Journal of

Personalized
Medicine

Review
Lipedema Research—Quo Vadis?
Anna M. Ernst 1, *, Hannelore Bauer 1 , Hans-Christian Bauer 1,2 , Marianne Steiner 1 , Anna Malfertheiner 3
and Anna-Theresa Lipp 4, *

1 Department of Environment & Biodiversity, Paris Lodron University of Salzburg, 5020 Salzburg, Austria
2 Institute for Tendon and Bone Regeneration, Paracelsus Medical University (PMU), 5020 Salzburg, Austria
3 Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar,
Technical University of Munich (TUM), 81675 Munich, Germany
4 Private Practice Dr. Lipp & Colleagues, 80331 Munich, Germany
* Correspondence: [email protected] (A.M.E.); [email protected] (A.-T.L.)

Abstract: When studying the current literature, one might get the impression that lipedema is a “mod-
ern” disease, with increasing incidence and augmenting prevalence throughout Western countries
during the last decade. However, a quick look into older textbooks shows that disproportionate accu-
mulation of fat in female bodies has long been known without being recognized as an independent
disease. Nevertheless, it was not until 1940 that Allen and Hines described a “syndrome characterized
by fat legs and orthostatic edema” in a seminal publication. The mere awareness that people who have
lipedema are not just overweight but suffer from a yet poorly defined pathological condition, may be
considered a decisive leap forward in the understanding of lipedema. A number of comprehensive
publications have since dealt with the clinical presentation of lipedema and have provided the first
clues towards the potential pathological mechanisms underlying its initiation and progression. Nev-
ertheless, despite all effort that has been undertaken to unravel lipedema pathology, many questions
have remained unanswered. What can be deduced with certainty from all experimental and medical
evidence available so far is that lipedema is neither a cosmetic problem nor is it a problem of lifestyle
but should be accepted as a serious disease with yet undetermined genetic background, which makes
women’s lives unbearable from both a physical and psychological point of view. To date, results
from clinical inspections have led to the categorization of various types and stages of lipedema,
describing how the extremities are affected and evaluating its progression, as demonstrated by skin
Citation: Ernst, A.M.; Bauer, H.; alterations, adipose tissue volume increase and physical and everyday-behavioral impediments.
Bauer, H.-C.; Steiner, M.; There is accumulating evidence showing that advanced stages of lipedema are usually accompanied
Malfertheiner, A.; Lipp, A.-T.
by excessive weight or obesity. Thus, it is not unreasonable to assume that the progression of li-
Lipedema Research—Quo Vadis? J.
pedema is largely driven by weight gain and the pathological alterations associated with it. Similarly,
Pers. Med. 2023, 13, 98. https://
secondary lymphedema is frequently found in lipedema patients at advanced stages. Needless to say,
doi.org/10.3390/jpm13010098
both conditions considerably blur the clinical presentation of lipedema, making diagnosis difficult
Academic Editors: Hisham Fansa and scientific research challenging. The present literature review will focus on lipedema research,
and Onno Frerichs based on evidence fromex vivo and in vitro data, which has accumulated throughout the last few
Received: 6 December 2022 decades. We will also open the discussion as to whether the currently used categorization of lipedema
Revised: 25 December 2022 stages is still sufficient and up-to-date for the accurate description of this enigmatic disease, whose
Accepted: 27 December 2022 name, strangely enough, does not match its pathologic correlate.
Published: 31 December 2022
Keywords: lipedema; adipose tissue; in vitro and ex vivo studies

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and
1.1. Background
conditions of the Creative Commons Lipedema (adiposis dolorosa, lipomatosis dolorosa of the legs, lipoedema) is a painful disorder
Attribution (CC BY) license (https:// characterized by the symmetrical accumulation of subcutaneous fatty tissue, particularly
creativecommons.org/licenses/by/ on the extremities (thighs, calves, upper arms) [1–8]. Lipedema affects predominantly, if not
4.0/). exclusively, women. Those cases in which lipedema was diagnosed in males were shown to

J. Pers. Med. 2023, 13, 98. https://doi.org/10.3390/jpm13010098 https://www.mdpi.com/journal/jpm

J. Pers. Med. 2023, 13, 98 2 of 15

be secondary to other diseases involving severe hormonal disturbances [9]. The worldwide
prevalence of lipedema among women and post-pubertal girls is estimated at 11% [10].
However, due to a lack of accurate diagnostics markers and potential misdiagnosis, all
statistical information must be viewed critically.
Diets and exercise as well as decongestive therapies (lymphatic drainage) and the
wearing of compression garments may improve the patients’ overall condition, but cannot
eliminate the pathological mechanism(s) underlying initiation and progression of the
disease. Currently, most women choose liposuction as the most causative treatment,
yielding long-lasting improvements [11–17].
It is generally agreed upon that lipedema manifests during periods of great hormonal
fluctuations, usually during puberty and pregnancy, and in rare cases during menopause.
A possible genetic predisposition appears plausible. A familial incidence of 15%, affecting
first-degree female relatives, has been documented although self-reports usually tend to
greatly exceed this percentage [9]. The mode of inheritance is considered to be either
X-linked dominant or autosomal dominant with incomplete penetrance and sex limitation,
but could also be oligogenic [9,18].
Currently, the visual inspection and an accurate survey of the patient’s medical history
is basic to routine diagnosis of lipedema and may be improved by sonographic examina-
tion to distinguish lipedema from lymphedema at advanced stages of the disease [19–21].
An interesting approach to narrow down the diagnosis of lipedema was reported previ-
ously [22]. Thereby, the regional body composition, comprising fat mass, lean mass and
bone mass in lipedema and non-lipedema women was measured using dual-energy X-ray
absorptiometry. The authors reported that across all body mass index (BMI) categories,
the fat mass of the legs related to the BMI was significantly higher in lipedema patients
compared to healthy controls.
Today diagnosis of lipedema relies on criteria, originally established by Wold et al. in
1951 [23]. Following modifications introduced by Herbst [1], these criteria are still applied
for routine diagnosis [7,24,25].
Based on the diagnostic criteria described so far, one might surmise that it should
not be too difficult to distinguish lipedema from other phenotypically similar diseases,
including obesity, lipohypertrophy or lymphatic and venous disturbances. In fact, the phe-
notypic appearance of normal weight patients is quite unambiguous, making an accurate
diagnosis easy. Problems arise, when phenotypically similar clinical pictures overlap with
lipedema [26]. The co-occurrence of lipedema and obesity is particularly frequent, which
has even led to the notion that the progression of lipedema is driven by weight gain rather
than by potential (yet undefined) lipedema-specific physiological alterations [27]. In this
respect, the question arises, what “advanced lipedema” means, and how it could be studied
without interference of overweight or obesity and obesity-associated pathologies.
The present literature review was conducted using PubMed to search for studies,
which depict the progress and current status of research into lipedema. A special focus was
placed on the results from ex vivo and in vitro studies.

