Novel 14S,21-dihydroxy-docosahexaenoic acid rescues wound healing and associated angiogenesis impaired by acute ethanol intoxication/exposure

Article abstract of DOI:10.1002/jcb.22709

Acute ethanol intoxication and exposure (AE) has been known to impair wound healing and associated angiogenesis. Here, we found that AE diminished the formation of novel reparative lipid mediator 14S,21-dihydroxy-docosa-4Z,7Z,10Z, 12E,16Z,19Z-hexaenoic acid (14S,21-diHDHA) and its biosynthetic intermediate 14S-hydroxy-DHA (14S-HDHA) from docosahexaenoic acid (DHA) in murine wounds. However, AE did not reduce the formation of DHA and the intermediate 21-HDHA. These results indicate that in the biosynthetic pathways of 14S, 21-diHDHA in wounds, AE suppresses the 14S-hydroxy-generating activity of 12-lipoxygenase-like (LOX-like), but does not suppress the 21-hydroxy-generating activity of cytochrome P450 and DHA-generating activities. The AE-suppression of 12-LOX-like activity was further confirmed by the diminished formation of 12-hydroxy-eicosatetraenoic acid in wounds under AE. Supplementing 14S,21-diHDHA to wounds rescued the AE-impaired healing and vascularization. 14S,21-diHDHA restored AE-impaired processes of angiogenesis in vitro: endothelial cell migration, tubulogenesis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). Taken together, the suppression of 14S,21-diHDHA formation is responsible, at least partially, for the AE-impairment of cutaneous wound healing and angiogenesis. Supplementing 14S,21-diHDHA to compensate its deficit in AE-impaired wounds rescues the healing and angiogenesis. These results provide a novel mechanistic insight for AE-impaired wound healing that involves the necessary roles of 14S,21-diHDHA. They also offer leads for developing 14S,21-diHDHA-related therapeutics to ameliorate AE-impairment of wound healing.

Full text of DOI:10.1002/jcb.22709

Journal of Cellular
Biochemistry
ARTICLE
Journal of Cellular Biochemistry 111:266–273 (2010)
Novel 14S,21-dihydroxy-docosahexaenoic Acid Rescues
Wound Healing and Associated Angiogenesis Impaired by
Acute Ethanol Intoxication/Exposure
*
Haibin Tian, Yan Lu, Shraddha P. Shah, and Song Hong
Louisiana State University, Health Sciences Center, Center of Neuroscience Excellence, New Orleans, Louisiana 70112
ABSTRACT
Acute ethanol intoxication and exposure (AE) has been known to impair wound healing and associated angiogenesis. Here, we found that AE
diminished the formation of novel reparative lipid mediator 14S,21-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21-
diHDHA) and its biosynthetic intermediate 14S-hydroxy-DHA (14S-HDHA) from docosahexaenoic acid (DHA) in murine wounds. However,
AE did not reduce the formation of DHA and the intermediate 21-HDHA. These results indicate that in the biosynthetic pathways of 14S,
21-diHDHA in wounds, AE suppresses the 14S-hydroxy-generating activity of 12-lipoxygenase-like (LOX-like), but does not suppress the
21-hydroxy-generating activity of cytochrome P450 and DHA-generating activities. The AE-suppression of 12-LOX-like activity was further
confirmed by the diminished formation of 12-hydroxy-eicosatetraenoic acid in wounds under AE. Supplementing 14S,21-diHDHA to wounds
rescued the AE-impaired healing and vascularization. 14S,21-diHDHA restored AE-impaired processes of angiogenesis in vitro: endothelial
cell migration, tubulogenesis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). Taken together, the suppression
of 14S,21-diHDHA formation is responsible, at least partially, for the AE-impairment of cutaneous wound healing and angiogenesis.
Supplementing 14S,21-diHDHA to compensate its deficit in AE-impaired wounds rescues the healing and angiogenesis. These results provide
a novel mechanistic insight for AE-impaired wound healing that involves the necessary roles of 14S,21-diHDHA. They also offer
leads for developing 14S,21-diHDHA-related therapeutics to ameliorate AE-impairment of wound healing. J. Cell. Biochem. 111: 266–
273, 2010. ß 2010 Wiley-Liss, Inc.
