sVEGF-R1 expression in the adult liver causes closure of sinusoidal fenestrations.

<p>(a) Scanning electron microscopy of sinusoids in control (‘off’) showing fenestrations arranged in sieve plates (white arrows) and loss of fenestration following one month of switch ‘on’ (sVEGF-R1 expression). RBC = Red Blood Cell (b) Quantification of the percent of fenestrations' surface area in the sinusoids. sinusoidal area. ‘off’−40.5%, ‘on’−8.5%.</p

Sinusoidal capillarization in sVEGF-R1 expressing livers without parenchymal damage.

<p>(a,b) Immunohistochemical staining for vWF (Von-Willebrand Factor) on liver sections (black arrows-sinusoids, white arrows-larger blood vessels). (c) H&E staining of liver sections showing normal appearance.</p

11β-HSD1 inhibiton reverses cardiac hypertrophy without effecting vascular density and cardiac function.

<p>(<i>A</i>) Real time PCR results for 11β-HSD1 after 12 weeks of sVEGF-R1 induction (dTg-ON), during the last 3 weeks of which in some of the dTg mice, sVEGF-R1 was de-induced (dTg-ON>OFF). (<i>B</i>) Cardiomyocyte area standardized to littermate controls, was determined after 12 weeks of sVEGF-R1 induction during the last 3 weeks of which some of the dTg mice were treated with 11β-HSD1 inhibitor (Roche Cpd A) or de-induced (dTg-ON>OFF). N = 4–5 mice and 25–30 cardiomyocytes were analyzed for each section. (<i>C</i>) Heart to body weight ratio in the same mice shown in B (<i>D</i>) MVD. N =  4–5 mice per treatment group and 3 HPFs were measured for each mouse. (<i>E</i>) End diastolic LV diameter. (<i>F</i>) Representative axial sections at the level of the papillary muscle. (<i>G</i>) Fractional shortening measured in mice treated as in B. N = 4–5 mice. * <i>P</i><0.05, ** <i>P</i><0.001, NS  =  not statistically significant.</p

Pharmacological inhibition of 11β HSD1 reverses cardiac hypertrophy in a model of Isoproterenol-induced heart failure.

<p>(<i>A</i>) Fractional shortening measured in mice treated for 5 weeks with Isoproterenol(ISO-5w) alone, Isoproterenol together with 11β-HSD1 inhibitor (ISO+CpdA-5w) or treated with Isoproterenol alone for 3 weeks and by Isoproterenol and 11β-HSD1 inhibitor for an additional 2 weeks (ISO-3w→ISO+CpdA-2w). N =  5–7 mice per treatment group. (<i>B</i>) End diastolic LV diameter in the same mice as in A. (<i>C</i>) Representative axial sections at the level of the papillary muscle. (<i>D</i>) Cardiomyocyte area standardized to littermate controls. N = 5–7 mice and 25–30 cardiomyocytes were analyzed for each section. (<i>E</i>) Heart to body weight ratio. (<i>F</i>) MVD. N =  5–7 mice per treatment group and 3 HPFs were measured for each mouse. * <i>P</i><0.05, ** <i>P</i><0.001, NS  =  not statistically significant.</p

A Transgenic Platform for Testing Drugs Intended for Reversal of Cardiac Remodeling Identifies a Novel 11βHSD1 Inhibitor Rescuing Hypertrophy Independently of Re-Vascularization

<div><p>Rationale</p><p>Rescuing adverse myocardial remodeling is an unmet clinical goal and, correspondingly, pharmacological means for its intended reversal are urgently needed.</p><p>Objectives</p><p>To harness a newly-developed experimental model recapitulating progressive heart failure development for the discovery of new drugs capable of reversing adverse remodeling.</p><p>Methods and Results</p><p>A VEGF-based conditional transgenic system was employed in which an induced perfusion deficit and a resultant compromised cardiac function lead to progressive remodeling and eventually heart failure. Ability of candidate drugs administered at sequential remodeling stages to reverse hypertrophy, enlarged LV size and improve cardiac function was monitored. Arguing for clinical relevance of the experimental system, clinically-used drugs operating on the Renin-Angiotensin-Aldosterone-System (RAAS), namely, the ACE inhibitor Enalapril and the direct renin inhibitor Aliskerin fully reversed remodeling. Remodeling reversal by these drugs was not accompanied by neovascularization and reached a point-of-no-return. Similarly, the PPARγ agonist Pioglitazone was proven capable of reversing all aspects of cardiac remodeling without affecting the vasculature. Extending the arsenal of remodeling-reversing drugs to pathways other than RAAS, a specific inhibitor of 11β-hydroxy-steroid dehydrogenase type 1 (11β HSD1), a key enzyme required for generating active glucocorticoids, fully rescued myocardial hypertrophy. This was associated with mitigating the hypertrophy-associated gene signature, including reversing the myosin heavy chain isoform switch but in a pattern distinguishable from that associated with neovascularization-induced reversal.</p><p>Conclusions</p><p>A system was developed suitable for identifying novel remodeling-reversing drugs operating in different pathways and for gaining insights into their mechanisms of action, exemplified here by uncoupling their vascular affects.</p></div

