Tissue-type Plasminogen Activator Gene Research Articles

Preventative effects of the albumin nano tissue-type plasminogen activator gene plasmid ultrasound microbubble carrier system on thrombosis after cardiac valve replacement

Mechanical valve replacement is a new method for treating rheumatic heart valve disease. However, patients require long-term anticoagulation therapy after surgery, thus reducing their use of anticoagulants, making the prevention of postoperative thrombosis of great research interest. We examined the potential preventative effects of the albumin nano tissue plasminogen activator (tPA) gene plasmid ultrasound microbubble carrier system on thrombosis. The average particle size of the nano tPA gene plasmid was 131.8 nm, the surface zeta potential was 30.65±40.34 mV, and the encapsulation efficiency was 76.56%, all meeting experimental requirements. One month after gene integration, the tPA content of group B had increased beyond pre-integration levels and above post-integration levels of group A. After valve replacement operations, the venous blood D-Dimer (D-D) content of group B increased significantly, whereas the venous blood D-D content of group A decreased. In addition, at 1, 2, and 4 weeks after operation, the venous blood D-D content of group B was greater than that of group A. These results suggest that the prepared albumin nano tPA gene plasmid ultrasound microbubble vector system can prevent thrombosis after heart valve replacement.

Retrovirus-Mediated Transfection of the Tissue-type Plasminogen Activator Gene Results in Increased Thrombolysis of Blood Clots.

Tissue-type plasminogen activator (tPA) is involved in the lysis of blood clots. In this study, we attempted to target thrombolysis and enhance blood clot lysis by generating a construct (pLEGFP-N1-tPA) to integrate tPA gene into the genome of different cell lines. pLEGFP-N1-tPA construct was generated and used to target the tPA gene in different cell lines. The thrombolytic effects mediated by the supernatant from transfected HeLa cells and Linx cells were assessed using plasma thrombus plates. Furthermore, enhanced green fluorescent protein (EGFP), which was fused to the tPA gene in the pLEGFP-N1-tPA construct, was analyzed under the fluorescent microscope to assess tPA localization. We also monitored tPA activity and expression in the transfected cell lines. As part of the study, we successfully generated the pLEGFP-N1-tPA construct. The sequence of this construct was verified and the construct was subsequently used to generate the PT67/pLEGFP-N1-tPA cell line. The pLEGFP-N1-tPA construct was also used to transfect HeLa cells and Linx cells. We observed that supernatants from transfected cells were capable of lysing thrombi. In addition, tPA activity and tPA concentration were elevated in the latter supernatants and tPA was rapidly and stably expressed in the transfected cell lines. These results reveal a potentially important thrombolytic role for tPA-targeted gene therapy following cardiac valve replacement.

Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression.

Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.

The Tissue Fibrinolytic System Contributes to the Induction of Macrophage Function and CCL3 during Bone Repair in Mice

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg –/–), urokinase-type plasminogen activator (uPA –/–) or tissue-type plasminogen activator (tPA –/–) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA –/– mice until day 6, compared with wild-type (uPA +/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA –/– mice at the bone injury site on day 4, compared with those in uPA +/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA –/– and Plg –/–, but not in tPA –/– mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1β, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA –/– and Plg –/–, but not in tPA –/– mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA +/+, but not in uPA –/– mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

Effects of Tissue-Type Plasminogen Site-Specific Transgene in Gelatin-Coated Dacron on the Fibrinolysis Activity of Rabbit Left Atrium.

To investigate the effects of tissue-type plasminogen activator (tPA) gene transfer with left-atrium local positioning on the fibrinolytic activity of rabbit left atrial blood. A total of 48 rabbits were randomly divided into 3 groups (n = 16): gene therapy, vector control, and blank control groups. Each group was divided into 2 subgroups (8 rabbits in each subgroup) according to the sacrifice time on the postoperative 3(rd) and 14(th) days. The tPA mRNA transcriptional level and exogenous tPA protein expression within regional myocardial tissues of the left atrium were detected on the postoperative 3(rd) and 14(th) days. After excluding the animals that died, 6 samples of each subgroup were randomly selected for the statistics (n = 6). The tPA activities in rabbit left atrial blood and peripheral blood were also detected. The tPAmRNA and tPA protein expressions within regional myocardial tissues were detected on the postoperative 3(rd) and 14(th) days. The tPA activity in left atrial blood in the gene therapy group was higher than the tPA activity of other groups (p < 0.02). No significant differences were observed in the tPA activity of peripheral blood among the 3 groups before surgery. A gelatin-coated Dacron piece, which carried the tPA gene, was implanted in the left atrial appendage. The gelatin-coated Dacron piece could express and secrete tPA proteins in the region, thus enhancing the fibrinolytic activity of left atrial blood. Fibrinolytic activity; Gelatin coating; Gene; Left atrium; Tissue-type plasminogen activator.

