Signaling In Endothelial Cells Research Articles

High concentrations of soluble endoglin can inhibit BMP9 signaling in non-endothelial cells

Endoglin (ENG) is a single-pass transmembrane protein highly expressed on vascular endothelial cells, although low expression levels can be detected in many other cell types. Its extracellular domain can be found in circulation known as soluble endoglin (sENG). Levels of sENG are elevated in many pathological conditions, in particular preeclampsia. We have shown that while loss of cell surface ENG decreases BMP9 signaling in endothelial cells, knocking down ENG in blood cancer cells enhances BMP9 signaling. Despite sENG binding to BMP9 with high affinity and blocking the type II receptor binding site on BMP9, sENG did not inhibit BMP9 signaling in vascular endothelial cells, but the dimeric form of sENG inhibited BMP9 signaling in blood cancer cells. Here we report that in non-endothelial cells such as human multiple myeloma cell lines and the mouse myoblast cell line C2C12, both monomeric and dimeric forms of sENG inhibit BMP9 signaling when present at high concentrations. Such inhibition can be alleviated by the overexpression of ENG and ACVRL1 (encoding ALK1) in the non-endothelial cells. Our findings suggest that the effects of sENG on BMP9 signaling is cell-type specific. This is an important consideration when developing therapies targeting the ENG and ALK1 pathway.

Programmed cell death 10 increased blood-brain barrier permeability through HMGB1/TLR4 mediated downregulation of endothelial ZO-1 in glioblastoma

Dysfunction of blood brain barrier (BBB) contributes to the development of peritumoral edema (PTE) and GBM progression. Programmed cell death 10 (PDCD10) exerts various influence on cancers, especially in glioblastoma (GBM). We previously found that PDCD10 expression was positively correlated with PTE extent in GBM. Thus, the present study aims to investigate the emerging role of PDCD10 in regulating BBB permeability in GBM. Here we found that in vitro indirect co-culture of endothelial cells (ECs) with Pdcd10-overexpressed GL261 cells resulted in a significant increase of FITC-Dextran (MW, 4000) leakage by reducing endothelial zonula occluden-1 (ZO-1) and Claudin-5 expression in ECs respectively. Overexpression of Pdcd10 in GBM cells (GL261) triggered an increase of soluble high mobility group box 1 (HMGB1) release, which in turn activated endothelial toll like receptor 4 (TLR4) and downstream NF-κB, Erk1/2 and Akt signaling in ECs through a paracrine manner. Moreover, Pdcd10-overexpressed GL261 cells facilitated a formation of abnormal vasculature and increased the BBB permeability in vivo. Our present study demonstrates that upregulation of PDCD10 in GBM triggered HMGB1/TLR4 signaling in ECs and significantly decreased endothelial ZO-1 expression, which in turn dominantly increased BBB permeability and contributed to tumor progression in GBM.

Amplification of protease-activated receptors signaling in sporadic cerebral cavernous malformation endothelial cells

In the central nervous system, thrombin-mediated activation of protease-activated receptors (PARs) results in neuroinflammation and increased vascular permeability. These events have been linked to cancer and neurodegeneration. Endothelial cells (ECs) isolated from sporadic cerebral cavernous malformation (CCM) specimens showed dysregulation of genes involved in “thrombin-mediated PAR-1 activation” signaling. CCM is a vascular disease involving brain capillaries. In CCM, ECs show defective cell junctions. Oxidative stress and neuroinflammation play a key role in disease onset and progression. In order to confirm the possible role of thrombin pathway in sporadic CCM pathogenesis, we evaluated PARs expression in CCM-ECs. We found that sporadic CCM-ECs overexpress PAR1, PAR3 and PAR4, together with other coagulation factor encoding genes. Moreover, we investigated about expression of the three familial CCM genes (KRIT1, CCM2 and PDCD10) in human cerebral microvascular ECs, following thrombin exposure, as well as protein level. Thrombin exposure affects EC viability and results in dysregulation of CCM gene expression and, then, in decreased protein level. Our results confirm amplification of PAR pathway in CCM suggesting, for the first time, the possible role of PAR1-mediated thrombin signaling in sporadic CCM. Thrombin-mediated PARs over activation results in increased blood-brain barrier permeability due to loss of cell junction integrity and, in this context, also the three familial CCM genes may be involved.

MEK inhibition reduced vascular tumor growth and coagulopathy in a mouse model with hyperactive GNAQ

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.

