Therapeutic applications of conditioned medium from adipose tissue

Abstract

For the past number of decades, adipose tissue has attracted significant interest due to its complicated composition and versatile functions. Adipose tissue is no longer considered to be just an energy‐storing fat pad, but is also a key ring player in interaction networks between various organs and tissues. A wide range of factors released by adipose tissue are responsible for regulation of adipose tissue and other distant target tissues and cells, such as kidneys, skeletal muscle, the cardiovascular system and the immune system, in an auto‐/paracrine manner. A mixture of bioactive molecules makes up the conditioned medium of adipose tissue. The beneficial role played by these bioactive molecules in angiogenesis, wound healing, tissue regeneration and immunomodulation has been demonstrated by various studies. Study of this conditioned medium helps deepen our understanding of underlying mechanisms and broadens the potential for therapeutic applications. In this review, we have aimed to improve fundamental understanding of conditioned medium from adipose tissue and to summarize recent efforts to study its therapeutic applications.

1. Introduction

Adipose tissue, an elaborate and heterogeneous tissue, is consisted of not only mature adipocytes and fibroblast pre‐adipocytes or stem cells but also of pericytes, endothelial cells and macrophages.1 Adipose tissue is also a storage of secretory factors, including cytokines, growth factors, hormones and extracellular matrix‐processing proteases.2 It can communicate with central and peripheral organs via the various secretory factors, which are known as adipose‐derived secreted factors or adipokines.3 The discovery of the secretory factors from adipose tissue has made the conditioned medium gain an increased interest from scientific and medical community. Plenty of the recent researches have demonstrated the beneficial effect of the conditioned medium from adipose tissue in angiogenesis and adipogenesis in vitro and in vivo.4, 5 Besides, conditioned medium from adipose tissue was illustrated to be involved in the vast amount of pathophysiological processes such as wound healing,6 immunoregulation7 and regeneration.8 The vast amount of processes that the conditioned medium from adipose tissue are involved in and the versatility of manner in which they can influence the target receptors make this bioactive mixture an interesting source for both therapeutic and diagnostic applications. Here, we review the recent developments in conditioned medium from adipose tissue and discuss the potential therapeutic applications of conditioned medium from adipose tissue.

2. Conditioned medium

Conditioned medium from adipose tissue is consisted of all the secretory factors from adipose tissue, including adipokines (such as leptin and adiponectin),9, 10 cytokines (such as TNF‐α and IL‐6),11, 12, 13 growth factors (such as VEGF)14 and extracellular vesicles (such as exosomes).15 The preparation method of conditioned medium has been compared in the previous studies.2, 16, 17 By the means of these secreted factors, adipose tissue can communicate with various organs and tissues and participate in multiple pathophysiological processes. Recent studies analyseis the composition of the conditioned medium in the background of a differentiating or pro‐inflammatory stimulus to get a deeper understanding of a set of factors secreted by adipose tissue.18, 19 Instead of classifying the adipokines as non‐classical, classical or ER/Golgi‐dependent secretory factors,18 we divided the adipokines into pro‐inflammatory and anti‐inflammatory to make an easier understanding of their biological effects. Sources and functions of key adipokines are summarized in Table 1. However, as a lack of the establishment of the standardized and effective isolation and detection methods, the quality of conditioned medium samples various in different studies. Zvonic et al. detected 101 proteins in the ADSCs (adipose‐derived stem cells) secretome, while Kim et al. detected 474 proteins.20, 21 Alvarez et al. cultured tissue block in media containing labelled lysine and found that 70 labelled proteins out of 259 identified proteins were considered secreted by adipose tissue.22 Furthermore, the composition of the conditioned medium varies under different conditions. In the presence of insulin, the up‐regulated factors contributed to ER stress response and extracellular matrix remodelling.1 Likewise, more inflammatory and extracellular matrix factors were found in obese adipose tissue.23 By means of performing a secretome mapping, Roca et al.24 characterized the rat visceral, subcutaneous and gonadal fat‐specific secretomes and found different levels of protein secretion between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT); they found adipokines or adipose tissue‐related proteins (such as thrombospondin‐1, galectin‐1) were at a higher level in VAT but vitamin D‐binding protein was on the contrary. These findings indicate that secretory factors from adipose tissue in different anatomical locations may have different biological functions, which is in accordance with the different functions of the widespread adipose tissue. Among the conditioned medium, a nanosized extracellular vesicle, exosome has attracted widespread attention. Exosome‐harbouring proteins, RNA and lipids from donor cells can be transferred to distant target cells to change the pathophysiological state of the target cells.25, 26, 27 So far, there are over 4000 different proteins that have been identified from purified exosomes.28 Except some proteins common to most exosomes (such as tumour suppressor gene 101 [Tsg 101], heat‐shock protein 70 [Hsp70], CD9, CD63 and CD81), others depend on the source of the cells and tissue type. Taking the biogenetic specificity of exosomes into consideration, adipose tissue‐derived exosomes are commonly involved in obesity and obesity‐related diseases through the ability of signalling responses when fused with target cells.15, 29, 30 The main characters and functions of exosomes were summarized in our previous review.31

Table 1.

