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Journal of Bodywork and Movement Therapies (2008) 12, 169–182
Bodywork and
Journal of
Movement Therapies
REVIEW AND BIOINFORMATICS RESEARCH
Expression of the endocannabinoid system
in fibroblasts and myofascial tissues
John M. McPartland, M.S., D.O.
Department of Osteopathic Manipulative Medicine, Michigan State University, East Lansing, MI, USA
Received 7 November 2007; received in revised form 29 December 2007; accepted 8 January 2008
KEYWORDS
Cannabinoids;
Endocannabinoids;
Ajulemic acid;
Osteopathic
medicine;
Chiropractic;
Myofascial release;
Fibromyalgia;
Myofascial trigger
points;
Biodynamics
Summary The endocannabinoid (eCB) system, like the better-known endorphin
system, consists of cell membrane receptors, endogenous ligands and ligand-
metabolizing enzymes. Two cannabinoid receptors are known: CB
1
is principally
located in the nervous system, whereas CB
2
is primarily associated with the immune
system. Two eCB ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are
mimicked by cannabis plant compounds. The first purpose of this paper was to
review the eCB system in detail, highlighting aspects of interest to bodyworkers,
especially eCB modulation of pain and inflammation. Evidence suggests the eCB
system may help resolve myofascial trigger points and relieve symptoms of
fibromyalgia. However, expression of the eCB system in myofascial tissues has not
been established. The second purpose of this paper was to investigate the eCB
system in fibroblasts and other fascia-related cells. The investigation used a
bioinformatics approach, obtaining microarray data via the GEO database
(www.ncbi.nlm.nih.gov/geo/). GEO data mining revealed that fibroblasts, myofi-
broblasts, chondrocytes and synoviocytes expressed CB
1
,CB
2
and eCB ligand-meta-
bolizing enzymes. Fibroblast CB
1
levels nearly equalled levels expressed by
adipocytes. CB
1
levels upregulated after exposure to inflammatory cytokines and
equiaxial stretching of fibroblasts. The eCB system affects fibroblast remodeling
through lipid rafts associated with focal adhesions and dampens cartilage
destruction by decreasing fibroblast-secreted metalloproteinase enzymes. In
conclusion, the eCB system helps shape biodynamic embryological development,
diminishes nociception and pain, reduces inflammation in myofascial tissues and
plays a role in fascial reorganization. Practitioners wield several tools that
upregulate eCB activity, including myofascial manipulation, diet and lifestyle
modifications, and pharmaceutical approaches.
&2008 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS
www.intl.elsevierhealth.com/journals/jbmt
1360-8592/$ - see front matter &2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jbmt.2008.01.004
Corresponding author at: 53 Washington Street Extension, Middlebury, VT 05753, USA. Tel.: +18023888304.
E-mail address: mcpruitt@verizon.net
Introduction
The introduction comprises a broad review of the
endogenous cannabinoid (eCB) system, in three
sections: 1. cannabinoid receptors, 2. eCB ligands,
3. clinical aspects of receptors and ligands. Ligands
that bind to receptors may activate receptors
(‘‘agonists’’) or deactivate receptors (‘‘inverse
agonists’’). The chemical concepts underlying eCB
research may raise anxiety in clinicians. However,
the realization that chemistry is structure (Ingber,
1998) makes many of these concepts readily
understood by bodyworkers. For example, the
pharmacological principle of structure–activity
relationships (SARs) is analogous to the anatomical
concept of structure–function relationships.
After the introduction, this paper investigates
the eCB system in fibroblasts, utilizing a bioinfor-
matics approach. Bioinformatics uses networks of
computers, software algorithms and internet-ac-
cessible databanks to organize, analyze, and pre-
dict biological structure and function. This
approach poses a new challenge to clinicians,
impelling them to grasp the utility and ease of
‘‘GEO,’’ the bioinformatics tool used in this
study. Bioinformatics democratizes the research
process; all one needs is computer access and
imaginative questions. Several pre-publication re-
viewers of this paper immediately grasped GEO to
answer questions of their own. The paper finishes
with a discussion of clinical applications. The
discussion delivers a unique perspective not here-
tofore presented in the literature—that our task as
clinicians who treat pain and myofascial dysfunc-
tion is to enhance endogenous eCB activity in our
patients.
Cannabinoid receptors
Cannabinoid receptors (CBRs) take their name from
the Cannabis plant. The Cannabis plant is a source
of exogenous ligands. The ligands are lipophilic
(i.e., water-insoluble), thus difficult to study, and
took 150 yr to elucidate. Finally, in 1964, Raphael
Mechoulam isolated D
9
-tetrahydrocannabinol (THC)
and cannabidiol (CBD). Since then, Raphael Me-
choulam, Roger Pertwee and many others have
identified more than 70 unique Cannabis com-
pounds, collectively called the cannabinoids (re-
viewed in Pertwee, 2005). Candice Pert’s co-
discovery of the m-opioid receptor in 1973 launched
a quest for CBRs. But CBR discovery awaited the
development of water-soluble synthetic THC ana-
logs, such as CP55, 940. In 1988 Allyn Howlett and
Bill Devane showed that [
3
H]CP55, 940 bound to a
receptor located in the cell membrane of the
neuronal (brain) cells. Two years later, Lisa Matsuda
cloned the gene for the CBR and decoded its DNA
sequence. The cDNA sequence translates into a
chain of 472 amino acids that weave back and forth
across the cell membrane. This topology is char-
acteristic of a G-protein-coupled receptor (GPCR).
GPCRs are named after their G-proteins, short for
guanine nucleotide binding proteins, which func-
tion as intracellular ‘‘molecular switches.’’ GPCRs
include opioid receptors, dopamine receptors,
serotonin receptors and many others (reviewed in
Howlett et al., 2002).
A second CBR was discovered in 1993, so the
receptors became known as CB
1
and CB
2
. The two
receptors express slightly different structures and
slightly different functions: CB
1
principally func-
tions in the nervous system, whereas CB
2
is
primarily associated with cells governing immune
function, such as white blood cells. Taken together,
CB
1
and CB
2
bridge the constituent parts of
psychoneuroimmunology and represent a micro-
cosm of mind-body medicine. CB
1
and CB
2
are
tensegrity structures that span the cell membrane.
A ligand that loads the receptor’s extracellular
surface will distort the shape of its transmembrane
weave of amino acids, thereby altering the in-
tracellular side of the receptor and its interface
with the G-protein. This shape-altering ‘‘conforma-
tional change’’ in the receptor activates the G-
protein, which disconnects from the receptor, splits
into subunits, and the subunits move around the
inside of the cell. The activated G-protein subunits
further transduce signal by reorganizing other
tensegrity structures (e.g., enzymes and ion chan-
nels), causing a ‘‘cascade’’ that ultimately governs
gene expression and cell behavior. Steve Ingber
characterized tensegrity structures as the hard-
ware behind living systems, and signal transduction
machinery as the software (Ingber, 1998).
CB
1
is the most common GPCR neuroreceptor in
the human brain, but it is distributed unevenly.
Highest densities of CB
1
are found in the hippo-
campus (affecting short-term memory) and parts of
the basal ganglia (e.g., the substantia nigra, globus
pallidus and the striatum (caudate and putamen)).
CB
1
in these nuclei coordinate movement, as does
CB
1
in the cerebellum. High densities in the
cerebral cortex, amygdala and dorsal horn of the
spinal cord affect cognition, mood and emotion,
and pain perception. Very low densities are found
in the brainstem cardiorespiratory centers, which
probably accounts for the lack of lethal effects
from cannabis overdose (reviewed in Howlett
et al., 2002).
ARTICLE IN PRESS
J.M. McPartland170
The genes for CB
1
and CB
2
are paralogs (genes
separated by a gene duplication event) with
orthologs (genes separated by a speciation event)
in all known vertebrate species. A CBR gene tree
within a species tree is illustrated in Figure 1. The
gene tree was constructed from ten species whose
entire genomes have been sequenced, specifically
chosen to obtain a balanced species divergence
within the evolutionary ‘‘tree of life.’’ The human,
mouse and puffer fish genomes all express CB
1
and
CB
2
genes, whereas the sea squirt and nematode
genomes expressed only one gene, which we called
the ancestral CBR gene (McPartland et al., 2006).
No CBR genes were found in the ‘‘lower’’ organisms
with deeper evolutionary roots. These findings
suggest that the gene duplication event that gave
rise to CB
1
and CB
2
occurred in the ancestor of
vertebrates. The ancestral CBR gene that preceded
the duplication event may have evolved in the last
common ancestor of nematodes and sea
squirts—600 million years ago (McPartland et al.,
2007).
eCBs and their enzymes
Animals likely did not evolve neuroreceptors for a
plant ligand. Indeed the CBR gene evolved eons
before the appearance of Cannabis, which is not
more than 34 million years old (McPartland and
Guy, 2004). The first endogenous cannabinoid,
anandamide (AEA), was discovered in 1992 by a
team including Devane (co-discoverer of CB
1
) and
Mechoulam (28 yr after discovering THC). Soon
2-arachidonoylglycerol (2-AG) was described along
with several less-understood eCBs (reviewed in
Mechoulam et al., 1998). AEA and 2-AG are
metabolites of arachidonic acid and do not resem-
ble THC. However, AEA and 2-AG are lipophilic, like
THC, and fit the binding pocket in CB
1
and CB
2
.
