Production Of Tissue Plasminogen Activator Research Articles

Pathophysiology of atherosclerosis: Association of risk factors and treatment strategies using plant-based bioactive compounds.

Under physiological conditions, endothelial cells act as protective barrier which prevents direct contact of blood with circulating factors via production of tissue plasminogen activator. Risk factors of metabolic disorders are responsible to induce endothelial dysfunction and may consequently lead to prognosis of atherosclerosis. This article summarizes the process of atherosclerosis which involves number of sequences including formation and interaction of AGE-RAGE, activation of polyol pathway, protein kinase C, and hexosamine-mediated pathway. All these mechanisms can lead to the development of oxidative stress which may further aggravate condition. Different pharmacological interventions are being used to treat atherosclerosis, however, these might be associated with mild to severe side effects. Therefore, plant-based bioactive compounds having potential to combat and prevent atherosclerosis in diabetic patients are attaining recent focus. By understanding process of development and mechanisms involved in atherosclerotic plaque formation, these bioactive compounds can be better option for future therapeutic interventions for atherosclerosis treatment. PRACTICAL APPLICATIONS: Atherosclerosis is one of major underlying disorders of cardiovascular diseases which occur through multiple mechanisms and is associated with metabolic disorders. Conventional therapeutic interventions are not only used to treat atherosclerosis, but are also commonly associated with mild to severe side effects. Therefore, nowadays, bioactive compounds having potential to combat and prevent atherosclerosis in diabetic patients are preferred. By understanding mechanisms involved in atherosclerotic plaque formation, bioactive compounds can be better understood for treatment of atherosclerosis. In this manuscript, we have focused on treatment strategies of atherosclerosis using bioactive compounds notably alkaloids and flavonoids having diverse pharmacological and therapeutic potentials with special focus on the mechanism of action of these bioactive compounds suitable for treatment of atherosclerosis. This manuscript will provide the scientific insights of bioactive compounds to researchers who are working in the area of drug discovery and development to control pathogenesis and development of atherosclerosis and its associated cardiometabolic disorders.

The Role of Abnormal Hemostasis and Fibrinolysis in Morbidity and Mortality of Acute Promyelocytic Leukemia.

Despite the improved therapeutic advances in the management of acute promyelocytic leukemia (APL), a significant early mortality during induction, also referred to as early death (ED), remains an obstacle for further improvement in outcome. Hemorrhagic complications are the most common cause of morbidity and mortality. Perturbed hemostatic dysfunction is present as the result of abnormalities in both the coagulation and the fibrinolytic systems. The activation of coagulation is distinct from the classical disseminated intravascular coagulation. Multiple abnormalities in the fibrinolytic system have recently been identified. The most significant change is increased production of tissue plasminogen activator (tPA) and its receptor annexin A2 by the APL promyelocytes. Among the hemorrhagic complications, intracranial hemorrhage predominates. The pathogenesis of this catastrophic event is elucidated by new evidence of adverse effect of tissue plasminogen activator (tPA) on the brain, including both the plasmin-dependent and plasmin-independent pathways. In order to address the hemorrhagic complications, a thorough understanding of the hemostatic dysfunction is essential. In this article, our current concept of the abnormal hemostasis in APL is reviewed. The failure to reduce the early death rate, despite the introduction of effective therapy, will also be discussed.

In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.

Proteomics Profiling of Chimeric-Truncated Tissue Plasminogen activator Producing- Chinese Hamster Ovary Cells Cultivated in a Chemically Defined Medium Supplemented with Protein Hydrolysates

Background:Culture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression.Methods:In this study, designing the optimum mixture of four soy and casein-derived hydrolysates was successfully performed by design of experiment and specific productivity increased in all predicted combinations. Protein profile of recombinant CHO (rCHO) cells producing tissue plasminogen activator in a serum-free medium (SFM) supplemented with designed hydrolysate additives was compared to that of rCHO cells cultivated in SFM.Results:Identification of differentially expressed proteins using two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF revealed the role of energy metabolism related proteins and importance of prevention of oxidative stress by this special media enrichment strategy. Up-regulation of mitochondrial enzymes, pyruvate dehydrogenase E1, and Peroxiredoxin-III, as well as other proteins involved in metabolic pathways, and uridine monophosphate/cytidine monophosphate kinase indicated higher metabolic activity. Furthermore, along with antioxidant effect of peptones, proteins with antioxidant function such as ferritin and peroxiredoxin-III were up-regulated.Conclusion:Understanding molecular mechanisms involved in enhancement of protein expression can provide new approaches for efficiently engineering rCHO cell. These results support the competence of proteomics studies in finding new insights to biochemical pathways for a knowledge-based optimization of media compositions.

