tPA mRNA Research Articles

Modulation of the expression of tissue plasminogen activator and its inhibitor by hypoxia in human peritoneal and adhesion fibroblasts.

Objective: While the pathophysiology leading to adhesion development remains poorly understood, a pivotal role has been ascribed to the ability of plasminogen activator activity (PAA) of the mesothelial cells lining the peritoneal cavity to limit adhesion development. Injury to the peritoneum, with loss of the mesothelial cells and reduction of PAA exposes underlying fibroblasts, and frequently results in adhesions.Design: The objective of this study was to determine whether normal peritoneal and adhesion fibroblasts express tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-I) and whether their expression is regulated by oxygen.Materials/Methods: We have utilized the multiplex RT/PCR technique to determine the effect of hypoxia on the expression of tPA and PAI-I in normal peritoneal (NPF) and adhesion (ADF) fibroblasts. Primary cultures of NPF and ADF were exposed to hypoxia (2% O2) for 24 hrs. Total RNA was extracted from cells and subjected to multiplex RT/PCR to quantitate relative change in mRNA levels of tPA and PAI-I in response to hypoxia treatment.Results: Basal tPA mRNA levels were 45% higher in NPF than ADF. Hypoxia decreased tPA in both NPF and ADF by 70% and 95%, respectively. Basal PAI-I mRNA levels were 64% higher in ADF than NPF. Hypoxia increased PAI-I mRNA levels by 67% and 53% in NPF and ADF, respectively.Conclusions: PAA of the peritoneum does not solely reside in the mesothelial cells, but also exists within fibroblasts, thus providing the potential to resolve postoperative fibrinous collections even at sites at which the mesothelial cells have been removed or destroyed. Furthermore, PAA in fibroblasts is regulated by oxygen; creation of a hypoxic state (as occurs with suture ligation, electrosurgery, etc.) markedly attenuates PAA, thereby leading to adhesion development. Objective: While the pathophysiology leading to adhesion development remains poorly understood, a pivotal role has been ascribed to the ability of plasminogen activator activity (PAA) of the mesothelial cells lining the peritoneal cavity to limit adhesion development. Injury to the peritoneum, with loss of the mesothelial cells and reduction of PAA exposes underlying fibroblasts, and frequently results in adhesions. Design: The objective of this study was to determine whether normal peritoneal and adhesion fibroblasts express tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-I) and whether their expression is regulated by oxygen. Materials/Methods: We have utilized the multiplex RT/PCR technique to determine the effect of hypoxia on the expression of tPA and PAI-I in normal peritoneal (NPF) and adhesion (ADF) fibroblasts. Primary cultures of NPF and ADF were exposed to hypoxia (2% O2) for 24 hrs. Total RNA was extracted from cells and subjected to multiplex RT/PCR to quantitate relative change in mRNA levels of tPA and PAI-I in response to hypoxia treatment. Results: Basal tPA mRNA levels were 45% higher in NPF than ADF. Hypoxia decreased tPA in both NPF and ADF by 70% and 95%, respectively. Basal PAI-I mRNA levels were 64% higher in ADF than NPF. Hypoxia increased PAI-I mRNA levels by 67% and 53% in NPF and ADF, respectively. Conclusions: PAA of the peritoneum does not solely reside in the mesothelial cells, but also exists within fibroblasts, thus providing the potential to resolve postoperative fibrinous collections even at sites at which the mesothelial cells have been removed or destroyed. Furthermore, PAA in fibroblasts is regulated by oxygen; creation of a hypoxic state (as occurs with suture ligation, electrosurgery, etc.) markedly attenuates PAA, thereby leading to adhesion development.

MRNA expression of urokinase and plasminogen activator inhibitor-1 in human crescentic glomerulonephritis.

