Identifying novel therapeutic inhibitors to target FMS-like tyrosine kinase-3 (FLT3) against acute myeloid leukemia: a molecular docking, molecular dynamics, and DFT study.

Around 30% of acute myeloid leukemia (AML) patients have triggering mutations in Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3), which has been suggested as a possible therapeutic candidate for AML therapy. Many tyrosine kinase inhibitors are available and have a wide variety of applications in the treatment of cancer by inhibiting subsequent steps of cell proliferation. Therefore, our study aims to identify effective antileukemic agents against FLT3 gene. Initially, well-known antileukemic drug candidates have been chosen to generate a structure-based pharmacophore model to assist the virtual screening of 217,77,093 compounds from the Zinc database. The final hits compounds were retrieved and evaluated by docking against the target protein, where the top four compounds have been selected for the analysis of ADMET. Based on the density functional theory (DFT), the geometry optimization, frontier molecular orbital (FMO), HOMO-LUMO, and global reactivity descriptor values have been evaluated that confirming a satisfactory profile and reactivity order for the selected candidates. In comparison to control compounds, the docking results revealed that the four compounds had substantial binding energies (-11.1 to -11.5 kcal/mol) with FLT3. The physicochemical and ADMET (adsorption, distribution, metabolism, excretion, toxicity) prediction results corresponded to the bioactive and safe candidates. Molecular dynamics (MD) confirmed the better binding affinity and stability compared to gilteritinib as a potential FLT3 inhibitor. In this study, a computational approach has been performed that found a better docking and dynamics score against target proteins, indicating potent and safe antileukemic agents, furthermore in-vivo and in-vitro investigations are recommended.Communicated by Ramaswamy H. Sarma.

Does HAART dysregulate angiogenesis in HIV infected preeclampsia?

A dysregulation of angiogenic mediators has been implicated in HIV infection. Inconsistent data exists on highly active antiretroviral therapy (HAART) usage in pregnancy and its association with PE development. In view of the high prevalence of HIV infection and PE in SA, this study was aimed at determining PlGF and sFlt-1 levels in HIV-infected normotensive and preeclamptic pregnancies treated with HAART. Both PlGF and sFlt-1 were quantified in serum from HIV positive [normotensive (N+) and preeclamptic (P+)]; and HIV negative [normotensive (N-) and preeclamptic (P-)] pregnancies, using a Milliplex Multiplex immunoassay. sFlt-1 was significantly upregulated in P+ vs the N+ groups. PlGF was significantly downregulated in PE vs normotensive groups, regardless of HIV status. sFlt-1/PlGF ratio was significantly increased in PE- vs the N- groups. We report an amplification of sFlt-1 in lieu of PlGF down-regulation in HIV-infected pregnancies receiving HAART .

FMS-like tyrosine kinase-3 (FLT3) inhibitors with better binding affinity and ADMET properties than sorafenib and gilteritinib against acute myeloid leukemia: in silico studies.

Over 30-35% of patients down with AML are caused by mutations of FLT3-ITD and FLT3-TKD which keeps the protein activated while it activates other signaling proteins downstream that are involved in cell proliferation, differentiation, and survival. As drug targets, many inhibitors are already in clinical practice. Unfortunately, the average overall survival rate for patients on medication suffering from AML is 5 years despite the huge efforts in this field. To perform docking simulation and ADMET studies on selected phytochemicals against FLT3 protein receptor for drug discovery against FLT3 induced AML, molecular docking simulation was performed using human FLT3 protein target (PDB ID: 6JQR) and 313 phytochemicals with standard anticancer drugs (Sorafenib and Gilteritinib in addition to other anticancer drugs). The crystal structure of the protein was downloaded from the protein data bank and prepared using Biovia Discovery Studio. The chemical structures of the phytochemicals were downloaded from the NCBI PubChem database and prepared using Open Babel and VConf softwares. Molecular docking was performed using PyRx on Autodock Vina. The ADMET properties of the best performing compounds were calculated using SwissADME and pkCMS web servers. The results obtained showed that glabridin, ellipticine and derivatives (elliptinium and 9-methoxyellipticine), mezerein, ursolic acid, formononetin, cycloartocarpesin, hypericin, silymarin, and indirubin are the best performing compounds better than sorafenib and gilteritinib based on their binding affinities. The top-performing compounds which had better binding and ADMET properties than sorafenib and gilteritinib could serve as scaffolds or leads for new drug discovery against FLT3 induced AML.Communicated by Ramaswamy H. Sarma.

