Increased synovial lipodystrophy induced by high fat diet aggravates synovitis in experimental osteoarthritis
Larrañaga-Vera et al. Arthritis Research & Therapy
Increased synovial lipodystrophy induced by high fat diet aggravates synovitis in experimental osteoarthritis
Ane Larrañaga-Vera 0 2
Ana Lamuedra 0 2
Sandra Pérez-Baos 0 2
Ivan Prieto-Potin 0 2
Leticia Peña 1
Gabriel Herrero-Beaumont 0 2
Raquel Largo 0 2
0 Bone and Joint Research Unit, IIS-Fundación Jiménez Díaz UAM , Avda. Reyes Católicos, 2, Madrid 28040 , Spain
1 Clinical Analysis Department, HU-Fundación Jiménez Díaz , Madrid , Spain
2 Bone and Joint Research Unit, IIS-Fundación Jiménez Díaz UAM , Avda. Reyes Católicos, 2, Madrid 28040 , Spain
Background: Metabolic syndrome (MetS) may be associated with knee osteoarthritis (OA), but the association between the individual components and OA are not well-understood. We aimed to study the effect of hypercholesterolemia on synovial inflammation in knee OA. Methods: OA was surgically induced in rabbits fed with standard diet (OA group, n = 10) or in rabbits fed with high fat diet (OA-HFD, n = 10). Healthy rabbits receiving standard diet (Control, n = 10) or fed with HFD (HFD, n = 6) were also monitored. Twelve weeks after OA induction, synovial membranes were isolated and processed for studies. Results: Animals fed HFD showed higher levels of total serum cholesterol, triglycerides and C-reactive protein than control rabbits. Twelve weeks after OA induction, synovial membrane inflammation and macrophage infiltration were increased in rabbits with OA, particularly in the OA-HFD group. Extensive decrease of synovial adipose tissue area, adipocyte size and perilipin-1A synthesis were observed in the OA-HFD group in comparison to the OA and control groups. The HFD further increased the proinflammatory mediators IL-1β, IL-6 and TNF in the OA synovium. However, the synovial gene expression of adipokines, such as leptin and adiponectin, were markedly decreased in the rabbits with OA, especially in the OA-HFD group, in correlation with adipose tissue loss. However, circulating leptin was upregulated in the HFD and OA-HFD groups. Conclusion: Our results indicate that a HFD is an aggravating factor worsening synovial membrane inflammation during OA, guided by increased infiltration of macrophages and removal of the adipose tissue, together with a remarkable presence of proinflammatory factors. Synovial adipocytes and dyslipemia could probably play pivotal roles in OA joint deterioration in patients with MetS, supporting that the link between obesity and OA transcends mechanical loading.
Osteoarthritis; Hypercholesterolemia; Synovial inflammation; Metabolic syndrome; Macrophages; Synovial adipose tissue; Adipokines
Osteoarthritis (OA) is the most common joint disorder
worldwide, characterized by joint pain, impaired mobility
and structural changes in the joints. Although cartilage
destruction is the main feature of the disease, every joint
structure such as the synovium, bone, meniscus or
muscle is affected, leading to the recognition of OA as a
whole-organ disease [
]. Synovial inflammation is present
in a substantial population of patients with OA and has
been associated with different signs and symptoms of the
disease, including increased pain and joint effusion, which
could promote more rapid cartilage degeneration [
Elevated thickness of the lining layer and greater presence
and activation of synovial macrophages have been
identified in cartilage degradation and osteophyte formation in
both human and experimental OA [
OA is not merely a local disease, but there are different
systemic processes that determine its progression. The
concept of metabolic OA, coined in recent years, identifies
a syndrome whereby the contribution of metabolic
dysregulation and low-grade systemic inflammation to the
progression of the disease has been firmly pointed out [
]. The metabolic syndrome (MetS) comprises a cluster of
conditions, including glucose intolerance, high blood
pressure, hypercholesterolemia and hypertriglyceridemia, and
]. The accumulation of the different
components of MetS has been related to both the occurrence
and progression of knee OA [
]. However, little is
known about the specific contribution of each of these
metabolic alterations in OA progression, and specifically
in the synovial damage associated with OA.