Weak staining for urokinase-plasminogen activator (uPA), tissue type plasminogen activator (tPA), or plasminogen activator inhibitor-1 (PAI-1) confined to crescents has been described in a few cases of severe crescentic glomerulonephritis. We evaluated the molecular mechanism by which these proteins are increased or induced within crescents. We examined uPA, tPA and PAI-1 mRNA expression in 12 renal biopsies with crescentic glomerulonephritis, and in six control renal biopsies with no detectable abnormalities by RNA in-situ hybridization. The expressions of uPA, tPA and PAI-1 proteins were also assessed by immunofluorescence. To better determine the cellular origin of uPA and PAI-1 transcripts, CD68 protein was studied by immunohistochemistry on the same sections on which in-situ hybridization had been performed. In controls, there were very low level signals of uPA and PAI-1 mRNAs in a few glomerular epithelial cells (GECs). Specific signals of uPA and PAI-1 mRNAs were detected in the cells forming crescents in all the cases with crescentic glomerulonephritis. However, weak expression of mRNA for tPA was detected in two cases only. Immunostaining for uPA and PAI-1 was positive in some but not all, cases of crescentic glomerulonephritis. A double-labelling study showed that the signal for PAI-1 and uPA mRNAs was mainly in CD68- cells. Local accumulation of uPA or PAI-1 in crescents is associated with enhanced mRNA expression of these proteins. The up-regulation of PAI-1 mRNA by GECs, in particular, could play a major role in the formation of persistent fibrin deposits and progression of the lesions in crescents. Whether up-regulation of uPA is an epiphenomenon or plays a pathogenic role in the formation of crescents remains to be clarified.

Induction of the Plasminogen Activator System Accompanies Peripheral Nerve Regeneration after Sciatic Nerve Crush

Peripheral nerve regeneration is dependent on the ability of regenerating neurites to migrate through cellular debris and altered extracellular matrix at the injury site, grow along the residual distal nerve sheath conduit, and reinnervate synaptic targets. In cell culture, growth cones of regenerating axons secrete proteases, specifically plasminogen activators (PAs), which are believed to facilitate growth cone movement by digesting extracellular matrices and cell adhesions. In this study, the PA system was shown to be specifically activated in sensory neurons after sciatic nerve crush in adult mice. The number of sensory neurons expressing urokinase PA receptor (uPAR) mRNA levels increased above sham levels by 8 hr after crush, whereas the number of sensory neurons expressing uPA and tissue PA (tPA) mRNAs was significantly increased by 3 d after crush. PA mRNA levels were also increased at the crush site, with uPA mRNA elevated by 8 hr after crush and tPA and uPAR mRNA levels markedly increased by 7 d. PA-dependent enzymatic activity was significantly increased from 1 to 7 d after crush in nerves that had been crushed compared with uncrushed nerves. Immunohistochemistry showed that tPA was localized within regenerating axons of the sciatic nerve. There were no significant changes in plasminogen activator inhibitor 1 activity between crush and sham after the injury. These results clearly demonstrated that after injury the PA system was rapidly induced in sensory neurons, where it may play an important role in nerve regeneration in vivo.

Dynamics of gene expression for immediate early- and late genes after seizure activity in aged rats

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c- fos and tissue plasminogen activator ( tPA), is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure, and the levels of c- fos and tPA mRNAs induction are decreased in the cortex and in the hippocampus of 30 month old rats, as compared to the levels expressed by 3 month old rats. In addition, at the peak induction, the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes, we found that there was a shift toward earlier times in the maximum expression of growth-related molecules, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18 month old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (1) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (2) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Tissue plasminogen activator as a key effector in neurobiology and neuropathology

Tissue plasminogen activator (tPA) is expressed by many types of neurons in the developing and adult rodent brain. We have now mapped tPA transcripts and protein in the human central nervous system using tissue arrays and find widespread expression, in particular in neocortical mantle, thalamus, amygdala, and hippocampal pyramidal neurons. The abundant presence of tPA protein in cellular vesicles implies that its acute release, e.g. upon ischaemic stroke or trauma, could play a role in neuronal damage. We also found in patients with multiple sclerosis (MS), and to a lesser extent patients with leukaemia and encephalitis, prominently elevated tPA activity in the cerebrospinal fluid and in MS in neurons in the proximity of areas of demyelination elevated tPA mRNA and antigen levels. In addition, we observed up-regulation of tPA expression in a mouse model of MS, experimental autoimmune encephalomyelitis. Accumulating evidence implies roles for tPA in normal neural function, as well as in neurodestructive processes in humans, such as occur in MS and brain tumours and warrant further studies on expression of tPA and its regulatory molecules in neurodegenerative diseases.