Sources and functions of key adipokines

	Source	Function	Reference
Pro‐inflammatory adipokines
Leptin	Adipocytes	Appetite regulation	9
Resistin	Mononuclear cells (human), adipocytes (rodent)	Induce insulin resistance	39, 40
RBP‐4	Liver, adipocytes, macrophages	Decrease insulin sensitivity	46, 47
TNF‐α	Vascular cells, adipocytes	Promote insulin resistance	11, 12
IL‐6	Adipocytes, vascular and muscular cells	Gain weight Insulin resistance	13
Visfatin	Visceral adipocytes	Effect insulin sensitivity	43
Anti‐inflammatory adipokines
Adiponectin	Adipocytes	Improve insulin resistance	10
Omentin	Omental adipose tissue stromal cells	Improve insulin resistance	59, 60
SFRP5	Adipocytes	Improve insulin resistance	29

RBP‐4, retinol‐binding protein‐4; TNF‐α, tumour necrosis factor‐α; IL‐6, interleukin‐6; SFRP5, secreted frizzled‐related protein5.

3. Therapeutic applications of conditioned medium

Studies have shown that the adipose tissue communicates with other central and peripheral organs by a variety of bioactive secreted molecules.3 The secretory function makes adipose tissue a complex and dynamic endocrine organ involving various pathophysiological processes. Given that adipose tissue is a heterogeneous tissue consisting of ADSCs, adipocytes, endothelial cells, macrophages, blood cells and immune cells,22, 32 every cell type may regulate the secretion pattern of the adipose tissue. It is more reasonable to investigate the conditioned medium of adipose tissue which contains a mixture of secretomes of different cell types to gain a wider picture into the potential applications of adipose tissue. The main therapeutic applications of conditioned medium are summarized in Table 2.

Table 2.

The main therapeutic applications of conditioned medium

Applications	Adipokines	Mechanism	Reference
T2DM	Adiponectin	Potential biomarkers	33, 34
	Leptin	Potential biomarkers	35
	SFRP5	Potential biomarkers	36
	TNF‐α	Potential biomarkers	42
	Resistin	Potential biomarkers	39, 40
	IL‐6	Potential biomarkers	41
	RBP‐4	Potential biomarkers	43, 44
	Omentin	Anti‐diabete	70
Wound healing	TNF‐α	Activate cells in inflammation phase	59, 61
	PDGF	Promote to form clot and branches	59, 60
	TGF‐β	Activate cells in inflammation phase	59, 61
	VEGF	Promote matrix production and angiogenesis	61, 62
	FGF	Promote matrix production and angiogenesis	61, 62
	α‐SMC	Matrix formation and tissue remodelling	64
Adipose tissue regeneration	VEGF	Promote early angiogenesis	8, 73
	FGF	Promote early angiogenesis	8, 73
Atherosclerosis	A‐FABP	Induce vascular inflammation	85
	Adiponectin	Prevent obesity‐induced endothelial dysfunction	85
	Omentin	Anti‐atherogenesis	87

T2DM, type 2 diabetes mellitus; α‐SMC, α‐smooth muscle actin; A‐FABP, adipocyte fatty acid‐binding protein.