The contribution of obesity to OA progression is
probably the most extensively studied association [
In fact, obesity has been pointed out as the main
contributing factor for the association between OA and
MetS, in studies showing a markedly attenuated
association after adjustment for body mass index .
However, OA is also common in non-weight bearing joints of
obese persons, suggesting a systemic mechanism rather
than a simply mechanic phenomenon [
The possible role of hyperlipidemia in mediating
obesity-related effects on OA has been explored in
different studies. Contradictory results have been published
on the relationship between serum lipids and OA
incidence in humans, probably due to the presence of
obesity and being overweight as confounding factors [
turn, different experimental studies have suggested that
hypercholesterolemia could be mainly associated with
osteophyte generation rather than to aggravation of
cartilage lesions [
]. Macrophages, endothelial cells
and fibroblasts are dominant cells within the synovium,
together with abundant adipose tissue that constitute
the synovial stroma, and every component is sensitive to
changes in lipid levels [
Adipokines have been considered at least partially
responsible for the link between systemic metabolic
alterations and OA [
]. Adipokines are essentially
released by adipocytes and exhibit pleiotropic functions
both in central and peripheral systems, including blood
pressure control, hemostasis, food intake, energy
expenditure, cell metabolism and inflammation, among others
]. They are also synthesized by joint cells during
OA, mainly by the synovium, cartilage and
intraarticular fat tissue, and have been demonstrated to play
proinflammatory and catabolic or anabolic roles in OA
pathophysiology. It has been hypothesized that the
altered circulating patterns of adipokines induced by obesity
could be responsible for the deleterious effect of this
disease on OA. However, it is not known whether expression
and release of adipokines in the joint could be modulated
by metabolic factors during OA, thus contributing to
Therefore, this work aimed to study the effect of
hypercholesterolemia, without any other component of
the MetS, on synovial inflammation in an experimental
model of knee OA. We have also determined the
synovial expression and systemic concentration of
adiponectin and leptin, two adipokines involved in joint
deterioration associated with metabolic OA.
Thirty-six New Zealand male white rabbits, 13–15 weeks
of age, weighing 2.5–3.0 kg (Granja San Bernardo, Navarra,
Spain) were housed individually in cages with transparent
walls (0.5 m cage height and 0.6 m2 floor space) exposed
to a 12-hour light/dark cycle.
After 2 weeks of adaptation to our facilities, 16 rabbits
started receiving a high fat diet (HFD) (0.5% cholesterol
+ 4% peanut oil; S9504-S010; 22% kJ from fat, 20% kJ
from proteins and 58% kJ from carbohydrates; Ssniff,
Soest, Germany) administered ad libitum (Fig. 1a, time
point 0). At this time point, there were no significant
differences between the group on HFD and the one that
remained at standard diet (112; 10% kJ from fat, 17% kJ
from proteins; 73% kJ from carbohydrates; Safe-Diets,
Augy, France) regarding body weight or age, as can be
observed in Fig. 1b. Six weeks later, bilateral
osteoarthritis (OA) was surgically induced in 10 of these 16
animals (OA-HFD group, n = 10) by anterior cruciate
ligament transection and partial medial meniscectomy
] (week 6, Fig. 1a). At this time point, OA was also
induced in 10 rabbits fed with standard diet (OA group,
n = 10). The surgery was always performed in the
morning after overnight fasting, under general anesthesia
(intramuscular administration 20 mg/ml xylazine
(Rompun, Bayer, Kiel, Germany) and 50 mg/ml ketamine
(Ketolar, Pfizer, Hameln, Germany) in a 3:1 ratio), under
aseptic conditions in an operating room. Besides, 10
rabbits fed with standard diet (control group, n = 10), and
six rabbits fed with the HFD (HFD group, n=6)
underwent no experimental intervention.
