Isolation and culture of MSC
MSC were isolated from subcutaneous adipose tissue from healthy human kidney donors (n = 5) that became available during kidney donation procedures after obtaining written informed consent as approved by the Medical Ethical Committee of the Erasmus University Medical Centre Rotterdam (MEC-2006-190).
MSC were cultured at 37° C, 5% CO2 and 20% O2 in minimum essential medium-α (Sigma Aldrich, St. Louis, MO, USA) supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml) (1% P/S; Lonza), 2 mM L-glutamine (Lonza) and 15% fetal bovine serum (Lonza). MSC were used at passage 3–6.
Culture of HUVEC
Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel, Switzerland) and cultured at 37° C, 5% CO2 and 20% O2 in endothelial growth medium 2 (EGM-2, Lonza). HUVEC were used at passage 3–6.
Immunophenotyping of MSC and HUVEC
HUVEC and MSC were phenotyped based on the expression of specific molecules on their cell surface. Damage and activation markers were also measured on the surface of HUVEC and MSC by flow cytometry (FACS Canto II, BD Biosciences, NJ, USA). Monoclonal antibodies conjugated with different fluorophores were used to measure the presence of these molecules.
Markers measured on MSC surface membranes were CD29 (Cat# 11-0299-42, eBioscience, Santa Clara, CA, USA), CD31 (Cat#555445, Becton Dickinson), CD38 (Cat# 562444, Becton Dickinson), CD44 (Cat# 553134, Becton Dickinson), CD45 (Cat#345809, Becton Dickinson), CD54 (Cat#559771, Becton Dickinson), CD62e (Cat#551145, Beckton Dickinson), CD73 (Cat# 550257, Becton Dickinson), CD144 (cat# 348510, Biolegend, San Diego, CA, USA), CD146 (Cat#747737, Becton Dickinson), TGF-β rII (Cat#FAB241P, R&D Sytems, Minneapolis, MN, USA ). PD-L1 (Cat# 557924, Becton Dickinson), HLA-II (Cat#347402, Becton Dickinson).
Markers measured on HUVEC membrane were CD31 (Cat#555445, Beckton Dickinson), CD54 (Cat#559771, Beckton Dickinson), CD62e (Cat#551145, Beckton Dickinson), CD105 (Cat# FAB10971F, R&D Sytems), CD106 (Cat#744309, Beckton Dickinson), CD144, (cat# 348510, Biolegend, San Diego, CA, USA), CD146 (Cat#747737, Beckton Dickinson), Tie-2 (Cat# FAB3131N, R&D Sytems), HLA-II (Cat#347402, Beckton Dickinson) and VEGF-r2 (Cat#560494, Beckton Dickinson). Data were analyzed using Kaluza Analysis 2.1 (Beckman Coulter).
In vitro hypoxia-reoxygenation injury model
HUVEC were cultured until complete confluence in EGM-2 medium (Lonza) at 37 °C, 5% CO2 and 20% O2. Oxygen was enzymatically removed from culture medium using bovine catalase (0.43 mg/ml, Sigma) and glucose oxidase (0.125 mg/ml, Sigma) as previously described (26) to quickly remove all oxygen from the medium. Oxygen percentage was measured using an universal perfusion solution monitor (version 1.10, Hugo Sachs Elektronik -harvard Apparatus GmbH, March-Hugstetten, Germany). Culture medium was removed and cells were washed with PBS prior to the addition of hypoxic medium. Hypoxia was maintained by culturing HUVEC in a hypoxia incubator during 24 hours at 37 °C, 5% CO2 and 0–1% O2. Additional file 1A shows that levels of oxygen were around 0% from the addition of the catalase and glucose oxidase and until O2 measurement after 24 hours. After this time point, cultures were washed with PBS and normoxic culture medium supplemented with human recombinant 20 ng/mL tumor necrosis factor- α (TNF-α; Peprotech, Rocky Hill, NJ, USA) was added for 24 hours to mimic the inflammatory response after ischemia-reperfusion (injured HUVEC).
