14S,21R-Dihydroxydocosahexaenoic Acid Remedies Impaired Healing and Mesenchymal Stem Cell Functions in Diabetic Wounds*

Treatment of diabetes-impaired wound healing remains a major unresolved medical challenge. Here, we identified suppressed formation of a novel reparative lipid mediator 14S,21R-dihydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21R-diHDHA) in cutaneous wounds of diabetic db/db mice. These results indicate that diabetes impedes the biosynthetic pathways of 14S,21R-diHDHA in skin wounds. Administration of exogenous 14S,21R-diHDHA to wounds in diabetic animals rescued healing and angiogenesis. When db/db mesenchymal stem cells (MSCs) were administered together with 14S,21R-diHDHA to wounds in diabetic animals, they coacted to accelerate wound re-epithelialization, granulation tissue formation, and synergistically improved vascularization. In the pivotal cellular processes of angiogenesis, 14S,21R-diHDHA enhanced VEGF release, vasculature formation, and migration of db/db dermal microvascular endothelial cells (DMVECs), as well as remedied paracrine angiogenic functions of db/db MSCs, including VEGF secretion and the promotion of DMVEC migration and vasculature formation. Our results show that 14S,21R-diHDHA activates the p38 MAPK pathway in wounds, db/db MSCs, and DMVECs. Overall, the impeded formation of 14S,21R-diHDHA described in this study suggests that diabetes could affect the generation of pro-healing lipid mediators in wound healing. By restoring wound healing and MSC functions, 14S,21R-diHDHA is a new lead for the development of better therapeutics used in treating wounds of diabetics.

However, diabetic hyperglycemia and the concomitant oxidative stress damage DNA, proteins, and lipids in various tissues resulting in dysfunction of cells and enzyme systems involved in wound healing and LM biosynthesis (12)(13)(14)(15). Therefore, diabetic complications are likely to dysregulate the biosynthesis of DHA-derived LMs in wound healing. Treatments that counteract this dysregulation may improve diabetic wound healing.
Recent studies have used bone marrow-derived mesenchymal stem cells (MSCs) to promote wound healing (16 -18). MSC transplants promote wound healing and angiogenesis by producing paracrine angiogenic cytokines (2, 19 -21) and by possibly differentiating into skin cells (2, 19 -24). Treating diabetics with their own MSCs can avoid side effects, including a graft versus host response associated with nonautologous transplantation. However, diabetes also impairs MSC pro-healing functions (24).
To date there are no reports regarding LM formation or the effects of LM either in diabetic wound healing and angiogenesis or in diabetes-impaired MSC functions. To identify dys-regulation in the formation of DHA-derived LMs in diabetic mice, we have conducted LC-MS/MS-based lipidomic studies (25) of wounded skin from diabetic db/db and nondiabetic db/ϩ mice. Here, we describe the identification of a novel DHA-derived mediator 14S,21R-diHDHA, which is suppressed in wounds of diabetic db/db mice and 12/15(or L-12)lipoxygenase gene knock-out mice (12/15-LOX is most related to human 15-LOX type-1) (26). We demonstrate that 14S,21R-diHDHA restored wound healing and angiogenesis in diabetic mice as well as the pro-healing functions of db/db MSCs and key cellular processes of angiogenesis. It activates the p38 MAPK but does not affect the ERK1/2, although signaling through both p38 MAPK and ERK1/2 is critical for wound healing and associated angiogenesis (27,28).

EXPERIMENTAL PROCEDURES
Studies were blinded. Animal protocols were approved by the Institutional Animal Care and Use Committee and Institutional Review Board of Louisiana State University Health Sciences Center, New Orleans.
Isolation and Identification of DMVECs from db/db and db/ϩ Mice-This was done as described previously (33) and as detailed in supplemental Fig. S1. The final DMVECs were 95% CD31 ϩ and VE-cadherin ϩ . The cellular identity of the DMVECs was confirmed by immunocytochemical analysis, which demonstrated their purity and expression of the endothelial markers CD31, VE-cadherin, von Willebrand factor, and Tie-2 (supplemental Fig. S1A).
