PAI-1 mRNA Levels Research Articles

Effects of triggers of senescence and senolysis in murine pancreatic cancer cells

BackgroundThe combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis. MethodsPanc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav. ResultsEto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive. Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable. ConclusionsIn vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.

Abstract TP300: Apolipoprotein-E Deficiency and High Fat Diet Alter Immunomodulatory Cellular and Molecular Determinants Promoting Atherosclerosis, a Stroke Risk Factor

Background: Most ischemic strokes are caused by atherosclerosis. Atherosclerotic plaque formation is modulated by genetic and environmental interactions. Apolipoprotein E (ApoE) regulates lipid metabolism, and its deficiency is associated with dyslipidemia and atherosclerosis. ApoE is abundantly expressed in glial cells and neurons of the brain, affecting the blood brain barrier and cognitive function. Poor dietary habits promote the onset of atherosclerosis. In this study, we examined the effects of ApoE deficiency and high fat diet (HFD) on plaque formation and immunomodulatory cellular and molecular mechanisms in mice models. Methods: Wild type and ApoE knockout (KO) mice were fed with normal or HFD. Carotid plaques were examined using hematoxylin and eosin staining. Targeted proteomic profiling identified the circulating inflammatory proteins. Pathway specific transcriptomic profiles were identified using PCR arrays and real-time PCR. Immunohistochemistry assays were done in brain sections using a neuronal marker (NeuN), an astrocyte marker (GFAP) and a microglia marker (Iba1). The expression of Pentraxin-3 (PTX3), lipocalin2, and PAI-1 were examined using immunohistochemistry. Images were acquired and analyzed using a Keyence fluorescence microscope and NIH ImageJ. Results: ApoE deficiency and HFD enhanced atherosclerotic plaque formation as a function of age in association with a significant increase in circulating pro-inflammatory cytokines in blood/plasma, including M-CSF, C-reactive protein, and PTX3. The number of microglial cells and astrocytes was increased with altered morphology in ApoE KO and aged animals’ brain cortex and dentate gyrus. Whereas neuronal cell number was decreased in the brains of ApoE deficient and aged mice. The mRNA levels of SELE, PAI-1, lipocalin2, and PTX3 were upregulated in ApoE deficient and WT animals fed with HFD. Higher levels of PTX3 were also observed in atherosclerotic plaques. Conclusion: ApoE deficiency and HFD may play a key role in altering immunomodulatory cellular responses and initiating pro-inflammatory molecular signaling. These events may promote atherosclerotic plaque formation, a risk factor for ischemic stroke and worsen brain damage.

Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

Anti-thrombotic activity of phenolic acids obtained from Salvia miltiorrhiza f. alba in TNF-α-stimulated endothelial cells via the NF-κB/JNK/p38 MAPK signaling pathway.

Over the past 100years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.

Regulatory Mechanism of MicroRNA-30b on Neonatal Hypoxic-Ischemic Encephalopathy (HIE)

ObjectiveThis study is to investigate the role of microRNA (miR)-30b in the pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonates. MethodsTotally 26 cases of neonatal HIE were included in this study. The protein expression levels of CD26P and PAI-1 were detected with ELISA. Serum levels of miR-30b and PAI-1 mRNA was measured by quantitative real-time PCR. Human brain microvascular endothelial cells (HBMECs) were cultured under hypoxic condition, and the intracellular expression levels of miR-30b and PAI-1 were evaluated. Dual-luciferase reporter assay was performed to confirm the interaction between miR-30b and PAI-1. ResultsCompared with the control group, both the mRNA and protein expression levels of PAI-1 in the serum were up-regulated in the neonates with HIE, together with up-regulated serum CD26P levels. However, the serum expression level of miR-30b was down-regulated in neonatal HIE. In hypoxia-induced HBMECs, the mRNA and protein expression levels of PAI-1 were significantly up-regulated, while the miR-30b expression level was significantly down-regulated. Dual-luciferase reporter assay showed that PAI-1 was the direct target of miR-30b. ConclusionNeonatal HIE is accompanied with abnormal platelet activation, significantly up-regulated serum PAI-1 expression levels, and down-regulated miR-30b expression. MiR-30b might regulate the disease pathogenesis and immune responses via modulating PAI-1.

