Reduced Plasminogen Activator Research Articles

Discovery and preclinical development of LY3473329, a potent small molecule inhibitor of lipoprotein(a) formation

Abstract Background Lipoprotein(a) [Lp(a)] is a lipoprotein particle consisting of a low-density lipoprotein (LDL) particle and apolipoprotein(a) [apo(a)]. Lp(a) is a causal risk factor for cardiovascular disease. The formation of Lp(a) requires an initial interaction of apo(a) Kringle IV Domains 7 and 8 (KIV7, KIV8) with apoB-100 on LDL, followed by formation of a disulfide bond between apo(a) and apoB-100. Blocking the initial apo(a)-apoB interaction is a viable therapeutic approach to reduce circulating levels of Lp(a). The apo(a) protein is encoded by the LPA gene that arose from the plasminogen gene through an evolutionary gene duplication event. Because of sequence conservation between apo(a) and plasminogen, compound selectivity for apo(a) is imperative for clinical development. Purpose We report the discovery and preclinical characterization of LY3473329, a novel selective small molecule inhibitor of Lp(a) formation. Methods Computational and synthetic chemistry approaches coupled with multiple biochemical and biophysical assays were used to create apo(a) KIV7,8 binders. Chemical optimization led to molecules with potent inhibition of in vitro Lp(a) formation. Both apo(a) and plasminogen binding assays were conducted to evaluate compound selectivity. The potential for LY3473329 to lower steady state Lp(a) levels was assessed in human Lp(a) transgenic mice and in cynomolgus monkeys, and its effect on plasminogen activity was assessed in rat and monkey. Results Computational modeling, synthetic chemistry and small molecule library screens identified compounds that bind apo(a) KIV7 and KIV8 domains, and subsequent chemical optimization yielded compounds with sub-micromolar Lp(a) inhibition potency. Exploration of compound multivalency created inhibitors with sub-nanomolar potency, including LY3473329. LY3473329 engages multiple apo(a) KIV motifs, allowing it to potently inhibit Lp(a) formation in vitro. Oral doses of LY3473329 given to human Lp(a) transgenic mice and cynomolgus monkeys caused dose-dependent decreases in plasma Lp(a). The key binding motif for LY3473329 is shared by two KIV domains in rat and monkey plasminogen, but only one KIV domain in human plasminogen. LY3473329 was non-selective for rat and monkey plasminogen, but showed ∼ 50-fold selectivity for human plasminogen in in vitro binding assays. In cynomolgus monkeys and rats, LY3473329 significantly reduced plasminogen activity levels without adverse effects. Given its selectivity against human plasminogen, LY3473329 is expected to lower Lp(a) in human without affecting plasminogen. LY3473329 has been evaluated in a human Phase 1 study and is currently in Phase 2 clinical development. Conclusions LY3473329 is a potent and selective small molecule inhibitor of Lp(a) formation and is the only orally administered Lp(a) investigational therapeutic in clinical development.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability

BackgroundMyeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. ObjectivesWe aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. MethodsWe used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. ResultsGlycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. ConclusionCollectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.

Enhanced clot lysis by a single point mutation in a reteplase variant.

Recombinant tissue-type plasminogen activator (rtPA) is the clot lysis drug approved for clinical use, and is characterised by a short half-life and substantial inactivation by plasminogen activator inhibitor-1 (PAI-1). We previously discovered that a tPA mutation (A419Y) at the protease domain led to enhanced fibrinolysis activity. In the present study, we studied the mechanism of such mutation in enhancing the proteolytic activity, and whether such enhancement persists in reteplase, an United States Food and Drug Administration-approved tPA truncated variant. We constructed and expressed a series of reteplase-based mutants, including rPAG (glycosylated rPA), rPAG -Y (with A419Y mutant at rPAG ), rPAG -A4 (tetra-alanine mutation at 37-loop of rPAG ), and rPAG -A4/Y (with both) and evaluated their plasminogen activation and PAI-1 resistance. Surface plasmon resonance analysis showed that the rPAG had fibrin affinity comparable to full-length tPA. Moreover, rPAG -Y had 8·5-fold higher plasminogen activation and stronger tolerance to PAI-1 compared to rPAG . We also found that the mutations containing tetra-alanine (rPAG -A4 and rPAG -A4/Y) had dramatically reduced plasminogen activation and impaired clot lysis. In a pulmonary embolism murine model, rPAG -Y displayed a more efficient thrombolytic effect than rPAG . These results identified a novel mutant reteplase variant of tPA with increased fibrinolytic activity, laying the foundation for the development of a new potent fibrinolytic agent.

