Phenotype stability under hypoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under hypoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Confocal microscopic observations showed positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that more than 80% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§)PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control, (#) PEMs <i>versus</i> collagen. (§,* and #: <i>p</i><0.05 and §§§ and ***: <i>p</i><0.001).</p

Morphological aspect of differentiated cell.

<p>Optical phase contrast microscopy visualization of differentiated cells seeded on type I collagen (A, B) and polyelectrolyte multilayer films (PEMs) (C, D) until confluence under normoxic (A, C) and hypoxic (B, D) environment. Objective×20, scale bar 55 µm. The morphological examination of the confluent cells showed cobblestone shape (A, C) in normoxia and a spindle like (B, D) shape in hypoxia.</p

Phenotype stability under normoxia.

<p>After the third passage, the smooth muscle cells phenotype stability of differentiated cell cultivated under normoxic conditions was investigated by confocal microscopy observation (A) and flow cytometry analyses (B, C). A: Microscopical observations show positive cells for contractile markers: α- Smooth Muscle Actin (α-SMA), Smooth Muscle Myosin Heavy Chain (SM-MHC) and Calponin confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)). Objective×40, NA = 0.8, scale bars 75 µm. B: Flow cytometry showed that about 90% cells expressed SMCs markers. C: Mean fluorescence intensity analyses showed a higher SMCs contractile markers expression for differentiated cells compared to control (mature SMCs) whatever the surface coating. (§) PEMs <i>versus</i> control, (*) Collagen <i>versus</i> control. (§ and *: <i>p</i><0.05, §§ and **: <i>p</i><0.01, and *** <i>p</i><0.001).</p

Vascular cell phenotype characterization.

<p>The endothelial cell were characterized by the expression of specific markers: CD31 (A–D) and von Willebrand Factor (E–H) and the smooth muscle cells by the expression of contractile markers: α- Smooth Muscle Actin (α-SMA: E–H), Smooth Muscle Myosin Heavy Chain (SM-MHC: I–L) and Calponin (M–P). Images were obtained by confocal microscopy observation at cell confluence on both coated surfaces (type I collagen and Polyelectrolyte Multilayer films (PEMs)) and cultivated under normoxic and hypoxic conditions. Objective×40, NA = 0.8, scale bars 75 µm. The figure showed the positive expression of specific ECs markers for cell differentiated under normoxic environment and positive expression of specific contractile SMCs markers for cell differentiated under hypoxic environment.</p

Renal parameters.

<p><b>KW</b>, Kidney Weight; <b>Na⁺</b>, sodium, <b>K⁺</b>, potassium, <b>eGFR</b>, estimated Glomerular filtration rate; Values are the mean±sem. Non-parametric ANOVAs analysis with two factors allowed the evaluation of interaction between aging and genotype. Student's T test *, ** and *** p<0.01, p<0.001 and p<0.0001 respectively when comparing SHHF ^+/?<i>vs.</i> SHHF^cp/cp at same time point; § p<0.05, §§ p<0.01 and §§§ p<0.001 to compare T14-mo <i>vs</i> T1.5-mo for a same genotype; <b>N</b> stands for the number of samples; <b>ns</b> stands for not significant.</p

Metabolic follow-up.

<p><b>A</b>- The monitoring of body weight shows that the onset of obesity occurs during the first three months after birth of SHHF^cp/cp rats. Progressively, the SHHF^cp/cp rats continue to gain weight accentuating their differences with the SHHF^+/? (n = 5 to 14 rats per genotype). (<b>B–C</b>) Paraffin embedded tissues dissected from SHHF^cp/cp and SHHF^+/? rats at 14 months of age showing metabolic disorder related-tissue alterations <b>B</b>- Peri-renal visceral fat of SHHF^cp/cp rats stained with Sirius red exhibited marked fibrosis (arrows) and hypertrophic adipocytes. <b>C</b>- Hemaetoxylin & Eosin staining shows the deposition of lipid droplets (arrows) in the liver dissected from SHHF^cp/cp rats suggesting the development of non-alcoholic hepatic steatosis. Pictures are representative of each analyzed group (n = 5 to 7 rats per genotype). Values are mean ± sem. Non-parametric ANOVA analysis with two factors allowed the evaluation of an interaction between aging and genotype. * p<0.05, ** p<0.01, *** p<0.001 for comparing SHHF^cp/cp vs. SHHF^+/? at the same time point.</p

Blood pressure follow-up in conscious SHHF rats.

