The adipokine leptin modulates adventitial pericyte functions by autocrine and paracrine signalling

Transplantation of adventitial pericytes (APCs) improves recovery from tissue ischemia in preclinical animal models by still unknown mechanisms. This study investigates the role of the adipokine leptin (LEP) in the regulation of human APC biological functions. Transcriptomic analysis of APCs showed components of the LEP signalling pathway are modulated by hypoxia. Kinetic studies indicate cultured APCs release high amounts of immunoreactive LEP following exposure to hypoxia, continuing upon return to normoxia. Secreted LEP activates an autocrine/paracrine loop through binding to the LEP receptor (LEPR) and induction of STAT3 phosphorylation. Titration studies using recombinant LEP and siRNA knockdown of LEP or LEPR demonstrate the adipokine exerts important regulatory roles in APC growth, survival, migration and promotion of endothelial network formation. Heterogeneity in LEP expression and secretion may influence the reparative proficiency of APC therapy. Accordingly, the levels of LEP secretion predict the microvascular outcome of APCs transplantation in a mouse limb ischemia model. Moreover, we found that the expression of the Lepr gene is upregulated on resident vascular cells from murine ischemic muscles, thus providing a permissive milieu to transplanted LEP-expressing APCs. Results highlight a new mechanism responsible for APC adaptation to hypoxia and instrumental to vascular repair.


Supplementary
: Ingenuity® Pathway Analysis (IPA) of genes modulated by hypoxia in human APCs, based on two metrics: z-score and p-value. A positive or negative z-score value indicates that a function is increased or decreased in hypoxic relative to normoxic cells. In order to enhance the stringency of the analysis, we considered only functions with a z-score > 1 or < -1.
The p-value (red dots), calculated with the Fischer's exact test, reflects the likelihood that the association between a set of genes in the dataset and a related biological function is significant. Results indicate that LEP is implicated in the majority of functions modulated by hypoxia. F u n c tio n a n n o ta t io n s F u n c tio n s

E 2 F 1 , G L I S 3 , L E P , L H X 3 , T L R 3 A D R A 2 C ,C 5 ,H I F 1 A , N A M P T , T R E M 1 , V E G F A C A S P 1 , H I F 1 A ,I F N A R 1 ,L E P , T L R 3 ,T R E M 1 , V E G F A A L D H 3 B 1 ,B N I P 3 ,C A S P 1 , E 2 F 1 , IF N A R 1 , N A M P T , P R D X 3 ,T F R C C 5 , C A S P 1 ,C X C L 2 , H IF 1 A , L E P , N A M P T , T R E M 1 C 5 , C A S P 1 ,C X C L 2 , H IF 1 A , L E P , N A M P T , T R E M 1 C 5 , C A S P 1 , C X C L 2 , L E P , N A M P T , T R E M 1
Supplementary Figure  Experiments were performed in different APC lines (n ≥ 3) for each control and experimental condition. All experimental conditions were compared with each respective control condition in the same gel. Antibodies used in this manuscript have been previously used in our laboratory as published previously. 1-3 Different titration was performed for the antibodies during the protocol optimization. All PVDF membranes were assessed for the loading control (-actin or occasionally /-tubulin) to confirm that different protein levels were not a result of pipetting error. Membranes were incubated up to 10 min with the ECL prime reagent (GE Healthcare) according to manufacturer's instructions. Only the best and representative images were captured for publication. Image capture was usually performed using ChemiDoc MP system (Bio-Rad) or by Li-cor image 700/800 wavelength (Biosciences). Original Western blotting images were digitalized at 600 dpi by using Image Lab 5.1 software (Bio-Rad). Band densitometry was performed by Image J online software for all bands including targets and loading controls. Ratios among the area calculated for the target and the area calculated for the loading control were assessed in each sample to calculate the relative area of the protein of interest. The area of the control condition was considered 1 and the fold change was calculated for the targeted protein.
Using fold change values, we compared the regulatory effect of the experimental condition vs. its respective control condition for each APC line at the protein level. After demonstrating no regulation of the selected loading control by the experimental conditions, new gels were often run to achieve representative and improved images for each sample which are included in the final version of this manuscript. Brightness processing were performed if necessary and always applied equally to control and experimental samples. No further improving image procedures were performed.
Panel 1 (refer to text Figure 2e. Hypoxia induces leptin production and secretion by human APCs). Representative original immunoblots performed in a single membrane to confirm LEPR regulation in hypoxic APCs. Bands displayed in this manuscript has been framed into red boxes. Figure 3a. Effect of exogenous rh-leptin on canonical signalling and functional assays in human APCs.). Original images captured using Licor image 700/800 wavelength. Images i and ii correspond to Li-cor imaging; Image iii corresponds to the grey-scale image for image i; Image iv corresponds to the grey-scale image for image ii. Bands displayed in this manuscript has been framed into red boxes. Figure 4a. Effect of normoxia and hypoxia on LEP-associated kinases and functional activities of human APCs). Sequential immunoblots performed in a single membrane are indicated in ordinal numbers starting from '1st'. Bands displayed in this manuscript has been framed into red boxes.

Panel 3 (refer to text
Panel 4 (refer to text Figure 4b. Effect of LEP silencing on LEP-associated kinases and functional activities of human APCs). Sequential immunoblots performed in a single membrane are indicated in ordinal numbers starting from '1st'. Bands displayed in this manuscript has been framed into red boxes.
Panel 5 (refer to text Figure 4c. Effect of LEPR silencing on LEP-associated kinases and functional activities of human APCs). Sequential immunoblots performed in a single membrane are showed. Bands displayed in this manuscript has been framed into red boxes. . Sequential immunoblots performed in a single membrane are showed. LEPR silencing in APCs exposed to hypoxia was evidenced by western blot. Bands displayed in this manuscript has been framed into red boxes.
Panel 9 (refer to Supplementary Figure IV d. Validation of LEP and LEPR silencing by siRNA). Sequential immunoblots performed in a single membrane are showed. LEPR silencing in HUVECs exposed to normoxia was evidenced by western blot. Bands displayed in this manuscript has been framed into red boxes.