Two animals in the OA-HFD group died during the
time of the study due to OA surgery complications.
Weight gain was monitored every week. Systolic blood
pressure (SBP) was measured before OA surgery and 1
week before euthanasia, using a High Definition
Oscillometry unit (DVM Solutions Houston TX, USA) adapted
to the hind paw of the rabbits. This non-invasive
method for SBP measurement has been validated in cats,
an animal physiologically and anatomically similar to
Twelve weeks after OA induction (Fig. 1),
overnightfasted rabbits were bled from their marginal ear vein in
the morning and killed by an intracardiac injection of
pentobarbital (50 mg/kg, Tiobarbital, Braun medical S.A.
Barcelona, Spain). The articular cavity of each rabbit was
accessed by sectioning the patellar tendon and taking
out the patella, thus the entire infrapatellar synovial
membrane (SM) was collected by the same operator
(AL-V), always taking the same specimen from each
animal (Additional file 1: Figure S1). The SM was not
separated from the adipose tissue [
]. Half of the SM
containing both stroma and lining was then fixed in 4%
paraformaldehyde for 24 h and then was embedded in
paraffin for histological studies; the other portion was
immediately frozen and used for molecular biology
studies. Femoral condyles were also removed and fixed in 4%
buffered paraformaldehyde, decalcified for 4 weeks in a
solution of 10% formic acid plus 5% paraformaldehyde,
and embedded in paraffin . The left and right SM
and condyles were analyzed as independent samples.
Serum and synovial measurements
Glucose, total cholesterol, HDL cholesterol and
triglyceride levels were assayed by automatic techniques as
previously described [
]. Adiponectin, leptin and plasma
C-reactive protein (CRP) were measured by ELISA using
commercial specific kits (SEA605Rb and SEA084Rb,
respectively, USCN, Houston TX, USA and ab157726,
Abcam, Cambridge, UK). Both adiponectin and leptin
were measured in synovial tissue homogenates. For this
purpose, total protein from the SM was extracted as
described elsewhere [
], and equal amounts of
proteins diluted in the same volume for each knee were
tested by specific ELISA for each adipokine.
Histological synovitis grading
The SM from both knees of each rabbit were sectioned
5-μm thick and stained with hematoxylin and eosin, and
Masson’s Trichrome. Synovitis was evaluated according
to the Krenn score [
] as previously described [
assessing lining hyperplasia, activation of synovial
stroma related to fibrosis, and tissue infiltration. Each
item was evaluated by a blinded observer using a
subscale of 0− 3 points, where 0 indicated absence, 1 mild,
2 intermediate and 3 strong evidence of synovitis. The
total score was obtained from the sum of partial grades
with a maximum total score of 9.
Histological cartilage grading
The decalcified femurs were cleaved in a sagittal plane
along the central portion of the articular surface of each
medial femoral condyle corresponding to the
weightbearing area, and subsequently embedded in paraffin
wax. Cartilage was sectioned 5-μm thick and stained
with hematoxylin/eosin and alcian blue to evaluate
cartilage abnormalities. These samples were evaluated using
a modified version of Mankin's grading score system,
which analyses four different parameters with a total
score up to 21: structure (0–8), proteoglycan staining
(0–6), loss of chondrocytes (0–4), and clone formation
SM infiltrating macrophages were visualized using
mouse anti-rabbit macrophage monoclonal antibodies
(mAb) (RAM11; Dako, Glostrup, Denmark) as
previously described [
], whereas adipocytes were identified
with anti-perilipin A1 (PLIN, Abcam, ab61682, 1/100
dilution) antibody. To evaluate RAM11-positive
immunoreactivity, five photographs were obtained using a Leica
DMD108 digital micro-imaging instrument (Leica,
Microsystems, Inc. Buffalo Grove, IL, USA) at × 10
magnification ensuring constant light exposure. Each image
was analyzed with ImageJ software (NIH, Bethesda, MD,
USA), and the percentage of positive area was calculated
with the Color Deconvolution plugin [
] in relation to
the total tissue area. For each SM, the percentage of
positive staining was calculated as the mean of these five
images corresponding to the same SM [
Adipose tissue area (%ATA) and adipocyte size were
analyzed in PLIN-stained slides using the Coreo Iscan Au
Scanner (Ventana Medical Systems, USA) and ImageJ
software. Five representative images at × 20
magnification were used to identify stained adipocyte boundaries.
