Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.
Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45–. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.
Endothelial cells (ECs) are squamous cells that line the inner surface of the blood vessel wall as a monolayer. They have many essential roles, including control of vascular tone, regulation of thrombosis, modulating the inflammatory response, and contributing to angiogenesis1. Given the importance of endothelial cells in cardiometabolic physiology, they are frequently used experimentally to further the understanding of pathophysiology and to examine new pharmacological treatments for cardiometabolic disease.
However, there is enormous heterogeneity in endothelial cell morphology, function, gene expression, and antigen composition depending on the origin of their vascular bed2. While endothelial cells from large arteries are best suited to atherosclerosis studies, endothelial cells from small vessels, known as microvascular endothelial cells (MVECs), are more suitable for angiogenesis studies2. Understanding the molecular basis for endothelial heterogeneity may provide valuable insights into vascular bed-specific therapies. Microvascular endothelial function also significantly differs in numerous diseases, including diabetes, cardiovascular disease, and systemic infection3,4. Therefore, the ability to isolate endothelial cells from defined patient groups allows direct comparison of their endothelial cell function and cellular cross-talk5.
In this paper, we describe a novel method of isolating human MVECs from subcutaneous adipose tissue (hSATMVEC) taken at the time of cardiac implantable electronic device (CIED) insertion. hSATMVEC isolated following enzymatic digestion of subcutaneous adipose tissue (SAT) were grown and passaged on gelatin-coated plates. We then describe a range of phenotyping assays that have been successfully applied to hSATMVECs in order to validate their phenotype and demonstrate use in routine endothelial cell assays. Finally, we describe an application of hSATMVECs in an experimental model of hSATMVECs-adipocyte cross-talk.
The samples of human tissue used in the technique described have been taken from patients undergoing guideline-indicated insertion of CIEDs according to routine clinical practice in Leeds Teaching Hospitals NHS Trust (Leeds, United Kingdom). The study protocol, along with all other documentation, was approved by the local ethics committee (11/YH/0291) prior to participant enrolment. The study was conducted in compliance with the principles of the Declaration of Helsinki.
1. Patient population
2. Endothelial cell isolation and culture
NOTE: A schematic for hSATMVEC isolation is shown in Figure 1.
3. Flow cytometry
4. Endothelial cell doubling time and cell proliferation (Figure 3)
5. Endothelial cell tube formation
6. Insulin stimulation of hSATMVEC
7. hSATMVEC-adipocyte co-culture set up (Figure 4)
NOTE: In the results below, commercially available human white subcutaneous preadipocytes at passage 2 from a single male Caucasian donor were used in all adipocyte assays. Preadipocytes were initially expanded from the vendor-supplied vial (passage 0) into twelve cryovials containing cryo-SFM freezing media (passage 1).
8. Adipocyte 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBD)-glucose uptake
hSATMVEC purity and phenotype
Isolated hSATMVEC from control patients (that is, those people without a history of cardiometabolic disease) were 99.5% CD31+CD144+CD45- on flow cytometry (Figure 2). Isolated hSATMVEC had a cobblestone-like morphology typical of ECs (Figure 3A). hSATMVECs had a mean population doubling time of 56.6 h ± 8.1 h (mean ± SEM, n = 10) (Figure 3B), and active DNA replication in hSATMVECs was confirmed using a cell proliferation imaging kit (Figure 3C).
hSATMVEC behaved like functioning MVECs and formed tubes in Matrigel (Figure 3D). The relative expression of key proteins involved in the insulin signaling Akt-eNOS pathway is shown in Figure 3E. Both eNOs and Akt demonstrated increases in insulin induced phosphorylation, represented as the ratio of phosphorylated protein/total protein normalized to ß-actin (Figure 3E).
hSATMVEC-adipocyte co-culture
An illustrative model of hSATMVEC-adipocyte co-culture can be seen in Figure 4A. As the human subcutaneous white preadipocytes become more differentiated, one will notice the development of lipid vacuoles (Figure 4B), which can be quantified by Oil Red O staining (Figure 4B).
