To obtain the expression of Isoleucine Zipper modified soluble CD40L (IZ-sCD40L) in Pichia pastoris, firstly, DNA fragment of IZ-sCD40L was obtained by PCR and over-lap PCR . Then the expression vector pPICZaA-IZ-sCD40L was constructed. Nucleotide sequencing analysis indicated that the DNA fragment of IZ-sCD40L was correctly inserted into the pPICZaA vector. Linearized pPICZ(alpha)A-IZ-sCD40L was introduced into Pichia pastoris GS115. Positive clone was selected by PCR and its phenotype was determined. The positive clone was introduced with methanol. The results of SDS-PAGE and Western blot showed that product was recombinant Isoleucine Zipper modified soluble CD40L fusion protein.
Angiotensin II ; CD40 Ligand ; biosynthesis ; genetics ; Cloning, Molecular ; Isoleucine ; analogs & derivatives ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic

BACKGROUNDAngiotensin II (Ang II) is a very important vasoactive peptide that acts upon hepatic stellate cells (HSCs), which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang II and angiotensin II type 1 receptor antagonist (AT(1)RA) on the proliferation, contraction and collagen synthesis in HSCs.

METHODSHSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by (3)H-thymidine incorporation. The effects of angiotensin II and AT(1)RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen I (Col I), collagen III (Col III) and transforming growth factor beta1 (TGF-beta1) by enzyme linked immunosorbent assay. HSC was harvested to measure collagen I, collagen III and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression.

RESULTSAng II ((1 x 10(-10) - 1 x 10(-4)) mol/L) stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT(1)RA inhibited angiotensin II induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin II (1 x 10(-9) - 1 x 10(-5) mol/L) and with time over 48 hours. AT(1)RA blocks angiotensin II induced contraction of collagen lattice. Col I, Col III and TGF-beta1 levels of the Ang II group were higher than those of control group and this increase was downregulated by AT(1)RA. The mRNA expressions of Col I, Col III and TIMP-1 were higher in HSCs from the Ang II group than the control group and downregulated by AT(1)RA.

CONCLUSIONSAngiotensin II increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col III and TGF-beta1 levels and stimulated Col I, Col III and TIMP-1 expression in HSCs. Angiotensin acts via the angiotensin II receptor because all of these effects are blocked by angiotensin II type 1 receptor antagonist.

Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Liver ; cytology ; drug effects ; metabolism ; Rats ; Transforming Growth Factor beta1 ; biosynthesis

OBJECTIVEAngiotensin II is an important pro inflammation factor in the cardiovascular system. This experiment is aimed to study the effects of angiotensin II on inducible nitric oxide synthase expression in human umbilical endothelial cells.

METHODSHuman umbilical endothelial cells were cultured in vitro and treated with angiotensin II alone or in combination with AT1, AT2 and NF-kappaB inhibitors respectively. The inducible nitric oxide synthase expressions at protein and mRNA levels were measured with Western blot and reverse transcription-polymerase chain reaction (RT-PCR), and the activity of NF-kappaB was analyzed with EMSA.

RESULTSAngiotensin II up-regulated inducible nitric oxide synthase expressions at the protein and mRNA levels at 5 h (P < 0.05), the activity of NF-kappaB was enhanced at 2 h (P < 0.05). These effects could be blocked by AT1 and NF-kappaB inhibitors but not by AT2 inhibitor.

CONCLUSIONAngiotensin II can upregulate the expression of inducible nitric oxide synthase through NF-kappaB pathway in human umbilical endothelial cells. AT1, other than AT2, play a key role in this process.

Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Angiotensin II Type 2 Receptor Blockers ; Cell Line ; Endothelial Cells ; chemistry ; drug effects ; Heart Failure ; metabolism ; Humans ; NF-kappa B ; metabolism ; Nitric Oxide Synthase Type II ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; cytology ; Up-Regulation

BACKGROUNDIntrarenal activation of the renin angiotensin system (RAS) plays an important role in mediating renal fibrosis. Both angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II (AngII) receptor antagonists have been shown to exert a protective role against diabetic and non-diabetic nephropathy. However, the exact mechanism of how blocking local RAS prevents renal fibrosis is unclear. The present study was to investigate the influence of a new AngII receptor antagonist, irbesartan (Irb), on AngII-induced hypertrophy in human proximal tubular cell line (HK-2).

