Thrombomodulin mRNA Expression Research Articles

GroEL of Porphyromonas gingivalis-induced microRNAs accelerate tumor neovascularization by downregulating thrombomodulin expression in endothelial progenitor cells.

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor-bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound-healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next-generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI3 K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI3 K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR-1248, miR-1291, and miR-5701. Losing the functions of miR-1248, miR-1291, and miR-5701 can effectively alleviate the GroEL-induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI3 K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs.

Fingolimod does not prevent syndecan-4 shedding from the endothelial glycocalyx in a cultured human umbilical vein endothelial cell model of vascular injury

BackgroundShedding of the endothelial glycocalyx (EG) is associated with poor outcomes in a range of conditions including sepsis. Fresh frozen plasma (FFP) restores the damaged EG to baseline thickness, however the mechanism for this effect is unknown, and some components of FFP have adverse effects unrelated to the EG. There is some limited evidence that sphingosine-1-phosphate (S1P) within FFP restores the EG by activating the endothelial cell S1P receptor 1 (S1PR1). However, there are disadvantages to using S1P clinically as an EG restorative therapy. A potential alternative is the S1PR agonist fingolimod (FTY720). The aim of this study was to assess whether FTY720 prevents EG shedding in injured cultured human umbilical vein endothelial cells.MethodsShedding of the EG was induced in cultured human umbilical vein endothelial cells (HUVECs) by exposure to adrenaline, TNF-α and H2O2. The cells were then assigned to one of six conditions for 4 h: uninjured and untreated, injured and untreated, injured and treated with FTY720 with and without the S1PR1 inhibitor W146, and injured and treated with 25% FFP with and without W146. Syndecan-4, a component of the EG, was measured in cell supernatants, and syndecan-4 and thrombomodulin mRNA expression was quantitated in cell lysates.ResultsThe injury resulted in a 2.1-fold increase in syndecan-4 (p < 0.001), consistent with EG shedding. Syndecan-4 and thrombomodulin mRNA expression was increased (p < 0.001) and decreased (p < 0.05), respectively, by the injury. Syndecan-4 shedding was not affected by treatment with FTY720, whereas FFP attenuated syndecan-4 shedding back to baseline levels in the injured cells and this was unaffected by W146. Neither treatment affected syndecan-4 or thrombomodulin mRNA expression.ConclusionsFTY720 did not prevent syndecan-4 shedding from the EG in the HUVEC model of endothelial injury, suggesting that activation of S1PR does not prevent EG damage. FFP prevented syndecan-4 shedding from the EG via a mechanism that was independent of S1PR1 and upregulation of SDC-4 production. Further studies to examine whether FTY720 or another S1PR agonist might have EG-protective effects under different conditions are warranted, as are investigations seeking the mechanism of EG protection conferred by FFP in this experimental model.

TLR3 promotes venous thrombosis through neutrophil recruitment

Venous thromboembolism (VTE) remains the third most common cause of mortality among all cardiovascular diseases. Innate inflammatory mechanisms contribute to the pathogenesis of VTE. Toll-like receptors (TLRs) play pivotal roles in the innate immune system by detecting “danger signals”. Endogenous RNA has been identified as a potent procoagulant factor in thrombosis and may possibly act through TLR3. To investigate how eRNA mediate procoagulant effects through endothelial TLR3 in venous thrombosis. The FeCl 3 model was used to induce venous thrombosis in WT or TLR3 deficient (-/-) mice. A specific fluorescent probe for RNA (Syto RNA Select), RNase1, poly(I:C) or RNA extracted from murine endothelial cells (eRNA) were injected to mice. HUVECs were treated with poly(I:C) or eRNA extracted from HEK 293 cells in the presence of a TLR3 antagonist. Gene expression and signaling pathways activated were analyzed using western blot and real-time quantitative PCR. Turbidimetric measurement of plasma clot formation was performed by incubating HUVECs treated with poly(I:C) or eRNA with human plasma. We found that FeCl 3 promoted RNA release in vivo and increased the RNA content within the thrombus. RNase1 treatment reduced thrombus size compared to control mice. In contrast, eRNA and poly(I:C) treatment increased thrombus size in WT mice whereas no modifications were observed in TLR3-/- mice. Hence, poly(I:C) and eRNA treatments bolstered neutrophil infiltration in WT but not in TLR3-/- mice. In vitro, eRNA stimulated the release of CXCL5 in WT but not in TLR3-/- endothelial cells. In addition, eRNA enhanced plasma clot formation by downregulating thrombomodulin mRNA expression. These effects are mediated through NFkB activation. These results suggest that eRNA through TLR3 enhances neutrophil recruitment and clot formation leading to venous thrombosis.