Homoplasmy stability of transplastomic tobacco plants (Nicotiana tabacum CV. Xhanti) containing human tissue-type plasminogen activator (K2S form) gene in T1 generation

The rates of recombinant protein in nuclear-transformed plants are often less than 1% of total soluble proteins. As the plant plastid is highly polyploidy, plastid transformation can lead to high-level production of recombinant protein. In addition, plastid transformation has several other advantages such as prevention of gene escape that has a high importance in molecular farming. Tissue-type plasminogen activator (tPA) is an important protein that is used to treating clots in cardiovascular diseases. Thus, production of tPA protein in plant system was considered. The tPA (K2S form) gene was transferred to tobacco chloroplast genomes. In this study, we analyzed expression and stability of tPA gene in transplastomic tobacco plants in T1 generation. The presence of tPA gene in transplastomic plants was confirmed with specific primer by PCR analysis. Homoplasmy, gene expression, and protein assay were confirmed with southern blot, RT-PCR, western blotting and ELISA analysis. Results showed that the tPA gene in T1 generation of transplastomic tobacco plants is stable and is expressed. In addition, the maximum amount of tPA protein was estimated up to 0.13% of total soluble protein.

Cardiac-targeting transfection of tissue-type plasminogen activator gene to prevent the graft thrombosis and vascular anastomotic restenosis after coronary bypass

AimTo observe the tissue-type plasminogen activator gene (t-PA) plasmid packaged with albumin nanoparticles crosslinked to albumin ultrasound microbubbles for targeting transfection to myocardium to prevent the graft thrombosis and vascular anastomotic restenosis after coronary bypass. MethodsA dog model of coronary bypass using the autoallergic saphenous vein as the graft was made. A highly expressive t-PA gene plasmid packaged with albumin nanoparticles crosslinked to albumin ultrasound microbubbles was constructed. Targeting myocardial transfection was performed with this gene vector under the aid of therapeutic ultrasound(1MHz, 1.5 w/cm2, 6minutes, intravenously) after the bypass. The expression of t-PA in myocardium was detected with a multiclonal antibody to t-PA by the indirect immunohistochemical method. Venous blood t-PA and D-dimer contents were tested before and 1, 2 and 4weeks after the operation. The effects of this gene vector on thrombosis of the grafts and the coronary intimal hyperplasia around the anastomotic stoma were observed using a routine pathological examination, a morphometry for intimal thickness and area and the immuno-histochemical stain with a monoclonal antibody to PCNA for estimating the intimal SMC proliferation. ResultsThe effective expression of t-PA protein by myocardium was obtained, followed by the persistent raises of blood t-PA and D-dimer 1, 2 and 4weeks after the transfection. Thrombosis of the grafts was successfully restrained. The expression of PCNA by coronary intimal vSMCs and intimal hyperplasia were remarkablely reduced. ConclusionThis t-PA gene targeting vector could be used to prevent the dog thrombosis, which provided the experimental identification for prevention on human thrombotic diseases.

Cloning and expression of hybrid streptokinase towards clot-specific activity

Streptokinase (SK) is a thrombolytic agent that is widely used to treat myocardial infarction and pulmonary embolism. The lack of fibrin specificity of SK for the clot lysis is one of the limitations of SK. In this study, we have incorporated the finger and Kringle 2 domains from the human tissue type plasminogen activator gene (t-PA) at the 5′ end of the SK gene. These domains are responsible for specific binding to fibrin. We have used the pRSETB vector in an attempt to express the hybrid streptokinase possessing specificity for fibrin. On this regard, three hybrid streptokinase were constructed and expressed in Escherichia coli BL21 (DE3): the finger domain with SK (FSK), the Kringle 2 domain with SK (KSK) and the finger domain+Kringle 2 with SK (FKSK). The activities of the hybrid SKs were assessed by caseinolytic assay and clot lysis assay. All hybrid SKs were found to activate plasminogen in the caseinolytic plate assay. In the clot lysis assay, KSK and FSK were able to dissolute human blood and artificial clots in a fibrin-dependent manner unlike the SK and FKSK proteins.