METHODSThe cell line, HK-2, was grown in Dulbeccos's Modified Eagle's Medium containing 10% heat-inactivated fetal calf serum. After rested in serum-free medium for 24 hours, the effects of Irb on AngII (10(-7) mol/L)-induced [(3)H]-leucine incorporation, total protein content (measured by the Coomassie brilliant blue G250 method), and change in cell size (determined by scanning electron microscopy) were observed. The influence of Irb on the cell cycle was analyzed by fluorescence activated cell sorter (FACS) flow cytometry.

RESULTSAngII induced cell hypertrophy in a time and dose dependent manner. Stimulation of cells with AngII for 48 hours resulted in a increase in [(3)H]-leucine incorporation [0 hour: (5584 +/- 1016) cpm/10(5) cells vs 48 hours: (10741 +/- 802) cpm/10(5) cells, P < 0.05], which was significantly attenuated by treatment with Irb. AngII significantly increased the total protein content in HK-2 cells [control: (0.169 +/- 0.011) mg/10(5) cells vs AngII group: (0.202 +/- 0.010) mg/10(5) cells, P < 0.05], which was also markedly inhibited by cotreatment with Irb (P < 0.01). Scanning electron microscopy showed that AngII induced an increase in average physical cell size, which was significantly inhibited by Irb [control: (11.92 +/- 1.62) microm; AngII group: (20.63 +/- 3.83) micro m; AngII + Irb group: (13.59 +/- 3.15) micro m; P < 0.01 vs control, respectively]. Furthermore, flow cytometry revealed that AngII arrested cells in the G(0)-G(1) phase, which was significantly reversed by treatment with Irb [G(0)-G(1) cells in AngII group: (76.09 +/- 1.82)%, in AngII + Irb group: (67.00 +/- 2.52)%, P < 0.05].

CONCLUSIONIrb can inhibit AngII-induced hypertrophy in HK-2 cells.

Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; Biphenyl Compounds ; pharmacology ; Cell Cycle ; drug effects ; Cells, Cultured ; Humans ; Hypertrophy ; Kidney Tubules, Proximal ; drug effects ; pathology ; ultrastructure ; Protein Biosynthesis ; Tetrazoles ; pharmacology

Angiotensin II ; biosynthesis ; genetics ; Animals ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; therapeutic use ; Liver Cirrhosis, Experimental ; chemically induced ; drug therapy ; metabolism ; Male ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 1 ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of angiotensin II (AngII) receptor (AT(1), AT(2)) antagonists on myocardial matrix metalloproteinases (MMPs) and fibronectin (FN) in rats with myocardial infarction (MI).

METHODSRat MI was induced by permanent ligation of the left coronary artery. Placebo, AT(1) receptor antagonist valsartan (10 mgxkg(-1)xd(-1)) or AT(2) receptor antagonist PD123319 (30 mgxkg(-1)xd(-1)) were given 7 days prior MI surgery. On the 1st, 3rd and 7th day after MI, Expressions of MMP-2, 3, 9, tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) and FN at protein level were determined by Western blot in left ventricular free wall (LVFW), interventricular septum (IS) and right ventricular (RV). Myocardial FN distribution was also assayed by immunofluorescence.

RESULTSTypical myocardial remodeling was shown in IS and LVFW 7 days after MI. MMP-2, 3, 9 expressions at protein level were significantly increased whereas TIMP-1 and FN expressions significantly decreased in IS 1, 3, 7 days post MI in a time-dependent manner compared to that of sham operated hearts. MMP-2, 3, 9 expressions was significantly increased and TIMP-1 and FN expression significantly decreased in LVFW at the 1st post MI day and maintained up to 7th post MI day compared to that of sham operated hearts. Up-regulated expressions of MMP-2, 3, 9 and down-regulated TIMP-1 and FN expressions in IS and LVFW could be significantly attenuated by valsartan but not by PD123319. Valsartan but not PD123319 also significantly reduced MI sizes (40.4% +/- 2.1% vs 49.5% +/- 2.1%, P < 0.05).

