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Prolonged inflammation and excessive neovascularization of the cornea due to severe injury can impair optical clarity and lead to vision impairment. Plasminogen kringle (K) fragments, known as angiostatins (AS), play a well-established role as inhibitors of neovascularization by suppressing pro-angiogenic signaling­. However, AS effects in the cornea, beyond inhibiting the angiogenesis, are still unexplored. In this study, we estimate the protective effect of two AS variants (K1-3 and K5) against alkali burn injury induced in rabbit and rat corneas. AS K1-3 in the single doses of 0.075 or 0.75 μg (0.1 or 1.0 μM, respectively) or 0.3 μg of AS K5 (1.0 μM) were applied locally as eye drops daily for 14 days after the injury. A significant regression of corneal vessels in-growth in injured eyes treated with AS was revealed. Western blot analysis of corneal tissue lysates revealed that injury-induced overexpression of protein markers­ of hypoxia (HIF-1α), angiogenesis (VEGF), tissue remodeling and fibrosis (MMP-9), autophagy (beclin-1) and endoplasmic reticulum stress (GRP-78) was significantly reduced under AS treatment. Besides, the level of tight junctions protein ZO-1 was shown to be up-regulated after the treatment of the damaged cornea with AS K1-3. Summarizing, our study uncovered novel biological functions of the kringle-containing plasminogen fragments indicating its beneficial effects during corneal healing in the experimental model of alkali burn. The data obtained can be helpful for the development of novel efficient formulations to manage complications of ocular surface injuries.

Aim. Plasminogen kringle 5 is an endogenous angiogenic inhibitor. The purpose of the present review was to highlight the potential biomedical application of kringle 5 in the regulation of angiogenesis and tumor growth. Methods. Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Since the uncontrolled growth of new blood vessels causes the progression of many common diseases, first of all, oncological diseases, autoimmune disorders, neovascular damage of the eye, the use of angiostatins can be a promising pharmacotherapeutic approach to the prevention and adjuvant therapy of these pathological conditions. The advantages of angiostatins application are their non-toxicity even at high doses, non-immunogenicity, lack of tolerance of target cells to their action. Angiostatins comprise a group of kringle-containing proteolytically-derived fragments of plasminogen/plasmin, which act as potent inhibitory mediators of endothelial proliferation and migration. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Results. Current literature data suggest that in addition to expressed and highly specific cytotoxicity in relation to endotheliocytes and some types of tumor cells, the kringle domain 5 of human plasminogen has other advantages as an antiangiogenic and antitumor regulator, including its specific inhibitory activity, which affects only activated, proliferating endothelial cells, and therefore is non-toxic to other types of normal cells. As an endogenous protein, which is formed in the human organism, K5 does not provoke an immune response. K5 as a small polypeptide molecule with a stable structure can be obtained as a recombinant protein in E. coli cells, and can also be used in pharmacokinetic systems of targeted delivery and sustained release. Conclusions. The prospect of successful use of K5 as a therapeutic agent to manage pathological processes associated with dysregulation of angiogenesis makes it necessary to develop and improve methods of its production and to further test its plausible pleiotropic biological activities.

Aim. Plasminogen kringle 5 is an endogenous angiogenic inhibitor. The purpose of the present review was to highlight the potential biomedical application of kringle 5 in the regulation of angiogenesis and tumor growth. Methods. Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Since the uncontrolled growth of new blood vessels causes the progression of many common diseases, first of all, oncological diseases, autoimmune disorders, neovascular damage of the eye, the use of angiostatins can be a promising pharmacotherapeutic approach to the prevention and adjuvant therapy of these pathological conditions. The advantages of angiostatins application are their non-toxicity even at high doses, non-immunogenicity, lack of tolerance of target cells to their action. Angiostatins comprise a group of kringle-containing proteolytically-derived fragments of plasminogen/plasmin, which act as potent inhibitory mediators of endothelial proliferation and migration. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Results. Current literature data suggest that in addition to expressed and highly specific cytotoxicity in relation to endotheliocytes and some types of tumor cells, the kringle domain 5 of human plasminogen has other advantages as an antiangiogenic and antitumor regulator, including its specific inhibitory activity, which affects only activated, proliferating endothelial cells, and therefore is non-toxic to other types of normal cells. As an endogenous protein, which is formed in the human organism, K5 does not provoke an immune response. K5 as a small polypeptide molecule with a stable structure can be obtained as a recombinant protein in E. coli cells, and can also be used in pharmacokinetic systems of targeted delivery and sustained release. Conclusions. The prospect of successful use of K5 as a therapeutic agent to manage pathological processes associated with dysregulation of angiogenesis makes it necessary to develop and improve methods of its production and to further test its plausible pleiotropic biological activities. [ABSTRACT FROM AUTHOR]