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h−1) and low dilution rate (0.012 h−1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

Bacterial Lipoprotein TLR2 Agonists Broadly Modulate Endothelial Function and Coagulation Pathways In Vitro and In Vivo

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

Endothelial Differentiation of Adipose-Derived Stem Cells from Elderly Patients with Cardiovascular Disease

Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50-89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI(3)K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α(5)β(1) expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering.

Relationship between tissue plasminogen activator production and specific growth rate in Chinese Hamster Ovary cells cultured in mannose at low temperature

Chinese hamster ovary (CHO) cells, producing human recombinant tissue plasminogen activator (tPA), were grown with mannose (5, 20 and 40 mM) instead of glucose at 31, 33 and 37 degrees C. The highest tPA concentration (1.5 mg l(-1) at 144 h of cultivation) and tPA specific production rate (47 ng 10(-6) cell h(-1)) were obtained at 31 degrees C and 40 mM mannose. Regardless of the temperature or mannose concentration used, an inverse relationship between the specific growth rate and tPA specific production rate was observed, suggesting that tPA production rate would be directly controlled by the growth rate.

Kallikrein gene ‘knock-down’ by small interfering RNA transfection induces a profibrotic phenotype in rat mesangial cells

Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by 'knocking down' its expression using specific small interfering RNAs (siRNA). Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed. Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein-kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-beta protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance. These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.

Tissue plasminogen activator induced by dengue virus infection of human endothelial cells.

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of dengue virus (DV) infection. However, the pathogenesis of hemorrhage induced by dengue virus infection is poorly understood. Since endothelial cells play a pivotal role in the regulation of hemostasis, we studied the effect of DV infection on the production of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in vitro using both primary isolated endothelial cells, human umbilical cord veins cells, and a human microvascular endothelial cell line. DV infection significantly induced the secretion of tPA but not PAI-1 of human endothelial cells. In addition, tPA mRNA of endothelial cells was induced by DV as demonstrated by RT-PCR. Antibody against IL-6 but not control antibody inhibited DV-induced tPA production of endothelial cells. Furthermore, a good correlation between sera levels of IL-6 and tPA was found in DHF but not DF patients. These results suggest that IL-6 can regulate DV-induced tPA production of endothelial cells, which may play important roles in the pathogenic development of DHF/DSS.

Repeated addition of insulin for dynamic control of apoptosis in serum-free culture of Chinese hamster ovary cells

Repeated addition of insulin, together with an initial addition of the reduced form of glutathione (GSH) and a chelator, aurintricarboxylic acid (ATA), was proposed as a strategy for the dynamic control of apoptosis in serum-free culture of Chinese hamster ovary (CHO) cells. Insulin suppressed cell death in serum-free culture by means of lowering intracellular reactive oxygen species levels via an increase in the specific glucose consumption rate. This effect of insulin addition was found not only at 0 h but also at 48 h during serum-free cultivation. In consideration of the reduction of insulin by GSH, insulin was again added at 24 h following the initial addition during the serum-free cultivation. The repeated addition of insulin decreased cell death and increased the production of tissue plasminogen activator compared with the single addition at 0 h.

Repeated Addition of Insulin for Dynamic Control of Apoptosis in Serum-Free Culture of Chinese Hamster Ovary Cells

Repeated addition of insulin, together with an initial addition of the reduced form of glutathione (GSH) and a chelator, aurintricarboxylic acid (ATA), was proposed as a strategy for the dynamic control of apoptosis in serum-free culture of Chinese hamster ovary (CHO) cells. Insulin suppressed cell death in serum-free culture by means of lowering intracellular reactive oxygen species levels via an increase in the specific glucose consumption rate. This effect of insulin addition was found not only at 0 h but also at 48 h during serum-free cultivation. In consideration of the reduction of insulin by GSH, insulin was again added at 24 h following the initial addition during the serum-free cultivation. The repeated addition of insulin decreased cell death and increased the production of tissue plasminogen activator compared with the single addition at 0 h.

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes.

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.

Prevention Conference VI: Diabetes and Cardiovascular Disease: Writing Group II: pathogenesis of atherosclerosis in diabetes.