Isolation and Identification of MSCs from Mice-MSCs were isolated as published previously (22) and described in supplemental Fig. S1. To verify the identity of the MSCs, we first confirmed their differentiation ability according to a previous report (18). The isolated MSCs exhibited a spindle-shaped morphology (supplemental Fig. S1Bi). After culturing the MSCs for 10 days in osteogenic or adipocytic differentiation media, the cells differentiated into the osteoblastic (supplemental Fig. S1Bii) or adipocytic lineages (supplemental Fig. S1Biii), respectively. We also analyzed the MSCs by flow cytometry as in Ref. 18 and confirmed that more than 95% of cells expressed MSC marker Sca-1 (supplemental Fig. S1Biv) (18,19,22).
Splinted Excisional Wound Healing Model-This model was established as described previously (34) with minor modification. Briefly, paired 5-mm circular, full-thickness wounds were made symmetrically along the midline on the dorsal skin of mice. For each wound on a diabetic db/db mouse, 14S,21R-diHDHA in DMEM (50 ng/wound), MSCs (db/db or db/ϩ, 10 6 cells/wound), or 14S,21R-diHDHA (50 ng) plus MSCs (db/db, 10 6 cells) was applied to the wound bed (10 l) and injected intradermally at four points (10 l/site) distributed evenly near the wound edge (50 l total/wound). A donutshaped silicone splint was adhered around the wound to prevent skin contraction and to allow wounds to heal through re-epithelialization and granulation (34). This model closely parallels human wound healing (34). In another experiment, wounds were imposed to the skin of db/ϩ mice, and 14S,21R-diHDHA in DMEM (50 ng/wound), MSCs (db/ϩ, 10 6 cells/ wound), or 14S,21R-diHDHA (50 ng) plus MSCs (db/ϩ, 10 6 cells) was applied to the wound bed and wound edge. Skin wounds were also generated in 12/15-LOX-KO and control C57/6J mice and collected for the analysis of lipids.
Analysis of Wound Healing-Wounds rimmed with 2 mm of normal skin were excised from db/db mice sacrificed at day 8 post-wounding and db/ϩ mice at day 5 post-wounding, fixed, and embedded (34). Cryosections (10 m thick/section) of wounds were analyzed to determine relative epithelial gap (recorded as a percent relative to that of the initial wound) and granulation tissue area after hematoxylin and eosin (H&E) staining. Results were expressed as decreased percentage of relative epithelial gap compared with control ((relative epithelial gap in control Ϫ relative epithelial gap in treatment)/relative epithelial gap in control ϫ 100%) and increased percentage of granulation tissue area compared with control ((granulation tissue area in treatment Ϫ granulation tissue area in control)/granulation tissue area in control ϫ 100%). Wound vascularity, as determined by CD31 Ϫ positive area per field/total wound bed area per field ϫ 100% (% vascularity), was assessed by staining vessel endothelial cells with monoclonal rat anti-mouse CD31 antibody (BD Biosciences) followed by anti-rat IgG horseradish peroxidase (HRP) detection kits (BD Biosciences) and then by hematoxylin for nuclei. Images were captured using a microscope. Results are depicted as increased percentage of wound vascularity compared with control ((% vascularity in treatment Ϫ % vascularity in control)/% vascularity in control ϫ 100%).
Preparation of MSC-conditioned Media-Quiescent db/db MSCs (3 ϫ 10 5 cells) were prepared by starvation in DMEM containing high glucose (25 mM) and 0.5% FBS (Invitrogen) for 12 h. The cells were then cultured in DMEM containing high glucose (25 mM) without or with 14S,21R-diHDHA (100 nM) for 24 h. MSCs from nondiabetic db/ϩ mice were cultured similarly in the absence of 14S,21R-diHDHA. After centrifugation at 3,000 ϫ g for 15 min at 4°C, the supernatant became "MSC-conditioned medium." DMVEC Migration-Quiescent db/db and db/ϩ DMVECs at 80% confluence were resuspended in DMEM containing 25 mM (for db/db cells) or 5 mM (for db/ϩ cells) glucose and added to the upper chamber of a 24-transwell plate (8-m pore, BD Biosciences) at 1 ϫ 10 5 cells/well. DMEM containing high glucose (25 mM) or low glucose (5 mM) in the presence or absence of 14S,21R-diHDHA (100 nM) or MSC-conditioned media were added to the lower chamber. The cells were allowed to migrate for 4 h at 37°C in 5% CO 2 . Migrated cells, which were attached to the undersides of membranes, were stained with Giemsa. The images were captured by a microscope. Results were expressed as increased percentage of migrated DMVECs compared with control using the equation ((migrated DMVECs per field in treatment Ϫ migrated DMVECs per field in control)/migrated DMVECs per field in control ϫ 100%).
