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To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) and study their expressions in human microvascular endothelial cells (HMVECs).Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1(+)-HIF-1alpha-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1alpha mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting.DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1+-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one.The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.

To investigate the signal transduction mechanism underlying the effects of angiotensin II (Ang II) and aldosterone (Aldo) on the signal passageway of active protein-1 (AP-1).In vitro, Hepatic stellate cells (HSCs) of the line HSC-T6 were cultured and treated with Ang II or Aldo, the principal effector molecules of the renin-angiotensin-aldosterone system (RAAS) for 10, 30, 60, 120, and 180 minutes respectively. The protein expression of phospho-P42/44 was detected by Western blotting. In addition, HSC-T6 cells were preincubated for 60 min with U0126, an inhibitor of MAPK/ERK kinase, irbesartan, an AT-1 receptor blocker, N-acetylcysteine (NAC), antioxidant, angiotensin converting enzyme inhibitor (ACEI), or tumor necrosis factor alpha (TNFalpha) prior to exposure to Ang II or Aldo. Then the protein expression of phospho-P42/44 was measured by Western blotting. The DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, the mRNA expression of alpha1 (I) procollagen was detected.The levels of phopho-ERK1/2 protein increased after the treatment of Ang II and Aldo at all time points and both peaked 10 minutes after (both P0.01). The levels of phopho-ERK1/2 protein of the irbesartan + Ang II and U0126 + Ang II groups were significantly lower than that of the Ang II group (both P0.01). The level of phopho-ERK1/2 protein of the Ang II group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Ang II + TNFalpha group (P0.01). The level of phopho-ERK1/2 protein of the U0126 + Aldo group was significantly lower than that of the Aldo group (P0.01). The phopho-ERK1/2 protein level of the NAC + Aldo group was not significantly different from that of the Aldo group (P0.05). The phopho-ERK1/2 protein level of the Aldo group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Aldo + TNFalpha group (P0.01). The AP-1 DNA binding protein increased after the treatment of Ang II and peaked 30 min after. U0126, irbesartan, and NAC, as well as ACEUI, significantly inhibited the increased AP-1 DNA binding activity induced by Ang II. The AP-1 DNA binding protein increased after the treatment of Aldo and peaked twice, 30 min and 240 min after. U0126 and NAC significantly and NAC partly inhibited the increased AP-1 DNA binding activity induced by Aldo.Stimulation of HSC by Ang II and Aldo results in activation of AP-1 via ERK1/2 pathway leading to up-regulation of AP-1 target gene alpha1 (I) procollagen mRNA expression.

It is known that intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in liver fibrogenesis. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and on the platelet-derived growth factor-B (PDGF-B).In vitro, hepatic stellate cell (HSC)-T6 cell line was treated with Aldo for 10 min, 0.5 h, 1 h, 2 h and 3 h. Protein expression of phospho-p42/44 was detected by Western blot. In addition, HSC-T6 were preincubated for 1 h or not at all with U0126 (an inhibitor of the MAPK/ERK kinase), and antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expressions of phospho-p42/44 and PDGF-B were measured by Western blot. DNA biding activity of EGR-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of immunohistochemistry, expression of PDGF-B was detected.Aldo induced phospho-p42/44 expression could be abrogated by U0126; NAC did not inhibit phospho-p42/44 expression. Gel shift study showed that stimulation of HSC by Aldo markedly increased the EGR-1 DNA binding activity, which was abrogated by U0126, reaching a maximum at 60 minutes, and then declined progressively. NAC did not reduce the EGR-1 activity. Aldo increased the PDGF-B protein level in HSC, which was not attenuated by NAC and U0126.Stimulation of HSC by Aldo results in activation of EGR-1 via ERK1/2 pathway, leading to up-regulation of PDGF-B expression.