Illustrative phase contrast and fluorescence imaging (excitation 440-480 nm/emission 504-544 nm) of differentiated adipocytes following 30 min of incubation with 20 µM 2-NBD glucose is shown in Figure 4C. Glucose uptake can be quantified by measuring green area as a percentage of total cell area.
Figure 1: Schematic demonstrating the harvesting and processing of hSATMVECs Please click here to view a larger version of this figure.
Figure 2: Typical output of flow cytometry analysis of cultured hSATMVECs. (A) Scatter plot of side scatter area (SSC-A) and forward scatter area (FSC-A) showing gating (red box) of cultured hSATMVECs around specific cell population density. (B) Scatter plot of forward scatter width (FSC-Width) and FSC-A showing gating (red box) of cultured hSATMVECs around specific cell population density. (C) Histogram showing fluorescence of gated cells to CD45-FITC. (D) Scatter plot of fluorescence intensity of CD144-PE (x-axis) and CD31-PerCP (y-axis). (E) Histogram showing fluorescence of gated cells to CD144-PE. (F) Histogram showing fluorescence of gated cells to CD31-PerCP. Please click here to view a larger version of this figure.
Figure 3: hSATMVEC phenotype. (A) Light microscopy image of hSATMVEC near confluence showing cobblestone-like appearance. (B) hSATMVEC doubling time – Light microscopy of HATEC taken 24 h apart demonstrating the extent of cell proliferation. (C) hSATMVEC proliferation. Fluorescence microscopy of hSATMVEC with propidium iodide and EdU/Alexa-fluor 488 from control subjects. (D) hSATMVEC tube formation from control subjects (images taken at 4x magnification). (E) Insulin stimulation of HATECs – showing relative expression of phosphorylated Akt (serine 473) to total Akt (left panel) phosphorylated eNOS (serine 1177) to total eNOS (right panel) at increasing insulin concentrations with illustrative western blots beneath standardized to B-actin. Data are shown as mean ± SEM. Sample sizes are beneath each panel. Abbreviations: 5-Ethynyl-2´-deoxyuridine (EdU), human subcutaneous adipose tissue microvascular endothelial cells (hSATMVEC), standard error of the mean (SEM). Please click here to view a larger version of this figure.
Figure 4: hSATMVEC-adipocyte co-culture. (A) Schematic image of hSATMVEC-adipocyte co-culture. (B) Differentiation of adipocytes. The left panel shows preadipocytes at day 0 and the right panel shows day 10 differentiated adipocytes after the addition of PDM. The bottom figure shows the amount of lipid stored in adipocytes and preadipocytes stained with Oil Red O. (C) Glucose uptake assay. The left panel shows phase imaging of adipocytes following co-culture and incubation with 20 µM of 2-NBD glucose for 30 min. The right panel shows green imaging from which glucose uptake (as % green of total area) can be quantified. Data are shown as mean ± SEM. Abbreviations: Day 10 (D10), human subcutaneous adipose tissue microvascular endothelial cells (hSATMVEC), preadipocyte differentiation media (PDM) Please click here to view a larger version of this figure.
Supplementary Table 1: Staining cocktail for flow cytometry. Please click here to download this File.
This study describes a technique of isolating hSATMVEC taken from SAT during routine implantation of CIEDs. We demonstrate that the hSATMVEC isolated has high purity, expresses EC-specific transmembrane proteins CD144 and CD31, and shows no significant expression of the leukocyte CD45. We go on to show that, in a reproducible and reliable manner, isolated hSATMVEC proliferate and can be used experimentally to study the intracellular machinery involved in insulin signaling and angiogenesis. In addition to being able to culture them in isolation, they can also be used in co-culture to study hSATMVEC-adipocyte cross-talk.