Rescue of hypertrophy is associated with reversing the hypertrophic gene signature, including of the Myosin heavy chain isoform shift.

<p>(<i>A</i>) Real time PCR results of selected genes in control and dTg (dTg ON) mice after 12 weeks of sVEGF-R1 induction, with or without treatment in the last 3 weeks. Treatment groups included either 11βHSD1 inhibitor (Roche Cpd A), Enalapril (ACE-I), Pioglitazone (PIO) or VEGF-driven neo-vascularization by sVEGF-R1 withdrawal (dTg ON>OFF). Results are normalized relative to litter-mate controls (designated as 1). N = 4-6 mice. Genes examined: Brain naturetic peptide (BNP). Glucose transporter 1 (Glut1) and Periostin. See text for further elaboration. (<i>B</i>) Real time PCR results of MyHCβ and MyHCα in control and dTg (dTg ON) mice after 12 weeks of sVEGF-R1 induction, with or without treatment in the last 3 weeks. N = 4–6 mice. Quantification (right diagram in <i>B</i>) and representative images (C) of immunohistochemistry for MyHCβ in the same mice as in B, depicting MyHCβ protein level elevation in dTg mice and rescue by both treatments. Scale bars: 50 µm. * <i>P</i><0.05, ** <i>P</i><0.001, NS  =  not statistically significant.</p

sVEGF-R1 expression in the adult liver causes activation of HSCs.

<p>(a) Scanning electron microscopy of sinusoids in ‘off’ (a control littermate) vs. switch ‘on’ liver for one month showing prominent HSCs surrounding sinusoids (arrows) (b) Scanning electron microscopy showing transformation of HSCs from lipid droplets (L) containing cells (‘off’) to myofibroblasts like cells (arrows). (c) Quantification of the surface area of HSCs. ‘off’-5.8%, ‘on’ (sVEGF-R1 expression for one month)-43.7%. (d) Western blot analysis with anti α-smooth muscle actin antibody performed on liver extracts (e) Goldner staining for collagen fibers (green) indicating perisinusoidal accumulation of extra-cellular matrix.</p

Complications of portal hypertension resulting from sVEGF-R1 expression in the liver.

<p>(a) Anesthetized mice showing abdominal distension. ‘on’-sVEGF-R1 expression for one month. (b) Abdominal ultrasonography documenting ascites after one month of sVEGF-R1 expression in the liver. L-liver lined with pink line, ascites (hypoechogenic) is marked by a yellow asterisk and a yellow line. (c) Representative spleens taken from control (‘off’) and after one month of sVEGF-R1 expression (‘on’) in the liver. Average spleen weight is 100 mg and 193 mg in control and transgenic mice, respectively.</p

A bi-transgenic system for conditional and reversible suppression of VEGF signaling in the liver.

<p>(a) A schematic representation of the driver and responder transgenes used in the bi-transgenic system (b) Northern blot analysis of hepatic and renal RNA with a probe specific for transgenic (human) VEGF-R1. The term “off” indicates control littermates; “on” indicates that sVEGF-R1 expression was induced for 1 month in two transgenic animals. Note the tissue specific expression of the transgene.</p

Liver phenotype is reversible upon relieving sVEGF-R1.

<p>One week after sVEGF-R1 shut off (on→off) (a) Quantification of the percent of fenestrations' surface area in the sinusoids. ‘on’−8.5%, ‘on→off’−34.4% (b)-quantification of area covered by HSCs ‘on’−43.7%, ‘on→off’ (‘on’ for one month then ‘off’ for one week)−14.4% (c) Goldner staining highlighting collagen fibers in green showing reduced extracellular-matrix deposition perisinusoidally compared to ‘on’ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021478#pone-0021478-g003" target="_blank">fig 3E</a>).</p