Stimulation of Plasminogen Activator Activity and Matrix Metalloproteinases of Human Dental Pulp–Derived Cells by Tumor Necrosis Factor–α

The effects of tumor necrosis factor (TNF)-alpha on the plasminogen activator (PA)/plasmin system in human dental pulp (HDP) cells were examined. TNF-alpha treatment induced a significantly high level of PA activity in the conditioned medium of HDP cells in a time- and dose-dependent manner, compared with untreated control cells. Western-blot analysis revealed that tissue type (t)PA protein in conditioned medium was increased by TNF-alpha when compared with control medium. Furthermore the tPA mRNA level had increased in HDP cells treated with TNF-alpha, as determined by reverse transcription-polymerase chain reaction, but urokinase PA and PA inhibitor-1 mRNA levels did not increase. We examined the effects of TNF-alpha against activities of matrix metalloproteinases (MMPs) using zymography. TNF-alpha stimulated MMP-2 activity in conditioned medium and stimulated MMP-9 activity with addition of plasminogen into conditioned medium. The present results suggested that TNF-alpha stimulates PA activity via an enhancement of tPA gene expression in HDP cells and MMP-2 activity, and further that tPA-activated TNF-alpha stimulated MMP-9.

Effects of aging on luteinizing hormone secretion, ovulation, and ovarian tissue-type plasminogen activator expression.

This study examined the effects of aging on LH surge magnitude, ovulation, and ovarian expression of tissue-type plasminogen activator (tPA), a protease implicated in follicular rupture. While mean LH levels and ovulation rates were similar in middle-aged cyclic and young groups, there was a significant correlation between peak LH levels and ovulation rates in individual rats, such that females with lower LH surges ovulated fewer ova. In a separate experiment, proestrous LH levels were characterized in young and middle-aged rats, followed by in situ hybridization analysis of ovarian tPA mRNA. In young proestrous rats, tPA expression was observed in thecal-interstitial cells and oocytes, but not granulosa cells, prior to the LH surge. After the LH surge, there was a marked increase in tPA mRNA levels in granulosa cells of preovulatory, but not smaller follicles, peaking at 0200 hr estrus. By 0500 hr estrus, ovarian tPA expression declined, and ovulation had occurred. In contrast, LH-induced follicular tPA mRNA levels were dramatically lower in middle-aged rats with attenuated LH surges, and persisting preovulatory follicles were common in ovaries of these females on estrus morning. These findings suggest that age-related declines in ovulatory function result in part from altered induction of ovarian tPA expression, likely due to decreased proestrous LH secretion.

Plasminogen Activators and Inhibitors are Transcribed during Early Macaque Implantation

Plasminogen activators and inhibitors may be important early in primate implantation but evidence for this is sparse in non-human primates. We define the expression of urokinase type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1) and type 2 (PAI-2), the receptor for uPA (uPAR) and fibrin/fibrinogen in monkey implantation sites. In situ hybridization and immuno-histochemical localization of rhesus monkey implantation sites (day 15–16 postovulation) indicate: (1) uPA mRNA is localized to placental trophoblast, epithelial plaque and endometrial stroma. (2) tPA mRNA is mainly expressed in glandular cells of endometrium. (3) PAI-1 expression is linked to a specific population of trophoblasts that confront maternal cells, adding support to our view that it has a regulatory role in trophoblast invasion. (4) Localization of tPA antigen confirms that uterine glands are the major source of tPA and that it is also closely associated with fibrin(ogen) suggesting its possible function during implantation is fibrinolysis. (5) Unlike uPA mRNA, however, the distribution of uPA protein and its cell surface receptor uPAR suggests that it mediates trophoblast invasion and plays a significant role in angiogenesis. (6) PAI-2, the inhibitor associated with pregnancy in humans, was found in unidentified cells located specifically along the maternofetal junction. This localization adjacent to areas of cell death at the maternofetal junction implies that it may have a role as a protective curtain with anti-apoptotic function. In conclusion our results suggest that gene expression of PAs and PAIs in early implantation sites are tissue-specific, location-sensitive and function-related.