Angiostatins (AS) are proteolytically derived fragments of plasminogen with the established antiangiogenic and anti-inflammatory capacities, which can be useful for ocular surface relief after injury. Aim. To investigate the effects of AS K1-3 and K5 on the protein levels of SARSCoV- 2 receptor, ACE2, and marker of activated satellite glia, GFAP, in the injured rat cornea, and to assess if AS could affect the viability of retinal pigment epithelial (RPE) cells. Methods. AS (K1-3 and K5) were produced by limited proteolysis of plasminogen isolated from human plasma followed by affine chromatography purification. AS (K1-3 0.1 or 1.0 µM, K5 0.1 µM) were applied topically as eye drops in rat model of alkali burn of cornea. The protein levels of ACE2 and GFAP were evaluated in corneal tissue lysates by western blot and expressed as arbitrary units (a.u.). The effects of AS on the viability of RPE cells were evaluated by MTT test. Results. The ocular injury caused by alkali burn induced overexpression of both ACE2 (by 7.7 fold vs. control, P < 0.001) and GFAP (by 62 folds vs. control, P < 0.001). Application of K5 or K1-3 (1.0 µM) reduced the expression level of ACE2 in injured corneas by two-folds (P < 0.05 vs. Burn group), whereas K1-3 (0.1 µM) lowered the content of ACE2 by 3.4-folds compared with the Burn group (P < 0.05) suggesting that AS can decrease a potential risk of SARS-CoV-2 entry due to the down-regulation of its receptor expression. Interestingly, K5 appeared to be more effective in suppressing the GFAP overexpression (by 10.9-fold vs. Burn group, P < 0.01) indicating that AS may act as neuroprotective substances via alleviating excessive response of satellite glia in injured cornea. AS had no cytotoxic effects on retinal pigment endothelium in the range of concentrations 2-100 nM. Conclusions. Collectively, our data may pave the way for a possible application of AS in designing an ophthalmic drug as an effective and safe supplementary with combined protective effects. [ABSTRACT FROM AUTHOR]

This article was focused on the evaluation of protein biomarkers related to thrombosis, hypoxia, angiogenesis, and tissue remodeling in tear fluid of patients with non-penetrating corneal trauma. 32 patients with non-penetrating corneal injures were enrolled in the study, the control group consisted of 15 healthy patients. Samples of tear fluid were collected from the patients and control volunteers with the use of a disposable end micropipette. Protein levels of D-dimer, hypoxia-inducible factor 1α (HIF-1α), angiostatins, and matrix metalloproteinase 9 (MMP-9) in tear fluids were determined by western blot analysis. Proteolytic activity values of MMP-9 were measured by gelatin zymography. Results of western blot and zymography assay were calculated by densitometry analysis and expressed as arbitrary units. Significant increase of D-dimer and HIF-1α levels in tear fluid of patients with injured cornea by 7.3 (p

Background and Objectives Microvascular remodeling and angiogenesis are elements of tissue remodeling characteristic of chronic inflammatory diseases, including nasal polyps (NPs). Angiogenesis reflects the balance between the actions of pro- and anti-angiogenic factors. Many pro-angiogenic factors are known, including vascular endothelial growth factor (VEGF). A number of anti-angiogenic factors (e.g., angiostatin and endostatin) also has been identified. Our objective was to assess the roles of VEGF, angiostatin, and endostatin in NP development. Methods The expression levels of VEGF, angiostatin, and endostatin were measured in NPs harvested during endoscopic endonasal surgery and compared with those in inferior turbinate mucosa (control) samples acquired from patients with hypertrophic rhinitis without allergy. Western blotting and immunohistochemical staining were used to analyze all samples. Results The levels of VEGF and angiostatin were significantly higher in the NP subjects than in the controls. Neither the VEGF/angiostatin ratio nor the endostatin level differed significantly between the two groups. However, the VEGF/endostatin ratio was significantly higher in the NP than in the control group. Both the NP and control tissues were diffusely immunoreactive for VEGF, angiostatin, and endostatin. Conclusion NP-associated hypoxia can elevate angiostatin level; moreover, an imbalance in the VEGF/endostatin ratio can contribute to NP formation.

This article was focused on the evaluation of protein biomarkers related to thrombosis, hypoxia, angiogenesis, and tissue remodeling in tear fluid of patients with non-penetrating corneal trauma. 32 patients with non-penetrating corneal injures were enrolled in the study, the control group consisted of 15 healthy patients. Samples of tear fluid were collected from the patients and control volunteers with the use of a disposable end micropipette. Protein levels of D-dimer, hypoxia-inducible factor 1α (HIF-1α), angiostatins, and matrix metalloproteinase 9 (MMP-9) in tear fluids were determined by western blot analysis. Proteolytic activity values of MMP9 were measured by gelatin zymography. Results of western blot and zymography assay were calculated by densitometry analysis and expressed as arbitrary units. Significant increase of D-dimer and HIF-1α levels in tear fluid of patients with injured cornea by 7.3 (p<0.05) and 56 (p<0.001) folds, respectively, was shown compared with control, indicating thrombotic events and hypoxia condition to be involved in pathogenesis of ocular trauma. Dramatically elevated levels/activity of MMP-9 enzyme (by 105 folds vs. control, p<0.001) suggest intense tissue remodeling and degradation of extracellular matrix in the damaged cornea. Up-regulation of angiostatin level, products of proteolytical cleavage of plasminogen, in tear fluid collected from patients with traumatic eye in comparison with healthy volunteers (by 7.3 folds, p<0.05), could represent an adaptive mechanism, which counteracts excessive hypoxia-induced neovascularization in injured cornea. It is summarized that there was a strong association between elevation of D-dimer, HIF-1α, angiostatins, and MMP-9 levels suggesting thrombosis- and hypoxia-mediated mechanisms triggering wound healing of injured cornea. The findings of this study are novel and provide a basis for further investigations of the reparation mechanisms during non-penetrating ocular trauma. Studied proteins of the tear fluid can serve as relevant biomarkers of corneal wound healing and are appropriate for diagnostic and prognostic purposes. [ABSTRACT FROM AUTHOR]