1.2. “Lymph Makes You Fat”—Could This Be Relevant for Lipedema?

The question of whether lipedema is associated with lymphedema or not is all the
more important as several lines of evidence have indicated that defective lymphatics may
contribute to fat accumulation [28–30]. In this context, deletion of the transcription factor
Prox-1 (prospero homeobox-1) in lymphatic endothelial cells in a mouse model led to
the leakage of lymph, stimulating adipocytes to store fat and enhancing adult-onset of
obesity [28]. Thus, a potential crosstalk between lymphatics and adipose tissue would be a
welcome explanation for the locally restricted fat gain in lipedema.
It is still being debated as to whether vascular disorders are integral to lipedema,
particularly at late stages. Since lipedema has originally been described as “a syndrome
characterized by fat legs and orthostatic edema” [23], special attention has been paid to this
important aspect from early on. Numerous imaging techniques have been used to study
J. Pers. Med. 2023, 13, 98 3 of 15

the lymphatic conditions of lipedema patients; however, with fairly inconsistent results.
This may be partly explained by the frequently found overlap of lipedema and obesity
within the patient cohorts studied.
While Bilancini et al. [31] reported a marked slowness of the lymphatic system in
lipedema patients compared to healthy controls, only a moderate impairment of the lym-
phatic system in lipedema patients was found by Haarwood [32], emphasizing that the
lymphatic function in lipedema was not comparably impaired as observed in lymphedema.
A subclinical status of lymphedema in lipedema patients was also described by Lohrmann
et al. [33] using magnetic resonance lymphangiography. In addition, during advanced
stages of lipedema, multiple microlymphatic aneurysms of lymphatic capillaries have been
described which might contribute to edema formation [34]. A morphological study using
skin biopsies from lipedema thigh tissue revealed increased dermal spaces and abnormal
vessel phenotype compared to controls, which could be an indication of a possible vascular
disorder [35]. Further evidence supporting the notion of a lymphatic defect associated with
lipedema came from Ma et al. [36], demonstrating elevated levels of PF4 (platelet-factor
4)/CXCL4) in circulating blood plasma exosomes from lipedema patients. According to
these results, PF4 may be considered a novel biomarker for lymphatic defects unrelated to
body weight.
On the other hand, considerable evidence has accumulated contradicting the assump-
tion that lymphatic insufficiency is involved in lipedema etiology, as has been summarized
in a recent publication [27]. For instance, magnetic resonance imaging (MRI) studies could
not verify the presence of edema in subcutaneous fat and muscle tissue of lipedema pa-
tients [37]. Similarly, no dermal edema could be observed by high-resolution cutaneous
ultrasonography in lipedema patients, focusing on the thigh or ankle region [19], and no
edematous condition could be found in lipedema adipose tissue upon quantification of the
local tissue water content [38].
In a recent study, dilated lymphatic vessels but no dermal backflow and no edema
in lower extremities of lipedema patients at early stages was shown, suggesting that
no lymphatic disturbance is involved in the etiology of early-stage (stage I and II) li-
pedema [39]. This further supports the notion that lymphedema may be induced by being
overweight/obese which accompanies lipedema at advanced stages [40].
Taking together all medical evidence available so far, it is reasonable to suggest that
edema formation due to lymphatic disorders appears to play only a minor, if any, role
in the initiation of lipedema. During advanced stages of lipedema, venous or lymphatic
insufficiency might exacerbate the clinical picture of lipedema. Whether or not these
vascular disturbances are merely a consequence of weight gain needs to be clarified in
more comprehensive systematic studies, with particular focus on the BMI and other weight-
associated parameters of the participants.
Increased fragility of blood capillaries in lipedema adipose tissue was shown by
Szolnoky et al. using angiosterrometry [41,42]. The authors demonstrated a substantial
improvement of capillary fragility in lipedema patients, following complete decongestive
therapy [42]. Morphological alterations of the microvasculature in lipedema adipose tissue
microvessels of the lower papillary dermal layer were reported by Al-Ghadban et al. [43],
showing increased capillary diameter in normal weight lipedema patients compared to
controls. Lymphatic vessel areas did not differ between lipedema and non-lipedema
adipose tissue from non-obese donors, but were significantly increased in adipose tissue
from obese donors. No alterations of vessel morphology but increased systemic levels
of VEGF-C in serum from lipedema patients and increased M2-polarized macrophage
infiltration into lipedema adipose tissue were re-ported by Felmerer et al. [44], indicating
a potential disturbance of the permeability properties in lipedema blood and lymphatic
vasculature.
Interesting results from a recent study support the notion that the endothelial barrier
is compromised in lipedema adipose vasculature [45]. The authors showed that soluble
factors released from cultured lipedema SVF (stromal vascular fraction) cells led to a
J. Pers. Med. 2023, 13, 98 4 of 15