MO322VENOUS THROMBOEMBOLISM AND NEPHROTIC SYDROME: A FAMOUS BUT STILL SURPRISING COUPLE

Abstract Background Venous thromboembolism (VTE) is a multifactorial disorder, accounting for high morbidity and mortality rates, due to a complex interplay of several variables classifiable as inherited (mutated Leiden V factor, prothrombin, protein C, protein S and antithrombin) and acquired (lupus anticoagulants, pregnancy, major surgery procedures, cancer and inflammatory diseases) risk factors. The association of VTE with the nephrotic syndrome, particularly deep vein and renal vein thrombosis (DVT and RVT, respectively) is tightly established. This risk is particularly high in patients with idiopathic membranous nephropathy. In fact, thromboembolic events occur with a frequency between <10% and 45% in this disease. The reason(s) underlying the hypercoagulable status in nephrotic patients are not clearly understood. Multiple hemostatic abnormalities have been described, including decreased levels of antithrombin and plasminogen (due to urinary losses), increased platelet activation, reduced plasminogen activation, overproduction of fibrinogen and factors V and VIII as a compensatory response to hypoalbuminemia. The risk of thrombosis seems to be related to the severity and duration of the nephrotic status and seems to be particularly increased with serum albumin concentrations ≤2.0 g/dl (20 g/L). Case report We report the case of a 28 year-old male with nephrotic syndrome due to membranous nephropathy positive for serum anti-phospholipase-A2 receptor antibody. The patient was asymptomatic for VTE, but abdominal ultrasound showed endoluminal obstruction of both renal veins. Abdominal Computer Tomography confirmed the extensive bilateral renal vein thrombosis and also revealed an extension of the thrombosis to the inferior cava vein and the left common iliac vein. He was treated with low-molecular weight heparin for six months. According to our internal protocol, a complete Thrombophilia Molecular Study was performed and showed a normal Leiden V factor, but a rare homozygous mutation of the G20210A gene encoding for prothrombin. The prevalence of this is less than 5% in the general population, but is highly variable with ethnicity. The G20210A mutation confers a mildly increased thrombotic risk that is amplified by the presence of other risk factors, such as nephrotic syndrome. Conclusions In our case report, the association of a nephrotic syndrome secondary to a primitive membranous glomerulonephritis and the mutation in homozygous of the G20210A prothrombin, a rare mutation associated with a high thrombotic risk, led to a severe VTE in an still asymptomatic 28-year-old patient. Based on this experience, we would highlight the importance of the genetic screening for polymorphisms associated with inherited thrombophilia in nephrotic patients complicated with VTE.

The Contribution of the Urokinase Plasminogen Activator and the Urokinase Receptor to Pleural and Parenchymal Lung Injury and Repair: A Narrative Review.