Impact of l-citrulline on nitric oxide signaling and arginase activity in hypoxic human pulmonary artery endothelial cells.

Impaired nitric oxide (NO) signaling contributes to the development of pulmonary hypertension (PH). The l-arginine precursor, l-citrulline, improves NO signaling and has therapeutic potential in PH. However, there is evidence that l-citrulline might increase arginase activity, which in turn, has been shown to contribute to PH. Our major purpose was to determine if l-citrulline increases arginase activity in hypoxic human pulmonary artery endothelial cells (PAECs). In addition, to avoid potential adverse effects from high dose l-citrulline monotherapy, we evaluated whether the effect on NO signaling is greater using co-treatment with l-citrulline and another agent that improves NO signaling, folic acid, than either alone. Arginase activity was measured in human PAECs cultured under hypoxic conditions in the presence of l-citrulline (0-1 mM). NO production and endothelial nitric oxide synthase (eNOS) coupling, as assessed by eNOS dimer-to-monomer ratios, were measured in PAECs treated with l-citrulline and/or folic acid (0.2 μM). Arginase activity increased in hypoxic PAECs treated with 1 mM but not with either 0.05 or 0.1 mM l-citrulline. Co-treatment with folic acid and 0.1 mM l-citrulline increased NO production and eNOS dimer-to-monomer ratios more than treatment with either alone. The potential to increase arginase activity suggests that there might be plasma l-citrulline concentrations that should not be exceeded when using l-citrulline to treat PH. Rather than progressively increasing the dose of l-citrulline as a monotherapy, co-therapy with l-citrulline and folic acid merits consideration, due to the possibility of achieving efficacy at lower doses and minimizing side effects.

Unraveling the Role of Peroxisome Proliferator-Activated Receptor β/Δ (PPAR β/Δ) in Angiogenesis Associated with Multiple Myeloma.

Growing evidence suggests a role for peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in the angiogenesis, growth, and metastasis of solid tumors, but little is known about its role in multiple myeloma (MM). Angiogenesis in the bone marrow (BM) is characteristic of disease transition from monoclonal gammopathy of undetermined significance (MGUS) to MM. We examined the expression and function of PPAR β/δ in endothelial cells (EC) from the BM of MGUS (MGEC) and MM (MMEC) patients and showed that PPAR β/δ was expressed at higher levels in MMEC than in MGEC and that the overexpression depended on myeloma plasma cells. The interaction between myeloma plasma cells and MMEC promoted the release of the PPAR β/δ ligand prostaglandin I2 (PGI2) by MMEC, leading to the activation of PPAR β/δ. We also demonstrated that PPAR β/δ was a strong stimulator of angiogenesis in vitro and that PPAR β/δ inhibition by a specific antagonist greatly impaired the angiogenic functions of MMEC. These findings define PGI2-PPAR β/δ signaling in EC as a potential target of anti-angiogenic therapy. They also sustain the use of PPAR β/δ inhibitors in association with conventional drugs as a new therapeutic approach in MM.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles.

Existing vascular endothelial growth factor-oriented antiangiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel antiangiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters, and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. SIGNIFICANCE STATEMENT: Systemic administration of antiangiogenic therapeutics induces side effects to non-targeted tissues. This study, among others, has shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, this study generated CD31-targeting liposomes with encapsulated Fasudil, a clinically relevant Rho kinase inhibitor, and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations, and angiogenic functions verify the benefits of site-targeted antiangiogenic therapy.

Circadian regulator CLOCK promotes tumor angiogenesis in glioblastoma.

Glioblastoma (GBM) is one of the most aggressive tumors in the adult central nervous system. We previously revealed that circadian regulation of glioma stem cells (GSCs) affects GBM hallmarks of immunosuppression and GSC maintenance in a paracrine and autocrine manner. Here, we expand the mechanism involved in angiogenesis, another critical GBM hallmark, as a potential basis underlying CLOCK's pro-tumor effect in GBM. Mechanistically, CLOCK-directed olfactomedin like 3 (OLFML3) expression results in hypoxia-inducible factor 1-alpha (HIF1α)-mediated transcriptional upregulation of periostin (POSTN). As a result, secreted POSTN promotes tumor angiogenesis via activation of the TANK-binding kinase 1 (TBK1) signaling in endothelial cells. In GBM mouse and patient-derived xenograft models, blockade of the CLOCK-directed POSTN-TBK1 axis inhibits tumor progression and angiogenesis. Thus, the CLOCK-POSTN-TBK1 circuit coordinates a key tumor-endothelial cell interaction and represents an actionable therapeutic target for GBM.

