Specific Activation of K-RasG12D Allele in the Bladder Urothelium Results in Lung Alveolar and Vascular Defects

K-ras is essential for embryogenesis and its mutations are involved in human developmental syndromes and cancer. To determine the consequences of K-ras activation in urothelium, we used uroplakin-II (UPK II) promoter driven Cre recombinase mice and generated mice with mutated KrasG12D allele in the urothelium (UPK II-Cre;LSL-K-rasG12D). The UPK II-Cre;LSL-K-rasG12D mice died neonatally due to lung morphogenesis defects consisting of simplification with enlargement of terminal air spaces and dysmorphic pulmonary vasculature. A significant alteration in epithelial and vascular basement membranes, together with fragmentation of laminin, points to extracellular matrix degradation as the causative mechanism of alveolar and vascular defects. Our data also suggest that altered protease activity in amniotic fluid might be associated with matrix defects in lung of UPK II-Cre;LSL-K-rasG12. These defects resemble those observed in early stage human neonatal bronchopulmonary dysplasia (BPD), although the relevance of this new mouse model for BPD study needs further investigation

ENPP1 enzyme replacement therapy improves blood pressure and cardiovascular function in a mouse model of generalized arterial calcification of infancy

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), which normally hydrolyzes extracellular ATP into AMP and pyrophosphate (PPi). The disease is characterized by extensive arterial calcification and stenosis of large- and medium-sized vessels, leading to vascular-related complications of hypertension and heart failure. There is currently no effective treatment available, but bisphosphonates – nonhydrolyzable PPi analogs – are being used off-label to reduce arterial calcification, although this has no reported impact on the hypertension and cardiac dysfunction features of GACI. In this study, the efficacy of a recombinant human ENPP1 protein therapeutic (rhENPP1) was tested in Enpp1asj-2J homozygous mice (Asj-2J or Asj-2J hom), a model previously described to show extensive mineralization in the arterial vasculature, similar to GACI patients. In a disease prevention study, Asj-2J mice treated with rhENPP1 for 3 weeks showed >95% reduction in aorta calcification. Terminal hemodynamics and echocardiography imaging of Asj-2J mice also revealed that a 6-week rhENPP1 treatment normalized elevated arterial and left ventricular pressure, which translated into significant improvements in myocardial compliance, contractility, heart workload and global cardiovascular efficiency. This study suggests that ENPP1 enzyme replacement therapy could be a more effective GACI therapeutic than bisphosphonates, treating not just the vascular calcification, but also the hypertension that eventually leads to cardiac failure in GACI patients

Normal kidney development in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-B: H&E stained kidney sections from day E17.5 showing normal development both in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls (X200).</p

Characterization of the <i>UPK II-Cre;Rosa-Stop-YFP</i> reporter mice.

<p>A–J: <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> reporter mice reveal fluorescence only in the bladder urothelium (x200) (<b>A,B</b>) and ureter urothelium (x200) (day 1, P1) (white arrow) (<b>C,D</b>), but not in lung (x200) (P1) (<b>E,F</b>), placenta (x200) (E19.5) (<b>G,H</b>) or yolk sac (x200) (E19.5) (<b>I,J</b>) among other negative tissues. <b>K</b>: PCR for recombinant UPK II-YFP was only positive in <i>UPK II-Cre;Rosa-Stop-YFP^+/+</i> bladder (E19.5).</p

Lung phenotype of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A–B: Representative H&E stained sections showing normality of lung (X200) morphology in <i>UPK II-Cre;LSL-K-ras^G12D</i> when compared with controls at E14.5 (pseudoglandular stage). <b>C–H</b>: Formation of air spaces was impaired in lungs (X100) of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (<b>C,D</b>) and E19.5 (<b>E,F</b>), reflecting a defective septation process. P1 lung morphology was also different between controls and transgenic mice, which exhibited an enlargement and simplification of sacculi (<b>G,H</b>). <b>I–J</b>: Morphometric analysis of lung sections showed a decreased total air space (<b>I</b>) and mean alveolar (saccular) area (<b>J</b>) in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice at E17.5 (n = 5); the impairment of air space development led to an increased total air space area and mean alvelolar area in the early postnatal period (P1; n = 12) in transgenic mice when compared to controls; bars, SEM. <b>K–L</b>: Masson tri-chrome staining of day P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> lungs (X100), showing absence of fibrosis both in cases and controls. <b>M</b>: The direct expression of K<i>-ras^G12D</i> was ruled out with specific PCR showing the absence of recombination between <i>K-ras</i> and the Lox sequence in lung and placenta.</p