CONCLUSIONAT(1) receptor antagonist involves in the pathology procession of myocardial remodeling and might lead to the development and progression of congestive heart failure by the increasing expressions of MMP-2, 3, 9, which contribute to degradative extracellular matrix FN in myocardium.

Angiotensin II Type 1 Receptor Blockers ; therapeutic use ; Animals ; Fibronectins ; biosynthesis ; Male ; Matrix Metalloproteinase 2 ; biosynthesis ; Matrix Metalloproteinase 3 ; biosynthesis ; Matrix Metalloproteinase 9 ; biosynthesis ; Myocardial Infarction ; drug therapy ; pathology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the renin-angiotensin system (RAS) in mesenteric adipose tissues and effect of angiotensin II on adipocyte differentiation.

METHODSThirty normal 8-week-old male Wistar rats were divided into groups on normal diet and high-fat diet. The rats on high-fat diet for 24 weeks developed the metabolic syndrome respectively. The mRNA and protein expression of mesenteric adipose tissue were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Lipid drop in 3T3-L1 preadipocytes and mature adipocytes were observed using oil-red O staining. The fluorescence microscope was used to detect cytosolic-free calcium in 3T3-L1 preadipocytes and mature adipocytes.

RESULTSThe expressions of angiotensinogen, angiotensin converting enzyme, angiotensin II receptor type 1 in mesenteric adipose tissue were significantly increased in rats with metabolic syndrome compared with those in rats on normal diet (P <0. 05, P <0. 01). After administration of angiotensin II , no lipid droplet in 3T3 -L1 preadipocytes and adipocytes were observed, however, intensive lipid droplet in adipocyte was found after administration of captopril and candesartan. Angiotensin II increased the intracellular-free calcium concentration in preadipocytes (P < 0. 01 ) , which was blocked by captopril and candesartan; in contrast, angiotensin II effect was blunt in mature adipocyte. Captopril and candesartan partially recovered the angiotensin II -mediated increase of cytosolic-free calcium.

CONCLUSIONRAS in the mesenteric adipose tissues is active in rats with metabolic syndrome, and antagonization of RAS can recover the lipogenesis of adipocyte.

Adipocytes ; metabolism ; Adipose Tissue ; metabolism ; Angiotensin II ; pharmacology ; Angiotensinogen ; biosynthesis ; Animals ; Benzimidazoles ; pharmacology ; Calcium ; metabolism ; Captopril ; pharmacology ; Cells, Cultured ; Male ; Metabolic Syndrome ; physiopathology ; Peptidyl-Dipeptidase A ; biosynthesis ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 2 ; biosynthesis ; Renin-Angiotensin System ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazoles ; pharmacology

Interleukin (IL)-6 is an autocrine growth factor for mesangial cells. It is not known whether high glucose influences IL-6 production in mesangial cells. Angiotensin II (AGII) is involved in the progression of renal diseases including diabetic nephropathy. Therefore, we evaluated the effects of high glucose in concert with AGII on IL-6 production in human mesangial cells and the modulation by blocking AGII. After 48 hr of culture, IL-6 mRNA expression was analyzed by reverse transcription and polymerase chain reaction (PCR). Quantitative determination of IL-6 concentrations in the culture supernatants of mesangial cells was performed using a sandwich enzyme immunoassay kit. Incubation of mesangial cells with high glucose (450 mg/dL) reduced the ratio of PCR products for IL-6 to beta-actin on densitometric results, while AGII (10(-7)M) increased it. The IL-6 secretion in the supernatant was also increased by AGII and decreased by high glucose. The IL-6 mRNA expression and IL-6 secretion in combination of high glucose and AGII were higher than those in high glucose and similar with those in control media. The addition of losartan (10(-6)M) or captopril (10(-6)M) to high glucose had no additional effects on IL-6 production. These results suggest that whereas AGII increases IL-6 production, high glucose decreases it. The IL-6 production of mesangial cells in diabetic milieu may be complicated and depend on the local effects of high glucose and/or AGII.
Angiotensin II/*pharmacology ; Captopril/pharmacology ; Cells, Cultured ; Gene Expression/drug effects ; Glomerular Mesangium/cytology/*metabolism ; Glucose/*pharmacology ; Humans ; Interleukin-6/*biosynthesis/genetics/secretion ; Losartan/pharmacology