KEY WORDS: ACUTE ETHANOL INTOXICATION/EXPOSURE; 14S,21-DI-HYDROXY-DOCOSAHEXAENOIC ACID; WOUND HEALING; ANGIOGENESIS;
VEGF; ENDOTHELIAL CELLS; RE-EPITHELIALIZATION; P38 MAPK
cute ethanol intoxication and exposure (AE) has been
reported to significantly impair wound healing and
We have recently revealed the molecular structure of novel
endogenous DHA-derived 14S,21-di-hydroxy-docosa-4Z,7Z,10Z,
12E,16Z,19Z-hexaenoic acid (14S,21-diHDHA) in skin wounds and
found that it is a reparative lipid mediator (LM) that promotes wound
healing [Lu et al., 2010]. It has been known that AE dysregulates the
biosynthesis of LMs [Shukla et al., 2001; Molina, 2008; Sozio and
Crabb, 2008]. However, there are no reports regarding the roles of
LMs in AE-impaired wound healing and angiogenesis, no mention
of 14S,21-diHDHA.
A
associated angiogenesis [Radek et al., 2005, 2008; Bird et al.,
2009]. Many patients hospitalized for traumatic wounds have AE
associated with their injuries [Rivara et al., 1993; Sommers, 1994;
Radek et al., 2008]. Angiogenesis or formation of new vessels is
paramount for supplying blood for optimal healing [Li et al., 2003;
Velazquez, 2007], and this is achieved through the processes
including endothelial cell (EC) migration and vascularization.
Vascular endothelial growth factor (VEGF) activities in wounds
regulate these processes [Brown et al., 1992; Shweiki et al., 1992;
Dvorak et al., 1995; Nissen et al., 1998; Ferrara, 2004] and AE
impairs VEGF-modulated vascularization [Radek et al., 2005, 2008].
Docosahexaenoic acid (DHA) is an endogenous v3 essential fatty
acid relatively abundant in skin (in full thickness) [Lu et al., 2010].
Here, we found that in skin wounds of mice, AE suppressed the
formation of 14S,21-diHDHA and its biosynthetic intermediate 14S-
hydroxy-DHA (14S-HDHA), which indicates that AE suppresses
the 14S-hydroxy-generating activity of 12-lipoxygenase-like
(12-LOX-like) for the biosynthesis of 14S,21-diHDHA in wounds.
However, AE did not reduce the formation of 21-HDHA (another
Additional Supporting Information may be found in the online version of this article.
NIH fund 1R01DK087800 (to S.H.); startup fund (to S.H.) from Center of Neuroscience Excellence, LSUHSC-New
Orleans; and Bridge and Supplemental Grant 2009 (To S.H.) from LSUHSC-New Orleans.
*Correspondence to: Song Hong, LSUHSC—Center of Neuroscience Excellence, Lions Building, 2020 Gravier St., Suite
D, New Orleans, LA 70112. E-mail: shong@lsuhsc.edu
Received 1 April 2010; Accepted 11 May 2010 ꢀ DOI 10.1002/jcb.22709 ꢀ ß 2010 Wiley-Liss, Inc.
Published online 19 May 2010 in Wiley Online Library (wileyonlinelibrary.com).
266

intermediate) and DHA (the precursor), suggesting that AE does not
suppress the 21-hydroxy-generating activity of cytochrome P450
and DHA-generating activity of lipases for the biosynthesis.
Furthermore, applying 14S,21-diHDHA to wounds of mice rescued
AE-impaired healing and associated proliferative-phase angiogen-
esis. We also found that 14S,21-diHDHA counteracts AE-impair-
ment of EC migration and vasculature formation. These results
provide novel leads for developing therapeutics useful for restoring
AE-impaired wound healing.
SAMPLE PREPARATION AND ANALYSIS OF LIPID MEDIATORS
Prostaglandin F2a-d4 (PGF2a-d4), DHA, and Leukocyte-12-LOX
(L-12-LOX) were purchased from Cayman (Ann Arbor, MI).
Wounded-skin was collected from mice immediately following
sacrifice at day 1 postwounding. Tissues were submerged in acetone
containing PGF2a-d4 as an internal standard (20 ng/sample; 48C).
