Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

BACKGROUND: A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. OBJECTIVE: To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. METHODS: Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. RESULTS: Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. CONCLUSIONS: DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

Phosphoglycerate kinase (PGK) 1 succinylation modulates epileptic seizures and the blood-brain barrier.

Epilepsy is the most common chronic disorder in the nervous system, mainly characterized by recurrent, periodic, unpredictable seizures. Post-translational modifications (PTMs) are important protein functional regulators that regulate various physiological and pathological processes. It is significant for cell activity, stability, protein folding, and localization. Phosphoglycerate kinase (PGK) 1 has traditionally been studied as an important adenosine triphosphate (ATP)-generating enzyme of the glycolytic pathway. PGK1 catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP, producing 3-phosphoglycerate (3-PG) and ATP. In addition to cell metabolism regulation, PGK1 is involved in multiple biological activities, including angiogenesis, autophagy, and DNA repair. However, the exact role of PGK1 succinylation in epilepsy has not been thoroughly investigated. The expression of PGK1 succinylation was analyzed by Immunoprecipitation. Western blots were used to assess the expression of PGK1, angiostatin, and vascular endothelial growth factor (VEGF) in a rat model of lithium-pilocarpine-induced acute epilepsy. Behavioral experiments were performed in a rat model of lithium-pilocarpine-induced acute epilepsy. ELISA method was used to measure the level of S100ß in serum brain biomarkers' integrity of the blood-brain barrier. The expression of the succinylation of PGK1 was decreased in a rat model of lithium-pilocarpine-induced acute epilepsy compared with the normal rats in the hippocampus. Interestingly, the lysine 15 (K15), and the arginine (R) variants of lentivirus increased the susceptibility in a rat model of lithium-pilocarpine-induced acute epilepsy, and the K15 the glutamate (E) variants, had the opposite effect. In addition, the succinylation of PGK1 at K15 affected the expression of PGK1 succinylation but not the expression of PGK1total protein. Furthermore, the study found that the succinylation of PGK1 at K15 may affect the level of angiostatin and VEGF in the hippocampus, which also affects the level of S100ß in serum. In conclusion, the mutation of the K15 site of PGK1 may alter the expression of the succinylation of PGK1 and then affect the integrity of the blood-brain barrier through the angiostatin / VEGF pathway altering the activity of epilepsy, which may be one of the new mechanisms of treatment strategies.

Extracellular vesicle therapy attenuates antiangiogenic signaling in ischemic myocardium of swine with metabolic syndrome.

OBJECTIVE: Our recent studies using a porcine model of metabolic syndrome (MS) and chronic myocardial ischemia show that extracellular vesicle (EV) therapy improves blood flow and arteriogenesis in ischemic myocardium, although mechanisms of these changes are unclear. We hypothesized that in the setting of MS, EV therapy would decrease antiangiogenic signaling to mediate increased blood flow to chronically ischemic myocardium. METHODS: Yorkshire swine were fed a high-fat diet for 4 weeks to induce MS, then underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic myocardial ischemia. Two weeks later, pigs underwent intramyocardial injection of vehicle (control, n = 6) or human bone marrow-derived EVs (n = 8). Five weeks later, left ventricular myocardium in ischemic territory was harvested. Protein expression was measured using immunoblot analysis, and data were analyzed using Wilcoxon rank sum test. Myocardial perfusion was measured with isotope-labeled microspheres, and correlation data were analyzed using Spearman rank correlation coefficient. RESULTS: EV treatment was associated with decreased expression of antiangiogenic proteins, angiostatin (P < .001) and endostatin (P = .043) in ischemic myocardium compared with control. In EV-treated pigs, there was a negative correlation between blood flow to ischemic myocardium and angiostatin (rs = -0.76; P = .037), but not endostatin expression (rs = .02; P = .98). EV treatment was also associated with decreased cathepsin D, which cleaves precursors to produce angiostatin and endostatin, in ischemic myocardium (P = .020). CONCLUSIONS: In the setting of MS and chronic myocardial ischemia, EV therapy is associated with decreased expression of antiangiogenic proteins, which might contribute to increased blood flow to chronically ischemic myocardium.