To investigate the changes and the effects of captopril on the renal blood flow and microvascular perfusion in dogs with acute cardiac insufficiency.Acute cardial insufficiency was induced by combining occlusion of the left anterior descending artery with right ventricular pacing in 12 mongrel dogs. The ascending aorta and left kidney were dissected and ultrasonic flow probes were placed on ascending aorta and renal artery to monitor cardiac output (CO) and renal blood flow (RBF). Contrast-enhanced ultrasound of the kidney was performed as CO was reduced to 25% (LCO25%) and 50% (LCO50%) from the basic measurement and microvascular flow velocity (beta), microvascular volume (A) and microvascular blood flow (renal cortex) were observed. After CO reduced to 50%, captopril 1 mg/kg and 2 mg/kg were injected successively and contrast-enhanced ultrasound of the kidney were performed again before and after injection.At baseline, CO, RBF, CXbeta (beta of renal cortex), A and A x beta were (1.46 +/- 0.16) ml/min, (107.5 +/- 35.7) ml/min, 1.39 +/- 0.14, 120.3 +/- 14.8 and 167.4 +/- 25.0, respectively. After the LCO25% was reached, RAF, CXbeta, A and A x beta decreased to (72.50 +/- 32.4) ml/min, 0.87 +/- 0.082, 117.6 +/- 13.1, and 102.6 +/- 15.5, respectively. The corresponding values after the LCO50% was reached were (44.1 +/- 17.2) ml/min, 0.61 +/- 0.039, 106.9 +/- 12.0, and 64.7 +/- 8.83, respectively. It is suggested that the volume of the renal microvasculature remained stable until the LCO50% was reached. When captopril 1 mg/kg and 2 mg/kg were injected successively at LCO50%, MAP decreased from (85.4 +/- 7.8) mm Hg to (78.7 +/- 7.3) mm Hg and to (69.1 +/- 6.3) mm Hg (P0.05), respectively, while CO increased from 0.73 +/- 0.084 to 0.83 +/- 0.065 and to 0.9 +/- 0.054 (P0.05), respectively. RBF increased from (44.1 +/- 17.2) ml/min to 60.3 +/- 17.8 and to 79.4 +/- 17.8 (P0.05), respectively. After captopril 1 mg/kg and 2 mg/kg were injected, the increased flow ratios with CO were 0.15 +/- 0.084 and 0.31 +/- 0.011, respectively, and with RBF were 0.29 +/- 089 and 0.522 +/- 0.040, respectively. The increased renal blood flow ratio was higher than that of CO after captopril was used. The corresponding increases were from 0.61 +/- 0.039 to 0.75 +/- 0.020 and to 0.86 +/- 0.027 for CX beta, from 106.9 +/- 11.9 to 115.4 +/- 11.1 and to 116.6 +/- 8.9 for A, from 64.7 +/- 8.83 to 87.0 +/- 8.6 and to 100.6 +/- 8.9 for A x beta, respectively.The renal microvasculature plays a role by keeping its volume stable in the protection against renal ischemia when acute cardiac output decreases slightly. The role of captopril to improve renal microvascular perfusion is independent of increased total cardiac output or increased systemic blood pressure.

In order to evaluate the clinical manifestations, management and outcome of childhood lung abscess, a retrospective chart review of 27 pediatric patients with International Classification of Diseases, Ninth Revision-Clinical Modification (ICD-9 CM) code of 503.1 (lung abscess) from August 1987 to August 2003 was conducted. Among the 27 patients (14 males and 13 females), 30% (8/27) were primary lung abscess and 70% (19/27) had underlying chronic diseases (secondary lung abscess). The predisposing factors of the primary group (n = 8) included 6 cases of respiratory tract infection, 1 with choking during swimming, and 1 with laceration wound. The underlying diseases in the secondary group (n = 19) included 10 cases of hematologic disorder (52%), 3 of congenital heart disease, 2 of central nervous system anomalies, and 1 each of hyperimmunoglobulin E syndrome, chronic lung disease, liver cirrhosis with fistula formation, and Swyer-James syndrome. Eleven patients (41%) underwent diagnostic tapping, including echo-guided aspiration (10 cases) and computed tomography-guided percutaneous needle aspiration (1 case). Positive yield rate from aspiration of lung abscess was 63.6% (7/11). Surgical intervention was performed in 8 (42%) of the secondary group and in 1 patient from the primary group. The pathogens were identified in 11 patients (41%): 5 with oral flora, 2 with Staphylococcus aureus plus other pathogens, 1 with S. aureus alone, 1 with Pseudomonas aeruginosa plus Proteus mirabilis, 1 with P. aeruginosa alone, and 1 with Aspergillus. The average duration of parenteral antibiotic use was 40 days. Five cases (18.5%) died due to poor control of the underlying diseases, and 4 of the patients (15%) had sequelae (2 with bronchiectasis and 2 with lung fibrosis). Seventy percent of lung abscess occurred in children with underlying medical conditions. Early percutaneous aspiration has an important role in identification of pathogens. Oral anaerobes and S. aureus are the core pathogens in primary lung abscess and gram-negative pathogens should also be considered in secondary lung abscess.