DMVEC Vasculature Formation-Quiescent db/db and db/ϩ DMVECs were cultured on Matrigel (BD Biosciences) in DMEM containing high glucose (25 mM, for db/db cells) or low glucose (5 mM, for db/ϩ cells) in the presence or absence of 14S,21R-diHDHA (100 nM) or MSC-conditioned media for 24 h at 37°C in 5% CO 2 . The vasculature length was measured using a microscope and ImageJ1.40 software (National Institutes of Health). Results were expressed as increased percentage of vasculature length compared with control using the equation ((vasculature length per field in treatment Ϫ vasculature length per field in control)/vasculature length per field in control ϫ 100%).
Bio-Plex Protein Array-Quiescent db/db DMVECs (3 ϫ 10 5 cells) were cultured in DMEM containing high glucose (25 mM) in the presence or absence of 14S,21R-diHDHA for 24 h. The supernatants were collected. VEGF in db/db DMVEC supernatants or MSC-conditioned media was quantified by the Bio-Plex Protein array kit (Bio-Rad).
Western Blot-14S,21R-diHDHA (50 ng/wound) was immediately injected into wound bed and edge in db/ϩ and db/db mice after wounds were made. 15 min later, wounds were collected for analysis of expressions of P-p38 and p38. Quiescent db/db DMVECs or db/db MSCs were incubated in DMEM with or without 14S,21R-diHDHA (100 nM) for 10 -120 min. Cells were lysed for analysis of expression of P-p38 and P-ERK1/2 (phosphorylated forms) as well as p38 and ERK1 (nonphosphorylated forms). Western blot was performed as described previously (27,28) with minor modification. Briefly, 30 g of total proteins from each lysed sample was resolved by SDS-PAGE on 4 -15% Tris-HCl gels (Bio-Rad). The electrotransferred protein bands were stained by primary antibodies for P-p38 or P-ERK1/2 (BD Biosciences), followed by fluorescent-labeled secondary antibodies (LI-COR, Lincoln, NB), and finally quantified using an Odyssey Imaging System (LI-COR). When necessary, blots were stripped and reprobed.
Statistical Analysis-Results were analyzed by one-way ANOVA analysis of variance followed by Fisher's LSD post hoc comparison and expressed as means Ϯ S.E. A value of p Ͻ 0.05 was considered significant.   (Fig. 1A), namely 14,21-diHDHAs existed in wounds (Fig. 1A, top).
The formation of 14S,21R-diHDHA and its biosynthetic precursor 14S-HDHA (11) was reduced, although the reduction of 14S-HDHA was modest, and 14S,21S-diHDHA was under the detection limit in wounds of diabetic mice compared with nondiabetic control animals (Fig. 1, A and B) based on LC-MS/MS quantification using deuterated internal standard of each compound. The DHA level was slightly reduced, but it was not significant. We also analyzed the skin wounds of 12/15-LOX-KO mice and controls. It was observed that the formation of 14S-HDHA was significantly reduced, and 14S,21R-diHDHA was found in the wounds of wild-type controls but was undetectable in the 12/15-LOX-KO mice, although DHA levels were not significantly different (Fig. 1B,  right panel). This implicates that 12/15-LOX participates in the formation of 14S,21R-diHDHA and 14S-HDHA.
14S,21R-diHDHA Remedies Diabetic Wound Healing and Vascularization-Because 14S,21R-diHDHA formation was decreased in diabetic wounds, we were motivated to study whether administration of exogenous of 14S,21R-diHDHA could enhance wound healing. 14S,21R-diHDHA was applied into the wounds of db/db mice. Interestingly, the administration of 14S,21R-diHDHA promoted wound healing significantly (Fig. 2). In comparison with vehicle control, 14S,21R-diHDHA accelerated re-epithelialization and promoted granulation formation. Analysis of hematoxylin and eosinstained cryosections of wounds collected at day 8 postwounding revealed that the relative epithelial gap was decreased 36.2% (Fig. 2B, left panel), whereas the granulation tissue area was increased 60.7% (Fig. 2B, right panel). Because impaired vascularization or angiogenesis is a critical hallmark of nonhealing wounds in diabetes (45,46), we investigated whether 14S,21R-diHDHA could promote vascularization during wound healing in diabetic mice. The data demonstrate that wounds from db/db mice treated with 14S,21R-diHDHA exhibited a 41.9% increase in vascularity compared with control animals at day 8 post-wounding (Fig. 2C). When 14S,21R-diHDHA was applied on wounds of db/ϩ mice, the same effects on re-epithelialization, granulation formation, and vascularization were observed (supplemental Figs. S4 and S5).