Co-exposure to PCB126 and PFOS increases biomarkers associated with cardiovascular disease risk and liver injury in mice

Polychlorinated biphenyl (PCB)126 and perfluorooctane sulfonic acid (PFOS) are halogenated organic pollutants of high concern. Exposure to these chemicals is ubiquitous, and can lead to potential synergistic adverse effects in individuals exposed to both classes of chemicals. The present study was designed to identify interactions between PCB126 and PFOS that might promote acute changes in inflammatory pathways associated with cardiovascular disease and liver injury. Male C57BL/6 mice were exposed to vehicle, PCB126, PFOS, or a mixture of both pollutants. Plasma and liver samples were collected at 48 h after exposure. Changes in the expression of hepatic genes involved in oxidative stress, inflammation, and atherosclerosis were investigated. Plasma and liver samples was analyzed using untargeted lipidomic method. Hepatic mRNA levels for Nqo1, Icam1, and PAI1 were significantly increased in the mixture-exposed mice. Plasma levels of PAI1, a marker of fibrosis and thrombosis, were also significantly elevated in the mixture-exposed group. Liver injury was observed only in the mixture-exposed mice. Lipidomic analysis revealed that co-exposure to the mixture enhanced hepatic lipid accumulation and elevated oxidized phospholipids levels. In summary, this study shows that acute co-exposure to PCB126 and PFOS in mice results in liver injury and increased cardiovascular disease risk.

The phosphodiesterase 5 inhibitor tadalafil has renoprotective effects in a rat model of chronic kidney disease.

Phosphodiesterase 5 inhibitors are widely used to treat erectile dysfunction and lower urinary tract symptoms with benign prostatic hyperplasia. Recent studies have indicated the renoprotective effects of this class of compounds. Whether renoprotection depends on blood pressure reduction remains controversial. In this study, we investigated the renoprotective effects of the phosphodiesterase 5 inhibitor, tadalafil, in a rat model of high‐salt induced kidney injury with hypertension. Dahl salt‐sensitive rats were fed a normal diet, high‐salt (8% sodium chloride) diet, or high‐salt diet with oral administration of either low‐ or high‐dose tadalafil (1 and 10 mg kg−1 day−1, respectively). Serum creatinine, urinary protein, and blood pressure were measured at baseline and after 8 weeks, at which point the rats were examined for glomerular injury and fibrosis. PAI1 mRNA levels were also evaluated. After 8 weeks, blood pressure, serum creatinine, and urinary protein levels were significantly higher in the high‐salt group than those in the normal‐salt group. Serum creatinine and urinary protein were significantly lower in both tadalafil groups than those in the high‐salt group, while only high‐dose tadalafil affected blood pressure. In addition, glomerulosclerosis and α‐smooth muscle actin expression significantly decreased in both tadalafil treatment groups. PAI1 mRNA increased significantly in the high‐salt group but decreased in both tadalafil‐treated groups. Our results indicated that both low‐ and high‐dose tadalafil prevented fibrosis and glomerular injury in a chronic kidney disease rat model. Mechanistically, these effects may be associated with PAI1 expression and glomerular structure protection.

Effects of arterial hemorrhage speed on the blood coagulation/fibrinolysis system and hemodynamics in rats.

: The effects of rapid hemorrhage on coagulopathy have been reported. However, the effects of different hemorrhage speeds on the blood coagulation/fibrinolysis system have not been investigated. This study aimed to compare different hemorrhage speeds for clarifying their effects on the coagulation/fibrinolysis system and circulation disorders in rats. Male Sprague-Dawley rats (301-396 g) were randomly assigned to five groups depending on hemorrhage speed and length of procedure: first, rapid (1.4 ml/min, 30-min bleeding); second, rapid-L (1.4 ml/min, 30-min bleeding and observation until 6 h); third, slow (0.1 ml/min, intermittently, 6-h bleeding); fourth, control (30-min observation); and fifth, control-L (6-h observation). Hemorrhage was induced by withdrawing blood until 40% of the estimated blood volume from the femoral artery. We measured vital signs, hematology, general chemistry, blood gas status, coagulation parameters, fibrinolytic markers [tissue-type plasminogen activator and plasminogen activator inhibitor one (PAI-1)], vascular endothelial damage (syndecan-1), and liver PAI-1 mRNA expression. Rapid hemorrhage induced elevation of lactate and syndecan-1 levels and prolonged prothrombin time and activated partial thromboplastin time in the rapid group. In contrast, slow hemorrhage did not induce these changes. Hemorrhage speed had no effect on plasma tissue-type plasminogen activator and hematology. Plasma PAI-1 levels were significantly increased in the rapid-L group, while liver PAI-1 mRNA levels were increased in the slow group. This study shows changes in the circulatory and fibrinolysis systems, depending on the hemorrhage speed. Hemorrhage might promote production of PAI-1, while tissue hypoxia due to rapid hemorrhage might promote release of PAI-1.

