Human Endothelial Cell Function Research Articles

Pseudomonas aeruginosa N-3-Oxododecanoyl Homoserine Lactone Disrupts Endothelial Integrity by Activating the Angiopoietin-Tie System.

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.

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

BackgroundVasculopathy 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.MethodsVascular 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.ResultsOur 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.ConclusionIn 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.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, β-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE−/− mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.

Tumor-Derived Exosomes Promote the Angiogenic Function of Vascular Endothelial Cells by Activating the miR-423-5p/EFNA3 Signaling Pathway

Angiogenesis plays a key role in promoting the growth and metastasis of breast tumors. Tumor exosomes (EXs) contribute to angiogenesis in various tumor tissues by transferring their carried RNAs. MiR-423-5p was enriched in multiple tumors and implicated in tumor growth. In this study, we investigated the roles and underlying mechanisms of tumor-derived EXs and their carried miR-423-5p in regulating human umbilical vein endothelial cell (HUVEC) functions. EXs derived from MCF-7 cells (MCF-7 EXs) or with miR-423-5p knockdown (MCF-7 EXsSimiR-423-5p) were collected and incubated with ECs, and then the proliferation, migration, and tube formation abilities of ECs were detected. We found that miR-423-5p was enriched in breast cancer, MCF-7 cell lines and their derived EXs. After coculture with HUVECs, MCF-7 EXs merged into HUVECs and subsequently increased the miR-423-5p expression, proliferation, migration, and tube formation abilities of HUVECs, paralleling the increased EFNA3 and Notch1 expression, which was partially abolished by miR-423-5p knockdown. Altogether, our data suggest that MCF-7 EXs enriched with miR-423-5p promote the angiogenic function of vascular endothelial cells by activating the miR-423-5p/EFNA3/Akt signaling pathway.

NLRC4-mediated pyroptosis was involved in coagulation disorders of acute pancreatitis.

Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1β, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.

Nuciferine reduces vascular leakage and improves cardiac function in acute myocardial infarction by regulating the PI3K/AKT pathway

The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen–glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.

Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture.

Connexin (Cx) 37, 40, and 43 are implicated in vascular function, specifically in the electrical coupling of endothelial cells and vascular smooth-muscle cells. In the present study, we investigated whether factors implicated in vascular dysfunction can modulate the gene expression of Cx37, Cx40, and Cx43 and whether this is associated with changes in endothelial layer barrier function in human microvascular endothelial cells (HMEC-1). First, HMEC-1 were subjected to stimuli for 4 and 8h. We tested their responses to DETA-NONOate, H2O2, high glucose, and angiotensin II, none of which relevantly affected the transcription of the connexin genes. Next, we tested inflammatory factors IL-6, interferon gamma (IFNγ), and TNFα. IFNγ (10ng/mL) consistently induced Cx40 expression at 4 and 8 h to 10-20-fold when corrected for the control. TNFα and IL-6 resulted in small but significant depressions of Cx37 expression at 4h. Two JAK inhibitors, epigallocatechin-3-gallate (EGCG) (100-250μM) and AG490 (100-250μM), dose-dependently inhibited the induction of Cx40 expression by IFNγ. Subsequently, HMEC-1 were subjected to 10ng/mL IFNγ for 60h, and intercellular and transcellular impedance was monitored by electric cell-substrate impedance sensing (ECIS). In response to IFNγ, junctional-barrier impedance increased more than cellular-barrier impedance; this was prevented by AG490 (5μM). In conclusion, IFNγ can strongly induce Cx40 expression and modify the barrier properties of the endothelial cell membrane through the JAK/STAT pathway. Moreover, the Cx37, Cx40, and Cx43 expression in endothelial cells is stable and, apart from IFNγ, not affected by a number of factors implicated in endothelial dysfunction and vascular diseases.