We further used in vitro models of DMVEC migration and vasculature formation to investigate the action of 14S,21R-diHDHA on the cellular processes of angiogenesis. In simulated diabetic hyperglycemia (25 mM glucose in medium) (45,46), 14S,21R-diHDHA promoted the cellular processes of angiogenesis through significantly enhancing db/db DMVEC migration 45.5% (Fig. 3A) and vasculature formation 40.8% (Fig. 3B). Moreover, diabetic db/db DMVECs treated with 14S,21R-diHDHA produced more vascular endothelial growth factor (VEGF), a potent angiogenic factor (47,48), than control-treated cells (Fig. 3C). We also compared migration and vasculature formation between db/db DMVECs and db/ϩ DMVECs and found db/ϩ DMVECs could form a longer tube than db/db DMVECs without any treatment, but no difference was observed for migration. In addition, 14S,21R-diHDHA could also promote db/ϩ DMVEC migration and vasculature formation (supplemental Fig. S6). FEBRUARY (2). To determine whether 14S,21R-diHDHA and db/db MSCs together could enhance wound healing in db/db mice better than either one alone, we administered 14S,21R-diHDHA (50 ng/wound) along with db/db MSC transplantation (10 6 cells/wound). This treatment resulted in a decrease in the relative epithelial gap of 75.2% (Fig.  4, A and B) and an increase in granulation tissue area of 107.8% (Fig. 4, A and C) at day 8 post-wounding. In particular, 14S,21R-diHDHA combined with db/db MSCs was more effective than db/db MSCs alone (Fig. 4, A-C) or 14S,21R-diHDHA alone (Fig. 2, A and B) in accelerating wound healing. In addition, administration of 14S,21R-diHDHA with db/db MSCs was as effective as db/ϩ MSCs alone (Fig. 4, A-C). Treatment with db/db and db/ϩ MSCs promoted re-epithelialization with decreased percentages of the relative epithelial gap of 25.6 and 61.7%, respectively, as well as increased percentages of the total granulation area by 52.0 and 82.4%, respectively, compared with control (Fig. 4,  A-C). Indeed, db/db diabetic MSCs are less efficient than db/ϩ nondiabetic MSCs in promoting wound healing, indicating impaired wound healing due to diabetes. Therefore, 14S,21R-diHDHA coacts with db/db MSCs to promote diabetic wound healing and compensates for the diabetesimpaired pro-healing functions of db/db MSCs. In addition, we also confirmed that 14S,21R-diHDHA coacts with db/ϩ MSCs to promote wound healing on db/ϩ mice (supplemental Fig. S4).

14S,21R-Dihydroxy-DHA for Diabetic Wound Healing
14S,21R-diHDHA Remedies Diabetes-impaired Functions of db/db MSCs and Acts Synergistically with db/db MSCs in Promoting Angiogenesis-We further investigated whether 14S,21R-diHDHA would improve the function of db/db MSCs in promoting diabetes-impaired angiogenesis. The angiogenic functions of db/db MSCs were impaired compared with db/ϩ MSCs even though db/db MSCs increased wound vascularity 78.5% (versus 130.1% for db/ϩ MSCs) relative to
MSCs secrete angiogenic cytokines such as VEGF to promote angiogenesis (22). As such, we hypothesized that 14S,21R-diHDHA promoted angiogenic functions of db/db MSCs partly as a result of increased angiogenic cytokine production by the cells. To test this, VEGF levels were quantified in media from db/db MSCs, 14S,21R-diHDHA-treated db/db MSCs, and db/ϩ MSCs. The data demonstrate that greater amounts of VEGF were released by db/ϩ MSCs than that by db/db MSCs (Fig. 5G), consistent with impaired angiogenesis in db/db MSCs. This impairment was restored by 14S,21R-diHDHA treatment, which induced db/db MSCs to secrete significantly more VEGF under simulated hyperglycemia (Fig.  5G). Taken together, these results suggest that 14S,21R-diHDHA can rescue the functions of db/db MSCs or synergize with db/db MSCs to promote angiogenesis by rescuing db/db MSC paracrine angiogenic functions.
