Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models.

The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential

Engineered microenvironments for synergistic VEGF - integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. it controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells

VEGF(164)-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF(164) increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF(164)-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF(164)-deficient (VEGF(120/188)) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF(+/+)) controls. In contrast, administration of a VEGFk-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF(164) selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined

Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-cc (TGF-a), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and aT3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta -estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas

Spatiotemporal release of VEGF from biodegradable polylactic-co-glycolic acid microspheres induces angiogenesis in chick chorionic allantoic membrane assay

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.While vascular endothelial growth factor (VEGF) is an acknowledged potent pro-angiogenic agent there is a need to deliver it at an appropriate concentration for several days to achieve angiogenesis. The aim of this study was to produce microspheres of biodegradable polylactic-co-glycolic acid (PLGA) tailored to achieve sustained release of VEGF at an appropriate concentration over seven days, avoiding excessive unregulated release of VEGF that has been associated with the formation of leaky blood vessels. Several formulations were examined to produce microspheres loaded with both human serum albumin (HSA) and VEGF to achieve release of VEGF between 3 and 10 ng per ml for seven days to match the therapeutic window desired for angiogenesis. In vitro experiments showed an increase in endothelial cell proliferation in response to microspheres bearing VEGF. Similarly, when microspheres containing VEGF were added to the chorionic membrane of fertilised chicken eggs, there was an increase in the development of blood vessels over seven days in response, which was significant for microspheres bearing VEGF and HSA, but not VEGF alone. There was an increase in both blood vessel density and branching – both signs of proangiogenic activity. Further, there was clearly migration of cells to the VEGF loaded microspheres. In summary, we describe the development of an injectable delivery vehicle to achieve spatiotemporal release of physiologically relevant levels of VEGF for several days and demonstrate the angiogenic response to this. We propose that such a treatment vehicle would be suitable for the treatment of ischemic tissue or wounds

Angiotensin II and VEGF are Involved in Angiogenesis Induced by Short-Term Exercise Training

Results from our laboratory have suggested a pathway involving angiotensin II type 1 (AT1) receptors and vascular endothelial growth factor (VEGF) in angiogenesis induced by electrical stimulation. The present study investigated if similar mechanisms underlie the angiogenesis induced by short-term exercise training. Seven days before training and throughout the training period, male Sprague-Dawley rats received either captopril or losartan in their drinking water. Rats underwent a 3-day treadmill training protocol. The tibialis anterior and gastrocnemius muscles were harvested under anesthesia and lightly fixed in formalin (vessel density) or frozen in liquid nitrogen (VEGF expression). In controls, treadmill training resulted in a significant increase in vessel density in all muscles studied. However, the angiogenesis induced by exercise was completely blocked by either losartan or captopril. Western blot analysis showed that VEGF expression was increased in the exercised control group, and both losartan and captopril blocked this increase. The role of VEGF was directly confirmed using a VEGF-neutralizing antibody. These results confirm the role of angiotensin II and VEGF in angiogenesis induced by exercise

Regulation of vascular endothelial growth factor bioactivity in patients with acute lung injury

Background: Reduced bioactive vascular endothelial growth factor (VEGF) has been demonstrated in several inflammatory lung conditions including the acute respiratory distress syndrome (ARDS). sVEGFR-1, a soluble form of VEGF-1 receptor, is a potent natural inhibitor of VEGF. We hypothesised that sVEGFR-1 plays an important role in the regulation of the bioactivity of VEGF within the lung in patients with ARDS. Methods: Forty one patients with ARDS, 12 at risk of developing ARDS, and 16 normal controls were studied. Bioactive VEGF, total VEGF, and sVEGFR-1 were measured by ELISA in plasma and bronchoalveolar lavage (BAL) fluid. Reverse transcriptase polymerase chain reaction for sVEGFR-1 was performed on BAL cells. Results: sVEGFR-1 was detectable in the BAL fluid of 48% (20/41) of patients with early ARDS (1.4– 54.8 ng/ml epithelial lining fluid (ELF)) compared with 8% (1/12) at risk patients (p = 0.017) and none of the normal controls (p = 0.002). By day 4 sVEGFR-1 was detectable in only 2/18 ARDS patients (p = 0.008). Patients with detectable sVEGFR-1 had lower ELF median (IQR) levels of bioactive VEGF than those without detectable sVEGFR-1 (1415.2 (474.9–3192) pg/ml v 4761 (1349–7596.6) pg/ml, median difference 3346 pg/ml (95% CI 305.1 to 14711.9), p = 0.016), but there was no difference in total VEGF levels. BAL cells expressed mRNA for sVEGFR-1 and produced sVEGFR-1 protein which increased following incubation with tumour necrosis factor a. Conclusion: This study shows for the first time the presence of sVEGFR-1 in the BAL fluid of patients with ARDS. This may explain the presence of reduced bioactive VEGF in patients early in the course of ARDS

Vascular Endothelial Growth Factor (VEGF) Prevents the Downregulation of the Cholinergic Phenotype in Axotomized Motoneurons of the Adult Rat

Vascular endothelial growth factor (VEGF) was initially characterized by its activity on the vascular system. However, there is growing evidence indicating that VEGF also acts as a neuroprotective factor, and that its administration to neurons suffering from trauma or disease is able to rescue them from cell death. We questioned whether VEGF could also maintain damaged neurons in a neurotransmissive mode by evaluating the synthesis of their neurotransmitter, and whether its action would be direct or through its well-known angiogenic activity. Adult rat extraocular motoneurons were chosen as the experimental model. Lesion was performed by monocular enucleation and immediately a gelatine sponge soaked in VEGF was implanted intraorbitally. After 7 days, abducens, trochlear, and oculomotor nuclei were examined by immunohistochemistry against choline acetyltransferase (ChAT), the biosynthetic enzyme of the motoneuronal neurotransmitter acetylcholine. Lesioned motoneurons exhibited a noticeable ChAT downregulation which was prevented by VEGF administration. To explore whether this action was mediated via an increase in blood vessels or in their permeability, we performed immunohistochemistry against laminin, glucose transporter-1 and the plasmatic protein albumin. The quantification of the immunolabeling intensity against these three proteins showed no significant differences between VEGF-treated, axotomized and control animals. Therefore, the present data indicate that VEGF is able to sustain the cholinergic phenotype in damaged motoneurons, which is a first step for adequate neuromuscular neurotransmission, and that this action seems to be mediated directly on neurons since no sign of angiogenic activity was evident. These data reinforces the therapeutical potential of VEGF in motoneuronal diseases.España, MINECO and FEDER BFU2015-64515-PJunta de Andalucía and FEDER : P10-CVI605