Elimination of hydrocortisone from the medium enables tissue plasminogen activator gene expression by normal and immortalized nonmalignant human epithelial cells.

Human cervical epithelial cells transfected and immortalized with human papillomavirus type 16 DNA (HCE16/3) can be, like many other epithelial cells, normally grown in medium supplemented with epidermal growth factor, cholera toxin, hydrocortisone, insulin, transferrin, thyroid hormone and serum. We found that hydrocortisone diminished tissue plasminogen activator (tPA) production to an undetectable level. The removal of hydrocortisone increased urokinase plasminogen activator (uPA) activity within 24-48 h and tPA activity within 48-72 h, and converted the cells to a more elongated and fibroblastic phenotype. Upregulation of uPA mRNA was seen as early as at 3 h and of tPA mRNA within 48-72 h. Higher molecular weight forms (97-110 kDa) of plasminogen activators were seen in zymograms, apparently complexed with PAI-1, starting at 6 h both in the presence and absence of hydrocortisone. Immunoprecipitation with a PAI-1 monoclonal antibody confirmed that both uPA and tPA were complexed. We also studied normal diploid human bronchial epithelial cells (NHBE) and NHBE cells transformed with an adeno-12/SV40 hybrid virus (BEAS-2B). In both types of nonmalignant epithelial cells, the removal of hydrocortisone increased uPA activity. The omission of hydrocortisone increased tPA levels significantly in BEAS-2B cell cultures, and in NHBE cell cultures tPA became detectable at 72 h. No PA complexes were seen in these two cell types. We conclude that normal and immortalized nonmalignant epithelial cells produce tPA, but only if hydrocortisone is omitted in the growth medium.

Enhanced Expression of Plasminogen Activator Inhibitor 1 in Patients with Nephrotic Syndrome

Hypercoagulability is present in patients with nephrotic syndrome. However, alterations in coagulation and fibrinolysis reflected in the glomeruli and urine are not fully understood. We examined plasma and urine concentrations of tissue-type plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) in 33 patients with nephrotic syndrome (nephrotic group). We compared these concentrations with the concentrations in 30 nonnephrotic patients with chronic glomerulonephritis (nonnephrotic group) and with the concentrations in 30 healthy volunteers (control group). We also examined fibrin/fibrinogen degradation products in serum and urine and plasma D-dimers. The expression of tPA and PAI-1 was examined in isolated glomeruli using RT-PCR methods. Deposition of fibrinogen/fibrin-related antigen was observed by direct immunofluorescence. The incidence of fibrinogen/fibrin-related antigen deposition in the nephrotic group was significantly higher than that in the nonnephrotic group. The concentrations of fibrin/fibrinogen degradation products in serum and urine and of plasma D-dimers were significantly elevated in the nephrotic group as compared with the nonnephrotic and control groups. The plasma concentrations of tPA in the nephrotic group were significantly higher than those in the control group. The urinary excretion of tPA in the nephrotic group was also significantly higher than in the nonnephrotic and control groups. The urinary excretion of PAI-1 in the nephrotic group was higher than that in the control group. The ratio of PAI-1 mRNA to tPA mRNA in glomeruli was increased in the nephrotic group as compared with the nonnephrotic group. These results indicate that the fibrinolytic activity is increased in patients with nephrotic syndrome despite urinary losses of tPA. However, a relatively enhanced expression of PAI-1 may be involved in the intraglomerular fibrinogen/fibrin-related antigen deposition seen in nephrotic syndrome.

Stimulation of Plasminogen Activator and Inhibitor in the Lymphatic Endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at Mr of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of ∼110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.