Thus, the effects of THC, AEA and 2-AG substan-
tially overlap, because they all activate CB
1
and CB
2
.
AEA and 2-AG are not stored in vesicles like
classic neurotransmitters. Rather they are synthe-
sized ‘‘on demand’’ from precursor phospholipids in
the neuron cell membrane and immediately re-
leased into the neural synapse (Pertwee, 2005).
AEA is cleaved from its precursor, N-arachidonoyl
phosphatidylethanolamine (NAPE) by the enzymes
NAPE-PLD and ABHD4; 2-AG is cleaved from
diacylglycerol (DAG) by two DAG lipase enzymes,
DAGLaand DAGLb. After the release into the
synapse, AEA and 2-AG activate CB
1
, and then
other enzymes break down AEA (FAAH, FAAH2 and
NAAA) and 2-AG (MAGL and COX2). For a full
description of these acronyms, see Table 1.
Recently a FAAH-blocking agent was described,
which prolonged AEA activity in the synapse,
analogous to a serotonin uptake inhibitor (reviewed
in Pertwee, 2005).
In the CNS, the eCB system serves as a negative
feedback mechanism and dampens excessive sy-
naptic release of other neurotransmitters. For
example, persistent activation of a nociceptor
causes excessive glutamate release in the dorsal
horn synapses (see Figure 2). This maladaptively
upregulates glutamate receptors in the post-synap-
tic cell (in this case a wide dynamic neuron that
ascends to the brain). However, DAGLaenzymes are
located in the post-synaptic cell, and influx of Ca
2+
from upregulated glutamate receptors causes
DAGLato synthesize 2-AG (Figure 2A). The 2-AG
moves retrograde (opposite the direction of gluta-
mate) across the synapse to CB
1
located on the
presynaptic neuron (Figure 2B). Activated CB
1
closes presynaptic Ca
2+
channels, which halts
glutamate vesicle release. This newly discovered
phenomenon is called ‘‘depolarization-induced
suppression of excitation’’ (Ma´tya´s et al., 2007).
The eCB system ‘‘mellows the synapse,’’ and
requires neuroscientists to rewrite textbooks that
describe the synapse as a ‘‘one-way street.’’
At CB
2
in white blood cells, AEA and 2-AG act as
autocrine, paracrine or endocrine modulators and
circulate in the blood stream for short periods of
time. The eCBs (and THC) are immunomodulators
and not simply immunosuppressors as character-
ized in the 1970s (Klein, 2005). They do indeed
suppress production of Th1 (T-helper1, cellular
ARTICLE IN PRESS
Figure 1 The cannabinoid receptor gene tree within a
species tree. The species tree consists of ten organisms
whose entire genomes have been sequenced, it is
represented by thin tubular lines. The gene tree is
represented by thicker lines, either solid (representing
ancestral CBR gene orthologs and CB
1
gene orthologs
after the duplication event) or dashed (representing CB
2
gene orthologs).
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 171
immunity) cytokines such as interleukin (IL)-2 and
interferon gamma (INFg), as well as tumor necrosis
factor alpha (TNFa). However, they increase secre-
tion of T-helper2 (Th2), humoral immunity) cyto-
kines (IL-4, IL-5, IL-10). Other subsets of
lymphocytes including B cells (MZ, B1a) and natural
killer NK cells require eCBs and CB
2
to function
properly (Ashton, 2007). Cannabis has been de-
scribed as an adaptogen along with Echinacea and
other plant products that stimulate resistance to
disease and stress (Emboden, 1976). The alkyla-
mide compounds in Echinacea are potent agonists
of CB
2
(not CB
1
); Echinacea compounds are not
psychoactive because CB
2
is rare in the brain
(Raduner et al., 2006).
Clinical aspects of the eCB system
CB
1
receptors have been detected as early as
gestational day 2 in mouse embryos, so the eCB
system is fully functional at every stage of devel-
opment (Park et al., 2004). Aspects of the eCB
system inform the work of Erich Blechschmidt, a
biodynamic embryologist studied by many body-
workers. Blechschmidt (1977) claimed that the
embryo is fully functional at every stage of
development. The embryo develops in motion,
guided by fluid dynamics, and each motion impacts
the development of each subsequent development.
Fluids moving in channels establish a matrix, a
pressure-generated framework, and this directs the
formation of connective tissues (Freeman, 2004).
Only after the structure is cast by the fluid forces
does genetic expression play a role in the embryo-
nic development. The genes do not act, but react
to the external forces, especially hydrostatic
pressures (reviewed in McPartland and Skinner,
2005). If chemistry is structure, then gene tran-
scription (at least its initiation) is mechanotrans-
duction.
Axon migration in the embryonic brain is guided
initially by the fluid dynamics, a fluid within a
fluid (Newman et al., 1985). Subsequent migra-
tion is guided by the genetic expression of UNC5
and EPHA1, which are cell membrane receptors
found in the tip of axon growth cones. UNC5
and EPHA1 are activated by ligands (netrins and
ephrins) found in the extracellular fluids. The
activated receptors begin a signal cascade via
FAK and Rho. FAK is a focal adhesion-associated
enzyme involved in cellular adhesion. Rho is an
enzyme (a GTPase) that regulates intracellular
actin dynamics. Together, FAK and Rho direct
cytoskeletal dynamics, thereby regulating growth
cone motility (Dickson, 2002). The eCBs modulate
this cascade (by activating Rho), making eCBs vital
ingredients in the chemotropic soup that guides
neurons to their destinations (Berghuis et al.,
2007).
ARTICLE IN PRESS
Table 1 Fibroblast expression of cannabinoid receptors or endocannabinoid ligand enzyme documented by
charts deposited in the GEO profiles database.
Protein acronym,
gene symbol
a
Protein full name, synonyms (if any), enzyme protein function Number of GEO
profile charts
located in search
CB
1
,CNR1 Cannabinoid receptor 1 165
CB
2
,CNR2 Cannabinoid receptor 2 142
FAAH,FAAH Fatty acid amide hydrolase 1, catabolic enzyme of AEA 82
COX2,PTGS2 Cyclooxygenase 2, aka prostaglandin-endoperoxide synthase 2,
catabolic enzyme of 2-AG
66
NAAA,ASAHL N-acylethanolamine acid amidase, catabolic enzyme of AEA 55
DAGLb,DAGLB Diacylglycerol lipase beta, aka KCCR13L, synthetic enzyme of 2-AG 36
NAPE-PLD,NAPE-
PLD
NAPE-selective phospholipase D, biosynthetic enzyme of AEA 31
ABHD4,ABHD4 Abhydrolase domain-containing protein 4, aka FLJ12816, synthetic
enzyme of AEA
28
DAGLb,DAGLA Diacylglycerol lipase alpha, aka NSDDR, C11ORF11, synthetic enzyme of
2-AG
21
MAGL,MGLL Monoacylglycerol lipase, catabolic enzyme of 2-AG 14
FAAH2,FAAH2 Fatty acid amide hydrolase 2, aka AMDD,FLJ31204, catabolic enzyme of
AEA
9
a
Protein acronym, gene symbol or synonym that received the greatest number of hits in the GEO profiles database is listed in
bold print.
J.M. McPartland172
Biodynamic practitioners claim that the fluid
forces that organize embryological development
are present throughout our life span, ready for our
cooperation in harnessing their therapeutic po-
tency. In other words, the forces of embryogenesis
become the forces of healing after birth (McPart-
land and Skinner, 2005). This axiom is evoked by the
fact that adult neurogenesis by neural stem cells is
guided by the aforementioned ‘‘embryonic’’ axon
guidance molecules (Koeberle and Bahr, 2004).
Adult neural stem cells express CB
1
(Curtis et al.,
2006), and neurogenesis by these cells is governed
by the eCB system (Aguado et al., 2007).
Effects on neurogenesis and retrograde transmis-
sion illustrated in Figure 2 regulate neural plasti-
city, thereby affecting adaptive learning, emo-
tional memory and nociception pain. Via these
mechanisms and others the eCB system provi-
des neuroprotection in Alzheimer’s, Parkinson’s,
Huntington’s, multiple sclerosis, seizure disorders
and limits infarct size following cerebral ischemia
(reviewed in Pacher et al., 2006). The eCB system
balances sympathetic–parasympathetic tone, im-
parts anti-emetic and antihypertensive benefits,
and favorably modulates stress in the HPA axis
(reviewed in Pertwee, 2005). AEA and 2-AG (as well
as THC) are anti-carcinogenic and inhibit tumor
growth in breast, prostate, and lung carcinomas,
gliomas, melanomas, lymphomas, and other can-
cers (Guzman, 2003). Cannabinoids induce apopto-
sis (programmed cell death) in cancer cells via a
CB
1
-mediated ceramide–caspase pathway. In non-
cancer cells, eCBs actually promote cell survival,
via the ERK pathway (Guzman, 2003).
