scholarly journals The recovery of some components of the renin angiotensin system in the rat pancreas after chronic exposure to hypoxic condition

2003 ◽  
Vol 31 (3) ◽  
pp. 563-571 ◽  
Author(s):  
SP Ip ◽  
TP Wong ◽  
SJ Tsai ◽  
PS Leung
Keyword(s):  
Angiotensin Ii ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chronic Exposure ◽  
Chronic Hypoxia ◽  
Renin Angiotensin System ◽  
Rt Pcr ◽  
Angiotensin System ◽  
Ace Activity

Previous studies have shown that the expression of the major components from a local pancreatic renin-angiotensin system (RAS) was upregulated after chronic exposure to oxygen deprivation (10% oxygen). In the present study, the reversibility of expression for the pancreatic RAS affected by chronic hypoxia was investigated in the pancreas. Rats were first subject to hypoxia for one Month and they were then returned to normoxic conditions for a varying period of time (1, 2, 3 and 4 weeks). The degree of recovery in the expression of RAS components was analyzed with standard curve-quantitative competitive-reverse transcription-polymerase chain reaction (SC-QC-RT-PCR), Western blot analysis and a specific assay for angiotensin-converting enzyme (ACE) activity. Results from SC-QC-RT-PCR showed that the upregulated expression of angiotensin II type 1 (AT(1)) receptor mRNA following chronic hypoxia could be completely restored to the control level after the rats were returned to the normoxic condition for 3 weeks. The reversibility of mRNA expression for angiotensin II type 2 (AT(2)) receptor and angiotensinogen was observed after the return to normoxic conditions for 2 and 3 weeks respectively when compared with that of their respective controls. Results from Western blot analysis further confirmed that the expression of AT(1) receptor protein was also reversible after return to normoxic conditions for 4 weeks. In addition, the activation of ACE activity returned to its normal level in a time-dependent manner. These data indicate that the upregulation of a local pancreatic RAS affected by chronic hypoxia could be recoverable. The significance of its reversibility and adaptability following chronic hypoxia may be of physiological relevance to the pancreas.

scholarly journals Advanced glycation end-products activate the renin-angiotensin system through the RAGE/PI3-K signaling pathway in podocytes

2012 ◽  
Vol 35 (5) ◽  
pp. 282 ◽  
Author(s):  
Cailan L. Cheng ◽  
Ying Tang ◽  
Zhenda Zheng ◽  
Xun Liu ◽  
Zengchun C. Ye ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Western Blot ◽  
Advanced Glycation End Products ◽  
Renin Angiotensin System ◽  
Ang Ii ◽  
Advanced Glycation ◽  
Angiotensin System ◽  
Glycation End Products ◽  
End Products ◽  
Ace Activity

Purpose: The purpose of this study was to investigate the effects of advanced glycation end-products (AGEs) on the components of the renin-angiotensin system (RAS) in podocytes and to understand the mechanism of these effects. Methods: Immortalized mouse podocytes were exposed to various concentrations of AGEs for different time intervals. The expression levels of angiotensinogen (AGT), angiotensin II type 1 and 2 receptors (AT1R and AT2R) and renin were examined by real-time PCR and western blot; the receptor for AGEs (RAGE) and both Akt and phosphorylated Akt were examined by western blot; levels of angiotensin II (Ang II) were assayed by ELISA, and the activity of angiotensin-converting enzyme (ACE) was evaluated by measuring the production of hippuric acid in vitro. Results: Treatment with AGEs resulted in significant increases in the expression of AGT (62%, P=0.002) and AT1R (59%, P=0.01). Moreover, Ang II levels increased significantly in both cell lysates (70%, P=0.018) and conditioned media (65%, P=0.01). ACE activity was also significantly higher in cell lysates (68% , P= 0.035) and conditioned media (65%, P=0.023). There were no changes in renin or AT2R expression (P > 0.05). AGEs did increase the expression of RAGE by 50% (P=0.012) and the phosphorylation of Akt by 100% (P=0.001). When podocytes were pretreated with anti-RAGE antibody (50 µg/ml) or the phosphoinositide 3-kinase (PI3-K) inhibitor, LY294002 (10 µM), the AGEs-induced increases in AGT and AT1R expression were reduced. Likewise, Ang II levels and ACE activity decreased significantly. Conclusion: AGEs activate the RAS in podocytes through the RAGE-PI3-K/Akt-dependent pathway and lead to an increase in podocyte apoptosis.

