**2. Mechanisms**

While there are a number of cell types, environmental factors, and cellular pathways that contribute to the development of adipose tissue fibrosis, the overarching cause is obesity (Figure 1) [18–23]. However, the link between obesity and fibrosis is complex and incompletely understood. While there is an increased prevalence of adipose tissue fibrosis in cases of severe obesity [19,24], not all obese patients will develop fibrotic tissue. Primarily based on the correlative nature of human studies, there remains many open questions surrounding the link between obesity and fibrotic tissue. This synopsis aims to introduce several mechanisms that have been thoroughly researched.

Proper adipose tissue ECM composition and remodeling is crucial for cellular function. During the development of fibrosis, excess ECM proteins are produced while ECM degradation is limited. Several cell types in adipose tissue including adipocyte progenitors, adipocytes, fibroblasts, and myofibroblasts are responsible for the production of ECM proteins [25].

Fibronectin and collagens are the most prevalent ECM proteins found in healthy and fibrotic adipose tissue. Comparing obese and diabetic mice, it was determined that type I, III, V, and VI collagens are present in higher concentrations compared to healthy mice [26]. Collagen I (COL1) is responsible for providing structure within the tissue [14], while collagen V (COL5) in high concentrations inhibits angiogenesis in adipose tissues [27]. Specific to adipose tissue fibrosis, collagen VI (COL6) has been investigated for its fibrotic role, with the absence of COL6 (in a knockout model) resulting in lower inflammation and uninhibited enlargement of individual adipocytes [28]. Furthermore, a component of COL6, COL6a3 named endotrophin, stimulates fibrotic collagen deposition, inflammation, and insulin resistance [22]. In times of energy surplus, adipocytes undergo hypertrophy (enlargements of cells) and hyperplasia (an increase in the number of cells) [29]. As adipocytes become larger, they not only exhibit morphological changes but also become metabolically dysfunctional [30,31], producing several proinflammatory chemokines. These chemokines include tumor necrosis factor-alpha (TNF α), inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), interleukin 8 (IL-8), C-reactive protein, Transforming growth factor beta 1 (TGFβ1), soluble intercellular adhesion molecule (ICAM), and monocyte chemoattractant protein 1 (MCP-1) [31–40]. Additionally, adipocytes exhibit increased gene expression for ECM production under a high fat diet [41]. Similar to mature adipocytes, adipose derived stem cells (ASCs) also undergo significant changes during obesity. After being fed a high fat diet, mice had higher concentrations of ASCs that expressed platelet-derived growth factor receptor α (PDGFRα) [42]. PDGFRα activation opposes adipogenesis and causes cells to differentiate into a profibrotic phenotype [43]. The PDGFRα<sup>+</sup> ASCs were found to promote fibrosis and are associated with insulin resistance [42].

**Figure 1.** Schematic showing changes to adipose tissue during obesity and fibrosis development.

Hypoxia is another major contributor in the development of adipose tissue fibrosis. As adipocytes expand due to lipid accumulation, they eventually reach the diffusion limit of oxygen. This results in hypoxia that causes stress signals to increase angiogenesis and remodel ECM proteins in an attempt to mitigate the oxygen shortage. In hypoxic adipose tissue, hypoxia-inducible factor 1-alpha (HIF1α) is expressed. Instead of initiating proangiogenic conditions in adipocytes, it enhances synthesis of ECM components. HIF1α is also suggested to play a role in collagen crosslinking and stabilization [7]. Both adipocytes and adipocyte progenitor cells produce angiogenic factors, such as vascular endothelial growth factor (VEGF), leptin, fibroblast growth factor 2 (FGF-2), and hepatocyte growth factor (HGF) [44,45]. Interestingly, endothelial cells derived from obese adipose tissue have increased gene expression related to inflammation and senescence, and impaired angiogenesis [46], suggesting that initial signals to promote angiogenesis are reversed if hypoxia is unresolved. Therefore, prolonged hypoxic conditions cause injury and long-term damage, fibrosis, cellular senescence, and necrotic adipocyte death [47].

Unresolved hypoxia can also attract proinflammatory cell types to the area. A population of cells that have a high concentration in fibrotic adipose tissue are M1 macrophages. Resident macrophages comprise between 10–15% of stromal cells in the adipose tissue of healthy, lean individuals [48]. This concentration jumps to between 45–60% of stromal cells in obese individuals [35,49,50]. Macrophages are typically observed in "crown-like structures" surrounding dead or dying adipocytes in obese adipose tissue [51,52]. The increase in macrophage concentration causes an increased recruitment of monocytes to the area; where the monocytes then differentiate into macrophages [35,48]. In the presence of M1 macrophages, ASCs are predisposed to differentiate into proinflammatory cells. This change occurs due to pro-inflammatory cytokines secreted by the macrophages [49,53], where it has been shown that they are the primary producers of TNF α, iNOS, and IL-6 [44].

ECM degradation is regulated by metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). MMPs have the ability to solubilize ECM components while specific TIMPs are able to inhibit MMPs activity. Obesity and type 2 diabetes have been linked to increased circulating TIMPs concentration causing MMP activity to be lower than in healthy individuals [54,55]. The increase in proinflammatory cytokines that occurs during hypoxia and inflammation has also been linked to regulation of MMPs expression [56].

Another cell population that has been linked to adipose tissue fibrosis is mast cells [57,58]. Mast cells can promote fibroblast growth and collagen production by releasing cytokines, chemokines, proteases, etc. [59,60]. By comparing adipose tissue from obese patients with the metabolic syndrome to healthy patients, it was determined that there is an accumulation of mast cells in the subcutaneous adipose tissue of obese patients. Related to fibrotic changes, there were positive correlations to collagen, leptin, and glucose concentrations, as well as waist circumference [57,61]. Overall, more research is needed to determine the role of mast cells and macrophages in the fibrotic transition of adipose tissues.

One method of better understanding the role of di fferent cell types in ECM production, immune reactions, and fibrosis is single-cell RNA (scRNA) sequencing. When looking at adipocytes specifically single nuclei adipocyte RNA sequencing (SNAP-seq) is used [62]. These techniques have been used to better understand variability in cells from di fferent fat deposits or pathological settings [63–67]. It o ffers a method of characterizing di fferent subpopulations of adipose tissue cells and detecting heterogeneity in the populations [68].

#### **3. In Vitro Adipose Tissue Fibrosis Modeling**

Whenever possible, in vitro models are utilized to refine, reduce, and replace animal models. In vitro models can be quickly developed (compared to animal models), are easily manipulated with highly defined conditions, and have a high likelihood of clinical translation (when developed with human cells). Having accurate biomimetic in vitro adipose tissue fibrosis models can serve as useful tools for generating a better understanding of the mechanisms behind fibrosis development and progression, as well as drug screening for e ffective anti-fibrotic drugs.
