Apratyramide, a Marine-Derived Peptidic Stimulator of VEGF-A and Other Growth Factors with Potential Application in Wound Healing

Article abstract of DOI:10.1021/acschembio.7b00827

A novel linear depsipeptide enriched with tyrosine-derived moieties, termed apratyramide, was isolated from an apratoxin-producing cyanobacterium. The structure was determined using a combination of NMR spectroscopy, mass spectrometry, and chiral analysis of the acid hydrolyzate and confirmed by total synthesis. Apratyramide up-regulated multiple growth factors at the transcript level in human keratinocyte (HaCaT) cells and induced the secretion of vascular endothelial growth factor A (VEGF-A) from HaCaT cells, suggesting the compound's potential wound-healing properties through growth factor induction. Transcriptome analysis and sequential validation supported the hypothesis and indicated its mode of action (MOA) through the unfolded protein response (UPR) pathway, which is functionally related to wound healing and angiogenesis. The conditioned medium of HaCaT cells treated with apratyramide induced angiogenesis in vitro. An ex vivo rabbit corneal epithelial model was applied to confirm the VEGF-A induction in this wound-healing model.

Full text of DOI:10.1021/acschembio.7b00827

Articles
Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
Apratyramide, a Marine-Derived Peptidic Stimulator of VEGF‑A and
Other Growth Factors with Potential Application in Wound Healing
Weijing Cai,†,‡ Lilibeth A. Salvador-Reyes,†,∥ Wei Zhang,†,⊥ Qi-Yin Chen,†,‡ Susan Matthew,†
Ranjala Ratnayake,†,‡ Soo Jung Seo,∇ Simon Dolles,† Daniel J. Gibson,∇ Valerie J. Paul,#
and Hendrik Luesch*,†,‡
‡
†Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development (CNPD3), and ∇The
Institute for Wound Research at the University of Florida, Department of Obstetrics & Gynecology, University of Florida,
Gainesville, Florida 32610, United States
∥Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City 1100, Philippines
⊥School of Pharmacy, Fudan University, Shanghai 200433, China
#Smithsonian Marine Station, Fort Pierce, Florida 34949, United States
S
* Supporting Information
ABSTRACT: A novel linear depsipeptide enriched with
tyrosine-derived moieties, termed apratyramide, was isolated
from an apratoxin-producing cyanobacterium. The structure
was determined using a combination of NMR spectroscopy,
mass spectrometry, and chiral analysis of the acid hydrolyzate
and confirmed by total synthesis. Apratyramide up-regulated
multiple growth factors at the transcript level in human
keratinocyte (HaCaT) cells and induced the secretion of
vascular endothelial growth factor A (VEGF-A) from HaCaT
cells, suggesting the compound’s potential wound-healing
properties through growth factor induction. Transcriptome
analysis and sequential validation supported the hypothesis
and indicated its mode of action (MOA) through the unfolded
protein response (UPR) pathway, which is functionally related to wound healing and angiogenesis. The conditioned medium of
HaCaT cells treated with apratyramide induced angiogenesis in vitro. An ex vivo rabbit corneal epithelial model was applied to
confirm the VEGF-A induction in this wound-healing model.
market for the treatment of DFUs: recombinant platelet-
ound healing is a complex biological process and
Wconsists of a series of events including inflammation, cell derived growth factor, rhPDGF-BB, Becaplermin.7 There are
also other growth factors under clinical trials, including VEGF,
bFGF, and GM-CSF.7 One of the limitations for topical
administration of exogenous growth factors is low absorption
due to the protein nature of these growth factors.8−10
Therefore, an alternative therapeutic method aimed at
stimulating the production and secretion of endogenous growth
factors from wound tissue by small molecules might be more
promising.