PH-responsive, self-sculptured Mg/PLGA composite microfibers for accelerated revascularization and soft tissue regeneration

Biodegradable Mg/polymer composite fibers offer a promising therapeutic option for tissue injury because of bioactive Mg2+ and biomimetic microstructure. However, current studies are limited to the contribution of Mg2+ and the single microstructure. In this study, we designed Mg/poly (lactic-co-glycolic acid) (Mg/PLGA) composite microfibers that significantly enhanced angiogenesis and tissue regeneration synergistically by Mg2+ and self-sculptured microstructure, due to spontaneous in situ microphase separation in response to the weakly alkaline microenvironment. Our composite microfiber patch exhibited superior performance in the adhesion, spreading, and angiogenesis functions of human umbilical vein endothelial cells (HUVECs) due to the joint contribution of the hierarchically porous microstructure and Mg2+. Genomics and proteomics analyses revealed that the Mg/PLGA composite microfibers activated the cell focal adhesion and angiogenesis-related signaling pathways. Furthermore, the repair of typical soft tissue defects, including refractory urethral wounds and easily healed skin wounds, validated that our Mg/PLGA composite microfiber patch could provide favorable surface topography and ions microenvironment for tissue infiltration and accelerated revascularization. It could cause rapid urethral tissue regeneration and recovery of rabbit urethral function within 6 weeks and accelerate rat skin wound closure within 16 days. This work provides new insight into soft tissue regeneration through the bioactive alkaline substance/block copolymer composites interactions.

The Inhibition of Fibrosis and Inflammation in Obstructive Kidney Injury via the miR-122-5p/SOX2 Axis Using USC-Exos.

Background: Fibrosis and inflammation due to ureteropelvic junction obstruction substantially contributes to poor renal function. Urine-derived stem-cell-derived exosomes (USC-Exos) have therapeutic effects through paracrine. Methods: Invitro, the effects of USC-Exos on the biological functions of HK-2 and human umbilical vein endothelial cells were tested. Cell inflammation and fibrosis were induced by transforming growth factor-β1 and interleukin-1β, and their anti-inflammatory and antifibrotic effects were observed after exogenous addition of USC-Exos. Through high-throughput sequencing of microRNA in USC-Exos, the pathways and key microRNAs were selected. Then, the antifibrotic and anti-inflammatory effects of exosomal miR-122-5p and target genes were verified. The role of the miR-122-5p/SOX2 axis in anti-inflammatory and antifibrotic effects was verified. Invivo, a rabbit model of partial unilateral ureteral obstruction (PUUO) was established. Magnetic resonance imaging recorded the volume of the renal pelvis after modeling, and renal tissue was pathologically analyzed. Results: We examined the role of USC-Exos and their miR-122-5p content in obstructive kidney injury. These Exos exhibit antifibrotic and anti-inflammatory activities. SOX2 is the hub gene in PUUO and negatively related to renal function. We confirmed the binding relationship between miR-122-5p and SOX2. The anti-inflammatory and antifibrotic effects of miR-122-5p were inhibited, indicating that miR-122-5p has anti-inflammatory and antifibrotic effects by inhibiting SOX2 expression. Invivo, the PUUO group showed typical obstructive kidney injury after modeling. After USC-Exo treatment, the shape of the renal pelvis shown a remarkable improvement, and inflammation and fibrosis decreased. Conclusions: We confirmed that miR-122-5p from USC-Exos targeting SOX2 is a new molecular target for postoperative recovery treatment of obstructive kidney injury.

M2 macrophage-derived exosomes induce angiogenesis and increase skin flap survival through HIF1AN/HIF-1α/VEGFA control

BackgroundSkin flap transplantation is a routine strategy in plastic and reconstructive surgery for skin-soft tissue defects. Recent research has shown that M2 macrophages have the potential for pro-angiogenesis during tissue healing. MethodsIn our research, we extracted the exosomes from M2 macrophages(M2-exo) and applied the exosomes in the model of skin flap transplantation. The flap survival area was measured, and the choke vessels were assessed by morphological observation. Hematoxylin and eosin (H&E) staining and Immunohistochemistry were applied to assess the neovascularization. The effect of M2-exo on the function of Human umbilical vein endothelial cells (HUVECs) was also investigated. We also administrated 2-methoxyestradiol (2-ME2, an inhibitor of HIF-1α) to explore the underlying mechanism. We tested the effects of M2-Exo on the proliferation of HUVECs through CCK8 assay and EdU staining assay. ResultsThe survival area and number of micro-vessels in the skin flaps were increased in the M2-exo group. Besides, the dilation rate of choke vessels was also enhanced in the M2-exo group. Additionally, compared with the control group, M2-exo could accelerate the proliferation, migration and tube formation of HUVECs in vitro. Furthermore, the expression of the pro-angiogenesis factors, HIF-1α and VEGFA, were overexpressed with the treatment of the M2-exo. The expression of HIF1AN protein level was decreased in the M2-exo group. Finally, treatment with HIF-1α inhibitor reverses the pro-survival effect of M2-exo on skin flaps by interfering with the HIF1AN/HIF-1α/VEGFA signaling pathway. ConclusionThis study showed that M2-exosomes promote skin flap survival by enhancing angiogenesis, with HIF1AN/HIF-1α/VEGFA playing a crucial role in this process.

