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Y. H. Pita-Juarez et al.
downstream analysis. Hence, for all assays, cells (or spots in
the case of Visium and areas of interest, in the case of GeoMx
DSP) are ltered out if they contain a low number of genes,
or a very low number of expressed transcripts. For many
single-cell spatial transcriptomics methods based on imaging, morphology can also be utilized to keep or discard areas,
such as the shape of the cells and the probability of incorrect
cell segmentation, as well as specic surface markers, such
as Pan-cytokeratin, DAPI delineating nuclei, and CD45. To
ensure that no erroneous data is included for downstream
analyses, spots/areas/cells with many negative probes in the
case of imaging single-cell spatial transcriptomics and spots
exhibiting high contamination, assessed with tools like
SpotClean, for spot-level spatial transcriptomics can also be
excluded from downstream analysis [87]. Finally, similarly
to scRNA-seq, cells/spots with high numbers of transcripts
that are known to indicate low quality data, such as high
mitochondrial reads are usually discarded from the analysis.
Technical differences between samples owed to processing batches or technology differences can also be a major
issue in spatial transcriptomics and extra care should be
taken during integrating different samples, especially across
modalities. Normalization accounts for differences in
sequencing and capture depth between cells (spots or areas
of interest, depending on the experiment) and is further complicated by variations in cellular density across the tissue.
Several methods for normalization and sample integration
that are available for single-cell RNA-seq have been adapted
to spatial transcriptomics with remarkable success [88]. The
selection of a dimensionality reduction method depends on
the main goals: summarization (e.g., PCA) or visualization
(e.g., UMAP) [89].
Downstream Analysis
The goal of downstream analysis after preprocessing is to
identify spatial domains with coherent gene expression proles, such as tissue niches and cell–cell interactions (CCIs)
within, or pathogenic domains in diseased tissues where
aberrant interactions may occur. Methods such as clustering
as well as integration with scRNA-seq data, such mapping,
deconvolution, and ligand-receptor studies [90], can be used
to achieve this goal (Fig.11.4b–d). However, in contrast to
single-cell RNA-seq, there are many different methods that
infer spatially aware embeddings [91] or clustering [92, 93]
and can integrate features from histology. This paradigm is
carried on to downstream analysis, such as cell-cell communication, which can also be performed in a spatially-aware
manner [94, 95], where interactions between faraway cells/
spots have less weight than between adjacent ones. Trajectory
analysis can also be done in a spatially aware manner [96,
97] and an analysis specic to spatial transcriptomics is
neighborhood [98, 99] and spatially variable gene identication [100], to identify higher-level features that might differ
in their spatial localization, either between conditions of
interest or treatments.
Finally, the integration of single cell with spatial transcriptomics data has been repeatedly shown to be able to
enhance the insight generated from each modality alone. For
spot-level spatial transcriptomics, this is often done through
cell type deconvolution, where given a single-cell reference,
each spot is broken down into the cell types it contains [101].
Many tools then offer the ability to perform downstream
analysis taking into account that information, such as celltype specic differential expression using distances [102,
103], identication of spatial domains with similar cell type
compositions [104], imputation of unseen gene expression,
based on single-cell RNA-seq [105], and inference of cellcell communication between cell types in each spot [106].
Even when lacking an appropriate reference for cell type
deconvolution, methods exist that allow for reference-free
cell type deconvolution into cell types which can then be
annotated using expression [107, 108]. For single-cell spatial
transcriptomics, the different feature space between singlecell RNA-sequencing and spatial transcriptomics can pose
issues during integration, which can be solved through specialized pipelines [109, 110].
Conclusion andFuture Directions
RNA transcriptomics at bulk, single cell, and spatial level
have been proven invaluable assets for studying the gene
expression patterns involved in wound healing. By analyzing
the transcriptomes of different cell types in wounded tissue,
researchers identify the genes and signaling pathways that
are activated or suppressed during the healing process. This
information has already provided insights into the cellular
and molecular mechanisms underlying tissue repair, as well
as potential therapeutic targets for promoting or accelerating
healing [1].