To investigate the changes in the expressions of ATP-binding cassette transporter A1 (ABCA1), intercellular cell adhesion molecule type 1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) at mRNA and protein levels after the treatment of the vascular endothelial ECV304 cells with oxidized low-density lipoprotein (Ox-LDL) and 8-Br-cAMP, thereby to explore possible mechanisms by which ABCA1 affects the formation of atherosclerosis (AS).Resuscitated and cultured ECV304 cells were incubated in serum-free medium for 12 h to induce the quiescence phase of growth, which were subsequently treated with Ox-LDL (30 ng/ml) and 8-Br-cAMP (0.5 mmol/L) respectively for 3, 6, 12, and 24 h. After the cells were harvested at the specified time points, the mRNA and protein levels of ABCA1, ICAM-1 and MCP-1 were detected by RT-PCR and Western blotting, respectively.The mRNA and protein expression levels of ABCA1, ICAM-1 and MCP-1 all increased after treatments with Ox-LDL and 8-Br-cAMP. The highest expression of ICAM-1 occurred in cells with a 12-hour treatment, and those of ABCA1 and MCP-1 occurred following 6-hour incubation with Ox-LDL. The expression peaks of ABCA1, ICAM-1 and MCP-1 all took place after 6-hour incubation with 8-Br-cAMP.The mRNA and protein expressions of ICAM-1 and MCP-1 in vascular endothelial ECV304 cells increase in response to Ox-LDL treatment, in the event of which ABCA1 is also up-regulated to offer protective effects against AS. cAMP not only enhances the expression of ABCA1 but also those of ICAM-1 and MCP-1.

To observe the effects of recombinant hk2a, a novel neurotoxin from the sea anemone Anthopleura sp. on the left ventricular function in New Zealand rabbits with chronic congestive heart failure(CCHF).CHF model was established using New Zealand rabbits by abdominal aortic coarctation. The left ventricular ejection fraction (LVEF) was measured by Acuson ultrasound systems (Sequoia 512) at 0, 5, 15, 30, 45 and 60 min respectively after intravenous injection with a single dose of recombinant hk2a (20 microg/kg x b x w.). The response of the rabbits to hk2a treatment was observed in comparison with that of the control group.Recombinant hk2a immediately and significantly increased the left ventricular ejection fraction of the rabbit models of CCHF after intravenous injection (P0.05), and its effects were maintained for more than 1 h without affecting the heart rate.recombinant hk2a significantly increases the left ventricular ejection fraction in New Zealand rabbits with CCHF.

The aim of the present study was to determine the effects of angiotensin-converting enzyme inhibitor, perindopril, on the progression of rat hepatic fibrosis induced by CCl4.Male wistar rats weighting about 250g were treated with perindopril (2mg/kg, daily gavage), except for model group and control group. After 4, 6 weeks, morphological examination was based on microscopy. RT-PCR was utilized to detect gene expression of angiotensin II type 1 receptor (AT1 receptor) in the liver. Meanwhile, the protein expressions of AT1 receptor, transforming growth factor beta 1 (TGF-beta1) and platelet-derived growth factor-BB (PDGF-BB) in liver tissue were examined by Western blot. The activity of matrix metalloproteinase-2 (MMP-2) was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radio-immunity technique.RT-PCR and Western blot revealed that there was a up-regulation in AT1 receptor expression in model group compared with control group. Perindopril treatment significantly reduced mean fibrosis score, messenger RNA and protein levels of AT1 receptor, protein levels of TGF-beta1 and PDGF-BB, Serum levels of HA and LN, and MMP-2 activity.These results suggest that angiotensin II may play an important role in fibrosis of liver. Perindopril may have a inhibiting effect on CCl4-induced hepatic fibrogenesis of rat.