<p><b>SBP</b>, Systolic Blood Pressure; <b>HR</b>, Heart Rate; <b>bpm</b>, beats per minute. Values are mean ± sem. Non-parametric ANOVAs analysis with two factors allowed the evaluation of interaction between aging and genotype. <b>N</b> stands for the number of rats; <b>ns</b> stands for not significant. Student's T test * p<0.05 to compare SHHF^cp/cp<i>vs.</i> SHHF^+/? at same age.</p

Hemodynamic phenotyping.

<p>Invasive blood pressure measurements were obtained on anesthetized rats during vascular phenotyping of the animals. <b>A</b>- Systolic (SBP), Diastolic (DBP) Blood Pressures and Pulse Pressure (PP) were measured at both the earliest and latest ages. Results showed that over time only SHHF^cp/cp rats increased their SBP and PP becoming significantly higher at 14 months of age compared to SHHF^+/? animals. <b>B</b>- Distensibility, <b>C</b>- Incremental Elastic modulus (E_inc) to Wall Stress (WS) curves and <b>D</b>- WS at Einc 600 kPa were recorded. Values are mean ± sem of 5 to 14 measurements depending on the genotype and age. Fisher's LSD Multiple-Comparison Test * p<0.05 for comparing of SHHF^cp/cp<i>vs.</i> SHHF^+/? rats at the same age; § p<0.05 for comparison of 14 month-old <i>vs.</i> 1.5-month-old rats of the same genotype.</p

Cardiac parameters.

<p><b>HW</b>, Heart Weight; <b>LV</b>, Left Ventricle; <b>LVIDd</b>, Left Ventricle Internal Diameters at diastole; <b>E</b> and <b>A</b>, early and late filling waves; <b>EDT</b>, E-vel Deceleration Time. These echocardiographic parameters are the mean ± sem of the average of three to four consecutive cardiac cycles for each rat. % of Myocardial fibrosis was determined on Sirius red stained heart sections by measuring the percentage of fibrotic area to whole heart section area using Image J software. Non-parametric ANOVAs analysis with two factors allowed the evaluation of interaction between aging and genotype. Student's T test * p<0.05; ** p<0.01, *** p<0.001 to compare SHHF^+/?<i>vs.</i> SHHF^cp/cp at same time point; ; § p<0.05, §§ p<0.01 and §§§ p<0.001 to compare T14-mo <i>vs</i> T1.5-mo for a same genotype; <b>N</b> stands for the number of rats; <b>ns</b> stands for not significant.</p

Cardiac follow-up.

<p>Transthoracic echocardiograms were performed on isoflurane-anesthetized SHHF at different time points throughout the protocol (1.5; 6; 9 and 14 months of age, n = 5 to 10 rats per genotype). <b>A</b>- Fractional Shortening (FS) and <b>B</b>- Ejection Fraction (EF) showed the progressive but faster decline of heart systolic function in SHHF^cp/cp rats compared to SHHF^+/? controls. <b>C</b>- LV mass as well as <b>D</b>- Left Ventricular (LV) Internal Diameters at end systole (LVIDs) were significantly higher in the SHHF^cp/cp group from 6 months and continued to rise till 12 and 14 months of age respectively demonstrating LV hypertrophy and dilation <b>E</b>- Red Sirius staining performed on heart sections obtained from SHHF^+/? and SHHF^cp/cp rats at 1.5 and 14 months of age showed greater myocardial fibrosis in 14-month-old SHHF^cp/cp rats compared to SHHF^+/? from the same age (n = 5 to 7 rats per genotype). Mean ± sem. Non-parametric ANOVAs analysis with two factors allowed the evaluation of an interaction between aging and genotype. * p<0.05, ** p<0.01, *** p<0.001 for comparing SHHF^cp/cp<i>vs.</i> SHHF^+/? at the same age.</p