Molecular mechanism whereby paraoxonase-2 regulates coagulation activation through endothelial tissue factor in rat haemorrhagic shock model.

We investigated the molecular mechanism of paraoxonase-2 (PON-2) in regulating blood coagulation activation in rats with haemorrhagic shock through endothelial tissue factor (TF). Thirty adult Sprague Dawley rats were randomly divided into three groups: healthy control group (group A), the haemorrhagic shock PON-2 treatment group (group B), and the haemorrhagic shock group (group C). After the model was established, blood was withdrawn from the inferior vena cava of all rats. The difference in plasma thrombomodulin (TM) levels of the three groups was determined by Western blotting. The expression of transcription factors Egr-1 and Sp1 was detected by Western blotting assays. reverse transcription-polymerase chain Reaction (RT-PCR) was used to determine the mRNA expression of t-PA, PAI-1, TM, and PON-2 in the serum of three groups of rats. Endothelial TF was measured by enzyme linked immunosorbent assay (ELISA), and coagulation assay was used to detect the activity of coagulation factor VIII. Histopathological examination of the arteries of the rats was performed. The molecular mechanism of PON-2 in regulating blood coagulation activation in haemorrhagic shock model rats by endothelial tissue factor was analysed. The expression of thrombin was determined by electrophoresis. Compared with the healthy control group, the expression of TM in groups B and C decreased, both 188.64 ± 12.47 and 137.48 ± 9.72, respectively, with a significant difference. The mRNA expression of TM and PON was determined by RT-PCR. The mRNA expression of TM and PON in group B was 0.97 ± 0.07 and 1.14 ± 0.09, compared with the control group, and the mRNA expression of TM and PON in group C was 0.86 ± 0.38 and 1.12 ± 0.41, both of which increased, and there were significant differences. By measuring the expression of endothelial TF, the expression of TF in groups B and C was elevated to 12.69 ± 1.07 and 11.59 ± 0.87, with significant differences. The enzyme activities of PON-2 in groups B and C, which were 110.34 ± 14.37 and 52.37 ± 8.06, respectively, were increased compared with the healthy control group and there were significant differences. PON-2 regulates the activation of coagulation in rats with haemorrhagic shock by regulating the expression of endothelial tissue-related genes such as plasma TM and endothelial TF under hypoxic and ischaemic conditions.

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells.

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences.

Effect of Salvia Miltiorrhiza on Tmmrna Expression in Coronary Artery Tissue of Rats with High Salt Induced Blood Stasis

Objective To understand the effect of Salvia miltiorrhiza on the cardiovascular system by the expression of tissue TMmRNA, and to provide the scientific basis for the use of Salvia miltiorrhiza and a low salt diet for the prevention and treatment of cardiovascular and cerebrovascular diseases caused by high salt intake. Methods Thirty SD rats were randomly divided into a blank group, high salt group and Salvia miltiorrhiza group. The blank group was given intragastric distilled water and the high salt and the Salvia miltiorrhiza groups were treated with intragastric administration of 12% NaCl, for 30 consecutive days. Seven days before finishing this model, sterile water was injected intraperitoneally every afternoon in the blank group and the high salt group while, in the Salvia miltiorrhiza group, an injection of Salvia miltiorrhiza was given every afternoon. Then the animals were killed and the coronary arteries were removed. The expression of thrombomodulin mRNA (TMmRNA) in the coronary arteries of the rats in each experimental group was detected by the RT-PCR technique. Result Compared with the blank group, the expression of TMmRNA in the coronary tissue of the high salt group was obvious (p 0.05). Compared with the high salt group, the expression of TMmRNA in the coronary tissue of the Salvia miltiorrhiza was significantly different (p Conclusion Salvia miltiorrhiza can reduce the expression of TMmRNA in the tissue of blood stasis caused by high salt, promote the repair of blood vessel endothelium and blood movement, and can effectively improve the damaged coronary artery. Acknowledgements Supported by a project grant from Natural Science Foundation of China (Grant No. 81503476).