MSC were added to HUVEC to assess their effect on HUVEC injury observed after hypoxic and inflammatory injury. In order to study the effect of MSC on HUVEC, MSC were added to HUVEC in three different manners (Additional file 1D-F). Firstly, MSC were directly incubated with HUVEC for 24 hours after injuring HUVEC, allowing cell-to-cell physical and paracrine communication. Secondly, to determine the effect of physical MSC-HUVEC interaction, MSC were inactivated by warming them to 50° C during 30 minutes as previously described (22). After this procedure the metabolism of MSC is completely stopped and they lose their ability to secrete soluble factors, but the cell surface membrane and its associated proteins remain intact. Lastly, to assess the effect of cytokines and growth factor released by MSC on HUVEC, MSC were incubated with HUVEC in a transwell system. The transwell had a porous membrane of pore size 0.4 µm (Greiner Bio-One, Kremsmunster, Austria) that allows communication through soluble factors but prevents physical contact or cell migration. In all three conditions, the incubation of MSC with HUVEC started 24 hours after reoxygenation and culture with TNF-α to test their reparative role.
Assessment of HUVEC viability
HUVEC viability was assessed by Annexin-V (PE) and ViaProbe (PercP) staining using the Annexin-V apoptosis detection kit I (Beckton Dickinson, Franklin Lakes, NJ, USA) and measured by flow cytometry (Additional file 1B and C). Data were analyzed using Kaluza Analysis 2.1 software (Beckman Coulter, Brea, CA, USA).
Measurement of LDH release
HUVEC vitality was measured using a colorimetric assay to measure lactate dehydrogenase (LDH) release to the medium as a marker for cell injury. HUVEC were cultured in 96-well plates and an LDH activity assay kit (Sigma) was used according to the manufacturer’s protocol. The results were obtained by measuring the absorbance of the reagent that is formed at 450 nm with a spectrophotometer (Victor2, PerkinElmer, Waltham, MA, USA).
Migration of MSC toward HUVEC monolayer
Migration of MSC towards injured HUVEC was assessed by culturing a monolayer of HUVEC in the lower well of a transwell system and injuring them as described above. MSC were added on top of a porous membrane with 3 µm pore size and cultured for 6 hours. After this, both upper and lower wells were trypsinized and cells counted by flow cytometry after staining them with CD31 antibody to discriminate endothelial cells from MSC. Stromal cell-derived factor 1α (SDF-1α, 10 ng/ml) was used as a positive control for MSC migration.
MSC adhesion to HUVEC
MSC adhesion to HUVEC was assessed under static and flow conditions. MSC were fluorescently labelled with PKH26 (Sigma) following the manufacturer’s protocol in order to enable later identification. HUVEC were injured as described above and MSC were added on top at a ratio of 1:2 or 1:10 and incubated for 10, 30 or 60 minutes. After these time points, supernatant was collected and wells were washed to eliminate all non-adherent MSC. Attached cells were trypsinized and analyzed by flow cytometry. Fluorescent signal detected by flow cytometry allowed the determination of the percentage of attached MSC by comparing this number to the initial number of added MSC.
To analyze MSC adhesion under flow conditions, HUVEC were seeded in Ibidi® µ-Slide I Luer slides (Gräfelfing, Germany) grown to complete confluence and injured as described above. Subsequently, the slide was connected to a rolling pump (REGLO Analog, Ismatec, Wertheim, Switzerland) and culture media was perfused at 37° C at a rate of 0.77 ml/min. PKH-labelled MSC were added to the perfusion system in different fashions: one time infusion of 200,000 MSC during flow, two times infusion of 100,000 MSC each during flow, one time infusion of 200,000 MSC followed by a 10 minutes stop in the flow to facilitate adhesion as in the adhesion experiment under static conditions or one time infusion of 200,000 MSC which was recirculated for 10 minutes. After each infusion, 10 additional minutes of flow were maintained. To analyze MSC adhesion to HUVEC during flow conditions, the slides were inspected by fluorescence microscopy to identify the adhesion of PKH-labelled MSC. To calculate the percentage of adherent MSC, the content of the slide was trypsinized and MSC were counted by flow cytometry using their fluorescence to identify them and comparing this number to the initial number of added MSC.