Having inventoried this list of benefits, a dys-
functional eCB system may nevertheless cause
harm. The autonomic effects of eCBs have been
implicated in hemorrhagic and endotoxic shock,
cardiac reperfusion injury, doxorubicin-induced
cardiotoxicity and advanced liver cirrhosis (Pacher
et al., 2006). Mutations in CB
1
and FAAH genes have
been linked with obesity and schizophrenia (Pacher
et al., 2006), and the genes for DAGLaand NAPE-
PLD share an evolutionary signature associated with
genes that harbor mildly deleterious alleles and
disease-related phenotypes (McPartland et al.,
2007).
Levels of AEA in cerebrospinal fluid are increased
in schizophrenics, but the elevated levels are
negatively correlated with psychotic symptoms
(Giuffrida et al., 2004). This suggests that abnormal
stimulation of post-synaptic D
2
receptors triggers
release of AEA and retrograde signaling via CB
1
,
thus homeostatically attenuating presynaptic
dopamine release. Note that CB
1
and CB
2
expo-
sed to high doses of THC become desensitized
ARTICLE IN PRESS
Figure 2 (A and B) The eCB system dampens excessive
nociception at the dorsal horn. (A) Persistent firing of a c-
fiber nociceptor opens voltage-gated calcium channels
(VGCCs) in the presynaptic axon terminal. Calcium influx
causes presynaptic vesicles of glutamate to release into
the synaptic cleft. Excessive activation and upregulation
of glutamate receptors in the postsynaptic cell causes the
opening of calcium channels. (B) Open calcium channels
in the postsynaptic cell stimulate DAGLa enzymes to
synthesize 2-AG, which is released into the synapse and
activates CB
1
in the presynaptic cell. The G-proteins from
activated CB
1
close VGCCs, thereby halting release of
presynaptic glutamate vesicles.
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 173
(transported to intracellular compartments via
endocytosis). High doses of THC may therefore
provoke psychiatric illness in susceptible indivi-
duals by desensitizing CB
1
receptors and diminish-
ing retrograde signaling (Giuffrida et al., 2004). On
the other hand, cannabidiol, a nonpsychoactive
ingredient in cannabis, shows promise as an
antipsychotic agent (Zuardi et al., 2006).
Marijuana famously causes the ‘munchies’, and
this behavior teleologically begins in utero, during
blastocyst implantation. The blastocyst makes
active contact with the endometrium followed by
an uptake of nourishment from the endometrial
mucosa, an act characterized as the ‘‘earliest
suckling function’’ (Blechschmidt, 1977), and blas-
tocyst implantation requires a functional eCB
system (Park et al., 2004). When newborn mice
are given rimonabant, a drug that blocks CB
1
, they
stop suckling and die (Fride, 2004).
Obesity leads to excessive production of eCBs by
adipocytes, which drives CB
1
activity in a feed-
forward dysregulation via ghrelin, leptin and orexin
signaling pathways (Matias and Di Marzo, 2007).
Last year a pharmaceutical company sought ap-
proval of the CB
1
blocker rimonabant (Accomplia
s
,
Zimulti
s
) for the treatment of obesity. The US Food
and Drug Adminstration rejected the drug because
subjects in rimonabant studies suffered depressed
mood, anxiety, headache, nausea and diarrhea.
Given the myriad benefits of a fully functioning eCB
system, it should be no surprise that rimonabant
unmasked previously silent multiple sclerosis and
seizure disorders and doubled the risk for suicid-
ality (Food and Drug Administration, 2007). Com-
plete blockade of CB
1
might approximate the
phenotype expressed by genetically engineered
‘‘knockout mice.’’ Mice lacking CB
1
suffer increased
morbidity and premature mortality, and show
greater aggression, epilepsy, age-related neuron
loss, anxiogenic-like behavior, depressive-like be-
havior, anhedonia and fear of newness (Zimmer et
al., 1999;Martin et al., 2002).
From a materialistic viewpoint, the aforemen-
tioned moods and emotions represent the rhythmic
entrainment of synchronously firing CNS neurons.
The eCB system (and Cannabis) alters consciousness
by modulating these biological oscillators (Crystal
et al., 2003;O’Leary et al., 2003;Galarreta et al.,
2004). This materialistic perspective has been
challenged by some neuroscientists, who argue
that consciousness is ‘‘nonlocal’’ and not housed
within a specific neural substrate (reviewed by
Dossey, 2007). This concept echoes Leonardo Da
Vinci, who believed consciousness occupied the
‘‘void’’ of the ventricular system, rather than
brain parenchyma (Pevsner, 2002). The ventricular
system centers another oscillatory phenomenon
known as the primary respiratory mechanism
(Sutherland et al., 1967). The ventricular ‘‘void’’
is filled with cerebral spinal fluid (CSF), awash with
eCBs (Giuffrida et al., 2004). Cells lining the
ventricular system express CB
1
and eCB enzymes
(Curtis et al., 2006), which modulate the rhythmic
production of CSF (Mancall et al., 1985) and control
the eCB levels in the CSF (Ashton et al., 2004).
Surprisingly little has been published about the
eCB system and fascia. Indeed the very presence of
CB
1
,CB
2
, AEA and 2-AG in fascia-related cells has
not been established in the peer-reviewed litera-
ture. The investigational purpose of this paper was
to search for evidence of the eCB system in fascia-
related cells and tissues. The investigation used a
bioinformatics approach.
Methods
Bioinformatics experiments are described as in
silico, rather than in vitro or in vivo experiments.
Bioinformatics is particularly adept with genomic
and molecular data. For example, the genomic
data that created Figure 1 were downloaded from
the Entrez ‘‘PubMed’’ server (www.ncbi.nlm.nih.-
gov/sites/entrez). The upper left corner of the
homepage contains a pull-down menu with
‘‘PubMed’’ as the default item. Instead, ‘‘Genome’’
was selected and gene sequences resembling hu-
man CB
1
were sought in other species genomes.
Then a freeware application, ClustalX, was used to
construct the gene tree (Frazer et al., 2003). The
results were published (McPartland et al., 2006).
For the investigation of fascia, ‘‘GEO Profiles’’
was selected from the same pull-down menu. GEO
is a repository of data derived from microarray
experiments. Microarray (‘‘gene chip’’) technology
uses a robot to apply thousands of droplets of
different DNA sequences on a grid. Microarray
experiments generate far more data than can be
published in a scientific paper. For example, a
microarray experiment might identify a disease-
related gene by comparing gene expression in the
normal versus the diseased cells; thousands of
other genes were examined in the study and go
unreported in the scientific literature but are
deposited in the GEO database.
The GEO Profiles database (www.ncbi.nlm.nih.
gov/projects/geo/) was queried by combining an
eCB name (e.g., CB
1
) and a cell type name.
‘‘Fibroblast,’’ the predominant cell type in fascia,
was chosen for the search. GEO results are
presented as charts with red histograms providing
a quantitative signal value (‘‘how much of this gene
ARTICLE IN PRESS
J.M. McPartland174
is expressed’’). However, the signal value is
expressed in arbitrary units and may not be reliable
due to variability in the quantity of DNA deposited
in each droplet and the efficiency of cDNA–mRNA
hybridization. Therefore, chip data are secondarily
analyzed by a qualitative measure (‘‘has this gene
been expressed or not?’’). This statistically derived
Detection Call denotes gene transcripts as ‘‘Pre-
sent’’ (i.e., statistically valid), ‘‘Marginal’’ or
‘‘Absent’’ (i.e., statistically unreliable and dis-
played in GEO profile as faded red histograms).
Unfortunately, the Detection Call may contradict
the signal value; genes marked as absent may have
higher quantitative signals than genes marked as
present. This discrepancy becomes acute in genes
with low expression levels. Expression levels are
gauged by blue squares appearing in the red
histograms. The blue squares represent the per-
centile ranked signal value of a gene compared to
thousands of genes on that chip. In cases of
discrepancy genes with measurable signal values
nevertheless marked as absent were counted
as present if expression levels came close to the
limits of detection (percentile rank o20%). GEO
results were statistically analyzed with two online
calculators, GraphPad (http://graphpad.com/
quickcalcs) and VassarStats (http://faculty.vassar.
edu/lowry/VassarStats.html).
Results
The query ‘‘CB
1
and fibroblast’’ located 165 GEO
profile charts, whereas ‘‘CB
2
and fibroblast’’ lo-
cated 142 charts (Table 1). The signal values in
these charts were relatively low, judging by the
Detection Call results: signal values in 34 of the 165
CB
1
charts (20.6%) were low enough to be statisti-
cally unreliable (i.e., Detection Call: absent).