significant reduction of VE-cadherin (cadherin-5) expression in endothelial cells in an

in vitro model. This effect could not be elicited by conditioned medium from control
SVF cells, suggesting that the microenvironment of blood vessels in lipedema and non-
lipedema adipose tissue differs substantially. VE-cadherin is an essential component of
endothelial cell-to-cell adherens junctions and is critically involved in maintaining vascular
permeability in addition to orchestrating numerous intracellular signaling pathways [46].
Thus, it may be speculated that a potential downregulation of VE-cadherin expression in
the adipose tissue vasculature in vivo could lead to even more far-reaching effects than just
alterations in permeability properties.

1.3. In Search of a Molecular Marker for Lipedema Diagnosis

There is still a risk that lipedema is being under- or over-diagnosed, particularly due
to the above-mentioned co-occurrence of obesity, lymphedema and other pathological
conditions that potentially mask lipedema. This has fueled a quest for the identification of
unique, unambiguous molecular diagnostic markers of lipedema.
In this context, a number of studies have been conducted, applying histomorphology
as well as cell-based molecular and immunological techniques (Tables 1 and 2). Results
from these studies show that lipedema manifests itself at many levels (Figure 1).

Table 1. Histological/morphological, histochemical and immunocytochemical studies.

METHODS TISSUE/CELLS RESULTS AUTHORS

Increased infiltration of CD68+ macrophages in
Paraffin-embedded lipedema adipose tissue and occurrence of “crown-like
Immunostaining adipose tissue structures”; [47]
Histological sections large number of KI67+ /CD34+ proliferating cells in
lipedema tissue
Hypertrophy of adipocytes in adipose tissue from
non-obese lipedema donors;
Paraffin-embedded increased macrophage density in lipedema skin and fat;
Histochemical staining
adipose tissue increased numbers of dermal blood vessels in lipedema [43]
Immunostaining
Histological sections adipose tissue;
increased dilatation of capillaries in non-obese lipedema
adipose tissue compared to non-obese controls
Increased M2-macrophage infiltration into lipedema
adipose tissue;
Paraffin-embedded no morphological changes in lymphatic and
Immunostaining adipose tissue blood vasculature; [44]
Histological sections no changes in number, size or percentage coverage of
lymphatic vessels or blood vessels in lipedema
tissue sections
Increased dermal spaces and abnormal vessel
Paraffin-embedded
Histochemical staining phenotype (rounded endothelial cells; perivascular
adipose tissue [35]
Immunostaining spaces, perivascular immune cell infiltrate) in lipedema
Histological sections
specimens compared to controls
Increased epidermal thickness in lipedema patients;
Paraffin-embedded
Histochemical staining adipocyte hypertrophy, increased fibrosis and
adipose tissue [48]
Immunostaining significant increase in CD68+ macrophages in
Histological sections
lipedema tissue
Paraffin-embedded
Histochemical staining
adipose tissue Significant hypertrophy of lipedema adipocytes [49]
Immunostaining
Histological sections
Immunohistochemistry Paraffin-embedded Significantly increased number of CD29/CD34 positive
BODIPY staining of adipose tissue cells in lipedema adipose tissue; [50]
droplets Histological sections enhanced adipogenic potential of lipedema ASCs
J. Pers. Med. 2023, 13, 98 5 of 15

Table 1. Cont.

METHODS TISSUE/CELLS RESULTS AUTHORS

No difference in epidermal thickness of thigh tissue
between lipedema and control tissue;
Histochemical staining Paraffin-embedded no signs of fibrosis; no alterations in lymphatic
Immunostaining adipose tissue endothelial cells in lipedema adipose tissue;
[45]
Machine learning Histological sections higher number of CD68+ macrophages in
analysis SVF, ASCs CD31+/podoplanin- areas of lipedema tissue;
morphological alterations of interendo-thelial junctions
between lipedema en-dothelial cells in vitro
SVF, ASCs Increased occurrence of myofibroblast-like cells in
Immunostaining
In vitro differentiated lipedema adipocytes from normal weight and [51]
Histochemical staining
adipocytes overweight lipedema donors

Table 2. Molecular studies and cell-based functional assays.

METHODS TISSUE/CELLS RESULTS AUTHORS

Increased parameters of oxidative stress (plasma MDA
Blood samples
HPLC and plasma protein carbonyl concentrations) in [52]
Plasma
lipedema patients compared to controls
Lipoaspirates Enhanced SVF cell yield in lipedema preparations with
Immunophenotyping
SVF/ASCs increased CD90 and CD146-positive cells;
Flow cytometry [53]
In vitro differentiated reduced in vitro differentiation capacity of
OilRed O staining
adipocytes lipedema ASCs
Increased proliferative activity of lipedema ASCs;
Lipoaspirates increased IL-8 levels in supernatants from
ELISA
SVF/ASCs lipedema ASCs;
OilRedO staining [54]
In vitro differentiated reduced adipokine and aromatase levels in supernatants
Cell counting
adipocytes from in vitro differentiated lipedema adipocytes;
reduced differentiation capacity of lipedema ASCs
Significant increase in CFU potential and higher
adipogenic potential of lipedema ASCs;
Proliferation assay Lipoaspirates increased expression of leptin and PPARγ in
CFU fibroblast assay SVF/ASCs (2D cultures) lipedema adipocytes;
[55]
qPCR In vitro differentiated no change in proliferation rate of lipedema ASCs
OilRedO staining adipocytes compared to controls;
comparable inflammatory gene expression in lipedema
and control ASCs and adipocytes
No difference in adipogenic gene expression (ADIPOQ,
LPL, PPARγ, Glut4) between lipedema and healthy
3D-differentiated adipocytes;
qPCR ASCs spheroids (3D
upregulation of IL-6 expression in 3D cultures of [56]
OilRedO staining cultures)
lipedema ASCs and adipocytes;
elevated CFU activity and adipogenic potential of ASCs
grown as spheroids
Increased levels of VEGF-C in serum from
lipedema patients;
qPCR Adipose tissue increased expression of VEGFR-3 in lipedema
[44]
ELISA Serum adipose tissue;
significant decrease in VEGF-A and VEGF-D, and Tie-2
expression in lipedema adipose tissue
J. Pers. Med. 2023, 13, 98 6 of 15

