Data supporting that adipose-derived mesenchymal stem/stromal cells express angiotensin II receptors in situ and in vitro

This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1].


a b s t r a c t
This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1].
& 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Value of the data Data regarding the in situ expression of angiotensin II receptors will help to pursue the role of angiotensin II in the adipose tissue growth and renewal

Specifications
Angiotensin II triggers intracellular Ca2 þ mobilization in about 5% of ADSCs Co-expression of angiotensin II receptors labels particular ADSCs subpopulation

Data
Using antibodies against AngII receptors (AT1, AT2 and MAS1) we have detected their distribution in human adipose tissue [1]. Specifically, we demonstrated that AT1 and AT2 receptors are co-localized with stromal cells expressing PDGF receptor beta (Fig. 1). Furthermore, AT1 expressing cells were found in the close proximity to CD31-positive endothelial cells (Fig. 2). We also found that adipose tissue contains cells expressing MAS1 receptor (Fig. 3).
We isolated ADSCs from adipose tissue and examined their ability to respond to AngII. Using fluorescent probe Fluo-8 Ca2 þ transients were recorded in ADSCs incubated with AngII in the presence or absence of AT1 inhibitor losartan. We showed that only 5.2 7 2.7% of cells responded not to single but serial Ang II applications (Fig. 4), whereas in the rest of cells receptors underwent rapid internalization upon the ligand binding [1].
To evaluate the proportion of ADSCs stably expressing AT1 receptor we provoked its internalization by incubating cells with antibodies against AT1 receptor at the room temperature instead of þ 4°C. we found that in these conditions only 2.8 7 0.9% of cells were positively stained for AT1 receptor (Fig. 5). We also examined the expression of AT2 receptor and found that proportions of cells expressing AT2 receptors varied substantially between different donors (Table 1). Using flow cytometry we demonstrated that AT2 receptor was predominantly expressed on ADSCs stably expressing AT1 receptor rather than on the other cells in the population (Fig. 6).

Immunofluorescent detection of angiotensin II receptors in situ
Subcutaneous fat tissue was obtained from 18 donors during abdominal surgery. All donors gave their informed consent and the local ethics committee of city clinical hospital #31 (Moscow, Russia) approved the study protocol. Part of each sample was placed in O.C.T. Compound (Sakura Inc., Tokyo, Japan) and frozen in liquid nitrogen for immunofluorescent analysis. Other part of each sample was  used for ADSC isolation (see below). Angiotensin receptors were visualized by immunofluorescent staining of 10 μm frozen sections using mix of antibodies against AT1 receptor (rabbit polyclonal, PA5-20812, ThermoFisher Scientific) or AT2 receptors (rabbit polyclonal, Alomon) and mouse PDGF receptor beta to stromal cells or CD31 antibodies (BD Pharmingen) to endothelial cells. This was followed by incubation with Alexa594-conjugated donkey anti-rabbit antibody and Alexa488-conjugated anti-mouse antibody (Molecular Probes). Cell nuclei were counterstained with DAPI (Molecular Probes), and sections were mounted in Aqua Poly/Mount (Polysciences Inc). For negative controls mouse or rabbit non-specific IgGs were used in appropriate concentration. Images were obtained using confocal microscope LSM 780 and ZEN2010 software (Zeiss).

Ca2þ imaging
AT1 receptor activation was assessed using Ca2þ imaging in individual cells as previously described [4]. Briefly, cells were seeded in 24-well plate at low density to prevent cell-to-cell communications during the imaging. ADSCs were loaded with Fluo-8 (ab142773, Abcam), 4 μM in Hanks Balanced Salt Solution with 20 mM HEPES, for 1 h. To stimulate Ca2þ transients in serial mode ADSCs were treated by Ang II (ab120183, Abcam, 10 nM) alone or together with AT1 receptor antagonist losartan (ab120997, Abcam, 1 μM). Losartan was added 30 min before Ang II addition for metabolisation in the cells. Cells  were stimulated with Ang II for 4 min, and then washed by Hanks solution 3-5 times for 5 min following by the next stimulation with Ang II. Threshold concentration was determined using addition of 0.1 nM -10 μM Ang II in series. Ca2þ transients were measured in individual cells using fluorescent microscope Nikon Eclipse Ti (objective 10×) equipped with camera Andor iXon 897 (Andor Technology). Movies were analyzed using NIS-Elements (Nikon) and ImageJ software. Alterations of cytosolic Ca2þ from the resting level were quantified as a difference of Fluo-8 fluorescence intensity (ΔF/F0) recorded from an individual cell.