The eld of single-cell multi-omics has been expanding
rapidly, and spatial tools are following closely behind as the
newest and most comprehensive modality. Single-cell RNA
transcriptomic studies have revealed the heterogeneous skin
cell types involved in the wound healing, identied novel
gene expression patterns, such as the upregulation of extracellular matrix (ECM) proteins, growth factors, and cytokines, as well as the activation of inammatory and
angiogenic pathways [18]. On the other end, spatial transcriptomics has emerged as a groundbreaking tool for studying the spatial organization of gene expression patterns in
wounded tissue.
Combining scRNA-seq with ST can be a powerful technique, allowing researchers to map the transcriptomes of
11 High Content Single Cell and Spatial Tissue Proling Modalities for Deciphering the Pathogenesis and Treatment of Wound…
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215
individual cells in their native spatial context, providing a
more comprehensive understanding of the cellular interactions and signaling pathways involved in tissue repair [111].
Recent publications have described spatial versions of highthroughput single-cell assays such as single-cell assays of
transposase-accessible chromatin with sequencing (ATACseq) [112], chromatin landscapes in single cells (CUT&Tag)
[113], and whole-genome sequencing [114], along with
imaging techniques for epigenomic elements. Spatial biology now includes proteomics and functional CRISPR-based
perturbations to study the spatial effects of gene knockouts
[115]. Joint proling of multiple measurements from the
same single cell, such as joint scRNA-seq and ATAC-seq, is
expected to be included in spatial tools soon. To keep pace
with these rapid technological advances, innovations such as
sample barcoding and targeted sequencing methods have
been widely adopted in scRNA-seq workows to increase
throughput and efciency, reduce sequencing depth requirements, and decrease costs [116].
While many of these discoveries are still in the research
phase, their implications for therapy can be signicant [117–
122]. The development of computational algorithms to inte-
grate these techniques with clinical data has the potential to
accelerate the translation of knowledge into clinical care.
Further improvements in standardization and accessibility
have already led to the integration of these complex assays
into clinical trials, fostering the development of personalized
medicine approaches for optimized treatments in wound
healing. Ultimately, the continued advancements in these
techniques have the potential to signicantly enhance our
understanding of wound healing mechanisms and lead to
more effective treatments.
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Structural andFunctional Changes
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inSkin oftheDiabetic Foot
PaschalisChatzipantelis, EleftheriaAngelikiValsami,
AntoniosKafanas, andAristidisVeves
12
Abstract
Dermatologic conditions which are related to diabetes
include acanthosis nigricans, characterized by a hyperpigmented, velvety, cutaneous thickening that appears
predominantly in the neck, axilla, and groin areas; necrobiosis lipoidica (NL), a chronic, necrotizing, granulomatous skin disease; granuloma annulare; diabetic bullae
and diabetic dermopathy; and infections. As these conditions can be present in the lower extremity, they should be
sought and easily recognized by the health care providers
who manage the diabetic lower extremity.
Anatomy oftheSkin
The normal adult skin consists of epidermis, dermis, and subcutaneous tissue. The epidermis shows in most anatomic
places, thin downward projections, called rete ridges. The epidermis contains four stratied squamous epithelium layers
from supercial to deep (Fig. 12.1) [1, 2]. The rst layer,
which is called stratum corneum, is composed of multiple layers of anucleate keratinocytes. The stratum lucidum is a special layer of stratum corneum and is located just above the
stratum granulosum and below the stratum corneum. We can
nd this thin layer only in the thick skin of the palms, soles,
and digits. It is mainly composed of eleidin, a protein rich in
lipids which act as a barrier to water. The second layer, stratum
granulosum, consists of one to three layers of attened cells
P. Chatzipantelis
Medical School, Democritus University of Thrace,
Alexandroupolis, Greece
E. A. Valsami · A. Veves (*)
The Rongxiang Xu, MD, Center for Regenerative Therapeutics,
Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA, USA
e-mail: aveves@bidmc.harvard.edu
A. Kafanas
Lincoln County Hospital, Lincoln, Lincolnshire, UK
positioned parallel to the surface. Their cytoplasm contains
keratohyline granules consisting of keratin, prolagrin, loricrin, and trichohyalin, proteins which play an important role in
the process of cornication. When the keratinocyte differentiation process begins, these granules maturate and become
enlarged, which results to the conversion of keratin tonolaments into a homogenous keratin matrix, which is considered
a very important step in cornication. The keratohyalin granules are essential for the skin moisturization and UV protection. Stratum spinosum, the third layer, consists of ve to ten
layers of keratinocytes with desmosomes. These are adhesive
intercellular junctions that mechanically integrate adjacent
cells by coupling adhesive interactions mediated by desmosomal cadherins to the intermediate lament cytoskeletal network. The desmosomal cadherins are connected to intermediate
laments by a dense clusters of cytoplasmic plaque proteins
which consist of members of the armadillo gene family,
including plakoglobin and plakophilin and members of the
plakin family of cytolinkers, of which desmoplakin is one of
them. The Langerhan cells are interspersed among the keratinocytes of this layer. These cells are tissue resident dendritic/
macrophages. They transmit to the immune system the microenvironmental context in which they encounter foreign proteins and play a crucial role in helping the immune system to
respond appropriately [3]. The innermost layer of the epidermis is the stratum basalis. This is a single layer of cells, which
is mainly made of basal precursor of the keratinocytes of the
epidermis. Melanocytes and Merkel cells are the two other cell
types which are found dispersed among the basal cells in the
stratum basalis. Melanocytes are highly differentiated cells
that produce melanin pigment inside melanosomes. Melanin
provides pigmentation to the skin, eyes, and hair and also
absorbs harmful UV (ultraviolet) rays and protects cells from
damage. Melanocytes are dark and dendritic in shape [4].
