Targeted Antagonism of Vascular Endothelial Growth Factor Reduces Mortality of Mice with Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is associated with a mortality of 45%. Our previous research indicated that anti-vascular endothelial growth factor (VEGF) could maintain the normal structure and function of the respiratory barrier. However, systemic application of VEGF antagonists would lead to animal death. This study attempts to study the targeted drug delivery for ARDS. In this study, we used soluble fms-like tyrosine kinase-1 (sFlt)-targeted ultrasound microbubbles to antagonize the effect of VEGF on lung tissue. Ninety male BALB/c mice were randomly assigned to 6 groups: phosphate buffer saline (PBS) group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). After administration of LPS or PBS in the corresponding groups, the sFlt-targeted microbubbles or empty microbubbles were injected into the blood circulation. Then the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and helped release drugs to the lung tissues targeted. The lung injury score, lung wet/dry ratio (W/D), liver and kidney functions, and the mortality of the mice in all groups were investigated at the predetermined time point. The difference in mortality between groups was examined by Fisher test. Other data were analyzed by one-way analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The results showed that the PaO2 levels were normal in the PBS group, the blank group, and the control group. The LPS group and ARDS group showed significant hypoxia. PaO2 was improved significantly in the treatment group. The lung injury score and W/D were normal in the PBS group, the blank group, and the control group. The lung injury score and W/D increased significantly in the LPS group and ARDS group and decreased in the treatment group (P<0.05). The mortality rate of the ARDS model was 60% (95% confidence interval 47.5%-72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%-52.5%, P<0.05). It was concluded that anti-VEGF with sFlt targeted ultrasound microbubbles attenuated the lung injury and ultimately reduced the 7-day mortality effectively. It might be a suitable therapeutic tool for the treatment of ARDS.

Therapeutic potential of combination therapy of soluble VEGF receptor 1 and conventional chemotherapy for ovarian cancer growth.

AIM: This study aimed at evaluating the additional anti-tumor effects of exogenous rVEGFR1 (sFlt1) on conventional chemotherapy in ovarian cancer cell lines. METHODS: We utilized cells from two ovarian cancer cell lines, SKOV3 and HeyA8, and treated them with a combination of rVEGFR1 (sFlt1) and carboplatin as well as rVEGFR1 (sFlt1) alone. First, we evaluated cell survival after treatment by using cell counting and MTS assays. Next, we performed Ki67 staining for evaluating the inhibitory effects of the treatment on cell proliferation, and a lactate dehydrogenase (LDH) assay for evaluating cytotoxicity. Finally, to determine whether MAP kinase signaling is involved in this process, we performed western blot analysis of extracellular signal-regulated kinase (ERK), phospho-ERK, c-jun n-terminal kinase (JNK) and phospho-JNK. RESULTS: The cytotoxic and growth-restriction effects were more pronounced in the group co-administered with rVEGFR1 (sFlt1) and carboplatin than in cells treated with either rVEGFR1 (sFlt1) or carboplatin alone. Quantitative analysis of Ki67-positive cells also showed a decreased proportion of Ki67-positive cells in SKOV3 cells treated with a combination of exogeneous rVEGFR1 (sFlt1) and carboplatin compared to that in cells treated with either rVEGFR1 (sFlt1) or carboplatin alone. In the LDH assay, we also found significantly enhanced cell toxicity from the combination therapy. Finally, western blotting analysis showed that the MAPK signaling pathway was not affected by sFlt1 treatment. CONCLUSION: This study confirmed the additive effects of rVEGFR1 (sFlt1) combined with conventional chemotherapy for ovarian cancer growth in in vitro assays, thus suggesting the combination of rVEGFR1 (sFlt1) and carboplatin as a potential novel therapeutic option for ovarian cancer.

Increased soluble fms-like tyrosine kinase 1 after ischemia reperfusion contributes to adverse clinical outcomes following kidney transplantation.