Ultrasound-targeted transfection of tissue-type plasminogen activator gene carried by albumin nanoparticles to dog myocardium to prevent thrombosis after heart mechanical valve replacement

BackgroundThere are more than 300,000 prosthetic heart valve replacements each year worldwide. These patients are faced with a higher risk of thromboembolic events after heart valve surgery and long-term or even life-long anticoagulative and antiplatelet therapies are necessary. Some severe complications such as hemorrhaging or rebound thrombosis can occur when the therapy ceases. Tissue-type plasminogen activator (t-PA) is a thrombolytic agent. One of the best strategies is gene therapy, which offers a local high expression of t-PA over a prolonged time period to avoid both systemic hemorrhaging and local rebound thrombosis. There are some issues with t-PA that need to be addressed: currently, there is no up-to-date report on how the t-PA gene targets the heart in vivo and the gene vector for t-PA needs to be determined.AimsTo fabricate an albumin nano-t-PA gene ultrasound-targeted agent and investigate its targeting effect on prevention of thrombosis after heart mechanic valve replacement under therapeutic ultrasound.MethodsA dog model of mechanical tricuspid valve replacement was constructed. A highly expressive t-PA gene plasmid was constructed and packaged by nanoparticles prepared with bovine serum albumin. This nanopackaged t-PA gene plasmid was further cross-linked to ultrasonic microbubbles prepared with sucrose and bovine serum albumin to form the ultrasonic-targeted agent for t-PA gene transfection. The agent was given intravenously followed by a therapeutic ultrasound treatment (1 MHz, 1.5 w/cm2, 10 minutes) of the heart soon after valve replacement had been performed. The expression of t-PA in myocardium was detected with multiclonal antibodies to t-PA by the indirect immunohistochemical method. Venous blood t-PA and D-dimer contents were tested before and 1, 2, 4, and 8 weeks after the operation.ResultsThe high expression of t-PA could be seen in myocardium with increases in blood t-PA and D-dimer contents and thrombosis was prevented 8 weeks after operation.ConclusionWe successfully fabricated an albumin nano-t-PA gene ultrasound-targeted agent that could prevent dog thrombosis after mechanical heart valve replacement. Our study provides an experimental basis for prevention of human thrombosis-related diseases.

Circadian mRNA expression of coagulation and fibrinolytic factors is organ-dependently disrupted in aged mice

To evaluate the effects of aging on the circadian gene expression of coagulation and fibrinolytic factors in the mouse tissues, we examined temporal mRNA expression profiles of plasminogen activator inhibitor-1 ( PAI-1), tissue-type plasminogen activator ( tPA), tissue factor ( TF), and thrombomodulin ( TM) genes together with circadian clock genes in the brains, hearts and livers of young (5 weeks old) and aged (15 months old) mice. Cardiac mRNA expression of β-myosin heavy chain ( β-MHC), a molecular marker of cardiac hypertrophy, was obviously increased in the aged mice. Rhythmic expression of the clock genes mPer2 and BMAL1 in these organs was almost identical between young and aged mice, whereas that of PAI-1, TF and TM mRNAs and of clock-controlled genes such as DBP and Dec1 were damped to low levels in the livers of aged mice. Expression levels of tPA mRNA were significantly decreased and those of TF were significantly elevated throughout the day in the brain of aged mice. Expression levels of PAI-1 in the heart of aged mice were continuously elevated over 2-fold the peak levels of young mice throughout the day. However, day/night fluctuations in plasma PAI-1 levels were unaffected by aging. Aging tissue- and time-dependently affects the mRNA expression of coagulation and fibrinolytic factors. Aging-dependent constitutive PAI-1 induction in the heart might be a risk factor for cardiovascular diseases that is independent of plasma PAI-1 levels.

Preparation of ultrasound microbubbles crosslinked to albumin nanoparticles packaged with tissue-type plasminogen activator gene plasmid and method of in vivo transfection.

AimsTo observe the effect of constructed ultrasound microbubble crosslinked to albium nanoparticles packaged with tissue-type plasminogen activator (tPA) gene plasmid on the in vivo transfection.MethodsThe rabbits were chosen for all experiments. A highly expressive gene plasmid for tPA was constructed and packaged into a prepared nanoparticle with bovine serum albumin (BSA). This albium nanoparticle packaged with tPA gene plasmid was crosslinked to an ultrasound microbubble prepared with BSA and sucrose to form a nano-targeting vector system for tPA gene transfection. The transfection and effective expression of tPA in heart, liver, leg skeletal muscle and the cervical rib were detected with polyclonal antibodies to tPA using immunohistochemical method; the tPA level and D-dimer content of blood were also tested.ResultsThe expression of tPA could be seen in the tissues mentioned above, with the increase in blood tPA level and D-dimer content from 0.20 ± 0.05 µg/L and 81.76 ± 9.84 µg/L before the operation, to the higher levels of 0.44 ± 0.05 µg/L and 669.28 ± 97.74 µg/L after transfection.ConclusionThe nano-targeting vector system for tPA gene was contructed successfully. This provides a new theory and experimental method for the nano-targeted transgene.