To construct the eukaryotic expression vector for human hypoxia-inducible factor-1alpha (HIF-1alpha) gene and examine its expression.Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on the total RNA extracted from HT29 cells to obtain the cDNA of HIF-1alpha, which was inserted into T vector pUC18. DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1+ identified by endonuclease digestion. This recombinant vector was transfected into HEK293 cells by means of liposome and its expression examined.The amplified products were confirmed as the cDNA of HIF-1alpha by DNA sequencing, and pcDNA3.1+ -HIF-1alpha obtained was verified by endonuclease digestion, being capable of expression in HEK293 cells.We have successfully constructed the eukaryotic expression vector for HIF-1alpha, which can be expressed in HEK293 cells.

The aim of the present study was to determine the effects of angiotensin-converting enzyme inhibitor (ACE-I) and angiotensin II type 1 receptor (AT-1 receptor) blocker on the progression of rat hepatic fibrosis induced by CCl4.60 male wistar rats weighting about 250 g were divided into 4 groups. Model group (Mo): The rats were injected with 40% CCl(4) 0.25 ml/100 g subcutaneously three times a week. Perindopril group (Pe): The rats were injected with 40% CCl(4). Perindopril, equivalent to 2 mg x kg(-1) x d(-1), was given i.g. Losartan group (Lo): The rats were injected with 40% CCl(4). Losartan, equivalent to 50 mg x kg(-1) x d(-1), was given i.g. Control group (Nc): the rats were injected with olive oil only. After 4, 6 weeks, morphological examination was based on microscopy. RT-PCR was utilized to detect gene expression of AT-1 receptor in the liver. Meanwhile, the protein expressions of AT-1 receptor, TGF-beta1 and PDGF-BB in liver tissue were examined by Western blot. The activity of matrix metalloproteinase-2 (MMP-2) was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radioimmunoassays.RT-PCR and Western blot revealed that there was a up-regulation in AT-1 receptor expression in model group compared with control group. Both of perindopril and losartan treatment significantly reduced mean fibrosis score, messenger RNA and protein levels of AT1 receptor, protein levels of TGF-beta1 and PDGF-BB, Serum levels of HA and LN, and MMP-2 activity.These results suggest that angiotensin IImay play an important role in fibrosis of liver. Perindopril and losartan may have inhibiting effects on CCl(4)-induced hepatic fibrogenesis of rat.

To investigate the value of the echocardiographic observation of the left ventricular hypertrophy (LVH) in diagnosing myocardial microvascular damage in patients with essential hypertension (EH).After intravenous injection with Quanfuxian (a contrast agent consisting of albumin and C3F8 prepared by Nanfang Hospital), the values of A (the maximum number of microbubbles accumulating in the local tissues for assessing the density of local microvessels), beta (the filling velocity of contrast agent for evaluating local blood flow velocity) and A x beta (the product of A and beta for estimating local myocardial blood flow) at rest and after dipyridamole injection were measured by intermittent harmonic imaging with myocardial contrast echocardiography (MCE). The ratios of A and beta along with the microvascular coronary flow velocity reserve (CFVR) were also calculated.Compared with the control group, the rest values of A, beta and A.beta in EH patients were higher, especially in those with LVH. After dipyridamole injection, the values of A, beta, A x beta and the ratios of A and beta, along with CFVR as well, were significantly lowered (P0.01), and the reductions were especially obvious in LVH cases. As the hypertension exacerbated, the values of A and A x beta tended to increase in positive correlation with systolic and diastolic blood pressure (P0.01), while the ratio of A and CFVR were decreased, the latter was inversely correlated with diastolic blood pressure (r=-0.55, P0.01). Positive correlations were noted of the values of A and A x beta with the left ventricular mass and left ventricular mass index (P0.01).EH patients, especially those with LVH, are characterized by increased rest myocardial microvascular blood flow, impaired myocardial microvascular flow reserve and endothelium independent vasodilation relaxing ability, and reduced capillary density, and these conditions tend exacerbate as the disease worsens. Microvascular function impairment should be suspected when complication of LVH arises in the EH patients. As a new important noninvasive technique, MCE can be a promising modality for diagnosing microvascular disease in essential hypertension.