Renal ischemia reperfusion injury (IRI) adversely affects clinical outcomes following kidney transplantation. Understanding the cellular mechanisms and the changes in gene/protein expression following IRI may help to improve these outcomes. Serum soluble fms-like tyrosine kinase 1 (sFlt-1), a circulating antiangiogenic protein, is increased in the first week following kidney transplantation. We evaluated the casual relationship of elevated sFlt-1 levels with renal microvascular dysfunction following IRI in a longitudinal study of 93 kidney transplant recipients and in several animal models. Transplant recipients with higher sFlt-1 levels had higher odds of delayed graft function, graft rejection, impaired graft function, and death. In a subgroup of 25 participants who underwent kidney biopsy within 4 months of kidney transplantation, peritubular capillary area was lower in those with elevated serum sFtl-1 levels. The administration of recombinant sFlt-1 into rodents resulted in significant structural and functional changes of the renal microvasculature, including reduced peritubular capillary density and intracapillary blood volume, and lead to increased expression of inflammatory genes and increased fibrosis. In a murine model of IRI, the kidney was a site of sFlt-1 production, and systemic neutralization of sFlt-1 preserved peritubular capillary density and alleviated renal fibrosis. Our data indicate that high sFlt-1 levels after IRI play an important role in the pathogenesis of microvascular dysfunction, thereby contributing to adverse clinical outcomes following kidney transplantation.

Arginase controls soluble vascular endothelial growth factor receptor 1 (sFlt1) to maintain pregnancy homeostasis.

Excessive soluble fms-like tyrosine kinase-1 (sFlt-1) has been strongly implicated in preeclampsia. An increase in the serum sFlt-1 level occurs before the onset of preeclampsia, and the sFlt-1 level is already higher in women who are predisposed to preeclampsia than in normotensive pregnant women. This study aimed to investigate the relation between arginase and sFlt-1 in the plasma of preeclamptic women and normotensive pregnant women. We suggested that a regulatory mechanism exists that suppresses the level of sFlt-1. The relationship between arginase, one of the nitric oxide (NO) modulators, and sFlt-1 was examined. First, the pregnant women were divided into 4 groups: group 1, sFlt-1 <6000 pg/ml and arginase activity <110 U/L; group 2, sFlt-1 ≥6000 pg/ml and arginase activity <110 U/L; group 3, sFlt-1 ≥6000 pg/ml and arginase activity ≥110 U/L; and group 4, sFlt-1 <6000 pg/ml and arginase activity ≥110 U/L. Groups 2 and 3 comprised preeclamptic women. The preeclampsia/normotensive ratio increased from groups 1 to 3. Under the higher sFlt-1 condition, lower arginase activity was associated with lower occurrence of preeclampsia. Next, in human umbilical endothelial vein cells (HUVECs), a slightly higher concentration of sFlt-1, as in group 2, reduced arginase expression and arginase activity, and S-(2-boronoethyl)-l-cysteine (BEC; arginase inhibitor) impaired sFlt-1 secretion. In contrast, a higher level of sFlt-1 increased arginase expression and activity in HUVECs, as in group 3. These results showed that arginase controlled sFlt-1 elevation to some extent. In conclusion, our results suggest the existence of a mechanism to maintain the level of sFlt-1. Soluble Flt-1 negatively regulated itself against increasing serum sFlt-1 in preeclampsia. Moreover, this study revealed that arginase inhibitors are a potential treatment option for preeclampsia.

sFLT-1 inhibits proliferation, migration, and invasion of colorectal cancer SW480 cells through vascular mimicry formation suppression.

To investigate the effects of soluble fms-like tyrosine kinase-1 on the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells. The recombinant plasmid pBLAST49-sFLT-1 or pBLAST49 control plasmid was transfected into SW480 cells to obtain hsFLT-1-SW480 or Ctrl-SW480 cells. The three-dimensional model culture, sulforhodamine B assay, scratch assay, and Transwell assay were performed to detect the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells, respectively. Western blotting was used to detect the expression of vascular endothelial-cadherin protein. Compared with Ctrl-SW480 cells, vascular mimicry formation ((0.85 ± 0.04) vs (7.40 ± 0.69), p < 0.05) and vascular endothelial-cadherin expression ((1.25 ± 0.08) vs (1.89 ± 0.03), p < 0.05) were significantly decreased, and the growth rate was also significantly decreased in hsFLT-1-SW480 cells ((32.54 ± 5.12) vs (88.13 ± 11.52), p < 0.05). Moreover, the migration ((0.46 ± 0.08) vs (0.94 ± 0.03), p < 0.05) and invasion capacity ((59.14 ± 3.64) vs (134.85 ± 10.16), p < 0.05) of SW480 cells were significantly inhibited upon soluble fms-like tyrosine kinase-1 transfection. soluble fms-like tyrosine kinase-1 inhibits cell proliferation, migration, and invasion of colorectal cancer SW480 cells through suppression of vascular mimicry formation, which provides a good basis for the development of new drugs for the treatment of colorectal cancer by targeting both angiogenesis and vascular mimicry formation.