MSC-EC molecular interaction mechanism
In order to assess the role of specific adhesion molecules on MSC and HUVEC interaction, two molecules on the MSC cell surface were blocked. CD29 and CD44 were blocked by incubating MSC with CD29 polyclonal antibody (Cat# AF1778, R&D Systems) and CD44 polyclonal antibody (Cat# AF3660, R&D Systems) at a concentration of 2.5 µg/106 MSC for 20 minutes. The effective blockage of these molecules was assessed by staining MSC with the previously described CD29 and CD44 antibodies and measuring fluorescence by flow cytometry. MSC with either blocked CD29, CD44 or both were added to a monolayer of injured HUVEC for 10, 30 or 60 minutes. After these time points, wells were washed to eliminate all non-adherent MSC. Attached cells were trypsinized and analyzed by flow cytometry. Fluorescent signal detected by flow cytometry allowed the determination of the percentage of MSC attached.
Measurement of oxidative stress
Oxidative stress of HUVEC was measured using CellRox reagent (ThermoFisher, Manhattan, NY, USA) according to the manufacturer’s manual. Briefly, the cell-permeant CellROX reagent enters the cell and there it is oxidized by ROS, exhibiting red fluorescence. The production of ROS was quantified by measuring the fluorescence of oxidized CellRox inside the cell by flow cytometry. Data were analyzed using Kaluza Analysis 2.1 (Beckman Coulter).
Measurement of HUVEC monolayer permeability
HUVEC were grown to complete confluence on a transwell insert and cultured for 48 hours to allow the formation of tight intercellular junctions. The membrane of the insert had 0.4 µm size pores, which prevented cell migration but allowed soluble factor exchange with the lower well. FITC-conjugated dextran (100 mg/mL) diluted in PBS was added to the insert and incubated for 2 hours, while 200 µl of PBS was added to the lower well. HUVEC monolayer permeability was assessed by measuring the amount of FITC-conjugated dextran in the lower well after 2 hours by measuring fluorescence at 515 nm with a spectrophotometer (Victor2, PerkinElmer).
A scratch assay was performed on a confluent HUVEC monolayer. HUVEC were grown on 24-well plates until complete confluence and with the tip of a 200 µl pipette a scratch was made from top to bottom of the well, removing the cells in this area. After 2, 6- and 24-hours pictures were taken with an Axiovert 40 C microscope (Zeiss, Oberkochen, Germany) coupled to a Zeiss CanonSLR camera (Zeiss) to observe the closing of the scratched area. The size of the scratch area was measured using the plugin MRI Wound Healing Tool for Image J (National Institutes of Health, Bethesda, MD, USA).
A tube formation assay was performed to evaluate the effect of hypoxic and inflammatory injury on the angiogenic potential of HUVEC. Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix was kept at 4° C overnight prior to the experiment to allow complete thawing. At the start of the experiment, 50 µl Geltrex was added to each well of an ice-cold 96-well plate using cold pipette tips to avoid premature Geltrex solidification. The plates were incubated for 30 minutes at 37° C to allow Geltrex solidification. Cells were added to the wells in a concentration of 2 × 104 per well. During 6 hours, pictures were taken hourly with an Axiovert 40 C microscope (Zeiss) coupled to a Zeiss CanonSLR camera (Zeiss) to evaluate the formation of tube-like structures. To evaluate the angiogenic capacity, the total length of the tubes formed during the assay was measured. The images were analyzed by Wimasis Image Analysis (Cordoba, Spain).
Migration of MSC through a HUVEC monolayer
This setup was modified to assess MSC transmigration through a HUVEC monolayer. HUVEC were grown on top of the porous membrane of the upper well of the transwell system. After injury, HUVEC monolayer confluence was checked by microscopy and MSC were added directly on top of the HUVEC monolayer and incubated for 6 hours. During this incubation, SDF-1α (10 ng/ml) was added to the bottom well to elicit a chemotactic response for MSC. Both wells were trypsinized and cells counted by flow cytometry.