VEGF is one of the most potent angiogenic growth factors
and promotes all steps in the angiogenic cascade.11 In the
VEGF family, VEGF-A is the best studied angiogenic growth
factor, regulating both physiological and disease processes, such
as tumor growth, psoriasis, and wound healing.12−14 VEGF-A is
produced by keratinocytes, fibroblast smooth muscle cells,
platelets, neutrophils, and macrophages during wound healing,
proliferation, tissue granulation, and remodeling of scar tissue,
which involves the coordinated efforts of several cell types, such
as keratinocytes, fibroblasts, endothelial cells, macrophages, and
platelets.1−3 A wide variety of growth factors and cytokines are
involved in each stage of the wound-healing process: platelet-
derived growth factors (PDGFs), vascular endothelial growth
factors (VEGFs), basic fibroblast growth factors (bFGFs), and
granulocyte-macrophage colony stimulating factor (GM-CSF),
and many studies have shed light on the crucial roles of these
growth factors on initiating and facilitating wound healing.2,4
Dysregulation of these growth factors could delay wound
closure and result in chronic wounds (e.g., diabetic foot ulcers
[DFUs], pressure ulcers [PUs], and chronic venous leg ulcers
[VLUs]), which represent a major healthcare burden in the
US.5,6
Despite many efforts that have been spent on the
development of growth factors as therapeutic agents, to date,
this field has been disappointing. There is only one Federal
Drug Administration (FDA) approved growth factor on the
Received: September 23, 2017
Accepted: November 13, 2017
© XXXX American Chemical Society
A
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Figure 1. (A) Structure of apratyramide (1). (B) ESIMS fragmentation pattern of 1.
Scheme 1. (A) Retrosynthetic Analysis of 1. (B) Convergent Synthesis of 1
and keratinocytes are thought to be a major source of VEGF-A
after injury.15−17 VEGF-A stimulates angiogenesis by acting on
endothelial cells in the wound sites.18 It has been found that
VEGF-A gene expression is up-regulated in the skin after
wounding.16 Furthermore, the altered expression pattern of
VEGF mRNA during skin repair in genetically diabetic (db/db)
mice suggested that the impairment in VEGF synthesis and
release at the wound site might contribute to chronic wounds.16
In agreement with these observations, many in vitro and in vivo
studies have shown that administration of VEGF-A topically or
by gene transfer accelerates experimental wound healing
through stimulation of angiogenesis, re-epithelialization,
collagen deposition, and synthesis and maturation of
extracellular matrix.19−22 Therefore, the above information
strongly suggests the therapeutic application of VEGF-A
inducers in the treatment of chronic wounds.
recognized the healing properties of herbs, honey, leaves, oil,
etc.23 So far, some but not all of the active components of these
natural wound healers have been identified and they fall into
several structural classes: vitamins,24−26 terpenes or terpe-
noids,27−30 polyphenols,31−34 and alkaloids.35,36 These com-
pounds enhance wound healing through various mechanisms,
including promoting skin cells proliferation and migration,
angiogenesis, and collagen synthesis, as well as exerting anti-
inflammatory and antiseptic activities.37 In addition to these
traditional natural sources, marine organisms are becoming a
rich source for new drugs. For example, pseudopterosins are a
series of diterpene-pentoseglycosides from gorgonian corals
that enhance wound healing through anti-inflammation.38,39
Marine cyanobacteria produce various secondary metabolites
which belong to peptides, polyketides, or hybrid of peptide-
polyketides.40 Despite the fact that marine cyanobacteria
produce compounds with a broad spectrum of biological
activities, including anticancer, antimicrobial, protease inhib-
The use of natural products for the treatment of wounds and
injuries is as old as civilization. Since ancient times, people have
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Figure 2. Apratyramide (1) is an inducer of VEGF-A and other growth factors. (A) Transcript level of VEGF-A in HCT116 (human colon cancer)
cells, 16 h. (B) Transcript level of VEGF-A in CCD-18Co (human normal colon) cells, 16 h. (C) Antiproliferative effect of 1 on HCT116 and CCD-
18Co cells, 48 h. (D) Transcript level of VEGF-A in HaCaT cells after 4 and 12 h treatment with 30 μM of 1. (E) Transcript level of VEGF-A in
HaCaT cells after 16 h. (F) Level of VEGF-A secretion from HaCaT after 24 h. (G) Antiproliferative activity of 1 on HaCaT cells, 24 h. (H)
Transcript level of PDGFB in HaCaT cells, 16 h. (I) Transcript level of bFGF in HaCaT cells, 16 h. Data are presented as mean + SD, *P < 0.05, **P
< 0.01, ***P < 0.001, compared to control using unpaired t test (n = 3).
20
itory, immunomodulatory, and neuromodulatory properties,
and are considered a valuable source for medicinal therapeutic
use, they have not yet been linked to activities associated with
wound healing, to the best of our knowledge. We report a novel
linear depsipeptide isolated from marine cyanobacteria as a
growth factor inducer with potential wound-healing properties.
