molecular mechanism responsible for the antitumor activity of 3-Hydroxytanshinone (3-HT), a type of tanshinone, has not been fully understood. Therefore, our study aimed to investigate the mechanism by which 3-HT regulates the expression of HIF-1α. Our findings demonstrate that 3-HT inhibits HIF-1α activity and expression under hypoxic conditions. Additionally, 3-HT inhibits hypoxia-induced angiogenesis by suppressing the expression of VEGF. Moreover, 3-HT was found to directly bind to α-enolase, an enzyme associated with glycolysis, resulting in the suppression of its activity. This inhibition of α-enolase activity by 3-HT leads to the blockade of the glycolytic pathway and a decrease in glycolysis products, ultimately altering HIF1-α expression. Furthermore, 3-HT negatively regulates the expression of HIF-1α by altering the phosphorylation of AMP-activated protein kinase (AMPK). Our study's findings elucidate the mechanism by which 3-HT regulates HIF-1α through the inhibition of the glycolytic enzyme α-enolase and the phosphorylation of AMPK. These results suggest that 3-HT holds promise as a potential therapeutic agent for hypoxia-related angiogenesis and tumorigenesis.

TCF12 Transcriptionally Activates SPHK1 to Induce Osteosarcoma Angiogenesis by Promoting the S1P/S1PR4/STAT3 Axis.

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.

ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.