Every white area showing no immunoreactivity to PLIN
was manually removed. Finally, the area of each
adipocyte was measured and the average size was calculated
for each SM sample.
Briefly, total protein was extracted from the SM as
described elsewhere [
]. Protein extracts were separated
by SDS-PAGE and transferred to a polyvinylidene fluoride
membrane. The following primary antibodies were applied
overnight at 4 °C: anti-human collagen type I (Col I,
Merck Millipore, Billerica, MA, USA); anti-human PLIN
(Abcam), anti-rabbit IL-1, anti-rabbit IL-6, anti-rabbit
TNF (Cloud-Clone Corp, Houston TX, USA), and
antihuman cyclooxygenase-2 (COX-2) (Santa Cruz
Biotechnology, Dallas TX, USA). Loading control was performed
employing EZBlue gel staining reagent (Sigma-Aldrich).
Results were normalized relative to total protein presence
and expressed as arbitrary densitometric units  (AU).
Total RNA was extracted from SM using TriPure Isolation
Reagent (Roche Diagnostics, Indianapolis, IN, USA),
according to the manufacturer’s instructions. RNA was
reverse-transcribed and RNA expression was quantified
using the StepOnePlus™ detection system and StepOne™
software v2.2 (Applied Biosystems) as previously described
]. TaqMan® primers and probes were used to
measure adiponectin (Oc03823307_s1), leptin (Oc03395809_s1)
and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH
Oc03823402_g1) as endogenous control. Target genes
were normalized relative to the expression of the
Histological analyses were carried out by two observers
(AL-V and RL) in a blinded fashion. Scoring and
quantitative analyses were averaged for the images and sections
from the same SM to calculate the value per sample for
statistical analyses. Each limb was analyzed as an
independent sample for the studies of synovial tissue. All
statistical analyses were performed using GraphPad
Prism version 5.0 for Windows (GraphPad Software, San
Diego, CA, USA). We employed the non-parametric
Kruskal-Wallis test with a post-hoc correction for
(Dunn’s procedure) for comparisons between multiple
groups, and the Mann-Whitney U test for comparisons
between two groups. P values less than 0.05 were
considered significant. Data are expressed as the mean ±
95% confidence interval (CI).
We first studied the effect of the HFD in rabbits over an
18-week period in order to ensure the different
characteristics that have been associated with MetS, such as
being overweight, hypertension, basal glucose and
dyslipidemia. There were no significant differences between
the different groups in weight gain at week 6, the time
point of surgery to induce OA (Fig. 1b). At the end of
the study after 18 weeks of HFD feeding, rabbits fed a
HFD gained less weight than controls (Table 1). Animals
in the OA and OA-HFD groups also gained less weight
than controls, probably due to discomfort associated
with knee surgery. Rabbits in the HFD, OA and
OHFDA groups maintained similar SBP to control animals
during the whole study period (Table 1). After 18 weeks
of HFD, rabbits did not have any alteration in basal
Group Weight gain (kg) SBP (mmHg) Basal glucose (mg/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) HDL (mg/dl) CRP (μg/ml)
Control (n = 10) 1.9 (1.7–2.0) 100 (93–107) 109 (101–116) 32.2 (8.9–55.5) 48 (32–64) 11.1 (8.6–13.6) 15.95 (8.2–23.7)
HFD (n = 6)
OA (n = 9)
Measures were obtained from serum or plasma samples taken just before animals were killed. Values represent mean with 95% confidence interval
HFD high-fat diet, OA osteoarthritis, SBP systolic blood pressure, HDL high-density lipoprotein, CRP C-reactive protein
*P < 0.05 vs. Control
glucose levels or in oral glucose tolerance (data not
shown) in comparison to control animals (Table 1).
