Grußwort

<p>The flow-chart shows the experimental procedures performed at certain time-points during the study. GD, gestational day; PPD, postpartum day; PFU, plaque forming unit.</p

Functional evaluation of the kidneys.

<p>(<b>A</b>) Chart depicts albumin/creatinine ratios (in log scale) in urine specimens from mice in the GFP, msFlt-1(1-3) and hsFlt-1-e15a-treated groups. Mean urine albumin/creatinine ratios were higher in hsFlt-1-e15a-treated mice (GD18, p = 0.04 and PPD8, p = 0.03) and msFlt-1(1-3)-treated mice (PPD8, p = 4×10⁻⁵) than in controls. Proteinuria was extremely high in the mouse overexpressing msFlt-1(1-3) with chronic hypertension. (<b>B</b>) Subgroup analysis showed that hsFlt-1-e15a expressed under the CMV promoter in the adenovirus led to significant increase in albumin/creatinine ratio on PPD8 (p = 0.003); hsFlt-1-e15a expressed under the CMV promoter in the fiber-mutant adenovirus led to significant increase in albumin/creatinine ratio on GD18 (p = 0.04); while hsFlt-1-e15a expressed under the CYP promoter in the fiber-mutant adenovirus led to a marginally significant increase in albumin/creatinine ratio on GD18 (p = 0.056) and a significant increase on PPD8 (p = 0.04).</p

Blood pressure monitoring.

<p>X-axes show gestational days (GDs) and postpartum days (PPDs). Mean arterial pressure changes (ΔMAP) are depicted on the Y-axes. Blue dots represent ΔMAP values for given time-points, blue error bars show +/-standard errors. Red lines depict the ΔMAP patterns, fitted from the linear mixed effects models. During pregnancy (left sides of the sub-figures), there was no significant blood pressure elevation in control mice (A,C; ΔMAP slope = 0.513 mmHg/day; p = 0.187), whereas hsFlt-1-e15a treatment significantly increased blood pressure (B,D; ΔMAP slope = 2.05 mmHg/day; p = 8.09x10^-8). The ΔMAP slope in the hsFlt-1-e15a group was higher compared to the controls (1.53 mmHg/day; p = 0.0043). The ΔMAP at parturition (GD18) was 13.2 mmHg higher in hsFlt-1-e15a-treated mice than in the control mice (p = 0.00107). After cesarean delivery (right sides of sub-figures), a similar quadratic pattern of ΔMAP was found in both control and hsFlt-1-e15a-treated mice, but ΔMAP dropped below the baseline in control mice (-1.96 mmHg; p = 0.560), while it remained above this in hsFlt-1-e15a treated mice (6.88 mmHg; p = 0.0346). There was no dose effect of the number of viral construct injections (1x10⁹ PFU vs. 2x10⁹ PFU) in hsFlt-1-e15a-treated or in control mice either before delivery (dose effect: -1.06 mmHg, p = 0.693) or in the postpartum period (dose effect: 1.30 mmHg, p = 0.763).</p

Histopathological evaluation of the kidneys.

<p>(<b>A,B,G</b>) Representative H&E (A: Ad-RGD-CMV-GFP, B: Ad-RGD-CYP-GFP) and Jones (G: Ad-RGD-CMV-GFP) stained sections show morphologically normal glomeruli in control animals. (<b>C,D,F,H</b>) Representative H&E (C: Ad-RGD-CMV-sFlt-1-e15a, D: Ad-RGD-CYP-sFlt-1-e15a, F: Ad-CMV-sFlt-1-e15a) and Jones (H: Ad-RGD-CMV-sFlt-1-e15a) stained sections show glomeruli with signs of swollen capillary endothelial cells and occlusion of glomerular capillaries in mice overexpressing sFlt-1-e15a. (<b>E,I</b>) Representative H&E (E) and Jones (I) stained sections show glomeruli with signs of swollen capillary endothelial cells and occlusion of glomerular capillaries in the dam overexpressing msFlt-1(1-3) with chronic hypertension. 400x magnifications. (<b>J</b>) High magnification image (1200x) from sub-image G shows a normal capillary structure. (<b>K</b>) High magnification image (1200x) from sub-image I shows thickened capillary loops. (<b>M</b>) The glomerular damage score was significantly higher in all treatment groups compared to the combined control group. Mice treated with msFlt-1(1-3) had an odds ratio (OR) of 2.4 for glomerular damage (p = 0.01). The OR for glomerular damage was 3.1 in hsFlt-1-e15a-treated mice (2.8×10⁻⁵). Among hsFlt-1-e15a-treated mice, mice in the Ad-CMV-hsFlt-1-e15a group had the largest OR (3.9, p = 4.8×10⁻⁵) for glomerular damage.</p

Litter sizes, maternal, placental and fetal weights, and placental/fetal weight ratios.