Altered fibrinolytic system in rat models of depression and patients with first-episode depression

Tissue plasminogen activator (tPA) is a serine protease involved in cleavage of neurotrophic factors. In addition, tPA and neuroserpin can also directly bind to low density lipoprotein receptor-related protein 1 (LRP1), promoting neurogenesis and neurite outgrowth. Given both the cleavage and non-cleavage actions of the fibrinolytic system are crucial in neurological functions, the present study, for the first time, systematically detected the changes of fibrinolytic system factors in rats exposed to chronic unpredictable mild stress (CUMS) or lipopolysaccharide (LPS) and patients with depression. In general, our data demonstrated that both CUMS and LPS reduced tPA but elevated plasminogen activator inhibitor-1 (PAI-1; SERPINE1) mRNA expression. Intriguingly, decreased expression of neuroserpin and LRP1 was also observed in rats exposed to CUMS or LPS. The down-regulated neuroserpin and LRP1 signaling were confirmed by western blotting and immunoflurence data. Likewise, elevated PAI-1 but a significant reduction of neuroserpin and LRP1 mRNA expression were observed in the peripheral blood mononuclear cells (PBMCs) of patients with first-episode depression, and the mRNA levels of PAI-1, neuroserpin and LRP1 were correlated with the Beck Depression inventory (BDI) scores, further strengthening the clinical significance and involvement of the fibrinolytic system in depression. Collectively, the present study demonstrated the alterations of fibrinolytic system in stressed and inflamed brain and in patients with first-episode depression, firstly showing that not only the cleavage actions, but also the non-cleavage actions of the system may play an essential role in the development of depression.

Sepsis causes right ventricular myocardial inflammation independent of pulmonary hypertension in a porcine sepsis model.

IntroductionRight ventricular (RV) myocardial dysfunction is a common feature in septic shock. It can worsen outcome, but the etiology is poorly understood. Pulmonary artery hypertension (PAH) plays a part in the pathogenesis of the right heart dysfunction in sepsis but its importance is unknown. In pigs, PAH in sepsis is substantial and the translational value of porcine sepsis models therefore questioned. We hypothesized that porcine sepsis causes a myocardial inflammatory response which leads to myocardial dysfunction independent of PAH.Materials and methodsSepsis was induced by Escherichia coli-infusion in 10 pigs resulting in PAH and increased right ventricular pressure (RVP). The same degree of RVP was achieved by external pulmonary artery banding (PAB) in a consecutive series of 6 animals.ResultsSepsis, but not PAB, led to increase in endothelial damage marker PAI-1 and cytokines TNF and IL-6 (all p<0.05) in plasma. In myocardium, TNF and IL-6 were significantly elevated in sepsis, TNF in both ventricles and IL-6 mostly in RV, while IL-1β, IL-18 and C5a were significantly higher in RV compared to LV after PAB (all p<0.05). Myocardial mRNA levels of IL-1β, IL-6, IL-18, IP-10, E-selectin and PAI-1 were significantly elevated in RV and LV during sepsis compared to PAB, while Caspase-1 was decreased in septic compared to PAB animals (all p<0.05). Cathepsin L activity was increased in RV by PAB, while sepsis inhibited this response. Escherichia coli-induced sepsis caused myocardial inflammation independent of PAH.ConclusionProminent PAH should therefore not exclude porcine sepsis models to further our understanding of human sepsis.