The samples were extracted with acetone and extracts were purified
via C18-solid-phase extraction, and then analyzed via LC–MS/MS
(LTQ; Thermo, Waltham, MA) equipped with a chiral column (AD-
RH, 150 mm ꢂ 2.1 mm ꢂ 5 mm; Chiral Tech., West Chester, PA)
[Schneider et al., 2007]. The mobile phase had a flow-rate of 0.15 ml/
min, eluted as D (acetonitrile:H2O:acetic acid ¼ 45:55:0.01) from 0
to 45 min, ramped to acetonitrile from 45.1 to 60 min, flowed as
acetonitrile for 10 min, and then run as D again for 10 min.
Conditions for mass spectrometry were: electrospray, 4.2 kV;
heating capillary, ꢃ45 V and 2908C; tube lens offset, 70 V; and
sheath N2 gas, 1.2 L/min; and helium gas, 0.1 Pa as a collision gas.
The collision energy was between 20% and 35%. A relative
collision-energy of 0% to 100% corresponds to a high frequency
alternating voltage for resonance excitation from 0 to 5 V
maximum. The raw data of LC–MS or MS/MS were acquired in
full-scan modes (m/z 100–500 for MS and MS/MS) and used for
structure identification and quantification of lipids [Lu et al., 2007].
The quantification is based on the peak areas of LC–MS/MS or
LC–MS extracted ion chromatograms of the identified compounds
[Bazan et al., 2008].
MATERIALS AND METHODS
SPLINTED EXCISIONAL-WOUND HEALING MODEL
Published procedures were followed [Galiano et al., 2004a, b; Radek
et al., 2005] with minor modifications. Briefly, PBS (sterile) with or
without ethanol (20%) were injected into each mouse (150 ml, i.p.,
C57BL/6J, female, 8–10 weeks old, 17–21 g; Jackson Laboratory,
Bar Harbor, ME). After 30 min, the ethanol-treated mice had a blood
ethanol of ꢁ100 mg/dl, just above the legal limit in most states
[Radek et al., 2005]. After 24 h the blood ethanol was undetectable
[Radek et al., 2005]. Under sterile conditions, paired 4-mm circular,
full-thickness wounds were made symmetrically along the midline
on the dorsal skin of the anesthetized mice with and without AE. For
each wound, PBS or PBS with 14S,21-diHDHA (50 ng) was applied
to the wound-bed (10 ml) and injected intradermally to four points
(10 ml/site) evenly distributed near the wound edge (50 ml total). A
donut-shaped 0.5 mm-thick silicone splint (Grace Bio-Laboratories,
Bend, OR) was adhered to the skin and the wounds were centered
within the splint [Galiano et al., 2004a,b]. The splints prevent murine
skin contraction to allow wounds to heal through granulation and
re-epithelialization, which closely parallels human wound healing
[Galiano et al., 2004a,b].
14S,21-diHDHA preparation. LM 14S,21-diHDHA was prepared
by incubating 14S-hydroxy-DHA (14S-HDHA; 50 mg) with human
P450 (BioCatalytics, Pasadena, CA). 14S-HDHA was prepared by
incubating DHA (50 mg) with porcine L-12-LOX (1,000 unit;
Cayman) in Tris buffer (pH 7.4, with 0.01% X-114 Triton, 378C,
20 min). The incubations were extracted with two volumes of
ethyl acetate three times. The extracts were reconstituted into
methanol. 14S,21-diHDHA or 14S-HDHA in final solutions was
separated and quantified by the LC–MS/MS as above [Hong et al.,
2003, 2007].
Histological analysis of wound healing. The established proce-
dures were followed [Galiano et al., 2004a,b; Radek et al., 2005; Wu
et al., 2007]. Briefly, wounds with 2 mm normal skin rims were
excised from mice sacrificed at days 5 and 7 postwounding [Galiano
et al., 2004a,b], fixed in 4% paraformaldehyde, and then embedded
in OCT (Tissue-Tec, Torrance, CA). Cryosections (10 mm thick/
section) of wounds at day 5 postwounding were analyzed to
determine the relative epithelial gap (% of initial wounds) and
granulation tissue area after hematoxylin and eosin (H & E) staining.
