Background: Claudin‑5, claudin‑9, and claudin‑11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeabilit... More
Background: Claudin‑5, claudin‑9, and claudin‑11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin‑5, claudin‑9, and claudin‑11 in hypoxia‑stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported. Methods: In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin‑5, claudin‑9, and claudin‑11 was confirmed. Then, the protein content and distribution of claudin‑9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin‑9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up‑regulates claudin‑9 in hypoxia‑stimulated HCMECs. Results: We found that hypoxia‑suppressed claudin‑9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia‑suppressed claudin‑9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high‑dose TXL promoted the cytomembrane localization of claudin‑9 in hypoxia‑stimulated HCMECs, with attenuation of cell injury. Furthermore, high‑dose TXL elevated the hypoxia‑inhibited H3K9ac in the claudin‑9 gene promoter (F = 37.766; P = 0.000), activating claudin‑9 transcription. Conclusions: The results manifested that TXL reversed the hypoxia‑suppressed claudin‑9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs Less
Astragalus polysaccharide is a major component of radix astragali, a vital qi-reinforcing herb medicine with favorable immune-regulating effects. In a previous animal exp... More
Astragalus polysaccharide is a major component of radix astragali, a vital qi-reinforcing herb medicine with favorable immune-regulating effects. In a previous animal experiment, we demonstrated that astragalus polysaccharide effectively alleviates ischemia-reperfusion injury (IRI) of cardiac muscle through the regulation of the inflammatory reactions. However, the relationship between this herb and the cohesion molecules on the cell surface remains controversial. In this study, human cardiac microvascular endothelial cells (HCMECs) were used to validate the protective effects of astragalus under an IRI scheme simulated through hypoxia/reoxygenation in vitro. The results indicated that astragalus polysaccharide inhibited the cohesion between HCMECs and polymorphonuclear leukocyte (PMN) during IRI through the downregulation of p38 MAPK signaling and the reduction of cohesive molecule expression in HCMECs. Less
Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) ar... More
Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM. Less
Although the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However... More
Although the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extrahepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. Because physiologic stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it after treatment with agonists. Microvascular endothelial cells from lung, heart, intestine, and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it after treatment with phorbol-myristate acetate and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and umbilical vein did not constitutively secrete FVIII or release it after treatment with agonists, probably because of a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions. Less
Aims: Highly proliferative, CD34+/CD45+ fibroblasts derived from monocytic, blood-borne precursor cells play a critical role in the development of fibrosis in a murine is... More
Aims: Highly proliferative, CD34+/CD45+ fibroblasts derived from monocytic, blood-borne precursor cells play a critical role in the development of fibrosis in a murine ischaemic/reperfusion cardiomyopathy (I/RC) model. The differentiation of human monocytes into fibroblasts in vitro occurs after transendothelial migration (TEM) induced by monocyte chemoattractant protein 1 (MCP-1). Because Rho-associated kinase-1 (ROCK-1) has been implicated in fibrosis and leukocyte TEM, we investigated its involvement in I/RC.Methods and resultsWe subjected mice with genetic deletion of ROCK-1 to I/RC. We found that ROCK-1−/− mice did not develop the fibrosis and cardiac dysfunction characteristic for I/RC: compared with wild-type, ROCK-1−/− hearts showed markedly lower numbers of I/RC-induced α-smooth muscle actin+ fibroblasts and CD34+/CD45+ fibroblast precursors. Isolated cardiac fibroblasts from ROCK-1−/− mice undergoing I/RC were large and slowly proliferating, similar to fibroblasts isolated from sham-treated hearts. We also performed in vitro assays in which human peripheral blood mononuclear cells (PBMC) migrated through endothelial cells in response to MCP-1. Prior to migration, PBMC were incubated with ROCK-1-targeting small interfering RNA to silence ROCK-1 expression. We found that an 80% reduction of ROCK-1 protein did not inhibit TEM, but significantly reduced the amount of mononuclear cells that differentiated into fibroblasts by >20-fold. Conclusion: Our data implicate an important role for ROCK-1 in the differentiation, but not in the TEM of monocytes that mature into cardiac fibroblasts. These cells mediate non-adaptive fibrosis. KEYWORDS: Cardiac fibroblasts, Monocytes, Rho-associated kinase-1, Endothelial transmigration, Fibrosis Less
The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circul... More
The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IkappaB kinase (IKK)-NF-kappaB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-kappaB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation. Less
The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell ... More
The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell (EC) death. Treatment with interleukin (IL)-18 increased NF-kappaB activation and PTEN induction, suppressed Akt activation, and stimulated EC death. While ectopic expression of p65 enhanced PTEN transcription, adenoviral transduction of dnIkappaB-alpha, dnp65, or dnIKKbeta was inhibitory. Furthermore, IL-18 suppressed HO-1 mRNA expression via enhanced mRNA degradation. Overexpression of HO-1, treatment with HO-1 inducer hemin, or the CO donor cobalt (III) protoporphyrin IX all reversed IL-18-mediated NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Furthermore, hemin induced HO-1 expression, and HO-1 knockdown, HO-1 inhibition, or CO scavengers all reversed the prosurvival effects of hemin. In addition, the CO donors CORM-1 and CORM-3 and the heme metabolites biliverdin and bilirubin attenuated IL-18-induced EC death via a similar signaling pathway. IL-18 induced p38alpha MAPK activation, and suppressed p38beta isoform expression. While p38alpha knockdown attenuated, p38beta knockdown potentiated IL-18-mediated EC death. Hemin and HO-1 reversed IL-18-mediated p38alpha induction and restored p38beta levels. These results demonstrate that IL-18 suppresses HO-1 expression and induces EC death. HO-1 overexpression, HO-1 induction, or treatment with heme metabolites all reverse IL-18-mediated p38alpha MAPK and NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Thus, HO-1 inducers and CO donors may have the therapeutic potential to effectively block IL-18 signaling and reduce IL-18-dependent vascular injury and inflammation. Less
Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endoth... More
Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury. Less
Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induc... More
Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences. Less