This study provides new insights into the role of small peptides
in wound healing and broadens the spectrum of activities of
compounds from marine cyanobacteria.
compound 1 as a minor metabolite (2.0 mg, [α]D −101.9 (c
0.59, MeOH)).
The HRESIMS of 1 in the positive mode showed a molecular
ion peak at m/z 805.4388 [M + H]+, suggesting a molecular
formula of C44H60N4O10 with 17 degrees of unsaturation. The
1H NMR spectrum of 1 (Supporting Information Figure S1)
displayed characteristic peptide signals for several α-protons
(δH 3.4−5.3), an exchangeable proton of amide (δH 8.08),
four N-methyls (δH 2.2−2.8), and two O-methyls (δH 3.5−
3.7). A signal at δC 75.09 in the 13C NMR spectrum
(Supporting Information Figure S2) corresponding to a typical
oxygenated sp3 carbon suggested the presence of a hydroxy acid
in addition to amino acids. Following the interpretation of 1D
and 2D NMR experiments, 1H and 13C NMR signals
(Supporting Information Table S1) were assignable into five
partial structures: three modified tyrosines [N-Me-Tyr, N-Me-
Tyr(1-OMe), and N,N-diMe-Tyr(OMe)], one proteinogenic
amino acid (Val), and one α-hydroxy acid moiety [2-hydroxy-3-
RESULTS AND DISCUSSION
■
Isolation and Structure Determination. Five different
Guamanian collections of freeze-dried apratoxins-producing
Moorea bouillonii were collected from Fingers Reef, Guam.41
Each was individually extracted with CH2Cl2 and MeOH (2:1)
followed by solvent partitioning, silica chromatography, and
reversed-phase HPLC purification to yield optically active
C
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Figure 3. Ingenuity pathway analysis (IPA) for transcriptome profiling of apratyramide (1). (A) Heat map for transcript changes in HaCaT cells
after 3 and 12 h treatment with 30 μM 1. Red indicates up-regulation. Green indicates down-regulation. (B) Top regulator effect network. (C) Top
related molecular network associated with the function of cellular compromise and cellular maintenance. (D) Validation of selected hits from
transcriptome profiling using immunoblot analysis.
methylpentanoic acid (Hmpa)] (Figure 1A). The sequence of
these units was established on the basis of HMBC and NOESY
correlations (Supporting Information Table S1) and was
further verified by ESIMS fragmentation (Figure 1B). Due to
its structural enrichment of tyrosine units as well as its origin
from Apra Harbor, we named this compound apratyramide (1).
The absolute configuration of 1 was determined by
enantioselective HPLC analysis and comparison with authentic
standards.
Total Synthesis of Apratyramide. Owing to the limited
supply of apratyramide from nature, we performed the total
synthesis in order to obtain more material for biological
evaluation. Following a retrosynthetic analysis of apratyramide
(Scheme 1A), a convergent synthesis (Scheme 1B) was
conducted by obtaining two building blocks: an ester (2) and
a tripeptide (3). To construct the ester 2, two commercially
available amino acids were obtained as starting materials. The
α-hydroxy carboxylic acid 4 was prepared from L-isoleucine by a
published protocol.42 The esterification of N-Boc-O-Me-
tyrosine with acid 4 provided ester 5 in 96% yield by
Mitsunobu reaction (Ph3P/DEAD).43 The Boc group of ester 5
was removed using 4 M HCl in ethyl acetate. Then, the desired
N,N-dimethylated amino ester 2 was formed by a reductive
alkylation reaction of the free amine in 5 using a mixture of
aqueous HCHO and NaBH3CN.44 The end acid group of ester
2 was liberated by hydrogenation with Pd/C/H2 in MeOH.
The tripeptide 3 was constructed smoothly by sequential
coupling of N-Boc-N-Me-tyrosine(OBn) with the methyl ester
of N-Me-tyrosine(OBn) and then with N-Boc-valine using the
coupling system EDCI/HOAt.45 The Boc group in 3 was
cleaved by 4 M HCl in ethyl acetate to obtain acetate free
amine, which was then coupled with the free acid in ester 2 to
provide precursor 7 by coupling combination HATU/HOAt in
DMF. Finally, the hydrogenation of 7 by Pd/C/H2 in MeOH
gave final product 1 in 63% yield. The NMR spectra for natural
and synthetic apratyramide (1) were identical (Supporting
Information Figure S7−8). Comparison of the optical activity
and HR-MS of natural and synthetic 1 further confirmed the
structural assignment for the natural product.