Wound vascularity (vessel density) at day 7 postwounding was
assessed [Radek et al., 2005]. Cryosections were pretreated with 0.3%
hydrogen peroxide for 15 min at room temperature to block
endogenous peroxidase activity, and then blocked in PBS contain-
ing 1% fetal bovine serum for 1 h. Cryosections were stained with
primary antibody, rat anti-mouse CD31 (BD Biosciences, San Jose,
CA), and then with anti-rat Ig horseradish peroxidase (HRP)
detection kits (BD Biosciences). Nuclei were stained with hematox-
ylin. The images were recorded by Nikon TS-100 microscope with a
CCD color camera (Nikon, Tokyo, Japan). Three fields in wound-bed
were analyzed for each cryosection. The CD31-positive area per field
within the wound bed was assessed by NIH Image J1.40 software and
averaged for each wound, and the percent vascularity was calculated
as (CD31-positive area per field/total wound bed area per
field) ꢂ 100%.
PROCESSES OF ANGIOGENESIS: MIGRATION AND VASCULATURE
FORMATION
Isolation and identification of murine microvascular endothelial
cells (mMVECs). This was done as described previously [Cha et al.,
2005] and as detailed in Supplemental materials. The final mMVECs
were 95% CD31þ and VE-cadherinþ.
mMVEC migration. Cell migration was performed as described
previously [Bajpai et al., 2007]. Cell culture inserts containing
membranes with 8 mm pore size were placed in a 24-well tissue
culture plate (BD Biosciences). The underside of the membrane was
coated with 0.5% gelatin at 48C overnight and then blocked with
0.1% BSA at 378C for 1 h. Quiescenced mMVECs were prepared by
starvation in DMEM containing 0.5% FBS with or without AE
(100 mg/dl ethanol) for 24 h, then suspended in 100 ml DMEM in the
upper chamber of a 24-transwell plate (1 ꢂ 105 cells/well). The lower
chamber was added with 800 ml DMEM containing AE, 14S,21-
diHDHA, murine VEGF isoform 164 (mVEGF164; Invitrogen),
mVEGF164 þ AE, or mVEGF164 þ AE þ 14S,21-diHDHA, where AE
was 100 mg/dl, 14S,21-diHDHA was 100 nM, and mVEGF164 was
JOURNAL OF CELLULAR BIOCHEMISTRY
14S,21-diHDHA, ACUTE ETHANOL-IMPAIRED HEALING 267

50 ng/ml. DMEM alone was used as non-AE control. Transmigration
was carried out for 4 h (378C, 5% CO2). Migrated cells on the
underside of the membrane were stained with Giemsa, and then
counted in five randomly selected fields/well using a microscope.
Results were expressed as migrated cells/field.
where AE was 100 mg/dl, 14S,21-diHDHA was 100 nM, and
mVEGF164 was 50 ng/ml. DMEM alone was used as the non-AE
control. Cells were lyzed for the analysis of expression of P-p38
(phosphorylated form of p38) and p38 (non-phosphorylated form).
Total protein (30 mg) of each sample was separated by electrophor-
esis on a 10% polyacrylamide gel, transferred onto a polyvinylidene
fluoride membrane (Millipore, Billerica, MA), and then stained with
specific antibodies: P-p38 as well as p38 (BD biosciences). The
images was analyzed using Odyssey Imaging System (LI-COR,
Lincoln, NE) [Tian et al., 2009].
mMVEC tubulogenesis. The in vitro tubulogenesis parallels the
in vivo vasculature formation. Published procedures were followed
with minor modifications [Radek et al., 2005, 2008]. Briefly,
quiescenced mMVECs (2 ꢂ 104) adhered to prepared Matrigel in a
96-well culture plate were cultured (378C, 5% CO2, 6 h) in DMEM
containing AE, 14S,21-diHDHA, mVEGF164, mVEGF164 þ AE,
or mVEGF164 þ AE þ 14S,21-diHDHA, where AE was 100 mg/
dl, 14S,21-diHDHA was 100 nM, and mVEGF164 was 50 ng/ml.
DMEM alone was used as the non-AE control. In another
experiment, mMVECs on prepared Matrigel were cultured in DMEM
containing 0–1,000 nM 14S,21-diHDHA under AE (100 mg/dl) and
non-AE. The tubular length was measured using a microscope and
NIH Image J1.40 software. Tubular length of each group was
normalized to the value of non-AE control.
