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. Author manuscript; available in PMC: 2008 Oct 16.
Published in final edited form as: FEBS Lett. 2007 Sep 24;581(25):4927–4931. doi: 10.1016/j.febslet.2007.09.032

Production of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol by Endothelial Progenitor Cells

Christiane A Opitz 1, Neta Rimmerman 2, Yanmin Zhang 1, Laura E Mead 3, Mervin C Yoder 3, David A Ingram 3, J Michael Walker 2, Jalees Rehman 1
PMCID: PMC2072933  NIHMSID: NIHMS32713  PMID: 17904123

Abstract

Recent studies have highlighted the importance of paracrine growth factors as mediators of pro-angiogenic effects by endothelial progenitor cells (EPCs), but little is known about their release of lipid-based factors like endocannabinoids by EPCs. In the current study, the release of the endocannabinoids anandamide and 2-arachidonoylglycerol by distinct human EPC sub-types was measured using HPLC/tandem mass-spectrometry. Anandamide release was highest by adult blood colony-forming EPCs at baseline and they also demonstrated increased 2-arachidonoylglycerol release with TNF-α stimulation. Treatment of mature endothelial cells with endocannabinoids significantly reduced the induction of the pro-inflammatory adhesion molecule CD106 (VCAM-1) by TNF-α.

Keywords: endothelium-derived factors, inflammation, endothelium, endothelial progenitor cells, endocannabinoids

Introduction

Circulating Endothelial Progenitor Cells (EPCs) appear to play a critical role in vascular repair and angiogenesis[1,2]. However, circulating EPCs demonstrate significant heterogeneity in their proliferative rates and surface markers: Endothelial-like cells that appear within a few days of culturing blood-derived mononuclear cells proliferate minimally, express multiple monocytic markers and are referred to as monocytic EPCs (M-EPCs) or cultured angiogenic cells (CAC)[3,4]. On the other hand, endothelial-like cells appearing after multiple weeks of culture are highly proliferative colony-forming EPCs (CF-EPCs), that do not express monocytic markers and are found in much greater number in the umbilical cord blood than in adult blood[4-6].

It appears that EPCs can replenish dysfunctional endothelium and promote vascular growth by providing new endothelial “building blocks,” however, recent studies suggest that EPCs may also exert beneficial effects via paracrine mechanisms[2,3,7-9]. Previous attempts to define the paracrine function of EPCs have mostly focused on protein mediators such as growth factors and cytokines[3,7,9]. The production and release of lipid mediators with cardiovascular activity by EPCs has not yet been studied. Endocannabinoids such as N-arachidonoylethanolamine (AEA or anandamide) and 2-arachidonoylglycerol (2- AG) are such endogenous lipid-based ligands of cannabinoid receptors and their cardiovascular effects include the dilation of blood vessels and cardioprotection in the setting of cardiac ischemia or infarction[10-13].

We wanted to determine whether the paracrine activity of EPCs included the release of potentially cardioprotective endocannabinoids. Our findings not only demonstrate that multiple EPC subtypes produce and release endocannabinoids, but also that the production can be modulated by the cytokine Tumor Necrosis Factor-α (TNF-α) and that endocannabinoids can suppress the expression of the pro-inflammatory cell adhesion molecule VCAM-1 (Vascular Cell Adhesion Molecule-1, CD106) on endothelial cells.

Methods

Isolation and Characterization of EPCs and HAECs

Human monocytic EPCs and human colony-forming EPCs (derived from adult peripheral blood or umbilical cord blood) were isolated as previously published[3,5]. Human aortic endothelial cells (HAEC) were obtained from Cambrex (Baltimore, MD) and cultured in EGM-2 growth medium as per manufacturer instructions.

Measurement of the endocannabinoids anandamide and 2-AG in conditioned cell medium

To measure the release of endocannabinoids by EPCs (monocytic EPCs, cord-blood derived colony-forming EPCs, adult blood derived colony forming EPCs) as well as mature HAECs, cells were switched to a standardized endothelial basal medium EBM-2 culture medium, antibiotics, and 0.2% bovine serum albumin. Fetal bovine serum was not used in order to exclude contamination of potential endocannabinoids contained in the serum. Some samples were treated with 10 ng/ml of TNF-α to assess the effects of inflammatory stimulation. After 24 hours, cell numbers were determined and cell media was collected, flash frozen and kept at -80°C. Control flasks containing cell-free media were incubated under the same conditions and further analyzed with test samples.