Table 2. Cont.

METHODS TISSUE/CELLS RESULTS AUTHORS

Aberrant lipid metabolic profile, increased cholesterol,
Gene array of triglycerides and LDL and ApoB in lipedema serum;
adipose-tissue related Adipose tissue no alteration in cytokine profile (IL-6, IL-18, lipocalin-2
[48]
genes Serum and leptin);
ELISA upregulation of CCND1/cyclinD1 and downregulation
of CEBP, CFD, NCOR2, KLF4 in lipedema adipocytes
Lipoaspirates;
Identification of lipedema-relevant miRNAs
conditioned medium
Analysis of extracellular preferentially in sEVs; potential involvement of
from SVF cells; small [57]
miRNAs from SVF differentially expressed miRNAs in Notch, Wnt
extracellular vesicles
SMAD/TGFß-pathway, oxidative stress and senescence
(sEVs)
Mass spectrometry Blood plasma exosomes Increase platelet factor 4 (PF4) levels in circulating
[36]
analysis (mouse and human) exosomes from patients with lipedema
Whole exome
Discovery of a missense variant in the AKR1C1 gene
sequencing Blood samples (germline
encoding an aldo-keto reductase involved in [58]
qPCR DNA)
progesterone metabolism
Molecular modeling
Lipidomic analysis (lipid Significant increase in IL-11, IL-28A, IL29 expression in
mass spectrometry) Adipose tissue biopsies lipedema serum;
Cytokine profiling Lipoaspirates no significant alteration in lipid composition in adipose
[49]
(Multiplex SVF tissue and serum from lipedema donors;
immunoassay) Serum significantly increased oxidative metabolism (enhanced
Mitochondrial stress test mitochondrial function) of lipedema SVF cells
Differential expression of >4400 genes partly involved in
cell cycle/cell proliferation and lipid metabolism, in
lipedema adipose tissue,
>900 changes in lipid composition and >600
Transcriptional profiling Whole adipose tissue
differentially altered metabolites in lipedema adipocytes:
Lipidomic and biopsies
differential expression of >3400 genes, partly involved
metabolomic analyses ASCs [50]
in extracellular matrix, cell-cycle/proliferation signaling
Functional assays In vitro differentiated
pathways, in lipedema ASCs;
BODIPY staining adipocytes
upregulation of the cell cycle regulator Bub1 and
enhanced activation of histone H2A in lipedema ASCs;
enhanced proliferation and differentiation of
lipedema ASCs
Lipoaspirates
Whole adipose tissue
(AT)
Significantly increased ZNF423 in lipedema SVF, EC and
SVF
qPCR PC compared to controls;
human primary ECs
Protein array significant upregulation of aromatase expression in
(hECs) [45]
Endothelial permeability lipedema whole adipose tissue;
SVF-derived sorted
assay lipedema SVF cell-induced dysfunction of the vascular
EC/PC
endothelial barrier in vitro
SVF cell-derived
conditioned medium
(CM)
Lipoaspirates Significant upregulation of PPARγ, CD36 and FABP4 in
ASCs differentiated adipocytes from non-obese lipedema
RT-PCR, qPCR [51]
In vitro differentiated donors; reduced adiponectin/leptin ratio in obese but
adipocytes not non-obese lipedema adipocytes
Identification of 21 deleterious variants in genes linked
Next-generation to syndromic fat accumulation (ALDH18A1, GHR) and
Genomic DNA from
sequencing; multi-gene differential diagnosis (PLIN1, LIPE, PPARγ, POMC, [59]
peripheral blood
panel NR0B2, GCKR, NPC1), as well as lipedema candidate
genes (RYR1, PPARA)
J.J.Pers.
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17

Adipogenic comittment Cell proliferation

Adipogenic differentiation Modulation of progesterone and
estrogen biosynthesis

LIPEDEMA

Chronic inflammation
Hypervascularity
Macrophage infiltration
Mitochondrial impairment
Fibrosis
Oxidative Stress
Endothelial barrier dysfunction

Figure1.1.Biological
Figure Biologicalprocesses
processessuggested
suggestedto
tobe
beaffected
affectedin
inlipedema
lipedemapathology,
pathology,as
asevidenced
evidencedby
bydata
data
fromex
from exvivo
vivoand
andininvitro
vitroresearch.
research.