RESULTS
THE FORMATION OF 14S,21-diHDHA WAS REDUCED IN WOUNDS
UNDER AE
14S,21-diHDHA was identified in wounds of non-AE control C57BL/
6J mice (Fig. 1A), and its formation was diminished in wounds of
mice under AE (Fig. 1A left inset, right panel) based on the LC–MS/
MS analysis. 14S,21-diHDHA structure (Fig. 1A right inset) is
revealed by its LC–MS/MS ions at mass to charge ratios (m/z) 359
[MꢃH]ꢃ, 323 [M-H-2H2O]ꢃ, 315, 279 [M-H-CO2-2H2O]ꢃ, 271 [315-
CO2]ꢃ, 253 [315-CO2-H2O]ꢃ, 233, 205, 161 [205-CO2]ꢃ, and 215
[233-H2O]ꢃ (Fig. 1A); as well as its LC retention time (RT); both
matched those of 14S,21-diHDHA generated from DHA by
Western blot. This was performed according to published speci-
fications [Siebert et al., 2001]. Briefly, quiescent mMVECs
were incubated in DMEM containing AE, 14S,21-diHDHA,
mVEGF164, 14S,21-diHDHA þ AE, mVEGF164 þ AE, mVEGF164 þ
14S,21-diHDHA, or mVEGF164 þ AE þ 14S,21-diHDHA for 30 min,
Fig. 1. AE reduced the formation of novel 14S,21-diHDHA and its biosynthetic intermediate 14S-HDHA in skin wounds. Splinted-excisional-wounds were made to C57BL/6J
mice that underwent AE or non-AE control (PBS alone). Wounded skin was collected from mice at day 1 postwounding (n ¼ 6), then analyzed as detailed in Materials and
Methods Section. A: The LC–MS/MS spectrum (left), chromatogram (left inset), structure elucidation (middle inset), and quantification (right) of 14S,21-diHDHA from wounds.
B: LC–MS/MS chromatograms of 14S-HDHA (left), 21-HDHA (middle), and DHA (right) from wounds. For comparison, the ion abundance of each MS/MS chromatogram was
divided by the extraction recovery of internal standard spiked to the sample, and then normalized for per gram of skin. Results are expressed as mean ꢄ SEM. ꢅP < 0.05 for the
comparison between AE and non-AE control.
268 14S,21-diHDHA, ACUTE ETHANOL-IMPAIRED HEALING
JOURNAL OF CELLULAR BIOCHEMISTRY

sequential catalysis of L-12-LOX and P450 [Lu et al., 2010]. The LC
peak area of 14S,21-diHDHA under AE is smaller than that under
non-AE (Fig. 1A left inset). Additionally, from wounds, we
found 14S-HDHA, 21-HDHA, and DHA, the procusors for
the 14S,21-diHDHA biosynthesis [Lu et al., 2010], whose LC–MS/
MS spectra and RTs matched those published before (Fig. 1B) [Lu
et al., 2010]. The LC peak area of 14S-HDHA in wounds with AE was
much less than that without AE (Fig. 1B left), while the peak areas for
21-HDHA (Fig. 1B middle) and DHA (Fig. 1B right) were near the
same, which indicates that AE reduced the formation of 14S-HDHA
in wounds, but did not affect the formation of 21-HDHA and DHA.
ethanol) (74.8 ꢄ 2.5 cells/field for AE treatment vs 73.6 ꢄ 3.9 cells/
field for non-AE control, Fig. 3A,B). 14S,21-diHDHA significantly
promoted mMVEC migration (105.8 ꢄ 5.6 cells/field) when
mVEGF164 and AE were absent. AE (100 mg ethanol/dl) signifi-
cantly inhibited mVEGF164-induced mMVEC migration (95.4 ꢄ 5.5
for [AE þ mVEGF164] vs 152.4 ꢄ 4.7 cells/field for mVEGF164
alone). 14S,21-diHDHA significantly increased the AE-impaired
VEGF-modulated mMVEC migration (140.5 ꢄ 5.0 cells/field;
Fig. 3A,B).