scholarly journals Losartan protects against intermittent hypoxia-induced peritubular capillary loss by modulating the renal renin–angiotensin system and angiogenesis factors

2019 ◽  
Vol 52 (1) ◽  
pp. 38-48 ◽  
Author(s):  
Jiqiang Wu ◽  
Yao Chu ◽  
Zhenxiu Jiang ◽  
Qin Yu
Keyword(s):  
Angiotensin Ii ◽  
Western Blot ◽  
Blot Analysis ◽  
Intermittent Hypoxia ◽  
Renin Angiotensin System ◽  
Tubular Epithelial Cells ◽  
Angiotensin Ii Receptor ◽  
Peritubular Capillary ◽  
Angiotensin System ◽  
Angiogenesis Factor

Abstract Obstructive sleep apnea is characterized by chronic intermittent hypoxia (CIH), which is a risk factor for renal peritubular capillary (PTC) loss, and angiotensin II receptor blockers can alleviate PTC loss. However, the mechanism by which losartan (an angiotensin II receptor blocker) reduces CIH-induced PTC loss and attenuates kidney damage is still unknown. Thus, in this study, we examined the protective effects of losartan against CIH-induced PTC loss and explored the underlying mechanisms in rat CIH model. The immunohistochemical staining of CD34 and morphological examination showed that CIH reduced PTC density and damaged tubular epithelial cells. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR, and western blot analysis results revealed that CIH increased the expression of hypoxia inducible factor-1α (HIF-1α), angiotensin II (Ang II), angiotensin II type 1 receptor (AT1R), pro-angiogenesis factor vascular endothelial growth factor (VEGF), and anti-angiogenesis factor thrombospondin-1 (TSP-1) in the renal cortex of rats. CIH may up-regulate VEGF expression and simultaneously increase TSP-1 production. By histopathological, immunohistochemistry, ELISA, RT-qPCR, and western blot analysis, we found that the expressions of renal renin–angiotensin system (RAS), HIF-1α, VEGF, and TSP-1 were decreased, and PTC loss and tubular epithelial cell injury were attenuated with losartan treatment. Losartan ameliorated CIH-induced PTC loss by modulating renal RAS to improve the crosstalk between endothelial cells and tubular epithelial cells and subsequently regulate the balance of angiogenesis factors. Our study provided novel insights into the mechanisms of CIH-induced kidney damage and indicated that losartan could be a potential therapeutic agent for renal protection by alleviating CIH-induced PTC loss.

scholarly journals NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. G197-G206 ◽  
Author(s):  
J. Praetorius ◽  
D. Andreasen ◽  
B. L. Jensen ◽  
M. A. Ainsworth ◽  
U. G. Friis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Intracellular Ph ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Level ◽  
Rt Pcr ◽  
Acid Extrusion ◽  
Molecular Expression ◽  
Nhe Isoforms

Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusion, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pHi) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na+ concentration ([Na+]o) during pHirecovery decreased H+ efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na+. The Na+/H+exchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na+]o and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na+/H+ exchange by isoforms NHE1, NHE2, and NHE3.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals Melatonin Reduces Inflammatory Injury through Inhibiting NF-κB Activation in Rats with Colitis

2005 ◽  
Vol 2005 (4) ◽  
pp. 185-193 ◽  
Author(s):  
Jun-Hua Li ◽  
Jie-Ping Yu ◽  
Hong-Gang Yu ◽  
Xi-Ming Xu ◽  
Liang-Liang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Colon Tissue ◽  
Inflammatory Injury ◽  
Proinflammatory Mediators ◽  
Proinflammatory Molecule ◽  
Colitis Model

Proinflammatory mediators are important in the pathogenesis of IBD, which are regulated by activation of NF-κB. The aim of this study was to investigate whether melatonin reduces inflammatory injury and inhibits proinflammatory molecule and NF-κB in rats with colitis. Rat colitis model was established by TNBS enema. NF-κB p65, TNF-α, ICAM-1, and IκBα in colon tissue were examined by immunohistochemistry, EMSA, RT-PCR, and Western blot analysis. Expression of proinflammatory molecule and activation of NF-κB were upregulated and IκB level decreased in rats with colitis. Melatonin reduces colonic inflammatory injury through downregulating proinflammatory molecule mediated by NF-κB inhibition and blockade of IκBα degradation.