Angiostatins comprise a group of kringle-containing proteolytically-derived plasminogen/plasmin fragments, which act as potent inhibitory mediators of endothelial sells proliferation and migration. Angiostatins are involved in modulation of vessel growth in healthy tissues and various pathological conditions associated with aberrant neovascularization. The aim of the present paper was to summarize available information, including our own experimental data, on prospects of angiostatin application for treatment of ocular neovascular diseases (OND), focusing on retinal pathologies and corneal injury. In particular, literature data on prospective and retrospective studies, clinical trials and animal models relating to the pathophysiology, investigation and management of OND are described. Special emphasis was made on the laboratory approaches of production of different angiostatin isoforms, as well as comparison of antiangiogenic capacities of native and recombinant angiostatin polypeptides. Several studies reported that angiostatins may completely abolish pathologic angiogenesis in diabetic proliferative retinopathy without affecting normal retinal vessel development and without exhibiting adverse side effects. Angiostatins have been tested as a tool for corneal antiangiogenesis target therapy in order to manage diverse ocular surface pathological conditions induced by traumas, chemical burns, previous surgery, chronic contact lens wear, autoimmune diseases, keratitis and viral infections (herpes, COVID-19), corneal graft rejection, etc. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Application of adenoviral genetic construct encoding angiostatin K5 as a promising tool for OND treatment illustrates a vivid example of upcoming revolution in local gene therapy. Further comprehensive studies are necessary to elucidate the clinical potential and optimal regimes of angiostatin-based intervention modalities for treating ocular neovascularization.

Platelets store, produce and release a variety of angiogenesis regulators, which can contribute to both normal tissue repair and angiopathy-associated pathologies. Plasminogen has been earlier shown to regulate some platelet functions, but if it is able to modulate angiogenic capacities of platelets is still poorly studied. Thus, the aim of the present study was to evaluate the effects of different plasminogen forms on the formation and secretion of angiogenic protein regulators by platelets. Human washed platelets were obtained by gel-filtration on Sepharose-2B. The levels of P-selectin (CD-62P) exposed on the plasma membrane of untreated and activated platelets was monitored by flow cytometry. Secretion of platelet-derived vascular endothelial growth factor (VEGF) as well as plasminogen fragmentation and angiostatin formation by intact platelets and platelet plasma membranes were analyzed by immunoblotting. It was shown that thrombin or collagen exposure resulted in enhanced P-selectin surface expression by platelets, while Lys-form of plasminogen reduced agonist-induced platelet secretion. Lys-plasminogen, but not Glu-form, inhibited agonist-induced VEGF release from platelets. Activation of platelets significantly accelerated plasminogen cleavage and angiostatin formation. Anti-actin antibodies inhibited plasminogen fragmentation during incubation with platelet plasma membranes indicating surface-exposed actin participation in plasminogen conversion to angiostatins. The present study uncovers a novel function of plasminogen to limit angiogenic potential of platelets via angiostatin formation and inhibition of VEGF secretion.

BackgroundMedulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis.MethodsOncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease.ResultsMedulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus' cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers of tumor-associated blood vessels and a trend for increased survival compared to mice treated with the control virus.ConclusionsThese data suggest that oncolytic measles viruses encoding anti-angiogenic proteins may have therapeutic benefit against medulloblastoma and support ongoing efforts to target angiogenesis in medulloblastoma.