14S,21R-diHDHA Activates the p38 but Not ERK1/2 Signaling Pathway-Activation of MAPK pathways is essential in wound healing and associated angiogenesis (28,48,49). Therefore, we sought to determine whether 14S,21R-diHDHA treatment led to activation of the MAPKs. Our results showed that 14S,21R-diHDHA treatment triggered phosphorylation of p38 in db/db DMVECs and MSCs, but had no effect on phosphorylation of ERK1/2. The ratio of phosphorylated to nonphosphorylated p38 in db/db DMVECs treated with 14S,21R-diHDHA remained higher than that of the control for at least 120 min (Fig. 6, A and B). In addition, 14S,21R-diHDHA treatment increased the levels of phosphorylated p38 in wounds of db/ϩ and db/db mice, and there was no significantly different levels of phosphorylated p38 in wounds of db/ϩ and db/db mice (Fig. 6, C and D).
It has been reported that the knock-out of 12/15(or L-12)LOX gene from mice reduced DHA 14-hydroxylation by peritoneal macrophages by Ͼ95% (5). There was no report before to study of the effect of 12/15-LOX knock-out on DHA FIGURE 6. 14S,21R-diHDHA activated p38 MAPK but not ERK1/2 signaling pathway. Quiescent subconfluent db/db DMVECs and MSCs were treated with 100 nM 14S,21R-diHDHA for 10 -120 min, and wounds of db/ϩ and db/db mice were treated with 14S,21R-diHDHA (50 ng/wound) for 15 min. Whole tissue and cell lysates were analyzed by Western blot with antibodies specific to phospho-p38 (P-p38) and phospho-ERK1/2 (P-ERK1/2) as well as total p38 and ERK1. Densitometric ratios of P-p38 to p38 and P-ERK1/2 to ERK1 are presented as a percentage of control (i.e. without 14S,21R-diHDHA treatment). Representative Western blot images of P-p38, p38, P-ERK1/2, and ERK1 as well as densitometric ratios of P-p38 to p38 and P-ERK1/2 to ERK1 in db/db DMVECs and MSCs (A and B), and in wounds of db/ϩ and db/db mice (C and D) are shown. Results are mean Ϯ S.E. (n ϭ 3). *, p Ͻ 0.05 compared with control.
Moreover, treating wounds with 14S,21R-diHDHA improved healing as well as vascularization or angiogenesis in diabetic mice (Fig. 2). Treatment with 14S,21R-diHDHA also rescued the key cellular processes of healing-associated angiogenesis: DMVEC transmigration and vasculature formation (Fig. 3). Angiogenesis is critical for growing new vessels that are essential for optimal healing (1). Endothelial cells in the vascular network adjacent to wounds migrate, proliferate, and undergo vascularization and neovessel formation within the granulation tissue (62). Diabetes impairs angiogenesis and causes microcirculatory deficiencies in skin wounds (1,2). Thus, new approaches toward ameliorating impaired angiogenesis would significantly contribute to efforts to develop better treatments for diabetic wounds (2,63). 14S,21R-diH-DHA represents a new lead for this approach. The promotion of re-epithelialization and granulation tissue formation by 14S,21R-diHDHA in wounds suggests that this lipid mediator may also enhance other cellular processes of healing that involve epithelial cells and fibroblast cells (Scheme 1) (64).