Particular
Particular attention
attention has been paid paid toto the
thecharacterization
characterizationofofa adistinct distinctpopulation
population of
of adipose
adipose tissue
tissue resident
resident mesenchymal
mesenchymal stem stem cells,
cells, referred
referred to astoadipose
as adipose tissue-derived
tissue-derived stro-
stromal/stem
mal/stem cellscells (ASCs),
(ASCs), from from lipedema
lipedema subcutaneous
subcutaneous adipose
adipose tissue
tissue [60,61].
[60,61].
Since
Sinceliposuction
liposuctionisiscurrently
currentlythe themost
mostcausative
causativetreatment
treatmentof oflipedema,
lipedema,lipoaspirates
lipoaspirates
from
fromsubcutaneous
subcutaneousadipose adipose tissue
tissueareare
available
availablein sufficient
in sufficientquantity for research
quantity purposes,
for research pur-
which may be considered a key benefit. Lipoaspirates are mainly
poses, which may be considered a key benefit. Lipoaspirates are mainly used for isolation used for isolation of the
“stromal vascular fraction” (SVF), but also of mature adipocytes,
of the “stromal vascular fraction” (SVF), but also of mature adipocytes, residing in a dif- residing in a different
layer
ferentoflayer
the liposuction material.material.
of the liposuction These cellular
These fractions may alsomay
cellular fractions be isolated
also befrom biopsied
isolated from
(solid) adipose
biopsied (solid)tissues,
adipose though
tissues, cell yield tends
though to betends
cell yield lower. to be lower.
The
The SVFSVF isis aa clinically
clinically interesting
interesting adipose
adipose tissue
tissue fraction,
fraction, which
which is is usually
usually sedi-
sedi-
mented from the aqueous layer of enzymatically digested
mented from the aqueous layer of enzymatically digested adipose tissue. It contains adipose tissue. It contains
a het-
aerogeneous
heterogeneous population
population of cells,
of cells, including
including endothelial
endothelial cells cells and endothelial
and endothelial precursor
precursor cells,
cells, ASCs, pre-adipocytes, pericytes, smooth muscle cells, fibroblasts,
ASCs, pre-adipocytes, pericytes, smooth muscle cells, fibroblasts, lymphocytes and mac- lymphocytes and
macrophages
rophages [62–64].[62–64].
ASCs
ASCsaccount
accountfor forless
lessthan
than0.1%0.1%of of the
the SVF
SVF and
and areare grown
grown in in 2D-
2D- oror 3D
3D cultures
cultures [65].
[65].
They are characterized by their growth behavior, multi-lineage differentiation
They are characterized by their growth behavior, multi-lineage differentiation potential potential and
immunophenotypic
and immunophenotypic markersmarkers[66–70]. ASCsASCs
[66–70]. may maybe kept be in
keptaninundifferentiated
an undifferentiated state state
and
may be expanded in vitro and serially passaged up to a limited
and may be expanded in vitro and serially passaged up to a limited passage number. passage number. Long-term
expansion
Long-termleads to alterations
expansion leads toinalterations
morphology and proliferation
in morphology kinetics [71].kinetics
and proliferation These cells
[71].
are easy to handle since they adhere well to the culture dish and do not show spontaneous
These cells are easy to handle since they adhere well to the culture dish and do not show
differentiation (lipogenesis) when cultured in basic medium without adipogenic stimuli.
spontaneous differentiation (lipogenesis) when cultured in basic medium without adipo-
In contrast, freshly isolated mature adipocytes float and would need to be kept in “ceiling
genic stimuli. In contrast, freshly isolated mature adipocytes float and would need to be
cultures” [72].
kept in “ceiling cultures” [72].
1.4. Focusing on Adipogenesis
1.4. Focusing on Adipogenesis
Adipogenic differentiation is a complex multi-step process that involves the stimula-
tion ofAdipogenic differentiation
a transcriptional is a complex
cascade and multi-step
can be divided into process that involves
two separate phases, the stimula-
adipogenic
tion of a transcriptional
commitment and terminal cascade and can be[73–75].
differentiation dividedUpon
into two separate
treatment phases,
with adipogenic
appropriate in-
commitment and terminal differentiation [73–75]. Upon treatment with appropriate
ductive media uptake of glucose is enhanced and adipogenesis/lipogenesis is initiated. in-
ductive media uptake of glucose is enhanced and adipogenesis/lipogenesis
Inductive media basically contain the glucocorticoid analog dexamethasone, insulin, in- is initiated.
Inductive media
domethacin basically
and the cAMPcontain the IBMX
stabilizer glucocorticoid analog dexamethasone, insulin,
(3-isobutyl-1-methyl-xanthine). Other op- in-
domethacin and the cAMP stabilizer IBMX (3-isobutyl-1-methyl-xanthine). Other
tional other ingredients, leading to the augmentation of lipid droplet formation may be optional
other ingredients,
added [69,76,77]. leading to the augmentation of lipid droplet formation may be added
[69,76,77].
 J. Pers. Med. 2023, 13, x FOR PEER REVIEW 9 of 17

J. Pers. Med. 2023, 13, 98 8 of 15

The progression of adipogenic differentiation is usually evaluated bythe accumula-

tionThe
of lipid droplets of
progression (cytoplasmic
adipogenictriglycerides)
differentiationandisthe corresponding
usually evaluatedalterations in ad-
bythe accumu-
ipogenic/lipogenic gene expression [78,79]. Lipid droplets (LD) are dynamic
lation of lipid droplets (cytoplasmic triglycerides) and the corresponding alterations in organelles,
responsible for storage
adipogenic/lipogenic geneof free-fatty
expressionacids andLipid
[78,79]. neutral triglycerides.
droplets (LD) areThey also orchestrate
dynamic organelles,
responsible for storage of free-fatty acids and neutral triglycerides. They also[80].
the degradation of excess triglycerides by activating lipolysis and lipophagy orchestrate
In vitro studies
the degradation of lipedema
of excess adipose
triglycerides derived stem
by activating cells and
lipolysis andpreadipocytes
lipophagy [80].aremainly
performed
In vitro in 2D cultures
studies and inadipose
of lipedema vitro differentiation
derived stem of ASCs
cells andupon adipogenic
preadipocytes stimula-
aremainly
tion is usually followed after 21–28 days of cultivation. Interestingly, in
performed in 2D cultures and in vitro differentiation of ASCs upon adipogenic stimulationvitro differenti-
isation,
usuallyparticularly in 2D
followed after cultures,
21–28 leads
days of to the accumulation
cultivation. of vitro
Interestingly, in smalldifferentiation,
multilocular lipid
par-
droplets, while freshly isolated mature adipocytes contain a single large
ticularly in 2D cultures, leads to the accumulation of small multilocular lipid droplets, unilocular LD
while
[81] (Figure 2a–f).
freshly isolated mature adipocytes contain a single large unilocular LD [81] (Figure 2a–f).