The tubulogenesis for AE alone is not significantly different from
that in non-AE control. However, under mVEGF164 stimulation, AE
significantly decreased the tubular length compared to mVEGF164
alone, which is consistent with previous report [Radek et al., 2005,
2008]. This impairment was rescued by 14S,21-diHDHA treatment
that increased the AE-impaired and VEGF-regulated tubulogenesis
of mMVECs. It is observed that 14S,21-diHDHA alone also promoted
tubulogenesis without the addition of VEGF (Fig. 3C,D). In
addition, 14S,21-diHDHA enhanced the effect of mVEGF164 on
EC migration and tubulogenesis (Fig. 3A–D). Higher concentration
of 14S,21-diHDHA resulted in larger mMVEC tubular lengths, that
is, more tubulogenesis, except that the difference on tubulogenesis is
small for 100 and 1,000 nM or for 0 and 1 nM (Fig. 3E). AE reduced
the enhancement by14S,21-diHDHA (10, 100, or 1,000 nM) on
mMVEC tubulogenesis. However, AE did not affect the response to 0
or 1 nM 14S,21-diHDHA (Fig. 3E). Taken together 14S,21-diHDHA is
able to rescue the AE-impaired cellular processes of angiogenesis by
significantly increasing mMVEC tubulogenesis and migration.
14S,21-diHDHA RESCUED AE-IMPAIRED WOUND HEALING
Because AE impairs wound healing [Radek et al., 2005] and 14S,21-
diHDHA formation (Fig. 1), we tested whether applying 14S,21-
diHDHA to wounds could reduce AE-impairment of the healing.
Compared to wounds of non-AE control, wounds under AE had
larger relative epithelial gap (77.6 ꢄ 4.0% for AE control vs
53.9 ꢄ 3.5% for non-AE control) and less granulation tissue
formation (4493.3 ꢄ 520.6 for AE control vs 8599.0 ꢄ 552.9
pixels/section for non-AE control) at day 5 postwounding based
on the analysis of cryosections with H & E staining (Fig. 2A–C).
However, 14S,21-diHDHA treatment of wounds under AE sig-
nificantly promoted would healing compared to AE control: it
accelerated re-epithelialization for having smaller relative epithelial
gap (59.0 ꢄ 2.9%; Fig. 2A,B), and increased granulation tissue area
(7272.5 ꢄ 373.2 pixels/section; Fig. 2A,C). In addition, 14S,21-
diHDHA treatment of wounds without AE had smaller epithelial gap
and more granulation tissue compared to non-AE control (Fig. 2A–
C). The relative epithelial gap and granulation tissue area of 14S,21-
diHDHA-treated wounds under AE were not significantly different
with those of non-AE control, which indicates that 14S,21-diHDHA
rescues AE-impaired wound healing (Fig. 2A–C).
14S,21-diHDHA RECOVERED AE-IMPAIRED P38
PHOSPHORYLATION
Activation of MAPK pathways is essential in wound healing and
associated angiogenesis [Sharma et al., 2003; Bajpai et al., 2007; Gee
et al., 2010]. Since AE impairs VEGF-induced EC migration
and vasculature formation as well as 14S,21-diHDHA rescues
AE-impaired processes of angiogenesis (Fig. 3), we postulated
that 14S,21-diHDHA could recover AE-impaired phosphorylation
signalings. 14S,21-diHDHA directly triggered p38 phosphorylation
under AE and non-AE (Fig. 3F,G). Although AE alone had no effect
on p38 phosphorylation in mMVECs compared to non-AE control, it
significantly decreased the levels of the phosphorylated-p38
induced by mVEGF164 or 14S,21-diHDHA (Fig. 3F,G). 14S,21-
diHDHA recovered AE-impaired p38 phosphorylation induced by
mVEGF164 in mMVECs. However, AE did not significantly affect
p38 phosphorylation in mMVECs modulated by mVEGF164
and 14S,21-diHDHA together (see last two bars in Fig. 3G), which
further indicates that 14S,21-diHDHA meliorate the AE impairment
of VEGF-modulated p38 phosphorylation (see last four bars in
Fig. 3G). Furthermore, our studies indicate that 14S,21-diHDHA did
not affect the phosphorylation of VEGF receptor 2 (data not shown).