Compound extraction from cell medium

Samples were thawed on ice and HPLC grade water and methanol (VWR international, Plainview, NY) were added to each sample to create a 70% aqueous solution. [2H8] N-arachidonoyl dopamine (NADA, Cayman Chemical, Ann Arbor, MI) was added to the sample and used as an internal standard to control for the recovery of the test compounds. It was chosen because it possesses structural, chromatographic, and mass spectroscopic (MS) properties that are similar to those of AEA and 2-AG. Target lipids were extracted using 500mg Bond Elut C18 solid phase columns (Varian, Harbor City, CA) and samples were eluted with 100% methanol [14].

High performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis and endocannabinoid quantification

Analysis of endogenous cannabinoids was carried out as described previously [14]. Briefly, an aliquot of the final elution was loaded onto a reversed phase Zorbax 2.1 × 50 mm C18 column with an Agilent 1100 autosampler (Agilent, Wilmington, DE). Gradient formation was achieved by an HPLC system comprised of a Shimadzu SCL10Avp controller and two Shimadzu LC10ADvp Pumps. Cannabinoid levels in the sample were analyzed in multiple reaction monitoring mode on a triple quadrupole mass spectrometer API 3000 (Applied Biosystems/MDS SCIEX, Foster city, CA) with electrospray ionization. The following molecular ion/fragments were used: positive ion mode 2-AG 379.3→287.3, anandamide 348.3→62.1; Negative ion mode: [2H8] NADA 446.4→123.3. All calculations were based on calibration curves using synthetic standards.

Flow cytometry

For flow cytometric analysis, cells were detached using EDTA, re-suspended in PBS and incubated with fluorescent monoclonal antibodies against VEGF-Receptor 2, CD14 and VCAM-1 or isotype-matched negative controls (Becton Dickinson, San Jose, CA) following manufacturer instructions. Cells were washed, fixed in 20% formaldehyde (Tousimis, Rockville, MD) and analyzed on a FACS-Calibur Instrument (Becton-Dickinson, San Jose, CA).

To measure the effects of endocannabinoid treatment on the expression of endothelial VCAM-1, HAECs were exposed to 10 ng/ml TNF-α or control medium for 6 hours and co-treated with varying doses of anandamide, 2-AG, or the vehicle dimethyl-sulfoxide (DMSO).

Statistical Analysis

The analysis and comparison of endocannabinoid release between cell types was performed by ANOVA using Bonferroni corrections. Differences between baseline conditions and TNF-alpha stimulation were assessed with a paired t-test. The analysis of dose-dependent effects of endocannabinoids on VCAM-1 expression was performed by repeated-measures ANOVA also using Bonferroni corrections for multiple comparisons.

Results

Distinct subtypes of endothelial progenitor cells

We analyzed the surface expression of CD14 and VEGFR-2 by flow cytometry on all three EPC subtypes (monocytic EPCs, adult blood derived colony forming EPCs, cord-blood derived colony forming EPCs) and mature human aortic endothelial cells (HAECs). As can be seen in Fig. 1, all four cell types express the endothelial marker VEGFR-2, but only monocytic EPCs additionally express the marker CD14. As previously published, this marker characterizes monocytic EPCs, while no specific surface markers are known to distinguish CF-EPCs and mature endothelial cells[3,5,15,16]. The latter three cell-types (adult blood CF-EPCs, cord blood CF-EPCs, HAECs) are distinguished by their source of isolation (adult peripheral blood, umbilical cord blood, human aorta tissue, respectively).

Fig 1. Flow cytometric phenotyping of the four distinct endothelial cell types.

Fig 1

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The expression of the monocyte/macrophage marker CD14 (y-axis) and the endothelial marker VEGF-receptor-2 (x-axis) was assessed on monocytic EPCs (Panel A), adult mature human aortic endothelial cells (Panel B), adult blood colony-forming EPCs (Panel C) and cord-blood colony forming EPCs (Panel D). Only monocyte-derived cells were positive for CD14 and VEGF-receptor-2; the remaining three other cell types were positive only for VEGF-receptor-2.

Endothelial progenitor cells produce and release endocannabinoids

Using liquid chromatography/tandem mass spectrometry (LC/MS/MS) we determined the release of anandamide and 2-AG into the conditioned medium of EPCs (Fig. 2 A). All subtypes of EPCs as well as mature HAECs produced and released the endocannabinoids anandamide and 2-AG. The amount of released anandamide was highest in adult CF-EPCs (32.0±10.4 pmol per million cells; p=0.007, Fig. 2B) when compared to cord CF-EPCs (6.3±2.1 pmol per million cells; p<0.05 vs adult CF-EPCs), monocytic EPCs (2.2±1.1 pmol per million cells; p<0.05 vs adult CF-EPCs) and HAECs (5.6±1.7 pmol per million cells; p<0.05 vs adult CF-EPCs). There was no significant difference in the anandamide release between cord CF-EPCs, monocytic EPCs and HAECs.