Figure2.2.(a–c):
Figure (a–c):InInvitro
vitrodifferentiated
differentiatedadipocytes
adipocytesshow showmultilocular
multilocular lipid
lipid droplets
droplets after
after 21
21 days
days ofof
adipogenic stimulation from (a) light microscopy (live cell imaging) (scale bar: 100 µm);
adipogenic stimulation from (a) light microscopy (live cell imaging) (scale bar: 100 µm); (b) scanning (b) scanning
electron microscopy (scale bar: 50 µm); (c) immunofluorescence staining of perilipin (red),
electron microscopy (scale bar: 50 µm); (c) immunofluorescence staining of perilipin (red), Lipid-
LipidToxTM (green) and DAPI (blue) (scale bar: 50 µm). (d–f): Mature adipocytes in/from subcuta-
ToxTM (green) and DAPI (blue) (scale bar: 50 µm). (d–f): Mature adipocytes in/from subcutaneous
neous adipose tissue show unilocular lipid droplets from (d) semithin section from a resin-embed-
adipose tissue showfixed
ded biopsy-tissue, unilocular lipid droplets
with Karnovsky’s from and
fixative (d) semithin
post-fixedsection from stained
with OsO4 a resin-embedded
with meth-
biopsy-tissue, fixed with Karnovsky’s fixative and post-fixed with OsO4
ylene blue/azurII; (e) immunofluorescence staining of a paraffin section from biopsied stained with adipose
methylene tis-
blue/azurII;
sue. Perilipin(e)(red),
immunofluorescence
DAPI (blue) (scalestaining of a (f)
bar: 50 µm); paraffin section from biopsied
immunofluorescence stainingadipose
of mature tissue.
adi-
pocytes (red),
Perilipin isolated
DAPIfrom liposuction
(blue) material.
(scale bar: 50 µm);LipidToxTM (green), DAPI
(f) immunofluorescence (blue) (scale
staining bar: adipocytes
of mature 50 µm).
isolated from liposuction material. LipidToxTM (green), DAPI (blue) (scale bar: 50 µm).
So far, information concerning the biogenesis of lipid droplets in lipedema adipo-
cytes isfar,
So information
lacking but couldconcerning
contributethetobiogenesis of lipid
the elucidation of droplets in lipedema
the mechanisms adipocytes
underlying ad-
isipocyte
lackinghypertrophy
but could contribute to
in lipedema. the elucidation of the mechanisms underlying adipocyte
hypertrophy in lipedema.
Only limited information exists concerning adipogenic gene expression in lipedema
ASCsOnlyandlimited information
adipocytes compared exists concerning
to healthy adipogenic
controls. gene expression
A significant increase ininexpression
lipedema
ASCs
of theand adipocytes
master compared
transcriptional to healthy
regulator PPARγ controls. A significant
(peroxisome increase in expression
proliferator-activated receptor
ofgamma)
the master
and transcriptional
a higher adipogenicregulator PPARγ
potential (peroxisome
of lipedema ASCsproliferator-activated
compared to controls recep-
was
tor gamma) and a higher adipogenic potential of lipedema ASCs compared
reported by Al-Ghadban et al. using 2D cultures [55]. This is partly supported by data to controls
was reported
showing by Al-Ghadban
significant et al. using
upregulation 2D cultures
of various [55]. This
adipogenic is partly
genes, supported
including by
PPARγ,
data showing significant upregulation of various adipogenic genes,
CD36/fatty acid translocase, and the fatty acid binding protein-4 (FABP4) in in vitro dif-including PPARγ,
CD36/fatty
ferentiated acid translocase,
lipedema and from
adipocytes the fatty acid binding
non-obese donorsprotein-4
compared(FABP4) in in vitrocon-
to non-lipedema dif-
ferentiated lipedema
trols [51]. In contrast,adipocytes fromdid
other studies non-obese
not revealdonors compared
significantly to non-lipedema
increased expressioncon-
of
trols [51]. In contrast, genes
adipogenesis-related other in
studies did not
lipedema reveal
adipose significantly
cells [45,48]. A increased
significantexpression
upregulationof
adipogenesis-related genesZNF423
of the transcription factor in lipedema adipose
(Zfp423) cells [45,48].
in lipedema SVF A significant
cells compared upregulation
to controls
ofwas
the reported
transcriptionrecently [45]. Since ZNF423 is critically involved in earlytoadipocyte
factor ZNF423 (Zfp423) in lipedema SVF cells compared controls
was reported recently [45]. Since ZNF423 is critically involved in early adipocyte commit-
ment [82], a potential dysregulation of lipedema adipose tissue formation at an early stage
of mesenchymal stem cell differentiation is possible.
J. Pers. Med. 2023, 13, 98 9 of 15