Expression of the plasminogen activator system in the human vascular wall

In this paper we describe the expression of the tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and the uPA receptor (uPAR), in normal and atheromatous human vascular tissue obtained at coronary and peripheral vascular surgery. tPA, uPA, PAI-1 and uPAR antigens were localised by immunohistochemistry. Vessel homogenates were used to quantitate tPA, uPA and PAI-1 antigens as well as uPA and PAI-1 activities using immunoassay and immunoactivity assays, respectively. Quantitative reverse transcription polymerase chain reaction assays (PAI-1 and uPA) were developed and used to quantify PAI-1 and uPA mRNA. In-situ hybridisation (tPA, uPA and PAI-1) was used to localise mRNA. In normal saphenous vein or internal mammary artery, expression of tPA, uPA and PAI expression is associated with endothelium and with intimal or medial smooth muscle cells, but expression is at a low level. uPAR protein was seen on the endothelium of normal saphenous vein or internal mammary artery but absent on the smooth muscle cells. In complex atheroma tPA, uPA, PAI and uPAR proteins were associated with the endothelium, groups of smooth muscle cells (in the intima and around vascular channels, but not with the media), infiltrating mononuclear cells, and also with acellular areas. PAI-1, tPA and uPA mRNA were demonstrated in atheroma in endothelial cells and smooth muscle cells, as well as in areas rich in macrophages. In stenosing saphenous vein grafts there was strikingly increased tPA and uPA (but not PAI-1) expression in neointimal smooth muscle cells and migrating SMC at the intima/media border. A major difference between complex atheroma and either normal vessel or saphenous vein grafts was greatly increased expression of PAI-1 mRNA associated with smooth muscle cells (SMC) in the former. In spite of the greatly increased PAI-1 mRNA expression in atheromatous lesions, the immunoactivity assay showed PAI-1 activity to be low compared to normal internal mammary artery. Our findings would be compatible with previous reports implicating the plasminogen activator/inhibitor system in the initiation and control of matrix remodelling during normal and pathological vessel growth and repair, but also emphasize the complexity of this process in human vessels.

Elevation of mRNA levels of tissue-type plasminogen activator and urokinase-type plasminogen activator in hippocampus and cerebral cortex following middle cerebral artery occlusion in rats

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) have been used for thrombolitic therapy. In contrast, it is suggested that these compounds might be involved in neuronal cell damage. The activation and the function of tPA and uPA are less understood in ischemic brain tissue. Therefore, changes in tPA and uPA mRNA in rat brain tissue after MCA occlusion and in the neuronal cell line, PCI2 cells, during the hypoxic stimulation were examined. Permanent middle cerebral artery (MCA) occlusion was induced by advancing a filament into the internal carotid artery in 36 adult male Sprague-Dawley rats. The ischemic cerebral cortex and contralateral cortex of MCA area, and bilateral hippocampus were collected at 0 (controls), 1, 3, 6, 12 and 24 h after occlusion. Hypoxia was induced in PCI2 cells with a multigas incubator (set to 1% 02). The quantitative reverse transcription-polymerase chain reaction acted as a measurement of alteration in mRNA levels. The mRNA levels of tPA and uPA were significantly increased after MCA occlusion in the ischemic cerebral cortex. The magnitude of the increase in tPA and uPA mRNA in 24 h after occlusion was twice the value in sham-operated rat (0 h). The increases oftPA mRNA were time-dependent in insult and contralateral hippocampus. The increase ofuPA mRNA was also seen in the hippocampus bilaterally, although the increase was more significant on the ipsilateral side. In PCI2 cells, necrotic (~ 35%) and apoptotic cells (~65%) could be distinguished by hypoxic stimulus for 24 h, and the mRNA for tPA was significantly increased for 6 h-12h, while the mRNA for uPA was not detected at any point in the study. Our results suggest that focal ischemia might result in the activation of these proteases not only in the insult but also in the contralateral brain tissue. [Neurol Res 2000; 22: 413-419]

Hypertriglyceridemic VLDL downregulates tissue plasminogen activator gene transcription through cis-repressive region(s) in the tissue plasminogen activator promoter in cultured human endothelial cells.