Abstract TP209: Role Of Endoglin In The Regulation Of Organic Anion Transporting Polypeptide 1a4 At The Blood-brain Barrier: Relevance To The Treatment Of Ischemic Stroke

Objectives: We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signaling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in brain uptake of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). To date, the involvement of endoglin (ENG), a co-receptor that can modulate the TGF-β/ALK1 pathway, in regulation of Oatp1a4 expression/activity has not been elucidated. The objective of this study is to evaluate whether ENG plays a role in the control of BBB transport mechanisms for stroke therapeutics. Methods: Our studies were conducted using the immortalized mouse brain endothelial cell line bEnd.3. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were then treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and in vitro uptake assays using the known Oatp substrate drug [ 3 H]atorvastatin. Additionally, ENG expression in bEnd.3 cells was assessed under normoxic and oxygen/glucose deprivation (OGD) conditions (i.e., 1% O 2 in glucose-free medium). Results: BMP-9 treatment in non-transfected bEnd.3 cells resulted in increased Oatp1a4 protein expression and increased cellular uptake of [ 3 H]atorvastatin. These enhancements in Oatp1a4 functional expression were attenuated in ENG-shRNA knockdown bEnd.3 cells treated with BMP-9. Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection. Under OGD conditions, expression of ENG in bEnd.3 cells was significantly increased. Conclusions: Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 by TGF-β/ALK1 signaling in brain microvessel endothelial cells. These observations have clear implications for CNS delivery of drugs relevant to treatment of stroke. Studies are ongoing in our laboratory to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Abstract WMP113: Red Blood Cells From Stroke Patients With Admission Hyperglycemia Induces Brain Endothelial Dysfunction

Background: Beyond its traditional role as oxygen carriers, red blood cells (RBCs) are increasingly recognized to participate in regulating vascular function. Many RBC disorders, such as sickle cell disease, are associated with vascular complications resulting in stroke. Our recent study revealed that admission hyperglycemia in stroke patients intensifies oxidative stress in RBCs. We hypothesize that RBCs that are altered by hyperglycemia may perturb endothelial function. Method: Brain endothelial cells were incubated with RBCs isolated from stroke patients with admission hyperglycemia (hyper-RBC) and normoglycemia (normo-RBC) (n=3/group). Mass spectrometry was used to profile the changes in endothelial cells. Functional alterations were assessed by gene set enrichment analysis (GSEA). Significant changes were verified using qPCR and western blot. Result: Compared to normo-RBC, hyper-RBC activates cell adhesion process and toll like receptor signaling in brain endothelial cells (Fig A, B). Cell adhesion molecules (ICAM1, VCAM1) and toll like receptor TLR4 were upregulated at both mRNA and protein levels (Fig C, D), which may promote vaso-occlusion and inflammation. In contrast, vasoregulation was deactivated by hyper-RBC (Fig A, B). The expression of eNOS was downregulated and the bioavailability of vasodilator NO was decreased, consistent with impaired vasodilation (Fig C, D). Notably, the changes induced by hyper-RBC could be reversed by pretreating hyper-RBC with ROS scavenger TEMPOL. The upregulation of ICAM1, VCAM1 and TLR4 in hyper-RBC-treated cells was attenuated by TEMPOL pretreatment, and eNOS expression and NO bioavailability were also restored by TEMPOL (Fig C, D). Conclusion: Intensified oxidative stress in RBCs induced by admission hyperglycemia in stroke patients can cause endothelial dysfunction. Further studies are underway to validate these targets, and ask whether these mechanisms may exacerbate brain injury in stroke patients.