Similarly, signal values in 34 of the 142 CB
2
charts
(23.9%) contained only ‘‘Detection Call: absent’’
results.
Although signal values for CB
1
and CB
2
varied
from one chart to the next, some microarray
studies compared CB
1
and CB
2
within the same
experiment, making their comparisons more reli-
able. Signal values of fibroblast CB
1
and CB
2
were
directly compared in 21 studies amenable to
pooling (i.e., studies using identical microarray
platforms and protocols). CB
1
signal values (mean
10.4, SEM 1.4) exceeded CB
2
signal values (mean
6.4, SEM 0.85), a significant difference
(p¼0.0002, paired ttest).
Seven studies, identified in Table 2, directly
compared fibroblast signal values to signal values in
other cell types. Additionally, other microarray
experiments reported interesting results:
CB
1
expression in cardiac fibroblasts (mean 3.2,
SEM 1.30) doubled following mechanical stretch-
ing (mean 6.3, SEM 1.47), but the difference in
this small study (n¼3) fell short of significance
(p¼0.075, paired ttest) (GEO accession num-
ber GDS1035, CB
2
not tested).
CB
1
and CB
2
expression in human fibroblasts was
similar to CB
1
and CB
2
expression in gorilla and
bonobo fibroblasts (GDS340).
CB
1
expression in synovial fibroblasts (‘‘synovio-
cytes’’) equaled 1.5 (GDS386, an n¼1 experi-
ment), which increased 5-fold (to 8.3) following
exposure to the inflammatory cytokine TNFa.In
the same study, CB
2
levels surprisingly decreased
(baseline ¼4.1, TNFaexposure ¼3.5). These
results conflicted with an analogous n¼1 study
of synovial fibroblasts (GDS1796), where CB
1
decreased following exposure to IL-1b(base-
line ¼16.3, IL-1bexposure ¼7.5), but CB
2
slightly increased (baseline ¼6.9, IL-1bex-
posure ¼8.9).
Skeletal muscle CB
1
expression in the vicinity of
the neuromuscular junction (mean 325.4, SEM
106.29) was greater than CB
1
expression in
skeletal muscle away from the neuromuscular
junction (mean 262.7, SEM 48.40), but the
difference in this small study (GDS1838, n¼4)
was not significant (p¼0.61, unpaired ttest).
Querying GEO with eCB ligand enzymes located
fewer GEO Profile charts, because many microarray
experiments did not include gene expression of
these enzymes, especially the most recently
discovered enzymes, such as FAAH2 (Table 1).
Nevertheless, GEO profile chart results demon-
strated that all nine AEA- and 2AG-metabolic
enzymes were expressed in fibroblasts (Table 1).
Discussion
‘‘Data mining’’ describes the process of knowledge
discovery or retrieval of hidden information from
databases. This study used ‘‘top-down’’ data
mining, where databases were searched to test a
hypothesis (‘‘the eCB system is present in fibro-
blasts’’). In contrast, ‘‘bottom-up’’ data mining
(dredging databases to generate a new hypothesis)
may carry pejorative implications amongst experi-
mental scientists. Nevertheless, bottom-up data
mining generates worthwhile results, for example
the human genome project.
ARTICLE IN PRESS
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 175
Data mining of GEO Profiles provided robust
evidence of CB
1
and CB
2
expression in fibroblasts
and related cell types, such as myofibroblasts and
synoviocytes. This evidence agreed with Bensaid
et al. (2003), who detected CB
1
in mouse embryo
3T3-F442A cells, although Bensaid and colleagues
described 3T3-F442A cells as ‘‘undifferentiated
adipocytes,’’ instead of fibroblasts.
Three microarray studies in Table 2 compared
fibroblasts and adipocytes, and collectively re-
ported a 1:1 ratio in CB
1
expression between the
two cell types. In contrast, Bensaid et al. (2003)
reported CB
1
levels in differentiated adipocytes
were 4.35-fold greater than those in undifferen-
tiated 3T3-F442A cells. Similarly, Matias et al.
(2006) described a 4-fold increase in CB
1
levels
after 3T3-F442A cells (‘‘preadipocytes’’) under-
went 4 days of insulin-induced differentiation into
adipocytes. However, 8 and 12 days later, there was
only a 2-fold difference between the cell types.
Most recently (while this paper was in review), a
study by Gasperi et al. (2007) reported that 3T3-L1
cells have the machinery to bind, synthesize and
degrade AEA, and that their differentiation into
adipocytes increases by approximately 2-fold and
3-fold, respectively, the binding efficiency of CB
1
and the catalytic efficiency of FAAH. Engeli et al.
(2005) compared spinal cord tissue and adipose
tissue, and reported a 1:1 ratio in CB
1
expression
(at levels significantly greater than those in other
non-neural tissues). We surmise that fibroblast CB
1
levels are significant as well, if the fibroblast-to-
adipose CB
1
ratio is 1:1 (Table 2) or 1:2–4(Bensaid
et al., 2003;Matias et al., 2006;Gasperi et al.,
2007).
Results with GEO indicated that fibroblasts
also expressed AEA- and 2-AG-metabolic enzymes
(Table 1). Thus, fibroblast CB
1
and CB
2
may be
signaled by eCBs in an autocrine fashion. This
evidence was supported by Matias et al. (2006),
who extracted AEA and 2-AG from undifferentiated
3T3-F442A cells. Additionally, fibroblast CB
1
and
CB
2
may be signaled in a paracrine fashion, by eCBs
secreted from neighboring leukocytes, a rich source
of AEA and 2-AG (reviewed in Ashton, 2007).
The importance of eCB signaling in fibro-
blasts can be deduced by studies of other cell
types. Fibroblasts share many signal transduction
ARTICLE IN PRESS
Table 2 GEO charts of fibroblast CB
1
and CB
2
signal values that were directly compared to signal values
expressed by other cell types.
CB
1
or CB
2
GEO accession number
a
,
difference between
means
b
Tissue type
c
(mean signal value, SEM, nreplicates)
CB
1
GDS951
d
, n.s.d.
e
F
1
(120.2, 20.75, 6)¼A (104.7, 32.35, 6)
CB
1
GDS1784, n.s.d.
e
F
2
(117.3, 4.82, 4)¼A (132.3, 5.64, 4)
CB
1
GDS1298, n.s.d.
e
F
3
(82.7, 11.11, 36)¼A (79.5, 10.9, 34)
CB
1
GDS857
d
, n.s.d.
e
F
4
(7.3, 1.8, 3)¼M (7.3, 1.1, 3)
CB
1
GDS2091, po0.01
f
F
3
(67.0, 4.53, 6)¼C (63.9, 9.63, 6)4O (53.7, 2.69, 6)
CB
1
GDS1505, p¼0.0026
e
F
5
(8.0, 0.31, 12)4K (6.0, 0.85, 12)
CB
1
GDS1402, po0.01
f
S (0.6494, 0.211, 6)4F
6
(0.20, 0.009, 7)¼SmM (0.19, 0.023,
26)¼Ep (0.19, 0.042, 6)¼End (0.15, 0.044, 16)
CB
2
GDS951
d
, n.s.d.
e
F
1
(292.3, 146.66, 6)¼A (263.4, 101.30, 6)
CB
2
GDS1784, n.s.d.
e
F
2
(23.8, 0.57, 4)¼A (26.0, 1.61, 4)
CB
2
GDS1298, n.s.d.
e
F
3
(119.4, 19.80, 36)¼A (133.7, 20.40, 34)
CB
2
GDS857
d
,p¼0.0027
e
F
4
(5.5, 0.33, 3)4M (1.3, 0.95, 3)
CB
2
GDS2091, n.s.d.
f
F
3
(3.2, 0.79 3)¼C (4.11, 3)4O (9.7, 8.63, 3),
CB
2
GDS1505, n.s.d.
e
F
5
(7.9, 0.05, 4)¼K (7.8, 0.11, 4)
CB
2
GDS1402, n.s.d.
f
SmM (1.67, 0.261, 26)¼S (1.65, 0.541, 6)¼F
6
(1.50, 0.087,
7)¼Ep (1.32, 0.071, 6)¼End (1.31, 0.042, 16)
a
GEO profiles accession number.
b
Means testing: n.s.d., no significant difference.
c
Tissue types: F
1
, mouse embryo fibroblast (C3H10T1/2 preadipocyte); F
2
, mouse embryo fibroblast; F
3
, mouse embryo
fibroblast (NIH-3T3); F
4
, mouse corneal fibroblast (NIH-3T3); F
5
, human skin fibroblast; F
6
, human skin, liver, and lung
fibroblasts; C, chondrocyte; End, endothelial cell; Ep, epithelial cell; O, osteoblast; K, keratinocyte; M, myofibroblast; S,
stroma cell (connective tissue from heart, breast, prostate, and skeletal muscle); SmM, smooth muscle cell.
d
A GEO data set whose numerical signal values were statistically unreliable (Detection Call ‘‘Absent’’).
e
Unpaired ttest.
f
One-way ANOVA with post-hoc Turkey test.