However, there was increased total serum cholesterol
and triglycerides in the rabbits fed HFD in comparison
to controls. Although no significant differences were
observed in circulating CRP levels between either the HFD
or OA-HFD groups and controls (Table 1), there was a
significant increase in CRP in animals fed with HFD vs.
those fed with the standard diet, as a result of grouping
rabbits into HFD plus OA-HFD and control plus OA
(27.1 ± 7.3 vs 10.5 ± 1.9, p = 0.026).
Histological synovial inflammation and cartilage damage
Rabbits fed HFD had mild lining hyperplasia, discrete
presence of infiltrating cells and a slight increment in
stromal fibrosis, and thus the synovitis score was
significantly higher than that observed in healthy controls
(Fig. 2b, 2i). The OA group had a higher synovitis score
than the control and HFD groups, with similar lesions to
those described in synovitis in humans with advanced
OA: mild to moderate lining hyperplasia, discrete
presence of inflammatory cells, and stromal activation. The
OA-HFD group had mild lining thickening, a clear
increment in stromal cellularity, presence of infiltrating
cells and inflammatory foci. All samples had enlarged
stroma with an intense cell density (Fig. 2d). The
synovitis score in the OA-HFD group was significantly higher
than in the other groups (Fig.2i).
The HFD administration did not modify the
histological appearances of cartilage damage, with the HFD
group having a similar score to control animals (HFD
2.8 ± 1.5 vs. control 2.9 ± 1.0; p not significant (NS)). In
addition, HFD did not significantly modify the
histopathological damage in the cartilage in the OA-HDF
group in comparison to the damage observed in the OA
group (OA 15.2 ± 2.1 vs. OA-HFD 14.0 ± 2.7; p NS).
Macrophage infiltration and presence of foam cells
There was moderate presence of macrophages in the SM
of rabbits fed HFD, which were especially localized in
the lining layer (Fig. 2f, j). Lipid droplets were identified
in their cytoplasm and their morphological shape
resembled to pro-atherosclerotic foam cells, as previously
] (Fig. 2f ). RAM11 staining was scarce in
the SM in the OA group, whereas there was extensive
infiltration of RAM11-positive cells in the OA-HFD
group to a much greater extent than in the HFD and OA
groups (Fig. 2g, h, j). They were both consistently localized
in the lining and sub-lining layers in every sample, and had
the characteristic phenotype of foam cells [
] (Fig. 2h).
Characterization of synovial stroma
Whereas healthy SM was mainly composed of
adipocytes with little surrounding matrix, we observed patchy
distribution of some fibrotic areas in the HFD group
(Fig. 3a, b). OA membranes had a highly vascularized
fibrotic stroma with some lax and dense stents,
greencolored on Masson’s Trichrome staining (Fig. 3).
OAHFD samples also had highly vascularized fibrotic
membranes. The quantification of col I protein revealed
a clear increase in the fibrotic content of the SM in the
OA and OA-HFD groups (Fig. 3e-f ) in comparison to
control and HFD groups.
Adipose tissue area and adipocyte size in the SM
We quantified the adipose tissue fraction using PLIN
staining, a distinguishing marker of adipocytes [
A clear diminution in the percentage of adipose tissue
area (%ATA) in the SM in the OA and OA-HFD samples
in comparison to control and OA groups was observed,
which was even lower in the OA-HFD than in the OA
group (Fig 4a-e). Furthermore, SM adipocytes were
significantly smaller in both the OA and OA-HFD groups
than in the controls (Fig. 4f ). The shape of these cells in
control tissues was regular (Fig. 4a), whereas we
observed high heterogeneity in the appearance of these
cells in the SM in the OA and OA-HFD groups (Fig. 4c, d).