1.5. Focusing on Hypertrophy and Hyperplasia

Accumulation of adipose tissue in vivo relies principally on hyperplasia (cell number
increase) or hypertrophy (cell size increase) of adipocytes [83]. The hyperplastic expan-
sion is accomplished by the constant replenishment of ASCs and preadipocytes, which
differentiate into mature adipocytes upon environmental cues. From a metabolic point of
view, hyperplastic expansion is considered more favorable for the organism compared to
hypertrophy. The latter occurs in response to excessive accumulation of triglycerides, which
may also be triggered by a disturbed balance of lipogenesis and lipolysis [84]. Without
having a sufficiently accurate picture based on scientific data, it appears plausible that both
hyperplasia and hypertrophy contribute to the fat accumulation in lipedema adipose tissue,
although the initial triggering signal is unknown.
A significant increase in adipocyte size of adipose tissue from non-obese lipedema
donors compared to non-obese controls was reported recently [43]. Interestingly, adipocyte
areas did not differ between samples from obese lipedema patients compared to obese
controls, suggesting that it is a sine qua non to study lipedema-specific characteristics
exclusively in non-obese patients. Adipocyte hypertrophy and an aberrant lipid metabolism
in lipedema adipose tissue was reported by Felmerer et al. [48]. Accordingly, hypertrophy
of lipedema adipocytes in paraffin-embedded adipose tissue compared to controls was also
observed by Wolf et al. [49].
Hyperproliferation of cultured lipedema ASCs was reported by Bauer et al., describing
a proliferative boost after a lag period of comparable growth between lipedema and non-
lipedema ASCs [54]. Accelerated proliferation of lipedema ASCs compared to controls
was also observed by Musarat Ishaq [50]. Moreover, the authors demonstrated increased
expression of Bub1, a cell-cycle regulator involved in cell proliferation and increased
activation of histone H2A, in lipedema ASCs. A significantly elevated SVF cell yield
isolated from lipoaspirates from lipedema patients compared to non-lipedema controls was
also reported by Priglinger et al., [53], though cell doubling time did not differ between
lipedema and non-lipedema ASCs in culture. Increased expression of CCND1/cyclin D1,
a cell cycle regulator that is usually increased upon proliferative signals, was detected in
lipedema but not control adipocytes [48]. This finding further supports the notion of a
potential hyperproliferative activity of lipedema ASCs.

1.6. Chronic Inflammation and Oxidative Stress—Primary or Secondary to Lipedema?

Adipose tissue hypertrophy is usually associated with increased cell death and the
concomitant invasion of macrophages scavenging adipocyte debris. Macrophages engulfing
dying adipocytes are histologically presented as “crown-like structures” and are actually
considered a pathologic hallmark of obesity [85]. Although crown-like structures have
often been associated with lipedema, there is more evidence for assigning this histological
finding to obesity rather than to lipedema.
In addtion to increased macrophage infiltration, upregulation of inflammatory genes
and proteins in lipedema adipose cells and tissue has also been reported [48,54]. A signifi-
cant upregulation of IL-8 levels in supernatants of cultured lipedema ASCs compared to
controls was observed by Bauer et al. [54], suggesting that ASC-derived proinflammatory
cytokines may promote the local inflammatory milieu in lipedema adipose tissue. Upon
adipogenic induction, IL-8 levels dropped to equally high basal levels in lipedema and non-
lipedema adipocytes [54]. Serum samples from lipedema patients exhibited increased levels
of IL-28A and -29 and the adipocyte-derived IL-11 [49]. Liposuction led to a significant
reduction of systemic INFα2 and IL-34 [49], the latter being associated with obesity-related
inflammation and other obesity-related pathologies [86]. A trend of elevated expression of
VEGF, IL-6, IL-1ß, and TNFα in differentiated lipedema adipocytes compared to controls
was observed by Al-Ghadban [55], principally in adipose tissue derived from the thigh but
not abdomen of donors. A significant decrease of IL-8 but not IL-6, IL-1ß or TNFα was
found in supernatants from lipedema SVFs [45]. It is particularly important to emphasize
J. Pers. Med. 2023, 13, 98 10 of 15

that inflammatory cytokines play a critical role in endothelial-barrier function [87–91] and
thus may be important players in the initiation and progression of lipedema.
Particularly limited information exists concerning the assumption that lipedema is
caused by or accompanied by mitochondrial disturbances. Using a mitochondrial stress
test, a significant increase of the maximal possible oxygen consumption after stimulation
with the uncoupler FCCP of lipedema SVF cells compared to controls was found [49]. Basal
mitochondrial respiration did not differ between lipedema and control cells. This supports
earlier findings suggesting mitochondrial dysfunction and oxidative stress in lipedema
adipose tissue. The authors reported increased serum concentrations of malondialdehyde
and plasma protein carbonyls compared with healthy control persons [52].

2. Future Considerations
There is no doubt that experienced physicians are able to correctly diagnose lipedema
in daily clinical routine. On a scientific basis, however, the clarity to diagnose lipedema
upon specific “markers” is currently not given, be it on a histological, cell biological or
molecular level.
A recent publication states that facts and myths about lipedema are often confused and
passed on in a kind of tradition [27]. This is even more astonishing since it would be very
easy to verify assumptions and claims with scientific evidence or to prove them wrong.
As summarized above, considerable scientific data based on ex vivo and in vitro stud-
ies have accumulated during the last couple of years. Most of these studies focused on
various aspects of adipose tissue differentiation and the cellular and molecular microenvi-
ronment of adipose cells in vivo and in vitro. Why has so little success been achieved in
the case of lipedema?
In the following, some possible explanations are given, and an attempt is made to
provide an improved approach for future studies. Knowledge, which has accumulated
during the last few years, should be harnessed to develop optimized strategies for ex vivo
and in vitro study on lipedema, which can then represent a real and important complement
to the daily clinical routine.