Experimental study on anti angiogenesis of recombinant mouse angiostatin gene in mice with gallbladder carcinoma.

Gallbladder cancer is one of the gastrointestinal tumors with an extremely poor prognosis. Its incidence rate is gradually increasing worldwide, and the rate of radical resection surgery is extremely low. Not sensitive to radiotherapy and chemotherapy, with a very poor prognosis. This study aimed to investigate whether the recombinant mouse angiostatin gene transfected anti-angiogenic gallbladder cancer cells can express angiostatin protein with the activity of inhibiting the growth of vascular endothelial cells and the inhibitory effect on the growth of gallbladder cancer. The recombinant mouse angiostatin gene eukaryotic expression plasmid was transfected into the gallbladder cancer cell line by applying liposome LIPOFECTAMINE 2000, and its activity was detected by vascular endothelial cell proliferation analysis. The results show that angiostatin can inhibit the growth of transplanted gallbladder cancer, and as the number of injections increases, the inhibition rate of gallbladder cancer growth also increases. At the end of the experiment, the total inhibition rate of gallbladder cancer growth reached 95% 5%, 20%, 30%, 40% gradually increase. Therefore, angiostatin has potential clinical application value in gene therapy of gallbladder cancer.

The essential anti-angiogenic strategies in cartilage engineering and osteoarthritic cartilage repair.

In the cartilage matrix, complex interactions occur between angiogenic and anti-angiogenic components, growth factors, and environmental stressors to maintain a proper cartilage phenotype that allows for effective load bearing and force distribution. However, as seen in both degenerative disease and tissue engineering, cartilage can lose its vascular resistance. This vascularization then leads to matrix breakdown, chondrocyte apoptosis, and ossification. Research has shown that articular cartilage inflammation leads to compromised joint function and decreased clinical potential for regeneration. Unfortunately, few articles comprehensively summarize what we have learned from previous investigations. In this review, we summarize our current understanding of the factors that stabilize chondrocytes to prevent terminal differentiation and applications of these factors to rescue the cartilage phenotype during cartilage engineering and osteoarthritis treatment. Inhibiting vascularization will allow for enhanced phenotypic stability so that we are able to develop more stable implants for cartilage repair and regeneration.

Exercise training ameliorates cognitive dysfunction in amyloid beta-injected rat model: possible mechanisms of Angiostatin/VEGF signaling.

Vascular endothelial growth factor (VEGF) regulates angio/neurogenesis and also tightly links to the pathogenesis of Alzheimer's disease (AD). Although exercise has a beneficial effect on neurovascular function and cognitive function, the direct effect of exercise on VEGF-related signaling and cognitive deficit in AD is incompletely understood. Therefore, the purpose of this study was to investigate the protective effect of exercise on angiostatin/VEGF cascade and cognitive function in AD model rats. Wistar male rats were randomly divided into five groups: control (CON), injection of DMSO (Sham-CON), CON-exercise (sham-EX), intrahippocampal injection of Aß (Aß), and Aß-exercise (Aß-EX). Rats in EX groups underwent treadmill exercise for 4 weeks, then the cognitive function was measured by the Morris Water Maze (MWM) test. mRNA levels of hypoxia-induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and angiostatin were determined in hippocampus by RT-PCR. We found that spatial learning and memory were impaired in Aß-injected rats, but exercise training improved it. Moreover, exercise training increased the reduced mRNA expression level of VEGF signaling, including HIF1α, VEGF, and VEGFR2 in the hippocampus from Aß-injected rats. Also, the mRNA expression level of angiostatin was elevated in the hippocampus from Aß-injected rats, and exercise training abrogated its expression. Our findings suggest that exercise training improves cognitive function in Aß-injected rats, possibly through enhancing VEGF signaling and reducing angiostatin.

The Role of Fibrinolytic Regulators in Vascular Dysfunction of Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by vascular dysfunction and extensive fibrosis of the skin and visceral organs. Vascular dysfunction is caused by endothelial cell (EC) apoptosis, defective angiogenesis, defective vasculogenesis, endothelial-to-mesenchymal transition (EndoMT), and coagulation abnormalities, and exacerbates the disease. Fibrinolytic regulators, such as plasminogen (Plg), plasmin, α2-antiplasmin (α2AP), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and angiostatin, are considered to play an important role in the maintenance of endothelial homeostasis, and are associated with the endothelial dysfunction of SSc. This review considers the roles of fibrinolytic factors in vascular dysfunction of SSc.