Metformin Ameliorates Renal Fibrosis in Mice with Unilateral Ureteral Obstruction and Inhibits TGF-ß1-Induced Upregulation of Cadherin-6 in Renal Proximal Tubule Epithelial Cells

Background: Metformin has been reported of nephroprotective effects by suppressing TGF-β1-induced renal tubulointerstitial fibrosis. The mechanisms have not been fully clarified. Cadherin-6(CDH6) is a TGF-β1 target gene related to epithelial-to-mesenchymal transition(EMT). This study aimed to examine the effects of metformin on TGF-β1-induced expression of CDH6 and renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and HK-2 human renal proximal tubule epithelial cells. Methods: Six-week-old male Kunming mice(n=18) were randomly divided into three groups: 1. Sham: sham-operated mice; 2. UUO: mice underwent UUO; 3. MET+UUO: UUO mice received metformin by gavage for 21 days after UUO. Kidneys were harvested for histopathologic examination and urine was collected for creatinine evaluation. HK-2 cells were stimulated with either TGF-β1 and/or metformin. RT-qPCR and Western Blot were used to evaluate mRNA and protein level respectively. Results: Masson’s Trichrome staining showed significant tubulointerstitial accumulation of extracellular matrix in obstructed kidneys, which could be reduced by metformin. Meanwhile, urinary creatinine was increased with metformin treatment. Upregulation of CDH6, COL1A1, PAI-1 mRNA level was observed in obstructed kidneys, which was reduced in MET+UUO group. Immunohistochemistry showed increased expression of CDH6, α-SMA in obstructed kidneys, which was alleviated in metformin treated group. In HK-2 cells, TGF-β1 upregulated mRNA level of CDH6, vimentin, PAI-1 and protein level of CDH6. Pretreatment with metformin attenuated TGF-β1-induced upregulation of CDH6, vimentin, PAI-1 mRNA level. Conclusion: Metformin prevents renal fibrosis partly through inhibition of TGF-β1-induced increase of CDH6 expression and EMT in renal proximal tubule epithelial cells. Disclosure D. Liang: None. S. Liu: None. Z. Song: None. W. Liang: None. R. Chang: None. Y. Li: None.

Thrombotic characteristics of extracellular vesicles derived from prostate cancer cells.

Prostate cancer (PC) patients in advanced stages of the disease have high risk of blood coagulation complications. The procoagulant molecule Tissue factor (TF), and the fibrinolysis inhibitor plasminogen activator inhibitor-1 PAI-1 play important role in this complication. Extracellular vesicles (EV) shed from cancer cells may contribute to the regulation of TF and PAI-1. The procoagulant activity of EV can be associated with the oncogenic and metastatic characteristics of their cells. We have expressed EGFRvIII in DU145 cells to assess the role of this oncogene in the procoagulant activity of EV. The intercellular exchange of TF via EV was assessed by downregulating its expression in DU145 cells using shRNA vector, and determining the transfer of TF via EV enriched with the protein. Two PC cell lines with different metastatic potential were used to assess the correlation between the procoagulant activity of EV and the metastatic potential of PC cells. Photometric assays were used to determine FXa-activity and thrombin generation as indicators for the procoagulant activity of EV. Double-tagged proteinase-activated receptor 1(PAR-1) expressed in CHO cells to assess its activation by EV. The expression of EGFRvIII in DU145 cells led to increased mRNA levels for TF and PAI-1, but the increase in these proteins expression was detected mostly in the EV. EV with enhanced levels of TF protein conferred higher TF procoagulant activity on the acceptor cells by intercellular exchange of this protein. Procoagulant activity of EV, assessed by FXa activity, and thrombin generation, was correlated with the oncogenic and metastatic potential of PC cells. The ability of EV to generate thrombin led to the activation of PAR-1, which was evident by the truncation of tagged-PAR-1. The active oncogene EGFRvIII increases the concentration of TF and PAI-1 in EV. The procoagulant activity of EV is associated with the oncogenic and metastatic characteristics of their PC cells. Also, EV may contribute to the high procoagulant activity in the tumour microenvironment by the intercellular exchange of TF. Finally, through the generation of thrombin, EV can activate PAR-1, which evidently contributes to cancer progression, linking the coagulation system to tumor progression.

Characterization of TGF-β expression and signaling profile in the adipose tissue of rats fed with high-fat and energy-restricted diets

Transforming growth factor beta (TGF-β) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-β and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-β, TGF-β receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-β and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-β and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-β mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-β receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-β expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine.