Adipocyte size further decreased in the SM in the
OAHFD group in comparison to the OA group (Fig. 4f ). PLIN
content in the SM was also evaluated by western blot. In
correlation with the %ATA, there was diminution in the
SM PLIN in the OA and OA-HFD groups in comparison
to the controls (Fig. 4g, h). Of note, the synthesis of PLIN
was also significantly diminished in the OA-HFD group in
comparison to the OA group.
Adipokine gene expression and concentration in SM and serum
Rabbits in both the HFD and OA groups had a clear
decrease in leptin and adiponectin gene expression in the
SM in comparison to controls (Fig. 5a, b). We observed
an additive effect of these interventions in the OA-HFD
group, where the gene expression of both leptin and
adiponectin was significantly lower to that observed in the
HFD and OA groups (Fig. 5). Interestingly, there was
significant correlation between adipokine gene expression
and the %ATA (R = 0.746; p = 0.001 for leptin expression;
R = 0.732; p = 0.002 for adiponectin expression). Leptin
levels in the SM measured by ELISA were decreased in
the HFD group in comparison to controls, and it was also
significantly reduced in the SM in the OA-HFD group in
comparison to controls, the HDF and the OA groups
(Fig. 5c). Adiponectin concentration only significantly
diminished in the HFD group in comparison to controls
(Fig. 5d). There was no correlation between the presence
of these proteins and the %ATA in the SM.
However, HFD increased the circulating concentration
of both adipokines, and there were no significant
(See figure on previous page.)
Fig. 2 Histopathological and macrophage analysis in the synovial membrane (SM). a-d Representative sections of SM stained with hematoxylin-eosin
or e-h stained with a monoclonal anti-rabbit macrophage antibody (RAM11) from Control rabbit (a and e); rabbit fed with a high-fat diet (HFD) (b and f);
osteoarthritic (OA) rabbit (c and g); and OA rabbit fed with a HFD (OA-HFD) (d and h). a-d Scale bar = 100 μm. e-f Scale bar = 100 μm. i Synovitis
score quantified as described in “Methods. j Quantification of RAM11-positive area represented as percentage of total area. Data from individual
measurements and mean for each group are shown. n = 12–20 SM per group for histopathological analysis; n = 10–16 SM per group for
differences between the HFD and OA-HFD group in the
serum concentration of these mediators (Fig. 5e, f ).
Synovial proinflammatory mediators
We then explored whether the HFD was able to modify
the presence of different proinflammatory cytokines,
such as IL-1β, IL-6, TNF and COX-2 in the SM of
rabbits in the OA group. As expected, western blot
studies that OA induced a marked increase in the presence
of all the studied proinflammatory mediators in
comparison to control animals. The presence of
hyperlipidemia further increased the presence of IL-1 β, IL-6 and
TNF in the SM in the OA-HFD group in comparison to
the OA group (Fig. 6).
In this study, we have shown that HFD aggravated
OA synovitis, by inducing severe tissue architecture
disorganization of the synovium, along with
remarkable intensification of the proinflammatory cytokines
IL-1β, IL-6 and TNF, and extensive infiltration of
macrophages. However, HFD did not have any effect
on the aggravation of the pathologic change in
cartilage associated with OA. A relevant histological
synovial alteration was the significant loss of synovial
adipose tissue content, in correlation with decreased
leptin and adiponectin gene expression.
In order to isolate the effect of hyperlipidemia, we
employed an experimental model of HFD intake that
was not associated with weight gain [
]. The lack of
significant weight gain in the HFD group has been
previously reported and attributed to the animal
selfregulation of caloric intake [
]. In fact, animals in both
the HFD and OA-HFD groups had a significant decrease
in weight gain, which was probably related to the
increase in systemic inflammation induced by the diet
]. Different studies using lipid-rich diets have not
been able to adequately apportion the contribution of
added mechanical load and hyperlipidemia in OA, a
factor that was avoided in our experiments.