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

Effect of titanium-prepared platelet-rich fibrin treatment on the angiogenic biomarkers in gingival crevicular fluid in infrabony defects of patients with chronic periodontitis: A randomized controlled clinical trial.

AIM: The aim of this double-blinded, randomized, controlled clinical study was to investigate the effect of titanium-prepared platelet-rich fibrin (T-PRF) treatment on the angiogenic biomarkers in gingival crevicular fluid (GCF) in infrabony defects of patients with chronic periodontitis. MATERIALS AND METHODS: Twenty five systemically healthy participants who complied with inclusion criteria with periodontal infrabony defects were recruited. In each patient, the infrabony defect of one side of arch was designated as control group (allograft), whereas the infrabony defect on the contralateral side of same arch was designated as test group (allograft + T-PRF). The therapy methods (test or control) were randomly decided. GCF samples were collected at baseline (presurgery) and then the 3rd, 7th, 14th, and 30th days after surgery. Platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF)-2, anjiogenin (ANG), angiostatin (ANT) in the GCF samples were measured using human enzyme-linked immunosorbent assay kits. RESULTS: In both groups, total amounts of PDGF-BB, VEGF-A, FGF-2, ANG, and ANT peaked in the GCF samples obtained at the early postoperative day (day 3) and decreased over time in the samples obtained at the 7th, 14th, and 30th days postsurgery. There were no significant differences between groups for the total amounts of PDGF-BB, VEGF-A, FGF-2, ANG, and ANT at all evaluation periods. CONCLUSION: Application of T-PRF combined with allograft in infrabony defects of patients with chronic periodontitis had no significant effects on angiogenic biomarkers in GCF.

Safety and Efficacy of OXB-202, a Genetically Engineered Tissue Therapy for the Prevention of Rejection in High-Risk Corneal Transplant Patients.

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye, corneal transplantation is one of the most successful clinical transplant procedures. However, in high-risk patients, which account for >20% of the 180,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient cornea. The main reason for graft failure is irreversible immunological rejection, and it is therefore unsurprising that neovascularization (NV; both pre and post grafting) is a significant risk factor for subsequent graft failure. NV is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the equine infectious anemia virus called pONYK1EiA, which subsequently prevents rejection by suppressing NV. Previously, it has been shown that rabbit donor corneas treated with pONYK1EiA substantially suppress corneal NV, opacity, and subsequent rejection in an aggressive rabbit model of cornea graft rejection. Here, efficacy data are presented in a second rabbit model, which more closely mirrors the clinical setting for high-risk corneal transplant patients, and safety data from a 3-month good laboratory practice toxicology and biodistribution study of pONYK1EiA-modified rabbit corneas in a rabbit corneal transplant model. It is shown that pONYK1EiA-modified rabbit corneas (OXB-202) significantly reduce corneal NV and the rate of corneal rejection in a dose-dependent fashion, and are tolerated with no adverse toxicological findings or significant biodistribution up to 13 weeks post surgery in these rabbit studies. In conclusion, angiogenesis is a valid target to prevent corneal graft rejection in a high-risk setting, and transplanted genetically modified corneas are safe and well-tolerated in an animal model. These data support the evaluation of OXB-202 in a first-in-human trial.

Filamin A interacting protein 1-like expression inhibits progression in colorectal cancer.