High-density lipoprotein protects vascular endothelial cells from indoxyl sulfate insults through its antioxidant ability

ABSTRACT Chronic kidney disease (CKD) patients have a high risk of cardiovascular disease. Indoxyl sulfate (IS) is a uremic toxin that has been shown to inhibit nitric oxide production and cause cell senescence by inducing oxidative stress. High-density lipoprotein (HDL) has a protective effect on the cardiovascular system; however, its impacts on IS-damaged endothelial cells are still unknown. This study aimed to explore the effects of exogenous supplement of HDL on vascular endothelial cells in a uremia-mimic environment. Tube formation, migration, adhesion, and senescence assays were used to evaluate the cell function of human aortic endothelial cells (HAECs). Reactive oxygen species generation was measured by using Amplex red assay. L-NAME and MCI186 were used as a nitric oxide synthase inhibitor and a free radical scavenger, respectively. HDL exerted anti-inflammatory and antioxidant effects via HIF-1α/HO-1 activation and IL-1β/TNF-α/IL-6 inhibition in IS-stimulated HAECs. HDL improved angiogenesis ability through upregulating Akt/eNOS/VEGF/SDF-1 in IS-stimulated HAECs. HDL decreased endothelial adhesiveness via downregulating VCAM-1 and ICAM-1 in IS-stimulated HAECs. Furthermore, HDL reduced cellular senescence via upregulating SIRT1 and downregulating p53 in IS-stimulated HAECs. Importantly, the above beneficial effects of HDL were mainly due to its antioxidant ability. In conclusion, HDL exerted a comprehensive protective effect on vascular endothelial cells against damage from IS through its antioxidant ability. The results of this study might provide a theoretical basis for potential HDL supplementation in CKD patients with endothelial damage.

Protective effects of villi mesenchymal stem cells on human umbilical vein endothelial cells by inducing SPOCD1 expression in cases of gestational diabetes mellitus

BackgroundGestational diabetes mellitus (GDM) is characterized by a lack of response to insulin in pregnancies, and often accompanied by severe complications. GDM is associated with structural and functional alterations, particularly endothelial dysfunction, in various tissues. This study is aimed to investigate the effect of placental mesenchymal stem cells (MSCs) on the endothelial biological function of human umbilical vein endothelial cells (HUVECs) and their molecular mechanisms. MethodsVilli mesenchymal stem cells (VMSCs) were co-cultured with HUVECs, and transcriptomic analysis of differential genes was performed in HUVECs under high-glucose induction. Lentiviral transfection was performed to construct HUVECs with stable knockdown or overexpression of SPOCD1. The immunohistochemical assays were used to detect the expression of SPOCD1 in GDM patients. TUNEL fluorescence staining was applied for detection of the HUVEC apoptosis. β galactosidase staining assay was performed to detect the cell senescence. Electron microscopy was used to detect the cell pyroptosis. qRT-PCR and western blot assays were conducted for identifying the mRNA & protein expressions of genes. ResultsVMSCs, when co-cultured with HUVECs, could inhibit the apoptosis, pyroptosis and senescence induced by high-glucose condition in HUVECs. Transcriptomic results showed an upregulation of SPOCD1 expression induced by VMSCs in HUVECs. Overexpression of SPOCD1 inhibited high-level glucose-induced apoptosis, pyroptosis and senescence in HUVECs via the β-catenin pathway. ConclusionVMSCs induce β-catenin activation by upregulating the expression of SPOCD1 in HUVECs, which ultimately inhibits high-level glucose-induced apoptosis, pyroptosis and senescence in HUVECs. This observation provides potential therapeutic insight for future GDM treatment.

Investigating whether exosomal miR-205-5p derived from tongue squamous cell carcinoma cells stimulates the angiogenic activity of HUVECs by targeting AMOT.

