The novel mineralocorticoid receptor antagonist finerenone attenuates neointima formation after vascular injury

<div><p>Background</p><p>The novel nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone holds promise to be safe and efficient in the treatment of patients with heart failure and/or chronic kidney disease. However, its effects on vascular function remain elusive.</p><p>Purpose</p><p>The aim of this study was to determine the functional effect of selective MR antagonism by finerenone in vascular cells <i>in vitro</i> and the effect on vascular remodeling following acute vascular injury <i>in vivo</i>.</p><p>Methods and results</p><p><i>In vitro</i>, finerenone dose-dependently reduced aldosterone-induced smooth muscle cell (SMC) proliferation, as quantified by BrdU incorporation, and prevented aldosterone-induced endothelial cell (EC) apoptosis, as measured with a flow cytometry based caspase 3/7 activity assay.</p><p><i>In vivo</i>, oral application of finerenone resulted in an accelerated re-endothelialization 3 days following electric injury of the murine carotid artery. Furthermore, finerenone treatment inhibited intimal and medial cell proliferation following wire-induced injury of the murine femoral artery 10 days following injury and attenuated neointimal lesion formation 21 days following injury.</p><p>Conclusion</p><p>Finerenone significantly reduces apoptosis of ECs and simultaneously attenuates SMC proliferation, resulting in accelerated endothelial healing and reduced neointima formation of the injured vessels. Thus, finerenone appears to provide favorable vascular effects through restoring vascular integrity and preventing adverse vascular remodeling.</p></div

Functional effects of finerenone <i>in vitro</i>.

<p>Human smooth muscle cells (SMC) and human endothelial cells (EC) were incubated either with aldosterone alone or with aldosterone and different concentrations of finerenone, each dissolved in dimethylsulfoxide (DMSO, final concentration 0.1%) for 24 hours. A-D, Cell proliferation was determined by BrdU incorporation assays (n = 10 for SMCs/n = 6 for ECs, *<i>P</i><0.05 to serum-free, #<i>P</i><0.05 to DMSO by ordinary 1way ANOVA followed by multiple comparisons using the Tukey method). E-F, Apoptosis was determined by flow-cytometry-based caspase 3/7 activity measurement (n = 3, **<i>P</i><0.01 to serum-free, #<i>P</i><0.05 and ##<i>P</i><0.01 to DMSO by ordinary 1way ANOVA followed by multiple comparisons using the Tukey method, aldosterone 10 nM was used for B and D-F).</p

Finerenone reduces the intimal and medial leukocyte content.

<p>Wire-induced femoral artery dilation was performed in 10-week-old C57BL/6 mice. Finerenone or vehicle was daily delivered as oral gavage. A, Ten days after injury, leukocyte content was assessed by immunfluorescence staining for the pan-leukocyte marker CD45 (red). Co-immunostaining for CD31 (green) and staining of nuclei with DAPI (blue) was performed to assess the endothelial lining and the overall cell number for better morphological orientation and to allow quantification. B, The amount of leukocytes was determined as the total number of CD45⁺ cells (n = 6, *<i>P</i><0.05, **<i>P</i><0.01 by ordinary 1way ANOVA followed by multiple comparisons using the Tukey method).</p

Finerenone prevents medial and intimal cell proliferation.

<p>Wire-induced femoral artery dilation was performed in 10-week-old C57BL/6 mice. Finerenone or vehicle was daily delivered as oral gavage. A, Ten days after injury, cell proliferation was assessed by immunfluorescence staining for DAPI (blue), α-smooth muscle actin (α-SMA, red), and Ki-67 (green). B, The amount of proliferating cells was determined as Ki-67⁺ cells/DAPI⁺ cells (n = 6, **P<0.01, ***P<0.001 by ordinary 1way ANOVA followed by multiple comparisons using the Tukey method).</p

Finerenone promotes early endothelial recovery.

<p>Electrical denudation of the carotid artery was performed in 10 weeks old C57BL/6J mice. Finerenone or vehicle was daily delivered as oral gavage. A, Three days following injury, endothelial regeneration was evaluated by injection of a 5% Evan’s blue solution and en face microscopy. B, The re-endothelialized distance was calculated by substraction of the deendothelialized distance from 4 mm (standardized denudated area, n = 9, **<i>P</i><0.01 by ordinary 1way ANOVA followed by multiple comparisons using the Tukey method).</p

Blood values in mice treated with vehicle or finerenone.