Apratyramide Induces Transcription of VEGF-A in
HCT116 Cells. In our search for modulators of VEGF-A and
angiogenesis from marine cyanobacteria, we previously
identified apratoxins from the same cyanobacterium as potent
inhibitors by preventing cotranslational translocation of VEGF-
A and other secreted proteins.46,47 Apratyramide (1), however,
had the opposite effect. Using human colon cancer HCT116
cells, we observed that 1 up-regulated VEGF-A, while displaying
minimal cytotoxicity (Figures 2A,C). Fifty micromolar
apratyramide doubled the VEGF-A transcript level in
HCT116 cells (Figure 2A). Apratyramide (1) also exerted a
D
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a
Table 1. Selected Groups of Top Up- and Down-Regulated Genes after 12 h Treatment with 30 μM Apratyramide (1)
Down-regulated genes, FDR
correction, 12 h
Up-regulated genes, FDR correction, 12 h
Growth factor
Cytokine
AREG, VEGFA, HBEGF, EREG
IL1A, IL1B, MYDGF
SCGB1A1, CXCL1, CCL2,
TNFSF10
Transcription
regulator
DDIT3, CREB3L2, XBP1, ETV5, ATF4, JUND, ZNF165, ETV4, ATF3, MYC, NFE2L1, KLF6, SQSTM1, DAP, GMNN, UHRF1, GTF2I,
PREB, NFKB2, FOXE1, MEF2D, MAGED1, IRF2BP2, BHLHE40
VGLL1, DLX5, E2F8,
SMAD6, ID3, GRHL3
G-protein coupled GPR1
receptor
P2RY2
MicroRNA
MIR-3143, MIR-554,
MIR-548
a
The P values obtained were controlled for multiple testing (false discovery rate) using the Benjamini−Hochberg method. Differentially expressed
genes were then ranked by the P values; genes with P < 0.05 (with FDR correction) and fold change >1.5 or <0.67 were considered as differentially
expressed genes at a statistically significant level, which were grouped based on functions. Selected groups are presented above. See Supporting
Information Table S2 for a full list of top up- and down-regulated genes.
similar effect in the corresponding normal colon cells (CCD-
18Co) with negligible effects on cell viability (Figures 2B,C).
Therefore, we aimed to further explore apratyramide’s
therapeutic applications where VEGF-A upregulation might be
beneficial.
Analysis (IPA) (Figures 3B,C). In accordance with our
hypothesis, the global changes of transcript levels are associated
with increased downstream phenotypic effects, including
angiogenesis, mitogenesis, differentiation of epithelial tissue,
and formation of skin, and decreased effects, such as apoptosis
of liver cells and hypoplasia of organs (Figure 3B). IPA analysis
of 371 microarray hits indicated the unfolded protein response
(UPR) (Supporting Information Figure S21) as the top
canonical pathway with a p-value of 1.45 × 10−16. The IPA
also elucidated that the 371 hits were most closely related to a
molecular network associated with the function of cellular
compromise and cellular maintenance (Figure 3C). The
network contains molecular components from the UPR
pathway (ATF4, INSIG1, CHOP, DNAJC3, PP1R15A, JINK1/
2), NRF2-mediated oxidative stress response signaling (ATF4,
DNAJC3, JINK1/2, Akt, HERPUD1, DNAJB9), as well as
glucocorticoid receptor signaling (ADRB, Akt, JINK1/2,
PEPCK, PCK2).