Inhibition of T-cell activation attenuates hypertension, TNFα, IL-17, and blood-brain barrier permeability in pregnant rats with angiogenic imbalance.

PROBLEM: Angiogenic imbalance during pregnancy is associated with immune activation, hypertension, increased T cell infiltration, and neurological insults. METHOD OF STUDY: On gestational day (GD) 12, timed-pregnant rats were infused with anti-angiogenic factors sFlt-1 and sEndoglin (4.7 and 7 µg/kg) to create HELLP syndrome via mini-osmotic pumps for 8 days, with a subset of these rats having Orencia (2 mg/kg) infused on GD13. On GD19, blood-brain barrier (BBB) permeability was evaluated via Evan's Blue infusion, blood was collected for T-cell measurements, inflammatory cytokine secretion. Brain tissues were also collected to examine inflammatory cytokine infiltration. RESULTS: T-cell attenuation with Orencia decreased circulating CD4(+) and CD8(+) T cells, circulating tumor necrosis factor alpha (TNFα) and IL-17, BBB permeability and significantly decreased biochemical evidence of HELLP compared to untreated HELLP rats. CONCLUSIONS: These data support the hypothesis that T cells have a critical role in contributing to the pathophysiology that is seen in angiogenic imbalance during pregnancy.

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.

Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

BACKGROUND: Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. METHODS: In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. FINDINGS: From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of rAAV.sFLT-1 was highly reproducible. No drug-related adverse events were noted; procedure-related adverse events (subconjunctival or subretinal haemorrhage and mild cell debris in the anterior vitreous) were generally mild and self-resolving. There was no evidence of chorioretinal atrophy. Clinical laboratory assessments generally remained unchanged from baseline. Four (67%) of six patients in the treatment group required zero rescue injections, and the other two (33%) required only one rescue injection each. INTERPRETATION: rAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. FUNDING: National Health and Medical Research Council of Australia, Richard Pearce Bequest, Lions Save Sight Foundation, Brian King Fellowship, and Avalanche Biotechnologies, Inc.

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

OBJECTIVE: Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. METHODS: Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. RESULTS: Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 µg/mg vs. 19.3 ± 5.6 µg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). CONCLUSIONS: A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.

Phase I clinical trial of peptide vaccination with URLC10 and VEGFR1 epitope peptides in patients with advanced gastric cancer.

Peptide vaccine treatment has attracted attention in recent years as a new therapy option for chemotherapy­resistant, advanced, unresectable cancer. The safety of peptide vaccination with HLA-A*2402-restricted URLC10-A24-177 and VEGFR1-A12-9 1084 epitope peptides (fixed 2-mg dose) was investigated in a phase I clinical trial of patients with advanced gastric cancer who were refractory to chemotherapy. We determined the HLA genotype of the subjects after enrollment, results of which were held by the evaluation committee and kept from both patients and investigators until completion of the study. The primary end­point was safety of the peptide vaccination. The secondary end­points were immunological responses and clinical outcome, which were compared between the HLA-A*2402-positive and HLA-A*2402-negative groups. The peptides were subcutaneously administered on day 1, 8, 15 and 22 within a 28-day treatment cycle. A total of 14 patients was enrolled in this study; 12 of the 14 patients received 4 or more vaccinations (at least 1 course). No patient had a severe treatment-related adverse event. Findings from evaluation of clinical responses after a single course showed that 4 cases had stable disease and 8 cases had progressive disease. The median overall survival time (MST) for the 12 patients was 3.9 months. The MSTs in the HLA-A*2402­positive and HLA-A*2402­negative groups were, 4.2 and 3.6 months (p=0.9164), respectively. The results of this study showed that vaccination with URLC10 and VEGFR1 peptides was a safe treatment for advanced gastric cancer. This trial was registered with University Hospital Medical Information Network (UMIN, no. 000002409).