Placental growth factor reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1 and MMP-7 and collagen types I and IV in hypertensive pregnancy.

Preeclampsia is a complication of pregnancy manifested as maternal hypertension (HTN) and fetal intrauterine growth restriction, with unclear mechanisms. Placental ischemia increases antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) relative to angiogenic placental growth factor (PlGF); however, the molecular targets are unclear. To test the hypothesis that placental ischemia-induced changes in sFlt-1 and PlGF target vascular and uteroplacental matrix metalloproteinases (MMPs), we tested whether raising the sFlt-1-to-PlGF ratio by infusing sFlt-1 (10 µg·kg-1·day-1) in pregnant (Preg) rats increases blood pressure (BP) and alters MMPs and whether correcting sFlt-1/PlGF by infusing PlGF (20 µg·kg-1·day-1) in Preg rats with reduced uterine perfusion pressure (RUPP) improves BP and reverses the changes in MMPs. On gestational day 19, BP was higher and the litter size and uterine, placenta, and pup weight were less in Preg + sFlt-1 and RUPP than Preg rats and restored in RUPP + PlGF versus RUPP rats. Gelatin and casein zymography and Western blots revealed decreases in MMP-2 and MMP-9 and increases in MMP-1 and MMP-7 in the aorta, uterine artery, uterus, and placenta of Preg + sFlt-1 and RUPP versus Preg rats, which were reversed in RUPP + PlGF versus RUPP rats. Collagen types I and IV were more abundant in Preg + sFlt-1 and RUPP versus Preg rats and were reversed in RUPP + PlGF versus RUPP rats. Thus, PlGF reverses decreased vascular and uteroplacental MMP-2 and MMP-9 and increased MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and sFlt-1 in HTN in pregnancy. Angiogenic factors and MMP modulators could rectify changes in MMPs and collagen, restore vascular and uteroplacental remodeling, and improve HTN and intrauterine growth restriction in preeclampsia. NEW & NOTEWORTHY Understanding the mechanisms of preeclampsia could help in its prevention and management. This study shows that correcting soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) imbalance by infusing PlGF reverses the decreases in vascular and uteroplacental matrix metalloproteinase (MMP)-2 and MMP-9 and the increases in MMP-1, MMP-7, and collagen types I and IV induced by placental ischemia and antiangiogenic sFlt-1 in hypertension in pregnancy. Angiogenic factors and MMP modulators could rectify changes in vascular and uteroplacental MMPs and collagen content and ameliorate hypertension and intrauterine growth restriction in preeclampsia.

Anti-sFlt-1 Therapy Preserves Lung Alveolar and Vascular Growth in Antenatal Models of Bronchopulmonary Dysplasia.

RATIONALE: Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. OBJECTIVES: Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. METHODS: To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. RESULTS: With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. CONCLUSIONS: We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.

Multiple therapeutic peptide vaccines for patients with advanced gastric cancer.

We performed a clinical trial using HLA-A24-binding peptide vaccines containing a combination of novel cancer-testis antigens and anti-angiogenic peptides for advanced gastric cancer (GC). Thirty-five GC patients who had shown resistance to the standard therapy were enrolled in this clinical trial using vaccinations with a mixture of multiple peptides derived from DEPDC1, URLC10, FoxM1, Kif20A and VEGFR1. The safety, the overall survival (OS), and the immunological responses based on an ELISPOT assay were determined to assess differences in patients who were HLA-A24-positive [24(+)] and HLA-A24-negative [24(-)]. No severe adverse effects were observed except for severe skin reactions in 4 patients. The differences in OS were not significant between patients who were 24(+) and 24(-). In the 24(+) group, patients who showed T cell responses specific to antigen peptides had a tendency towards better survival than those who showed no response, especially to the DEPDC1 peptide. The patients with local skin reactions had significantly better OS than the others. Peptide vaccine therapy was found to be safe and is expected to induce specific T cell responses in patients with advanced GC. The survival benefit of peptide vaccine monotherapy may not have been shown and further trials are needed to confirm these results.

Dual targeting of vascular endothelial growth factor and bone morphogenetic protein-9/10 impairs tumor growth through inhibition of angiogenesis.

Clinical development of anti-angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP-9/10. Single targeting of either VEGF or BMP-9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP-9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti-VEGF and BMP-9/10 therapies.

Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects.

BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2-induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co-delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co-delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF-binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1461-1469, 2017.

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo.

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Two-photon microscopy of a Flt1 peptide-hyaluronate conjugate.

AIM: Two-photon microscopy was performed to visualize ocular distribution of Flt1 peptide-hyaluronate (HA) conjugate micelles for eye drop treatment of corneal neovascularization. MATERIALS & METHODS: Flt1 peptide-HA conjugate micelles were topically administered to the eye for two-photon microscopy and antiangiogenic effect assessment after silver nitrate cauterization. RESULTS: In vivo two-photon microscopy revealed that Flt1 peptide-HA conjugate micelles were absorbed and remained on the corneal epithelia with an increased residence time, facilitating the corneal delivery of carboxyfluorescein succinimidyl ester (CFSE) as a model drug. Furthermore, repeated eye drops of Flt1 peptide-HA conjugate micelles showed comparable therapeutic effect to the subconjunctival injection on the corneal neovascularization. DISCUSSION & CONCLUSION: We confirmed the feasibility of Flt1 peptide-HA conjugate micelles for eye drop treatment of corneal neovascularization.

A novel engineered VEGF blocker with an excellent pharmacokinetic profile and robust anti-tumor activity.

BACKGROUND: Relatively poor penetration and retention in tumor tissue has been documented for large molecule drugs including therapeutic antibodies and recombinant immunoglobulin constant region (Fc)-fusion proteins due to their large size, positive charge, and strong target binding affinity. Therefore, when designing a large molecular drug candidate, smaller size, neutral charge, and optimal affinity should be considered. METHODS: We engineered a recombinant protein by molecular engineering the second domain of VEGFR1 and a few flanking residues fused with the Fc fragment of human IgG1, which we named HB-002.1. This recombinant protein was extensively characterized both in vitro and in vivo for its target-binding and target-blocking activities, pharmacokinetic profile, angiogenesis inhibition activity, and anti-tumor therapeutic efficacy. RESULTS: HB-002.1 has a molecular weight of ~80 kDa, isoelectric point of ~6.7, and an optimal target binding affinity of <1 nM. The pharmacokinetic profile was excellent with a half-life of 5 days, maximal concentration of 20.27 µg/ml, and area under the curve of 81.46 µg·days/ml. When tested in a transgenic zebrafish embryonic angiogenesis model, dramatic inhibition in angiogenesis was exhibited by a markedly reduced number of subintestinal vessels. When tested for anti-tumor efficacy, HB-002.1 was confirmed in two xenograft tumor models (A549 and Colo-205) to have a robust tumor killing activity, showing a percentage of inhibition over 90% at the dose of 20 mg/kg. Most promisingly, HB-002.1 showed a superior therapeutic efficacy compared to bevacizumab in the A549 xenograft model (tumor inhibition: 84.7% for HB-002.1 versus 67.6% for bevacizumab, P<0.0001). CONCLUSIONS: HB-002.1 is a strong angiogenesis inhibitor that has the potential to be a novel promising drug for angiogenesis-related diseases such as tumor neoplasms and age-related macular degeneration.

Cambios moleculares en los márgenes de resección en el carcinoma de células escamosas de cavidad oral / Molecular changes in the resections borders in squamous cell cancer of oral cavity

Evaluación molecular de márgenes de resección en pacientes con carcinoma de células escamosas de cavidad oral sometidos a cirugía. 16 pacientes con carcinoma escamoso de cavidad oral, en cualquiera de sus localizaciones, sin tratamientos previos, intervenidos quirúrgicamente en el 2011. La pieza operatoria fue procesada por anatomía patológica a través del método tradicional, realizándose cortes adicionales que incluían: tumor y 0,5 cm de margen no tumoral. Se realizó hematoxilina-eosina y complementó con inmunomarcaje para p53, PCNA, Ki-67, factor de crecimiento epidérmico y receptor de crecimiento endotelial vascular. De los 16 pacientes en estudio la mayoría eran del género masculino, la edad promedio fue cercana a los 60 años, la mayoría eran pacientes consumidores de tabaco y alcohol. La lengua fue la localización más frecuente y los tumores se encontraban en un estadio avanzado (estadio III y IV). Estudio molecular: todos los marcadores evaluados se encontraban positivos en los márgenes de resección en el 93,75% de los pacientes. Los marcadores de proliferación celular como el PCNA y Ki-67 así como el p-53 se encontraban positivos entre 1,5 cm a 2 cm del tumor con un marcaje intenso. Por el contrario, el factor de crecimiento epidérmico el receptor de crecimiento endotelial vascular se encontraban positivos hasta 1,5 cm pero con menor intensidad. En el cáncer oral podemos observar con frecuencia cambios moleculares en el tejido aparentemente sano que rodea el tumor hasta por lo menos 15 mm.