Pleural and parenchymal lung injury have long been characterized by acute inflammation and pathologic tissue reorganization, when severe. Although transitional matrix deposition is a normal part of the injury response, unresolved fibrin deposition can lead to pleural loculation and scarification of affected areas. Within this review, we present a brief discussion of the fibrinolytic pathway, its components, and their contribution to injury progression. We review how local derangements of fibrinolysis, resulting from increased coagulation and reduced plasminogen activator activity, promote extravascular fibrin deposition. Further, we describe how pleural mesothelial cells contribute to lung scarring via the acquisition of a profibrotic phenotype. We also discuss soluble uPAR, a recently identified biomarker of pleural injury, and its diagnostic value in the grading of pleural effusions. Finally, we provide an in-depth discussion on the clinical importance of single-chain urokinase plasminogen activator (uPA) for the treatment of loculated pleural collections.

Investigation of Supercharging as A Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase.

Background: Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase.Objective: To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity.Materials and Methods: Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity.Results: SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activity of the supercharged variant than the wild type protein (P < 0.001). The stability of the supercharged variant was also comparable to the wild type.Conclusion: Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.

Effect of Rivaroxaban on hemostasis in patients with alcoholic cirrhosis of the liver in combination with obesity

The effect of rivaroxaban on hemostasis was investigated in patients with alcoholic liver cirrhosis (ALC) in combination with obesity and non-valvular atrial fibrillation (NVAF). The study included 48 obese patients with ALC of class A (according to the Child-Pugh score) with NVAF, who were not previously treated with direct oral anticoagulants. There are 43 men and 5 women aged 35 to 64 years, mean age is (54.2±4.8) years. Depending on the treatment, patients were divided into two groups: group I included 22 patients who received oral anticoagulant rivaroxaban at a dose of 10 mg/day for 12 months, group II included 26 patients who received basic therapy without rivaroxaban for 12 months. Assessment of the patients’ condition was performed before treatment, 12, 24 and 48 weeks after treatment onset. The severity of LC was assessed according to the Child-Pugh and the MELD scores. Stratification of the risk of bleeding was performed according to the HAS-BLED score. It is shown, that in obese patients with ALC of class A according to the Child-Pugh score in the presence of NVAF, the use of rivaroxaban in combination therapy for 48 weeks contributed to the positive dynamics of hemostasis: decreased factor VIII activity, von Willebrand factor activity, reduced plasminogen activator inhibitor-1, D-dimer levels (p&lt;0.05) and an increase in the activity of protein C (p&lt;0.05), which was accompanied by a moderate decrease in the Child-Pugh and the MELD scores, and this was not observed in the comparison group. The use of rivaroxaban in this cohort of patients for 48 weeks did not increase the risk of major bleeding according to the clinical course and the HAS-BLED score. It is proved that the use of rivaroxaban in the complex treatment of patients with ALC of class A (according to the Child-Pugh score) in combination with obesity and NVAF, contributes to the positive dynamics of hemostasis with a reduced risk of hypercoagulation. Such therapy does not increase the risk of major bleeding at the stage of compensation of ALC in combination with obesity and NVAF.

Combined but not single treatment with ethinylestradiol/levonorgestrel and spironolactone reduces plasminogen activator inhibitor-1 in insulin-resistant ovariectomised rats.

Objective:Increased circulating level of plasminogen activator inhibitor-1 (PAI-1) is associated with menopausal oestrogen deficiency. We therefore hypothesised that the combined oral contraceptive (COC) with spironolactone (SPL) improves insulin resistance (IR) in ovariectomised (OVX) rats by reducing circulating PAI-1.Methods:Twelve-week-old female Wistar rats were divided into sham-operated (SHM), OVX, OVX+SPL (0.25 mg/kg), COC (1.0 µg ethinylestradiol and 5.0 µg levonorgestrel) and OVX+COC+SPL rats treated with COC and SPL daily for eight weeks. IR was assessed by homeostatic model assessment of IR (HOMA-IR).Results:Data showed that OVX rats had a higher HOMA-IR value that is associated with increased visceral adiposity, triglycerides (TG), total cholesterol/high-density lipoprotein cholesterol (HDL-C), TG/HDL-C, plasma insulin, GSK-3, corticosterone and decreased 17β-oestradiol. However, these effects were attenuated in OVX+COC, OVX+SPL and OVX+COC+SPL rats compared to OVX rats. OVX rats had lower PAI-1 than SHM rats, whereas the beneficial effect on IR and other parameters by COC or SPL was accompanied with increased PAI-1. Improvement of IR and other parameters with combined COC and SPL in OVX rats was accompanied with reduced PAI-1.Conclusion:Taken together, COC or SPL improves IR independent of PAI-1, whereas a combination of COC and SPL in OVX rats ameliorates IR in a PAI-1-dependent manner.

