Angiopoietin-1 Mimetic Nanoparticles for Restoring the Function of Endothelial Cells as Potential Therapeutic for Glaucoma

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm’s canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP–cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma.

Effects of Alginate Encapsulation on the Vertebral Bone Adherent Mesenchymal Stromal Cell Paracrine Secretome

Background/Objectives: Mesenchymal stromal cells (MSCs) are spindle-shaped multipotent stem cells that can be found in any vascularized organ especially bone marrow, adipose tissue, and cord blood. MSCs have pro-angiogenic and myogenic properties which have been proposed as a potential treatment for preventing limb amputations in patients with critical limb threatening ischemia (CLTI) due to peripheral artery disease (PAD) and diabetes mellitus. Allogeneic MSCs from a young healthy donor may decrease the risk of amputation by promoting angiogenesis and muscle regeneration. However, their potency may be limited by host immune reactions to allogeneic cells. Encapsulating MSCs in a hydrogel may help to protect transplanted MSCs from the immune system of the host. Additionally, encapsulation may enhance the secretion of anti-inflammatory and pro-angiogenic factors from MSCs. Some of the molecules involved in fighting inflammation and promoting angiogenesis include interleukin (IL)-10, IL-33, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and angiopoietin-1. Methods. For the encapsulation group, MSCs were centrifuged in a 2% alginate solution through a needle and into a calcium chloride bath. Both unencapsulated and encapsulated MSCs were incubated for 48 hours in various conditions including normoxia, normoxia with high glucose (diabetic mimic), hypoxia, and hypoxia with high glucose. The media was then harvested and analyzed using enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, MN) for IL-10, HGF, VEGF, and angiopoietin-1. Results. No detectable levels of IL-10, HGF, or angiopoietin-1 were found in either cell media group. VEGF levels were significantly elevated in hypoxic high glucose conditions compared to normoxic and normoxic high glucose conditions within the unencapsulated group. Discussion. These results demonstrate that hypoxic high glucose conditions duplicating ischemic diabetic muscle amplify MSC VEGF secretion. The next step is to run a VEGF ELISA on the encapsulated cell media group and compare those results to the unencapsulated cell media group. WC: 300

Differential expression of angiopoietin 1 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding angiopoietin 1, ANGPT1, when comparing primary tumors of the breast to the tissue of origin, the normal breast. ANGPT1 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of ANGPT1 in primary tumors of the breast was correlated with overall survival in patients with luminal B and normal-like subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. ANGPT1 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Angiogenesis, Metabolism, Endothelial And Platelet Markers In Diabetes And Cardiovascular Disease

Introduction: Diabetes is a leading risk factor for cardiovascular disease (CVD), the pathophysiology of both being linked to metabolic, endothelial, renal, angiogenic and platelet abnormalities. We hypothesised that abnormalities in these systems are more adverse in those whose CVD is compounded by diabetes, compared to those with diabetes or CVD alone.Materials and methods: Serum or plasma from 66 patients with diabetes alone, 76 with CVD alone, and 70 with both diabetes and CVD i.e. diabetic cardiovascular disease, was probed for markers of angiogenesis [angiopoietin 1 and 2, vascular endothelial growth factor (VEGF) and endoglin], metabolic [sRAGE, leptin, lipocalin-2, interleukin-8, and cystatin-C], the endothelium (von Willebrand factor, endothelial microparticles and soluble E selectin]), and the platelet (platelet microparticles and soluble P selectin) by ELISA, Luminex or flow cytometry.Results: VEGF (p=0.04), von Willebrand factor (p=0.001) and endothelial microparticles (p=0.042) were all higher in diabetic cardiovascular disease than in diabetes alone and cardiovascular disease alone. Soluble E selectin was higher in diabetic cardiovascular disease than in diabetes alone (p=0.045), whilst cystatin-C (p=0.004) and soluble P selectin (p<0.001) were higher in diabetes and diabetic cardiovascular disease than in cardiovascular disease alone. There were no differences in angiopoietin 1 or 2, endoglin, sRAGE, leptin, lipocalin-2, or interleukin-8.Conclusion: Angiopoietin 1 or 2, endoglin, sRAGE, leptin, lipocalin-2, interleukin-8, and cystatin-c cannot differentiate diabetes from cardiovascular disease, or both conditions combined. Our data point to a more adverse endothelial (von Willebrand factor, endothelial microparticles), and angiogenic profile (VEGF) in those with diabetic cardiovascular disease, supporting the view that this group should be targeted more aggressively.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Aim: The role of platelets in atherosclerosis remains incompletely understood. Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here we sought to investigate the contribution of platelet sGC to atherosclerosis and the therapeutic potential of targeting sGC in atherosclerosis. Methods and Results: We genetically deleted sGC in platelets of atherosclerosis-prone Ldlr-/- mice. By intravital fluorescence microscopy such Pf4-Cre+Gucy1b1flox/floxLdlr-/- mice displayed enhanced leukocyte adhesion to atherosclerotic plaques in comparison with their litter mates. Moreover, histological and flow cytometry analyses revealed more numerous inflammatory leukocytes and larger plaque sizes in aortic tissue of Ldlr-/- mice lacking sGC in platelets. In vitro, supernatant from activated platelets lacking sGC promoted leukocyte adhesion to endothelial cells (EC) via enhanced EC activation. Using cytokine profiling, we identified reduced angiopoietin-1 release by Pf4-Cre+Gucy1b1flox/flox and human GUCY1A1 risk allele carrier platelets to be responsible for enhanced activation of EC and subsequent leukocyte adhesion. Pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced recruitment of adoptively transferred leukocytes in Ldlr-/- mice fed a Western diet. Pharmacological sGC stimulation further reduced atherosclerotic plaque formation and vascular inflammation. Conclusion: Loss of sGC in platelets contributes to atherosclerotic plaque formation via reduced release of the soluble factor angiopoietin-1 and, subsequently, enhanced leukocyte recruitment. Pharmacological sGC stimulation might represent a novel therapeutic strategy to prevent and treat CAD.