3.1. Diagnosis

As the complicated composition of the conditioned medium and its dynamic variation in pathological state, conditioned medium become a source of biomarkers for disease diagnosis. Type 2 diabetes mellitus (T2DM) is one of the major health problems of the modern world crying out for new diagnostic tools to assess early metabolic disorders. A number of bioactive substances secreted from adipose tissue provide us an alternative biomarker such as adiponectin,33, 34 leptin35 and SFRP536 to diagnose diabetes and its complications. Most of the T2DM are characterized by insulin resistance, accompanied by obesity.37 Insulin resistance can be induced by excessive release of free fatty acids (FFA) from adipose tissue mass and secretion of inflammatory factors and adipokines.38 Considering the close connection of obesity to diabetes, the detection of the secretory function of adipose tissue can be reasonable for the early diagnosis, rather than the diagnosis of hyperglycaemia, a symptom of the diabetes. Studies have shown an increased concentration of resistin,39, 40 leptin,35 IL‐641 and TNF‐α42 or a decreased concentration of adiponectin and visfatin in the case of insulin resistance and T2DM.43, 44, 45 Besides, animal experiments show a higher level of retinol‐binding protein‐4 (RBP‐4) in animals with insulin resistance46 and the injection of recombinant RBP‐4 in healthy mice can cause insulin resistance.47 Studies on chemerin show that there is a higher chemerin concentration in diabetic mice and serum chemerin concentration is significantly elevated in patients with T2DM and cardiovascular diseases.48, 49 In addition to the secreted proteins, the level of circulating exosomes in T2DM also shows a significant increase.50, 51 Moreover, there is also a close relationship between the levels of specific miRNAs and proteins in exosomes and diseases (such as obesity, pancreatic cancer, glioblastoma and melanoma).52, 53, 54, 55, 56 The evaluation of the factors in conditioned medium with high sensitivity methods may be a promising tool for the early diagnosis.

3.2. Wound healing

Acute and chronic wounds, such as extensive burns, can cause not only physical and mental suffering in patients but also heavy financial burdens. Treatments focused on accelerating the wound healing process are urgently needed. Recent studies have demonstrated that adipose tissue can create a henotic microenvironment to benefit wound healing57 (Fig. 1a), which is in accordance with the result that the speed and quality of wound healing were significantly improved when treated with adipose tissue extract.6 Furthermore, Campbell et al.58 used conditioned medium from adipose tissue to co‐culture with fibroblast feeder cells and keratinocytes and gained an elevated formation of cultured epidermal autografts. In vitro study showed that the conditioned medium can significantly enhance ADSCs proliferation and induce cell sprouting of endothelial cells.16 More precisely, in haemostasis phase, platelet‐derived growth factor (PDGF), transforming growth factors A1 and 2 (TGF‐A1 and TGF‐2), epidermal growth factor and insulin‐like growth factors in the conditioned medium can induce endothelial cells and platelets to form clot and branches.59, 60 TGF‐β, TNF‐α, FGF and IL‐1 can activate keratinocytes, leucocytes, neutrophils and macrophages to remove foreign materials, bacteria, dead cells in inflammation phase.59, 61 When in proliferation phase, factors such as interleukins, VEGF, bFGF, PDGF and angiogenin can promote the matrix production and angiogenesis.61, 62 Finally, in remodelling phase, PDGF, TGF‐β, α‐smooth muscle actin and FGFs play a leading role in matrix formation and tissue remodelling.63, 64 Above all, conditioned medium, a cell‐free extract, can be involved in the whole process of wound healing and exhibit a promising potential on wound healing.

Figure 1.

The mechanisms of main therapeutic applications of the conditioned medium from adipose tissue. (a) Factors such as VEGF, PDGF and α‐SMC accelerate the processes of wound healing by activating cells such as endothelial cells, neutrophils and macrophages. (b) Factors like adiponectin promote the pre‐adipocytes to differentiate into adipocyte, which contribute to the adipose tissue regeneration. (c) Factors in the conditioned medium influence the glycometabolism of liver and muscle, which attributes to the occurrence of insulin resistance. (d) Conditioned medium cause endothelial dysfunction, vascular inflammation and plaque formation, which are the main characters of cardiovascular diseases

3.3. Adipose tissue regeneration

Reconstructions of soft tissue defects caused by trauma, tumour ablation or congenital abnormalities need a good deal of adipose tissue substitutes to replace the lost soft tissue. Traditionally, tissue regeneration needs three crucial components: stem cells, biomaterial scaffolds and the microenvironment. Plenty of studies focused on the effects of ADSCs in adipose tissue regeneration.65, 66, 67 But the traditional adipose tissue regeneration was limited by the insufficient generated volume, time‐consuming isolation procedures and the potential tumourigenic feature of the stem cells.68 As indicated in the following recent studies, the contribution of stem cells to regenerated tissue is limited, and that the stimulation of local healing processes does play an important role.69, 70, 71, 72 Sarkanen et al. found a novel cell‐free extract from mature adipose tissue (ATE), a form of conditioned medium from adipose tissue, contains various adipogenic and angiogenic factors and have a beneficial effect on adipose tissue regeneration both in vitro and in vivo2, 5 (Fig. 1b). Lu et al.8 also demonstrated that the application of ATE can significantly improve the regeneration of adipose tissue in an engineering chamber. Additionally, they found the injection of ATE can up‐regulate the expression of angiogenic growth factors (such as VEGF and bFGF), which results in an early vascularization to provide nutrients, oxygen and recruit stem cells.8, 73 This approach provides us with new avenues for adipose tissue engineering.