Merkel cells are special type of cells derived from neural crest
cells. These cells are located in the basal epidermal layer. They
are very close to the nerve endings which receive the sensation
of touch. Merkel cells also contain neuroendocrine substances
that may act as hormones [5].
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
A. Veves et al. (eds.), The Diabetic Foot, Contemporary Diabetes, https://doi.org/10.1007/978-3-031-55715-6_12
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Sublamina densa r
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P. Chatzipantelis et al.
The epidermis is based on basement membrane which is
better visible with histochemical stains such as PAS and Alcian
Blue and silver nitrate stains. These special stains show a band
of polysaccharides and reticulum. The role of the basement
membrane is to connect and functionally separate the epidermis and the dermis. As it has become evident from transmission electron microscopy, we can see that the basement
membrane consists of Lamina lucida, an electron density
region, containing laminins. Below it is the Lamina densa an
electron dense region, of which a major component is collagen
IV and the Sublamina densa which is located in upper papillary dermis and contains loops of type VII collagen and
Fig. 12.1 Normal skin. H&E stain
anchoring brils (Fig.12.2) [6]. The basement membrane contains hemidesmosomes that are attached to the basal cell keratinocytes tonolaments. Hemidesmosomes are multiprotein
complexes that enable the stable adhesion of basal epithelial
cells to the underlying basement membrane [7].
Hemidesmosomes contain plaque proteins which are involved
in intermediate lament anchoring including BPAG1, BPAG2,
integrin α6β4, and p200. Lamina lucida also contains variable
protein structures, associated with anchoring laments. Any
defects in basement membrane zone adhesive molecules
attributable to autoantigens or gene defects can play a crucial
role in bullous dermatoses and wound healing [8].
Meissner’s corpuscles which are encapsulated nerveendings are responsible for sensitivity to light pressure. They
are mostly concentrated in thick hairless skin, especially on
the nger pads and they are primarily located in glabrous
skin just beneath the epidermis in papillary dermis. The
Vater-Pacini corpuscles, which are located in the deeper
level of the dermis, are large nerve-ending organelles that
generate a sense of pressure. They are found commonly in
the nipple and anogenital region. Pain, temperature, and itching sensation are transmitted by unmyelinated nerve bers
that end around hair follicles and in the papillary dermis. The
skin is supplied with sensory nerves and autonomic nerves,
which permeate the entire dermis with nerve bers and
exhibit frequent branching. Intra-epidermal nerve endings
are conventionally described as passing freely in between
keratinocytes. These are responsible for cutaneous sensation
of temperature, itch, touch, and pain. They show variable
distribution in different anatomical sites. Studies in human
skin biopsies conducted by confocal laser scanning micro-
Fig. 12.2 Protein micro- anatomy in basement
membrane. (Reproduced from The Biology of
the Basement Membrane. Plastic Surgery key.