S-Propargyl-Cysteine Ameliorates Peripheral Nerve Injury through Microvascular Reconstruction

Microvascular reconstruction is essential for peripheral nerve repair. S-Propargyl-cysteine (SPRC), the endogenous hydrogen sulfide (H2S) donor, has been reported to promote angiogenesis. The aim of this study is to utilize the pro-angiogenic ability of SPRC to support peripheral nerve repair and to explore the potential mechanisms. The effects and mechanisms of SPRC on angiogenesis and peripheral nerve repair were examined under hypoxic condition by establishing a sciatic nerve crushed injury model in mice and rats, and a hypoxia model in human umbilical vascular endothelial cells (HUVECs) in vitro. We found that SPRC accelerated the function recovery of the injured sciatic nerve and alleviated atrophy of the gastrocnemius muscle in mice. It facilitated the viability of Schwann cells (SCs), the outgrowth and myelination of regenerated axons, and angiogenesis in rats. It enhanced the viability, proliferation, adhesion, migration, and tube formation of HUVECs under hypoxic condition. SPRC activated sirtuin1 (SIRT1) expression by promoting the production of endogenous H2S, and SIRT1 negatively regulated Notch signaling in endothelial cells (ECs), thereby promoting angiogenesis. Collectively, our study has provided important evidence that SPRC has an effective role in peripheral nerve repair through microvascular reconstruction, which could be a potentially effective medical therapy for peripheral nerve injury.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36−/−) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36−/− mice were resistant to develop an obesity phenotype. Subjecting ECCD36−/− mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36−/− mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.

EPHB4-RASA1-Mediated Negative Regulation of Ras-MAPK Signaling in the Vasculature: Implications for the Treatment of EPHB4- and RASA1-Related Vascular Anomalies in Humans.

Ephrin receptors constitute a large family of receptor tyrosine kinases in mammals that through interaction with cell surface-anchored ephrin ligands regulate multiple different cellular responses in numerous cell types and tissues. In the cardiovascular system, studies performed in vitro and in vivo have pointed to a critical role for Ephrin receptor B4 (EPHB4) as a regulator of blood and lymphatic vascular development and function. However, in this role, EPHB4 appears to act not as a classical growth factor receptor but instead functions to dampen the activation of the Ras-mitogen activated protein signaling (MAPK) pathway induced by other growth factor receptors in endothelial cells (EC). To inhibit the Ras-MAPK pathway, EPHB4 interacts functionally with Ras p21 protein activator 1 (RASA1) also known as p120 Ras GTPase-activating protein. Here, we review the evidence for an inhibitory role for an EPHB4-RASA1 interface in EC. We further discuss the mechanisms by which loss of EPHB4-RASA1 signaling in EC leads to blood and lymphatic vascular abnormalities in mice and the implications of these findings for an understanding of the pathogenesis of vascular anomalies in humans caused by mutations in EPHB4 and RASA1 genes. Last, we provide insights into possible means of drug therapy for EPHB4- and RASA1-related vascular anomalies.

A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3

Pulmonary arterial hypertension (PAH) features pathogenic and abnormal endothelial cells (ECs), and one potential origin is clonal selection. We studied the role of p53 and toll-like receptor 3 (TLR3) in clonal expansion and pulmonary hypertension (PH) via regulation of bone morphogenetic protein (BMPR2) signaling. ECs of PAH patients had reduced p53 expression. EC-specific p53 knockout exaggerated PH, and clonal expansion reduced p53 and TLR3 expression in rat lung CD117+ ECs. Reduced p53 degradation (Nutlin 3a) abolished clonal EC expansion, induced TLR3 and BMPR2, and ameliorated PH. Polyinosinic/polycytidylic acid [Poly(I:C)] increased BMPR2 signaling in ECs via enhanced binding of interferon regulatory factor-3 (IRF3) to the BMPR2 promoter and reduced PH in p53-/- mice but not in mice with impaired TLR3 downstream signaling. Our data show that a p53/TLR3/IRF3 axis regulates BMPR2 expression and signaling in ECs. This link can be exploited for therapy of PH.

Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus

The risk of hepatocellular carcinoma (HCC) remains after achieving a sustained virological response (SVR) in patients with chronic hepatitis C (CHC). Epigenetic abnormalities might be key regulators in the development of HCC. This study aimed to identify the genes involved in hepatocarcinogenesis after an SVR. DNA methylation in liver tissue was compared between 21 CHC patients without HCC and 28 CHC patients with HCC, all of whom had achieved an SVR. Additional comparisons with 23 CHC patients before treatment and 10 normal livers were performed. The characteristics of a newly identified gene were explored invitro and invivo. We found that the transmembrane protein no. 164 (TMEM164) gene was demethylated by hepatitis C virus infection and HCC development after achieving an SVR. TMEM164 was expressed mainly in endothelial cells, alpha smooth muscle actin-positive cells, and some capillarized liver sinusoidal endothelial cells. TMEM164 expression was significantly correlated with liver fibrosis and relapse-free survival in HCC patients. TMEM164 was induced by shear stress, interacted with GRP78/BiP, accelerated ATF6 (activating transcription factor 6)-mediated endoplasmic reticulum (ER) stress signaling, and activated interleukin-6/STAT3 (signal transducer and activator of transcription 3) signaling in the TMNK1 liver endothelial cell line. Therefore, we termed TMEM164 "shear stress-induced transmembrane protein associated with ER stress signaling" (SHERMER). SHERMER knockout mice were protected against CCL4-induced liver fibrosis. SHERMER overexpression in TMNK1 cells accelerated HCC growth in a xenograft model. We identified a new transmembrane protein, SHERMER, in CHC patients with HCC after achieving an SVR. SHERMER was induced by shear stress and accelerated ATF6-mediated ER stress signaling in endothelial cells. Thus, SHERMER is a novel endothelial marker associated with liver fibrosis, hepatocarcinogenesis, and progression of HCC.

PS-BPB06-7: NOVEL MOLECULAR MECHANISMS OF CARDIO-RENAL INTERACTION MEDIATED BY INCREASED SERUM MODIFIED NUCLEOSIDES ASSOCIATED WITH RENAL FAILURE

Objective: The number of patients with chronic kidney disease (CKD) continues to rise by population aging. Moreover, CKD patients progress to end-stage renal failure and require renal replacement therapy. Many cohort studies have shown that impaired renal function itself is a risk factor for the development of cardiovascular disease, rather than comorbidities such as diabetes or hypertension. However, the molecular mechanisms are not fully understood. We focused on chemical modifications in RNA and their pathophysiological significance. Previously, we have shown that modified nucleosides, the metabolites of modified RNA, have unique physiological effects. For example, we reported that N6-methyladenosine is a true ligand for the A3 adenosine receptor, and that 1-methylguanosine promotes Zika virus replication in the host. In addition, it was found that a number of modified nucleosides are present in serum and excreted into extracellular spaces like urine. Therefore, we hypothesized that the decreased urinary excretion of modified nucleosides upon renal failure may cause abnormally high levels of modified nucleosides in the blood, which may exert detrimental effects on the heart and vasculature. Methods and Results: First, serum samples were collected from 210 CKD patients, and the correlation between serum modified nucleoside levels and eGFR was investigated. The results showed that several modified nucleosides such as 5-methylcytidine and pseudouridine were inversely correlated with renal function (r2 = -0.76, p < 0.0001; r2 = -0.78, p < 0.0001, respectively). Moreover, we found that 1-methylinosine was inversely correlated with renal function and positively correlated with BNP, a typical marker for heart failure (r2 = -0.82, p < 0.0001 vs. eGFR; r2 = 0.41, p = 0.010 vs. BNP). Next, to explore the possibility of cardiovascular injury associated with this elevation of 1-methylinosine, we examined its effect on vascular endothelial cells. We found that the addition of 1-methylinosine significantly increased cell proliferation using cultured human aortic endothelial cells (p = 0.004, n = 6). Conclusions: Through this study, we found that multiple modified nucleosides are significantly elevated in serum of CKD patients. Furthermore, the elevation of 1-methylinosine in serum can induce abnormal proliferative signals in vascular endothelial cells, which may thereby lead to endothelial damage. In conclusion, these results suggest that the elevated blood levels of modified nucleosides could be a novel marker for vascular injury, and might become a potential target for drug intervention in the cardio-renal interaction.

Cellular and molecular mechanisms of Notch signal in pulmonary microvascular endothelial cells after acute lung injury.

This study focused on the effect and mechanism of Notch signal on pulmonary microvascular endothelial cells (PMVECs) following acute lung injury. PMVECs were cultured in vitro and randomly divided into eight groups. Grouping was based on whether cells were co-cultured with T cells (splenic CD4+T cells were isolated using MACS microbeads) and the level of Notch expression: Normal group and Normal+T cells group, Model group and Model+T cells group, Notch low-expression group and Notch low-expression+T cells group, and Notch overexpression group and Notch overexpression+T cells group. Except for the Normal group and Normal+T cells group, all other groups were treated with 500 μL lipopolysaccharide (1 μg/mL). The expression of VE-cadherin and Zo-1 protein in the Model group (with or without T cells) was lower than that in the normal group (with or without T cells), their expression in the Notch low-expression group (with or without T cells) was significantly increased, and their expression in the Notch overexpression group (with or without T cells) was significantly decreased. Compared with the normal+T cells group, the number of Treg cells in the Notch low-expression+T cells group decreased significantly (P<0.01). The number of Th17 cells in the Notch overexpression+T cells group was higher than that in the Model+T cells group (P<0.01), while the number of Treg cells decreased (P<0.01). Our results demonstrated that activated Notch signal can down-regulate the expression of the tight junction proteins VE-Cadherin and Zo-1 in PMVECs and affect Th17/Treg immune imbalance. Autophagy was discovered to be involved in this process.