Fresh frozen plasma (Octaplas) and topical heparin in the management of ligneous conjunctivitis

Ligneous conjunctivitis is a rare form of chronic recurrent membranous conjunctivitis with reduced plasminogen activity. It is characterized by the formation of characteristic firm ("woody") membranes on the tarsal conjunctiva. Similar lesions may occur on other mucous membranes. When treated with local excision, ligneous conjunctivitis is invariably associated with recurrences. Various therapeutic modalities, including topical heparin, cyclosporine, fresh frozen plasma (FFP), plasminogen, and amniotic membrane transplantation have been reported to reduce postoperative recurrences. We present 2 cases of recurrent ligneous conjunctivitis in children successfully managed with surgical excision under cover of FFP transfusion, amniotic membrane grafting, and combined with concomitant postoperative administration of topical heparin, steroids.

Effects of gastric bypass followed by a randomized study of physical training on markers of coagulation activation, fibrin clot properties, and fibrinolysis

BackgroundThe risk of cardiovascular disease is reduced by bariatric surgery, but it is unknown if exercise after bariatric surgery reduces this risk even further. ObjectiveTo investigate if Roux-en-Y-gastric bypass (RYGB) and supervised physical training after RYGB improve cardiovascular disease risk markers within coagulation activation, fibrin clot properties, and fibrinolysis. SettingBariatric center, Hospital of Southwest Jutland, Denmark. MethodsSixty obese patients underwent RYGB and 6 months after RYGB were randomized to 26 weeks of physical training or a control group. Biomarkers within coagulation activation, fibrin clot properties, and fibrinolysis were measured presurgery, and 6, 12, and 24 months postsurgery. ResultsSix months after RYGB, the endogenous thrombin potential decreased from 1744 (1603–2003) to 1416 (1276–1582) nM × min (P<.001). Alterations in fibrin clot properties resulted in an increased clot lysis from 23.8% (16.1%–38.9%) to 40.3% (28.5%–59.35; P<.0001). Furthermore, fibrinogen was reduced from 12.6 (11.1–14.7) to 11.5 (9.90–13.3) µM (P<.001), and plasminogen activator inhibitor antigen was reduced from 40.5 (28.4–49.4) to 24.4 (15.4–32.7) ng/mL (P<.0001). Physical training after RYGB increased fibrinolytic activity from 58.0 (36.0–75.5) to 88.0 (66.0–132.0) IU/mL compared with 52.5 (30.0–80.0) to 64.0 (49.0–100.0) IU/mL in controls (P<.01) and reduced plasminogen activator inhibitor antigen from 23.5 (16.7–35.4) to 18.1 (14.3–25.4) ng/mL compared with 24.4 (13.9–28.7) to 24.2 (14.1–29.6) ng/mL in controls (P<.05). No effects of physical training were observed on markers of coagulation activation and fibrin clot properties. ConclusionWe observed favorable long-term reductions in markers of thrombin generation, improved fibrin clot properties, and increases in fibrinolysis after RYGB. Supervised physical training after RYGB further increased fibrinolysis.