2019. https://plasticsurgerykey.com/
the- biology- of- the- basement- membrane/)
“LAMINATED” MODEL OF THE EPIDERMAL BASEMENT MEMBRANE
Basal keratinocyte
Keratin intermediate
filaments
Hemidesmosome
Plasma membrane
Lamina lucida
Anchoring filaments
Lamina densa
egion
Anchoring fibrils
Microfibrils
Microthread-like fibers
Interstitial collagens
Anchoring plaques
Keratin 5
Keratin 14
Plectin, BPAG1
BPAG2, integrin a
HSPG
Laminins 5, 6, & 10*
Type IV collagen
Nidogen, HSPG
Type VII collagen
Linkin, fibulins
Fibrillins, LTBP, elastin
Type IV collagen
Types I and III collagen
6b4
12 Structural andFunctional Changes inSkin oftheDiabetic Foot
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scope show that intra-epidermal nerve endings may play an
important role in interactions between nervous system, keratinocytes, and immune system [9].
The papillary dermis is the uppermost layer of the dermis,
intertwines with the rete ridges and is composed of ne and
loosely arranged collagen bers mostly type I and some type
III.It also contains elastic bers which are typical sparse, thin,
and branching. Terminal capillary vessels, small nerve bers,
Meissner corpuscles in acral skin, and touch receptors are
present in papillary dermis. Positioned under the papillary dermis is the much thicker reticular dermis which is composed of
densely-packed collagen bers. At least eight different types
of collagen are found in human skin. Reticular dermis is the
primary location of dermal elastic bers. The dermis contains
many cell types. Fibroblasts are the principal cell types of the
dermis, and they are responsible for the synthesis of collagen,
elastic, and reticular bers. Histiocytes are tissue macrophages
present within the dermis that assist the immune system. Mast
cells are inammatory cells predominantly located in the perivascular areas of the dermis. Mast cells secrete vasoactive and
proinammatory mediators important in inammatory reactions, collagen remodeling, and wound healing.
Dermal adnexal structures such as eccrine and apocrine
glands, sebaceous glands, hair follicles, and their arrector
pili muscle are present in papillary dermis. The vessels
responsible for the blood supply of the skin are deep in the
hypodermis. The branches of small arterioles and venules
head upwards to form a deep and a supercial plexus. The
deep plexus is located at the dermal/hypodermal junction. It
provides the hypodermis and the deeper parts of the dermis
with fatty tissue, including the capillaries for hair follicles,
deep sebaceous glands, and sweat glands. The supercial
subpapillary plexus is located just beneath the dermal papillae and acts as a supplier in the dermal papillae. Lymphatics
are often inconspicuous in normal skin. Skin lymphatics are,
in fact, rather large vessels with extremely attenuated walls
principally lying as two plexuses which loosely follow the
arteriovenous plexuses. They are only detected in blood ow
stasis conditions when they become ectatic [10].
The subcutis also called the subcutaneous layer is a layer
directly below the dermis and serves to connect the skin to the
underlying tissue. It is not a part of the skin per se, although
the border between the hypodermis and dermis is difcult to
discern. The hypodermis consists of well- vascularized, loose
connective tissue and mature adipose tissue.
Dermal Matrix andInterstitial Fluid inDM
The dermal interstitial uid lls the spaces between the dermal matrix bers and contains mainly glycoproteins, water,
electrolytes, and plasma proteins. Collagen bers, which are
the major component of the dermis matrix, provide the skin
221
Fig. 12.3 Normal skin. Masson’s trichrome stain shows thick collagen
bundles in dermis (×100)
with tensile strength. Twenty-nine types of different collagen
have been described in humans; however, more than 90% of
the body’s collagen is represented by types I, II, III, IV, and
V.Type I accounts for approximately 80% of the total amount
of dermal collagen and is found in the large ber bundles of
the reticular dermis (Fig.12.3). Depletion of type I procollagen in human leg skin has been reported in diabetic
patients, both in the absence and in the presence of complications, with depletion being worse in patients with ulcers [11].
Additionally, unlike diabetic patients without complications,
signicant disarray of the dermal collagen bundles has been
reported after light microscopic analysis of the skin of
patients with foot ulcers [11]. The dermal interstitial uid
may be mechanistically important in explaining skin alterations in diabetes. In addition, interstitial uid is a rich source
of biomarkers that does not clot. It lls extracellular space in
tissues, which means that interstitial uid biomarkers provide systemic information due to their origins in blood and to
their contact with cells in dermis.