14S,21-diHDHA RECOVERED VASCULARIZATION/ANGIOGENESIS IN
AE-IMPAIRED WOUNDS
Considering that angiogenesis in wound healing is critical [Li et al.,
2003; Velazquez, 2007] and impeded by AE [Radek et al., 2005,
2008], we studied whether 14S,21-diHDHA recovers AE-impaired
angiogenesis during wound healing. 14S,21-diHDHA under non-AE
significantly promoted angiogenesis by increased wound vascular-
ity (15.2 ꢄ 0.7%) compared to non-AE control (11.0 ꢄ 0.9%;
Fig. 2D,E). Wounds under AE exhibited decreased wound vascularity
(5.1 ꢄ 0.5%) compared to non-AE control, which is consistent with a
previous report [Radek et al., 2005]. However, when 14S,21-diHDHA
was injected into wounds under AE, the wound vascularity
(12.5 ꢄ 0.9%) was significantly increased compared to that of
wounds from AE treatment. Therefore, 14S,21-diHDHA recovered
the angiogenesis in wounds.
14S,21-diHDHA RESCUED AE-IMPAIRED PROCESSES OF
ANGIOGENESIS
DISCUSSION
We questioned whether 14S,21-diHDHA could modulate the
processes of angiogenesis, EC migration, and vasculature formation.
Without mVEGF164 stimulation, AE treatment had no effect on
mMVEC migration compared to non-AE control (DMEM without
AE impeded the formation of 14S,21-diHDHA and 14S-HDHA in
wounds (Fig. 1), which correlated with AE-impaired healing and
angiogenesis (Figs. 2 and 3). 14S-HDHA is generated from DHA by
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14S,21-diHDHA, ACUTE ETHANOL-IMPAIRED HEALING 269

Fig. 2. Supplementing 14S,21-diHDHA rescued AE-impaired wound healing and associated vascularization or angiogenesis. The experiments of AE and wounding were
conducted as those in Figure 1. A: Micrographs and quantitative analysis of H & E stained cryosections of wounds, collected at day 5 postwounding, show that
supplementing 14S,21-diHDHA rescued AE-impaired B: re-epithelialization and C: granulation tissue formation by reducing epithelial gap and increasing the total
granulation tissue area (n ¼ 6, G ¼ granulation tissue, Double arrow ¼ epithelial gap); D: Microphotographs and (E) the quantitative analysis of wound vascularity (%) as
(CD31-positive area per field/total wound bed area per field) ꢂ 100 at day 7 postwounding show that 14S,21-diHDHA rescued vascularization in wounds by increasing
vascularity (n ¼ 12, original magnification: 100ꢂ, scale bar represents 100 mm). Results were mean ꢄ SEM with ꢅP < 0.05 for the comparisons marked in the graphs.
12-LOX-like in wounds or blood cells including platelets and
macrophages [Kim et al., 1990; Lu et al., 2010], and then
transformed by P450 to 14S,21-diHDHA [Lu et al., 2010]. 21-
HDHA was generated from DHA by P450 activity in tissues and
blood cells [VanRollins et al., 1984; Lu et al., 2010] and subsequently
converted to 14S,21-diHDHA by 12-LOX-like (Fig. 1) [Lu et al.,
2010]. DHA mainly results from the hydrolysis of esterified DHA by
lipases [Bazan, 2007]. The tentative biosynthetic pathways
for 14S,21-diHDHA from DHA in wounds are summarized
(Scheme 1), where AE suppresses the 14S-hydroxy-generating
activity of 12-LOX-like in wounds but does not suppress the 21-
hydroxy-generating activity of cytochrome P450 and DHA-
releasing activity of lipases. Consistent with these findings, in
wounds AE suppressed the formation of 12S-hydroxy eicosate-
traenoic acid, the marker of 12-LOX-like activity [Funk et al., 2002],
but did not decrease the generation of unesterified arachidonic acid
(Supplemental Fig. 1). L-12-LOX was used here for the enzymatic
synthesis of 14S-HDHA and 14S,21-diHDHA. AE is known to
greatly reduces leukocyte infiltration to wounds or inflammation
sites as well as leukocyte activation [Radek et al., 2009], which may
significantly contribute to the reduction of 14S-hydroxy-generating
L-12-LOX-like activity. P-12-LOX also has the14S-hydroxy-
generating activity [Kim et al., 1990]. AE could also affect
the 14S-hydroxy-generating activity of Platelet-12-LOX. Further
270 14S,21-diHDHA, ACUTE ETHANOL-IMPAIRED HEALING
JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 3. 14S,21-diHDHA promoted the AE-impaired processes of wound healing-associated angiogenesis: mMVEC migration, tubulogenesis, and p38 MAPK phosphorylation.