Diabetes is associated with increased atherosclerosis and other causes of myocardial dysfunction. The pathogenesis of cardiovascular disease (CVD) in diabetes is multifactorial and can be affected by metabolic and other factors. Under physiological conditions, the endothelial cell (EC) layer acts as a barrier to separate circulating factors and cells from the arterial intima and media. It also serves as an anticoagulant and fibrinolytic surface producing tissue plasminogen activator, which counters the effects of procoagulant factors such as fibrinogen and plasminogen activator inhibitor-1 (PAI-1). Endothelial cells produce nitric oxide (NO), which is a vasodilator and restrains smooth muscle cell (SMC) migration and proliferation. Circulating factors (hyperglycemia, increased free fatty acids, altered lipoproteins, and derivatives of glycation and oxidation) and hypertension, all of which are common in diabetes, can damage ECs, leading to their dysfunction. Plasma proteins, among them lipoproteins, cross the endothelial barrier, where they can be retained by subendothelial matrix molecules such as collagen and proteoglycans.1 These and other matrix molecules are produced by ECs and SMCs. Blood components can be modified, eg, by oxidation and glycation.2 Modified proteins and lipids can alter EC and SMC gene expression, leading to increased production of procoagulants, adhesion molecules, chemotactic factors, and cytokines. The net effect is the adhesion and penetration of circulating monocytes into the arterial intima, where they undergo differentiation and activation to macrophages. Lipids can accumulate intracellularly after uptake of modified lipoproteins (glycation, oxidation, and glycoxidation) by different scavenger receptors on macrophages and SMCs, as well as extracellularly by attaching to matrix molecules.3 The resulting lesion is termed the fatty streak. Both ECs and macrophages produce cytokines and growth factors that permit SMCs to migrate from the media to the intima. In the intima, SMCs proliferate in response to several growth factors. These SMCs and the matrix …

High prevalence of polymorphisms of angiotensin-converting enzyme (I/D) and endothelial nitric oxide synthase (Glu298Asp) in patients with systemic sclerosis

Purpose Systemic sclerosis is characterized by progressive microvascular occlusion and fibrosis and by an imbalance in the fibrinolytic system. In vivo and in vitro studies suggest that the renin-angiotensin system partly regulates vascular fibrinolytic balance. Angiotensin II increases the production and secretion of plasminogen activator inhibitor-1, while angiotensin-converting enzyme (ACE) contributes to reduced production of tissue plasminogen activator and endothelial nitric oxide synthesis by bradykinin degradation. The aim of our study was to investigate the effects of ACE insertion/deletion (I/D) and endothelial nitric oxide synthase (eNOS) Glu298Asp (G894→T) and T–786→C polymorphisms in patients with systemic sclerosis. Subjects and methods We studied 73 consecutive patients (47 with limited and 26 with diffuse cutaneous systemic sclerosis) and 112 control subjects. ACE I/D and eNOS polymorphisms were genotyped by polymerase chain reaction–restriction fragment length polymorphism analysis. Results The ACE I/D and the eNOS G894→T polymorphisms were more common in patients than in controls (for the ACE D allele: odds ratio [OR] = 3.4; 95% confidence interval [CI]: 1.5 to 7.9; P = 0.003; for the eNOS T allele: OR = 1.9; 95% CI: 1.0 to 3.4; P = 0.04). There was no association between the eNOS T–786→C polymorphism and systemic sclerosis. Conclusion Our findings of an increased risk of systemic sclerosis in ACE D and eNOS 894T allele carriers suggest that these polymorphisms may contribute to the pathogenesis of the disease.

Yeast mannan in combination with high concentration of Ca2+ enhances production of tissue plasminogen activator by human diploid fibroblasts stimulated with proteose peptone

The efficient production of tissue plasminogen activator by human diploid fibroblasts is described. Confluent fibroblasts lost their tissue plasminogen activator-producing ability when incubated in a serum-free control medium containing proteose peptone (10 mg ml−1) for longer than 4 to 7 d. In comparison, the cells continued producing the enzyme for more than 14 d when incubated in an experimental medium containing yeast mannan (2 mg ml−1) and CaCl2 (3.6 mM) in addition to peptone. The productivity of the enzyme maximally increased when the ratio of the medium volume to the monolayer area decreased. Such an improved culture system yielded 29 times the productivity achieved under the control conditions. The use of mannan is practically important for large-scale production and subsequent purification of the enzyme.

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control.

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t-PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long-duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t-PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems.

Computer simulation for large scale bioprocess design

Two bioprocess simulators, Aspen BPS™ and SuperPro Designer ®, were used to simulate and compare two flowsheets for production of tissue plasminogen activator (t-PA) from Chinese hamster ovary (CHO) cells. The flowsheets were designed to compare the economics of using serum and serum-free medium in the bioreactor. The base case flowsheet used a medium with 10% serum while the alternative flowsheet used a serum-free medium. Affinity chromatography was the major step in the downstream processing of the base case flowsheet. The alternate flowsheet was designed for two-step cell culture growth and production. Ion-exchange chromatography was considered sufficient for purification of the low protein medium for this case. At a price of US$ 25/l for serum-free medium, the alternate flowsheet was found to have an return on investment (ROI), of 115% compared to 65% for the base case flowsheet.

Interleukin 6 and Haemostasis

Interleukin 6 and Haemostasis

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.