2.1. The Issue of Patients’ Weight and the Dilemma of Assembling a Representative Cohort
It is generally agreed upon that obesity/weight gain accompanies lipedema particu-
larly during advanced stages. Thus, a normal weight patient exhibiting lipedema stage 3 is
very rare. The most serious mistake found in various reports of cohort studies is the high
BMI range within the observed groups/cohorts.
It is known that the influence of weight, in particular obesity, is decisive for many
histological and cell-biological properties. Thus, the interpretation of data emerging from
studies with a donor population whose BMI ranges from normal weight or overweight to
obesity, becomes difficult, if not impossible.
In addition, it is rightly being debated as to whether the BMI is a suitable tool to
evaluate the body mass of lipedema patients. Due to the strong inequality of the upper and
lower body, the indication of the total weight can be misleading. It would be advisable to
use both the BMI and the hip/waist ratio to estimate and categorize the donor cohorts.

2.2. The Dilemma with Edema and Vascular Disturbances/Dysfunctions

As previously mentioned, this topic has extensively been discussed, probably due
to the phenotypically similar picture of lymphedema and advanced lipedema stages.
However, it is striking that the data from the literature are contradictory. Why are data
concerning a prospective involvement of vascular disturbances in lipedema pathology
so inconsistent? Why could the question concerning a potential connection of vascular
dysfunction in lipedema not be answered satisfactorily for so long?
Here, too, the reason seems to lie in the poor selection of the subjects. Again, numerous
studies concerning vascular conditions of lipedema patients were performed with patients
J. Pers. Med. 2023, 13, 98 11 of 15

exhibiting excessive weight (obesity) or with cohorts comprising a mixture of normal

weight and obese patients.
It cannot be stressed enough that any overlap of lipedema and obesity is an enormous
obstacle in generating a clear picture of the vascular conditions of lipedema patients and to
identify lipedema-specific alterations.
Further, it cannot be entirely ruled out that lipedema adipose tissue could harbor
microedema, which is undetectable by standard methods and can only be seen with high-
resolution ultrastructural imaging. It would therefore be advisable to significantly expand
the range of methods for the histological examination of the lipedema adipose and vascular
tissue to include electron microscopic analyses.

2.3. The Dilemma with Age and Interdonor Variability

When reviewing the literature, it is noticeable that patient cohorts with a large age
range are included in many studies. Since the development and progression of lipedema is
obviously triggered by hormonal conditions, it would be advantageous to narrow down
the age range of study cohorts to ensure a comparable hormonal status of participants as
this is crucial for the development of adipose tissue.
This could also have a positive effect in reducing the high interdonor variability, which
very often leads to valueless data because they are outside statistical significance.

2.4. The Dilemma with “Controls”

Most of the ex vivo and in vitro studies include age- and BMI-matched controls in
order to pinpoint lipedema-specific characteristics. It is reasonable to assume that people
undergoing cosmetic liposuction suffer from excessive localized fat accumulation, whether
due to overweight/obesity or lipohypertrophy. Neither condition is an optimal control
for lipedema. This is all the more important to reconsider since it cannot be excluded that
lipohypertrophy and lipedema share similar molecular mechanisms underlying adipose
tissue accumulation. These considerations are of secondary importance for the surgeon but
play a crucial role when it comes to evaluating and interpreting scientific data.

2.5. Is the Current Classification in Light of the Data and Experience Collected from Recent Years
Still Sufficient?
Since increasing medical and scientific interest has been focused on lipedema during
the last couple of years, many observations have been made which would eventually
justify a revision of the current staging of lipedema. Each lipedema stage must be seen as a
complex entity since it represents the sum of several conditions. Thus, it would be advisable
to extend the current staging by indicating additional information such as the occurrence
of co-morbidities (obesity, lymphedema, and others), the patient’s original weight (in case
of multiple operations), and the family history. A more differentiated classification would
correspond to the heterogeneous presentation of this clinical picture.

3. Summary
In recent years, lipedema research has evolved from purely clinical testing to molecular
science. In this short review, we have tried to give an overview of the diverse laboratory-
technical approaches and the findings obtained from them. Excellent and time-consuming
procedures have shown that lipedema manifests itself on many levels and have once again
confirmed that in vitro and ex vivo studies are essential to understand the multifaceted
nature of the disease.
However, what has also become clear is that many questions are still unanswered, and
a lot of preliminary data still needs to be deepened. We also addressed the main obstacles
hampering lipedema research. Currently, there is no mouse model, no cell lines which could
be used to achieve reproducible results. Therefore, one of the biggest handicaps, the high
interdonor variability, that often makes scientific results unusable, most likely will persist.
It is also evident that too little attention has been paid to the problem of accompanying
J. Pers. Med. 2023, 13, 98 12 of 15

obesity for too long. This does not mean that we have to start from scratch, but we need
to start discarding some unverifiable opinions about lipedema. As outlined above, there
are already valuable indications regarding in which direction(s) lipedema research should
develop. Implementing new findings and merging data from different disciplines will pave
the way to the successful elucidation of this enigmatic disease.

Author Contributions: Conceptualization, A.M.E., H.B., H.-C.B., A.-T.L.; investigation, A.M.E., H.B.,
H.-C.B., M.S., A.M., A.-T.L.; writing—original draft preparation, H.B., H.-C.B., A.-T.L.; writing—
review and editing, A.M.E., H.B., H.-C.B., M.S., A.M., A.-T.L.; visualization, A.M.E., H.B., M.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Land Salzburg (project number 20204-WISS/240/3-
2019) (Austria).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.

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