Although exosomal microRNAs (exo-miRNAs) regulate angiogenesis, they are not sufficient for the development of anti-vascular drugs for tongue squamous cell carcinoma (TSCC). miR-205-5p is an exo-miRNA that is highly expressed in the saliva of patients with oral SCC. We aimed to clarify the role and molecular mechanism of exosomal miR-205-5p in regulating TSCC angiogenesis. Effect of exosomes derived from TSCC cells on human umbilical vein endothelial cell (HUVEC) function was determined using the CCK-8, Transwell, Transwell-Matrigel, and Matrigel-based tube formation assays. Protein levels were detected by western blot. The binding between miR-205-5p and the 3'UTR of AMOT was verified using a luciferase reporter assay. Exosomal miR-205-5p (exo-miR-205-5p) promoted the proliferation, migration, and invasion of HUVECs, increased the number of tubes formed by HUVECs, and increased the vascular endothelial growth factor receptor 2 levels in HUVECs. Exo-miR-205-5p downregulated the AMOT level in HUVECs. Results of the luciferase reporter assay showed that miR-205-5p could bind to the 3'UTR of AMOT. AMOT overexpression blocked the effect of exo-miR-205-5p on HUVEC functions. Exo-miR-205-5p derived from TSCC regulates the angiogenic activity of HUVECs by targeting AMOT and might be a new molecular target for the development of anti-vascular drugs for TSCC.

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 μM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

Breakdown of endothelial barrier integrity determines organ dysfunction and outcome of patients with sepsis. Increased levels of soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) have previously been linked with inflammation-induced loss of endothelial barrier function. We provide evidence for a causative role of sVE-cadherin to induce loss of endothelial barrier function. In patients with sepsis, sVE-cadherin levels were associated with organ dysfunction and the need for volume resuscitation. Similarly, LPS-induced systemic inflammation in rats with microvascular dysfunction was paralleled by augmented sVE-cadherin levels. Newly generated recombinant human sVE-cadherin (extracellular domains EC1-5) induced loss of endothelial barrier function in both human microvascular endothelial cells invitro and in rat mesenteric microvessels invivo and reduced microcirculatory flow. sVE-cadherinEC1-5 disturbed VE-cadherin-mediated adhesion and perturbed VE-protein tyrosine phosphatase (VE-PTP)/VE-cadherin interaction resulting in RhoGEF1-mediated RhoA activation. VE-PTP inhibitor AKB9778 and Rho-kinase inhibitor Y27632 blunted all sVE-cadherinEC1-5-induced effects, which uncovers a pathophysiological role of sVE-cadherin via dysbalanced VE-PTP/RhoA signaling.

Angiogenic Function of Human Placental Endothelial Cells in Severe Fetal Growth Restriction Is Not Rescued by Individual Extracellular Matrix Proteins.

Severe fetal growth restriction (FGR) is characterized by increased placental vascular resistance resulting from aberrant angiogenesis. Interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are critical to the complex process of angiogenesis. We have previously found that placental stromal abnormalities contribute to impaired angiogenesis in severe FGR. The objective of this research is to better characterize the effect of individual ECM proteins on placental angiogenic properties in the setting of severe FGR. ECs were isolated from human placentae, either control or affected by severe FGR, and subjected to a series of experiments to interrogate the role of ECM proteins on adhesion, proliferation, migration, and apoptosis. We found impaired proliferation and migration of growth-restricted ECs. Although individual substrates did not substantially impact migratory capacity, collagens I, III, and IV partially mitigated proliferative defects seen in FGR ECs. Differences in adhesion and apoptosis between control and FGR ECs were not evident. Our findings demonstrate that placental angiogenic defects that characterize severe FGR cannot be explained by a singular ECM protein, but rather, the placental stroma as a whole. Further investigation of the effects of stromal composition, architecture, stiffness, growth factor sequestration, and capacity for remodeling is essential to better understand the role of ECM in impaired angiogenesis in severe FGR.

Adrenomedullin, transcriptionally regulated by vitamin D receptors, alleviates atherosclerosis in mice through suppressing AMPK-mediated endothelial ferroptosis.