Effects of ultra-low-dose versus standard hormone therapy on fibrinolysis and thrombin generation in postmenopausal women

ObjectiveTo assess the effects of different doses of oral hormone therapy (HT) on thrombin generation and fibrinolysis. Study designOne hundred fifty postmenopausal women were assigned in a randomized controlled study in which the effect of standard dose (1mg 17β-estradiol/5mg dydrogesterone), ultra-low-dose HT (0.5mg 17β-estradiol/2.5mg dydrogesterone) on fibrinolysis and coagulation was compared to controls. Factors measured included plasma clot lysis time (CLT), fibrinolysis activators and inhibitors, thrombin generation (prothrombin fragments 1+2 [F1+2], endogenous thrombin potential [ETP]), normalized activated protein C sensitivity ratio (nAPCsr), and factor (F)VIII activity and were determined before and after 24 weeks of HT. ResultsIn ultra-low-dose group posttreatment CLT tended to be shorter when compared to standard HT (−7%) and control group (−4%) with reduced plasminogen activator inhibitor-1 (PAI-1) antigen (by −16% vs both groups). Higher mean changes from baseline between ultra-low-HT group and standard group in CLT (−7.4 vs 3.05) and PAI-1 antigen concentration (−5.1 vs 0.9) were observed. Standard HT, compared with the ultra-low-dose HT, led to higher F1+2 (+9%) and FVIII activity (+37%). ConclusionsIn contrast to the standard HT, ultra-low-dose HT may enhance fibrinolysis through reduced PAI-1 levels.

Food Extrusion Technology: Initiatives to Address Food and Nutrition Insecurity in South Africa

The use of extrusion can be regarded as beneficial due to its short production time and wide variety of foods produced by this method. South Africa as a developing country has been involved in food extrusion since the 1980’s and this technology is gaining momentum in academic research areas. A number of research efforts related to extrusion in South Africa have shown the consumption of extruded dry beans can reduce plasminogen activator inhibitor levels in hyperlipidaemic men; the production of sorghum-cowpea extruded instant porridge resulted in a nutritional acceptable product and can be used to supplement the diet of young children to assist with protein deficiencies. Furthermore, research has proven extruder parameters play a role in the outcome of the product and can influence product properties. Based on these research initiatives, Vaal University of Technology/Centre of Sustainable Livelihoods (VUT/CSL) has acquired an Extrusion Pilot Plant to implement interdisciplinary research of nutrition and engineering science. The research will look at process optimisation studies to obtain maximum product output and evaluating nutritional compositions of the products under various conditions. It is hoped the future research efforts at VUT/CSL will address food and nutrition insecurity and showcase the pilot plant as a testing facility and potential advancement to commercialisation.

Arm-Cranking Exercise Training Reduces Plasminogen Activator Inhibitor 1 in People With Spinal Cord Injury

ObjectiveTo investigate the effects of arm-cranking exercise training on plasminogen activator inhibitor 1 (PAI-1) as a risk factor of deep vein thrombosis, along with general physical parameters such as muscle strength, aerobic capacity, and hemodynamics, in individuals with spinal cord injury (SCI) and control subjects. DesignLongitudinal study. SettingCommunity-based supervised intervention. ParticipantsParticipants (N=17) comprised individuals with SCI (n=9) who volunteered for this study, and able-bodied individuals (n=8) matched for age, height, and body mass index who were assessed at baseline only. InterventionThe arm-cranking exercise program was performed for 10 weeks with 4 sessions per week. Sessions consisted of 2 sets of warmup (5min) and arm crank exercises (25min) with a 10-minute recovery at an intensity of 50% to 70% of heart rate reserve. Main Outcome MeasuresBody mass (BM), waist circumference (WC), aerobic capacity (peak oxygen consumption [Vo2peak]), PAI-1, blood pressure, glucose metabolism, and lipids. ResultsPAI-1, BM, WC, systolic blood pressure, and triglycerides (TG) decreased, and Vo2peak increased after training (P<.05, respectively). Spearman rank-order analysis revealed that changes in PAI-1 were related to changes in Vo2peak, BM, WC, TG, and high-density lipoprotein cholesterol. Multiple linear regression analysis revealed that WC was the most sensitive factor for predicting changes in PAI-1 (P=.038). ConclusionsThese results suggest that 10 weeks of arm-cranking exercise training for people with SCI may help to reduce the risk factors of cardiovascular disease. In addition, changes in abdominal fat may be related to changes in PAI-1 in the SCI population.

