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Fetal growth restriction (FGR) occurs in ∼8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterized by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad)-mediated overexpression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from midgestation correlates with reduced lamb birthweight near term. Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high intake (n=45) or control intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomized midgestation to receive bilateral UtA injections of 5×10¹¹ particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14), or control saline (n=13). Fetal growth/well-being were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularization within the perivascular adventitia, and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry, and qRT-PCR, respectively. Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3-4 weeks postinjection (p=0.016-0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) after Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter-to-abdominal circumference and brain-to-liver weight ratios). No effects were observed on UBF or neovascularization; however, Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal-to-placental weight ratio) and FLT1/KDR mRNA expression were increased in the maternal but not fetal placental compartments, suggesting downstream effects on placental function. Ad.VEGF gene therapy improves fetal growth in a sheep model of FGR, although the precise mechanism of action remains unclear.

Background: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia. Methods: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP –/– , global natriuretic peptide receptor (NPR)-B –/– and NPR-C –/– animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease. Results: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP –/– and NPR-C –/– , but not NPR-B –/– , mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of G i , ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level. Conclusions: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.

BackgroundThe normal development of the uteroplacental circulation in pregnancy depends on angiogenic and vasodilatory factors such as vascular endothelial growth factor (VEGF). Reduced uterine artery blood flow (UABF) is a common cause of fetal growth restriction; abnormalities in angiogenic factors are implicated. Previously we showed that adenovirus (Ad)-mediated VEGF-A165 expression in the pregnant sheep uterine artery (UtA) increased nitric oxide synthase (NOS) expression, altered vascular reactivity and increased UABF. VEGF-D is a VEGF family member that promotes angiogenesis and vasodilatation but, in contrast to VEGF-A, does not increase vascular permeability. Here we examined the effect of Ad.VEGF-DΔNΔC vector encoding a fully processed form of VEGF-D, on the uteroplacental circulation.MethodsUtA transit-time flow probes and carotid artery catheters were implanted in mid-gestation pregnant sheep (n = 5) to measure baseline UABF and maternal haemodynamics respectively. 7–14 days later, after injection of Ad.VEGF-DΔNΔC vector (5×1011 particles) into one UtA and an Ad vector encoding β-galactosidase (Ad.LacZ) contralaterally, UABF was measured daily until scheduled post-mortem examination at term. UtAs were assessed for vascular reactivity, NOS expression and endothelial cell proliferation; NOS expression was studied in ex vivo transduced UtA endothelial cells (UAECs).ResultsAt 4 weeks post-injection, Ad.VEGF-DΔNΔC treated UtAs showed significantly lesser vasoconstriction (Emax144.0 v/s 184.2, p = 0.002). There was a tendency to higher UABF in Ad.VEGF-DΔNΔC compared to Ad.LacZ transduced UtAs (50.58% v/s 26.94%, p = 0.152). There was no significant effect on maternal haemodynamics. An increased number of proliferating endothelial cells and adventitial blood vessels were observed in immunohistochemistry. Ad.VEGF-DΔNΔC expression in cultured UAECs upregulated eNOS and iNOS expression.ConclusionsLocal over-expression of VEGF-DΔNΔC in the UtAs of pregnant mid-gestation sheep reduced vasoconstriction, promoted endothelial cell proliferation and showed a trend towards increased UABF. Studies in cultured UAECs indicate that VEGF-DΔNΔC may act in part through upregulation of eNOS and iNOS.

In a model of growth-restricted sheep pregnancy, it was previously demonstrated that transient uterine artery VEGF overexpression can improve fetal growth. This approach was tested in guinea-pig pregnancies, where placental physiology is more similar to humans. Fetal growth restriction (FGR) was attained through peri-conceptual nutrient restriction in virgin guinea pigs. Ad.VEGF-A or Ad.LacZ (1 × 10vp) was applied at mid-gestation via laparotomy, delivered externally to the uterine circulation with thermosensitive gel. At short-term (3-8 days post surgery) or at term gestation, pups were weighed, and tissues were sampled for vector spread analysis, VEGF expression, and its downstream effects. Fetal weight at term was increased (88.01 ± 13.36 g; n = 26) in Ad.VEGF-A-treated animals compared with Ad.LacZ-treated animals (85.52 ± 13.00 g; n = 19; p = 0.028). The brain, liver, and lung weight and crown rump length were significantly larger in short-term analyses, as well as VEGF expression in transduced tissues. At term, molecular analyses confirmed the presence of VEGF transgene in target tissues but not in fetal samples. Tissue histology analysis and blood biochemistry/hematological examination were comparable with controls. Uterine artery relaxation in Ad.VEGF-A-treated dams was higher compared with Ad.LacZ-treated dams. Maternal uterine artery Ad.VEGF-A increases fetal growth velocity and term fetal weight in growth-restricted guinea-pig pregnancy.

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Our study aimed to target adenoviral gene therapy to the uteroplacental circulation of pregnant guinea pigs in order to develop a novel therapy for fetal growth restriction. Four methods of delivery of an adenovirus encoding β-galactosidase (Ad.LacZ) were evaluated: intravascular injection using phosphate-buffered saline (PBS) into (1) uterine artery (UtA) or (2) internal iliac artery or external administration in (3) PBS or (4) pluronic F-127 gel (Sigma Aldrich). Postmortem examination was performed 4 to 7 days after gene transfer. Tissue transduction was assessed by X-gal histochemistry and enzyme-linked immunosorbent assay. External vascular application of the adenovirus vector in combination with pluronic gel had 91.7% success rate in terms of administration (85% maternal survival) and gave the best results for maternal/fetal survival and local transduction efficiency without any spread to maternal or fetal tissues. This study suggests an optimal method of gene delivery to the UtAs of a small rodent for preclinical studies.