Oxygenated hypothermic machine perfusion after static cold storage improves endothelial function of extended criteria donor livers

BackgroundLack of oxygen and biomechanical stimulation during static cold storage (SCS) of donor livers compromises endothelial cell function. We investigated the effect of end-ischemic oxygenated hypothermic machine perfusion (HMP) on endothelial cell function of extended criteria donor (ECD) livers. MethodsEighteen livers, declined for transplantation, were transported to our center using static cold storage (SCS). After SCS, 6 livers underwent two hours of HMP, and subsequent normothermic machine perfusion (NMP) to assess viability. Twelve control livers underwent NMP immediately after SCS. mRNA expression of transcription factor Krüppel-like-factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) was quantified by RT-PCR. Endothelial cell function and injury were assessed by nitric oxide (NO) production and release of TM into the perfusate. ResultsIn HMP livers, mRNA expression of KLF2 (p = 0.043), eNOS (p = 0.028), and TM (p = 0.028) increased significantly during NMP. In parallel, NO levels increased during NMP in HMP livers but not in controls. At the end of NMP cumulative TM release was significantly lower HMP livers, compared to controls (p = 0.028). ConclusionA short period of two hours oxygenated HMP restores endothelial cell viability after SCS and subsequent normothermic reoxygenation of ECD livers.

Loss of placental thrombomodulin in oocyte donation pregnancies

To investigate whether thrombomodulin dysregulation is involved in the development of preeclampsia after oocyte donation (OD). Women who become pregnant after OD are prone to develop preeclampsia, a syndrome characterized by an aberrant immunologic response, hypercoagulability, and endothelial dysfunction. A mediator of inflammation and coagulation is thrombomodulin, which has a possible role to play in this syndrome. Case-control study. Not applicable. Placentas from 82 women with an uncomplicated pregnancy (48 naturally conceived, 21 IVF, and 33 OD pregnancies) and 9 women with an OD pregnancy complicated by preeclampsia have been studied. None. Abundances of thrombomodulin protein and vitamin D receptor (VDR) were determined using immunohistochemistry; mRNA expression was determined using quantitative polymerase chain reaction. Placental thrombomodulin protein abundance was lower in OD pregnancies (diffuse pattern in 45%) than in controls (diffuse pattern in 96%). Placental thrombomodulin mRNA expression was lower in OD pregnancies complicated by preeclampsia (0.72 ± 0.47) compared with in uncomplicated OD pregnancies (0.43 ± 0.18). Thrombomodulin expression correlated with inflammation and coagulation. VDR expression was decreased in OD pregnancies complicated by preeclampsia and was correlated with thrombomodulin mRNA. Pregnancies conceived through OD lose placental thrombomodulin expression. This loss is associated with an increased coagulation and inflammation and indicates that endothelial protection is diminished in OD pregnancies, which might be an explanation for the increased risk for preeclampsia. Vitamin D metabolism is dysregulated in OD pregnancies and might be a target for therapy.

Plasma homocysteine involved in methylation and expression of thrombomodulin in cerebral infarction

Homocysteine (Hcy) regulates endothelial injury and methylation status of key genes in cerebral ischemia. Thrombomodulin (TM) may be protective against cerebral ischemia by downregulating coagulation. However, it remains unclear whether Hcy involved in methylation and expression of TM in cerebral infarction (CI). Here, we find patients with cerebral infarction had a higher TM methylation level than controls (74.2% vs 47.5%, X2 = 14.724, P = 0.00), which are positively correlated with plasma levels of tHcy (r = 0.701, P = 0.00) and negatively related to mRNA expression of TM (r = −0.711, P = 0.00). Plasma levels of tHcy (t = 7.566, P = 0.00) and sTM (t = 17.268, P = 0.00) are significantly higher in cases than in controls. Our data indicate hyperhomocysteine leads to hypermethylation of the TM gene and further induces TM gene silencing, which may play an important role in the occurrence and development of CI. Plasma higher concentrations of sTM in cases are not caused by TM expression and may be only a result of Hcy induced endothelial injury.