Cytoprotective Roles of UPR and Its Modulatory
Effects on Growth Factors. The unfolded protein response
(UPR) pathway is a cytoprotective signaling cascade in
response to endoplasmic reticulum (ER) stress in cells. UPR
coordinates multiple signaling pathways and controls various
physiologies in cells and the whole organism including liver
development, plasma cell differentiation,48 bone develop-
ment,49,50 plasma cell differentiation,51,52 normal pancreatic
homeostasis,53 and placental development and embryonic
viability.54 Importantly, UPR is activated after skin injury,
suggesting the protective roles of UPR in rescuing wound
injuries.55 Therefore, intervening in ER stress and modulating
signaling components of UPR would provide promising
therapeutics for the treatment of chronic wounds.56
Apratyramide Induces Transcription, Secretion of
VEGF-A, and Transcription of Other Wound-Healing
Related Growth Factors in HaCaT Cells. Since VEGF-A
inducers are considered promising therapeutic agents for the
treatment of chronic wounds, we proposed that 1 may also
induce VEGF-A in normal cell types that are involved in the
wound-healing process. Thus, we logically moved on to a
commonly used in vitro wound-healing model: human
keratinocyte cells (HaCaT). As expected, the VEGF-A mRNA
level was induced 1.7-fold after 4 h treatment with 30 μM of 1
and a greater induction effect was observed after 12 h (Figure
2D). After 16 h, 50 μM of 1 increased VEGF-A transcript level
by 7-fold, while 31.6 μM of 1 led to a 5-fold increase (Figure
2E). Accordingly, 50 μM of 1 induced a 1.5-fold increase of
VEGF-A secretion from HaCaT cells after 24 h, and 31.6 μM of
1 induced a 1.3-fold increase without causing cytotoxicity
(Figures 2F,G). Around ninety-percent cell viability was
observed at 50 μM of 1 after 24 h (Figure 2G).
Multiple growth factors and cytokines are involved in a
complex integration of signals for regulating wound-healing
processes.7 This information prompted us to test whether 1
also induces other growth factors which might work
cooperatively with VEGF-A during wound healing. RT-qPCR
data indicated that PDGFB and bFGF were all stimulated by 1
(Figures 2H,I). Importantly, rhPDGF-BB is the only FDA-
approved growth factor on the market for the treatment of
DFUs.7
Transcriptome Profiling and Ingenuity Pathway
Analysis Indicate That UPR Plays a Role in Mechanisms
of Apratyramide-Induced VEGF-A. To further elucidate the
mode of action of apratyramide through which multiple growth
factors are induced, we performed microarray profiling using
the Affymetrix GeneChip Human Transcriptome Array 2.0 and
determined global changes in transcript levels in HaCaT cells
treated with apratyramide. Comparative analysis identified 371
differentially expressed genes after 12 h treatment with 30 μM
of 1 (P < 0.05, FDR corrected, fold change >1.5 or <0.67)
(Figure 3A). Consistent with our previous data, VEGF-A
appeared to be one of the most up-regulated genes (Table 1).
To examine the molecular functions and genetic networks, the
12 h microarray data was analyzed using Ingenuity Pathways
Interestingly, studies have unveiled the modulatory effects of
the UPR on VEGF-A. The UPR contributes to the transcrip-
tional, protein processing, and transportation of VEGF-A in the
ER through activation of Activating Transcription Factor 4
(ATF4), Inositol-requiring enzyme 1 (IRE1), and 150-kDa
oxygen-regulated protein (ORP150), which were all up-
regulated by apratyramide at transcript and protein levels
(Figure 3C,D).57−59 These findings suggest that apratyramide
may induce VEGF-A through the UPR pathway. Besides up-
regulating VEGF-A, the UPR has also been reported to enhance
angiogenesis by up-regulating a number of other pro-angiogenic
factors, such as FGFs, PDGFs, and IL-8.60 These observations
are also in accordance with our microarray and RT-qPCR
results where many other pro-angiogenic factors (e.g., bFGF,
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Figure 4. Conditioned medium from HaCaT cell culture treated with apratyramide (1) induced angiogenesis in vitro. (A) Conditioned medium
(CM) from HaCaT culture with the presence of compound 1 or solvent control DMSO (0.3%), 24 h, induced angiogenesis in vitro, determined by
matrigel assay using HUVECs (scale bar 200 μm), 14 h. VEGF-A protein, 100 ng/mL was used as positive control. Complete growth medium
(EGM, Lonza) for HUVEC was also used as a positive control. (B) Branch point counting was used as quantification method. Three random
microscope view-fields were counted and the number of branch points was averaged for each well. Error bars indicate mean + SEM of eight replicates
from two independent experiments. P-values were calculated relative to control (CM-DMSO) using unpaired t test (n = 8), **P < 0.01, ***P <
0.001.
PDGFB, HB-EGF) were also up-regulated after treatment with
apratyramide (Figure 2, Table 1, Supporting Information Table
S2). Collectively, improving ER homeostasis by activating the
UPR pathway independent of ER stress may be a promising
tool to accelerate wound closure, especially diabetes-associated
chronic wounds closure,55 and apratyramide has the promising
attributes to be one such therapeutic agent.