Filamin A interacting protein 1-like (FILIP1L) expression, which is decreased in various cancers, may inhibit carcinogenesis. In this study, we evaluated the effects of FILIP1L on oncogenic behavior and prognosis in colorectal cancer. siRNA-mediated FILIP1L knockdown enhanced tumor cell migration and invasion and inhibited apoptosis and cell cycle arrest in COLO205 cells. pcDNA-myc vector-mediated FILIP1L overexpression suppressed tumor cell migration and invasion and induced apoptosis and cell cycle arrest in HCT116 cells. FILIP1L knockdown enhanced angiogenesis by increasing VEGF-A and HIF-1α levels and decreasing angiostatin level. FILIP1L overexpression suppressed angiogenesis by decreasing VEGF-A and -D l level and increasing angiostatin and endostatin levels. Phosphorylated ß-catenin levels decreased and phosphorylated Akt and GSK-3ß levels increased following FILIP1L knockdown. FILIP1L overexpression had the opposite effects. FILIP1L expression was associated with reductions in tumor size, cell differentiation, lymphovascular invasion, stage, invasion depth and lymph node metastasis, and with longer overall survival. Mean Ki-67 labeling indexes and microvessel density values were lower in FILIP1L-positive tumors than in FILIP1L-negative tumors. These results indicate that FILIP1L suppresses tumor progression by inhibiting cell proliferation and angiogenesis in colorectal cancer.

Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors Reduce Reactive Gliosis and Improve Angiostatin Levels in Retina of Diabetic Rats.

Diabetic retinopathy (DR) is a multifactorial disease characterized by reactive gliosis and disbalance of angiogenesis regulators, contributing to endothelial dysfunction and microvascular complications. This study was organized to elucidate whether poly(ADP-ribose) polymerase-1 (PARP-1) inhibition could attenuate diabetes-induced damage to macroglia and correct angiogenic disbalance in diabetic rat retina. After 8 weeks of streptozotocin (STZ)-induced diabetes, Wistar male rats were treated with PARP-1 inhibitors, nicotinamide (NAm) or 3-aminobenzamide (3-AB) (100 and 30 mg/kg/daily i.p., respectively), for 14 days. After the 10-weeks experiment period, retinas were undergone an immunohistochemical staining for glial fibrillary acidic protein (GFAP), while western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of PARP-1, poly(ADP-ribosyl)ated proteins (PARs), GFAP, and angiostatin isoforms. Diabetes induced significant up-regulation and activation of retinal PARP-1, reactive gliosis development, and GFAP overexpression compared to non-diabetic control. Moreover, extensive fragmentation of both PARP-1 and GFAP (hallmarks of apoptosis and macroglia reactivation, respectively) in diabetic retina was also observed. Levels of angiostatin isoforms were dramatically decreased in diabetic retina, sustaining aberrant pro-angiogenic condition. Both NAm and 3-AB markedly attenuated damage to macroglia, evidenced by down-regulation of PARP-1, PARs and total GFAP compared to diabetic non-treated group. PARP-1-inhibitory therapy prevented formation of PARP-1 and GFAP cleavage-derived products. In retinas of anti-PARP-treated diabetic animals, partial restoration of angiostatin's levels was shown. Therefore, PARP-1 inhibitors counteract diabetes-induced injuries and manifest retinoprotective effects, including attenuation of reactive gliosis and improvement of angiogenic status, thus, such agents could be considered as promising candidates for DR management.

Aerobic training and l-arginine supplementation promotes rat heart and hindleg muscles arteriogenesis after myocardial infarction.

Arteriogenesis is a main defense mechanism to prevent heart and local tissues dysfunction in occlusive artery disease. TGF-ß and angiostatin have a pivotal role in arteriogenesis. We tested the hypothesis that aerobic training and l-arginine supplementation promotes cardiac and skeletal muscles arteriogenesis after myocardial infarction (MI) parallel to upregulation of TGF-ß and downregulation of angiostatin. For this purpose, 4 weeks after LAD occlusion, 50 male Wistar rats were randomly distributed into five groups: (1) sham surgery without MI (sham, n = 10), (2) control-MI (Con-MI, n = 10), (3) l-arginine-MI (La-MI, n = 10), (4) exercise training-MI (Ex-MI, n = 10), and (5) exercise and l-arginine-MI (Ex + La-MI). Exercise training groups running on a treadmill for 10 weeks with moderate intensity. Rats in the l-arginine-treated groups drank water containing 4 % l-arginine. Arteriolar density with different diameters (11-25, 26-50, 51-75, and 76-150 µm), TGF-ß, and angiostatin gene expression were measured in cardiac (area at risk) and skeletal (soleus and gastrocnemius) muscles. Smaller arterioles decreased in cardiac after MI. Aerobic training and l-arginine increased the number of cardiac arterioles with 11-25 and 26-50 µm diameters parallel to TGF-ß overexpression. In gastrocnemius muscle, the number of arterioles/mm(2) was only increased in the 11 to 25 µm in response to training with and without l-arginine parallel to angiostatin downregulation. Soleus arteriolar density with different size was not different between experimental groups. Results showed that 10 weeks aerobic exercise training and l-arginine supplementation promotes arteriogenesis of heart and gastrocnemius muscles parallel to overexpression of TGF-ß and downregulation of angiostatin in MI rats.

