FXR Agonist INT-747 Upregulates DDAH Expression and Enhances Insulin Sensitivity in High-Salt Fed Dahl Rats

<div><p>Aims</p><p>Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.</p><p>Methods and Results</p><p>In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.</p><p>Conclusion</p><p>Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.</p></div

Blood glucose and insulin measurements to assess insulin sensitivity: Measurement of a) blood glucose \and b) plasma insulin concentration over time during GTT in Dahl rats fed with low or high-salt diet for 5-weeks prior to the glucose challenge test.

<p>Values are Mean ± SEM for: Control (n  = 6); Vehicle (n  = 7); INT-10 mg/kg/day (n  = 5) and INT-30 mg/kg/day (n = 9). *p<0.05 versus high-salt diet data. ANOVA followed by Bonferroni post-test. GTT = glucose tolerance test. In c), the effect of INT-747 treatment on insulin sensitivity is shown. Insulin Resistance (IR) index was calculated as described in the text. Data is expressed as Mean±SEM. (*p<0.05 versus low-salt diet data. ANOVA followed by Bonferroni post-test). LS = low salt; HS = high salt; V = vehicle; INT-10 = INT-747 at 10 mg/kg/day and INT-30 = INT-747 at 30 mg/kg/day.</p

Schematic showing the time frame and experimental protocol followed to induce hypertension by high-salt feeding.

<p>Measurements period of BP, glucose challenge, urinary collection and blood draw are also shown. BP = blood pressure.</p

Assessing renal morphology and fibrosis.

<p><b>a:</b> H&E staining to assess the renal morphology of Dahl rats following: LS-diet with nearly normal morphology (A) or HS-diet and administration of vehicle, showing fibrinoid arteriolar necrosis with extravasation of erythrocytes and thrombosis of glomerular capillaries (B); INT-747 at 10 mg/kg/day shows similar findings (C) as does INT-747 at 30 mg/kg/day (D) for 6 weeks. Representative images are shown. (400X mag). <b>b:</b> Masson's Trichrome staining to evaluate renal fibrosis (seen as blue-colored expansion of the interstitium between the tubules) in Dahl rat kidneys following: LS-diet (A) or HS-diet and administration of vehicle (B); INT-747 at 10 mg/kg/day (C) or INT-747 at 30 mg/kg/day (D) for 6 weeks. Representative images are shown. (100X mag).</p

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization <i>in vivo</i>, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000–1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies

The effect of INT-747 treatment on DDAH1 expression in Liver.

<p>Animals were fed low (control)- or high-salt diet and treated with vehicle or INT-747 at 10 or 30 mg/kg/day for 6 weeks. Liver lysates were compared for DDAH1 expression by Western blot. rDDAH1: purified recombinant human DDAH1 described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060653#pone.0060653-Ghebremariam1" target="_blank">[53]</a>. The DDAH1 expression was normalized to β-Actin (ACTB). DDAH = dimethylarginine dimethylaminohydrolase. Data is expressed as Mean±SEM. (*p<0.05 versus control value. ANOVA followed by Bonferroni post-test).</p