Different patterns of synoviopathy have been described
in patients with OA, both in late and early disease, such
as those with an increased fibrotic component or those
essentially characterized by augmented inflammatory
]. In our rabbits, OA synovitis was
associated with a significant increment of fibrotic tissue and
partial loss of adipose tissue, and scarce presence of
macrophages. A HFD induced both qualitative and
quantitative changes in the SM in the rabbits with OA.
However, HFD did not significantly aggravate cartilage
damage in either the HFD group or the OA-HFD group.
These data are in line with previously published results
], and suggest that the aggravation in synovial
inflammation induced by HFD is not a secondary event
induced by more severe pathological change in the
The higher grade of synovial inflammation in the
OA-HFD group was characterized by the remodeling of
adipose tissue and by adipocyte loss. The remaining
adipocytes had heterogeneous morphology and were
significantly smaller in comparison to the OA and control
groups, confirmed by the decreased presence of PLIN.
HFD further increased synovial macrophages, most of
them with the appearance of foam cells, whereas the
fibrotic component was similar to that observed in the
OA group. To our knowledge, this is the first report of
correlation between synovial inflammation and loss of
adipose tissue in this localization. Contradictory reports
have been published on the contribution of the volume
or area of the intra-articular fat tissue to joint
deterioration and OA symptoms [
]. However, the
alterations in adipocyte size, morphology, loss of adipose
tissue with increased fibrosis and inflammatory content
have been well-described in inflamed adipose tissue in
other anatomic localizations, and described as
]. The study of the synovial fat pad as an
independent adipose intra-articular tissue has
contributed to its identification as a crucial player in OA
progression , although lack of recognition of the
synovium as a whole, integrated, functional and
structural unit hampers the understanding of the
mechanisms involved in the synovial alterations in OA.
Histologically, synovial lining, adipose sub-lining and
synovial fat pad represent a continuum. Sub-lining
adipose tissue and the fat pad seem to share a common
inflammatory state, both in cell content and cell
phenotype, induced by the disease process more than
by tissue-specific signals [
The mechanisms by which adipose tissue can be
replaced by fibrotic tissue in the OA synovium have not
been fully elucidated. However, the increase in the
hypoxia-associated mediators, induced by biomechanical
alterations and proinflammatory cytokines, could be at
least partially responsible for this phenomenon. An
increase in hypoxia-induced factor-1 (HIF-1)α has been
described in the OA synovium, in correlation with
greater joint destruction [
]. In adipose tissue, with
a similar structure and cellular component to that
observed in the stroma of the SM, HIF-1α induces tissue
fibrosis and inhibits pre-adipocyte differentiation .
Furthermore, in inflamed adipose tissue from mice fed a
HFD, HIF-1α-stimulated macrophages form highly
hypoxic structures called crown-like structures (CLS),
comprising macrophages encircling dead or dying adipocytes
]. Indeed, we have previously identified CLS in the
OA synovium in both human and hypercholesterolemic
rabbits with synovial inflammation [
Hyperlipidemia in rabbits in the OA group did not
seem to enhance the presence of fibrosis-associated
proteins, such as col I. However, it evoked a dramatic
increase in macrophage infiltration in the synovium and
greater decrease in adipose tissue content. Dyslipemia
has been directly related to macrophage infiltration and
inflammation in the synovium and adipose tissue [
In hyperlipidemic mice with OA, the synovial
proinflammatory macrophage subset was identified as responsible
for an increase in TNF synthesis and extracellular matrix
remodeling in the synovial membrane [
]. In line with
these data, we identified greater TNF expression in the
synovium in the OA-HFD group that paralleled the
increased macrophage density in this tissue. Although little
is known about the metabolic regulation of synovial
macrophages, prolonged lipid exposure could result in
failure of the lipid-handling mechanisms, leading to
different lipotoxic events, such as those described in
obesity-associated insulin resistance, atherosclerosis and
other inflammatory diseases related to MetS [
hyperlipidemia could drive M1 macrophage polarization
in the OA synovium, resulting in a major presence of
proinflammatory cytokines, as has been described in
adipose tissue [
]. Furthermore, adipocyte apoptosis
and impaired adipogenesis have been also associated
with the increased lipolysis induced by over-nutrition or
HFD feeding . Although hyperlipidemia could
aggravate OA synovial inflammation, increasing macrophage
density and adipose tissue destruction, the presence of
hyperlipidemia per se could only have limited effects on
SM alterations, as recently reported in HFD-fed mice [
Leptin and adiponectin gene expression diminished in
the SM in the OA and OA-HFD group in comparison to
control animals. These results appear to correlate with
the amount of intra-articular adipose tissue rather than
with the presence of a proinflammatory milieu.