Endothelial cells used in basic and translational research are commonly sourced from large vessels, such as the aorta and human umbilical vein, or microvasculature. These sources both have their own respective limitations7,8; endothelial cells from large vessels are either difficult to access (in the case of aortic tissue) or are derived from neonatal tissue with potentially differing physiology and environmental exposure9. Using endothelial cells isolated from tissue taken during CIED implantation allows for the investigation and experimentation of cellular physiology within specific real-world patient groups. CIEDs are implanted for a variety of indications, including in patients with bradyarrhythmias, heart failure and primary and secondary prevention of ventricular tachyarrhythmias10. These patients often have multiple co-morbidities, including diabetes, obesity, and coronary artery disease, which are a major global focus of cardiovascular research11,12,13. Moreover, while the illustrative data in this paper pertains to control patients, we have applied these techniques to isolate and study SATMVEC from a range of patients, including those with advanced heart failure and/or type 2 diabetes mellitus.
Not infrequently, we encounter problems with poor hSATMVEC yields following attempted cell isolation. This risk can be significantly reduced by using a larger starting volume of SAT to isolate hSATMVEC. In addition, we encounter this more frequently in SAT from people with cardiometabolic disease, and in particular, diabetes.
One limitation of this technique is that isolated hSATMVEC can only undergo a limited number of passages. In our experience after passage 5, regardless of patient phenotype, hSATMVEC proliferation slows significantly. In addition, hSATMVEC isolated using this technique do not proliferate well when too sparsely populated; therefore, we recommend not passaging hSATMVEC at a ratio greater than 1:6. as mentioned on page 1. We have successfully thawed and reanimated hSATMVEC stored in liquid nitrogen for up to 4 years, and in our experience, the chance of reanimation is greater when cryopreserved at a lower passage number (we usually cryopreserve hSATMVEC at passage 2).
Tissue taken at CIED insertion is freely available and can be harvested at no detriment to the patient. Therefore, an easy-to-access, relatively non-invasive source of endothelial cells from these patient groups is of great benefit in conducting targeted research. While the representative images in this paper are derived from 'control' patients (that is, patients without a diagnosis of heart failure or diabetes, albeit with an indication for CIED implantation), we have successfully isolated, cultured, and co-cultured SATMVECs from patients with heart failure, diabetes, and a combination of these pathologies. Moreover, these techniques can also be applied to other microvascular beds, including skeletal muscle, and we are currently optimizing a model of skeletal muscle MVEC-myocyte crosstalk.
hSATMVECs can be isolated from human tissue taken at the time of CIED insertion and are of sufficient purity to be used experimentally to study microvascular dysfunction and endothelial cell-adipocyte cross-talk in people with and without cardiometabolic disease.
The authors have nothing to disclose.
We are very grateful to the Faculty of Biological Sciences Bioimaging Department (University of Leeds, United Kingdom) for the use of the flow cytometry facility, which was supported by Biotechnology and Biological Sciences Research Council grant funding (BBSRC BB/R000352/1). SS was supported by a British Heart Foundation Clinical Research Training Fellowship (FS/CRTF/20/24071). CL was supported by a British Heart Foundation PhD studentship (FS/19/59/34896). LDR was supported by the Diabetes UK RD Lawrence Fellowship award (16/0005382). RMC was supported by a British Heart Foundation Intermediate Clinical Research Fellowship (FS/12/80/29821). MTK is a British Heart Foundation Professor of Cardiovascular and Diabetes Research (CH/13/1/30086) and holds a British Heart Foundation program grant (RG/F/22/110076)