The functionalized human serine protease granzyme B/VEGF121 targets tumor vasculature and ablates tumor growth.

The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF121 binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF121 fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. GrB/VEGF121 localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF121 induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF121 localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF121 to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF121 showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF121 at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.

[Novel gene transfer using micellar nanovectors inhibits choroidal neovascularization].

The treatment of age-related macular degeneration (AMD) caused by choroidal neovascularization (CNV) is difficult. More effective therapy for regulating CNV is needed. We demonstrated that intravenous nonviral vectors based on the complex of plasmid DNA with synthetic cationic polymers accumulate in choroidal neovascularization (CNV) with high efficiency through an enhanced the permeability and retention (EPR) effect. This review shows the results of in vivo angiogenic control by intravenous injection of a polyplex micelle-encapsulating plasmid vector using a mice CNV model. Polyion complex (PIC) micelles consisting of plasmid DNA and poly (ethylene glycol)-b-poly (N-[N-(2-aminoethyl)-2-aminoethyl] aspartamidef block copolymers [PEG-b-PAsp (DET)] were used. These show minimal cytotoxicity and high transfection efficiency both in vitro and in vivo, and have been utilized for gene therapy against a mouse corneal neovascularization model by local administration of plasmid-encoding soluble vascular endothelial growth factor receptor 1 (soluble Fms-like tyrosine kinase-1: sFlt-1). Transfection of plasmid-expressing sFlt-1 with PEG-C6-P[Asp (DET)] polyplex micelles by intravenous injection into mice CNV models showed significant inhibition of developing CNV. We found that nonviral gene therapy has significant potential for regulation of CNV using plasmids with PEG-C6-P [Asp (DET)] polyplex micelles.

VEGF receptor binding peptide-linked amphiphilic peptide with arginines and valines for endothelial cell-specific gene delivery.

An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch (R3V6) has been previously reported to deliver plasmid DNA (pDNA) into cells with no toxicity. Here, the vascular endothelial growth factor receptor binding peptide (VRBP) was linked to R3V6 to promote endothelial-specific gene delivery. The pDNA/VRBP-linked R3V6 (VRBP-R3V6) complex was physically characterized via various methods. In a gel retardation assay, pDNA was completely retarded by VRBP-R3V6 at a weight ratio of 1:2 (pDNA:peptide). VRBP-R3V6 also protected pDNA from DNase I for longer than 60 min. Heparin competition assay showed that the pDNA/VRBP-R3V6 complex did not release pDNA when heparin was introduced at a two-fold weight excess of pDNA. In vitro transfection showed that VRBP-R3V6 had transfection efficiency into endothelial cells approximately 200 times greater than that of R3V6. In addition, the transfection efficiency was further enhanced into hypoxic endothelial cells. However, in human embryonic kidney 293 and neuroblastoma N2A cells, VRBP-R3V6 only achieved a transfection rate 10 times higher than R3V6, indicating that VRBP-R3V6 has high specificity for endothelial cells. VRBP-R3V6 was also shown to be nontoxic in a cytotoxicity assay. The data presented here suggest that VRBP-R3V6 may prove useful for specific gene delivery to endothelial cells.

Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia.

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.

Hyperoxia therapy of pre-proliferative ischemic retinopathy in a mouse model.

PURPOSE: To investigate the therapeutic use and mechanisms of action of normobaric hyperoxia to promote revascularization and to prevent neovascularization in a mouse model of oxygen-induced ischemic retinopathy. METHODS: Hyperoxia treatment (HT, 40%-75% oxygen) was initiated on postnatal day (P) 14 during the pre-proliferative phase of ischemic retinopathy. Immunohistochemistry, ELISA, and quantitative PCR were used to assess effects on retinal vascular repair and pathologic angiogenesis in relation to glial cell injury, VEGF protein, and mRNA levels of key mediators of pathologic angiogenesis. Effects of intravitreal injections of VEGF and the VEGF inhibitor VEGFR1/Fc fusion protein were also studied. RESULTS: Administration of HT during the ischemic pre-proliferative phase of retinopathy effectively accelerated the process of revascularization while preventing the development of vitreous neovascularization. HT enhanced the formation of specialized endothelial tip cells at the edges of the repairing capillary networks and blocked the overexpression of several molecular mediators of angiogenesis, inflammation, and extracellular proteolysis. HT markedly reduced the reactive expression of GFAP in Müller cells and improved the morphology of astrocytes in the avascular region of the retina. Exogenous VEGF administered into the vitreous on P14 was not sufficient to cause vitreous neovascularization in the HT mice. Injection of the VEGF antagonist VEGFR1/Fc blocked both pathologic and physiological angiogenesis and did not rescue astrocytes. CONCLUSIONS: HT may be clinically useful to facilitate vascular repair while blocking neovascularization in the pre-proliferative stage of ischemic retinopathy by correcting a broad range of biochemical and cellular abnormalities.