Diabetes increases the degradation of collagen and elastic
bers resulting alterations in the appearance such as loss of
elasticity, increase in epidermal thickness and alterations in
the in the form of wrinkles, and reduced ability to retain
moisture, similar to skin aging [12, 13]. The results of the
histology research were applied to the mode. A generally
disorganized dermis has also been visualized by scanning
electron microscopy of diabetic skin of 12-week-old
Tsumura-Suzuki obese diabetic mice with smaller and less
dense bers [14]. These data, in addition to indicating pathological deposition, also indicate collagen reduction in diabetic skin. Similarly, decreased expression and production of
dermal type I collagen has recently been described in
Alloxan-treated mice with overt DM and also in mice with
blood glucose uctuations [15]. Type III, also known as fetal
collagen or reticulum bers, represents up to 10% of dermal
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P. Chatzipantelis et al.
collagen. This type of collagen is prevalent during fetal life;
however, in post fetal life, it is limited to the papillary and
adventitial dermis. Furthermore, it serves as a framework on
which type I collagen is synthesized. Decreased collagens I
and III content in diabetic skin are associated with a reduced
collagen I/III ratio. However, this decreased collagen content
is associated with increased gene expression of enzymes
involved in collagen synthesis and decreased production of
factors that promote collagen degradation. This suggests that
the defect in collagen protein content in diabetic skin at baseline is at the post-transcriptional level [16]. A lower I/III collagen ratio has been associated with reduced connective
tissue stability [17], which might explain the lower mechanical stability of diabetic skin. Studies showed that rapid initiation of insulin treatment is clearly benecial for collagen
recovery in the diabetic skin, offering an important source
for the recommendation of early glycemic control in diabetic
patients [18].
Elastic bers in the papillary dermis are thin and oriented
perpendicularly to the skin surface (Fig.12.4), while in the
reticular dermis, they are thicker and parallel to the skin surface. Elaunin and oxytalan bers are names for the elastic
bers in the papillary dermis. Elaunin bers are bundles of
microbrils that form a plexus oriented parallel to the
dermal- epidermal junction. From this plexus, cross-linked
elastic bers called oxytalan run upward and terminate at the
basement membrane. Studies show decreased levels and
degeneration of the elastic bers in diabetic patients [19–21].
Another important component of the human dermal matrix/
interstitial uid, namely hyaluronic acid, has been studied in
human skin in patients affected by insulin-dependent DM.A
considerable reduction in hyaluronic acid, particularly in the
region of the dermal epidermal junction, has been found in
the dermis of patients with low joint mobility, whereas in
Fig. 12.4 Normal vulval skin. Orcein stain highlights brown stained
elastic bers (×100)
patients with little or no impairment of joint mobility, hyaluronic acid distribution predominantly resembles that of the
normal condition [22].
Skin Inammation inDM
The normal mammalian response to skin injury occurs in
three overlapping but distinct stages: inammation, new tissue formation, and remodeling. Inammation, the rst stage
of wound repair, occurs immediately after tissue damage,
and components of the coagulation cascade, inammatory
pathways, and immune system are needed to prevent ongoing blood and uid losses, to remove dead and devitalized
tissues, and to prevent infection. Dysregulated inammation
is one of the primary pathologies associated with chronic
wounds; thus, understanding of the causes and consequences
of dysregulated inammation in diabetes is key to developing effective treatments [23].
Dermatologic Conditions inDM
Acanthosis Nigricans
Acanthosis nigricans (AN) is one of the most recognized
skin manifestation of diabetes. AN prevalence is currently on
the rise particularly due to obesity and diabetes. AN is
observed in fully 74% of obese patients and becomes a reliable cutaneous marker of hyperinsulinemia in obese individuals [24, 25]. AN is a symmetric eruption characterized
by a hyper-pigmented, velvety, cutaneous thickening that
appears predominantly in the neck, axilla, and groin areas.