Oxygenated hypothermic machine perfusion after static cold storage improves endothelial function of extended criteria donor livers

Background: Lack of oxygen and biomechanical stimulation during static cold storage (SCS) of donor livers compromises endothelial cell function. This study investigated the effect of 2 hours of end-ischemic oxygenated hypothermic machine perfusion (HMP) on endothelial cell function of extended criteria donor (ECD) livers. Method: Sixteen human livers that were declined for transplantation were transported to our center using conventional static cold storage (SCS; 4oC) and subjected to normothermic machine perfusion (NMP) to assess viability and function. Six livers underwent 2 hours oxygenated HMP (12°C) after SCS and prior to NMP. Ten control livers underwent NMP immediately after SCS. Endothelial cell function was assessed by quantification of mRNA expression encoding for transcription factor Krüppel-like-factor-2 (KLF-2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) using real-time PCR. Nitric oxide (NO) production (nitrite/nitrate levels) and release of thrombomodulin (TM) into the perfusion fluid were determined during NMP. Results: In livers that underwent end-ischemic HMP, mRNA expression of KLF-2 (p = 0.04), eNOS (p = 0.03) and TM (p = 0.03) increased significantly during NMP. This response was not observed in the control livers. In parallel, NO levels in the perfusate increased during NMP of livers that first underwent HMP, but not in control livers. Moreover, at the end of NMP cumulative TM release into the perfusate was significantly higher in control livers compared to livers first subjected to HMP (p = 0.03). Conclusion: A short period of 2 hours oxygenated HMP restores endothelial cell function and integrity after conventional static cold preservation and subsequent reoxygenation of ECD livers.

Administration of bone marrow stromal cells in sepsis attenuatessepsis-related coagulopathy

Introduction Coagulopathy plays an important role in sepsis. The aim of this study was to determine whether bone marrow stromal cell (BMSC) administration could attenuate coagulopathy in sepsis.Materials and methods In vitro: endothelial cells were cultured with/without BMSCs for 6 h following LPS stimulation and were collected for thrombomodulin (TM) and endothelial protein C receptor (EPCR) measurements. In vivo: Thirty-six mice were randomized into sham, sepsis, and sepsis + BMSC groups (n = 12 each group). Sepsis was induced through cecal ligation and puncture (CLP). BMSC infusion was started at 6 h after CLP. Lung tissues and plasma samples were collected at 24 h after CLP for enzyme-linked immunosorbent assay (ELISA), quantitative real-time RT-PCR, western blot, and immunohistochemistry analysis.Results In vitro: BMSCs attenuated the decrease in TM and EPCR mRNA and protein expression levels in LPS-stimulated endothelial cells. In vivo: BMSC treatment decreased lung injury and mesenteric perfusion impairment, and ameliorated coagulopathy, as suggested by the reduction in elevated TF, vWF, and TAT circulation levels. BMSC infusion decreased TF mRNA transcription and protein expression levels in lung tissues, and increased TM and EPCR mRNA transcription and expression levels.Discussion BMSC administration attenuated coagulopathy, and decreased lung injury and mesenteric perfusion impairment in sepsis.Key messagesBMSCs increased the expression of TM and EPCR from endothelium cells exposed to LPS in vitro.BMSC treatment attenuated lung injury and coagulopathy in the mice cecal ligation and puncture (CLP) model.BMSC administration-attenuated coagulopathy is related to the reduced expression of TF and increased expression of TM and EPCR.

Loss of Thrombomodulin in Placental Dysfunction in Preeclampsia.

Preeclampsia is a pregnancy-specific syndrome characterized by placental dysfunction and an angiogenic imbalance. Systemically, levels of thrombomodulin, an endothelium- and syncytiotrophoblast-bound protein that regulates coagulation, inflammation, apoptosis, and tissue remodeling, are increased. We aimed to investigate placental thrombomodulin dysregulation and consequent downstream effects in the pathogenesis of preeclampsia. Placentas from 28 preeclampsia pregnancies, 30 uncomplicated pregnancies, and 21 pregnancies complicated by growth restriction as extra controls were included. Immunohistochemical staining of thrombomodulin, caspase-3, and fibrin was performed. Placental mRNA expression of thrombomodulin, inflammatory markers, matrix metalloproteinases 2 and 9, and soluble Flt-1 were measured with quantitative polymerase chain reaction. Thrombomodulin mRNA expression was determined in vascular endothelial growth factor-transfected trophoblast cell lines. Thrombomodulin protein and mRNA expression were decreased in preeclampsia as compared with both control groups (P=0.001). Thrombomodulin mRNA expression correlated with maternal body mass index (P<0.01) and diastolic blood pressure (P<0.05) in preeclampsia. An increase in placental apoptotic cells was associated with preeclampsia (P<0.001). Thrombomodulin expression correlated positively with matrix metalloproteinase expression (P<0.01) in preeclampsia, but not with fibrin deposits or inflammatory markers. Placental soluble Flt-1 expression correlated with decreased thrombomodulin expression. Vascular endothelial growth factor induced upregulation of thrombomodulin expression in trophoblast cells. Decreased thrombomodulin expression in preeclampsia may play a role in placental dysfunction in preeclampsia and is possibly caused by an angiogenic imbalance. Hypertension and obesity are associated with thrombomodulin downregulation. These results set the stage for further basic and clinical research on thrombomodulin in the pathogenesis of preeclampsia and other syndromes characterized by endothelial dysfunction.