Fig 2. Release of the endocannabinoids Anandamide and 2-Arachidonoylglycerol (2-AG) by mature human aortic endothelial cells (HAECs) and endothelial progenitor cell sub-types.

Fig 2

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Cells were incubated in basal medium for 24 hours without fetal bovine serum to avoid serum contamination. Endocannabinoids were measured in conditioned medium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). A typical spectrum is shown in Panel A. Cell numbers were determined and the data are presented as pmol endocannabinoid release per million cells (mean ± SEM; n=4; Panel B: Anandamide, Panel C: 2-AG). Statistical comparisons between cell types were performed using ANOVA with Bonferroni corrections (NS= not significant, *=p<0.05 vs all other groups).

Monocytic EPCs showed a trend towards higher production of 2-AG (Fig. 2C) compared to adult CF-EPCs, cord CF-EPCs and HAECs, however the differences in the measured release of 2-AG from all the cell groups were not significantly different from each other (p=0.25).

Inflammatory stimulation with TNF-α can upregulate 2-AG release by adult CF-EPCs

To test whether inflammatory stimulation could augment the release of endocannabinoids in endothelial cells and endothelial progenitor cells, we exposed EPCs and HAECs to the pro-inflammatory cytokine TNF-α and measured the release of endocannabinoids by each cell type. CF-EPCs and HAECs showed a non-significant trend towards increased release of anandamide, with increases ranging from 36.7 % (cord blood CF-EPCs) to 68.8% (HAECs), while monocytic EPCs showed a decrease of −60.4% (Fig. 3A). The release of 2-AG by adult blood CF-EPCs was increased significantly by 268.2% after stimulation with TNF-α (p<0.05), while monocytic EPCs again showed a trend towards decreased 2-AG release, similar to that observed for anandamide release (Fig. 3B).

Fig 3. Endocannabinoid release in response to TNF-α stimulation.

Fig 3

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Stimulation of mature endothelial cells and EPCs with the pro-inflammatory cytokine TNF-alpha was performed in serum-free basal medium for 24 hours and the data are presented as percent change of endocannabinoid release over baseline (n=4). Stimulation with TNF-α showed a trend towards increased release of both anandamide (Panel A) and 2-AG in colony forming EPCs and mature endothelial cells. A trend towards decreased release of both endocannabinoids in TNF-α stimulated monocytic EPCs was evident, thus underscoring their phenotypic differences from the other three endothelial cell types. Statistical comparisons between cell types were performed using ANOVA with Bonferroni corrections (NS= not significant, *=p<0.05 vs all other groups).

Endocannabinoids can directly modulate induction of the pro-inflammatory adhesion molecule VCAM-1 on endothelial cells by TNF-α

While it is known that endocannabinoids suppress inflammation by acting on leukocytes[17], the modulatory effects of endocannabinoids on endothelial cell adhesion molecules have not been studied. We therefore stimulated HAECs with the pro-inflammatory cytokine TNF-α for 6 hours and co-treated them with varying doses of either anandamide or 2-AG (Fig. 4). The mean intensity of fluorescence of VCAM-1 determined by flow cytometry increased 10-fold with six hours of TNF-α stimulation (from 6.0±1.6 to 63.3±5.6 fluorescence units; p=0.001). Addition of anandamide to the TNF-α treated cells decreased the VCAM-1 induction in a dose-dependent manner, with a drop of nearly 30% from a mean VCAM-1 intensity of 63.3±5.6 fluorescence units to 45.5±6.8 fluorescence units at a dose of 10 μM of AEA (p<0.05 vs TNF only, Fig. 4A). Similarly, 2-AG showed a trend towards reduced VCAM-1 expression at 1 μM 2-AG and at a significant drop in VCAM-1 levels of about 20% (p<0.05 vs TNF only) with 10 μM 2-AG (Fig. 4B).

Fig 4. Endocannabinoids reduce VCAM-1 expression on endothelial cells.

Fig 4

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To measure the effect of endocannabinoids on the expression of the pro-inflammatory endothelial surface molecule VCAM-1, endothelial cells were co-stimulated with TNF-α and varying doses of the endocannabinoids anandamide (Panel A) and 2-AG (Panel B) for 6 hours. Expression of VCAM-1 was measured by flow cytometry and is depicted as mean fluorescent units (geometric mean ± SEM; n=4). Treatment of endothelial cells with endocannabinoids could partially inhibit the induction of the inflammatory adhesion molecule VCAM-1 by TNF-α

At baseline in the absence of TNF-alpha stimulation, endothelial cells did not show any significant expression of VCAM-1. After treatment with endocannabinoids (6 hours), there was still no significant VCAM-1 expression as is shown in a representative experiment (Supplemental Figure S1).