Tongqiaohuoxue decoction ameliorates obesity-induced inflammation and the prothrombotic state by regulating adiponectin and plasminogen activator inhibitor-1

Ethnopharmacological relevanceTongqiaohuoxue decoction (THD), a water extract of a mixture of eight species of medicinal herbs, has been used for the treatment of blood stasis and hypercoagulation in traditional East Asian medicine since 18th century. Aim of the studyTo investigate the in vivo efficacy of THD using high-fat diet (HFD)-induced obese mice with chronic inflammation and a prothrombotic state as an early vascular model. Materials and methodsTHD was prepared by hot water extraction and freeze-drying. Male C57BL/6 mice were divided into three groups. Group 1 (NC) mice were fed normal chow. Mice in group 2 (HFD) and 3 (HFD+THD) were fed with HFD for 12 weeks. In addition, Group 3 mice were administered with 100mg/kg body weight THD for 4 weeks after onset of obesity by HFD for 8 weeks. Glucose tolerance tests and histological tissue examinations were performed. The levels of adipokines, inflammatory markers, and prothrombotic markers were assessed. ResultsThe oral administration of THD for 4 weeks had no effect on the liver, adipose tissue, or total body weight when the HFD and HFD+THD groups were compared. Nevertheless, mice treated in THD interestingly showed a significant increase in adiponectin in blood and adipose tissue. To verify the effect of THD on adiponectin, 3T3-L1 adipocytes were treated with THD; it stimulated adiponectin production in a dose-dependent manner. In the HFD+THD group, pro-inflammatory cytokines were significantly down-regulated in the blood, adipose tissue, and liver. Insulin resistance was also notably improved by THD. Simultaneously, THD significantly reduced plasminogen activator inhibitor-1 (PAI-1) levels in serum, adipose tissue, and liver. Fibrin deposition and tPA activity, downstream targets of PAI-1, were also notably reduced in the HFD+THD group compared to the HFD group. ConclusionsTHD improved obesity-induced inflammation and insulin resistance by increasing adiponectin production. Additionally, THD administration exerted an anti-thrombotic effect through the regulation of PAI-1 and fibrinolysis. This study demonstrates the efficacy of a traditional East Asian medicine by providing scientific evidence and suggesting a possible mechanism of action.

Effects of combination PPARγ agonist and angiotensin receptor blocker on glomerulosclerosis

We previously observed that high-dose angiotensin receptor blocker (ARB) can induce regression of existing glomerulosclerosis. We also found that proliferator-activated recepto-γ (PPARγ) agonist can attenuate glomerulosclerosis in a nondiabetic model of kidney disease, with specific protection of podocytes. We now assessed effects of combination therapy with ARB and pioglitazone on established glomerulosclerosis. Sprague-Dawley male rats underwent 5/6 nephrectomy (5/6 Nx) at week 0 and renal biopsy at week 8. Rats were randomized to groups with equal starting moderate glomerulosclerosis, and treated with ARB, PPARγ agonist (pioglitazone), combination or vehicle from weeks 8 to 12. Body weight, systolic blood pressure (SBP), and urinary protein (UP) were measured at intervals. In rats with established sclerosis, SBP, UP, and GS were equal in all groups at week 8 before treatment by study design. Untreated control rats had hypertension, decreased GFR, and progressive proteinuria and glomerulosclerosis at week 12. Only combination therapy significantly ameliorated hypertension and proteinuria. ARB alone or pioglitazone alone had only numerically lower SBP and UP than vehicle at week 12. Both pioglitazone alone and combination had significantly less decline in GFR than vehicle. Combination-induced regression of glomerulosclerosis in more rats from weeks 8 to 12 than ARB or pioglitazone alone. In parallel, combination treatment reduced plasminogen activator inhibitor-1 expression and macrophage infiltration, and preserved podocytes compared with vehicle. These results were linked to increased AT2 receptor and Mas1 mRNA in the combination group. PPARγ agonists in combination with ARB augment regression of glomerulosclerosis, with downregulation of injurious RAAS components vs PPARγ alone, with increased anti-fibrotic/healing RAAS components, enhanced podocyte preservation, and decreased inflammation and profibrotic mechanisms.