Vitamin D receptors (VDR) play important roles in cardiovascular, immune, metabolic and other functions. Activation of VDR may help improve endothelial dysfunction, atherosclerosis, vascular calcification, and cardiac hypertrophy. However, the specific target genes and mechanisms of VDR in improving Human Umbilical Vein Endothelial Cell (HUVEC) functions remain unclear. This study aims to investigate the function and mechanism of VDR in HUVECs. Endothelial dysfunction cell model was constructed by oxidized low-density lipoprotein (ox-LDL). An animal model of atherosclerosis was established in male homozygous Apoe-/- mice (6 weeks) on a high fat diet for 6 weeks. The relationship between VDR and adrenomedullin (ADM) was studied by bioinformatics analysis, ChIP, and luciferase reporter gene analysis. Endothelial cell function was evaluated by Transwell migration and Tube Formation tests. Ferroptosis was detected by measuring intracellular iron content, levels of oxidative stress markers, and ferroptosis related proteins. Overexpression of VDR in HUVECs inhibits ox-LDL-induced endothelial dysfunction and ferroptosis. VDR binds to the ADM promoter sequence and regulates the transcription of ADM. Inhibition of ADM promotes ox-LDL-induced endothelial dysfunction and ferroptosis. ADM regulates ox-LDL-induced endothelial dysfunction and ferroptosis through the AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice inhibited lipid deposition and plaque area in atherosclerotic mice. VDR inhibits ox-LDL-induced endothelial dysfunction and ferroptosis by regulating ADM transcription and acting on AMPK signaling pathway. Overexpression of VDR in Apoe-/- mice reduced lipid deposition and plaque area in the thoracic aorta of atherosclerotic mice.

Sodium Butyrate as Key Regulator of Mitochondrial Function and Barrier Integrity of Human Glomerular Endothelial Cells.

The gut microbiota has emerged as an important modulator of cardiovascular and renal homeostasis. The composition of gut microbiota in patients suffering from chronic kidney disease (CKD) is altered, where a lower number of bacteria producing short chain fatty acids (SCFAs) is observed. It is known that SCFAs, such as butyrate and acetate, have protective effects against cardiovascular diseases and CKD but their mechanisms of action remain largely unexplored. In the present study, we investigated the effect of butyrate and acetate on glomerular endothelial cells. Human glomerular microvascular endothelial cells (hgMVECs) were cultured and exposed to butyrate and acetate and their effects on cellular proliferation, mitochondrial mass and metabolism, as well as monolayer integrity were studied. While acetate did not show any effects on hgMVECs, our results revealed that butyrate reduces the proliferation of hgMVECs, strengthens the endothelial barrier through increased expression of VE-cadherin and Claudin-5 and promotes mitochondrial biogenesis. Moreover, butyrate reduces the increase in oxygen consumption induced by lipopolysaccharides (LPS), revealing a protective effect of butyrate against the detrimental effects of LPS. Taken together, our data show that butyrate is a key player in endothelial integrity and metabolic homeostasis.

Cu(ATSM) Increases P-Glycoprotein Expression and Function at the Blood-Brain Barrier in C57BL6/J Mice.

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aβ), a contributor to Alzheimer's disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aβ levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aβ in AD.

Substance P promote macrophage M2 polarization to attenuate secondary lymphedema by regulating NF-kB/NLRP3 signaling pathway

Secondary lymphedema often occurs after filariasis, trauma, lymph node dissection and radiation therapy, which is manifested by infiltration of inflammatory cells and fibrosis formation in pathologically. Substance P is a widely used neuropeptide in the field of tissue repair, while the regenerative potential of the substance P has not been proven in the secondary lymphedema. In this study, animal model of secondary lymphedema was constructed by excising the skin and subcutaneous lymphatic network in the tail of mice, and the degree of swelling in the tail of mice was evaluated after 6 weeks under the treatment with substance P. Immunofluorescence staining was also performed to assess immune cell infiltration, subcutaneous fibrosis and lymphangiogenesis. The results revealed that substance P significantly alleviated post-surgical lymphedema in mice. Furthermore, we found that substance P promoted macrophages M2 polarization, a process associated with downregulation of the NF-kB/NLRP3 pathway. After application of disodium clodronate (macrophage scavenger, CLO), the positive effect of substance P in lymphedema is significantly inhibited. In vitro experiments, we further demonstrated the polarizing effect of substance P on bone marrow-derived macrophages (BMDMs), while substance P inhibited the activation of the NF-kB/NLRP3 pathway in BMDMs after the treatment of lipopolysaccharide (LPS). In addition, polarized macrophages were demonstrated to promote the proliferation, tube-forming and migratory functions of human lymphatic endothelial cells (hLEC). In conclusion, our study provides preliminary evidence that substance P alleviates secondary lymphedema by promoting macrophage M2 polarization, and this therapeutic effect may be associated with downregulation of the NF-kB/NLRP3 pathway.