J.M. McPartland176
mechanisms with neuronal cells, and the effects of
eCBs upon neuronal cells are well known (see FAK
and Rho described in the Introduction section). For
example, fibroblast growth factor (FGF) obviously
stimulates fibroblast growth (hence its name).
However, FGF also stimulates neuronal growth,
and it does so via a CB
1
-dependent mechanism
(Williams et al., 2003;Aguado et al., 2007).
Fibroblasts also share signal transduction mechan-
isms with cells that migrate or exert traction, such
as macrophages (including monocytes and micro-
glia), B- and T-lymphocytes, eosinophils, astro-
cytes, interneurons, human embryonic kidney 293
cells, HL60 cells, and trabecular meshwork cells.
The effects of eCBs upon migration and cytoskele-
ton activity in these cells are also well known
(reviewed in He and Song, 2007). In this case, CB
2
may play a larger role than CB
1
(Gokoh et al., 2005;
Scutt et al., 2007).
Within the neuron cell membrane, CB
1
is loca-
lized to a scaffolding microdomain known as the
‘‘lipid raft’’ (Rimmerman et al., 2007). This is true
for other cell types (Sarnataro et al., 2005;Bari
et al., 2007), and probably true for fibroblasts as
well. Fibroblast lipid rafts anchor integrins, which
are transmembrane receptors that link extracellu-
lar ECM ligands (such as collagen and fibronectin) to
the intracellular cytoskeleton (Gaus et al., 2006).
Integrin receptors transmit signal via intracellular
enzymes discussed previously (e.g., FAK, Rac and
Rho), and these in turn regulate the actin–micro-
tubule–cytoskeleton system. The integrin-centered
cluster of signaling proteins is known as a ‘‘focal
adhesion,’’ and it regulates fibroblast growth,
remodeling and migration (Gaus et al., 2006). It is
easy to speculate that focal adhesions are modu-
lated by a mechanism that is FGF- (Abe et al., 2007)
and CB
1
-dependent (Aguado et al., 2007). Con-
sidering the prominence of FGF during embryogen-
esis (Williams et al., 2003), FGF- and CB
1
-induced
fascial reorganization is another example of bring-
ing the ‘‘embryonic tool kit’’ back into action to
restore health.
CB
1
may affect other aspects of fibroblast
function. Fibroblast-like synovial cells exposed to
inflammatory TNFasecrete metalloproteinase en-
zymes, which facilitate articular cartilage destruc-
tion (Johnson et al., 2007). Johnson and colleagues
decreased metalloproteinase secretion by treating
the cells with ajulemic acid (AjA). These authors
hypothesized that AjA worked via PPARgreceptors.
Our GEO results, however, showed that synovial
cells exposed to TNFahad a 5-fold increase in CB
1
levels (GDS386). AjA binds and activates CB
1
(Vann
et al., 2007), and CB
1
activation dampens the
effects of TNFa(Ashton et al., 2007). Related
research has shown that articular cartilage destruc-
tion and nitric oxide-induced proteoglycan degra-
dation and collagen breakdown are decreased by
AEA (Mbvundula et al., 2005).
Clinical discussion
Practitioners wield several tools that upregulate
eCB activity, including bodywork, diet and lifestyle
modifications, and pharmaceutical approaches.
Many bodyworkers induce ‘‘cannabimimetic’’
changes in their patients, such as anxiolysis,
easement of suffering, increased sense of well-
being and even euphoria. We conducted a
randomized, blinded, controlled clinical trial
(McPartland et al., 2005) that measured AEA levels
twice, pre- and post-osteopathic manipulative
treatment (OMT). The OMT intervention consisted
of myofascial release, muscle energy technique,
and thrust techniques. OMT subjects experienced
cannabimimetic effects (based upon a question-
naire), which correlated with an increase in post-
OMT serum AEA levels (more than double pre-OMT
levels). Neither cannabimimetic effects nor
changes in AEA levels occurred in control subjects.
A smaller OMT trial reported little change in AEA
levels, but showed significant post-OMT augmenta-
tion of N-palmitoylethanolamine (PEA), a short-
chain analog of AEA (Degenhardt et al., 2007). PEA
is discussed below.
The cellular mechanisms underlying OMT have
been modeled by in vitro stretching of fibroblasts
(Dodd et al., 2006). An aforementioned GEO study
(GDS1035) used an identical Flexercell apparatus,
and reported a doubling of CB
1
expression in
fibroblasts following cyclic equiaxial stretching.
Speculatively, the stretching of CB
1
may activate
the receptor in the absence of ligand. ‘‘Constitu-
tive activity,’’ the activation of G-proteins in the
absence of ligand, has been measured in CB
1
(reviewed in Howlett et al., 2002). Correspond-
ingly, hydrostatic pressure applied to smooth
muscle cells stretches the angiotensin 1 receptor
into an active conformation (Zou et al., 2004). Cells
that line the cerebral ventricles express CB
1
(Curtis
et al., 2006), and these cells may be compressed by
hydrostatic pressure generated during the osteo-
pathic CV4 technique, possibly releasing eCBs or
directly activating CB
1
(McPartland and Skinner,
2005). Pert (2000) hypothesized that energy thera-
pists heal patients by inducing a vibrational tone
that shifts neuroreceptors into constitutively active
states, or the vibrational tone triggers release
of endorphins that activate the neuroreceptors.
Oschman (2000) described crystalline materials
ARTICLE IN PRESS
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 177
within biological structures that generate piezo-
electric fields when compressed or stretched.
Examples of crystalline materials applicable to
our study include the phospholipids that surround
CB
1
within cell membranes, and collagen in the ECM
that surrounds fibroblasts.
Fibroblasts react to acupuncture needle rotation,
a response modulated by Rho and Rac signaling
(Langevin et al., 2006). Langevin and colleagues
conducted a microarray study, but have not
deposited their results in the GEO databank. The
eCB system works through Rho and Rac (e.g.
Berghuis et al., 2007;He and Song, 2007), and
acupuncture may work through the eCB system (Li
et al., 2007), rather than the endorphin system as
assumed previously (Harbach et al., 2007). Simi-
larly, the eCB system may be responsible for
‘‘runner’s high’’—running on a treadmill raises
serum AEA levels (Sparling et al., 2003;Dietrich
et al., 2004). Chronic stress downregulates CB
1
expression, so stress reduction may enhance the
eCB system (Hill et al., 2005). Acute ethanol
ingestion decreased AEA and 2-AG in most brain
regions (Gonzales et al., 2002), and chronic ethanol
downregulated CB
1
expression (Ortiz et al., 2004).
Dietary inclusion of fish oils containing DHA
(docosahexaenoate 22:6 w-3) and other polyunsa-
turated fatty acids increased AEA and 2-AG levels in
the brain (Berger et al., 2001;Watanabe et al.,
2003). Oral administration of Lactobacillus upregu-
lated CB
2
in intestinal epithelial cells, and relieved
symptoms of irritable bowel syndrome (Rousseaux
et al., 2007).
Turning to pharmaceuticals, acetaminophen
(paracetamol) is converted into N-arachidonoyl-
phenolamine by the liver, a compound that acti-
vates CB
1
(Ho¨gestatt et al., 2005). Ibuprofen and
other non-steroidal anti-inflammatory drugs
(NSAIDs) inhibit COX2, an enzyme that breaks down
2-AG. So NSAIDs may prolong 2-AG activity. NSAIDs
also inhibit FAAH and therefore enhance AEA
activity (Fowler, 2004). The tricyclic antidepressant
desipramine increased CB
1
levels in the brain (Hill
et al., 2006), whilst fluoxetine decreased CB
1
expression (Oliva et al., 2005). THC and cannabidiol
may widen their own therapeutic windows by
increasing AEA levels, and THC surprisingly upregu-
lated CB
1
expression when administered acutely
(reviewed in McPartland and Guy, 2004). Adelmi-
drol, a synthetic analog of PEA, has been topically
applied to improve wound healing in animals
(Panagiotis et al., 2007). Ultrasound sonification
of adelmidrol gel also showed efficacy in the
treatment of lateral epicondylitis (Sioutis et al.,
2004).
Fibromyalgia, a disorder involving diffuse myo-
fascial pain, may be a syndrome of eCB deficiency
(Russo, 2004). During the menstrual cycle, AEA
decreases during the luteal phase (circa day 21)
and rises during the follicular phase (circa day 10),
due to the progesterone-induced upregulation of
FAAH (enzyme that breaks down AEA) in the luteal
ARTICLE IN PRESS
Figure 3 Schematic illustration of a polymodal c-fiber nociceptor, with its proximal terminal in the dorsal horn (DH),
cell body in the dorsal root ganglion (DRG), and an enlarged view of the distal terminal. A suture loop separates the
enlarged view from the rest of the nociceptor. Below the nociceptor is a peripheral sympathetic postganglionic neuron.