<p>Blood values in mice treated with vehicle or finerenone.</p

Role of the Phosphatase PTEN in Early Vascular Remodeling

<div><p>Background</p><p>The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN's effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury <i>in vivo</i> and <i>in vitro</i>.</p> <p>Methods and Results</p><p>Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H₂O₂ robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; <i>P</i><0.001) as determined by TUNEL assay. In contrast, siRNA-mediated knock-down of PTEN attenuated the basal as well as H₂O₂-induced apoptosis of VSMC. Mechanistically, overexpression of PTEN prevented serum-induced Akt-phosphorylation, whereas siRNA-mediated knock down of PTEN augmented Akt-activation. Moreover, co-transfection of PTEN and a constitutive active Akt mutant prevented PTEN-dependent augmentation of VSMC apoptosis, indicating, that PTEN regulates VSMC apoptosis by inhibition of Akt phosphorylation/activation.</p> <p>Conclusion</p><p>By interfering with the PI3-K/Akt-dependent survival signalling, the oxidative stress-induced up regulation of PTEN in VSMC of injured arteries augments the sensitivity of VSMC to apoptotic stimuli in the early phase following vascular injury, augmenting the initial injury and cell loss of the injured vessel wall. Thus, these data add to our understanding of PTEN's role during vascular remodelling.</p> </div

PTEN expression in human coronary VSMC <i>in vitro</i>.

<p><b>A</b>, PTEN-expression is not upregulated by mechanical stress in VSMC. Lysates of cells exposed to mechanical forces using a stretching device were analyzed by western blotting using specific antibodies. <b>B</b>, Lysates of SMC exposed to growth medium (FCS). PTEN expression was not upregulated after 24 h. Protein expression was determined using specific antibodies. Detection of Cdk4 served as loading control. <b>C</b>, Lysates of SMC exposed to oxidative stress using 500 µM H₂O₂. PTEN upregulation was triggered by oxidative stress within 24 h. Detection of p53 and Cdk4 served as apoptotic marker and loading control, respectively. <b>D</b>, The upregulation of PTEN protein levels was quantified by densitometric analysis of immunoblots (n = 3; *<i>P</i><0.05). <b>E</b>, The upregulation of PTEN activity is mediated by H₂O₂-induction. Shown is a phosphatase activity assay of immunoprecipitated protein from lysates of HcASMC with and without 24 h H₂O₂–treatment. Immunoprecipitations from lysates employing an IgG iso-antibody without H₂O₂-treatment with and without bpV supplementation, an anti-PTEN-antibody without H₂O₂ and bpV treatment and an anti-PTEN-antibody with H₂O₂– and bpV-treatment served as controls. Results are expressed as mean OD650 ± SD using an ELISA plate reader (^#<i>P</i><0.001, *<i>P</i><0.001; n = 4).</p

PTEN overexpression prevents serum-induced Akt phosphorylation after 15 min (A) and 30 min (B) serum induction using 10% FCS as determined by western blotting using an anti-pAkt-antibody.

<p>An antibody against total Akt was used as control. The potent PI3-K-inhibitor Ly294002 (50 nM) was supplemented to the medium for not-transfected cells. HcASMC were transfected as follows: non transfected (NT); transfected with a plasmid coding for GFP (pControl); transfected with an empty plasmid without PTEN-cDNA (pControl); transfected with a plasmid coding for PTEN (pPTEN); mock transfected (without plasmid, but with transfection reagent). PTEN- and Akt co-overexpression reverses PTENs pro-apoptotic effect (<b>C</b>). HcASMC were co-transfected with a WT form of PTEN and a constitutively active form of Akt. An empty plasmid was used as control. Following the magnetic separation of positively transfected SMC, apoptosis was determined after cell growth for 24 h and expressed as mean OD405 ± SD using a cell death detection ELISA (^#<i>P</i><0.001, *<i>P</i><0.001; n = 4).</p

PTEN knock down attenuates SMC apoptosis under basal conditions or oxidative stress.

<p><b>A</b>, PTEN expression following siRNA-mediated knock down. Protein expression was determined by western blotting using specific antibodies. Detection of Cdk4 served as a loading control. SMC were transfected as follows: non transfected (NT); mock transfected (without siRNA, but with lipid carrier); transfected with a non-targeting (scrambled) siRNA (Control siRNA); transfected with a targeting siRNA against PTEN (PTEN siRNA). SMC transfected with siRNA targeting PTEN or a scrambled control were incubated in basal medium in the absence (<b>B</b>) or presence of H₂O₂ (<b>C</b>). SMC apoptosis is expressed as mean OD405 ± SD using a cell death detection ELISA (*<i>P</i><0.05; n = 3).</p