Combinatorial anti-angiogenic gene therapy in a human malignant mesothelioma model.

Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos with poor prognosis. In the present study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F and MSTO-C were confirmed by western blot analysis and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, co-culture of MSTO-A, MSTO-E and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E and MSTO-F significantly enhanced efficacy. These results suggest that combinatorial anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen.

[Role of angiostatins in diabetic complications].

Angiogenesis is a process through which new blood vessels form from pre-existing vessels. Angiogenesis is regulated by a number of factors of peptide nature. Disbalance of angiogenic system appears to be the major causative factor contributing vascular abnormalities in diabetes mellitus, resulting in various complications. Angiostatins, which are kringle-containing fragments of plasminogen/plasmin, are known to be powerful physiological inhibitors of neovascularization. In the present review, current literature data on peculiarities of production of angiostatins and their functioning at diabetes mellitus are summarized and analyzed for the first time. Also, role of angiostatins in the pathogenesis of typical diabetic complications, including retinopathies, nephropathies and cardiovascular diseases, is discussed. Data presented in this review may be useful for elaboration of novel effective approaches for diagnostics and therapy of vascular abnormalities in diabetes mellitus.

Pigment epithelium-derived factor mediates impaired lung vascular development in neonatal hyperoxia.

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Effects of p75 neurotrophin receptor on regulating hypoxia-induced angiogenic factors in retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) exerts critical roles in the maintenance of the normal functions of the retina, whereas RPE dysfunction can induce retina neovascularization. p75 neurotrophin receptor (p75(NTR)) has been shown to play essential roles in angiogenesis. However, the function of p75(NTR) in the RPE remains unclear. In the present study, we demonstrated that p75(NTR) was highly expressed in the human choroidal neovascularization membranes. For in vitro study, RPE was exposed to hypoxia, and a knockdown of p75(NTR) was achieved via lentivirus-mediated RNA interference. The results showed that hypoxia induced the expression of p75(NTR) in the RPE, and the knockdown of p75(NTR) rescued RPE proliferation activity and inhibited apoptosis which induced by hypoxia. After the deletion of p75(NTR), RPE-secreted pro-angiogenic factors (vascular endothelial growth factor and platelet-derived growth factor), inflammatory factors [interleukin 1 beta (IL1ß), IL18, and stromal cell-derived factor 1], and matrix metalloproteinases (MMPs) (MMP3 and MMP9) were down-regulated under hypoxic conditions. While the RPE secreted anti-angiogenic factors (pigment epithelium-derived factor) and angiostatin, the tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1 and TIMP-3) were up-regulated after the knockdown of p75(NTR). The human umbilical vein endothelial tube formation ability can be inhibited when it is co-cultured with the supernatant extract from p75(NTR)-knockdown RPE under hypoxic induction. These results suggest that the knockdown of p75(NTR) suppressed pro-angiogenic factors which induced by hypoxia while promoting the anti-angiogenesis-related factors in the RPE. It is indicated that p75(NTR) could be a potential therapeutic target for RPE hypoxia or oxidative stress diseases.

PLASMINOGEN AND ANGIOSTATIN LEVELS IN FEMALE BENIGN BREAST LESIONS.