3.4. Metabolic diseases

The prevalence rate of obesity has rapidly and globally increased in the last 20 years and has reached epidemic proportions.74 It is well accepted that adipose tissue is an organ for lipid storage and mobilization. So, adipose tissue has been extensively used as a model system to study metabolic diseases, such as obesity, diabetes and dyslipidaemia. The discovery of the endocrine function of adipose tissue has provided new insights into the underlying mechanisms of many metabolic diseases (Fig. 1c). Vaspin, a serine protease inhibitor produced by adipose tissue, is considered as a potential insulin‐sensitizing factor.69 Studies in mice have shown that recombinant vaspin injection can lead to euglycaemia and improve insulin sensitivity.70 Omentin, which is synthesized mainly by adipose tissue stromal cells, is involved in metabolic process and show a significant inverse relation with metabolic syndrome due to its anti‐inflammatory, anti‐atherogenic and anti‐diabetic properties.75, 76 Decreased omentin expression in adipose tissue are always associated with overweight and obesity, as well as with insulin resistance and diabetes.71, 72 Besides, fibroblast growth factor 21 (FGF21), dipeptidyl peptidase 4 (DPP4) and adipocyte fatty acid‐binding protein 4 (FABP4) also show tight relationship with metabolic diseases.77, 78, 79, 80, 81, 82 All the above findings show that the secretory factors may serve as a potential target for the cure of various metabolic diseases.

3.5. Cardiovascular diseases

Metabolic syndrome is usually associated with risk factors for cardiovascular diseases, such as glucose and lipid metabolism impairment and atherosclerosis.83 The secretory factors of adipose tissue have also been found to play a role in the pathological processes (Fig. 1d). Sharma et al.84 found that in obese subjects, by modulating vasculature, metabolism and inflammatory through secretory factors, adipose tissue can negatively affect metabolic situation and cardiovascular function. Specifically, adipose tissue located on the surface of the heart surrounding large coronary arteries (such as epicardial perivascular adipose tissue, PVAT) has a close relationship with coronary artery disease through the production of vascular reactivity factors (leptin, resistin, TNF‐α, adiponectin, visfatin and omentin).85, 86, 87 Many studies show the relationship between PVAT inflammation and vasa vasorum neovascularization and consequently, endothelial dysfunction and atherosclerosis.88, 89 While Gao et al. focused on the role of PVAT in vascular tone and wall remodelling, they also found that PVAT regulate vascular tone by releasing different adipokines which is location and species specific, such as hydrogen peroxide, hydrogen sulphfide, adiponectin, leptin and methyl palmitate.90, 91, 92, 93 Furthermore, current studies have demonstrated that PVAT can play a protective role of vascular remodelling by the secretion of vasodilator adipokines such as adiponectin.94 Antonopoulos et al.95 found the expression level of adiponectin produced by PVAT of internal mammary artery (IMA) is associated with the level of NADPH oxidase activity in IMA and this crosstalk could be deficient in patients with atherosclerosis. Ricardo et al.96 showed that the low ORM (epicardial adipose tissue releases orosomucoid) levels have a protective role against hypoxia‐induced apoptosis in H9C2 cells.

4. Conclusions and prospects

There is overwhelming evidence that conditioned medium, a cell‐free bioactive substrate, plays a pivotal role in various pathophysiological processes through the autocrine/paracrine mechanism. With the development of protein detection and analysis technology, such as SingalP, SecretomeP and TargetP, more and more novel proteins have been discovered and studied. By now, the secretion productions of adipose tissue have provided us an abundant source for the identification of biomarkers associated with diseases like obesity and T2DM. Moreover, the beneficial effect of conditioned medium played in wound healing and regeneration also provide new insights into regenerative medicine. All these studies showed the tremendous potential application prospects of conditioned medium from adipose tissue. Combined with the abundant sources and time‐saving procedures, conditioned medium holds a great potential to be widely applied to biomedicine. As the disequilibrium among the adipokines can be found in various pathological processes, restoring the balance among the adipokines could represent a therapeutic strategy to treat or prevent many diseases. However, as a lack of the establishment of standardized and effective isolation and detection methods, the quality of conditioned medium in various samples in different studies and the results become incomparable. It is not conductive to summarize the findings to get a further understanding of the conditioned medium. A unified standard is strongly needed to make the findings unitive and comparable. In addition, as the complex composition of conditioned medium, an important step in this direction would include the study of each component, underlying mechanism in specific conditions, potential diagnostic and therapeutic applications. Maybe in the near future, the conditioned medium from adipose tissue can be made into a commercial product off the shelf and benefit various patients.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.