Martorell hypertensive ischemic leg ulcers (HYTILU) represent a unique form of lower extremity non-healing ulcers that develop in association with poorly controlled high blood pressure. The present study was performed in order to assess levels of protein regulators of angiogenesis (vascular endothelial growth factor, or VEGF, and angiostatins) and to evaluate activities of matrix metalloproteinases (MMPs) (gelatinases MMP-2 and -9) in wound cutaneous tissue in the case of patient with 2-years HYTILU history. VEGF and angiostatin levels were analyzed by Western blot, MMP activities were evaluated by gelatin zymography. We report here for the first time that wound tissue in HYTILU is characterized with increased levels of VEGF (by 75 folds vs. histologically normal tissue, P < 0.01) and dramatic overproduction of angiostatin levels, which are undetectable in healthy cutaneous tissue. Approximately 10-fold elevation in MMP-2 and -9 activities is observed in wound tissue as compared with uninjured cutaneous tissue. Obtained results indicate that increased production of angiogenic inhibitors, angiostatins, may counteract VEGF-induced pro-angiogenic signaling, and together with MMP overactivation, contributes to failed healing of ischemic ulcer. Further extended studies are needed to clarify how changes of angiogenic profile and imbalance of proteolytic activities in non-healing Martorell ulcers can be considered during their management procedures to improve efficacy of surgery debridement and/or skin grafting.

Tissue engineering for fibrocartilage regeneration using mesenchymal stromal cells (MSC) and biomaterial scaffolds is emerging as a promising strategy, but inhibiting vascularization to prevent endochondral ossification is important to develop stable implants. The objective of this study was to investigate the effect of angiostatin on inhibition of angiogenesis and promotion of chondrogenesis by collagen scaffolds with or without MSC implanted subcutaneously in rats. One scaffold from the following groups was implanted in each animal: Collagen scaffolds only, scaffolds functionalized with angiostatin, scaffolds loaded with MSC and scaffolds functionalized with angiostatin and loaded with MSC. The various scaffolds were harvested after 2 and 8 weeks for histological analysis, Real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence quantification. Results demonstrated significantly decreased expression of inflammatory (interleukin 1 alpha and beta) and angiogenic genes (platelet and endothelial cell adhesion molecule 1) in scaffolds functionalized with angiostatin after 2 weeks in vivo. Histologically, after 8 weeks, the scaffolds with angiostatin had less inflammatory cells and more collagen matrix formation, but no fibrocartilage formation was detected. Thus, although angiostatin suppressed angiogenesis, it did not stimulate ectopic chondrogenesis in tissue engineered constructs in vivo. [ABSTRACT FROM AUTHOR]

Background/aim: The clinical effect of angiostatin in diabetes mellitus (DM) patients receiving insulin is a meaningful gap in the literature. In this study, we aimed to show the levels and the clinical significance of angiostatin in DM patients receiving insulin. Materials and methods: This is a case-control study. Serum angiostatin levels were determined by ELISA. A total of 83 people consisting of healthy subjects (n = 36) and patients with a diagnosis of DM receiving insulin therapy (n = 47) were included in this study. Results: The mean angiostatin levels of the DM group were significantly higher than those of the control group (86.0 ± 68.1 ng/mL and 58.0 ± 22.4 ng/mL, respectively; P = 0.011). Significantly lower angiostatin levels were determined in the DM patients receiving metformin with respect to those not receiving metformin (97.2 ± 74.4 ng/mL and 49.3 ± 7.0 ng/mL, respectively; P = 0.021). Significantly higher levels of angiostatin were observed among the DM patients using a beta-blocker (BB) than the DM patients not using a BB (115.5 ± 78.71 ng/mL and 73.44 ± 60.08 ng/mL, respectively; p = 0.029). Conclusion: This is the first study evaluating and demonstrating the serum angiostatin levels in DM patients receiving insulin. Further studies are required to understand the effect of angiostatin in diabetics and the effect of medications on angiogenesis in these patients.q [ABSTRACT FROM AUTHOR]

Background: Fabry disease (FD) is characterized by early development of vasculopathy and endothelial dysfunction. However, it is unclear whether these findings also play a pivotal role in cardiac manifestation. As Fabry cardiomyopathy (FC) is the leading cause of death in FD, we aimed to gather a better insight in pathological mechanisms of the disease.Methods: Serum samples were obtained from 17 healthy controls, 15 FD patients with and 7 without FC. FC was defined by LV wall thickening of >12 mm in cardiac magnetic resonance imaging and serum level of proBNP, high sensitive Troponin T (hsT), and globotriaosylsphingosine (lyso-GB3) were obtained. A multiplex ELISA-Assay for 23 different angiogenesis markers was performed in pooled samples. Markers showing significant differences among groups were further analyzed in single samples using specific Elisa antibody assays. L-homoarginine (hArg), L-arginine, asymmetric (ADMA), and symmetric Dimethylarginine (SDMA) were quantified by liquid chromatography—mass spectrometry.Results: Angiostatin and matrix metalloproteinase 9 (MMP-9) were elevated in FD patients compared to controls independently of the presence of FC (angiostatin: 98 ± 25 vs. 75 ± 15 ng/mL; p = 0.001; MMP-9: 8.0 ± 3.4 vs. 5.0 ± 2.4 μg/mL; p = 0.002). SDMA concentrations were highest in patients with FC (0.90 ± 0.64 μmol/l) compared to patients without (0.57 ± 0.10 μmol/l; p = 0.027) and vs. controls (0.58 ± 0.12 μmol/l; p = 0.006) and was positively correlated with indexed LV-mass (r = 0.61; p = 0.003), hsT (r = 0.56, p = 0.008), and lyso-Gb3 (r = 0.53, p = 0.013). Accordingly, the ratio of L-homoarginine to SDMA (hArg/SDMA) was lowest in patients with FC (2.63 ± 1.78) compared to controls (4.16 ± 1.44; p = 0.005). For L-arginine, hArg and ADMA no significant differences among groups could be detected, although a trend toward higher ADMA and lower hArg levels could be observed in the FC group. Furthermore, a significant relationship between kidney and cardiac function could be revealed (p = 0.045).Conclusion: Elevated MMP-9 and angiostatin levels suggest an increased extracellular matrix turnover in FD patients. Furthermore, endothelial dysfunction may also be involved in FC, as SDMA and hArg/SDMA are altered in these patients.