A: Micrographs of migrated mMVECs. B: Number of migrated mMVECs/field (n ¼ 10). C: Representative images of mMVEC tube structures. D: The quantification of mMVEC
tubular lengths (% of non-AE control). E: mMVEC tubular lengths (% of non-AE control) for treatment with different concentrations (0–1,000 nM) of 14S,21-diHDHA under
AE (100 mg/dl) or non-AE (DMEM). F: Western blot images and G: relative amounts of phospho-p38 and p38 MAPK in mMVECs show that 14S,21-diHDHA triggered p38
phosphorylation and counteracted the AE impairment on this signaling activation. For (C) to (G), n ¼ 3. The lower chambers in (A,B), culture-wells for tubulogenesis in (C), or
culture-wells for p38 MAPK phosphorylation in (F,G) was added with DMEM containing AE, 14S,21-diHDHA, 14S,21-diHDHA þ AE, mVEGF164, mVEGF164 þ AE,
mVEGF164 þ AE þ 14S,21-diHDHA, or mVEGF164 þ 14S,21-diHDHA, where AE was 100 mg/dl, 14S,21-diHDHA was 100 nM, and mVEGF164 was 50 ng/ml. DMEM alone
was used as non-AE control. Original magnification: 100ꢂ, scale bar represents 100 mm. Results were mean ꢄ SEM. ꢅP < 0.05 for the marked comparisons.
JOURNAL OF CELLULAR BIOCHEMISTRY
14S,21-diHDHA, ACUTE ETHANOL-IMPAIRED HEALING 271

studies will be required. Nonetheless epidermis-12-LOX and 12R-
LOX are likely to be inefficient in the transformation of DHA [Siebert
et al., 2001].
such EC tubulogenesis in a dose-dependent manner (Fig. 3E).
14S,21-diHDHA recovers AE-impaired phosphorylation of p38
induced by VEGF [Radek et al., 2008] (Fig. 3F,G), which confirms
that it promotes AE-impaired angiogenesis by altering EC response
to angiogenic signals. More signaling transduction mechanisms for
the action of 14S,21-diHDHA are of our interest for future study. Our
new finding that AE inhibits VEGF-regulated mMVEC migration
parallels AE-impairment to wound angiogenesis because EC
migration is a pivotal step of angiogenesis; 14S,21-diHDHA not
only promotes EC migration, more importantly it restores AE-
impaired VEGF-regulated EC migration (Fig. 3A,B). In conclusion
(Scheme 1), AE suppresses the formation of 14S,21-diHDHA in
wounds. Supplementing 14S,21-diHDHA to compensate its deficit
in wounds rescues the AE-impaired healing and vascularization;
14S,21-diHDHA also restores the AE-impaired cellular processes of
angiogenesis and p38 MAPK phosphorylation signaling. Our studies
provide a mechanistic insight to the rescue of healing and
angiogenesis of AE-impaired wounds by the endogenous reparative
LM 14S,21-diHDHA.
Applying 14S,21-diHDHA to AE-impaired wounds to compensate
the 14S,21-diHDHA deficit (Fig 1A) restored the normal re-
epithelialization and granulation tissue formation (Fig. 2), which
defines the role of 14S,21-diHDHA for rescuing wound healing
(Fig. 2). Re-epithelialization is one of the main processes of wound
healing. It provides the basis for subsequent processes, such as
fibroblast proliferation and migration, and granulation tissue
formation [Braiman-Wiksman et al., 2007]. AE-impairment of
healing that we observed in a splinted-wound model is consistent
with those observed using a non-splinted-wound model [Radek
et al., 2005].
We also found that 14S,21-diHDHA restores AE-impaired wound
vascularization or angiogenesis in vivo (Fig. 2). 14S,21-diHDHA by
itself enhances EC tubulogenesis and migration in vitro. More
interestingly it can rescue AE-impaired VEGF-mediated EC
tubulogenesis and migration (Fig. 3, Scheme 1), as well as promote
ACKNOWLEDGMENTS
We thank Mr. Ryan R. Labadens for his expertise in editing and
manuscript preparation. This work is sponsored by NIH fund
1R01DK087800 (to S.H.); startup fund (to S.H.) from Center of
Neuroscience Excellence, LSUHSC-New Orleans; and Bridge and
Supplemental Grant 2009 (To S.H.) from LSUHSC-New Orleans.
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