Fondaparinux upregulates thrombomodulin and the endothelial protein C receptor during early-stage reperfusion in a rat model of myocardial infarction

IntroductionFondaparinux (FDX) was demonstrated to be cardioprotective in a rat model of myocardial ischemia reperfusion. In this model, FDX reduced infarct size after 2h of reperfusion, involving the activation of the survivor activating factor enhancement (SAFE) pathway as early as 30min post-reperfusion. Our aim was to study if this cardioprotection could be explained by anti-inflammatory mechanisms and a protective effect on vessels. MethodsWistar male rats were subjected to 40minutes (min) of myocardial ischemia, followed by 30min or 2h of reperfusion. Rats were randomized into four groups: control 30min (n=7), FDX 30min (n=7), control 2h (n=7), and FDX 2h (n=7). The FDX groups received 10mg/kg injection of FDX 10min prior to initiating reperfusion. We studied: 1) mRNA expression of endothelial markers, such as thrombomodulin (TM), endothelial protein C receptor (EPCR), and tissue factor (TF) and 2) proteic expression of ICAM-1, NF-κB, IκB, and JNK. Leukocyte infiltration was assessed by histochemistry. We also evaluated TM and EPCR mRNA expression in a model of isolated rat mesenteric arteries incubated with FDX. ResultsFDX upregulated the expression of TM and EPCR mRNA in the models of myocardial infarction and isolated mesenteric arteries. No difference was observed between the treated and control groups regarding the expression of pro-inflammatory signaling proteins, adhesion molecules, and leukocyte infiltration after 2h of reperfusion. ConclusionThe cardioprotective effect of FDX at early-stage reperfusion could be related to vascular protection, yet not to an anti-inflammatory effect.

Expression of thrombomodulin in maternal plasma and placental tissue in early onset severe preeclampsia

To explore the thrombomodulin (TM) expression levels changes in plasma and placenta in patients with early onset severe preeclampsia. Sixty cases of severe preeclampsia women who delivered in the affiliated Xuzhou Maternity and Child Health Care Hospital of Xuzhou Medical College were enrolled in the study from June 2012 to February 2014, including 30 patients with early onset severe preeclampsia (early onset group), and 30 patients with late onset severe preeclampsia (late onset group). Healthy pregnant women were divided into two control groups according to gestational weeks at delivery: early control group (n = 23, at 28-33⁺⁶ weeks), and late control group (n = 30, delivered after 34 weeks). ELISA was used to detect the levels of TM in plasma. Immunohistochemistry SP was applied to detect the TM protein expression on placenta. TM mRNA was determined by reverse transcription polymerase chain reaction (RT)-PCR technique. (1) TM level in plasma in early onset group and late onset group were (90.8 ± 6.9) and (87.5 ± 7.0) µg/L, and TM level in plasma in early control group and late control group were (37.7 ± 2.3) and (37.7 ± 2.5) µg/L. Plasma TM level in early onset group was higher than that in late onset group, early control group and late control group. The TM level had no statistically significant compare of early-onset group to late onset group. (P > 0.05). The plasma TM level in early onset group was significantly higher than that in early control group (P < 0.05), and the plasma TM level in late onset group was significantly higher than that in late control group (P < 0.05). (2) TM expressed mainly in the membrane and cytoplasm of placental syncytiotrophoblasts and endothelial cells. The expression of TM protein in early onset group was 47% (14/30), significantly lower than that in late onset group, early control group and late control group (P < 0.05), in which the positive rate were 90% (27/30), 91% (21/23) and 93% (28/30) respectively (P < 0.05). There was no difference between late onset group and late control group (P > 0.05). There was no difference between early control group and late control group (P > 0.05). (3) TM mRNA expression in early onset group, late onset group, early control group and late control group were 0.14 ± 0.06, 0.89 ± 0.23, 0.88 ± 0.22 and 0.93 ± 0.19, respectively. The expression of TM mRNA in early onset group was significantly lower than that in late onset group, early control group and late control group (P < 0.05), and the difference between early control group and late control group was not statistically significant (P > 0.05). There was no difference between late onset group and late control group (P > 0.05). There was no difference between early control group and late control group (P > 0.05). Decreased expression of TM in placenta may be associated with the pathogenesis of early onset severe preeclampsia, there may be different pathogenesis in early onset and late onset severe preeclampsia.