Purpose: Radiotherapy-induced gut toxicity (RIGT) is a debilitating effect of radiotherapy for cancer, often resulting in significant diarrhea and pain. Previous studies have highlighted roles of the intestinal microvasculature and matrix metalloproteinases (MMPs) in the development of RIGT. We hypothesized vascular mediators would be significantly altered in a dark agouti (DA) rat model of RIGT. Additionally, we aimed to assess the effect of MMP-2 and -9 inhibition on the response of tumor-associated microvascular endothelial cells (TAMECs) to radiation. Methods: DA rats were administered 2.5 Gy abdominal irradiation (3 times/week over 6 weeks). Vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ), von Willebrand factor (VWF), angiostatin, and endostatin expression was assessed at 3, 6, and 15 weeks. Additionally, DA rat mammary adenocarcinoma tumor-associated microvascular endothelial cells (TAMECs) were used to assess the effects of radiation (12 Gy) and the MMP inhibitor SB-3CT on MMP, VEGF, and TGFβ expression, and cell viability. Results: VEGF mRNA expression was significantly increased in the colon at week 15 (p = .0012), and TGFβ mRNA expression was significantly increased in both the jejunum and colon at week 3 (p = .0280 and p = .0310, respectively). Endostatin immunostaining was significantly increased at week 3 (p = .0046), and angiostatin at 3 and 6 weeks (p = .0022 and p = .0135, respectively). MMP-2 and -9 mRNA and total protein levels were significantly increased following irradiation of TAMECs. Although this increase was significantly attenuated by SB-3CT, it did not significantly alter endothelial cell viability or VEGF and TGFβ mRNA expression. Conclusions: Findings of this study support the involvement of VEGF, TGFβ, angiostatin, endostatin, and MMP-2 in the pathobiology of RIGT. However, the relationship between these mediators is complex and needs further investigation to improve understanding of their therapeutic potential in RIGT. [ABSTRACT FROM AUTHOR]

Corneal avascularity is maintained by angiogenic privilege, an active process involving the production of higher level of angiostatic factors to offset the effect of angiogenic factors. A wide range of pathological insults to the cornea can disrupt this intricate equilibrium and promote angiogenesis and corneal neovascularization with resultant visual impairment. Corneal neovascularization is also a major risk factor for graft failure after keratoplasty. Current treatment options for corneal neovascularization are restricted by limited efficacy, adverse effects, and a short duration of action. The unique anatomical position and relative immune privilege of cornea make it an ideal tissue for gene-based therapies. Gene transfer vectors have been used to deliver or target genes involved in the pathogenesis of corneal neovascularization in animal models. Several proangiogenic and antiangiogenic factors have been targeted and assessed in experimentally induced corneal neovascularization. Antisense oligonucleotides targeting corneal neovascularization have entered human clinical trials and have not required vector delivery systems. The emergence of these RNA-based strategies heralds a new era in the management of corneal neovascularization and ocular therapeutics. [ABSTRACT FROM AUTHOR]