Reduced homoarginine plasma levels are associated with unfavourable cardiovascular outcome in chronic kidney disease (CKD). Cardiovascular events in CKD are fostered by vascular calcification, an active process promoted by hyperphosphatemia and involving osteo-/chondrogenic transformation of vascular smooth muscle cells (VSMCs). The present study explored the effect of homoarginine on phosphate-induced osteo-/chondrogenic signalling and vascular calcification. Experiments were performed in hyperphosphatemic klotho-hypomorphic mice (kl/kl), in subtotal nephrectomy and vitamin D3-overload mouse calcification models and in primary human aortic smooth muscle cells (HAoSMCs). As a result, plasma homoarginine levels were lower in kl/kl mice than in wild-type mice and in both genotypes significantly increased by lifelong treatment with homoarginine. Surprisingly, homoarginine treatment of kl/kl mice and of mice with renal failure after subtotal nephrectomy augmented vascular calcification and enhanced the transcript levels of plasminogen activator inhibitor 1 (Pai1) and of osteogenic markers Msx2, Cbfa1, and Alpl. Similarly, homoarginine treatment of HAoSMCs increased phosphate-induced calcium deposition, ALP activity, as well as PAI1, MSX2, CBFA1, and ALPL mRNA levels. Homoarginine alone up-regulated osteo-/chondrogenic signalling and indicators of oxidative stress in HAoSMCs. Furthermore, homoarginine reduced citrulline formation from arginine by nitric oxide (NO) synthase (NOS) isoforms. NO formation by NOS was reduced when using homoarginine as a substrate instead of arginine. The osteoinductive effects of homoarginine were mimicked by NOS inhibitor L-NAME and abolished by additional treatment with the NO donors DETA-NONOate and PAPA-NONOate or the antioxidants TEMPOL and TIRON. Furthermore, homoarginine augmented vascular calcification and aortic osteo-/chondrogenic signalling in mice after vitamin D3-overload, effects reversed by the NO donor molsidomine. Homoarginine augments osteo-/chondrogenic transformation of VSMCs and vascular calcification, effects involving impaired NO formation from homoarginine.

Anethole improves the in vitro development of isolated caprine secondary follicles

The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 μg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM+), defined as the control treatment, α-MEM+ supplemented with ascorbic acid at a concentration of 100 μg/mL (AA), or α-MEM+ supplemented with three different concentrations of anethole (30, 300, 2000 μg/mL) for a period of 18 days. Treatments were named as α-MEM+, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 μm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM+ and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.

Gene expression in a rarely studied intraabdominal adipose depot, the round ligament, in severely obese women: A pilot study

ABSTRACTGene expression (qPCR) was compared in round ligament (RL), omental (OME) and mesenteric (MES) ATs from 48 severely obese women (BMI, 54±11 kg/m2; 38±9 yrs). The mRNA levels of enzymes of lipid metabolism (LPL, HSL, and PDE-3B), cortisol production (11βHSD-1), adipogenesis (PPAR-γ1/2), thrombosis and inflammation (PAI-1, IL-6, TNF-α and adiponectin) were determined. AT-LPL mRNA was highest in RL. The highest PDE-3B and lowest PAI-1 mRNA levels were observed in RL and MES. The lowest IL-6 and TNF-α and the highest adiponectin and PPAR-g1/2 mRNA levels were found in RL AT. 11βHSD-1 was highest in RL and OME. A higher lipogenic and adipogenic, and lower pro-inflammatory and pro-thrombotic profiles of the RL suggest a lesser deleterious impact on obesity-related complications.