To investigate the expression profile of angiotensin type 1 receptor (AT1R) mRNA in fibrotic and normal liver tissues.Semi-quantitative RT-PCR was used to detect the expression of AT1R mRNA in 18 specimens of fibrotic liver tissues adjacent to liver cancers and 12 normal liver tissue specimens. Specific AT1R product and GAPDH product used as internal control were both amplified by RT-PCR, and the ratio of their integrated optical densities calculated to estimate the relative quantity of AT1R mRNA expression.The relative mRNA expression of AT1R in fibrotic liver tissues was significantly higher than that of normal liver tissues (1.27+/-0.45 vs 0.71+/-0.21, P0.01).AT1R may play an important role in the development of hepatic fibrosis.

To investigate the effects of angiotensin II (AngII) and AT1a blocker losartan on growth and proliferation of rat hepatic stellate cells (HSCs).Rat HSCs were isolated, cultured and identified, followed by incubation with AngII or losartan at different concentrations. The cell growth and proliferation were assessed via cell counting and MTT assay, and the effects of the agents on HSC DNA synthesis evaluated by way of (3)H-thymidine incorporation ((3)H-TDR).AngII (1 x 10(-9) to 1 x 10(-7) mol/L) stimulated HSC proliferation as demonstrated by cell counting, MTT assay and thymidine incorporation test (P0.05), but such effect was not observed at lower doses (1 x 10(-9) mol/L). Losartan had significant inhibitory effect on HSC growth at the concentration of 1 x 10(-8) to 1 x 10(-6) mol/L (P0.05), but not at lower doses (1 x 10(-8) mol/L). Co-stimulation of the cells with losartan and AngII did not result in a significant increase in cell number as compared with the control group (P0.05).Rapid proliferation of rat HSCs occurs in response to AngII treatment, but is inhibited after AT1a receptor is blocked with the antagonist losartan.

To clone and construct the expression vector for human vascular endothelial growth factor (VEGF) genes.Total RNAs were extracted from human lung tissue of a 4-month-old fetus and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with the amplified products cloned into pMD18-T vector. Sequence analysis was performed before the amplified products were cloned into the expression plasmid pcDNA3.1-, the recombinant of which was verified by endonuclease digestion.After RT-PCR using a pair of primers (sense -21/7 bp and antisense 554/576 bp), two bands were identified. The band (487 bp) shorter in length was confirmed as VEGF121 (with the full length of VEGF121 being 444 bp) while the longer band (619 bp) was normal VEGF165 (with the full length of VEGF165 being 576 bp). Interestingly, another slightly longer VEGF165 nucleotide sequence was identified by sequencing analysis, which featured an unique 20 bp insertion precisely between exon 3 and exon 4 from the first ATG of human VEGF165 cDNA. The 20 bp insert was identified as the retaining intron 3 terminal nucleotides containing the splicing signal, which caused frame shift mutation in the reading frame and could probably give rise to a short polypeptides consisting of only 97 amino acid residuals due to the early appearance of stop code UAG in the middle of exon 4.We have successfully constructed the expression vector for VEGF121 and VEGF165 genes, and a new possible alternative splicing isoform of VEGF is identified in normal fetus whose molecular mechanism and physiological function needs further investigation.

To construct eukaryotic expression vector of thrombospondin-1 type I repeat sequences.Thrombospondin-1 type I repeat sequence gene was amplified from human fetal lung tissue by reverse transcriptase-PCR (RT-PCR) to construct both recombinant clone vector and expression vector through coupling reaction, followed by transforming these vectors into E.coli DH5alpha. The positive clones were selected for verification by double enzyme digestion and sequence analysis.The expected amplification product, thrombospondin-1 type I repeat gene sequence, was acquired by RT-PCR, which had a consistency up to 99% with the sequence from the GenBank.The eukaryotic expression vector PcDNA3.1(+)/TSP-1 type I repeat sequence was successfully constructed.