Introduction: Systemic sclerosis (SSc) is achronic connective tissue disease characterized by microangiopathy with inadequate angiogenesis. Angiostatin (AS) is a potent antiangiogenic factor specifically inhibiting proliferation and inducing apoptosis of vascular endothelial cells. Aim: To evaluate the level of angiostatin in the serum of patients with SSc. Material and methods: Serum levels of AS were measured in 20 SSc patients and 12 healthy controls. Results: A statistically significant difference in the serum levels of AS in SSc patients was observed compared to the control group (636.51 vs. 869.20 ng/ml; p = 0.012). Significant correlations between limited and disseminated SSc (lSSc/dSSc) were not found, however, a difference between lSSc and the control group was demonstrated (620.00 vs. 869.20 ng/ml; p = 0.011). The serum level of AS was not associated positively with organ changes caused by SSc. However, a statistically significant lower serum level of AS was observed in patients with SSc and no esophageal (p = 0.008) or pulmonary changes (p = 0.007) compared to the control group. Conclusions: Our results reveal significant differences in AS level in SSc patients compared to the healthy controls, and suggest that a low level of AS may occur as a result of impaired angiogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Aim: The goal of this study is to investigate how urinary angiostatin, vascular cell adhesion molecule 1 (VCAM‐1) and established measures of renal function relate to specific histologic findings in paired kidney biopsy samples from patients with lupus nephritis (LN). Method: Urine samples were collected from 54 LN patients together with paired kidney biopsy samples and examined for urinary angiostatin and VCAM‐1 protein levels. Nonparametric tests were used to examine the association of both urinary biomarkers and established traditional laboratory markers of renal function with nine specific renal histologic features seen in LN, including glomerular leukocyte infiltration, endocapillary proliferation, cellular crescents, fibrinoid necrosis, wire loops, interstitial inflammation, glomerulosclerosis, fibrous crescents, tubular atrophy and interstitial fibrosis. Results: Compared to traditional renal disease metrics, both urinary angiostatin and VCAM‐1 exhibited outstanding potential (area under the curve 0.97, 0.98, respectively) to predict renal biopsy activity index score ≥ 7, which is associated with poor long‐term prognosis. Whereas urine VCAM‐1 was most significantly associated with fibrous crescents, urine angiostatin was most significantly associated with endocapillary proliferation, cellular crescents, fibrinoid necrosis and fibrous crescents in concurrent renal biopsies. Conclusion: Urinary angiostatin and VCAM‐1 are predictive of specific histological changes in concurrent LN renal biopsies. Both urinary biomarkers are good candidates for use as noninvasive measures of renal pathology activity changes in LN. [ABSTRACT FROM AUTHOR]

Tumor angiogenesis is characterized by deregulated gene expression in endothelial cells (EC). While studies until now have mainly focused on overexpressed genes in tumor endothelium, we here describe the identification of transcripts that are repressed in tumor endothelium and thus have potential suppressive effects on angiogenesis. We identified nineteen putative angiosuppressor genes, one of them being bromodomain containing 7 (BRD7), a gene that has been assigned tumor suppressor properties. BRD7 was studied in more detail, and we demonstrate that BRD7 expression is inversely related to EC activation. Ectopic expression of BRD7 resulted in a dramatic reduction of EC proliferation and viability. Furthermore, overexpression of BRD7 resulted in a bromodomain-dependent induction of NFκB-activity and NFκB-dependent gene expression, including ICAM1, enabling leukocyte-endothelial interactions. In silico functional annotation analysis of genome-wide expression data on BRD7 knockdown and overexpression revealed that the transcriptional signature of low BRD7 expressing cells is associated with increased angiogenesis (a.o. upregulation of angiopoietin-2, VEGF receptor-1 and neuropilin-1), cytokine activity (a.o. upregulation of CXCL1 and CXCL6), and a reduction of immune surveillance (TNF-α, NFκB, ICAM1). Thus, combining in silico and in vitro data reveals multiple pathways of angiosuppressor and anti-tumor activities of BRD7. [ABSTRACT FROM AUTHOR]

Our aim was to develop a method for isolation of human plasminogen kringle 5 possessing functional activity. The proposed method includes the following steps: hydrolysis of plasminogen with elastase, separation of mini-plasminogen from kringle fragments 1–3 and 4 on Lys-Sepharose, mini-plasminogen hydrolysis with pepsin, affinity chromatography on AH-Sepharose and polyacrilamide gel electrophoresis. We obtained the electrophoretically pure fragment of human plasminogen kringle 5 showing functional activity towards the ligands with high and low molecular mass. Weight yield was 3.8% that corresponds to 25.3% of the theoretically possible. It was established that affinity chromatography on AH-Sepharose was the sufficient step to isolate kringle 5 from mini-plasminogen hydrolysate with pepsin. This approach does not require additional purification steps while the ability of kringle 5 to bind specifically to AH-Sepharose demonstrates the functional activity of the kringle.

Surgeons observed that primary tumors are capable of suppressing the growth of their metastases by generating anti-angiogenic factor angiostatin. A three-dimensional (3D) mathematical model of development of the metastatic tumor vasculature is presented to simulate the morphology and construction of 3D microvascular networks under the inhibitory effect of anti-angiogenic factor angiostatin excreted by the primary tumor. The simulation results demonstrate that metastatic tumor microvascular density (MVD) decreases by about 60%, 58% and 52%, respectively, at , 7 and 14 days under the effect of anti-angiogenic factor angiostatin. The abnormal geometric and morphological features of 3D microvasculature networks inside and outside the metastatic tumor improve in the presence of angiostatin. The present model may allow to simulate experimental tests and may provide theoretical models for clinical research of anti-angiogenic therapy strategies. [ABSTRACT FROM AUTHOR]