Furthermore, circulating leptin was significantly increased in
HDF-fed animals, probably due to the effect of the diet
on the extra-articular fat tissue [
]. Our data are in line
with previous reports indicating that hyperlipidemia
could be an aggravating factor for OA through the
stimulation of systemic proinflammatory mediators [
In the OA group we also found increased circulating
leptin as previously described in human and
experimental OA, related to joint damage [
]. Therefore, our data
do not support the hypothesis that hyperlipidemia could
be an aggravating factor in metabolic OA, stimulating
adipokine expression within the intra-articular adipose tissue.
Different joint cells, such as chondrocytes or bone cells,
could be responsible for adipokine synthesis in response
to biomechanical or proinflammatory stimuli [
In summary, these data show that HFD aggravates the
inflammation in the SM of rabbits with OA by inducing an
increase in the infiltrating macrophages in the synovium,
together with macrophage and metabolic-mediated
remodeling of adipose tissue, and further elevation of
proinflammatory cytokines. The lipotoxic effects induced by
dyslipemia in adipocytes and macrophages could play a
decisive role in the joint deterioration of patients with OA
and MetS, supporting the hypothesis of a plausible link
between obesity and OA going beyond mechanical loading.
Additional file 1: Figure S1. Synovial tissue collection. Photographs
show the precise locations where the rabbit joint was accessed and the
synovial membrane (SM) was collected. Half of the SM containing both
stroma and lining was then fixed and embedded in paraffin for histological
studies, and the other portion was immediately frozen and used for molecular
biology studies. (TIF 376 kb)
ATA: Adipose tissue area; CLS: Crown-like structures; Col I: Collagen I;
COX2: Cyclooxygenase-2; CRP: C-reactive protein; ELISA: Enzyme-linked
immunosorbent assay; HFD: High fat diet; IL-1β: Interleukin-1β;
IL6: Interleukin-6; MetS: Metabolic syndrome; OA: Osteoarthritis; PLIN: Perilipin-1A;
SBP: Systolic blood pressure; SM: Synovial membrane; TNF: Tumor necrosis factor
This work was supported by research grants from the Instituto de Salud
Carlos III [PI12/00144, PI15/00340, PI16/00065]; co-funded by Fondo Europeo
de Desarrollo Regional (FEDER).
Availability of data and materials
Conception and design: G H-B and RL; acquisition and assembly of data: A
LV; AL, S P-B, I P-P and LP; analysis and interpretation of the data: A L-V; AL, S
P-B, G H-B and RL; drafting of the article: A L-V, G H-B and RL; critical revision
of the article for important intellectual content: all authors; final approval of
the article: all authors.
The studies reported in this paper comply with the Animal Research: Reporting
of In Vivo Experiments (ARRIVE) guidelines. All animal care and experimental
protocols for this study complied with the Spanish regulations and the
Guideliness for the Care and Use of Laboratory Animals drawn up by the
National Institutes of Health (Bethesda, MD, USA) and were approved by the
Institutional Ethics Committee of the IIS-Fundacion Jimenez Diaz.
The authors have declared no competing interests.
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