The histological ndings are papillomatosis and hyperkeratosis, characterized by irregularly folded epidermis, exhibiting various degrees of acanthosis. Typically, the dermal
papillae are projected upward, and the valleys in between
them show mild to moderate acanthosis and lled with keratotic material. The epidermis at the top and at the sides of the
papillae appears thinned and the brown color of the lesions is
due to the thickening of keratinin-containing supercial epithelium [26]. There are eight types of acanthosis nigricans:
hereditary benign AN, obesity-associated, syndromic, malignant AN associated in particular with abdominal adenocarcinoma (gastric carcinoma), acral or benign AN, drug-induced
(nicotinic acid and corticosteroids), and mixed. AN is a
chronic but reversible condition. In obesity associated AN,
the pathogenesis is related to high levels of circulating insulin, bound with insulin-like growth factor receptors and stimulates keratinocyte and dermal broblast growth. In the
malignant form of AN, the associated growth factors secreted
by underlying malignancy are believed to result in cutaneous
changes of AN [27]. Acanthosis nigricans can occur in non-
12 Structural andFunctional Changes inSkin oftheDiabetic Foot
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obese patients with diabetes due to malfunction of receptor
binding. Three types have been described: Type A with insulin resistance due to a congenital defect of insulin receptors,
Type B with autoimmune diseases and antibodies against the
insulin receptor, and Type C insulin resistance are associated
with a postinsulin receptor defect.
Treatment consists of treating the underlying cause. In the
diabetic patient, weight control, dietary restrictions, and
increased physical activity are of primary importance and
have been proved to be most effective in controlling AN [28,
29]. Other treatment options include both laser therapy and
surgical excision. Topical keratolytics (e.g., salicylic acid,
retinoic acid, and ammonium lactate) and oral isotretinoin
can reduce thicker plaques in areas of maceration, decreasing odor and Psoralen plus UVA (PUVA) has been reported
as benecial for symptomatic relief in cases of paraneoplastic acanthosis nigricans [30].
Necrobiosis Lipoidica
Necrobiosis lipoidica (NL) is a chronic, necrotizing, granulomatous skin disease that occurs primarily in individuals
with diabetes, usually type I.It appears in the form of red
non-scaling patches or plaques sharply demarcated with
irregular contours. The edges are elevated, erythematous,
and slightly indurated; the center of the lesion is atrophic,
yellow-brownish, and may ulcerate (Fig.12.5). Lesions often
start out small, but have a tendency to grow to several centimeters in diameter. The major complication of the disease is
the formation of an ulcer, mainly occurring after trauma.
Infections can also occur. The ulceration is relatively frequent if lesions are large but perforation is generally rare.
They may be single or multiple, most commonly distributed
bilaterally on the lower extremities, particularly the pretibial
areas, but may occur on the face, trunk, and upper extremities as well. Histologically the whole of the dermis is affected
by palisaded granulomatous inammation sparing the epidermis. The inammation often spreads into subcutaneous
septae giving the false impression of subcutaneous panniculitis. Collagen degeneration without mucin component is
demonstrated in the central of the lesion (Fig. 12.6).
Furthermore, the periphery of the main lesion usually exhibits sclerosis and sometimes lipid droplets associated with
foam histiocytes are evident. In deep dermis, lymphoid follicles and plasma cells may be present. The latter are considered to be a strong histological nding conrming the
histological diagnosis. The differential diagnosis includes
palisaded granulomatous dermatitis, among them being
granuloma annulare, rheumatoid nodule, and necrobiotic
xanthogranuloma (NX). In NL the degenerated collagen is
pale, acellular, and horizontal in its distribution. This pattern
has been linked to the appearance of a layer cake. Rheumatoid
223
Fig. 12.5 NLD in a 16-year-old girl with T1DM and a patch on the leg
with an atrophic, depressed, slightly yellow center, and well-dened
raised purple edge
Fig. 12.6 Patchy lymphoplasmacytic inltration around blood vessels
(arrows point to plasma cells, H&E ×200)
nodule granulomas tend to be larger and are usually located
over bony prominence, near joints. Histologically they are
located in the deep dermis, or in subcutis enclosing a central
area with brin, which is homogeneously eosinophilic lucking mucin. NX typically shows a periorbital predilection.
Histological ndings for NX include an inammatory mixed
cellular population with Touton type giant cells, foamy histiocytes, and necrotic areas with neutrophilic debris involving the dermis and subcutaneous tissue [20]. The cause of
NL is unknown, but there are several proposed theories
behind the pathophysiology of NL such as microangiopathic
changes, abnormal collagen, altered lipid metabolism, and
impaired immunity. The combination of microangiopathy,
neuropathy, and the release of inammatory cytokines leads
to the destruction of the collagen matrix, resulting in sclerosis and granulomas formulation. Direct immunouorescence
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