<p>Controls (n = 17) and msFlt-1(1-3)-treated mice (n = 6) had a litter size consistent with the strain average published by the vendor (n = 11.5). The number of pups (13.8±0.4, p = 0.046) and living pups (13.6±0.45, p = 0.05) were higher in hsFlt-1-e15a-treated mice (n = 18) than in controls. The total weight of living pups (14.2±0.56 g, p = 0.04) and maternal weights (56.3±1.1 g, p = 0.04) were also higher in hsFlt-1-e15a-treated mice than in controls. Truncated msFlt-1(1-3)-treated mice did not differ in any parameters from the controls. Among hsFlt-1-e15a-treated mice, the total weights of living pups was higher in the Ad-RGD-CYP-hsFlt-1-e15a treated mice than in controls (15±0.48 g, p = 0.043). The number of pups (14.3±0.42, p = 0.047) and the number of living pups (14.1±0.46, p = 0.039) was higher in the Ad-RGD-CMV-hsFlt-1-e15a treated mice than in controls. Stars denote statistical significance.</p

The development of viral constructs for the various treatment groups.

<p>(<b>A</b>) Human and mouse Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase, from which the first three Ig-like domains are important in ligand-binding, while the 4–7th Ig-like domains in receptor dimerization. The most abundant placental sFlt-1 variant in humans, sFlt-1-e15a, contains six Ig-like domains and a unique C-terminus encoded by exon 15a, which is located within a primate-specific AluSeq retrotransposon. The truncated mouse sFlt-1 mutant [msFlt-1(1-3)] contains only the first three Ig-like domains of Flt-1. (<b>B</b>) Besides the replication deficient human adenovirus Type5 (dE1/E3), “RGD fiber-mutant” adenoviruses were also used. (<b>C</b>) Besides the CMV promoter that has a strong promoter activity, the human <i>CYP19A1</i> promoter was also used. <i>CYP19A1</i> is strongly and predominantly expressed in the placenta among 40 human tissues (left), and its expression increases during trophoblast differentiation (right). Relative gene expressions are shown on the Y-axes. (<b>D–E</b>) Various combinations of viruses, promoters and transgenes used in this study.</p

Aortic ring assays.

<p>(A) A light image of an aortic ring from a mouse injected with Ad-CMV-GFP. (B) An image of the same aortic ring, illustrating segmentation and classification, with the aortic ring shown in blue and the red color depicting the microvessel outgrowth area. (C) A light image of an aortic ring from a mouse injected with Ad-CMV-hsFlt-1-e15a. (D) An image of the same aortic ring, illustrating segmentation and classification, with the aortic ring shown in blue and the red color depicting the microvessel outgrowth area. (A-D) Scale bars depict 500μm. (E) The bar chart depicts the differences in mean microvessel outgrowth areas (pixels) between the GFP and hsFlt-1-e15a groups.</p

Profiling of mFlt-1 and msFlt-1-i13 expression.

<p>(<b>A</b>) Boxplots show the endogenous expression profile of the mouse transmembrane Flt-1 mRNA in placentas harvested on gestational day (GD) 18 and in five tissues harvested on postpartum day (PPD) 8. Endogenous Flt-1 mRNA expression was highest in the placenta in both the non-treated (saline) and the msFlt-1(1-3)-treated mice. (<b>B</b>) Boxplots show the endogenous expression profile of the mouse sFlt-1-i13 mRNA in placentas harvested on GD18 and in five tissues harvested on PPD8. Endogenous msFlt-1-i13 mRNA expression was restricted to the placenta in control animals, and transgenic msFlt-1(1-3) expression was detected in the placenta and the liver.</p