Protective Role of PEDF-Derived Synthetic Peptide Against Experimental Diabetic Nephropathy.

Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.

Treatment with oestrogen-receptor agonists or oxytocin in conjunction with exercise protects against myocardial infarction in ovariectomized rats.

What is the central question of this study? Could the activation of oxytocin or oestrogen receptors be protective against myocardial injury after ovariectomy? If so, would exercising have an additional ameliorating effect? What is the main finding and its importance? The results revealed that when accompanied by exercise, both oestrogen receptor agonists and oxytocin improved cardiac dysfunction, inhibited the generation of pro-inflammatory cytokines and reduced myocardial injury in ovariectomized female rats, suggesting a new approach for protecting postmenopausal women against ischaemia-induced myocardial injury. To investigate the putative protective effects of oxytocin or oestrogen receptor agonists against myocardial injury of ovariectomized sedentary or exercised rats, female Sprague-Dawley rats assigned to sham-operated control and ovariectomized (OVX) groups were kept sedentary or undertook swimming exercise for 4 weeks and were treated with saline, an oestrogen receptor (ER) β (DPN) or ERα agonist (PPT) or oxytocin. Ovariectomy increased weight gain and anxiety in sedentary rats, whereas exercise prevented weight gain. When accompanied by exercise, both ER agonists and oxytocin inhibited weight gain and anxiety; oxytocin, in the absence or presence of exercise, increased the left ventricular diastolic dimensions and ejection fraction, whereas ER agonists also increased left ventricular diameter when given to exercised rats. Upon the induction of myocardial ischaemia-reperfusion in the OVX rats, plasma creatine kinase-(muscle-brain) was depressed by PPT and oxytocin, whereas DPN, PPT and OT reduced plasminogen activator inhibitor-1 concentrations. The increased tumour necrosis factor-α concentration in OVX rats was also suppressed by exercise or DPN, PPT or oxytocin treatments, whereas the interleukin-6 concentration was diminished by all the treatments when given in conjunction with exercise. Disorganization of cardiac muscle fibres was reduced in all exercised rats. Oestrogen receptor agonists, as well as oxytocin, in conjunction with exercise may be effective new therapeutics to protect against myocardial ischaemia in postmenopausal women.

Anti-Diabetic Effects of Madecassic Acid and Rotundic Acid.

Anti-diabetic effects of madecassic acid (MEA) and rotundic acid (RA) were examined. MEA or RA at 0.05% or 0.1% was supplied to diabetic mice for six weeks. The intake of MEA, not RA, dose-dependently lowered plasma glucose level and increased plasma insulin level. MEA, not RA, intake dose-dependently reduced plasminogen activator inhibitor-1 activity and fibrinogen level; as well as restored antithrombin-III and protein C activities in plasma of diabetic mice. MEA or RA intake decreased triglyceride and cholesterol levels in plasma and liver. Histological data agreed that MEA or RA intake lowered hepatic lipid droplets, determined by ORO stain. MEA intake dose-dependently declined reactive oxygen species (ROS) and oxidized glutathione levels, increased glutathione content and maintained the activity of glutathione reductase and catalase in the heart and kidneys of diabetic mice. MEA intake dose-dependently reduced interleukin (IL)-1β, IL-6, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels in the heart and kidneys of diabetic mice. RA intake at 0.1% declined cardiac and renal levels of these inflammatory factors. These data indicated that MEA improved glycemic control and hemostatic imbalance, lowered lipid accumulation, and attenuated oxidative and inflammatory stress in diabetic mice. Thus, madecassic acid could be considered as an anti-diabetic agent.