ADAMTS13/VWF Axis Potentially Contributes to Diabetic Nephropathy By Regulating PAI-1 Levels

Background and objective: ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13) cleaves von Willebrand factor (VWF), a large multimeric protein that plays an important role in hemostasis and thrombosis. Severe deficiency or very low levels of ADAMTS13 in presence of external stimuli results in accumulation of thrombogenic ultra large VWF multimers (which are released from activated endothelium) known to trigger thrombotic microangiopathy. Activated endothelium/dysfunction is a prominent feature of diabetic nephropathy, and advanced diabetic glomerulopathy often exhibits thrombotic microangiopathy. Significantly reduced ADAMTS13 and increased plasma VWF levels have been found in diabetic patients with nephropathy. Although major site of ADAMTS13 synthesis is liver, ADAMTS13 is also expressed by podocytes in normal renal cortex. It remains unknown, however, whether VWF and ADAMTS13 imbalance plays a causal role in development of nephropathy in diabetic patients or rather is simply an associate marker of disease status, possibly secondary to endothelial function. We performed experiments in genetic models to determine whether ADAMTS13 and VWF axis contributes to diabetic nephropathy.Methods: Male, 8-10 weeks old wild-type (WT), Adamts13-/- and Vwf-/- mice were made diabetic by injecting multiple low doses of streptozotocin (60 mg/kg, i.p. for five consecutive days). Successful diabetes induction was tested after 2 weeks by measuring blood glucose. Mice having blood glucose levels above 300 mg/dL were included in the study. Controls were nondiabetic littermate mice treated with citrate buffer. The extent of renal injury was evaluated after 28 weeks of diabetes induction by measuring albuminuria and kidney to body weight ratio. Renal hypertrophy and extracellular matrix deposition was quantified by hematoxylin and immunostaining. PAI-1 mRNA and protein levels were measured by real time quantitative RT-PCR and ELISA.Results: Adamts13-/- diabetic mice exhibited significantly increased kidney to body weight ratio (P<0.05 vs. WT diabetic mice). Urine albuminuria, an index of renal injury was significantly elevated in Adamts13-/- diabetic mice (P<0.05 vs. WT diabetic mice). Increased renal injury in Adamts13-/- diabetic mice was concomitant with increased renal hypertrophy and extracellular matrix (ECM) deposition within glomeruli (P<0.05 vs. WT diabetic mice). Murine studies have shown that PAI-1 contributes to diabetic nephropathy by regulating TGF-beta and ECM deposition. A positive association exists between increased PAI-1 levels in glomeruli and microangiopathy in patients with diabetic nephropathy. We determined whether ADAMTS13 deficiency-induced microangiopathy in glomeruli increases PAI-1 levels. Adamts13-/- diabetic mice exhibited increased PAI-1 mRNA and protein levels (P<0.05 vs. WT diabetic mice). VWF remains the only known substrate of ADAMTS13 and increased plasma VWF levels have been associated with diabetic nephropathy. We determined the role of VWF in diabetic nephropathy. Vwf-/- diabetic mice exhibited significantly decreased kidney weight/body weight ratio, less urinary albuminuria, decreased kidney PAI-1 expression levels concomitant with improved kidney morphological changes (P<0.05 vs. WT diabetic mice).Conclusion: These findings provide experimental evidence for the first time that ADAMTS13/VWF axis potentially contributes to diabetic nephropathy, most likely by regulating PAI-1 levels. DisclosuresNo relevant conflicts of interest to declare.

Abstract 13535: The Synergistic Effects of Incretin-related Drugs for the Treatment of Pulmonary Arterial Hypertension

Introduction: A growing body of evidence suggests that inflammation plays a crucial role in the development of pulmonary arterial hypertension (PAH). Dipeptidyl peptidase-4 (DPP-4; also known as CD26), a protease which degrades incretin hormones such as glucagon-like peptide-1 (GLP-1), modulates inflammatory processes by regulating activity of T cells through co-stimulatory pathway. Hypothesis: Incretin-related drugs, including both DPP-4 inhibitors and GLP-1 receptor agonists, suppress the progression of PAH by attenuating inflammatory response of PA. Methods: Sprague Dawley rats were injected monocrotaline to induce PAH (N=80). After 14 days from PAH induction, these rats were treated with alogliptin, a DPP-4 inhibitor (M+A, N=20), liraglutide, a GLP-1 receptor agonist (M+L, N=20), both of these drugs (M+A+L, N=20), or vehicle (M, N=20). Results: A significant increase of survival was observed on days 30 after PAH induction both in the M+A and M+L compared to the M (M+A: 45%* vs. M+L: 60%* vs. M: 5%, * p &lt;0.05). Both alogliptin and liraglutide markedly improved right ventricular pressure (M+A: 33±4.1mmHg* vs. M+L: 26±1.9mmHg* vs. M: 85.9±1.2mmHg, * p &lt;0.05). mRNA levels of both CD28 and CD86, co-stimulators of T cells, significantly decreased and the level of caveolin-1 markedly increased in lung tissues of the M+A compared to those in the M. mRNA levels of both Tissue Factor (TF) and PAI-1 significantly decreased in lung tissues of the M+L than those in the M. Consistently, survival of rats in the M+A+L was the highest (65%) among all of the groups of PAH rats. Coimmunoprecipitation assays revealed that CD26 in T cells physically interacted with caveolin-1 in rat pulmonary vascular smooth muscle cells (rPASMCs). Immunoblot analyses showed that alogliptin suppressed caveolin-1 phosphorylation in rPASMCs. Furthermore, reporter gene assays demonstrated that alogliptin significantly inhibited the transcriptional activity of NF-κB. Conclusions: These results suggest that DPP-4 inhibition and stimulation of GLP-1 receptors synergistically mediate salutary effects on monocrotaline-induced PAH by modulating T cell activity and TF-associated signaling pathway. Thus, incretin-related drugs have a potential as a novel therapeutic tool for PAH.

Urinary Cell mRNA Profiles Demonstrate Persistent BKV Replication and a Profibrotic Milieu Despite Clearance of BK Viremia/Nephropathy.

Background: BKV nephropathy is not only associated with increased risk of proximate graft loss but also a significantly shortened graft survival compared to those who did not develop BKVN (KI 2005, vol 68 p1834). The basis for this progressive decline despite clearance of BKV viremia/nephropathy is not clear. We have previously reported that heightened expression of mRNAs associated with fibrosis at the time of BKVN predicts subsequent renal allograft loss. Herein we explored the changes in urinary cell mRNA profiles following clearance of BKVN and BK viremia. Methods: We prospectively studied 13 patients who developed BKVN and followed them until they had clearance of viremia and histology. Histological clearance was demonstrated by the absence of SV40 immunostaining of allograft biopsies. Urine specimens were collected at the time of BKVN diagnosis and at the time of BKVN clearance. Urine mRNA profiles at the time of BKVN diagnosis and following BKVN clearance were measured using real-time quantitative PCR. Results: BKVN clearance occurred at mean(SD) of 6±0.9 months. BKV copies in the blood decreased from 6.8x105±10x105 copies to 1x103 ± 9x102. Mean serum creatinine (mg/dL) levels at the time of BKVN diagnosis were not different from the levels obtained at the time of BKVN clearance (2.1±0.8 vs. 2.1±0.7).Table: No Caption available.Conclusion: Lower level of BKV replication in the urine persists following histological clearance of BKVN. Despite significant decrease in BKV replication, levels of TGFb1, PAI-1, vimentin, TIMP-1 and fibronectin mRNA increased significantly following clearance of BKVN. Our data generates the hypothesis that persistence of a profibrotic milieu following resolution of BKVN contributes to progressive decline in renal function.

The effects of TSH on human vascular endothelial cells and smooth muscle cells

Objective To study the effect of thyroid-stimulating hormone (TSH) on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used to determine the levels of endothelial nitric oxide synthase (eNOS),prostacyclin(PGI2),endothelin-1 (ET-1),plasminagen activator inhibitor(PAI-1) (mRNA) in endothelial cells and the phenotype transition of smooth muscle cells.The effect of TSH to the cycle of smooth muscle cells was detected by using flow cytometry.Immunocytochemistry and Western blot were used to investigate the expression of the cell cyclin A,D1,and the expression of endothelial cell associated factors eNOS and ET-1.Results Compared with the control group,eNOS and PGI2 mRNA levels decreased while ET-1 and PAI-1 mRNA levels increased when different concentrations of TSH were applied to endothelial cells(P＜0.05).The level of eNOS protein was decreased gradually while the level of ET-1 protein was gradually increased(P＜0.05).Different concentrations of TSH applied to smooth muscle cells could promote the transition of cell cycle phase G2 to phase M and increase the expression of cell cyclin A and D1.Conclusion TSH may damage the function of vascular endothelial cells and promote the proliferation of smooth muscle cells. Key words: Thyroid stimulating hormone; Vascular endothelial cells; Vascular smooth muscle cells; Atherosclerosis