The relationship between tissue plasminogen activator (tPA) levels and the potential regulation by hypertriglyceridemic very low density lipoprotein (HTG-VLDL) was examined in a human umbilical vein endothelial cell (HUVEC) culture model system. HUVEC cultures were incubated in the absence/presence of HTG-VLDL or normal (NTG)-VLDL (0 to 50 microg/mL) at 37 degrees C for various times (0 to 24 hours), followed by analyses of tPA antigen (ELISA), mRNA (reverse transcription-polymerase chain reaction), endothelial cell surface-localized plasmin generation assays, and nuclear transcription run-on assays. Secreted tPA antigen levels decreased approximately 53% (3.3+/-0.14 versus 6.97+/-0.42 microg/mL) and mRNA levels decreased approximately 70% in HTG-VLDL-treated HUVECs compared with NTG-VLDL-treated and culture medium control cells. Decreased tPA antigen and mRNA expression was associated with a concomitant approximately 98% decrease in tPA-mediated plasmin generation in HTG-VLDL-treated HUVEC cultures. Nuclear transcription run-on assays demonstrated that HTG-VLDL decreased tPA gene transcription approximately 73% (tPA mRNA/GAPDH mRNA) in cultured HUVECs. To identify and localize the repressive element(s) in the tPA promoter responsive to HTG-VLDL, a tPA promoter/luciferase construct (ptPA222/luc) was generated. HUVECs transiently transfected with this construct were incubated in the absence/presence of HTG-VLDL or NTG-VLDL (20 microg/mL). HTG-VLDL decreased promoter activity approximately 52% to 57% in the ptPA222/luc-transfected cells compared with NTG-VLDL-treated or buffer control cells. These results indicate that the 2.2-kb fragment of the promoter and 5' flanking region of the tPA gene contains the repressive sequences that direct the transcriptional downregulation of the tPA promoter. Data from these studies suggest that the repression of tPA gene expression by HTG-VLDL may contribute to the impaired fibrinolysis often associated with hypertriglyceridemia.

The Response of Adult Human Saphenous Vein Endothelial Cells to Combined Pressurized Pulsatile Flow and Cyclic Strain, In Vitro

Adult human saphenous vein endothelial cells (HVEC) were cultured in a compliant tubular device and evaluated by Northern hybridization for the effects of combined pressurized pulsatile flow and cyclic strain on the expression of mRNAs for endothelin-1 (ET-1), endothelial cell nitric oxide synthase (ecNOS), tissue plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1). The hemodynamic environment was designed to mimic shear stress conditions at the distal anastomosis of a saphenous vein graft, a common site of intimal proliferation. Steady-state mRNA levels in experimental tubes were expressed relative to that in controls. No changes were observed in ET-1 mRNA after 1 and 24 hr, but a 50% decrease in experimental cultures was observed after 48 hr in the vascular simulating device. Similar results were obtained for ecNOS mRNA, although a subgroup (4 of 11) showed a significant decrease (>50%) by 24 hr. For tPA mRNA, no change was observed after 1 hr, but a significant decrease (>60%) was measured after 24 hr and no message was detectable after 48 hr. Steady-state levels for PAI-1 mRNA remained unchanged through 48 hr of treatment. These results show that pressure, pulsatile flow, and cyclic strain, when applied in concert, differentially alter vasoactive and fibrinolytic functions in HVEC. Moreover, the dramatic decrease in steady-state levels of tPA mRNA is consistent with a shift toward an increased thrombotic state.

Elevated intraluminal pressure inhibits vascular tissue plasminogen activator secretion and downregulates its gene expression.