Extracellular DNA and histones as thrombus stabiliser.

Recent work suggests that extracellular (cell-free) nucleic acids, both DNA and RNA species, as well as extracellular nucleosomes can activate blood coagulation and promote thrombosis in vitro and in vivo (1, 2). Indeed, they substantially contribute to the development of microvascular thrombosis which can act as an intravascular immune response to circulating bacteria and has been termed immunothrombosis (3). However, they also foster pathological vessel occlusions as shown in animal models of large vessel thrombosis including deep-vein thrombosis and arterial thrombosis. Large filaments of extracellular nucleosomes that are released from activated neutrophils (neutrophil extracellular traps, NETs) and isolated extracellular nucleosomes support fibrin formation and platelet adhesion/activation via different mechanisms. These include for example stimulation of fibrin formation by degradation of the endogenous anticoagulant tissue factor pathway inhibitor and via activation of proteins involved in the contact pathway of blood coagulation such as factor XII (2, 3). The study by Varju et al. (4) adds a new aspect to the procoagulant role of nucleosome components and NETs. These authors show that isolated eukaryotic DNA and histone proteins such as HIIIS enhance the diameter of thrombus-forming fibrin fibers under in vitro conditions. Moreover, they are found to decrease the permeability of fibrin clots and to enhance the overall stability of such clots. Mechanistically, these effects are mediated by regulation of endogenous anticoagulant and fibrinolytic pathways. Indeed, histone proteins prevented the inactivation of thrombin by antithrombin and DNA reduced plasminogen activation. Furthermore, NETs released from isolated neutrophils suppressed t-PA-promoted fibrinolysis. Together with earlier work, these results further corroborate the conclusion that extracellular nucleosomes and their major components target the coagulation system at different levels and that their overall effect is to enhance fibrin formation. It will be of interest to clarify in the future whether the stabilization of fibrin clots by histones, DNA and nucleosomes as shown in (4) contributes to the prothrombotic role of NETs and extracellular nucleosomes in vivo.

Low activity of plasminogen activator: a common feature of non- iatrogenic comorbidities of schizophrenia.

Understanding the pathogenesis of non-iatrogenic comorbidities of schizophrenia may provide insights into the pathogenesis of schizophrenia itself. First-episode, drug-naïve schizophrenia patients are at high risk of thromboembolic events, diseases related to substance abuse, sexual dysfunction, reproductive disorders, inflammatory and autoimmune diseases, as well as complications of hyperinsulinemia or hyperhomocysteinemia. This review focuses on the role of reduced plasminogen activator activity in non-iatrogenic comorbidity of schizophrenia. By preventing thrombus dissolution, low tissue plasminogen activator activity increases the risk of thrombotic events. Components of the plasminogen activator system also play a key role in reproduction. Both illicit drugs and tobacco increase plasminogen activator levels in the central nervous system, which seems to relieve symptoms of the mental disorder. Chronic alcoholism, sexual dysfunction, inflammatory and autoimmune disorders, and complications of hyperinsulinemia or hyperhomocysteinemia are somehow related to low plasminogen activator activity. Plasminogen activator mediates several neurochemical processes that seem to prevent or reverse gray-matter atrophy seen in first-episode schizophrenia patients. Such processes include cleavage of brain-derived neurotrophic factor precursor to an anti-apoptotic neurotrophin and activation of N-methyl-D-aspartate receptor. Controlled, randomized studies are needed to determine if measures aimed at correcting plasminogen activator activity can improve the quality of life, reduce morbidity and mortality rates, and particularly improve the course of schizophrenia.