The relationship between nuclear factor-erythrocyte-related factor 2 and antioxidant enzymes in the placenta of patients with gestational diabetes and the metabolism of umbilical cord endothelial cells.

The paper aimed to explore the relationship between nuclear factor erythroid 2 related factor 2 (Nrf2), antioxidant enzymes and the metabolism of umbilical cord endothelial cells in the placenta of patients with gestational diabetes (GDM). A total of 200 pregnant women who underwent an obstetric examination at the Municipal Maternity and Child Health Hospital from March 2018 to December 2019 were selected, with an average age of 30.91±3.24. According to the plasma glucose level and oral glucose tolerance test (OGTT), pregnant women were divided into a control group and an observation group. Blood samples were collected from these pregnant women, serum was removed, put into a centrifuge tube and stored in the refrigerator of the laboratory at - 80 °C. Placental tissue was collected for biochemical analysis. GSH level was detected by absorbance kit, and serum MDA content was detected by spectrophotometry. The expression levels of Heme oxygenase-1(HO-1), Nrf2, and NQO1 protein in placental tissue were analyzed by gel electrophoresis. Western blot analysis was used to detect the protein expression levels of Bach1 and Keap1 in endothelial cells. PCR real-time analysis was used to detect the expression of GSH and NQO1 mRNA. Results showed that the SOD and GSH levels in the serum of the observation group were lower than those of the control group (P<0.05). The protein expression levels of HO-1, Nrf2 and NQO1 in the observation group were higher than those in the control group (P<0.05). The GSH level of the HNE+ observation group was lower than that of the HNE+ control group before stimulation (P<0.05). 1.5 hours after the stimulation, the GSH levels of the two groups of cells were decreased. After 6 hours, the GSH levels of the two groups began to increase. The GSH level of HUVEC in the HNE+ observation group was lower than that of the HNE+ control group after 48 hours. The expression level of Bach1 protein in the HNE+ observation group was lower than that in the HNE+ control group (P<0.05). The expression level of Keap1 protein in the HNE+ observation group and HNE+ control group did not change (P>0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 silence group were lower than that in the Nrf2 silence group (P<0.05). The expression levels of GSH and NQO1 mRNA in the HNE+Nrf2 overexpression observation group were higher than those of the HNE+Nrf2 silence group (P<0.05). The apoptosis of trophoblast in the placenta of GDM patients was significantly decreased. The continuous lack of redox signals in fetal endothelial cells in patients with gestational diabetes can destroy the defense ability of cells in the uterus against oxidative stress. Nrf2 antioxidant defense pathway can provide therapeutic targets for reducing oxidative stress associated with diabetes and aging.

Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol

Patients with Marfan syndrome (MFS) develop thoracic aortic aneurysms as the aorta presents excessive elastin breaks, fibrosis, and vascular smooth muscle cell (vSMC) death due to mutations in the FBN1 gene. Despite elaborate vSMC to aortic endothelial cell (EC) signaling, the contribution of ECs to the development of aortic pathology remains largely unresolved. The aim of this study is to investigate the EC properties in Fbn1C1041G/+ MFS mice. Using en face immunofluorescence confocal microscopy, we showed that EC alignment with blood flow was reduced, EC roundness was increased, individual EC surface area was larger, and EC junctional linearity was decreased in aortae of Fbn1C1041G/+ MFS mice. This modified EC phenotype was most prominent in the ascending aorta and occurred before aortic dilatation. To reverse EC morphology, we performed treatment with resveratrol. This restored EC blood flow alignment, junctional linearity, phospho-eNOS expression, and improved the structural integrity of the internal elastic lamina of Fbn1C1041G/+ mice. In conclusion, these experiments identify the involvement of ECs and underlying internal elastic lamina in MFS aortic pathology, which could act as potential target for future MFS pharmacotherapies.