CAMP‐signaling via EPAC1 mediates vascular endothelial cell adaptation to fluid‐shear stress

Herein, we report a novel role for cAMP‐signaling in human aortic endothelial cell (HAEC) adaptation to fluid‐mediated shear stress (FSS). Our results showed that increases in cAMP enhanced elongation and alignment of HAECs cultured under LSS for 48hrs and that this effect was largely mediated by the cAMP‐effector molecule, exchange protein activated by cAMP (EPAC)‐1, but not Protein Kinase A (PKA). Additionally, major HAEC cAMP‐hydrolyzing enzyme, phosphodiesterase (PDE)4D, was also shown to play a crucial role in integrating FSS signals in these cells.Our results demonstrated that flow‐mediated increases in atheroprotective genes, Kruppel‐like factor, endothelial nitric oxide synthase and thrombomodulin mRNA expression were reduced in EPAC‐1 and PDE4D knockdown HAECs exposed to both LSS and HSS. Furthermore, EPAC‐1 knockdown increased leukocyte extravasation across HAEC monolayers in vitro and activation of EPAC significantly reduced LPS‐induced leukocyte rolling flux in mice.VECs have numerous mechanosensors which facilitate the integration of FSS signals from the flowing blood. One such sensor consists of platelet‐endothelial cell adhesion molecule (PECAM)‐1, vascular endothelial‐cadherin (VECAD) and vascular endothelial growth factor receptor (VEFGR)‐2. Our current data suggests that EPAC1 and PDE4D can additionally mediate flow‐sensing through this mechanosensory complex. All together, this data provides important mechanistic insight providing therapeutic targets in endothelial dysfunction and atherosclerosis.

Microfluidic perfusion culture chip providing different strengths of shear stress for analysis of vascular endothelial function

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm(2) in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10(5)) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm(2). Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm(2).

Thrombomodulin modulates cell migration in human melanoma cell lines

Malignant melanoma cells are known to have altered expressions of growth factors as compared with normal melanocytes. Thrombomodulin (TM) is a thrombin receptor on endothelial cells that converts thrombin from a procoagulant to an anticoagulant enzyme. TM expression is downregulated in tumor cells, and this phenomenon correlates with tumor cell invasiveness and a poor prognosis in patients with cancer. In this study, we evaluated TM expression in two human melanoma cell lines that are known to have either low (WM35) or high (A375) aggressive phenotypes. Analysis by quantitative real-time PCR (qPCR) showed that the mRNA expression of TM is modestly (WM35) or dramatically (A375) downregulated in melanoma cells, as compared with human primary melanocytes. TM expression levels inversely correlated with in-vitro migration properties of tumor cells. In addition, interleukin-8 expression also correlated with the degree of aggressiveness, as indicated by high expression levels of this cytokine in A375 cells. Overexpression of TM in A375 cells by transient transfection reversed their aggressive phenotype and dramatically decreased interleukin-8 expression by these cells. Taken together, these results suggest that downregulation of TM plays a crucial role in melanocyte transformation and melanoma progression.

Decreased thrombomodulin mRNA expression on peripheral monocytes in disseminated intravascular coagulation patients relates to poor outcomes: The ex vivo effects of lipopolysaccharide and thrombin on monocyte thrombomodulin and CD14 mRNA