A potential for the use of propolis in preventive and therapeutic purposes has been acknowledged, but little attention has been paid to estimation of possible propolis cytotoxicity with respect to astrocytes. We tried to estimate how a propolis ethanol extract (PEE) affects rat brain astrocytes in vitro and also to uncover crucial molecular targets of the PEE action. Primary astrocytes were exposed to PEE in doses of 10, 25, or 100 μg/ml for 24 h, and then the cell viability was monitored by MTT assay. Levels of glial fibrillary acidic protein (GFAP), transcriptional nuclear factor-κB (NF-κB), poly(ADPribose) polymerase (PARP), and angiostatins were measured using Western blot to identify the molecular mechanisms underlying cell responses to PEE. The PEE treatment exerted a dose-dependent cytotoxic effect on cultured astrocytes. The PEE modulated astrocyte signaling pathways through inducing the expression of NF-κB and PARP. At the same time, the PEE stimulated GFAP synthesis and fibrillogenesis, which was indicative for activation of astrocytes preceding their suppression. The PEE significantly increased the production of angiostatin isoforms by astrocytes, thus contributing to an antiangiogenic potential of these cells. In summary, our results indicated that exposure to the PEE exerts certain cytotoxic effects on astrocytes in a dose-dependent manner; these effects are realized through modulation of cytoskeleton rearrangements and pro-apoptotic signaling pathways. A widely available, safe, and inexpensive substance, propolis, and its components and derivatives may be used in the prevention and treatment of neuronal impairments, including malignant tumors and neurodegenerative disorders associated with excessive astrocytic activation. [ABSTRACT FROM AUTHOR]

Cancer cells are often dependent on epigenetic pathways for their survival. Consequently, drugs that target the epigenome, rather than the underlying DNA sequence, are currently attracting considerable attention. In recent years, the first epigenetic drugs have been approved for cancer chemotherapy, mainly for hematological applications. Limitations in single-drug efficacies have thus far limited their application in the treatment of solid tumors. Nevertheless, promising activity for these compounds has been suggested when combined with other, distinctly targeted agents. In this review, we discuss the anti-angiogenic activity of histone deacetylase and DNA methyltransferase inhibitors and their combinations with other targeted (anti-angiogenic) therapeutics in treatment of solid tumors. The role that these inhibitors play in the inhibition of tumor angiogenesis, particularly in combination with other targeted agents, and the advantages they present over broad acting anticancer agents, are critically discussed. [ABSTRACT FROM AUTHOR]

Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat®). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 104 ( n = 3), 2.4 × 105 ( n = 3), or 8.0 × 105 transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 105 TU or 8.0 × 105 TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 105 TU. These data demonstrate that EIAV vectors provide a safe platform with robust and sustained transgene expression for ocular gene therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Components of plasminogen/plasmin system play crucial role in fibrinolytic rocesses. They are also involved in regulation of cell activity in normalcy and various pathological conditions. In particular, kringle-containing proteolityc plasminogen fragments, which are denoted as angiostatins, participate in processes during neovascularization, metastasis, tumor growth, inflammation. Angiostatins are considered to be potential markers of diseases associated with vascular pathologies. Therefore, elaboration of specific and sensitive methods for their detection is still important area of inquiry. In the present study, approaches for obtaining of plasminogen fragments through its limited proteolysis by pancreatic elastase and further purification of proteolytic derivates by means of gel filtration and affinity chromatography on Lys-Sepharose are described. Polyclonal antibodies raised to fragment K1-3 have been produced, and their principal immunochemical properties have been studied. It has been found that antibodies purified on immunoaffine sorbent demonstrate different affinity toward plasminogen and its fragment, as follows: Glu-Pg > Lys-Pg > К1-3 > mini-Pg > К4. Based on the data obtained, immunological features of plasminogen kringle domains are discussed. Antibodies against fragment K1-3 could be applied in immunochemical analysis, in particular, Western blot, for detection of angiostatins as markers of tumor growth, metastasis, cardiovascular diseases and inflammation processes, and used as molecular tool for investigation of plasminogen/plasmin system functioning in health and disease.