A number of mechanical cell stimulators have been used to study the effect of mechanical stimulation on cells in vitro. But the efficiency of these devices is not fully desirable. We recently developed a new device for mechanical cell stimulation, the centrifugal force stretcher, and compared its efficacy with that of the traditional Flexercell Strain Unit. When the mechanical stretcher circumrotates with certain speed, cardiac myocytes attached on the plate are stretched and elongated by centrifugal force. Neonatal rat cardiac myocytes were isolated by enzymatic dissociation from the hearts of 3~5 d old Sprague Dawley rats, and were mechanically stimulated by traditional 20% stretch and 180 r/min centrifugal force for 12 and 24 h. The effects of mechanical stimulation on the hypertrophic response of neonatal rat cardiac myocytes and production of angiotensin II (Ang II) were examined. Compared with the non-stretch group, the radioactivity of (3)H-leucine incorporated into the stretch-stimulated cardiac myocytes in the centrifugal force stretch group was significantly higher [(1295.17+/-51.19) vs (1122.67+/-51.63) in 12 h; (1447.5+/-35.96) vs (1210.67+/-90.92) in 24 h, P<0.05]. Ang II was also dramatically increased by 128% in 12 h (P<0.05) and 139% in 24 h (P<0.01). After the myocytes was stretched for 24 h, the LDH level in the medium in the Flexercell Strain Unit group was significantly higher than that in the centrifugal force group [(14.5+/-8.7) U/L vs (7.8+/-4.3) U/L, P<0.05]. The centrifugal force stretcher is a new and improved mechanical cell stimulator with the same effects on the protein synthesis and Ang II secretion of the cardiac myocytes, and the damage to the cells bronght by this stimulator is relatively slighter in comparison with the Flexercell Strain Unit.
Angiotensin II ; secretion ; Animals ; Cell Biology ; instrumentation ; Cells, Cultured ; Centrifugation ; Myocytes, Cardiac ; cytology ; metabolism ; Protein Biosynthesis ; Rats ; Rats, Sprague-Dawley ; Tensile Strength

OBJECTIVETo observe the effect of angiotensin II (Ang II) on calreticulin (CRT) expression and its association with mitochondrial dysfunction in cardiomyocytes.

METHODSPrimary neonatal rat cardiomyocytes were randomly divided into CRT siRNA group, control siRNA group, control group, Ang II+ CRT siRNA group, Ang II+ control siRNA group and Ang II group. The cell surface area, protein synthesis rate, mitochondrial membrane potential level, enzyme activities, and CRT expression were observed.

RESULTSCompared with those in the control group, the cell surface area and protein synthesis rate were both increased and mitochondrial membrane potential level and enzyme activities decreased in Ang II groups. CRT expression was significantly down-regulated in Ang II+ CRT siRNA group with increased cell surface area, protein synthesis rate, mitochondrial membrane potential level and enzyme activities as compared with those in Ang II+ control siRNA group.

CONCLUSIONAng II up-regulates CRT expression to induce mitochondrial injury, which may be an important mechanism of myocardial hypertrophy.

Angiotensin II ; pharmacology ; Animals ; Calreticulin ; metabolism ; Cardiomegaly ; Cells, Cultured ; Membrane Potential, Mitochondrial ; Mitochondria ; pathology ; Myocytes, Cardiac ; pathology ; Protein Biosynthesis ; RNA, Small Interfering ; Rats