Monocytes express substantial amounts of thrombomodulin, which is consumed throughout ongoing thrombin generation. The modulation of thrombomodulin may aggravate intravascular fibrin deposition and the clinical course of disseminated intravascular coagulation (DIC). Although thrombomodulin restoration has received considerable attention, no reports have been published on the in vivo expression status of thrombomodulin. CD14 expression on monocytes is important for regulation of the inflammatory response. We used an ex vivo stimulation study to evaluate the association of the levels of monocyte-expressed thrombomodulin and CD14 messenger RNA (mRNA) with the severity and prognosis of disseminated intravascular coagulation. A total of 78 patients with suspected DIC were enrolled. Thrombomodulin and CD14 mRNA levels were measured in peripheral blood by real-time quantitative reverse-transcription polymerase chain reaction. Thrombomodulin and CD14 mRNA were also assessed in ex vivo cultures of peripheral whole blood that were stimulated by lipopolysaccharide or thrombin. The levels of monocyte-expressed thrombomodulin mRNA were significantly lower in the non-survivors than in the survivors. A low level of monocyte-expressed thrombomodulin mRNA was a significant prognostic marker, but CD14 did not possess prognostic power. Monocyte-expressed CD14 mRNA correlated significantly with the severity of DIC in survivors. In addition, stimulation of ex vivo cultures of whole blood demonstrated that thrombin upregulates both thrombomodulin and CD14 mRNA, and lipopolysaccharide downregulates thrombomodulin mRNA. The downregulation of thrombomodulin on monocytes reflects the decompensated status of physiological defenses against hypercoagulopathy and represents the poor prognosis in DIC. The expression levels of thrombomodulin on monocytes may be a useful marker to screen for candidates eligible for recombinant thrombomodulin therapy in future.

Heme Oxygenase 1 Modulates Thrombomodulin and Endothelial Protein C Receptor Levels to Attenuate Septic Kidney Injury

This study investigated the effects of heme oxygenase 1 (HO-1) on thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression in sepsis-induced kidney injury. The role of HO-1 was evaluated in a cecal ligation and puncture (CLP)-induced model. Wistar rats were randomly assigned into four groups: sham, CLP, CLP + hemin (an HO-1 inducer), CLP + ZnPP (zinc protoporphyrin IX, an HO-1 inhibitor), and CLP + bilirubin. Compared with the sham group, the CLP group exhibited significantly elevated plasma levels of cystatin C, creatinine, urea nitrogen (blood urea nitrogen), tumor necrosis factor α, interleukin 1β, TM, and EPCR; lower plasma level of activated protein C, shorter prothrombin time and activated partial thromboplastin time; significantly increased microthrombus formation; and lower TM and EPCR mRNA and protein expression in the kidney. The administration of hemin lowered the plasma levels of cystatin C, creatinine, blood urea nitrogen, tumor necrosis factor α, interleukin 1β, TM, and EPCR; elevated plasma level of activated protein C; prolonged prothrombin time and activated partial thromboplastin time; attenuated microthrombus formation; and upregulated the expression of TM and EPCR and mRNA levels of TM and EPCR in the kidney in the CLP + hemin group. In contrast, ZnPP had the opposite effects. The results indicated that the enhanced induction of HO-1 increased the expression of TM and EPCR in the kidney and exerted an anticoagulant effect, thereby attenuating kidney injury in septic rats.

Thrombomodulin, a novel molecule regulating inorganic phosphate-induced vascular smooth muscle cell calcification

Hyperphosphatemia has emerged as a cardiovascular risk factor that stimulates calcification in vessels. We explored molecules that were induced by inorganic phosphate (Pi) at an early stage in vascular smooth muscle cells (VSMC). In the present study, we examined the role of thrombomodulin (TM) in Pi-induced VSMC calcification based on the results of DNA microarray analysis. Both mRNA and protein expression of TM were markedly augmented in Pi-induced calcification. Conversely, knockdown of TM by siRNA significantly inhibited calcification, in addition to Pi-induced apoptosis which plays critical roles in VSMC calcification. We further found that TM suppressed both of mRNA and protein expression of growth arrest-specific gene 6 (Gas6), a key molecule regulating apoptosis. Recombinant extracellular epidermal growth factor (EGF)-repeat domain of TM exaggerated calcification and this effect was abrogated by a neutralizing antibody for EGF receptor, suggesting that the cleaved and secreted form of TM may activate EGF receptor. We also found that downregulation of Gas6 by TM/EGF receptor axis was mediated by ERK in VSMC calcification. In the aorta of adenine-fed rat, a typical medial calcification model with hyperphosphatemia, we found that TM expression was increased. Furthermore, in human calcified aorta, increased TM expression was also observed. These results indicate that TM is a novel molecule that promotes apoptosis and vascular calcification by regulation of Gas6, presumably via EGF receptor/ERK axis.