The molecular evaluation of resection margins in patients with squamous cell carcinoma of oral cavity who underwent surgery. Field of cancerization concept. We included 16 patients with oral cavity squamous cell carcinoma in any of their locations,without pre treatment, surgically treated in our hospital in the 2011 year. The surgical specimen was processed by the pathology department of our institution, through the traditional method, additional sectioned including the tumor and at least 0.5 cm margin non tumorigenic. Study was performed hematoxylin eosin and was supplemented with immunostaining for p53, PCNA, Ki-67, epidermal growth factor receptor and vascular endothelial growth factor receptor. The most important features of the 16 patients studied were: The majorities were male, the average age was around 60 years old; most of them were tobacco and alcohol consumers. The tongue was the most frequent location and most of the tumors were in an advanced stage (stage III y IV). In molecular evaluation all the markers were positive in the resection margins in 93.75% of all patients. The cell proliferation markers suchas PCNA and Ki-67 and the p-53 were positive 1.5 cm to2 cm tumor with intense staining. Conversely, epidermal receptor grow factors and vascular endothelial grow factor receptor were positive up to 1.5 cm but with less intensity. In oral cancer can often observe molecular changes in the apparently healthy tissue surrounding the tumor to at least 15 mm.

Phase I/II study of S-1 plus cisplatin combined with peptide vaccines for human vascular endothelial growth factor receptor 1 and 2 in patients with advanced gastric cancer.

The aim of this study was to evaluate the safety and efficacy of vaccination with human leukocyte antigen (HLA)-A24-restricted human vascular endothelial growth factor receptor 1 (VEGFR1)-1084 and VEGFR2-169 combined with chemotherapy in patients with advanced gastric cancer. HLA-A 2402-positive patients with advanced or recurrent adenocarcinoma of the stomach were vaccinated with VEGFR1-1084 and VEGFR2-169 combined with S-1 and cisplatin. The study included 22 patients (median age 60.5 years) who received at least one cycle of the combination therapy. No severe adverse effects caused by the vaccine therapy were observed except for an inflammatory reaction at the site of injection in 6 patients. Twelve patients (55%) showed partial response and 10 had stable disease after two cycles of the combination therapy. The disease control rate (partial response and stable disease) was 100% after two cycles. The median time to progression was 9.6 months and median overall survival was 14.2 months. VEGFR1-1084-specific cytotoxic T lymphocyte (CTL) response was induced in 18 (82%) of the 22 patients and VEGFR2-169-specific CTL response was induced in 18 (82%) of the 22 patients. Patients showing CTL response to VEGFR2-169 peptide had significantly better prognosis than those without, as demonstrated by the overall survival (OS) and time to progression (TTP) (OS, p=0.028, TTP, p=0.006). The combination therapy was well tolerated and highly effective in advanced or recurrent gastric cancer. Substantial specific CTL for both peptides was frequently induced even under chemotherapy. Thus, cancer vaccination combined with standard chemotherapy warrants further analysis as a promising strategy for the treatment of advanced cancer.

AAV5-mediated sFLT01 gene therapy arrests retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice.

To test the effects of adeno-associated virus encoding sFLT01 (AAV5.sFLT01) on the retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice, a model for age-related macular degeneration (AMD), AAV5.sFLT01 was injected into the subretinal space of the right eyes and the left eyes served as controls. Histology found no retinal toxicity due to the treatment after 3 months. The treated eyes showed lesion arrest compared with lesion progression in the left eyes by fundus monitoring monthly and histological evaluation 3 months after treatment. Retinal ultrastructure showed fewer lipofuscin and better preserved photoreceptors after the treatment. A2E, a major component of lipofuscin, was lower in the treated eyes than in the control eyes. Molecular analysis showed that AAV5.sFLT01 lowered retinal extracellular signal-regulated kinase (ERK) phosphorylation and inducible nitric oxide synthetase expression, which suggested the involvement of reactive nitrogen species in the retinal lesions of Ccl2(-/-)/Cx3cr1(-/-). We concluded that local delivery of AAV5.sFLT01 can stabilize retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice. The findings provide further support for the potential beneficial effects of sFLT01 gene therapy for age-related macular degeneration.

