Ofra Benny

VEGF preconditioning leads to stem cell remodeling and attenuates age-related decay of adult hippocampal neurogenesis

Significance Generation of new neurons is maintained in the adult hippocampus throughout life. The process, which is driven by an exhaustible reservoir of neuronal stem cells (NSCs), greatly declines with age, however. We show that even a short, episodic exposure to the angiogenic factor VEGF and a resultant ramification/rejuvenation of the vasculature within the stem cell microenvironment (“niche”) is sufficient for neurogenesis to proceed at a markedly elevated rate for months later without accelerating the rate of NSC depletion. Importantly, this manipulation culminates in marked attenuation of age-dependent neurogenic decline. Long-term neurogenic enhancement via VEGF preconditioning was found to be associated with extensive NSC morphological remodeling resembling a “juvenile” pattern of NSC and blood vessel engagements. Several factors are known to enhance adult hippocampal neurogenesis but a factor capable of inducing a long-lasting neurogenic enhancement that attenuates age-related neurogenic decay has not been described. Here, we studied hippocampal neurogenesis following conditional VEGF induction in the adult brain and showed that a short episode of VEGF exposure withdrawn shortly after the generation of durable new vessels (but not under conditions where newly made vessels failed to persist) is sufficient for neurogenesis to proceed at a markedly elevated level for many months later. Continual neurogenic increase over several months was not accompanied by accelerated exhaustion of the neuronal stem cell (NSC) reserve, thereby allowing neurogenesis to proceed at a markedly elevated rate also in old mice. Neurogenic enhancement by VEGF preconditioning was, in part, attributed to rescue of age-related NSC quiescence. Remarkably, VEGF caused extensive NSC remodelling manifested in transition of the enigmatic NSC terminal arbor onto long cytoplasmic processes engaging with and spreading over even remote blood vessels, a configuration reminiscent of early postnatal “juvenile” NSCs. Together, these findings suggest that VEGF preconditioning might be harnessed for long-term neurogenic enhancement despite continued exposure to an “aged” systemic milieu.

Cellular mechanism of oral absorption of solidified polymer micelles

UNLABELLED Oral delivery of poorly soluble and permeable drugs represents a significant challenge in drug development. The oral delivery of drugs remains to be the ultimate route of any drugs. However, in many cases, drugs are not absorbed well in the gastrointestinal tract, or they lose their activity. Polymer micelles were recognized as an effective carrier system for drug encapsulation, and are now studied as a vehicle for oral delivery of insoluble compounds. We characterized the properties of monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles, and visualized their internalization in mouse small intestine. Using Caco-2 cells as a cellular model, we studied the kinetics of particle uptake, their transport, and the molecular mechanism of their intestinal absorption. Moreover, by inhibiting specific endocytosis pathways, pharmacologically and genetically, we found that mPEG-PLA nanoparticle endocytosis is mediated by clathrin in an energy-dependent manner, and that the low-density lipoprotein receptor is involved. FROM THE CLINICAL EDITOR Many current drugs used are non-water soluble and indeed, the ability to deliver these drugs via the gastrointestinal tract remains the holy grail for many researchers. The authors in this paper developed monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles as a drug nanocarrier, and studied the mechanism of uptake across intestinal cells. The findings should improve our current understanding and point to the development of more nanocarriers.

Vimocin and Vidapin, Cyclic KTS Peptides, Are Dual Antagonists of α1β1/α2β1 Integrins with Antiangiogenic Activity

Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys19 and Cys29, as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50–90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10–30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and “bioactive” space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1β1/α2β1 integrin antagonists in antiangiogenesis and cancer therapy.

Matrix metalloproteinase 12 promotes tumor propagation in the lung

Objective: Past studies are inconsistent with regard to the role of matrix metalloproteinase 12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor‐derived versus immune‐derived matrix metalloproteinase 12 expression. Our study aims to thoroughly dissect the role of matrix metalloproteinase 12 in lung tumorigenesis. Methods: We tested matrix metalloproteinase 12 expression and the association with prognosis using a tissue array and a published non–small cell lung cancer gene expression database. In addition, we characterized the contribution of matrix metalloproteinase 12 to tumor propagation in the lung using a series of in vitro and in vivo studies. Results: Tumor cells of a diverse set of human lung cancers stained positive for matrix metalloproteinase 12, and high matrix metalloproteinase 12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of matrix metalloproteinase 12 in lung cancer cells (human 460 lung cancer cell line, Lewis lung carcinoma). In vitro, matrix metalloproteinase 12 knockout Lewis lung carcinoma and Lewis lung carcinoma cells had the same proliferation rate, but Lewis lung carcinoma showed increased invasiveness. In vivo, deficiency of matrix metalloproteinase 12 in Lewis lung carcinoma cells, but not in the host, reduced tumor growth and invasiveness. Conclusions: We suggest that tumor cell–derived matrix metalloproteinase 12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies matrix metalloproteinase 12 should further be tested as a potential novel therapeutic target.

Rigidity of polymer micelles affects interactions with tumor cells.