Endorepellin, the C-terminal domain of perlecan, is an angiostatic molecule that acts as a potent inducer of autophagy via its interaction with VEGFR2. In this study, we examined the effect of endorepellin on endothelial cells using atomic force microscopy. Soluble endorepellin caused morphological and biophysical changes such as an increase in cell surface roughness and cell height. Surprisingly, these changes were not accompanied by alterations in the endothelial cell elastic modulus. We discovered that endorepellin-induced autophagic flux led to co-localization of mammalian target of rapamycin with LC3-positive autophagosomes. Endorepellin functioned upstream of AMPactivated kinase α, as compound C, an inhibitor of AMP-activated kinase α, abrogated endorepellin-mediated activation and co-localization of Beclin 1 and LC3, thereby reducing autophagic progression. Functionally, we discovered that both endorepellin and Torin 1, a canonical autophagic inducer, blunted ex vivo angiogenesis. We conclude that autophagy is a novel mechanism by which endorepellin promotes angiostasis independent of nutrient deprivation. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of recombinant human endostatin (Endostar) combined with hepatic artery interventional therapy on the progression-free survival (PFS) of patients with advanced hepatocellular carcinoma (HCC). MethodsA total of 86 patients with advanced HCC who were admitted to Fujian Provincial Tumor Hospital from March 2011 to May 2015 were selected and divided into treatment group and control group according to a matched pair design. The treatment group (43 patients) was given Endostar combined with hepatic artery interventional therapy, and the control group (43 patients) was given hepatic artery interventional therapy combined with oral administration of Ganfule. The chi-square test was applied for comparison of categorical data between the two groups, and the t-test was applied for comparison of continuous data between the two groups. The Kaplan-Meier method was applied for survival analysis, the Log-rank test was applied for univariate analysis, and Cox proportional hazards model was applied for multivariate analysis. ResultsThe median PFS in the treatment group and the control group was 154 d ［95% confidence interval (CI): 94-214 d］ and 70 d (95%CI: 39-101 d), respectively, with a significant difference between the two groups (χ2=10.741, P=0.001). Univariate analysis showed that the severity of liver cirrhosis, number of tumors, and main portal vein tumor thrombus/inferior vena cava tumor thrombus were the prognostic factors for patients with advanced HCC (χ2=8.182, 9.150, and 6.565, P=0.004, 0.027, and 0.038); multivariate analysis showed that the severity of liver cirrhosis and main portal vein tumor thrombus/inferior vena cava tumor thrombus were the independent prognostic factors for PFS in patients with advanced HCC who were treated with Endostar combined with hepatic artery interventional therapy (P=0.028 and 0.013). ConclusionEndostar can effectively prolong the PFS of patients with advanced HCC after hepatic artery interventional therapy, but it does not have a clear advantage in patients with severe liver cirrhosis or main portal vein tumor thrombus/inferior vena cava tumor thrombus. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have a role in the development and progression of human malignancy. The expression of miR-497 is decreased in malignant tumors, which suggests a role for miR-497 as a tumor suppressor. Angiomotin is encoded by the AMOT gene, which is a target for miR-497. Angiomotin has a role in angiogenesis, cell proliferation, and invasion in human malignancies, including osteosarcoma. However, the role of miR-497 in human osteosarcoma is unknown. This preliminary study included human osteosarcoma tissues and normal tissues from 20 patients, the osteosarcoma cell lines, MG-63, SAOS-2, U-2 OS, and the human osteoblast cell line hFOB (OB3). Western blots for angiomotin and quantitative real-time polymerase chain reaction for the expression of miR-497 and AMOT were performed. Knockdown studies were performed using RNA interference and transfection studies used miR-497 mimics. Quantitative cell migration assays were performed, and cell apoptosis was studied by flow cytometry. Osteosarcoma cells and cell lines showed reduced expression of miR-497 and increased expression of angiomotin. Transfection of osteosarcoma cells with miR-497 mimics suppressed the expression of angiomotin. Results from a dual-luciferase reporter system supported AMOT as a direct target gene of miR-497. Knockdown of AMOT using RNA interference resulted in inhibition of osteosarcoma cell proliferation, migration, and invasion. These preliminary studies support a role for miR-497 as a suppressor of AMOT gene expression in human osteosarcoma cells, resulting in suppression of tumor cell proliferation and invasion. Further studies are recommended to investigate the role of miR-497 in osteosarcoma and other malignant mesenchymal tumors. [ABSTRACT FROM AUTHOR]

Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development. [ABSTRACT FROM AUTHOR]

Aim Angiogenesis and chronic inflammation are known to be co-dependent in atherosclerosis and cardiovascular diseases. This study was undertaken to investigate whether simvastatin could affect serum levels of angiostatin, a potent endogenous inhibitor of neovascularization, in patients with ischemic heart disease (IHD). Main methods Twenty-six patients with clinically confirmed IHD and hypercholesterolemia were assigned 40 mg/day of simvastatin for 8 weeks. Levels of lipid metabolism, C-reactive protein (C-RP) and other biochemical parameters in serum samples were measured using biochemical analyzer. Serum angiostatin levels were determined by Western blot. Association of serum angiostatin levels with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and C-RP levels was evaluated. Key findings Simvastatin therapy improved the main parameters of lipid metabolism, including statistically significant (P < 0.05) reductions in TC (by 46%) and LDL-C (by 42%), and decreased inflammatory marker C-RP (by 32%), as compared with the baseline. Simvastatin treatment resulted in marked reduction of serum angiostatin level (by 80% in comparison with baseline, P < 0.05). Strong positive correlations between serum angiostatin level versus concentrations of TC, LDL-C, and C-RP were demonstrated before onset of the study (r = 0.48311, 0.6252, and 0.653, respectively) and after simvastatin therapy (r = 0.67752, 0.6485, and 0.8244, respectively). Significance We describe for the first time novel pleiotropic effect of statin therapy associated with decrease of serum angiostatin levels. Thus, circulating angiostatin represents an independent additional risk marker for cardiovascular events and could be applied as potential supplementary indicator for evaluation of statin therapy efficacy. [ABSTRACT FROM AUTHOR]

Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation ( p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis ( p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly ( p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases. [ABSTRACT FROM AUTHOR]