A closer investigation of these individual molecular
components of UPR further enabled us to identify several
lines of evidence demonstrating that they also directly attribute
to angiogenesis, wound healing, and VEGF-A up-regulation
dependent or independent of UPR.54,58,59,61−63
ATF4 is an important transcription factor in the UPR
signaling cascade which activates VEGF-A at both transcript
and protein levels.61−63 ATF4 promotes bone angiogenesis by
increasing VEGF expression and release in the bone environ-
ment.63 It has also been reported that ATF4 expression was
induced in smooth muscle cells after arteries injury in rats and
its overexpression further enhanced the expression of VEGF-A
by an interaction between ATF4 and a recognition element
located in the VEGF-A gene.62 The microarray data as well as
immunoblot analysis suggested that ATF4 is activated by
apratyramide at both the mRNA and protein level, which
subsequently leads to the induction of the transcription of a
number of its downstream molecular components: CHOP,
SLC6A9, CHAC1, ATF3, SARS, WARS, and others (Figure 3).
IRE1 is also an ER-located transmembrane protein, which
plays an essential role in physiological processes including
angiogenesis, placental development, and embryonic viabil-
ity.54,64 It has been shown that VEGF-A expression in the
placenta is partially dependent on IRE1.54 Another recent study
identified the deficiency of IRE1 in type 2 diabetic db/db mice
and that cell therapies using direct IRE1 gene transfer
significantly accelerated cutaneous wound healing in diabetic
mice through enhancing angiogenesis.64 These findings
strongly suggested the therapeutic strategy for diabetic wound
healing by enhancing IRE1 activity. In addition, IRE1 deletion
resulted in elevation of microRNAs, while the supply of IRE1
promoted the angiogenic potential of diabetic (bone marrow−
derived progenitor cells) BMPCs through modulating miRNA
biogenesis.64 Accordingly, we also observed down-regulation of
several microRNAs after 12 h treatment with apratyramide
(Table 1, Supporting Information Table S2).
ORP150 is an ER chaperone, the expression of which is
regulated by the UPR. Overexpression of the ORP150 gene by
adenovirus vectors accelerated wound healing by modulating
the intracellular transport of VEGF from ER to Golgi
apparatus.59 This observation implied that ORP150 was
involved in skin wound healing.
Conditioned Medium from HaCaT Cell Culture
Treated with Apratyramide Induced Angiogenesis in
Vitro. During wound healing, VEGF-A is secreted to stimulate
angiogenesis, which primarily acts on endothelial cells in the
wound area.18 Thus, we questioned whether the increased
VEGF-A secreted from HaCaT cells induced by 1 could
enhance angiogenesis. Here, conditioned medium (CM) was
collected from the HaCaT cell culture with or without the
presence of 1 at 24 h, when we previously detected an increase
of VEGF-A secretion. We incubated human endothelial cells
(HUVECs) with the obtained CM and monitored their
angiogenic activity using an in vitro angiogenesis assay (Figures
4A,B). As a positive control, the complete growth medium
(EGM) containing 2% FBS and bovine brain extract (BBE)
enabled HUVECs to form tubelike structures from individual
cells after 14 h. The CM from HaCaT cell culture (DMEM,
10% FBS) alone, however, had little effect on angiogenesis, as
most HUVECs remained as individual cells. This is possibly
due to a lack of growth supplement required for angiogenesis in
endothelial cells in the DMEM. The decreased angiogenesis in
the CM-DMSO group was rescued by the treatment with 30 or
50 μM of 1 in HaCaT culture, indicated by an increase of
tubelike structure formation. Similar to apratyramide’s effect,
VEGF-A protein, 100 ng/mL, also induced angiogenesis in
vitro. In contrast, the addition of VEGF-A at 100 ng/mL to the
complete growth medium did not significantly further induce
angiogenesis, probably due to the sufficient amount of
angiogenic factors in the BBE. The above results demonstrated
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Figure 5. Apratyramide induced VEGF-A in a rabbit corneal epithelial ex vivo model. Data are presented as mean + SEM, *P < 0.05.
that apraytramide indirectly enhanced angiogenesis, potentially
through an induction of VEGF-A secreted from HaCaT cells.