ABSTRACT Controlling the interaction of drug delivery systems (DDS) with tissues is critical for the success of therapies. Specifically in cancer, due to the high density of the tumors, tissue penetration of DDS is critical and may be challenging. In previous work we have shown that Solidified Polymer Micelles (SPMs) rapidly internalize into cells and tissues. Using AFM analysis, in the present work we measured differences in rigidity of SPM compared with Wet Polymer Micelles (WPM). We further examined whether the semi‐solid form of hydrated SPMs has an effect on the interaction with tumor cells both in mono‐layer systems and in multi‐layer clusters of cells as spheroids. For that we have performed detailed characterization of SPM compared to WPM, including examinations of particle size, stability, drug release kinetics and cell transcytosis, in melanoma A‐375 cells. Cell uptake measurements were done using fluorescent signal analysis, FACS and microscopy imaging, showing enhanced abilities of SPMs to penetrate cells and tissues. A simple physical model is presented that well agrees with the experiments and provides insight about the role of particle rigidity in the engulfment mechanism. We conclude that particle rigidity enhances cellular uptake and tissue penetration and that SPMs have a promising potential as an effective and highly permeable DDS. Our findings can be important in future rational design of DDS for particle adjustment to specific tissues and pathologies.

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Evolving multidimensional pharmacological approaches to CNV therapy in AMD

ABSTRACT Purpose: The leading cause of severe visual loss world-wide is age-related macular degeneration. Although anti-Vascular Endothelial Growth Factor agents have significantly led to the initial pharmacologic reversal of vision loss in many cases of exudative macular degeneration, there still has been recurrence of choroidal neovascularization, and/or the onset of chorioretinal atrophy with fibrosis.Materials and Methods: In this review we discuss the status of anti- Vascular Endothelial Growth Factor in age-related macular degeneration and describe different studies focused on new potential therapeutic targets beyond anti- Vascular Endothelial Growth Factor.Results: Further investigations have elicited that Vascular Endothelial Growth Factor is only one of many angiogenic, and pro-inflammatory factors that bring about the growth and leakage of active choroidal neovascularization. Various new multifaceted strategies, including inhibitors to down-stream targets of endothelial cell division, such as TNP-470, may lead to a more permanent inactivation of choroidal neovascularization.Conclusions: Based on the accumulated results in the treatment of age-related macular degeneration, it is hoped that the appropriate combination of anti-Vascular Endothelial Growth Factor agents with longer-acting and multidimensional pharmaceuticals, such as Methionine Aminopeptidase-2 inhibitors, will more effectively control choroidal neovascularization, prevent atrophy and fibrosis, and reduce the burden of frequent intraocular injections in age-related macular degeneration.

High mobility group box 1 antagonist limits metastatic seeding in the lungs via reduction of cell–cell adhesion

Metastatic spread is the leading cause for cancer-related mortality, with the lungs being a major site for metastatic seeding. Available therapies for patients with metastatic disease are extremely limited. Therefore, there is a desperate need for new strategies to prevent or limit metastatic dissemination and treat existing metastases. The metastatic cascade is highly complex and is affected by multiple factors related to both tumor cells themselves and the microenvironment in the future site of metastasis. We hypothesized that modifying the lung microenvironment by blocking central ubiquitous signals may affect metastatic seeding in the lungs. Given the high basal levels of the Receptor for Advanced Glycation End products (RAGE) in the pulmonary tissue, and its pro-inflammatory properties, we investigated the consequences of interfering with its ligand; High Mobility Group Box 1 (HMGB1). To this end, we tested the effect of Carbenoxolone, an HMGB1 antagonist, on primary tumor growth and metastatic progression in several murine tumor models. We show that antagonizing HMGB1 prevents the adhesion and colonization of cancer cells in the lungs through the reduction of their adhesion and cell–cell interaction both in vitro and in vivo. We demonstrated that these activities are mediated by downregulation of the adhesion molecule Intercellular Adhesion Molecule 1 (ICAM1) and ultimately result in reduced metastatic burden. Carbenoxolone decreases significantly lung metastases formation and can be used potentially as prophylactic therapy for metastatic diseases.

Analytical Method for Transdermal Delivery of the Anti-angiogenic CompoundTNP-470

Pathological angiogenesis is a critical component in cancer, in chronic systemic inflammatory diseases such as psoriasis and rheumatoid arthritis, and in ocular diseases. Anti-angiogenic drugs have the ability to prevent, inhibit, and regress newly formed blood vessels. The activity of TNP-470 (chloro acetylcarbamoylfumagillol), a potent anti-angiogenic drug, has been demonstrated in numerous preclinical studies and in eight clinical studies involving more than three hundred patients. Despite its encouraging efficacy, TNP-470 is unstable compound with short plasma half-life, and, as was found clinically it can cause neurotoxicity side-effects at high doses. In light of these limitations, developing a transdermal drug delivery for TNP-470, can offer a novel and promising clinical usage for this drug by improving its bioavailability, controlled dosage and safety profile. In this work, we developed a reliable method for skin permeation studies of TNP-470, using the pig skin in Franz diffusion cells and High-Performance Liquid Chromatography (HPLC) analysis. Additionally, we performed a broad stability and degradation studies of TNP-470 in different mediums and identify optimal stabilizing conditions in acetate buffer pH-4.5, which can be used for transdermal formulation. Our results demonstrated excellent permeability properties of TNP-470 through the pig skin, where 25% from the initial amount was crossed through the skin membrane after 72 hours. Our results suggesting that TNP-470 is a good candidate for transdermal drug delivery, whereas, an optimal dermal formulation would improve drug’s pharmacokinetic properties and toxicity profile by introducing it in a slow release system.