Disorganization of extracellular matrix and blood vessels in the lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A-F: Representative images of immunohistochemistry for pan-laminin (X400) (<b>A,B</b>), and immunofluorescence for entactin (X630) (<b>C,D</b>), showed a different pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with a less organized network and stronger expression in the stroma. Immunofluorescence for collagen IV (X630) (<b>E,F</b>) showed a similar, but less prominent, pattern. <b>G-J</b>: CD34 staining (<b>G,H</b>) and CD31 immunofluorescence (<b>I,J</b>) of lung vessels at E17.5 (X400) also exhibited a disorganized distribution in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice, with more mesenchymal vessels and a disruption of the normal subepithelial double capillary network (black arrows in <b>G</b>).</p

Fragmentation of ECM components in lungs of <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Total protein lysates from whole lung were analyzed by Western blot for for laminin β-1 from P1 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and control (Cre-) mice, showing an additional low molecular weight (mw) band (35 kDa) which suggest fragmentation. <b>B</b>: Representative images of immunofluorescence for laminin β-1 (X200) showed a disorganized membrane pattern in E17.5 lung from <i>UPK II-Cre;LSL-K-ras^G12D</i> mice. <b>C</b>: WB for lung E-cadherin in P1 lung, with an increase in low mw bands. (53 and 32 KDa) also in <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.</p

Urothelial hyperplasia in the <i>UPK II-Cre;LSL-K-ras^G12D</i> mice.

<p>A: Urothelial-restricted expression of K-ras^G12D. <b>B–E</b>: H&E analysis of bladders (X200) reveals a hyperplastic urothelium at E17.5 (<b>B,C</b>) and P1 (<b>D,E</b>) (black arrows). <b>F</b>: Differences in urothelial cellularity (cells/0.15 mm²) between <i>UPK II-Cre;LSL-K-ras^G12D</i> mice and controls were significant both at E17.5 (n = 2/group; *, <i>P</i> = 0.05) and P1 (n>6/group; **, <i>P</i><0.0001); bars, SEM. <b>G–H</b>: BRDU staining of bladder (X200) showing a higher proliferation in E17.5 <i>UPK II-Cre;LSL-K-ras^G12D</i> mice (urothelium limit is marked with a blue line). The mean number of BrdU positive nuclei/200 µm of urothelium was significantly higher than in controls (n = 10; 6±2 positive nuclei/200 µm vs 1.33±0.81; <i>P</i> = 0.01).</p

Pericyte Depletion Results in Hypoxia-Associated Epithelial-to-Mesenchymal Transition and Metastasis Mediated by Met Signaling Pathway

The functional role of pericytes in cancer progression remains unknown. Clinical studies suggest that low numbers of vessel-associated pericytes correlated with a drop in overall survival of patients with invasive breast cancer. Using genetic mouse models or pharmacological inhibitors, pericyte depletion suppressed tumor growth but enhanced metastasis. Pericyte depletion was further associated with increased hypoxia, epithelial-to-mesenchymal transition (EMT), and Met receptor activation. Silencing of Twist or use of a Met inhibitor suppressed hypoxia and EMT/Met-driven metastasis. In addition, poor pericyte coverage coupled with high Met expression in cancer cells speculates the worst prognosis for patients with invasive breast cancer. Collectively, our study suggests that pericytes within the primary tumor microenvironment likely serve as important gatekeepers against cancer progression and metastasis.National Institutes of Health (U.S.) (NIH Grant CA125550)National Institutes of Health (U.S.) (NIH grant CA155370)National Institutes of Health (U.S.) (NIH Grant CA151925)National Institutes of Health (U.S.) (NIH Grant DK81576)National Institutes of Health (U.S.) (NIH Grant CA163191)National Institutes of Health (U.S.) (NIH grant DK55001)Champalimaud Foundation (Champalimaud metastasis programme)Champalimaud Foundation (Champalimaud investigator)National Institutes of Health (U.S.) (NRSA F32 Ruth Kirschstein Postdoctoral Fellowship from NIH/NIDDK (5F32DK082119-02))National Institutes of Health (U.S.) (NIH Research Training Grant in Gastroenterology (2T32DK007760-11))National Institutes of Health (U.S.) (NIH Research Training Grant in Cancer Biology (5T32CA081156-08))United States. Dept. of Defense (DoD Breast Cancer Research Predoctoral Traineeship Award (W81XWH-09-1-0008))National Institutes of Health (U.S.) (NIH Research Training Grant in Cardiovascular Medicine (5T32HL007374-30))United Negro College Fund/Merck (Postdoctoral Science Research Fellowship)National Institutes of Health (U.S.) (NIH supplemental grant (CA125550)