Apratyramide Induces VEGF-A in a Rabbit Corneal
Epithelial Ex Vivo Model. In order to evaluate apratyramide
(1) in a more physiological context, we tested it in an ex vivo
rabbit corneal epithelial model, a validated wound-healing
model.65,66 The fresh rabbit eyes were obtained and wounds
were induced to the center of the corneas by a laser (Figure
5A). After that, eyeballs were trimmed to collect cornea tissues
which were then cultured in medium with or without the
presence of 1. Eighteen-hours later, total RNA was collected
from cornea tissues and subjected for RT-qPCR analysis.
Consistent with the effect in vitro, we have detected a dose-
dependent increase of VEGF-A mRNA in the cornea after
treatment with 1 (Figure 5B).
warrant further studies. We have also elucidated new
mechanisms for the unexplored wound-healing bioactivities of
marine cyanobacterial compounds.
MATERIALS AND METHODS
Details of experimental procedures are provided in the Supporting
Information.
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.7b00827.
■
S
Supplementary methods, including isolation, configura-
tional analysis, biological assays (cell viability, VEGF-A
secretion, immunoblot analysis, angiogenesis, RT-qPCR,
transcriptome profiling, ex vivo organ culture, and
mRNA measurements), total synthesis, supplementary
references, tables, figures, and NMR spectra (PDF)
CONCLUSIONS
■
We have discovered a novel linear depsipeptide, apratyramide,
from marine cyanobacteria as a growth factor inducer which has
the potential to rescue chronic wounds and accelerate wound-
healing processes by inducing growth factor secretion in the
wound area. Apratyramide was isolated through a standard
procedure, followed by total structural determination through a
combined analysis of NMR and MS spectra as well as chiral
analysis of the acid hydrolyzate. We conducted the total
synthesis of apratyramide through a convergent synthetic
strategy which provided us more material for further biological
evaluation. We performed a series of in vitro cell-based assays
to elucidate the biological activity as well as mechanism of
action, although the direct biological target remains elusive.
Apratyramide induced both transcription and secretion of
VEGF-A in human keratinocyte (HaCaT) cells, evident by RT-
qPCR and AlphaLISA analysis. Other wound-healing related
growth factors were also found to be induced at the
transcriptional level, including PDGFB and bFGF. Tran-
scriptome profiling using HaCaT cells identified 371 differ-
entially expressed genes after 12 h treatment with apratyramide.
Importantly, VEGF-A and other growth factors were up-
regulated, showing consistency with our previous in vitro data
and supporting our hypothesis of the potential wound-healing
properties of apratyramide through growth factor modulation.
IPA indicated that apratyramide induced growth factors
through or partially through the UPR pathway mediated by
ATF4, IRE1, and ORP150, three molecular components in the
UPR pathway that are functionally related to wound healing
and angiogenesis. Most importantly, the promising effects in a
phenotypic assay and molecular changes in the ex vivo model
AUTHOR INFORMATION
Corresponding Author
*E-mail: luesch@cop.ufl.edu.
■
ORCID
Wei Zhang: 0000-0002-4684-2911
Hendrik Luesch: 0000-0002-4091-7492
Author Contributions
L.A.S. and W.Z. contributed equally to this work. W.C., cell-
based assays, IPA analysis, ex vivo rabbit experiment,
experimental design, and manuscript writing; S.S., RT-qPCR
experiment of rabbit corneal samples; D.J.G., supervised ex vivo
rabbit studies; L.A.S., S.M., R.R., natural product chemistry;
W.Z., total synthesis; Q.Y.C., S.D., scale-up total synthesis;
V.J.P., sample collections and identification and edited
manuscript; H.L., designed and supervised experiments and
edited manuscript.
Notes
The authors declare the following competing financial
interest(s): H.L., W.C., L.A.S.R., and V.J.P. are listed as
inventors on a patent application by the University of Florida.
ACKNOWLEDGMENTS
■
Research was supported in part by the National Institutes of
Health, NCI grant R01CA172310, the Debbie and Sylvia
DeSantis Chair professorship (H.L.) and an institutional grant
by Research to Prevent Blindness. We thank Y. Zhang from the
G
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Articles
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Gene Expression and Genotyping Core of the Interdisciplinary
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from the China Scholarship Council (W.Z.) to work in the H.L.
lab.
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