Gadi Cohen

Vixapatin (VP12), a c-type lectin-protein from Vipera xantina palestinae venom: characterization as a novel anti-angiogenic compound.

A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.

Neurotherapeutic effect of cord blood derived CD45+ hematopoietic cells in mice after traumatic brain injury.

Treatment of traumatic brain injury (TBI) is still an unmet need. Cell therapy by human umbilical cord blood (HUCB) has shown promising results in animal models of TBI and is under evaluation in clinical trials. HUCB contains different cell populations but to date, only mesenchymal stem cells have been evaluated for therapy of TBI. Here we present the neurotherapeutic effect, as evaluated by neurological score, using a single dose of HUCB-derived mononuclear cells (MNCs) upon intravenous (IV) administration one day post-trauma in a mouse model of closed head injury (CHI). Delayed (eight days post-trauma) intracerebroventricular administration of MNCs showed improved neurobehavioral deficits thereby extending the therapeutic window for treating TBI. Further, we demonstrated for the first time that HUCB-derived pan-hematopoietic CD45 positive (CD45(+)) cells, isolated by magnetic sorting and characterized by expression of CD45 and CD11b markers (96-99%), improved the neurobehavioral deficits upon IV administration, which persisted for 35 days. The therapeutic effect was in a direct correlation to a reduction in the lesion volume and decreased by pre-treatment of the cells with anti-human-CD45 antibody. At the site of brain injury, 1.5-2 h after transplantation, HUCB-derived cells were identified by near infrared scanning and immunohistochemistry using anti-human-CD45 and anti-human-nuclei antibodies. Nerve growth factor and vascular endothelial growth factor levels were differentially expressed in both ipsilateral and contralateral brain hemispheres, thirty-five days after CHI, measured by enzyme-linked immunosorbent assay. These findings indicate the neurotherapeutic potential of HUCB-derived CD45(+) cell population in a mouse model of TBI and propose their use in the clinical setting of human TBI.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

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.

Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

Enhanced Therapeutic Anti-Inflammatory Effect of Betamethasone on Topical Administration with Low-Frequency, Low-Intensity (20 kHz, 100 mW/cm2) Ultrasound Exposure on Carrageenan-Induced Arthritis in a Mouse Model

The purpose of this work was to investigate whether low-frequency, low-intensity (20 kHz, <100 mW/cm(2), spatial-peak, temporal-peak intensity) ultrasound, delivered with a lightweight (<100 g), tether-free, fully wearable, battery-powered applicator, is capable of reducing inflammation in a mouse model of rheumatoid arthritis. The therapeutic, acute, anti-inflammatory effect was estimated from the relative swelling induced in mice hindlimb paws. In an independent, indirect approach, the inflammation was bio-imaged by measuring glycolytic activity with near-infrared labeled 2-deoxyglucose. The outcome of the experiments indicated that the combination of ultrasound exposure and topical application of 0.1% (w/w) betamethasone gel resulted in statistically significantly (p < 0.05) enhanced anti-inflammatory activity in comparison with drug or ultrasound treatment alone. The present study underscores the potential benefits of low-frequency, low-intensity ultrasound-assisted drug delivery. However, the proof of concept presented indicates the need for additional experiments to systematically evaluate and optimize the potential of, and the conditions for, tolerable low-frequency, low-intensity ultrasound-promoted non-invasive drug delivery.

The Effects of a Chactoid Scorpion Venom and Its Purified Toxins on Rat Blood Pressure and Mast Cells Histamine Release

The effect of the venom of the Chactoid family of scorpions on blood pressure was scantly investigated and was addressed in the present study using the venom of the Israeli scorpion, Scorpio maurus palmatus. Blood pressure in rats was monitored via cannulated femoral artery, while venom and toxins were introduced into femoral vein. Venom injection elicited a biphasic effect, expressed first by a fast and transient hypotensive response, which lasted up to 10 min, followed by a hypertensive response, which lasted up to one hour. It was found that these effects resulted from different venom components. Phospholipase A2 produced the hypotensive effect, while a non-enzymatic neurotoxic polypeptide fraction produced the hypertensive effect. Surprisingly, the main neurotoxic polypeptide to mice had no effect on blood pressure. In vitro experiments indicated that the hypertensive factors caused histamine release from the peritoneal mast cells, but this effect is assumed to be not relevant to their in vivo effect. In spite of the cytotoxic activity of phospholipase A2, it did not release histamine. These findings suggest that the effects of venom and isolated fractions on blood pressure parameters are mediated by different mechanisms, which deserve further pharmacological investigation.

Near Infrared Optical Visualization of Epidermal Growth Factor Receptors Levels in COLO205 Colorectal Cell Line, Orthotopic Tumor in Mice and Human Biopsies

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

Cytocompatibility of novel extracellular matrix protein analogs of biodegradable polyester polymers derived from α-hydroxy amino acids

One of the challenges in regenerative medicine is the development of novel biodegradable materials to build scaffolds that will support multiple cell types for tissue engineering. Here we describe the preparation, characterization, and cytocompatibility of homo- and hetero-polyesters of α-hydroxy amino acid derivatives with or without lactic acid conjugation. The polymers were prepared by a direct condensation method and characterized using gel permeation chromatography, 1H-nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, optical activity, and solubility. The surface charge of the polymers was evaluated using zeta potential measurements. The polymers were coated onto glass cover slips followed by characterization using nano-surface profiler, thin film reflectometry, and atomic force microscopy (AFM). Their interaction with endothelial and neuronal cells was assessed using adhesion, proliferation, and differentiation assays. Of the characterized polymers, Poly-HOVal-LA, but not Poly-(D)HOPhe, significantly augmented nerve growth factor (NGF)-induced neuronal differentiation of the PC12 pheochromcytoma cells. In contrast, Poly-HOLeu increased by 20% the adhesion of endothelial cells, but did not affect PC12 cell differentiation. NGF-induced Erk1/2 phosphorylation in PC12 cells grown on the different polymers was similar to the effect observed for cells cultured on collagen type I. While no significant association could be established between charge and the differentiative/proliferative properties of the polymers, AFM analysis indicated augmentation of NGF-induced neuronal differentiation on smooth polymer surfaces. We conclude that overall selective cytocompatibility and bioactivity might render α-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.