170L CO2 Incubator | GS Biotech | 170G-014 | |
2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) | Invitrogen | N13195 | |
4% PARAFIX buffered histological fixative | VWR Chemicals | PRC/R/38/1 | |
Akt (tAkt) Rabbit 1:1000 | Cell Signalling Technology | 9272 | |
BD Venflon Pro Safety 14 g x 45 mm, Orange, 50/pk | Medisave | 393230 | |
Bovine Serum Albumin solution, 7.5% | Merck | A8412-100ML | |
CD144 (VE-Cadherin) Antibody, anti-human, PE, REAfinity | Miltenyi Biotec | 130-118-495 | |
CD31 Antibody, anti-human, PerCP-Vio 700, REAfinity | Miltenyi Biotec | 130-110-811 | |
CD31 MicroBead Kit, human, 1 kit | Miltenyi Biotec | 130-091-935 | |
CD45 Antibody, anti-human, FITC, REAfinity | Miltenyi Biotec | 130-110-769 | |
Cell Extraction Buffer | Invitrogen | FNN0011 | |
Centrifuge 5804 R | Eppendorf | 5805000060 | |
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye | Invitrogen | C10337 | Cell proliferation imaging kit |
Collagenase/Dispase, 500 mg | Roche/Merck | 11097113001 | |
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 354230 | Basement Membrane Matrix |
Corning 100 mm TC-treated Culture Dish | Corning | 430167 | |
Costar 24-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile | Corning | 3526 | |
Costar 6-well Clear TC-treated Multiple Well Plates, Individually Wrapped, Sterile | Corning | 3516 | |
CytoFLEX S – 4 laser flow Cytometer | Beckman | ||
Dead Cell Removal Kit | Miltenyi Biotec | 130-090-101 | |
Dulbecco′s Phosphate Buffered Saline | Merck | D8537-500ML | |
EASYstrainer Cell sieve for 50 mL tubes, 70 µm mesh, Blue, sterile, 50/pk | Greiner Bio-One | 542070 | |
Endothelial Cell Growth Medium MV | PromoCell | C-22020 | |
Eppendorf Safe-Lock micro test tubes | Merck | EP0030120094 | |
Ethylenediaminetetraacetic acid solution | Merck | E8008-100ML | |
Falcon 24 Well TC-Treated Cell Polystyrene Permeable Support Companion Plate, with Lid, Sterile, 1/Pack, 50/Case | Appleton Woods | CF537 | |
Falcon Permeable Support for 24 Well Plate with 0.4 µm Transparent PET Membrane, Sterile, 1/Pack, 48/Case | Appleton Woods | CF521 | |
Freezing Medium Cryo-SFM | PromoCell | C-29912 | |
Gelatin solution, 2% in water | Merck | G1393-100ML | |
Gibco Antibiotic-Antimycotic (100x), 100mL | Fisher Scientific | 11570486 | |
Gibco TrypLE Select Enzyme (1x), no phenol red | Fisher Scientific | 12563029 | |
Hanks′ Balanced Salt solution | Merck | H6648-500ML | |
Human Subcutaneous Preadipocyte Cells | Lonza | PT-5020 | |
Incucyte ZOOM | Essen BioScience | Live-cell analysis system | |
Insulin solution human | Merck | I9278-5ML | |
LS Columns, 25/pk | Miltenyi Biotec | 130-042-401 | |
MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
MACS Tissue Storage Solution | Miltenyi Biotec | 130-100-008 | |
MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 | Tube Rotator |
MS Columns, 25/pk | Miltenyi Biotec | 130-042-201 | |
NuPAGE 4–12% Bis-Tris Gel | Invitrogen | NP0322BOX | |
OctoMACS Separator | Miltenyi Biotec | 130-042-109 | |
PGM-2 Preadipocyte Growth Medium-2 BulletKit | Lonza | PT-8002 | |
Phospho (Ser1177) eNOS Rabbit 1:1000 | Cell Signalling Technology | 9570 | |
Phospho-Akt (Ser473) Rabbit 1:1000 | Cell Signalling Technology | 4060 | |
Pre-Separation Filters 30 µm, 50/pk | Miltenyi Biotec | 130-041-407 | |
Propidium Iodide (PI)/RNase Staining Solution | Cell Signalling Technology | 4087 | |
QuadroMACS Separator | Miltenyi Biotec | 130-090-976 | |
Scalpel Disposable Sterile Style 10 | VWRI | 501 | |
Screw cap tube, 15 ml, (LxØ): 120 x 17 mm, PP, with print | Sarstetd | 62.554.502 | |
Screw cap tube, 50 ml, (LxØ): 114 x 28 mm, PP, with print | Sarstedt | 62.547.254 | |
Screw cap tube, 50 ml, (LxØ): 114 x 28 mm, PP, with print | Sarstetd | 62.547.254 | |
Total eNOS Mouse 1:1000 | BD Biosciences | 610297 | |
Triton X-100, BioUltra, for molecular biology | Merck | 93443-500ML | |
β-Actin (C4) Mouse 1:5000 | Santa Cruz Biotechnology | Sc-47778 |
.