Within the distal terminal are five receptors for activators (regular font) and five receptors for sensitizers (in italics),
named by their gene symbols. Also embedded in the distal terminal are two ion channels (Na
v
1.8 and GIRK) and CB
1
.A
lymphocyte expressing CB
2
is nearby.
J.M. McPartland178
phase. In a study of healthy women with normal
menstrual cycles, the decrease in AEA corre-
sponded with hypersensitivity to algometer-
induced pressure pain during the luteal phase.
Several subjects ‘‘changed’’ fibromyalgia diagnosis
during the course of a menstrual cycle, fulfilling the
tender point criterion (tenderness p4kg at X11
points) during the AEA-deficient luteal phase or
menstrual phase, but never during the AEA-rich
follicular phase (Dunnett et al., 2007).
Myofascial pain is a common reason why patients
self-medicate with cannabis (Ware et al., 2005).
This fact led us to hypothesize that myofascial
trigger points (MFTrPs) were endowed with CB
1
receptors (McPartland and Simons, 2007). The
etiology of MFTrPs has been attributed to abnormal
acetylcholine-related depolarization of motor end-
plates (i.e., the neuromuscular junction), followed
by release of inflammatory cytokines (Mense et al.,
2003;Shah et al., 2005). Our hypothesis that
MFTrPs were endowed with CB
1
receptors was
supported by a GEO study (GDS1838) that showed
greater CB
1
levels in skeletal muscle near the
neuromuscular junction. Two new papers confirmed
our results (Newman et al., 2007;Sa´nchez-Pastor
et al., 2007), showing that CB
1
activation in motor
endplates dampened acetylcholine release.
Myofascial dysfunction may recursively loop into
eCB system dysfunction: CB
1
receptors in a noci-
ceptor are synthesized in the dorsal root ganglion
and carried by axoplasmic flow to insertion sites in
the distal terminal of the nerve (Figure 3). In the
distal terminal, CB
1
activity dampens the activity of
activators and sensitizers. CB
1
activity closes Na
+
channels and opens K
+
(‘‘GIRK’’) channels, hyper-
polarizing the nociceptor (keeping it from firing),
and preventing peripheral sensitization and hyper-
algesia (Agarwal et al., 2007). However, mechan-
ical barriers that restrict axoplasmic flow will
prevent CB
1
receptors from reaching the distal
terminal (Hohmann and Herkenham, 1999). The
ligation loop in Figure 3 represents carpal tunnel
syndrome, thoracic outlet restriction, piriformis
syndrome, or any other mechanical barrier that
bodyworkers treat and eliminate. This restores
axoplasmic flow, facilitating CB
1
transport to its
peripheral site of action.
Conclusions
The eCB system exemplifies the osteopathic con-
cept that we possess self-regulatory mechanisms
that are self-healing in nature. The overall role of
the eCB system can be summarized as ‘‘resilience
to allostatic load,’’ a phrase synonymous with
health. The eCB system dampens nociception and
pain, decreases inflammation in myofascial tissues
and plays a role in fibroblast reorganization.
Understanding of the modulation of CB
1
,CB
2
and
eCBs represents new approaches for practitioners
to treat a variety of structural and functional
disorders.
Acknowledgments
This paper was presented, in part, at the Fascia
2007 conference in Boston. Dr. Jeffrey Bond,
Research Associate Professor of Microbiology and
Molecular Genetics, University of Vermont gener-
ously provided help with GEO search parameters.
Dr. McPartland serves as a scientific advisor for the
Cannabinoid Research Institute, a research division
of GW Pharmaceuticals (www.gwpharm.com).
References
Abe, M., Sogabe, Y., Syuto, T., Yokoyama, Y., Ishikawa, O., 2007.
Evidence that PI3K, Rac, Rho and Rho kinase are involved in
basic fibroblast growth factor-stimulated fibroblast—collagen
matrix contraction. Journal of Cellular Biochemistry 102,
1290.
Agarwal, N., Pacher, P., Tegeder, I., Amaya, F., Constantin, C.E.,
Brenner, G.J., Rubino, T., Michalski, C.W., Marsicano, G.,
Monory, K., Mackie, K., Marian, C., Batkai, S., Parolaro, D.,
Fischer, MJ., Reeh, P., Kunos, G., Kress, M., Lutz, B., Woolf,
CJ., Kuner, R., 2007. Cannabinoids mediate analgesia largely
via peripheral type 1 cannabinoid receptors in nociceptors.
Nature Neuroscience 10, 870–879.
Aguado, T., Romero, E., Monory, K., Palazuelos, J., Sendtner, M.,
Marsicano, G., Lutz, B., Guzman, M., Galve-Roperh, I., 2007.
The CB1 cannabinoid receptor mediates excitotoxicity-
induced neural progenitor proliferation and neurogenesis.
Journal of Biological Chemistry 282, 23892–23898.
Ashton, JC., 2007. Cannabinoids for the treatment of inflamma-
tion. Current Opinion in Investigational Drugs 8, 373–384.
Ashton, JC., Appleton, I., Darlington, CL., Smith, PF., 2004.
Cannabinoid CB1 receptor protein expression in the rat
choroid plexus: a possible involvement of cannabinoids in the
regulation of cerebrospinal fluid. Neuroscience Letters 364,
40–42.
Bari, M., Oddi, S., De Simone, C., Spagnolo, P., Gasperi, V.,
Battista, N., Centonze, D., Maccarrone, M., 2007. Type-1
cannabinoid receptors colocalize with caveolin-1 in neuronal
cells. Neuropharmacology 54, 45–50.
Bensaid, M., Gary-Bobo, M., Esclangon, A., Maffrand, JP., Le Fur,
G., Oury-Donat, F., Soubrie, P., 2003. The cannabinoid CB1
receptor antagonist SR141716 increases Acrp30 mRNA ex-
pression in adipose tissue of obese fa/fa rats and in cultured
adipocyte cells. Molecular Pharmacology 63, 908–914.
Berger, A., Crozier, G., Bisogno, T., Cavaliere, P., Innis, S., Di
Marzo, V., 2001. Anandamide and diet: inclusion of dietary
arachidonate and docosahexaenoate leads to increased brain
levels of the corresponding N-acylethanolamines in piglets.
ARTICLE IN PRESS
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 179
Proceedings of the National Academy of Sciences of the
United States of America 98, 6402–6406.
Berghuis,P.,Rajnicek,AM.,Morozov,YM.,Ross,RA.,Mulder,J.,
Urba´n,GM.,Monory,K.,Marsicano,G.,Matteoli,M.,Canty,A.,
Irving, AJ., Katona, I., Yanagawa, Y., Rakic, P., Lutz, B., Mackie,
K., Harkany, T., 2007. Hardwiring the brain: endocannabinoids
shape neuronal connectivity. Science 316, 1212–1216.
Blechschmidt, E., 1977. Beginnings of Human Life. Springer,
Berlin.
Crystal, J.D., Maxwell, K.W., Hohmann, AG., 2003. Cannabinoid
modulation of sensitivity to time. Behaviour and Brain
Research 144, 57–66.
Curtis, MA., Faull, RL., Glass, M., 2006. A novel population of the
progenitor cells expressing cannabinoid receptors in the
subependymal layer of the adult normal and Huntington’s
disease human brain. Journal of Chemical Neuroanatomy 31,
210–215.
Degenhardt, BF., Darmani, NA., Johnson, JC., Towns, LC.,
Rhodes, DC., Trinh, C., McClanahan, B., DiMarzo, V., 2007.
Role of osteopathic manipulative treatment in altering pain
biomarkers: a pilot study. Journal of the American Osteo-
pathic Association 107, 387–394.
Dickson, BJ., 2002. Molecular mechanisms of axon guidance.
Science 298, 1959–1964.
Dietrich, A., McDaniel, WF., 2004. Endocannabinoids and
exercise. British Journal of Sports Medicine 38, 536–541.
Dodd, JG., Good, MM., Nguyen, TL., Grigg, AI., Batia, LM.,
Standley, PR., 2006. In vitro biophysical strain model for
understanding mechanisms of osteopathic manipulative
treatment. Journal of the American Osteopathic Association
106, 157–166.
Dossey, L., 2007. PEAR Lab and Nonlocal Mind: Why They Matter.
Explore, New York, pp.191–196.
Dunnett, AJ., Roy, D., Stewart, A., McPartland, JM., 2007. The
diagnosis of fibromyalgia in women may be influenced by
menstrual cycle phase. Journal of Bodywork and Movement
Therapies 11, 99–105.
Emboden, WA., 1976. Adaptogens: a new formula for health?
Osteopathic Physician 73 (8), 68–73.
Engeli, S., Bohnke, J., Feldpausch, M., Gorzelniak, K., Janke, J.,
Batkai, S., Pacher, P., Harvey-White, J., Luft, FC., Sharma,
AM., Jordan, J., 2005. Activation of the peripheral endocan-
nabinoid system in human obesity. Diabetes 54, 2838–2843.