Allelic imbalance of tissue-type plasminogen activator (t-PA) gene expression in human brain tissue

We have identified a single-nucleotide polymorphism (SNP) in the t-PA enhancer (-7351C>T), which is associated with endothelial t-PA release in vivo. In vitro studies demonstrated that this SNP is functional at the level of transcription. In the brain, t-PA has been implicated in both physiologic and pathophysiologic processes. The aim of the present study was to examine the effect of the t-PA -7351C>T SNP on t-PA gene expression in human brain tissue. Allelic mRNA expression was measured in heterozygous post-mortem brain tissues using quantitative TaqMan genotyping assay. Protein-DNA interactions were assessed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Significantly higher levels of t-PA mRNA were generated from chromosomes that harboured the wild-type -7351C allele, as compared to those generated from the mutant T allele (for the hippocampus, C to T allelic ratio of ~1.3, p=0.010, n=12; and for the cortex, C to T allelic ratio of ~1.2, p=0.017, n=12). EMSA showed reduced neuronal and astrocytic nuclear protein binding affinity to the T allele, and identified Sp1 and Sp3 as the major transcription factors that bound to the -7351 site. ChIP analyses confirmed that Sp1 recognises this site in intact cells. In conclusion, the t-PA -7351C>T SNP affects t-PA gene expression in human brain tissue. This finding might have clinical implications for neurological conditions associated with enhanced t-PA levels, such as in the acute phase of cerebral ischaemia, and also for stroke recovery.

Study on Construction of Nano tPA Plasmid to Prevent Thrombosis After Mechanical Valve Replacement in Dogs

Mechanical valve is inclined to induce thrombosis. We intend to elucidate the transfection of nano tissue-type plasminogen activator (tPA) gene plasmid to prevent thrombosis after tricuspid mechanical valve replacement in dogs. A dog model of mechanical tricuspid valve replacement was constructed. A constructed chitosan nano tPA gene plasmid was used to transfect the dog cardiocytes at the same time of tricuspid valve replacement. The effect of this gene on the survival of dogs, anticoagulation (prothrombin time, INR and D-dimer contents, thrombosis in heart), and the expressions of tPA were observed. Nano tPA gene plasmid was successfully constructed and transfected into cardiocytes. The gene could significantly increase the survival of animals and the contents of D-dimer (P < 0.01), and could prevent thrombus formation on mechanical valves, but there was no significant difference of prothrombin time and INR between two groups (P > 0.05). The constructed nano tPA gene plasmid could prevent thrombus formation after mechanical valve replacement, which provides a clue for clinical use.

The Species-Specific Differences in the cAMP Regulation of the Tissue-Type Plasminogen Activator Gene between Rat, Mouse and Human is Caused by a One-Nucleotide Substitution in the cAMP-Responsive Element of the Promoters

In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.

Gene Response in Endothelial Cells Cultured on Engineered Surfaces Is Regulated by Shear Stress

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.

A complex, tissue-specific role for plasmin and its regulators in modulating fibrogenic activity

it has long been known that tissue injury often leads to the infiltration of interstitial spaces with plasma, resulting in the polymerization of fibrin as a provisional matrix to support the migration of cells that mediate healing process. When all goes well, during the course of tissue repair the

HDAC1 Acetylation Is Linked to Progressive Modulation of Steroid Receptor-Induced Gene Transcription

Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.

Experimental study of tissue-type plasminogen activator gene to prevent vein grafts stenosis