When 14S,21R-diHDHA was combined with db/db MSCs and used to treat the wounds of diabetic db/db mice, the combination promoted the crucial processes of wound healing: re-epithelialization and granulation tissue growth (Figs. 2 and 4) (34,65). Furthermore, 14S,21R-diHDHA was observed to restore diabetes-impaired angiogenic functions of db/db MSCs that promote wound vascularization, as well as db/db DMVEC migration and vasculature formation (Figs. 3 and 5). 14S,21R-diHDHA stimulated db/db DMVECs (Fig. 3C) and MSCs to increase VEGF secretion (Fig. 5G), which is at least partly responsible for accelerated angiogenesis in wound healing (Scheme 1). VEGF is one of the most important angiogenic cytokines (48) and promotes wound healing through enhancing angiogenesis (63). By producing VEGF, DMVECs promote their own angiogenesis processes, and MSCs promote vascularization achieved by endothelial cells and other type cells in wounds; thus DMVECs and MSCs manifest their autocrine and/or paracrine functions of promoting angiogenesis in wound healing (Scheme 1). The promotion of VEGF production represents a molecular mechanism for the proangiogenic functions of 14S,21R-diHDHA in wound healing 3 H. Tian, Y. Lu, S. P. Shah, and S. Hong, unpublished data. SCHEME 1. Diabetes reduces formation of 14S,21R-diHDHA in cutaneous wounds and application of exogenous 14S,21R-diHDHA counteracts the diabetic impairment on healing, angiogenesis, and associated functions of mesenchymal stem cells. Treatment with 14S,21R-diHDHA rescues healing, angiogenesis, and associated MSC functions that are impaired in diabetes. 14S,21R-diHDHA restores the diabetes-impaired cellular processes of angiogenesis and paracrine functions of MSCs, including endothelial cell (EC) migration, vasculature formation, and production of VEGF (autocrine and paracrine). This lipid mediator activates the p38 MAPK pathway in endothelial cells and MSCs. 14S,21R-diHDHA may also act directly to enhance re-epithelialization and granulation tissue formation in wound healing.
and also further indicates that 14S,21R-diHDHA remedies diabetes-impaired pro-angiogenic functions of db/db MSCs.
Treatment of nonhealing wounds in diabetic patients with autologous MSCs is of clinical significance, but its value is restricted by deficient functions of the MSCs due to diabetes. In this regard, being a remedy for diabetes-impaired pro-healing functions of db/db MSCs, 14S,21R-diHDHA is a promising new compound that could clinically restore these impaired MSC functions.
Activation of p38 and/or ERK1/2 is required for normal angiogenesis (27), including endothelial cell migration and vasculature formation (49,66). p38 and ERK1/2 activation also have important roles in VEGF expression in endothelial cells and MSCs (67,68). 14S,21R-diHDHA treatment results in immediate phosphorylation of p38 in wounds, db/db DMVECs, and MSCs, but not of ERK1/2, in db/db DMVECs and MSCs (Fig. 6), which suggests that the p38 signaling pathway is involved in 14S,21R-diHDHA promotion of healing and angiogenesis in diabetic wounds and MSC angiogenic functions. The receptor(s) of 14S,21R-diHDHA and mechanisms for 14S,21R-diHDHA activation of p38 signaling pathways are of interest for our future studies.
As summarized in Scheme 1, our studies reveal that diabetes impedes the formation of 14S,21R-diHDHA in wounds. Administration of exogenous 14S,21R-diHDHA rescues diabetes-impaired healing, angiogenesis, and associated MSC functions. These actions of 14S,21R-diHDHA appear to involve the activation of p38-MAPK signaling in endothelial cells and MSCs. This study identifies the novel lipid mediator, 14S,21R-diHDHA, as an important new lead for developing better therapeutics in the treatment of diabetic wounds.
The supplemental Fig. S1 shows the detailed isolation and identification of DMVECs and MSCs from mice. The supplemental Fig. S2 demonstrated that 14S,21R-diHDHA generated from 14S-HDHA by h-P450 and separated by chiral LC is highly pure, confirmed its structure by LC-MS/MS analysis of its hydrogenated product, and further justified its chiral analysis using the analogous diastereomers. The supplemental Fig.  S3 shows that several products are generated by h-P450 from 14S-HDHA and are separated from 14S,21R-diHDHA by the chiral LC. The supplemental Fig. S4 shows 14S,21R-diHDHA coacts with db/ϩ MSCs to promote wound healings in db/ϩ mice. The supplemental Fig. S5 shows the synergistic effect of 14S,21R-diHDHA and db/ϩ MSCs on angiogenesis in db/ϩ mice. The supplemental Fig. S6 shows 14S,21R-diHDHA improves the cellular angiogenic processes of db/ϩ DMVECs.