We recently discovered that patients with essential hypertension have a markedly impaired capacity for stimulated release of tissue plasminogen activator (tPA) from vascular endothelium. This defect may reduce the chance of timely spontaneous thrombolysis in case of an atherothrombotic event. We now investigated whether increased intraluminal pressure as such may depress vascular tPA release or downregulate its gene expression. Segments of human umbilical veins were studied in a new computerized vascular perfusion model under steady laminar flow conditions for 3 or 6 hours. Paired segments were perfused at high or physiological intraluminal pressure (40 versus 20 mm Hg) under identical shear stress (10 dyne/cm(2)). Quantitative immunohistochemical evaluation of cellular tPA immunoreactivity was performed on paraffin-embedded 5-microm vascular sections. tPA mRNA in endothelial cells was quantified with reverse transcription real-time TaqMan polymerase chain reaction with GAPDH as endogenous control. Secretion of tPA into perfusion medium was evaluated with SDS-PAGE and Western blotting, followed by densitometric quantification. High-pressure perfusion downregulated tPA gene expression with a 38% decrease in tPA mRNA levels (P=0.01) compared with vessels perfused under normal intraluminal pressure. tPA release into the perfusion medium was markedly suppressed by high pressure (P<0.01 ANOVA). The intracellular storage pool of tPA was reduced after 6 but not 3 hours. Thus, elevated intraluminal pressure downregulates tPA gene and protein expression and inhibits its release from the endothelium independently of shear stress. The defective capacity for stimulated tPA release that we demonstrated in patients with essential hypertension might thus be an effect of the elevated intraluminal pressure per se.

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles.

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.

Changes in tissue-plasminogen activator mRNA expression following cortical ablation in the rat brain.

Tissue plasminogen activator (tPA) has been used to treat acute thrombotic lesions. Roles other than the activation of fibrinolytic pathways have been suggested for tPA in the mature brain. We used the in situ hybridization technique to investigate the changes in tPA mRNA expression within the brain after cortical ablation. We found that expression of tPA mRNA started to increase diffusely in the cortex ipsilateral to the injury 6 h after ablation. This increase had become prominent 24 h after ablation. On d 5, the expression of tPA mRNA had returned to that of the control animals except for the area near the injury. We also found that administration of MK-801 before injury suppressed the increase of tPA mRNA in the ipsilateral cortex. These results suggest that the increase in tPA mRNA is likely to be mediated via activation of NMDA receptors.

Reduction of Plasminogen Activator Activity Stimulated by Lipopolysaccharide from Periodontal Pathogen in Human Gingival Fibroblasts by Low-energy Laser Irradiation

Human gingival fibroblasts (hGF) reside in gingival tissues which are challenged frequently by oral bacteria. Lipopolysaccharide (LPS) from periodontal pathogens can penetrate gingival tissues and stimulate plasminogen activator (PA) activity which is implicated in the PA/plasmin proteolytic system. The PA/plasmin system plays an important role in the degradation of the extracellular matrix and synthesis of kinin in the process of inflammation. Anti-inflammatory effects by low-energy laser irradiation have been reported. However, the mechanisms of biostimulatory effects have not been elucidated in detail. The primarily cultured hGF cells were challenged with LPS isolated from Campylobacter rectus which was known as a periodontal pathogen and Ga-Al-As diode low energy laser was irradiated (830 nm, 7.90 J/cm(2)). The cultured medium of hGF cells showed a marked elevation in PA activity by LPS, which was significantly inhibited by the laser irradiation in a dose-dependent manner. This effect was involved in the reduction of tPA mRNA levels.

Mechanical stress enhances expression and production of plasminogen activator in aging human periodontal ligament cells

Plasminogen activator (PA) converts plasminogen to plasmin, and plasmin activates the kinin cascade and latent extracellular matrix metalloproteases. The periodontal ligament serves to anchor the tooth to the alveolus and functions as a cushion between these hard tissues to migrate occlusal force during mastication. We reported previously that repeated mechanical tension force (MTF) as an experimental model of a traumatic occlusion, increased PA activity in human periodontal ligament derived fibroblast (hPLF) cells. In this study, the influence of in vitro cellular aging on MTF-stimulated PA activity in hPLF cells was studied. Aged hPLF cells produced a significantly higher PA activity when compared with those of young hPLF cells in response to MTF in a time- and magnitude-dependent manner. tPA mRNA levels in aged cells were higher than those in young cells, whereas PAI-1 mRNA remained unchanged and uPA mRNA was not detected. Because MTF-stimulated PA activity from hPLF cells was increased by in vitro cellular aging, aging of the periodontal ligament may affect the severity of the inflammation and the degradation of the extracellular matrix of periodontal ligament tissue by producing a large amount of PA in response to excessive force such as a traumatic occlusion.