It is known that benign breast tissue exhibit relatively low angiogenic capacity. Activation of angiogenesis in mammary pre-malignant lesions could be associated with disease progression and high risk of transformation into the breast cancer. However, insight into the underlying molecular mechanisms involved in angiogenesis regulation in non-cancerous breast pathologies is still poorly defined. The purpose of the present study was to determine levels of plasminogen and its proteolytic fragments (angiostatins) in mammary dysplasia (mastopathy and breast cyst) and benign neoplasms (fibroadenomas). Plasminogen and angiostatins were analyzed using immunoblotting and quantified by densitometric scanning. The significant increase in plasminogen levels was found in fibrocystic, cysts, and non-proliferatious fibroadenoma masses (4.7-, 3.7-, and 3.5-fold, respectively) compared to healthy breast tissues (control). In the same benign lesions, 6.7-, 4-, and 3.7-fold increase in plasminogen 50 kDa fragment (angiostatin) levels as compared with control were also observed. Activation of matrix metalloproteinase-9, which was detected using gelatine zymography, could be responsible for plasminogen cleavage and abundance of angiostatin infibrocystic and cyst masses. In contrast, dramatic decrease of both plasminogen and angiostatin levels (3.8- and 5.3-folds, respectively) was shown in tissues of proliferatious form of fibroadenoma in comparison with that of the dormant type of this neoplasm. Based on the obtained results, we concluded that angiostatin, a potent vessel growth inhibitor and anti-inflammatory molecule, can play a crucial role in pathophysiology of non-cancerous breast diseases. Further studies are needed to evaluate potential diagnostic and clinical implications of these proteins for prediction and therapy of benign breast pathologies.

Type IV collagen α1-chain noncollagenous domain blocks MMP-2 activation both in-vitro and in-vivo.

α1(IV)NC1 inhibits angiogenesis by regulating MAPK activation, this biological function was partly attributed α1(IV)NC1 binding to α1ß1-integrin. However, its potent antiangiogenic activity and the molecular targets of α1(IV)NC1 has not been investigated. In the present study, the regulation of MMP-2 activation by α1(IV)NC1 was evaluated. α1ß1-integrin which is required for inhibition of angiogenesis is not playing a role in cellular invasion and inhibition of MMP-2 activation by α1(IV)NC1. We found that α1(IV)NC1 binds the CBD of MMP-2 and forming a stable complex that prevents activation of MMP-2. The antiangiogenic activity of α1(IV)NC1 is mediated, in part, by this binding activity. In addition, up-regulation of TIMP-2 by α1(IV)NC1 led to saturation of MT1-MMP binding sites, which in turn led to inhibition of MMP-2 activation. In-vivo studies using α1-integrin null-mice treated with higher doses of α1(IV)NC1 showed integrin independent inhibition of tumor growth and active-MMP-2, without affecting MMP-9, MMP-7 and angiostatin.

Overexpression of angiomotin in sinonasal inverted papilloma.

BACKGROUND: Although inverted papilloma (IP) is one of the most common sinonasal tumors, its etiology and factors associated with tumor progression have not been fully determined. Generally, tumorigenesis or tumor growth requires angiogenesis to feed tumor cells. Angiomotin is a recently discovered protein that regulates migration and tubule formation in endothelial cells. It has been reported that angiomotin affects angiostatin (circulating inhibitor of angiogenesis), resulting in promotion of angiogenesis. Thus, we evaluated the expression and distribution of angiomotin in sinonasal IP, compared to normal control tissue. METHODS: The study included 10 subjects with sinonasal IP and 5 normal controls. Ethmoid sinus mucosa obtained during reduction of blowout fractures was used as a normal control. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, immunohistochemistry, and Western blot analysis were used to assess the expression, intensity, and distribution of angiomotin in tissues. RESULTS: Positive bands for angiomotin were seen in all specimens by RT-PCR. The expression level of angiomotin was significantly upregulated in IP tissues versus normal sinus mucosa by real-time PCR. Immunohistochemistry revealed positive reactions on endothelial cells of capillaries and small vessels within the tumor and normal tissues, but the positivity was significantly stronger in IP. Western blot analysis showed that expression levels of angiomotin were increased in IP compared to normal sinus mucosa. CONCLUSION: Angiomotin, a novel protein in angiogenesis, was overexpressed in IP. Although it is not an etiological or initiating factor in tumor development, it seems to be associated with progression and growth of IP via promoting angiogenesis.