Effect of HIV‐1 Infection on Angiopoietin 1 and 2 Levels and Measures of Microvascular and Macrovascular Endothelial Dysfunction

Background Individuals infected with HIV have an increased risk of developing cardiovascular disease; yet, the underlying mechanisms remain unknown. Recent evidence has implicated the Tie‐2 tyrosine kinase receptor system and its associated ligands ANG1 (angiopoietin 1) and ANG2 (angiopoietin 2) in maintaining vascular homeostasis. In the general population, lower ANG1 levels and higher ANG2 levels are strongly correlated with the development of cardiovascular disease. In this study, we aim to investigate the associations of HIV infection with angiopoietin levels and endothelial dysfunction. Methods and Results In this cross‐sectional study, we compared measures of ANG1, ANG2, and endothelial dysfunction using flow‐mediated vasodilation of the brachial artery in 39 untreated subjects infected with HIV, 47 treated subjects infected with HIV, and 46 uninfected subjects from the SCOPE (Observational Study of the Consequences of the Protease Inhibitor Era) cohort. Compared with uninfected controls, treated individuals infected with HIV had 53.1% lower mean ANG1 levels ( P <0.01) and similar ANG2 levels. On the other hand, untreated individuals infected with HIV had similar ANG1 levels, and 29.2% had higher ANG2 levels ( P <0.01) compared with uninfected controls. When compared with individuals with untreated HIV infection, those with treated HIV infection had 56% lower ANG1 levels ( P <0.01) and 22% lower ANG2 levels ( P <0.01).Both treated and untreated HIV infection were associated with significant impairment in hyperemic velocity, a key measure of microvascular dysfunction (median 61 versus 72 cm/s, P <0.01), compared with uninfected controls (median 73 cm/s). This difference persisted after adjustment for ANG1 and ANG2 levels. Interestingly, when compared with untreated individuals infected with HIV, treated individuals infected with HIV had worse hyperemic velocity (−12.35 cm/s, P =0.05). In contrast, HIV status, ANG1 levels, and ANG2 levels were not associated with macrovascular dysfunction as measured by flow‐mediated dilatation and brachial artery diameter, 2 other measures of vascular homeostasis. Conclusions HIV infection affects the balance between levels of ANG1 and ANG2 and may disturb endothelial homeostasis through disruption of vascular homeostasis. Individuals with treated HIV had decreased ANG1 levels and similar ANG2 levels, whereas individuals with untreated HIV had similar ANG1 levels and increased ANG2 levels, suggesting that treatment status may alter the balance between ANG1 and ANG2. HIV also promotes endothelial dysfunction via impairment of microvascular dysfunction, independent of the Tie‐2 receptor system; the finding of worse microvascular dysfunction in the setting of treated HIV infection may reflect the impact of viral persistence on the microvasculature or toxicities of specific antiretroviral regimens. Further research to clarify the mechanism of HIV‐mediated endothelial dysfunction is necessary to advance treatment of cardiovascular complications of HIV infection.

Update Overview of the Role of Angiopoietins in Lung Cancer

Angiogenesis is a biological process that involves the formation of new blood vessels from the existing vasculature, and it plays a fundamental role in the development and progression of several types of cancer, including lung cancer. The angiopoietin/Tie2 ligand/receptor system orchestrates vascular integrity. In particular, Angiopoietin-1 activates the endothelial cell (EC)-specific receptor tyrosine kinase,Tie2,which is essential for preserving endothelial quiescence. On the other hand, Angiopoietin-2 acts as an inhibitor of the Angiopoietin-1/Tie2 signaling pathways, thus facilitating the destabilization of quiescent endothelium in cases of inflammation and cancer. Clinical studies have proven that high levels of Angiopoietin-2 indicate the development of non-small-cell lung carcinomas (NSCLC), while high levels of Angiopoietin-2 are strongly related to tumor angiogenesis, lymphangiogenesis, metastasis, and poor prognosis. Interestingly, the association of Angiopoietin-2 levels with the type of surgical approach makes Angiopoietin-2 a valuable factor in selecting the most suitable therapeutic strategy for lung cancer patients. The role of the Angiopoietin-1 and Angiopoietin-4 levels in NSCLC development requires further investigation. The present review focuses on the clinical impact of the Angiopoietin-1, Angiopoietin-2, and Angiopoietin-4 levels in patients diagnosed with NSCLC, emphasizing the interaction between them, and how they affect the development, progression, and metastasis of lung disease. Finally, it estimates the role of angiopoietins levels in the effective therapy of lung cancer patients.