Food and Drug Administration, 2007. FDA Briefing Document:
New Drug Application 21-888. Zimulti (rimonabant). Food
and Drug Administration, Washington, DC Available at:
/www.fda.gov/ohrms/dockets/AC/07/briefing/
2007-4306b1-fda-backgrounder.pdfS.
Fowler, CJ., 2004. Possible involvement of the endocannabinoid
system in the actions of three clinically used drugs. Trends in
Pharmacological Science 25, 59–61.
Frazer, KA., Elnitski, L., Church, DM., Dubchak, I., Hardison,
RC., 2003. Cross-species sequence comparisons: a review of
methods and available resources. Genome Research 13, 1–12.
Freeman, B. (Ed.), 2004. The Ontogenetic Basis of Human
Anatomy, by Erich Blechschmidt. North Atlantic Books,
Berkeley, CA.
Fride, E., 2004. The endocannabinoid-CB(1) receptor system in
pre- and postnatal life. European Journal of Pharmacology
500, 289–297.
Galarreta, M., Erdelyi, F., Szabo, G., Hestrin, S., 2004. Electrical
coupling among irregular-spiking GABAergic interneurons
expressing cannabinoid receptors. Journal of Neuroscience
24, 9770–9778.
Gasperi, V., Fezza, F., Pasquariello, N., Bari, M., Oddi, S., Agro,
AF., Maccarrone, M., 2007. Endocannabinoids in adipocytes
during differentiation and their role in glucose uptake.
Cellular and Molecular Life Sciences 64, 219–229.
Gaus, K., Le Lay, S., Balasubramanian, N., Schwartz, MA., 2006.
Integrin-mediated adhesion regulates membrane order.
Journal of Cellular Biology 174, 725–734.
Giuffrida, A., Leweke, FM., Gerth, CW., Schreiber, D., Koethe,
D., Faulhaber, J., Klosterko¨tter, J., Piomelli, D., 2004.
Cerebrospinal anandamide levels are elevated in acute
schizophrenia and are inversely correlated with psychotic
symptoms. Neuropsychopharmacology 29, 2108–2114.
Gokoh, M., Kishimoto, S., Oka, S., Mori, M., Waku, K., Ishima, Y.,
Sugiura, T., 2005. 2-arachidonoylglycerol, an endogenous
cannabinoid receptor ligand, induces rapid actin polymeriza-
tion in HL-60 cells differentiated into macrophage-like cells.
Biochemical Journal 386, 583–589.
Gonzalez, S., Cascio, MG., Fernandez-Ruiz, J., Fezza, F., Di
Marzo, V., Ramos, JA., 2002. Changes in endocannabinoid
contents in the brain of rats chronically exposed to nicotine,
ethanol or cocaine. Brain Research 954, 73–81.
Guzman, M., 2003. Cannabinoids: potential anticancer agents.
Nature Reviews Cancer 3, 745–755.
Harbach, H., Moll, B., Boedeker, RH., Vigelius-Rauch, U., Otto,
H., Muehling, J., Hempelmann, G., Markart, P., 2007.
Minimal immunoreactive plasma beta-endorphin and de-
crease of cortisol at standard analgesia or different acu-
puncture techniques. European Journal of Anaesthesiology
24, 370–376.
He, F., Song, ZH., 2007. Molecular and cellular changes induced
by the activation of CB2 cannabinoid receptors in trabecular
meshwork cells. Molecular Vision 13, 1348–1356.
Hill, MN., Patel, S., Carrier, EJ., Rademacher, DJ., Ormerod, BK.,
Hillard, CJ., Gorzalka, BB., 2005. Downregulation of en-
docannabinoid signaling in the hippocampus following
chronic unpredictable stress. Neuropsychopharmacology 30,
508–515.
Hill, MN., Ho, WS., Sinopoli, KJ., Viau, V., Hillard, CJ., Gorzalka,
BB., 2006. Involvement of the endocannabinoid system in the
ability of long-term tricyclic antidepressant treatment to
suppress stress-induced activation of the hypothalamic–pitui-
tary–adrenal axis. Neuropsychopharmacology 31, 2591–2599.
Ho¨gestatt, ED., Jonsson, BA., Ermund, A., Andersson, DA., Bjork,
H., Alexander, JP., Cravatt, BF., Basbaum, AI., Zygmunt, PM.,
2005. Conversion of acetaminophen to the bioactive N-
acylphenolamine AM404 via fatty acid amide hydrolase-
dependent arachidonic acid conjugation in the nervous
system. Journal of Biological Chemistry 280, 31405–31412.
Hohmann, AG., Herkenham, M., 1999. Cannabinoid receptors
undergo axonal flow in sensory nerves. Neuroscience 92,
1171–1175.
Howlett, AC., Barth, F., Bonner, TI., Cabral, G., Casellas, P.,
Devane, WA., Felder, CC., Herkenham, M., Mackie, K.,
Martin, BR., Mechoulam, R., Pertwee, RG., 2002. Interna-
tional Union of Pharmacology. XXVII. Classification of canna-
binoid receptors. Pharmacological Reviews 54, 161–202.
Ingber, D., 1998. In search of cellular control: signal transduction
in context. Journal of Cellular Biochemistry Supplement
30–31, 232–237.
Johnson, D.R., Stebulis, J.A., Rossetti, R.G., Burstein, S.H.,
Zurier, R.B., 2007. Suppression of fibroblast metalloprotei-
nases by ajulemic acid, a nonpsychoactive cannabinoid acid.
Journal of Cellular Biochemistry 100, 184–190.
Klein, T.W., 2005. Cannabinoid-based drugs as anti-inflammatory
therapeutics. Nature Reviews Immunology 5, 400–411.
Koeberle, P.D., Bahr, M., 2004. Growth and guidance cues for
regenerating axons: where have they gone? Journal of
Neurobiology 59, 162–180.
ARTICLE IN PRESS
J.M. McPartland180
Langevin, H.M., Bouffard, N.A., Badger, G.J., Churchill, D.L.,
Howe, A.K., 2006. Subcutaneous tissue fibroblast cytoskele-
tal remodeling induced by acupuncture: evidence for a
mechanotransduction-based mechanism. Journal of Cellular
Physiology 207, 767–774.
Li, J.J., Chen, R.M., Liu, L., Wang, S.Y., Yu, P., Xie, Y., Li, M.,
Shi, J., 2007. Effects of electroacupuncture on the immunor-
eactivity of focal cutaneous CB2 receptor positive cells in
arthritis rats. Zhen Ci Yan Jiu 32, 9–15.
Mancall, A.C., DiGregorio, G.J., Brill, C.B., Ruch, E., 1985. The
effect of delta-9-tetrahydrocannabinol on rat cerebrospinal
fluid. Archives of Neurology 42, 1069–1071.
Martin, M., Ledent, C., Parmentier, M., Maldonado, R., Valverde,
O., 2002. Involvement of CB1 cannabinoid receptors in
emotional behaviour. Psychopharmacology (Berl) 159, 379–387.
Matias, I., Gonthier, M.P., Orlando, P., Martiadis, V., De
Petrocellis, L., Cervino, C., Petrosino, S., Hoareau, L., Festy,
F., Pasquali, R., Roche, R., Maj, M., Pagotto, U., Monteleone,
P., Di Marzo, V., 2006. Regulation, function and dysregulation
of endocannabinoids in models of adipose and beta-pancrea-
tic cells and in obesity and hyperglycemia. Journal of Clinical
Endocrinology and Metabolism 91, 3171–3180.
Matias, I., Di Marzo, V., 2007. Endocannabinoids and the control
of energy balance. Trends in Endocrinology and Metabolism
18, 27–37.
Ma´tya´s, F., Urba´n, GM., Watanabe, M., Mackie, K., Zimmer, A.,
Freund, T.F., Katona, I., 2007. Identification of the sites of 2-
arachidonoylglycerol synthesis and action imply retrograde
endocannabinoid signaling at both GABAergic and glutama-
tergic synapses in the ventral tegmental area. Neuropharma-
cology 54, 95–107.
Mbvundula, E.C., Bunning, R.A., Rainsford, K.D., 2005. Effects of
cannabinoids on nitric oxide production by chondrocytes and
proteoglycan degradation in cartilage. Biochemical Pharma-
cology 69, 635–640.
McPartland, J.M., Guy, G., 2004. The evolution of Cannabis and
coevolution with the cannabinoid receptor—a hypothesis. In:
Guy, G., Robson, R., Strong, K., Whittle, B. (Eds.), The
Medicinal Use of Cannabis. Royal Society of Pharmacists,
London, pp. 71–102.
McPartland, J.M., Simons, D.G., 2007. Myofascial trigger points:
translating molecular theory into manual therapy. Journal of
Manual and Manipulative Therapies 14, 232–239.
McPartland, J.M., Skinner, E., 2005. The biodynamic model of
osteopathy in the cranial field. Explore (NY) 1 (1), 21–32.
McPartland, J.M., Giuffrida, A., King, J., Skinner, E., Scotter, J.,
Musty, R.E., 2005. Cannabimimetic effects of osteopathic
manipulative treatment. Journal of the American Osteo-
pathic Association 105, 283–291.