A laboratory-scale device for the straightforward production of uniform, small sized cell microcapsules with long-term cell viability.

Microencapsulated and genetically engineered cells may be used for prolonged delivery of therapeutically active proteins. The objective of this study was to develop a simple, inexpensive and flexible laboratory-scale device for the production of cell microcapsules, especially capsules of small diameter (<300 µm). Many microencapsulation devices are expensive, difficult to assemble and to use, and often more suitable for large-scale experiments. However, the simplicity and low price of the encapsulation system should not limit the quality of capsules and reproducibility of the process: for successful in vitro and in vivo experiments it is important to be able to produce uniform, spherical microcapsules without deformities with high reproducibility. In addition, an advantage of the present procedure compared to other similar, co-axial laminar gas flow systems is the possibility to produce also small microcapsules, less than 200 µm in diameter, with narrow size distribution. First, design, optimization and reproducibility testing of this custom-built device were carried out. Second, microencapsulated retinal pigment epithelial cells (ARPE-19) capable of secreting soluble vascular endothelial growth factor receptor 1 (sVEGFR1) were engineered. The cells remained viable in alginate-poly-L-lysine-alginate microcapsules and secreted sVEGFR1 for prolonged periods.

Role of vascular endothelial growth factor-A in development of abdominal aortic aneurysm.

AIMS: Increased angiogenesis, chronic inflammation, and extracellular matrix degradation are the major pathological features of abdominal aortic aneurysm (AAA). We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, a potent angiogenic and proinflammatory factor, in the development of AAA. METHODS AND RESULTS: Human AAA samples showed increased VEGF-A expression, neovascularization, and macrophage infiltration compared with normal aortic walls. AAA was induced in mice by periaortic application of CaCl(2). AAA mice were treated with soluble VEGF-A receptor (sFlt)-1 or phosphate-buffered saline and sacrificed 6 weeks after the operation. Treatment with sFlt-1 resulted in reduced aneurysm size, restored wavy structure of the elastic lamellae, reduced Mac-2(+) monocytes/macrophages, CD3(+) T-lymphocytes, and CD31(+) vessels, and attenuated matrix metalloproteinase (MMP)-2 and 9 activity in periaortic tissue of AAA. Increased aortic mRNA expression of monocyte chemotactic protein-1, tumour necrosis factor-α, and intercellular adhesion molecule-1 in AAA was attenuated by sFlt-1 treatment. CONCLUSION: VEGF-A was overexpressed in the aortic wall of human and experimental AAA. Treatment with sFlt-1 inhibited AAA development in mice, in association with reduced neoangiogenesis, infiltration of inflammatory cells, MMP activity, and extracellular matrix degradation. These findings suggest a crucial role of VEGF-A in the development of AAA.

Gene transfer for neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex disease that has two phases: a degenerative phase often referred to as nonneovascular AMD (non-NVAMD) or dry AMD and a phase dominated by growth of new blood vessels in the subretinal space, referred to as NVAMD or wet AMD. Advances in the understanding of the molecular pathogenesis of NVAMD have led to new drug therapies that have provided major benefits to patients. However, those treatments require frequent intraocular injections that in many patients must be continued indefinitely to maintain visual benefits. Gene transfer to augment expression of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term stability in patients with NVAMD. Studies in animal models that mimic aspects of NVAMD have identified several possible transgenes, and a clinical trial in patients with advanced NVAMD has suggested that the approach may be feasible. Many important questions remain, but the rationale and preliminary data are compelling. The results of two ongoing clinical trials may answer several of the questions and help direct future research.

Antiangiogenic arming of an oncolytic vaccinia virus enhances antitumor efficacy in renal cell cancer models.

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.