The Spermatozoa Protein, SLLP1, Is a Novel Cancer-Testis Antigen in Hematologic Malignancies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4281-4281
Author(s):  
Zhiqing Wang ◽  
Yana Zhang ◽  
Arabinda Mandal ◽  
Jian Zhang ◽  
Francis J. Giles ◽  
...  
Keyword(s):  
Immune Responses ◽  
Western Blot ◽  
Tumor Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hematologic Malignancies ◽  
Myeloma Cell ◽  
Rt Pcr ◽  
Healthy Donors ◽  
Cancer Testis

Abstract SLLP1 is a unique non-bacteriolytic c-lysozyme-like protein isolated from human spermatozoa. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, suggesting that SLLP1 may be involved in sperm/egg adhesion. A recent study by dot blot analysis on RNA showed that SLLP1 was expressed only in the testis and in Burkitt lymphoma Raji cell line, suggesting that further studies are warranted to determine and characterize SLLP1 expression in tumor cells, in particular, fresh tumor specimens. Using a pair of sequence-specific primers in RT-PCR, we found that SLLP1 transcripts could be detected in 5/8 myeloma cell lines, suggesting that SLLP1 may be expressed in tumor cells from some hematologic malignancies. When we applied the investigations to 52 primary hematologic malignant specimens, SLLP1 transcripts were detected in 6/17 myeloma, 4/14 CML, 3/11 CLL, 2/9 AML and 0/1 hairy cell leukemia. In contrast, SLLP1 transcripts were not detected in the peripheral blood (n=12) or bone marrow (n=3) from any healthy donors. The specificity of the PCR products was confirmed by either sequence analysis or restriction digest with Pvu II. SLLP1 transcripts were translated into its corresponding protein in these tumor cells. Using tumor cell lysate in Western blot analysis, we detected SLLP1 protein in the myeloma cell lines and also in fresh malignant specimens, although positivities were only observed in specimens with high RT-PCR signals. All PCR-negative specimens were also negative in Western blot analysis. The specificity of the Western blot signals were confirmed in all cases by blocking assays with a high concentration of recombinant SLLP1 protein. We next investigated the expression of SLLP1 in a large panel of normal tissues using RT-PCR and real time quantitative PCR. Both approaches showed that SLLP1 is a novel Cancer-Testis antigen in hematologic malignancies. SLLP1 was detected, at a level of 8206 copies/0.25 mcg total RNA, only in normal testis. We also found that the SLLP1 mRNA copy numbers in fresh hematologic tumor specimens were up to 2316 copies/0.25 mcg total RNA, i.e. more than 25% of the level found in normal testis. We cloned and generated SLLP1 recombinant protein from E coli and used the purified recombinant SLLP1 in an ELISA system to detect anti-SLLP1 antibodies. Using sera from 24 healthy donors and the mean + 2SD as the cut-off signal intensities, we found that high titer IgG antibodies directed at SLLP1 could be detected in the sera from 2/9 AML, 5/23 CLL, 6/27 CML and 14/51 myeloma patients. The specificity of the antibodies was confirmed in Western blot analysis. Probably due to the decreased sensitivity of the detection system in Western blot analysis, only 1/2 AML, 3/5 CLL, 4/6 CML and 7/14 myeloma SLLP1 antibody+ sera produced a signal in the Western blot analysis. Interesting, IgG2 was by far the commonest SLLP1 antibodies in these patients. There was a good correlation between SLLP1 gene expression and immune responses. In summary, SLLP1 is a novel CT antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing patients. Our results support SLLP1 as a protein target appropriate for further in vitro study to define its suitability for immunotherapy.

Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1267-1267
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Richard A. Campbell ◽  
Melinda S. Gordon ◽  
Dror Shalitin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Early Stage ◽  
Vascular Endothelial ◽  
Cam Assay ◽  
Rt Pcr ◽  
Vessel Formation

Abstract We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4640-4640
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Low Grade ◽  
Adapter Proteins ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Sirna Knockdown

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

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