The effects of in vivo local expression of recombined human tissue-type plasminogen activator (t-PA) gene on the thrombosis and neointima formation of vein grafts were explored. Jugular vein-to-artery bypass grafting was performed on 72 New Zealand white rabbits. The rabbits were divided into 3 groups according to the different processing methods: transfected t-PA gene group (n = 24), vector group (n = 24) and blank control group (n = 24). Samples of vein grafts were harvested at different time points after surgery. The expression of t-PA gene in vein graft was detected by RT-PCR and the synthesis of t-PA protein by Western-Blot assay. The t-PA activity was measured by chromogenic substrate assay. The Cr51 labeled platelets accumulation in vein grafts was counted. The histopathological changes were compared in intima hyperplasia index among the three groups after operation. The results showed that at the 2nd, 5th, 14th and 28th day after operation, RT-PCR and Western-blot confirmed the expression of t-PA mRNA and protein at the site of gene transfer. The t-PA activity detected on the 2nd, 5th, 14th and 28th day in experimental group was 370.63 +/- 59.44, 344.13 +/- 48.47, 252.87 +/- 51.80 and 161.75 +/- 68.94 U/g respectively, and disappeared on the 60th day and undetected in the control groups. The number of platelets accumulated in the vein grafts in gene group, vector group and blank control group was (85.04 +/- 21.58) 10(6), (225.87 +/- 85.13) 10(6) and (211.7 +/- 78.02) 10(6) respectively. The number of platelets accumulated in gene group was significantly fewer than that in the control groups. Morphometric analysis revealed that intimal hyperplasia was markedly reduced in the t-PA gene group as compared with that in the control groups. It was suggested that the local expression of t-PA gene in vein graft significantly inhibited the accumulation of platelets, thrombosis and concomitant intimal hyperplasia, by which stenosis of bypass graft could be prevented effectively.

Degradation of maternal mRNA in mouse embryos: Selective degradation of specific mRNAs after fertilization

During oogenesis, mRNA is actively transcribed and accumulated in growing oocytes, but this transcription stops before the oocytes grow to their full size. The accumulated maternal mRNA is used for protein synthesis in the oocytes during meiotic maturation and even in the embryos to sustain development after fertilization. Therefore, the degradation of accumulated maternal mRNA starts during meiotic maturation, but its rate is slow. Nevertheless, some mRNA species should rapidly degrade after fertilization if they encode proteins that play a role in specific events during meiosis and are detrimental for development after fertilization. In this study, to identify the selective degradation of maternal transcripts after fertilization, we sought mRNAs that are degraded in the early hours after fertilization by constructing an oocyte cDNA library after subtracting the cDNA of embryos at the mid one-cell stage. H1oo, c-mos, tPA (tissue type plasminogen activator gene), and Gdf9 were identified as genes whose transcripts undergo rapid degradation after fertilization. RT-PCR analysis showed that none of these transcripts was expressed during pre-implantation development once they were eliminated, suggesting that the mRNA species that are required for oogenesis, but not for early pre-implantation development, are degraded rapidly after fertilization. Microinjection of chimeric mRNAs in which the coding and 3'-untranslated regions (3'UTR) were exchanged between c-mos and hypoxanthine phosphoribosyltransferase mRNAs revealed that the 3'UTR plays a role in the rapid degradation that occurs after fertilization. Cytoplasmic polyadenylation elements (CPEs) was found near a poly(A) signal in the 3'UTR of all the mRNA species identified as rapidly degrading mRNA. The mechanism for the selective degradation is discussed, in relation to its biological significance.

Effects of platelet derived growth factor antisense oligodeoxynucleotides and tissue-type plasminogen activator gene transfection on inhibition of intimal proliferation

To observe the effects of co-transfection of platelet derived growth factor antisense oligodeoxynucleotides (PDGF-AODN) and tissue-type plasminogen activator (tPA) gene on inhibition of intimal proliferation of auto-transplantion artery. One hundred male New Zealand rabbits were randomly divided into four groups (25 in each): Group A (control group), Group B (PDGF-AODN transfection group), Group C (tPA gene transfection group) and Group D (PDGF-AODN and tPA co-transfection group). The left and right external iliac arteries were transplanted reciprocally. The transplanted arteries were respectively soaked in PDGF-AODN, pBudCE4.1/tPA and PDGF-AODN plus pBudCE4.1/tPA solution about 15 minute before transplantation. The rabbits were sacrificed at 3d, 1w, 2w, 4w and 8w after operation. The specimens were harvested for pathologic examination, electron microscopy, chromogenic substrate test, 3H-TdR incorporation test and immunohistochemical staining. The scan electron microscopy showed that there were a few thrombocytes on vas-wall of Group C and D without thrombus, whereas there were abundant thrombocytes and thrombus forming on vas-wall of Group A and B. The intimal area, stenosis ratio of transplanted artery, 3H-TdR incorporation,the number of PDGF positive cell in Group D were significantly less than those in Group A (P<0.01),Group B and Group C (both P<0.05). The activity of tPA gene products in transplanted vas-wall of Group D was significantly higher than that of Group A (P<0.01). Local co-transfection of PDGF-AODN and tPA gene can effectively inhibit the proliferation of vascular smooth muscle cells, hyperplasia of intima and restenosis of transplanted artery.