Helicobacter pylori Infection Acts as an Independent Risk Factor for Intracranial Atherosclerosis in Women Less Than 60 Years Old

Background: Studies show inconsistent results regarding the relationship between Helicobacter pylori (H. pylori) infection and stroke. The present study assessed a potential association between H. pylori infection and an important risk factor for stroke, intracranial atherosclerosis.Methods: In total, 15,798 subjects with transcranial Doppler (TCD) and 13C-urea breath test (13C-UBT) were enrolled from March 2012 to March 2017. Intracranial atherosclerosis was further measured using intracranial carotid artery calcification (ICAC) on past or recent head CT, and 14,084 subjects were ultimately included in the study. Baseline demographics, atherosclerosis risk factors, and laboratory results were investigated. Since endothelial dysfunction is critical to the development of atherosclerosis, the role of H. pylori in migration, tube formation, and proliferation of human brain microvascular endothelial cells (HBMECs) was assessed in vitro.Results: The intracranial atherosclerosis group had a higher proportion of women and a greater rate of H. pylori infection than those without intracranial atherosclerosis. H. pylori infection was significantly more common in women with intracranial atherosclerosis than males. In addition, the incidence of intracranial atherosclerosis was significantly higher in women with H. pylori infection than uninfected women (53.8 vs. 46.4%, p < 0.001). In an adjusted model, H. pylori was shown to be an independent risk factor for intracranial atherosclerosis in women ≤ 60 years of age [odds ratio (OR) = 2.261, 95% CI = 1.839–2.780, p < 0.001]. Serum exosomes from patients with H. pylori infection had significantly reduced brain endothelial cell migration, tube formation, and proliferation in vitro.Conclusion:Helicobacter pylori infection may be an important independent risk factor for intracranial atherosclerosis in women ≤ 60 years of age.

Biphasic Effect of Pirfenidone on Angiogenesis

Pirfenidone (PFD), a synthetic arsenic compound, has been found to inhibit angiogenesis at high concentrations. However, the biphasic effects of different PFD concentrations on angiogenesis have not yet been elucidated, and the present study used an in vitro model to explore the mechanisms underlying this biphasic response. The effect of PFD on the initial angiogenesis of vascular endothelial cells was investigated through a Matrigel tube formation assay, and the impact of PFD on endothelial cell migration was evaluated through scratch and transwell migration experiments. Moreover, the expression of key migration cytokines, matrix metalloproteinase (MMP)-2 and MMP-9, was examined. Finally, the biphasic mechanism of PFD on angiogenesis was explored through cell signaling and apoptosis analyses. The results showed that 10–100 μM PFD has a significant and dose-dependent inhibitory effect on tube formation and migration, while 10 nM–1 μM PFD significantly promoted tube formation and migration, with 100 nM PFD having the strongest effect. Additionally, we found that a high concentration of PFD could significantly inhibit MMP-2 and MMP-9 expression, while low concentrations of PFD significantly promoted their expression. Finally, we found that high concentrations of PFD inhibited EA.hy926 cell tube formation by promoting apoptosis, while low concentrations of PFD promoted tube formation by increasing MMP-2 and MMP-9 protein expression predominantly via the EGFR/p-p38 pathway. Overall, PFD elicits a biphasic effect on angiogenesis through different mechanisms, could be used as a new potential drug for the treatment of vascular diseases.

Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics

Cell migration is a complex, tightly regulated multistep process in which cytoskeletal reorganization and focal adhesion redistribution play a central role. Core to both individual and collective migration is the persistent random walk, which is characterized by random force generation and resistance to directional change. We first discuss a model that describes the stochastic movement of ECs and characterizes EC persistence in wound healing. To that end, we pharmacologically disrupted cytoskeletal dynamics, cytochalasin D for actin and nocodazole for tubulin, to understand its contributions to cell morphology, stiffness, and motility. As such, the use of Atomic Force Microscopy (AFM) enabled us to probe the topography and stiffness of ECs, while time lapse microscopy provided observations in wound healing models. Our results suggest that actin and tubulin dynamics contribute to EC shape, compressive moduli, and directional organization in collective migration. Insights from the model and time lapse experiment suggest that EC speed and persistence are directionally organized in wound healing. Pharmacological disruptions suggest that actin and tubulin dynamics play a role in collective migration. Current insights from both the model and experiment represent an important step in understanding the biomechanics of EC migration as a therapeutic target.

Butein and Frondoside-A Combination Exhibits Additive Anti-Cancer Effects on Tumor Cell Viability, Colony Growth, and Invasion and Synergism on Endothelial Cell Migration

Despite the significant advances in targeted- and immuno-therapies, lung and breast cancer are at the top list of cancer incidence and mortality worldwide as of 2020. Combination therapy consisting of a mixture of different drugs taken at once is currently the main approach in cancer management. Natural compounds are extensively investigated for their promising anti-cancer potential. This study explored the anti-cancer potential of butein, a biologically active flavonoid, on two major solid tumors, namely, A549 lung and MDA-MB-231 breast cancer cells alone and in combination with another natural anti-cancer compound, frondoside-A. We demonstrated that butein decreases A549 and MDA-MB-231 cancer cell viability and colony growth in vitro in addition to tumor growth on chick embryo chorioallantoic membrane (CAM) in vivo without inducing any noticeable toxicity. Additionally, non-toxic concentrations of butein significantly reduced the migration and invasion of both cell lines, suggesting its potential anti-metastatic effect. We showed that butein anti-cancer effects are due, at least in part, to a potent inhibition of STAT3 phosphorylation, leading to PARP cleavage and consequently cell death. Moreover, we demonstrated that combining butein with frondoside-A leads to additive effects on inhibiting A549 and MDA-MB-231 cellular viability, induction of caspase 3/7 activity, inhibition of colony growth, and inhibition of cellular migration and invasion. This combination reached a synergistic effect on the inhibition of HUVECs migration in vitro. Collectively, this study provides sufficient rationale to further carry out animal studies to confirm the relevance of these compounds’ combination in cancer therapy.

Caveolin-1 Down-Regulation Reduces VEGF-A Secretion Induced by IGF-1 in ARPE-19 Cells

The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

Comparative assessment of electronic nicotine delivery systems aerosol and cigarette smoke on endothelial cell migration: the Replica Project

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations performed by the tobacco industries evaluated in vitro the effect of electronic cigarette (e-cig) compared to cigarette smoke demonstrating a significant reduction in endothelial cell migration inhibition following e-cig aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared to e-cig and heated tobacco products. We used a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study. Consistently with the original study, we observed a substantial reduction of the effects of e-cig and tobacco heated products on endothelial cell migration compared to cigarette smoke. In conclusion, our study further confirms the importance of e-cig and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

Wasp controls oriented migration of endothelial cells to achieve functional vascular patterning

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells as well as the resulting regularity of vessel caliber, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in human. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Inhibition of Neovascularization and Inflammation in a Mouse Model of Corneal Alkali Burns Using Cationic Liposomal Tacrolimus

Eye drops account for more than 90% of commercialized ophthalmic drugs. However, eye drops have certain shortcomings, such as short precorneal retention time and weak corneal penetration. The requirement of frequent instillation of eye drops also causes poor patient compliance, which may lead to further aggravation of the disease. We aimed to develop a cationic liposome formulation to increase the bioavailability of the therapeutic agent and solve the aforementioned problems. In the present study, we prepared cationic liposomal tacrolimus (FK506) with a surface potential of approximately +30 mV, which could bind to the negatively charged mucin layer of the ocular surface. Our results showed that the content of FK506 in the cornea was increased by 93.77, 120.30, 14.24, and 20.36 times at 5, 30, 60, and 90 min, respectively, in the FK506 liposome group (0.2 mg/ml) compared with the free drug group (0.2 mg/ml). Moreover, FITC-labeled FK506 liposomes significantly prolonged the ocular surface retention time to 50 min after a single dose. In addition, the results of the Cell Counting Kit-8 assay, live and dead cell assay, sodium fluorescein staining, and hematoxylin and eosin staining all indicated that FK506 liposomes had good biological compatibility in both human corneal epithelial cells and mouse eyeballs. Compared with the free drug at the same concentration, FK506 liposomes effectively inhibited vascular endothelial growth factor-induced green fluorescent protein-transduced human umbilical vein endothelial cell migration and tube formation in vitro. In a mouse corneal neovascularization model induced by alkali burns, FK506 liposomes (0.2 mg/ml) enhanced corneal epithelial recovery, inhibited corneal neovascularization, and reduced corneal inflammation, and its therapeutic effect was better than those of the commercial FK506 eye drops (1 mg/ml) and the free drug (0.2 mg/ml). Collectively, these results indicate that cationic FK506 liposomes could increase the efficacy of FK506 in the corneal neovascularization model. Therefore, cationic FK506 liposomes can be considered as a promising ocular drug delivery system.