Discussion

Endocannabinoid production and release by EPCs

To our knowledge, this is the first report of endocannabinoid production and release by endothelial progenitor cells. Since EPCs are found not only in the blood but also within the vessel wall [16], the paracrine activity of EPCs may create a local milieu that promotes vascular cell survival and suppresses the effects of noxious stimuli. Most studies investigating the paracrine activity of EPCs have focused on proteins released by EPCs [3,7-9,18], however, lipid-based paracrine mediators may offer the advantage of concentrating high levels of the hydrophobic mediators in the local interstitium, thus reducing systemic “leakage” which may be more likely with hydrophilic protein mediators. The endocannabinoids anandamide and 2-AG are such endogenous lipid-based mediators, which are synthesized and released. Both compounds initiate multiple signaling cascades, both are formed in the central nervous system as well as the periphery, and their potent vasodilatory, cardioprotective [10-13] and anti-inflammatory effects [17,19] make them an attractive pharmacological target for cardiovascular disease. Our experiments demonstrate that EPCs produce and release anandamide as well as 2-AG, although there are significant differences between some of the EPC types. Monocytic EPCs showed a trend towards secreting higher levels of 2-AG, while colony-forming EPCs secreted higher levels of anandamide. Remarkably, adult blood derived colony-forming EPCs secreted the highest levels of anandamide amongst all EPC cell types, thus underscoring the differences that may exist amongst EPC subtype physiology and paracrine activity. The finding that the endocannabinoid release was higher in adult blood CF-EPCs than in mature aortic endothelial cells derived from an aortic vessel wall (HAECs) also suggests that EPCs may have a distinct paracrine profile when compared to mature endothelial cells.

Stimulation with the pro-inflammatory cytokine TNF-α increased the release of 2-AG by adult blood CF-EPCs and CF-EPCs also showed a trend towards increased release of anandamide. Since endocannabinoids can suppress inflammation [17], the increase in anti-inflammatory endocannabinoid release by a pro-inflammatory cytokine may seem counter-intuitive. However, in CNS inflammation, endocannabinoids are similarly increased by inflammation and then act to reduce inflammation [20], thus serving as endogenous protective mediators to limit inflammatory damage.

The fact that monocytic EPCs were the only cell type that showed a trend towards a decrease in endocannabinoid release with TNF-alpha stimulation may point to their distinct physiology since monocytic EPCs, in contrast to the other three cell types were the only cell-type we studied that had a myeloid (leukocyte) origin [3,15,16]. Whether the observed differences in TNF-alpha response were due to differences in TNF signaling or differences in endocannabinoid production and release will need to be addressed in future studies.

Suppression of VCAM-1 by EPCs

Our studies demonstrated that endocannabinoids can suppress the induction of the pro-inflammatory endothelial adhesion molecule VCAM-1. Vascular inflammation is now an established etiological factor in the development and progression of atherosclerosis [21,22]. Endothelial VCAM-1 induction plays a central role in this process by allowing for the recruitment of inflammatory leukocytes [23]. Our finding that endocannabinoids act on endothelial inflammation complements previous studies showing anti-inflammatory effects of endocannabinoids on leukocytes [17, 19]. Since a recent study has shown that exogenous cannabinoids reduce the progression of atherosclerosis in vivo [24], we propose that endogenous cannabinoids may serve as physiological suppressants of endothelial inflammation and atherosclerosis. Future studies will be required to unravel the potential mechanisms by which exogenous or endogenous cannabinoids can improve vascular function.

Supplementary Material

01. Supplementary Figure S1: No significant expression of VCAM-1 on endothelial cells at baseline in the presence or absence of endocannabinoids.

Representative histograms showing flow cytometric analysis of baseline (no TNF-alpha added) endothelial VCAM-1 surface expression as well as the negative isotype antibody control (gray in each histogram) after 6 hours of treatment with DMSO or 10 micromolar of anandamide or 2-AG. There was no significant expression of VCAM-1 after treatment with the DMSO control or the endocannabinoids, as the negative control antibody nearly completely overlaps with the VACM-1 antibody.

Acknowledgments

This work was supported by in part by intramural funds of Indiana University, Department of Medicine and in part by NIH-K08-HL080082 to Jalees Rehman (PI).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplementary Figure S1: No significant expression of VCAM-1 on endothelial cells at baseline in the presence or absence of endocannabinoids.

Representative histograms showing flow cytometric analysis of baseline (no TNF-alpha added) endothelial VCAM-1 surface expression as well as the negative isotype antibody control (gray in each histogram) after 6 hours of treatment with DMSO or 10 micromolar of anandamide or 2-AG. There was no significant expression of VCAM-1 after treatment with the DMSO control or the endocannabinoids, as the negative control antibody nearly completely overlaps with the VACM-1 antibody.

NIHMS32713-supplement-01.tif (177.6KB, tif)

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