McPartland, J.M., Matias, I., Di Marzo, V., Glass, M., 2006.
Evolutionary origins of the endocannabinoid system. Gene
370, 64–74.
McPartland, J.M., Norris, R.W., Kilpatrick, C.W., 2007. Tempo
and mode in the endocannabinoid system. Journal of
Molecular Evolution 65, 267–276.
Mechoulam, R., Fride, E., Di Marzo, V., 1998. Endocannabinoids.
European Journal of Pharmacology 359, 1–18.
Mense, S., Simons, DG., Hoheisel, U., Quenzer, B., 2003. Lesions
of rat skeletal muscle after local block of acetylcholinester-
ase and neuromuscular stimulation. Journal of Applied
Physiology 94, 2494–2501.
Newman, S.A., Frenz, D.A., Tomasek, J.J., Rabuzzi, D.D., 1985.
Matrix-driven translocation of cells and nonliving particles.
Science 228, 885–889.
Newman, Z., Malik, P., Wu, T.Y., Ochoa, C., Watsa, N., Lindgren,
C., 2007. Endocannabinoids mediate muscarine-induced
synaptic depression at the vertebrate neuromuscular junc-
tion. European Journal of Neuroscience 25, 1619–1630.
O’Leary, D.S., Block, R.I., Turner, B.M., Koeppel, J., Magnotta,
V.A., Ponto, L.B., 2003. Marijuana alters the human
cerebellar clock. Neuroreport 14, 1145–1151.
Oliva, J.M., Uriguen, L., Perez-Rial, S., Manzanares, J., 2005.
Time course of opioid and cannabinoid gene transcription
alterations induced by repeated administration with fluox-
etine in the rat brain. Neuropharmacology 49, 618–626.
Ortiz, S., Oliva, J.M., Perez-Rial, S., Palomo, T., Manzanares, J.,
2004. Chronic ethanol consumption regulates cannabinoid
CB1 receptor gene expression in selected regions of rat brain.
Alcohol and Alcoholism 39, 88–92.
Oschman, J.L., 2000. The electromagnetic environment: im-
plications for bodywork: Part 2: Biological effects. Journal of
Bodywork and Movement Therapies 4 (2), 137–150.
Pacher, P., Ba´tkai, S., Kunos, G., 2006. The endocannabinoid
system as an emerging target of pharmacotherapy. Pharma-
cological Reviews 58, 389–462.
Panagiotis, M., Lloyd, DH., Pfeiffer, D., Stevens, K., Auxilia, S.,
Noli, C., Abramo, F., Miolo, A., 2007. Veterinary wounds:
assessment of the effect of an aliamide-containing topical gel
by evaluation of the reduction of wound volume measured by
high-resolution ultrasound biomicroscopy. Wounds 19 (5),
113–119.
Park, B., McPartland, J.M., Glass, M., 2004. Cannabis, cannabi-
noids and reproduction. Prostaglandins, Leukotrienes and
Essential Fatty Acids 70, 189–197.
Pert, C., 2000. Foreword. In: Oschman, J.L. (Ed.), Energy
Medicine. Churchill Livingstone, Edinburgh pp. ix–xi.
Pertwee, R.G., 2005. The therapeutic potential of drugs that
target cannabinoid receptors or modulate the tissue levels or
actions of endocannabinoids. AAPS Journal 7, E625–E654.
Pevsner, J., 2002. Leonardo da Vinci’s contributions to neu-
roscience. Trends in Neuroscience 25, 217–220.
Raduner, S., Majewska, A., Chen, J.Z., Xie, X.Q., Hamon, J.,
Faller, B., Altmann, K.H., Gertsch, J., 2006. Alkylamides
from Echinacea are a new class of cannabinomimetics.
Cannabinoid type 2 receptor-dependent and -independent
immunomodulatory effects. Journal of Biological Chemistry
281, 14192–14206.
Rimmerman, N., Hughes, H.V., Bradshaw, H.B., Pazos, M.X.,
Mackie, K., Prieto, A.L., Walker, J.M., 2007. Compartmenta-
lization of endocannabinoids into lipid rafts in a dorsal root
ganglion cell line. British Journal of Pharmacology 153,
380–389.
Rousseaux, C., Thuru, X., Gelot, A., Barnich, N., Neut, C.,
Dubuquoy, L., Dubuquoy, C., Merour, E., Geboes, K., Chamail-
lard, M., Ouwehand, A., Leyer, G., Carcano, D., Colombel, J.F.,
Ardid, D., Desreumaux, P., 2007. Lactobacillus acidophilus
modulates intestinal pain and induces opioid and cannabinoid
receptors. Nature Medicine 13, 35–37.
Russo, E.B., 2004. Clinical endocannabinoid deficiency (CECD):
can this concept explain therapeutic benefits of cannabis in
migraine, fibromyalgia, irritable bowel syndrome and other
treatment-resistant conditions? Neuro Endocrinology Letters
25, 31–39.
Sa´nchez-Pastor, E., Trujillo, X., Huerta, M., Andrade, F., 2007.
Effects of cannabinoids on synaptic transmission in the frog
neuromuscular junction. Journal of Pharmacology and Ex-
perimental Therapeutics 321 (2), 439–445.
Sarnataro, D., Grimaldi, C., Pisanti, S., Gazzerro, P., Laezza, C.,
Zurzolo, C., Bifulco, M., 2005. Plasma membrane and
lysosomal localization of CB1 cannabinoid receptor are
dependent on lipid rafts and regulated by anandamide in
human breast cancer cells. FEBS Letters 579, 6343–6349.
ARTICLE IN PRESS
Expression of the endocannabinoid system in fibroblasts and myofascial tissues 181
Scutt, A., Williamson, E.M., 2007. Cannabinoids stimulate
fibroblastic colony formation by bone marrow cells indirectly
via CB2 receptors. Calcified Tissue International 80, 50–59.
Shah, J.P., Phillips, T.M., Danoff, J.V., Gerber, L.H., 2005. An in
vivo microanalytical technique for measuring the local
biochemical milieu of human skeletal muscle. Journal of
Applied Physiology 99, 1977–1984.
Sioutis, J., Roussos, N., Manoleas, J., Patergianakis, N.,
Charalabidis, C., Karantonis, G., Lagogiannis, N., 2004.
The memory phenomenon and recurrent musculoskeletal
pain. Spinal Cord Journal 42 (Suppl.), S163 Available at:
/www.iscos.org.uk/abstracts25.htmlS.
Sparling, P.B., Giuffrida, A., Piomelli, D., Rosskopf, L., Dietrich,
A., 2003. Exercise activates the endocannabinoid system.
Neuroreport 14, 2209–2211.
Sutherland, W.G., Sutherland, A.S., Wales, A. (Eds.), 1967.
Contributions of Thought, the Collected Writings of William
Garner Sutherland. Sutherland Cranial Teaching Foundation,
Kansas City, MO.
Vann, R.E., Cook, C.D., Martin, B.R., Wiley, J.L., 2007.
Cannabimimetic properties of ajulemic acid. Journal of
Pharmacology and Experimental Therapeutics 320, 678–686.
Ware, M.A., Adams, H., Guy, G.W., 2005. The medicinal use of
cannabis in the UK: results of a nationwide survey. Interna-
tional Journal of Clinical Practice 59, 291–295.
Watanabe, S., Doshi, M., Hamazaki, T., 2003. N-3 polyunsatu-
rated fatty acid (PUFA) deficiency elevates and n-3 PUFA
enrichment reduces brain 2-arachidonoylglycerol level in
mice. Prostaglandins, Leukotrienes and Essential Fatty Acids
69, 51–59.
Williams, E.J., Walsh, F.S., Doherty, P., 2003. The FGF receptor
uses the endocannabinoid signaling system to couple to an
axonal growth response. Journal of Cellular Biology 160,
481–486.
Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M.,
Bonner, T.I., 1999. Increased mortality, hypoactivity and
hypoalgesia in cannabinoid CB1 receptor knockout mice.
Proceedings of the National Academy of Sciences of the
United States of America 96, 5780–5785.
Zou, Y., Akazawa, H., Qin, Y., Sano, M., Takano, H., Minamino,
T., Makita, N., Iwanaga, K., Zhu, W., Kudoh, S., Toko, H.,
Tamura, K., Kihara, M., Nagai, T., Fukamizu, A., Umemura,
S., Iiri, T., Fujita, T., Komuro, I., 2004. Mechanical
stress activates angiotensin II type 1 receptor without
the involvement of angiotensin II. Nature Cell Biology 6,
499–506.
Zuardi, A.W., Crippa, J.A., Hallak, J.E., Moreira, F.A., Guimar-
aes, F.S., 2006. Cannabidiol, a Cannabis sativa constituent,
as an antipsychotic drug. Brazilian Journal of Medical and
Biological Research 39, 421–429.
ARTICLE IN PRESS
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