Placental growth factor upregulation is a host response to antiangiogenic therapy.

PURPOSE: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. EXPERIMENTAL DESIGN: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. RESULTS: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non-tumor-bearing mice. CONCLUSION: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others.

Inhibition of ascites tumor growth in vivo by sTie-2 is potentiated by a combinatorial therapy with sFLT-1.

BACKGROUND: Inhibition of tumor angiogenesis is a promising approach for cancer therapy and the Tie-2/angiopoietin pathway appears to play an important role. In the present study, we have developed strategies to explore the therapeutic potential of blocking the Tie-2/angiopoietin pathway by sTie-2. METHODS: Ehrlich ascites tumor (EAT) cells were stably transfected to overexpress a truncated form of sTie-2. Transfectants were characterized for their in vitro growth behavior and transplanted into nude mice. Furthermore, recombinant sTie-2 produced by the baculovirus expression system was used to sequester angiopoietins in the murine ascites carcinoma model. The effect of sTie-2 treatment alone or in combination with sFLT-1 on the weight of the animal, ascites cell number and volume was studied. RESULTS: EAT cells stably transfected with a truncated form of sTie-2 showed no change in cell proliferation in vitro and colony forming in soft agar compared to control cells. However, sTie-2 transfected EAT cells transplanted into nude mice reduced tumor burden and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Recombinant sTie-2 showed angiogenic activity in the tube formation and wound healing assay in vitro. sTie-2 treatment alone or in combination with sFLT-1 in an ascites tumor mouse model resulted in reduced peritoneal angiogenesis, with a concomitant decrease in tumor cell number, volume of ascites and the number of invasive tumor cells, as assayed by CD31 staining. CONCLUSIONS: The findings of the present study demonstrate an important role for the Tie-2/angiopoietin pathway in the formation of tumor vasculature and suggest that sTie-2 might yield useful anticancer therapy.

Role of vascular endothelial growth factor in kidney disease.

Vascular endothelial growth factor (VEGF) is a main regulator of blood vessel growth and plays an important role in promoting endothelial survival and maintaining the microvasculature. The kidney is a highly vascularized organ and has two important microvasculatures; glomerular and peritubular capillaries. Loss of these capillaries is strongly associated with the progression of chronic kidney disease (CKD) to end-stage renal disease. In several kidney disease animal models, VEGF expression in the kidney is decreased and administration of VEGF is protective. Recent clinical observations revealed that blocking VEGF by endogenous inhibitor (soluble Flt-1) in preeclampsia and monoclonal antibody against VEGF in cancer patients cause proteinuria and renal dysfunction. However, plasma VEGF levels in diabetic nephropathy patients are increased and blocking VEGF improved diabetic nephropathy in animal models. Increased plasma VEGF levels have been reported in CKD patients. Deleterious effects of VEGF have been demonstrated in atherosclerosis and sepsis, which are frequent complications in CKD patients. Although administrating VEGF or novel drugs that activate VEGF pathway may improve the progression of CKD, careful monitoring will be required when CKD patients have complications of diabetes, atherosclerosis or sepsis.

Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature.

Vascular functions of PlGF remain poorly understood and controversial. Here, we show that tumor cell-derived PlGF-1 and PlGF-2 displayed significant remodeling effects on the tumor vasculature, leading to a normalized vascular phenotype and improved functions against leakage. In two murine tumor models, that is, T241 fibrosarcoma and Lewis lung carcinoma, stable expression of PlGF-1 and PlGF-2 in tumor cells resulted in significant reduction of tumor microvascular density and branch formation. Markedly, the vasculature in PlGF-expressing tumors consisted of relatively large-diameter microvessels with substantial improvement of pericyte coverage. Similarly, PlGF-induced vascular normalization and remodeling were also observed in a spontaneous human choriocarcinoma that expressed endogenous PlGF. Our findings shed light on functions of PlGF as a vascular remodeling factor that normalizes the tumor vasculature and thus may have conceptual implications of cancer therapy.

Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo.

BACKGROUND: Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation. METHODS: As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. RESULTS: The sFLT-1 produced by the baculovirus system showed potent anti-angiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. CONCLUSIONS: The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy.