Targeting endothelial permeability in the EPR effect.

The characteristics of the primary tumor blood vessels and the tumor microenvironment drive the enhanced permeability and retention (EPR) effect, which confers an advantage towards enhanced delivery of anti-cancer nanomedicine and has shown beneficial effects in preclinical models. Increased vascular permeability is a landmark feature of the tumor vessels and an important driver of the EPR. The main focus of this review is the endothelial regulation of vascular permeability. We discuss current challenges of targeting vascular permeability towards clinical translation and summarize the structural components and mechanisms of endothelial permeability, the principal mediators and signaling players, the targeted approaches that have been used and their outcomes to date. We also critically discuss the effects of the tumor-infiltrating immune cells, their interplay with the tumor vessels and the impact of immune responses on nanomedicine delivery, the impact of anti-angiogenic and tumor-stroma targeting approaches, and desirable nanoparticle design approaches for greater translational benefit.

Antibody affinity and cross-variant neutralization of SARS-CoV-2 Omicron BA.1, BA.2 and BA.3 following third mRNA vaccination.

There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in naïve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with naïve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with naïve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants.

An in vitro model of glucose transporter 1 deficiency syndrome at the blood-brain barrier using induced pluripotent stem cells.

Glucose is an important source of energy for the central nervous system. Its uptake at the blood-brain barrier (BBB) is mostly mediated via glucose transporter 1 (GLUT1), a facilitated transporter encoded by the SLC2A1 gene. GLUT1 Deficiency Syndrome (GLUT1DS) is a haploinsufficiency characterized by mutations in the SLC2A1 gene, resulting in impaired glucose uptake at the BBB and clinically characterized by epileptic seizures and movement disorder. A major limitation is an absence of in vitro models of the BBB reproducing the disease. This study aimed to characterize an in vitro model of GLUT1DS using human pluripotent stem cells (iPSCs). Two GLUT1DS clones were generated (GLUT1-iPSC) from their original parental clone iPS(IMR90)-c4 by CRISPR/Cas9 and differentiated into brain microvascular endothelial cells (iBMECs). Cells were characterized in terms of SLC2A1 expression, changes in the barrier function, glucose uptake and metabolism, and angiogenesis. GLUT1DS iPSCs and iBMECs showed comparable phenotype to their parental control, with exception of reduced GLUT1 expression at the protein level. Although no major disruption in the barrier function was reported in the two clones, a significant reduction in glucose uptake accompanied by an increase in glycolysis and mitochondrial respiration was reported in both GLUT1DS-iBMECs. Finally, impaired angiogenic features were reported in such clones compared to the parental clone. Our study provides the first documented characterization of GLUT1DS-iBMECs generated by CRISPR-Cas9, suggesting that GLUT1 truncation appears detrimental to brain angiogenesis and brain endothelial bioenergetics, but maybe not be detrimental to iBMECs differentiation and barriergenesis. Our future direction is to further characterize the functional outcome of such truncated product, as well as its impact on other cells of the neurovascular unit.

Cross-reactive immunity against the SARS-CoV-2 Omicron variant is low in pediatric patients with prior COVID-19 or MIS-C.

Neutralization capacity of antibodies against Omicron after a prior SARS-CoV-2 infection in children and adolescents is not well studied. Therefore, we evaluated virus-neutralizing capacity against SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron variants by age-stratified analyses (<5, 5-11, 12-21 years) in 177 pediatric patients hospitalized with severe acute COVID-19, acute MIS-C, and in convalescent samples of outpatients with mild COVID-19 during 2020 and early 2021. Across all patients, less than 10% show neutralizing antibody titers against Omicron. Children <5 years of age hospitalized with severe acute COVID-19 have lower neutralizing antibodies to SARS-CoV-2 variants compared with patients >5 years of age. As expected, convalescent pediatric COVID-19 and MIS-C cohorts demonstrate higher neutralization titers than hospitalized acute COVID-19 patients. Overall, children and adolescents show some loss of cross-neutralization against all variants, with the most pronounced loss against Omicron. In contrast to SARS-CoV-2 infection, children vaccinated twice demonstrated higher titers against Alpha, Beta, Gamma, Delta and Omicron. These findings can influence transmission, re-infection and the clinical disease outcome from emerging SARS-CoV-2 variants and supports the need for vaccination in children.

Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Omicron and Other Variants in Serum From Children With Vaccination-Induced Myocarditis.

Our study demonstrates that children who developed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination-induced myocarditis and may not receive another vaccination, could be susceptible to infection with Omicron and emerging variants. We observed higher neutralizing antibody titers in myocarditis patients vs. healthy vaccinated children, but significantly lower neutralization titers against Omicron in both groups.

Angiopoietin-2-induced lymphatic endothelial cell migration drives lymphangiogenesis via the ß1 integrin-RhoA-formin axis.

Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on ß1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments.

Role of bFGF in Acquired Resistance upon Anti-VEGF Therapy in Cancer.

Anti-angiogenic approaches targeting the vascular endothelial growth factor (VEGF) signaling pathway have been a significant research focus during the past decades and are well established in clinical practice. Despite the expectations, their benefit is ephemeral in several diseases, including specific cancers. One of the most prominent side effects of the current, VEGF-based, anti-angiogenic treatments remains the development of resistance, mostly due to the upregulation and compensatory mechanisms of other growth factors, with the basic fibroblast growth factor (bFGF) being at the top of the list. Over the past decade, several anti-angiogenic approaches targeting simultaneously different growth factors and their signaling pathways have been developed and some have reached the clinical practice. In the present review, we summarize the knowledge regarding resistance mechanisms upon anti-angiogenic treatment, mainly focusing on bFGF. We discuss its role in acquired resistance upon prolonged anti-angiogenic treatment in different tumor settings, outline the reported resistance mechanisms leading to bFGF upregulation, and summarize the efforts and outcome of combined anti-angiogenic approaches to date.

Advanced bioinformatic analysis and pathway prediction of NSCLC cells upon cisplatin resistance.

This study aims to identify pathway involvement in the development of cisplatin (cis-diamminedichloroplatinum (II); CDDP) resistance in A549 lung cancer (LC) cells by utilizing advanced bioinformatics software. We developed CDDP-resistant A549 (A549/DDP) cells through prolonged incubation with the drug and performed RNA-seq on RNA extracts to determine differential mRNA and miRNA expression between A549/DDP and A549 cells. We analyzed the gene dysregulation with Ingenuity Pathway Analysis (IPA; QIAGEN) software. In contrast to prior research, which relied on the clustering of dysregulated genes to pathways as an indication of pathway activity, we utilized the IPA software for the dynamic evaluation of pathway activity depending on the gene dysregulation levels. We predicted 15 pathways significantly contributing to the chemoresistance, with several of them to have not been previously reported or analyzed in detail. Among them, the PKR signaling, cholesterol biosynthesis, and TEC signaling pathways are included, as well as genes, such as PIK3R3, miR-34c-5p, and MDM2, among others. We also provide a preliminary analysis of SNPs and indels, present exclusively in A549/DDP cells. This study's results provide novel potential mechanisms and molecular targets that can be explored in future studies and assist in improving the understanding of the chemoresistance phenotype.

Low Dose of Penfluridol Inhibits VEGF-Induced Angiogenesis.

: Metastasis is considered a major burden in cancer, being responsible for more than 90% of cancer-related deaths. Tumor angiogenesis is one of the main processes that lead to tumor metastasis. Penfluridol is a classic and commonly used antipsychotic drug, which has a great ability to cross the blood-brain barrier. Recent studies have revealed that penfluridol has significant anti-cancer activity in diverse tumors, such as metastatic breast cancer and glioblastoma. Here, we aim to identify the effect of low doses of penfluridol on tumor microenvironment and compare it with its effect on tumor cells. Although low concentration of penfluridol was not toxic for endothelial cells, it blocked angiogenesis in vitro and in vivo. In vitro, penfluridol inhibited VEGF-induced primary endothelial cell migration and tube formation, and in vivo, it blocked VEGF- and FGF-induced angiogenesis in the matrigel plug assay. VEGF-induced VEGFR2 phosphorylation and the downstream p38 and ERK signaling pathways were not affected in endothelial cells, although VEGF-induced Src and Akt activation were abrogated by penfluridol treatment. When cancer cells were treated with the same low concentration of penfluridol, basal Src activation levels were mildly impaired, thus impacting their cell migration and wound healing efficiency. The potential of cancer-induced paracrine effect on endothelial cells was explored, although that did not seem to be a player for angiogenesis. Overall, our data demonstrates that low penfluridol levels, similar to the ones clinically used for anti-psychotic conditions, suppress angiogenic efficiency in the tumor microenvironment.

Pharmacogenetic Variants in MTHFR Gene are Significant Predictors of Methotrexate Toxicities in Bangladeshi Patients With Acute Lymphoblastic Leukemia.

BACKGROUND: The objective of this pharmacogenetic study was to investigate the relationship of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with methotrexate (MTX)-induced toxicities and plasma homocysteine level in patients with acute lymphoblastic leukemia (ALL) from Bangladesh. Several polymorphisms result in reduced MTHFR activity that causes impaired remethylation of homocysteine to methionine and abnormal MTX metabolism, especially in tissues with high turnover. Therefore, the risk of elevated plasma homocysteine as well as MTX-induced toxicities become higher with MTHFR polymorphisms. PATIENTS AND METHODS: We recruited 160 patients with ALL receiving MTX containing chemotherapeutic protocol, and they were genotyped for MTHFR C677T and A1298C polymorphisms with polymerase chain reaction-restriction fragment length polymorphism. We also measured the plasma homocysteine level of 51 patients by the AxSYM homocysteine assay method. RESULTS: We found 68.1% CC, 26.3% CT, and 5.6% TT genotype for MTHFR C677T polymorphism and 39.3% AA, 46.9% AC, and 13.8% CC genotype for MTHFR A1298C polymorphism in patients with ALL. Our study suggested that MTX-induced mucositis and diarrhea are significantly associated with MTHFR C677T as well as MTHFR A1298C polymorphisms (P < .05). CONCLUSION: The risk of elevated plasma homocysteine level was 5 to 6 times higher for both polymorphisms. This study may help to identify the patients who are at higher risk for MTX-related toxicities.

Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis.

Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.

In Vitro Spheroid Sprouting Assay of Angiogenesis.

Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation. Physiologically, angiogenesis is more prominent during development and wound healing and its dysregulation drives or is related to several diseases, including cancer. The endothelial cells are the main regulators of the angiogenic process, and thus the angiogenic outcome is assessed based on the effect on endothelial cell functions. Several in vitro and in vivo techniques have been developed to assess the effect of various factors on angiogenesis. Compared to the in vivo techniques, the in vitro techniques are considered less physiologically relevant. This has been partially overcome by the development of 3-dimensional (3D) in vitro models, one of which is the spheroid assay or 3D sprouting assay that exploits the effect of the extracellular matrix to endothelial cell functions. This chapter focuses on the description of the spheroid assay and mentions the variations and potential applications this assay can have.

Matrigel Plug Assay for In Vivo Evaluation of Angiogenesis.

Matrigel is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma in C57BL/6 mice, a tumor rich in extracellular matrix (ECM) proteins. It consists mainly of laminin (approximately 60%), collagen IV (approximately 30%), and nidogen-1/entactin (approximately 8%), while it also contains heparan sulfate proteoglycans, such as perlecan, other ECM proteins, as well as growth factors bound to the ECM. Matrigel mimics the physiological cell matrix and is the most commonly used matrix substrate to study in vitro and in vivo angiogenesis. Here, we describe the in vivo Matrigel plug assay and how it can be used for both qualitative and quantitative assessment of angiogenesis.

Mechanisms of angiogenesis in microbe-regulated inflammatory and neoplastic conditions.

Commensal microbiota inhabit all the mucosal surfaces of the human body. It plays significant roles during homeostatic conditions, and perturbations in numbers and/or products are associated with several pathological disorders. Angiogenesis, the process of new vessel formation, promotes embryonic development and critically modulates several biological processes during adulthood. Indeed, deregulated angiogenesis can induce or augment several pathological conditions. Accumulating evidence has implicated the angiogenic process in various microbiota-associated human diseases. Herein, we critically review diseases that are regulated by microbiota and are affected by angiogenesis, aiming to provide a broad understanding of how angiogenesis is involved and how microbiota regulate angiogenesis in microbiota-associated human conditions.